Instructions for use: the html version works with all tools of your browser. For searching press Ctrl-F, Back brings you to previous view, etc. Images with high resolution will expand upon a mouse click.

ISBN 978-80-260-6721-4

Copyright© 2014 Czechoslovak Microscopy Society

No parts of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means without the written permission of the publisher, except as stated below. Single photocopies of abstracts may be made for private study or research. Illustrations and short extracts from the text of individual abstracts may be copied provided that the source is acknowledged and permission of the authors has been obtained.

The publishers have made every effort to reproduce the figures at optimum contrast and with optimum resolution from the original material provided by the authors. The publishers accept no liability for the loss of image quality resulting from supply of low quality material or improperly prepared digital records. The content of the individual abstracts remains the responsibility of the respective authors.

The abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with consent of the author.

 

Table of content

↑ Go to the Table of content ↑

Plenary lectures

↑ Go to the Table of content ↑
Type of presentation: Plenary

IMC-PL-6095 Light Microscopy at the Nanoscale

Cremer C.1,2
1Superresolution Microscopy, Institute of Molecular Biology (IMB), Mainz, Germany, 2Kirchhoff Institute of Physics (KIP), and Institute of Pharmacy&Molecular Biotechnology (IPMB) University Heidelberg, Heidelberg, Germany
c.cremer@imb-mainz.de

Novel developments in optical technology and photophysics made it possible to radically overcome the diffraction limit (ca. 200 nm laterally, 600 nm along the optical axis) of conventional far-field fluorescence microscopy. Presently, three principal “nanoscopy” families have been established: “Nanoscopy” based on focused laser beams, like 4Pi-, STED- (STimulated Emission Depletion)-, and RESOLFT- (Reversible Saturable OpticaL Fluorescence depletion Transitions) microscopy; nanoscopy based on Structured Illumination Excitation (SIE), like SMI (Structured Modulated Illumination) microscopy, SIM (Structured Illumination Microscopy) and PEM (Patterned Excitation Microscopy); and nanoscopy based on various modes of Localization Microscopy, like PALM (PhotoActivated Localization Microscopy) and FPALM (Fluorescence Photoactivable Localization Microscopy), GSDIM (Ground State Depletion Imaging Microscopy), SPDM Spectral Precision Distance/Spatial Position Determination Microscopy), STORM (STochastic Optical Reconstruction Microscopy) and dSTORM (direct STORM). These and related far-field light microscopy methods have opened an avenue to image nanostructures down to single molecule resolution; they made possible to measure the size of molecule aggregates of few tens of nm diameter and to analyze the spatial distribution of individual molecules with a light optical resolution down to the few nanometer range, corresponding to ca. 1/100 of the exciting wavelength. Application examples obtained by focused, structured, and localization techniques cover a variety of biostructures, such as membrane complexes, neuronal synapses, cellular protein distribution, nuclear nanostructures, as well as the “nanoimaging” of individual viruses and lithographically generated nanostructures. Each of the nanoscopy methods described has its peculiar advantages; as a whole, they provide a tool set of light microscopy approaches to the nanoscale and open a wide range of perspectives in Biology, Medicine and the material sciences. Further improvements are expected to make possible a three-dimensional lightoptical resolution down to the 1 nm scale. The combination with Electron- and X-ray microscopy techniques is anticipated to provide further nanostructural insights.

C. Cremer, Optics far Beyond the Diffraction Limit: From Focused Nanoscopy to Spectrally Assigned Localization Microscopy (2012). In: Springer Handbook of Lasers and Optics, 2nd edition (F. Träger, Edit.), pp. 1351 – 1389.
C. Cremer, B.R. Masters (2013) Resolution enhancement techniques in microscopy. Eur. Phys. J. H 38: 281–344.

↑ Go to the Table of content ↑
Type of presentation: Plenary

IMC-PL-6096 Bioimaging at the nanoscale -- Single-molecule and super-resolution fluorescence microscopy

Zhuang X.1
1Department of Chemistry and Chemical Biology, Department of Physics, Howard Hughes Medical Institute, Harvard University, Cambridge
zhuang@chemistry.harvard.edu

Dissecting the inner workings of a cell requires imaging methods with molecular specificity, single-molecule sensitivity, molecular-scale resolution, and dynamic imaging capability such that molecular interactions inside the cell can be directly visualized. Fluorescence microscopy is a powerful imaging modality for investigating cells largely owning to its molecular specificity and dynamic imaging capability. However, the spatial resolution of light microscopy, classically limited by the diffraction of light to a few hundred nanometers, is substantially larger than typical molecular length scales in cells. Hence many subcellular structures and dynamics cannot be resolved by conventional fluorescence microscopy. We developed a super-resolution fluorescence microscopy method, stochastic optical reconstruction microscopy (STORM), which breaks the diffraction limit. STORM uses single-molecule imaging and photo-switchable fluorescent probes to temporally separate the spatially overlapping images of individual molecules. This approach has allowed multicolor and three-dimensional imaging of living cells with nanometer-scale resolution and enabled discoveries of novel sub-cellular structures. In this talk, I will discuss the general concept, recent technological advances and biological applications of STORM.                                                                                                                                                                                                                                                                                                                                                                               

↑ Go to the Table of content ↑
Type of presentation: Plenary

IMC-PL-6097 Imaging and Spectroscopy of Individual Atoms in Nanostructured Materials

Suenaga K.1
1National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan
suenaga-kazu@aist.go.jp

It has remained a challenge for scientists to image and discriminate individual atoms since Dalton first proposed distinct properties of atoms in his atomic theory. The requirements to analyze the atomic structures of matter with elemental information are nowadays increasing in importance of cutting-edge research. An elemental analysis down to the single atom limit was first demonstrated with the successful detection of a Gd dopant atom in carbon nano-peapods using a STEM-EELS technique at 100kV [1]. Specimen damage due to the high dose of the incident electron beam, which is required to isolate the signals from individual atoms, is an intrinsic problem for such a highly delicate analysis. Furthermore it is important to prevent the atoms from being kicked out during the observations. In order to reduce the atomic movements and also to enhance the EELS contrast, a lower accelerating voltage is preferred for single atom detection by STEM. Sawada et al. designed a new type of aberration corrector with triple dodecapole elements (the delta system) to reduce the higher-order geometric astigmatism [2, 3, 4], which is critical for the STEM performance operated at low accelerating voltages, i.e., 15 to 60 kV. Here, I demonstrate successful single-atom imaging and spectroscopy in nanostructured materials using STEM together with EELS and/or EDX.

Fig. 1 shows an example for chemical analysis of individual molecules. A carbon nanotube encapsulating two different metallofullerenes (La@C82 and Ce@C82) is examined at 30 kV operating voltage [5]. The annular dark-field (ADF) image clearly shows the molecular structures encapsulated inside the SWNTs (Fig. 1a). Each molecule carries one metal atom, appearing in brighter contrast, inside the cage. We can identify these atoms by simultaneous EELS. Fig. 1b shows two EELS spectra recorded from two atoms. The EELS spectrum shown in green corresponds to the atom indicated by the green arrow. This spectrum is the sum of four spectra, each of which had an acquisition time of 0.05 s. The resulting signal-to-noise ratio is high enough to isolate the La N-edge. On the other hand, the atom indicated by the blue arrow is assigned as Ce. Moreover, its peak position (≈122 eV) fits very well with that for Ce3+ [6]. Though the two edges of La N and Ce N overlap severely, we could identify the elements (La: Z = 57 and Ce: Z = 58) comprising the two encaged atoms. Fig. 1c shows the ADF image, and the elemental mappings for La, Ce, and carbon are shown in Figs. 1d, e, and f, respectively. A further comparison of simultaneous EELS and EDX measurement allows us to directly estimate the fluorescent yield of single atoms [7, 8].
The interrupted periodicities of 2D materials such as graphene, h-BN, and MX2 (dichalcogenides) are of great interest because they govern the physical/chemical properties. Atomic defects, such as a vacancy or impurity/dopant in single-layered materials are investigated with atomic precision. A single-layer of MoS2 exhibits interesting physical properties. The electrical conductivity of MoS2 can be further modulated by doping, such as Re (n-type) and Au (p-type). Typical ADF images of single-layered Re-doped and Au-doped MoS2 are presented in Fig. 2, respectively. The dopants, Re (Z = 75) and Au (Z = 79), appear in brighter contrast in the ADF images than both Mo (Z = 42) and S (Z = 16). Chemical analysis by means of EDX was also done to confirm the doping elements [9]. ADF image in the inset of Fig. 2(left) clearly shows that Re atoms sit at the Mo sites. The Re dopants are well dispersed in MoS2 layers and seldom form clusters on the host material. On the other hand, the Au dopants at similar concentration tend to aggregate on the MoS2 surface (Fig. 2 right). The Au atoms are indeed mobile under the electron beam [9].
A monovacancy in h-BN can be also examined by STEM-EELS (Fig. 3). Core-level spectroscopy on the nitrogen atoms in the vicinity of the boron vacancy was carried out [10]. As shown in Fig. 3a, a monovacancy is induced at the boron site by the knock-on effect, which can be proved by the fact that the darkest contrast appears in the middle of three nitrogen atoms showing brighter contrast. A line spectrum is recorded across the VB (boron monovacancy) along the yellow arrow. From the line spectrum, three typical spectra for the nitrogen K-edge were extracted, with probe positions corresponding to the yellow circles in Fig. 3b. While the first and third spectra are quite similar to the one for the sp2-bonded nitrogen atoms in h-BN with the known * peak at 401 eV, the second spectrum recorded near the VB indeed shows a sharp pre-peak around 392 eV. Although the spectra are rather noisy because of the minimized acquisition time, this pre-peak appears at the same energy level in many different experiments, and arises reproducibly at other VB sites and represents the lowered LUMO state [10].
Identification of individual atoms and examination of their electronic properties in materials are the ultimate goals of all microscopy-based analytical techniques. It is clear that the bonding/electronic states are now accessible from single atoms through EELS fine-structure analysis. For example the radical carbon atoms at the graphene edge have been successfully identified [11, 12, 13]. Moreover the active point defects in 2D materials can now be caught red-handed [14, 15, 16]. I will also show some of the atomic level observations of alloying behavior and phase transition phenomenon of 2D materials, that used to be investigated only by the macroscopic viewpoint [17, 18].                                                                                                                                                                              

References:
[1] K. Suenaga et al., Science, 290 (2000) 2280-2282
[2] H. Sawada, et al., J. Electron Microscopy, 58 (2009) 341-347
[3] H. Sawada, et al., Ultramicroscopy, 110 (2010) 958-961
[4] T. Sasaki, et al., J. Electron Microscopy, 59 (2010) S7-S13
[5] K. Suenaga, Y. Iizumi and T. Okazaki Eur. Phys. J. Appl. Phys., 54, 33508 (2011).
[6] K. Suenaga et al., Nature Chem., 1 (2010). 415-418
[7] K. Suenaga, et al., Nature Photonics, 6 (2012) 545-548
[8] L. Tizei et al., (in this conference)
[9] Y. C. Lin et al., Adv. Mater., (2014). DOI:10.1002/adma.201304985
[10] K. Suenaga, H. Kobayashi, and M. Koshino, Phys. Rev. Lett., 108 (2012). 075501
[11] K. Suenaga and M. Koshino, Nature 468 (2010) 1088-1090
[12] J. Warner et al., Nano Lett., 13 (2013) 4820-4826
[13] J. H. Warner et al., (unpublished)
[14] K. Suenaga et al., Nature Nanotech., 2, 358-360 (2007).
[15] Z. Liu et al., Nature Commun., 2, 213 (2011).
[16] O. Cretu, Y. C. Lin and K. Suenaga, Nano Lett., 14 (2014) 1064-1068
[17] D. O. Dumcenco et al., Nature Commun. 4 (2013) 1351 (5 pages)
[18] Y. C. Lin et al., Nature Nanotech., in press, (2014).

The present research is supported by a JST-CREST and Research Acceleration Programs. All my colleagues in AIST, Y.C. Lin, O. Cretu, L. Tizei, Z. Liu, M. Koshino, Y. Sato, and R. Senga, are gratefully acknowledged. Drs. H. Sawada, T. Sasaki, M. Mukai, Y. Kohno, M. Morishita and K. Kimoto are also acknowledged for the development of dedicated microscopes.

Fig. 1: Single molecular spectroscopy of mixed peapods (La@C82 and Ce@C82) at 30 kV[5]. (a) An ADF image with a rectangle showing where the spectrum image was taken. (b) Two EELS spectra recorded from two metal atoms. The atom indicated by the green arrow is assigned as La, and the other, indicated by the blue arrow, as Ce. (c) ADF image and (d, e, f) chemical maps for La, Ce, and carbon, respectively. Scale bar = 1 nm.

Fig. 2: Detection of single dopant atoms in single-layered MoS2[7]. (Left) An ADF image of Re-doped MoS2. The Re substitution at Mo site (Re@Mo) is pointed by a green arrow. (Right) An ADF images of Au-doped MoS2, where an Au adatom (indicated by a white arrow) located at the hollow-center (Au-HC). Scale bar = 0.3nm.

Fig. 3: Core-level spectroscopy of monovacancy in h-BN layer [8]. (a) ADF image shows a monovacancy in single-layer h-BN. Line spectrum was recorded along the yellow line. (b) Schematic presentation (red: nitrogen, blue: boron) of boron monovacancy. (c) Nitrogen K-edge fine structures extracted from the line-spectrum. Each of the three corresponds approximately to the probe positions marked in (b). A prominent pre-peak in the nitrogen K-edge can be found at 392 eV in the spectrum recorded at position 2, i.e., near the boron vacancy site.
↑ Go to the Table of content ↑
Type of presentation: Plenary

IMC-PL-6098 Electron Tomography for Nanoscale Materials Science

Midgley P.1
1Department of Materials Science and Metallurgy, University of Cambridge, Cambridge, UK
pam33@hermes.cam.ac.uk

The nanoscale complexity of modern materials and devices, be they structural or functional in design, requires high spatial resolution characterisation in all 3 dimensions. The remarkable power and flexibility of a modern TEM makes it the ideal tool for such 3D nanoscale imaging and analysis. Over the past 15 years or so, electron tomography (3D imaging) has grown from a niche technique to one which is now firmly established as an almost routine tool for the 3D study of materials. Early electron tomography used many of the ideas and practices established first in the life sciences. Here, a tilt series of bright-field (BF) images are acquired by rotating the sample about a single axis and recording images every 1-2°. Typically, in the electron microscope, the range of sample tilt is limited either by the sample itself (becoming too thick) or by the objective lens pole piece gap. As such, it is therefore likely that the full tilt range is not accessible, this leads to a ‘missing wedge’ of information and the reconstructions suffer from artefacts, especially an elongation parallel to the optic axis. Dual axis tomography can help in this regard, reducing the missing information through a second tilt series about an axis mutually perpendicular to the first.

For many materials problems, however, BF images may not be ideal and the introduction of STEM HAADF tomography offered materials microscopists an imaging mode that, in many cases, is much more suited for tomography, providing images with greatly reduced diffraction contrast, with a signal that in most cases varies monotonically with thickness (satisfying the projection requirement) and providing compositional contrast through the atomic number (Z) dependence of the high angle (Rutherford-like) scattering [1]. STEM tomography has now become for many the technique of choice for 3D nanoscale imaging in materials science. Fig. 1 shows two examples of STEM HAADF tomography [2,3]. In Fig. 1(a) we see Ge precipitates within an Al-rich matrix revealing a wide variety of morphologies and clear orientation relationships and in (b) the 3D distribution of Ru-Pt catalyst nanoparticles (1-2nm in size) decorating the surface of a mesoporous silica support – here we see only the external surface. The colour of the support indicates the surface curvature with a strong preference of the nanoparticles to be anchored at the ‘saddle points’. STEM tomography (both BF and ADF) has also been developed for the study of defects (especially dislocations) where the reconstruction (or 3D representation) of the dislocation resembles a ‘string’ running through space.

Although determination of the 3D morphology of materials at the nanoscale is now essentially routine, to achieve a high fidelity reconstruction typically ca. 100 images are needed across the tilt range. For many specimens long acquisition times, and thus extended exposure, can lead to damage. However, the number of images required in the tilt series can be reduced if there is prior knowledge about the specimen being reconstructed. Such prior knowledge can be used within a discrete tomography reconstruction (using the physical discreteness of the sample) or, perhaps more generally, within a compressed sensing framework where the primary requirement is that the sample may be described as being ‘sparse’ in some transform domain [4,5]. This sparsity constraint turns out to be very powerful and high fidelity reconstructions can be achieved with remarkably few images (in some cases an order of magnitude reduction compared to conventional reconstructions), see Fig. 2.

Coupling tomography acquisition with analytical techniques, such as EDX and EELS, allows a more detailed exploration of the sample’s chemistry as well as its morphology. Early efforts in this direction included the use of EFTEM, especially using the low loss regime (where loss probability is relatively high), EDX and core-loss EELS. Inevitably, although the speed and efficiency of spectrometers has improved greatly over the past few years, the acquisition time needed for multi-dimensional ‘spectrum-images’ is considerably higher than a conventional image. To keep the total exposure to a reasonable level, fewer images are recorded in the tilt series – ideally perhaps only every 10 or 20°. The reduction in data must be compensated by an increase in prior knowledge to achieve a high fidelity reconstruction; for such ‘multi-dimensional microscopy’ [6], compressed sensing offers an important framework to achieve this. As an example, Fig. 3(a) shows a composite figure illustrating the localised surface plasmon resonances from a silver nanocube. The reconstruction was undertaken on a series of spectrum-images recorded about a single tilt axis every 15°. The 4mm symmetry of the cube-substrate system was imposed at the reconstruction stage as well as a constraint that the reconstruction could be considered as being sparse in a wavelet domain. That constraint provided a reconstruction relatively free of artefact even when using few images [7]. Interpretation of the reconstruction seen in Fig.3(a) can be made within a quasi-static approximation and related back to the potential induced by the electron beam acting back on the electron. Mapping electro-magnetic potentials is also possible using electron holography and coupled with tomography was able to yield 3D reconstructions of the built-in potential near a p-n junction in a silicon device, see Fig. 3(b) [8]. 3D magnetic fields require an enhanced approach using dual axis geometry to determine all the components of the magnetic potential A (or induction B). Here, physical constraints (e.g. in the form of Maxwell’s equations), perhaps again within a compressed sensing framework, could be used to improve a reconstruction of the electro-magnetic potential.

So, what of the future? The electron tomography community is pushing in many directions. Atomic resolution tomography has been demonstrated in some cases: by assuming periodicity within a nanocrystal, the position of an isolated atom in a matrix can be determined and even the location of atoms around a dislocation core. Synergisitc studies with atom probe tomography have been demonstrated already and this may, in the future, develop into an important correlative approach. Mapping physical properties in 3D at the nanoscale continues to be an exciting prospect. Whilst early work showed this to be feasible, further development is needed to improve reconstruction quality. Given the almost ubiquitous use now, in materials-based tomography at least, of iterative techniques (e.g. SIRT, ART, etc) the conventional projection / back-projection approach could evolve into a more model-based one incorporating a detailed description of the beam’s interaction with the sample along its trajectory (e.g. dynamical effects). By iteratively refining an initial model, increased detail about the sample may be obtainable (e.g. strain, fields, induced charges). Lastly, industry requires a robust nanoscale metrology technique that provides reliable 3D measurements of length, porosity, distributions etc. We are still some way in many cases of being able to provide such data with statistical confidence (i.e. error bars!) on our 3D measurements. Improved reconstructions, with fewer artefacts, incorporating prior knowledge, should allow improved and unbiassed segmentation and thus will go a long way to providing a true 3D nanometrology technique.                                                                                                                                                    

References:
[1] P.A. Midgley et al., Chem. Commun. (2001) 907
[2] K. Kaneko et al., Ultramicroscopy 108 (2008) 210
[3] E.P.W. Ward et al., J. Phys. Chem. C 111 (2007) 11501
[4] Z. Saghi et al., Nano Letters 11 (2011) 4666
[5] R. Leary et al., Ultramicroscopy 2013 131 70-91
[6] P.A. Midgley and J.M. Thomas, Angewandte Chemie (2014) DOI: 10.1002/anie.201400625
[7] O. Nicoletti et al., Nature 502 (2013) 80
[8] A. Twitchett-Harrison et al., Nano Letters 7 (2007) 2020

The author thanks his many colleagues, past and present, who have contributed to the work presented here including most recently J.M. Thomas, R. Leary, Z. Saghi, D. Holland, K. Kaneko, S. Hata, O. Nicoletti, F. de la Peña, C. Ducati. PAM acknowledges funding from the European Research Council under FP7/2007-2013 / ERC grant agreement 291522-3DIMAGE.

Fig. 1: (a) 3D reconstruction of Ge precipitates in an Al-rich matrix showing colour-coded to highlight theor different morpholgy [2]; (b) Ru-Pt catalyst particles (red) shown on a colour-coded silica surface where the blue regions indicate positive Gaussian curvature (saddle points) [3].

Fig. 2: (a) Comparison of reconstructions using SIRT and compressed sensing (CS) codes for an iron oxide nanoparticle with a concavity; (b) the apparent concavity volume as a function of projection number [4].

Fig. 3: (a) Colour composite figure indicating five surface plasmon modes on a silver nanoparticle, 100nm in size [7]; (b) Reconstructed electrostatic potential near a p-n junction in a silicon device showing sub-surface carrier depletion [8].
↑ Go to the Table of content ↑

IFSM symposium

↑ Go to the Table of content ↑
Type of presentation: Plenary

IFSM-PL-1670 From Atomic Structure to Properties of Oxides: Applications of Aberration-corrected Transmission Electron Microscopy

Jia C. L.1,2
1Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Forschungszentrum Jülich GmbH, Jülich, Germany, 2International Centre for Dielectric Research, Xi'an Jiaotong University, Xi'an, China
c.jia@fz-juelich.de

Functional oxides provide an important part of the material basis for multifunctional devices as a result of their exceptional range of physical properties. These properties, in turn, depend strongly on the crystal structures, chemical compositions and defect configurations of the materials, which can be characterized on the atomic scale.

In a high-resolution transmission electron microscope equipped with an aberration corrector, the spherical aberration coefficient CS of the objective lens can be tuned to either a positive or a negative value. The use of a negative value of CS combined with an overfocus setting of the objective lens is used in the negative CS imaging (NCSI) technique [1]. Images obtained using the NCSI technique show superior atomic column contrast and intensity than corresponding positive CS images [2], especially for weakly scattering oxygen columns that are in close proximity to strongly scattering cation columns.

Using the NCSI technique, we have investigated the atomic details near 180° domain walls in thin films of PbZr0.2Ti0.8O3 [3,4]. The relative displacements of ions have been measured and on this basis the local polarization across the wall has been calculated. Using this technique we have studied the atomic structure of LaO-TiO2-type interfaces in epitactic LaAlO3/SrTiO3 heterostructures [5]. The prominent result is the oxygen octahedron rotation and the TiO6 octahedra distortion induced by LaAlO3 in SrTiO3 at the interface. The cation-oxygen octahedra represent the prominent structural element of perovskites, which can be modified by distortions, rotations, and particular atomic shifts. Small atomic rearrangements as they are expected to occur at the interfaces between perovskites of different structure can change dramatically the electronic system.

We have recently used the NCSI technique to perform quantitative comparisons between experimental and simulated images on an absolute intensity basis after taking into account the effects of the modulation transfer function of the camera and additional image spread [6]. This absolute intensity matching approach not only allows atomic column positions and defect structures to be determined with picometer precision, but also allows the local chemistry and the three-dimensional morphology of a crystal to be determined on the atomic scale.

Figure 1 shows results obtained from a study of the relationship between the atomic structure and properties of BiFeO3, a room temperature multiferroic material. In the rhombohedrally-distorted perovskite unit cell of BiFeO3 (shown in Fig. 1a), characteristic structural features include relative shifts between the cations and the oxygen anions along the [111] axis and rotations of oxygen octahedra about the [111] axis, which are related to the ferroelectric polarization and the antiferromagnetic properties of the material, respectively. Both the atomic shifts and the rotations of the octahedra can be quantified using the NCSI and ACM techniques and used to understand the electrical and magnetic properties of the material. Figure 1b shows an atomic-resolution image of a 109° domain boundary (thick arrow) between two domains. The use of NCSI conditions and a particular specimen thickness result in the atomic columns appearing bright on a dark background. The domains in the material can then be distinguished by measuring the positions of the atomic columns inside individual unit cells.

In Fig. 1(b), the domain above the boundary is oriented along the [110] direction. The O column positions are shifted upward and downward (Fig. 1c), corresponding to alternating rotations of octahedra. A corresponding off-centre displacement of Fe with respect to the middle point of the line connecting two neighbouring (left and right) O positions is visible and oriented in a downward direction. In this orientation, the [001] component (red arrow) of the [111] polarization vector can be measured and the octahedron rotation can be revealed. Below the boundary (Fig. 1d), the domain is viewed along the [1 ̅10] direction. The octahedron rotation is now not visible due to the overlap of atoms (Fig. 1d). However, the full vector (red arrow) of the atomic column displacement is now revealed. In this way, the polarization of the domain can be determined unambiguously.                                                                                                                       

References:

C.L. Jia, M. Lentzen, K. Urban, Atomic-Resolution Imaging of Oxygen in Perovskite Ceramics. Science 299, 870 (2003).
C.L. Jia L. Houben, A. Thust,and J. Barthel, On the benefit of the negative-spherical-aberration imaging technique for quantitative HRTEM. Ultramicroscopy 110, 500 (2010).
C.L. Jia et al., Atomic-scale study of electric dipoles near charged and uncharged domain walls in ferroelectric films. Nature Mater. 7, 57 (2008).
C.L. Jia et al., Direct observation of continuous electric dipole rotation in flux-closure domains in ferroelectric Pb(Zr,Ti)O3, Science 331, 1421 (2011).
C.L. Jia et al., Oxygen octahedron reconstruction in the SrTiO3/LaAlO3 heterointerfaces, Phys. Rev. B 79, 081405(R) (2009).
C.L. Jia et al., Atomic-scale measurement of structure and chemistry of a single-unit-cell layer of LaAlO3 embedded in SrTiO3. Microsc. Microanal. 19, 310 (2013).

This work was carried out in collaboration with L. Jin, S.B. Mi, K. Urban, A. Thust, J. Barthel, L. Houben, M. Lentzen, D. Hesse and M. Alexe.

Fig. 1: (a) Schematic diagram of the pseudocubic unit cell of BiFeO3. (b) Atomic-resolution image of a 109° domain wall (thick arrow) separating two domains: the domain above the wall and the magnification in (c) correspond to a [110] viewing direction, while the domain below the wall and the magnification in (d) correspond to a [1 ̅10] viewing direction. The red arrows denote the polarization.
↑ Go to the Table of content ↑
Type of presentation: Plenary

IFSM-PL-1780 Some surprises in electron diffraction physics and imaging.

Spence J. C.1
1Physics Department, ASU, Tempe , Az. USA. 85282, and LBNL USA.
spence@asu.edu

The multiple scattering theory on which modern electron microscopy (EM) is based had been fairly well worked out by about 1960, following work by Bethe, Sturkey, Heidenreich, Hirsch, Howie, Whelan, Cowley and Moodie and others. Nevertheless many surprises remained in the ensuing 50 years. For me the most important of these have been i) The finding that multiple energy-loss effects can be removed from EELS spectra, using earlier work on cosmic ray showers. ii) The richness of the "point-projection" geometry, championed by Gabor in 1949. In turn this has produced Ptychography, the theory of STEM lattice imaging for crystals and low-voltage field-emission point-projection imaging. It is remarkable that coherent overlapping convergent beam orders provide a solution to the phase problem, an atomic-resolution "shadow image", Talbot self-imaging, and in-line holography. iii) The discovery of "forbidden" termination reflections and their value for imaging surfaces and sub-surface dislocations and kinks. Their monolayer sensitivity is remarkable. iv) The detection of coherent bremstrahlung tunable X-ray emission lines in STEM EDX. It is remarkable that these lines can be indexed, and are absent when reflections are forbidden by symmetry. v) The explanation for dynamically forbidden reflections, which cancel due to symmetry-related paths for all thickness. vi) The usefulness of electron channelling effects (Alchemi) on EDX for locating foreign atoms in several fields (turbine blades, mineralogy), previously an academic curiosity. vii) The achievement of aberration correction. viii) The success of our TEM CCD camera, whose impact on cryo-em tomography we never anticipated. ix) The surprising sensitivity of low-angle scattering to atomic bonding, with the zero-order scattering the most sensitive quantity known. x) The finding that sufficiently short pulses of radiation can outrun radiation damage, thus breaking the nexus between damage, resolution and particle size if a large number of particles can be packed into a near delta-function pulse. xi) The information extracted from ELS spectra, with its unrivalled spatial resolution.

  The changing agenda of EM over this half-century, from the study of bulk defects such as dislocations, and atomic resolution imaging of interfaces, to nanoscience, cryo-electron and in-situ microscopy (liquid cells, catalysis) has been fascinating to watch. Recent developments - atomic resolution imaging with characteristic X-rays, direct injection detectors, sub-Angstrom resolution, high-resolution imaging in 3D, fast diffraction and imaging - continue to surprise. References in: High Resolution Electron Microscopy (Spence, 4th ed. 2014) and Electron Microdiffraction (Spence & Zuo, 1992).

To many colleagues and friends over half a century in many countries, and to the US funding agencies and Arizona State University.

↑ Go to the Table of content ↑
Type of presentation: Plenary

IFSM-PL-6099 Macromolecules in Motion: Visualization by 4-Dimensional Cryo-Em

Steven A. C.1
1Laboratory of Structural Biology Research, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, USA
stevena@mail.nih.gov

Cryo-electron microscopy offers a unique capability to determine the 3-D structures of macromolecular complexes. However, insight into biological activity requires understanding the structural transitions that the complex of interest undergoes. It is not possible, even in principle, to visualize the same molecule in successive states as this would involve the prohibitively difficult task of thawing the specimen, inducing the conformational change, re-vitrifying and re-locating the molecule. However, dynamics may be addressed by a statistical approach in which classification techniques are applied to data sets imaging conformationally mixed populations. Then, provided that there is a basis for ordering the various conformers in a temporal sequence, the reaction dynamics of the complex may be described and movies made.

The process of virus maturation is amenable this approach. With many viruses, the precursor particle undergoes radical structural changes as it matures into an infectious virion. We have investigated the maturation of bacteriophage HK97 capsid, an icosahedrally symmetric shell composed of 420 protein subunits, which expands from 45 nm to 55 nm and angularizes as it matures. These changes in morphology reflect large rotations of the protein subunits and local remodeling (1), and the pathway proceeds via three metastable intermediates (2). The capsid of herpes simplex virus, an animal virus, follows a similar pathway, which may be traced to a capsid protein domain similar in structure to that of HK97, but it passes through many more intermediates (3). Recently, we have found that bacteriophage phi6, which has a RNA genome rather than a DNA genome and an entirely different capsid protein fold from HK97, also undergoes massive subunit rotations and matures via two intermediates (4, 5) – Figure 1.

For this approach to visualization of conformational dynamics, several conditions must be met (6). The differences between states must be large if differences in the images that arise from viewing geometry are to be separated from real structural differences. The number of distinct conformational states must be relatively small. To establish a time-line, one must be able to induce the reaction of interest on a time-scale of seconds to hours. Notwithstanding, recent technical advances in automated collection of large data sets, the improved resolution and signal-to-noise ratio of direct detection cameras, and sophisticated classification techniques promise to expand the range of applicability.                                                                                                                                            

References

1. J.F. Conway et al. Science 292: 744-748 (2001)
2. R. Lata et al., Cell 100: 253-263 (2000)
3. J.B. Heymann et al., Nature Struct. Biol. 10, 334-341 (2003)
4. D. Nemecek et al., J. Mol. Biol. 414, 260-271 (2011)
5. D. Nemecek et al., Structure 21, 1384-83 (2013)
6. J.B. Heymann et al., J. Struct. Biol. 147, 291-301 (2004)

I thank many colleagues, particularly Drs N. Cheng, J.F. Conway, J.B. Heymann and D. Nemecek. This research has been supported by the intramural research program of NIAMS/NIH.

Fig. 1: Maturation dynamics of bacteriophage phi6 capsid visualized by 4-dimensional cryo-EM. The pathway progresses through four states: the initially assembled procapsid with its deeply indented facets; two expansion intermediates with near-planar facets; and the spherical mature nucleocapsid. The capsid is built from 60 P1A/P1B dimers, where P1A and P1B are chemically identical but conformationally distinct (non-equivalent) protein subunits. The top row shows renderings of the outer surfaces viewed along a 5-fold symmetry axis. The middle row shows models of the respective capsids consisting of a pentamer of P1A subunits (blue, green) and surrounding P1B subunits (red, yellow), viewed from above. The bottom row shows slabs through a portion of the structures, passing through one vertex (P1A’s blue, P1B’s blue). The procapsid-to-intermediate 1 transition is achieved by rotations of P1B subunits about the line connecting two 3-fold icosahedral axes. Further expansion to intermediate 2 is achieved by outward movement of the P1A subunits. The final step to nucleocapsid involves primarily local changes affecting the P1A subunits. Adapted from reference (5), where movies of the transition are available, courtesy of J.B. Heymann. Scale bars: 100 Å.
↑ Go to the Table of content ↑
Type of presentation: Plenary

IFSM-PL-6100 From the Prague Spring to a Spring in Electron Microscopy

Křivánek O. L.1
1Nion Co., Kirkland and Dept of Physics, ASU, Tempe, USA
krivanek@nion.com

Prague is my native city: I was born in Praha-Bubeneč, on the plateau behind the Prague Castle. I grew up in the era of the Czechoslovak Socialist Republic (ČSSR), when the Soviet Union and its satellites prided themselves on their space exploits and their education systems. Among the special efforts they made were competitions for talented youngsters in mathematics and physics, and I used to enjoy those. In my senior high school year, I qualified for the national round in both math and physics, and in physics I was invited onto the national team of three that represented Czechoslovakia at the 2nd International Physics Olympiad, held in Budapest in June 1968. Back then only the Soviet Union and its satellites participated – Western Europe, USA and other countries joined the Physics Olympiad later. Our team did well: we got a joint second place with the Hungarians and the East Germans, with the Soviets winning the first place. I have since then had the pleasure of working with one other former International Physics Olympian – Niklas Dellby, my partner at Nion.

That same summer I took the entrance exam to Charles University in Prague, to study physics. I passed and promptly took off on a trip I had planned: a vacation in the south of France, followed by a stay in London where I was planning to work in a summer job while improving my English. 1968 was the year of the famous Prague Spring, when “socialism with a human face,” which included many democratic measures, was introduced by a group of reformers led by Alexander Dubček (Fig. 1a), much to the displeasure of the old guard in the Kremlin. As I was boarding the train to France, my father told me: “If the Soviets invade, stay in the West.” I had not been following the political situation very closely, so this came as a surprise instruction to me. The Soviets invaded 4 weeks later (Fig. 1b), while my whole family happened to be in the West: my parents on vacation in Austria, my sister working in a summer job in France and me working as an office helper in London. We got together on the phone, and decided that none of us would go back to Prague, at least not for the time being. (See [1] for an especially lucid account of the Prague Spring.)

People were very sympathetic to citizens of a small country invaded by Soviet tanks, and the British National Union of Students had a special place in its London office for notices of available openings for prospective Czech and Slovak students. I was checking it daily while working in a new job, as a carpenter. Around the end of September, a small notice appeared, announcing that the University of Leeds was going to offer up to 5 scholarships to qualified Czechoslovak students. I called them up and caught the train to Leeds soon thereafter. There was an entrance interview during which it became clear that I knew my physics all right, and also that 3 years of high school English and a vocabulary of perhaps 3000 English words were not nearly enough for me to slot painlessly into the British university system.

Leeds took a chance on me, and at first they must have wondered how it would turn out. In my first year I got a First in math – understanding equations did not require much English – but only a Pass in physics, in which there were long textbook passages that I studied laboriously, with a dictionary in hand. I did better in later years, graduating with a First, at the top of my class. I was then accepted to do a physics Ph.D. in Cambridge, with Archie Howie as my inspiring supervisor. In my first year, our lab was not far from Ellis Cosslett’s, after whom the award I received is named, and who has been one of my heroes in electron microscopy, especially after I came to appreciate the pioneering nature of much of the work of his group.
I greatly enjoyed my time in Cambridge, both inside and outside the Physics Department. I learned a lot, made many friends, and made good use of Cambridge’s excellent extra-curricular facilities. I raced for Cambridge against Oxford in skiing and won the special and parallel slaloms at the 1975 Oxford-Cambridge ski race, in the Italian Dolomites. The 8-man boat crew I joined the previous spring (Fig. 1c) did three bumps and an overbump in the Cambridge May Races, and by Cambridge tradition, we got to keep our oars as souvenirs.

After Cambridge, I worked at Kyoto University for 3 months, and did post-docs at Bell Labs and UC Berkeley, where I joined the group of Gareth Thomas in the Materials Science Department. Being in Materials Science made me feel that I had to make a choice: I could concentrate on the materials we were studying and become a materials scientist, or on the instruments and techniques we were using and remain a physicist. I had done a little instrument design work and liked it, so the second option seemed more attractive. The technique I thought was especially interesting was a new one (to me) called Electron Energy Loss Spectroscopy (EELS). I got my first taste of it at the 1978 Cornell workshop, where I met people who became lifelong friends, such as Phil Batson, Christian Colliex, Ray Egerton, and Mike Isaacson. One was expected to build one’s own spectrometer in those days – there were no commercial models. When I got back to Berkeley, I climbed the stairs to Professor Thomas’s office and said: “I think I should build an energy loss spectrometer. It will allow us to study oxygen concentrations at grain boundaries in nitrogen ceramics.” – a subject the group was focusing on. Gareth asked just one question: “How much will it cost? ”, I replied “about $10k”, and I had my first OK to build a major instrument.

The spectrometer came together quickly and produced good results (Fig. 2a). In the summer and autumn of 1979, I was showing the results at various conferences. At one of them, at NBS in Washington, Nancy Tighe came up to me and said: “I think your spectrometer would interest Peter Swann of Gatan. You should give him a call.” This started my fruitful collaboration with Peter, from whom I learned on many fronts. Peter passed away in the summer of 2013, and many of us miss him very much.

Over the next year, Peter Swann, Joe Lebiedzik and I, with input from Mike Scheinfein, designed and built a second-generation serial EEL spectrometer. I also started in a new job, as Associate Director of the NSF-funded HREM facility at Arizona State University. With my collaborators at ASU, we applied the spectrometer to many interesting problems, and put together the EELS Atlas [2] that is used to this day. ASU was a great place to work. There were many good instruments, several leading researchers in electron microscopy, and stimulating annual schools and workshops (Fig. 1d), whose organization was my responsibility.
The pull of Gatan, however, proved irresistible when Peter moved its R&D facility from Pittsburgh to California, and in 1985 I became Director of Research at Gatan. A very productive period followed, during which I had the privilege of working with many talented researchers and designers: Dan Bui, Niklas Dellby, Garry Fan, Stuart Friedmann, Sander Gubbens (the current President of Gatan), Robert Keeney, Bernd Kraus, Mike Kundmann, Mike Leber, Chris Meyer, Paul Mooney, Ming Pan, Nils Swann, Peter Swann, Marcel Tence and Jacob Wilbrink, among others. We introduced a number of innovative products, including parallel EELS, imaging filters, CCD cameras, scanned image acquisition systems and DigitalMicrograph software. Gatan grew nearly 10x in size during this time, and I learned that developing instruments commercially can be a great way to fund instrumentation research, especially when working with like-minded researchers and lean and understanding administrations.
The next big change in my scientific life came when Peter decided to retire in 1992, and “professional managers” took over at Gatan. My freedom to do interesting projects was greatly restricted, and I started to look around. It had been clear to me since about 1990 that having managed to correct the second order aberrations of the quadrupole optics of imaging filters, I had a good chance of correcting third order aberrations – a classic problem in electron optics since Scherzer’s work on the subject in the 1930s and 40s. It seemed too speculative a project for Gatan, however, and so I explored doing it elsewhere. My first try for corrector funding was a chat with Uli Dahmen, the Berkeley NCEM director, who consulted with Bob Gottschall, his manager at DOE. Bob’s answer was apparently “over my dead body.” He had gotten burned funding Crewe’s corrector attempts, which never led to a working instrument.

I was more successful persuading Mick Brown of my Alma Mater, Cambridge University, who had a spare VG cold field emission (CFE) scanning transmission electron microscope (STEM), that we should jointly build a corrector for it. We applied for funding to the British Royal Society and secured the maximum allowed amount from the Paul Instrument Fund: £80k. I then moved to Cambridge with my family for two wonderful years. Niklas Dellby and others joined the project, and we had a working proof-of-principle STEM corrector about 2 years later [3], the same summer (1997) as the Heidelberg-Julich CTEM corrector started working.
The 100 kV VG STEM we built our corrector for was older than a research student who joined the project (Andy Lupini), and it had poor aberration coefficients (Cs~Cc~3.5 mm). We improved its resolution, but we did not beat any resolution records relative to the best uncorrected instruments. (The same was true for the Heidelberg effort – 1 MV microscopes were then giving higher resolution than their corrected 200 kV CTEM.) However, a corrector of an improved design we built for Phil Batson’s extensively modified VG at IBM Yorktown Heights achieved a double distinction: it led to the first STEM able to focus an electron probe to <1 Å diameter [4], and it was, as far as I know, the first commercial corrector (delivered in June 2000).

Aberration correction soon became a “hit”, with CEOS GmbH supplying correctors to all the regular manufacturers of electron microscopes, and the company Niklas Dellby and I started near Seattle, Nion, concentrating on correctors for CFE STEM and going it alone. Our idea was a somewhat crazy one: that we could extend our prowess in correctors by designing a whole new electron microscope, and that we would do it better than the regular manufacturers. Not many thought that we would succeed. But there were early believers to whom we owe a great deal, such as John Silcox, Andrew Bleloch, Steve Pennycook and Christian Colliex. Benchmarks established subsequently by Nion for resolution, stability, probe current, ultra-high vacuum, freedom from contamination and powerful software [5,6] have persuaded many others. 

Nion’s very capable team - Niklas Dellby, Neil Bacon, George Corbin, Peggy Cramer, Zeno Dellby, Russ Hayner, Petr Hrncirik, Tracy Lovejoy, Chris Meyer, Savath Phoungphidok, Michael Sarahan, Gwyn Skone, Zoltan Szilagyi, Janet Willis, Tad Yoo and myself for now, and growing, has done some amazing things. We first delivered 10 aberration correctors for VGs, then moved onto making whole electron microscopes. Currently we’re manufacturing Nion microscopes #10-13, and the interest in our instruments is on the rise. Building the instruments has been made easier by the close collaboration we enjoy with Czech Republic’s Delong Instruments, especially Vladimír Kolařík and Petr Homolka. Nion’s progress has also been helped by two simple facts: ordering an electron microscope from a small company is a gutsy thing to do, and gutsy scientists tend to be first-rate. (Figs 2b-3a,b) and references [7-13] show some of the revolutionary results they and their collaborators have obtained with Nion microscopes.

Aberration correction has ushered in an era of electron microscopy in which we can see the structure, composition and bonding of materials better than ever before. It amounts to a new spring in electron microscopy, best captured by the words of David Cockayne: “it is as though a veil of fog has lifted from our samples.” It is about to get better still, because of an exciting new development: studying energy losses with sub-20 meV energy resolution and sub-nm spatial resolution. This has been made possible by Nion’s new monochromator [14], which has been the subject of two separate talks at this congress [15], and which promises to make vibrational excitations in materials (phonons) readily observable (Fig. 4), at a high spatial resolution. It will probably also allow hydrogen to be mapped in the electron microscope, using energy losses that accompany high-angle scattering of fast electrons by hydrogen nuclei. 

My scientific instrumentation journey began with EELS and progressed onto aberration correction and high resolution STEM. It has now come back to EELS, with an energy resolution about 100x better than on my first try. My life’s journey began in Prague, and Prague is where this congress has been held. Both journeys are reminiscent of the famous lines by T.S. Eliot [16]:                                                                                

We shall not cease from exploration,
And the end of all our exploring
Will be to arrive where we started
And know the place for the first time.                                                                                                       

So let us celebrate exploration (also known as research) and knowing where we came from. And also congresses such as IMC, which enrich our knowledge of our field, and of ourselves.                                                                                                                                                        

[1] A. Levy, Rowboat to Prague (ISBN 0-670-60920-X), reprinted as So Many Heroes (ISBN 978-0933256125). See also http://en.wikipedia.org/wiki/Alan_Levy
[2] C.C. Ahn and O.L. Krivanek, EELS Atlas (1983) Gatan and the ASU HREM facility.
[3] O.L. Krivanek et al., Proc. EMAG 1997, IOP Conf. Ser. No 153 (J. Rodenburg, ed.) 35-40.
[4] P.E. Batson, N. Dellby and O.L. Krivanek, Nature 418 (2002) 617-620.
[5] O.L. Krivanek, et al., Ultramicroscopy 108 (2008) 179-195.
[6] N. Dellby et al., The European Physical Journal Applied Physics 54 (2011) 33505 (11 pages).
[7] D.A. Muller et al., Science 319 (2008) 1073-1076.
[8] O.L. Krivanek et al., Nature 464 (2010) 571-574.
[9] T.C. Lovejoy et al., Appl. Phys. Letts 100 (2012) 154101 to 154101-4.
[10] P.Y. Huang et al., Nano Letters 12 (2012) 1081-1086.
[11] W. Zhou et al., Microscopy and Microanalysis 18 (2012) 1342-1354.
[12] Q.M. Ramasse et al., Nano Letters 13 (2013), 4989–4995.
[13] J. Lee at al., Nature communications 4 (2013) 1650.
[14] O.L. Krivanek et al., Microscopy 62 (2013) 3-21.
[15] N. Dellby et al., these proceedings and O.L. Krivanek et al., these proceedings.
[16] T.S. Elliot, Four Quartets (1943) ISBN 978-0156332255.

Fig. 1: a) Alexander Dubček, who led the Prague Spring. b) Soviets tanks rolling through Prague’s Wenceslas square. c) Rowing on the river Cam. I am in seat #5 (bow = 1), holding the oar that does not quite match the others. d) 1981 ASU meeting. Front row: Mike Isaacson, Alan Craven, John Spence, John Venables, Albert Crewe, John Cowley, Bernard Jouffrey, Ian Wardell, Ondrej Krivanek, Colin Humphreys. Spot Ray Carpenter, Mark Disko, Murray Gibson, Sumio Iijima, Kazuo Ishizuka, Masashi Iwatsuki, Charlie Lyman, Peggy Mochel, Steve Pennycook, Jing Zhu and others in the photo.

Fig. 2: a) EEL spectrum of BaTiO3 recorded with the serial EEL spectrometer I built at Berkeley, at about 2 eV resolution. b) Reversible atomic motion in monolayer graphene: one of the 6 substitutional Si atoms moves right, left, right. Nion UltraSTEM100, 60 kV, 6 s between frames. (Ref. [13])

Fig. 3: a) EEL L2,3 spectrum from a single Si atom replacing a C atom in graphene (line) and theoretical fits (solid spectra). The right fit allowed the Si atom to “pop out” 0.65 Å from the graphene plane (inset) and gave better agreement. (Ref. [12]) b) Results from Nion microscopes. Nature vol. 464 (2010) issue 7288 cover: image of BN monolayer with impurities by Matt Chisholm, processing by the author and Tim Pennycook. Angewandte Chemie vol. 50 (2011) issue 43 cover: image of MoS2 by Quentin Ramasse. Nature Materials vol. 11 (2012) issue 10 cover: EELS elemental map by Julia Mundy.

Fig. 4: 60 kV results from the Nion High Energy Resolution Monochromated EELS-STEM (HERMES). a) Spectra obtained with the slit out and in, slit-in acquisition time 0.25 s, courtesy Niklas Dellby (Nion) and Philip Batson (Rutgers U.). b) spectrum from titanium hydride (acq. time 10 s), courtesy Peter Crozier and Jiangtao Zhu (ASU), and Tracy Lovejoy (Nion).
↑ Go to the Table of content ↑

IT-1. Electron optics and optical elements

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-1-IN-2012 Advances in electron vortex experiments in the TEM

Verbeeck J.1, Béché A.1, Clark L.1, Guzzinati G.1, Juchtmans R.1, Van Boxem R.1, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
jo.verbeeck@ua.ac.be

Electrons in a transmission electron microscope are successfully described by linear combinations of plane waves. The sample and the magnetic lenses deform the wavefronts of these waves in a way that transfers information from the sample onto a detection plane. Alternatives to this plane wave basis are however possible and especially cylindrical harmonics are an interesting option. The plane waves are replaced by waves which have a typical azimuthal phase factor exp(i m φ) with φ the angle in the plane perpendicular to the optical axis and m the so-called topological charge. Such waves are orbital angular momentum (OAM) eigenstates in the sense that a normalized cylindrical wave carries exactly mħ angular momentum around the cylinder axis. These waves are often referred to as vortex waves and they attract considerable attention in many different fields of physics including optics, acoustics, radio communication and quantum information [1].
Electron vortices were theoretically predicted to possess also a quantised magnetic moment mµB on top of the common OAM of mħ due to their electrostatic charge [2]. It took untill 2010 before pure electron vortex modes were demonstrated in a transmission electron microscope [3,4]. Since then, many different ways of producing these waves have followed (see e.g. fig.1), each with different advantages and disadvantages. The latest addition, sketched in fig.2, is the production of electron vortex waves making use of a thin single domain magnetic needle approximating a magnetic monopole [5]. This method holds great promise as it offers pure vortex modes at full beam current. Apart from producing single vortex modes, we also focused on the detection of the OAM in an arbitrary wave. Several methods are possible and will be discussed. In terms of the interaction with a sample we observed magnetic dependence in EELS spectra of ferromagnetic samples relating to electron magnetic chiral dichroism and its X-ray counterpart X-ray magnetic chiral dichroism. On top of this, we will discuss the use of vortex beams in elastic diffraction and the transfer of angular momentum to rotate nanoparticles.

References

[1] J. F. Nye and M. V. Berry., Proc. of the R. Soc. of London. A. 336/1605 (1974) 165.

[2] K. Bliokh et al., Phys. Rev. Lett. 99 (2007) 190404.

[3] M. Uchida and A. Tonomura., Nature, 464/7289 (2010) 737.

[4] J. Verbeeck et al., Nature 467/7313 (2010) 301.

[5] A. Béché et al., Nat. Phys.10/1 (2013) 26.

This work was financially supported by the European Union: ERC grant 246791 COUNTATOMS, ERC Starting Grant 278510 VORTEX, Integrated Infrastructure Initiative grant 312483-ESTEEM2.

Fig. 1: Using a probe aberration corrector and an annular condensor aperture, an approximation to an azimuthally varying phase plate can be obtained. This is one of the alternative ways to create electron vortex beams.

Fig. 2: Sketch of the creation of an electron vortex beam by impinging a plane wave to the end of a long bar magnet approximating a magnetic monopole.
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-1-IN-2597 Electron Optics for High-brightness High-beam-current Column Design --- extracting micro-amperes from point cathodes ---

Fujita S.1, Takebe M.1, Wells T.2, El-Gomati M. M.3, Shimoyama H.4
1SHIMADZU Corporation, Kyoto, Japan, 2York Probe Sources Ltd., York, United Kingdom, 3University of York, York, United Kingdom, 4Meijo University, Nagoya, Japan
fujita@shimadzu.co.jp

A principal goal of designing electron probe forming system is to focus desired beam current into as small a spot on the target as possible. Increasing demand for analytical measurements is making desired beam current higher than ever (Ib>10nA). This article describes strategies to design high-brightness high-beam-current electron optical columns.

Figure 1 shows Probe Property that relates the beam current Ib to the probe size d. The dotted curve assumes as electron source a thermionic gun while the dashed curve is for conventional ZrO/W (100) Schottky emitter (SE) gun system. A higher brightness of the latter makes the probe size substantially smaller in the middle beam current regime. However, the probe blurs fast once the current exceeds a certain threshold.

Probe Property is limited by three different mechanisms with increasing beam current order:

Beam Current Regime   “low”                “middle”          “high”
Limiting Mechanisms     wavelength       brightness       angular intensity
                                        chromatic(OL)   spherical(OL)   spherical(Gun)

Attempts were made to improve “high” beam current performance by increasing the source angular intensity and suppressing the gun spherical aberration. Extended Paraxial Trajectory Method is used to analyze electron rays starting from cathode surface with large slopes [1]. The emission characteristic of SE gun is then given by optical parameters familiar in lens designs.

The first strategy is to adopt an emitter whose tip radius is significantly larger [2]. Figure 2 compares a scaled-up emitter (giant SE = GSE) with a conventional SE. The tip size effect is reflected in “electron gun focal length,” f. The angular intensity is given by JΩ = f2*js where js is the cathode current density. Since the focal length is roughly proportional to the tip size, a large tip leads to an improved angular intensity.

The second strategy is to immerse the emitter in the condenser lens field, which is known to result in a suppressed spherical aberration.

Figure 3 compares the source emittance diagrams of conventional SE and GSE. GSE’s wide and less-distorted diagram demonstrates its high-beam-current capability. It is expected GSE’s improved emittance extends the “middle” beam current regime to Ib ~ 1μA (see Fig.1).

A test column was constructed by combining the GSE gun with an objective lens designed for efficient X-ray detection. SEM image observations at Vacc = 10kV over beam current range 100pA <Ib< 3μA confirmed semi-quantitatively the predicted probe property given in Fig.1.

[1] S.Fujita, M.Takebe, W.Ushio and H.Shimoyama, J.Electron Microsc. 59, 3 (2010).
[2] S.Fujita, T.R.C.Wells, W.Ushio, H.Sato, and M.M.El-Gomati, J.Microsc. 239, 215 (2010).

The authors thank Shimadzu Corporation for the support of this work as well as for the permission of the publication.

Fig. 1: Probe Properties with three different electron sources. Expected probe size is plotted against the beam current.

Fig. 2: Comparison of tip geometries of conventional SE and scaled-up emitter (GSE). Angular intensity can be increased by a larger tip radius.

Fig. 3: Source emittance diagrams of conventional SE and GSE. GSE’s wide and less-distorted emittance demonstrates an improved high-beam-current capability of the emitter.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-1-O-1664 Magnetic-Field-Superimposed Cold Field Emission Gun for 1.2-MV Transmission Electron Microscope

Kasuya K.1, Kawasaki T.1, Moriya N.1, Arai M.1, Furutsu T.1
1Central Research Laboratory, Hitachi, Ltd., Akanuma 2520, Hatoyama, Saitama, 350-0395, Japan
keigo.kasuya.bp@hitachi.com

     A magnetic-field-superimposed cold field emission gun (M-FEG) was developed for a 1.2-MV transmission electron microscope (TEM)[1]. This microscope is intended to have a point resolution of 40 pm and to take atomic-scale three-dimensional images by electron holography.

     Figure 1 shows the cross section of the developed M-FEG. The gun is designed to have a high brightness and stable emission current. The gun is equipped with a pre-accelerator magnetic lens placed close to the emitter [2]. The superimposed magnetic field causes the emitted electrons to converge so that the aberration-caused blurring with subsequent electrostatic lenses is minimized. As a result, the inherent high brightness of the cold field emitter can be obtained. The chambers of the gun are differentially evacuated with three non-evaporative getter (NEG) pumps and four ion pumps. The pressure of the first chamber, where the emitter is placed, was 3×10-10 Pa. This small pressure stabilizes time variations of the emission current [3].

     Figure 2 shows the measured time variations of the probe and total currents. After performing flashing of the emitter, the initial probe current of 1 nA was obtained at the total current of 1 µA. The probe current stayed almost constant for more than 10 hours during the initial period of the measurement. The 90% decrease time, at which the current falls to 90% of the initial value, was prolonged to 900 min in comparison with 3 min in a previous gun at 5×10-8 Pa [4]. The variation in the probe current over the course of the initial 8 hours was 5.2%.

     Another advantage of the pressure reduction is the increase in probe current. It increased two times higher than that of the conventional field emission gun operating at 10-8 Pa. This reason can be explained by the fact that the clean emitter surface has higher probe current density than the adsorbed surface. The gun provided large probe currents ranging from 1 to 170 nA for total currents ranging from 1 to 300 µA.

     The resulting current characteristics ensure that the 1.2-MV TEM will have fine resolution with a high S/N ratio. The illumination system of the microscope is discussed by Kawasaki in this conference.

[1] K. Kasuya et al., submitted to J. Vac. Sci. Technol. B.

[2] M. Troyon, Optik 57, 401 (1980).
[3] K. Kasuya et al., J. Vac. Sci. Technol. B 28, L55 (2010).
[4] T. Kawasaki et al., J. Elec. Microsc. 49, 711 (2000).

 

This research was supported by the Japan Society for the Promotion of Science through the FIRST Program, initiated by the Council for Science and Technology Policy.

Fig. 1: Cross-section of the developed magnetic-field-superimposed cold field emission gun (M-FEG). The pressure of the first chamber was 3 ×10-10 Pa.

Fig. 2: Measured time variations of probe and total currents. The 90% decrease time of the probe current was 900 min. The variation in the probe current over the initial 8 hours was 5.2%.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-1-O-1982 Challenges in Phase Plate Development and Applications

Sader K.1, Buijsse B.1, van Duinen G.1, Danev R.2
1FEI, Achtseweg Noord 5, 5651 GG Eindhoven, The Netherlands, 2Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
kasim.sader@fei.com

While there have been attempts to implement phase plates in transmission electron microscopes (TEMs) over a long period of time, a publication by Danev and Nagayama [1] renewed interest that functional phase plates could be produced. In particular in life sciences, the development of thin film vitrification techniques has enabled the examination of unstained macromolecules and thin cells in the electron microscope, but also created the need for phase contrast. Conventionally, contrast at low spatial resolutions has been generated by using a strong defocus, but with the added consequence of introducing oscillations in the contrast transfer function. A phase plate allows one to work in-focus, with a large increase in the contrast at low spatial resolutions.

Many types of phase plates have been proposed, but the most widespread implementation has been the original thin-film Zernike phase plate. This type of phase plate has shown practical performance, especially in life science applications. The most widely tested film type is amorphous carbon, but these suffer from aging problems, making frequent exchanges of the phase plate necessary. Alternatives to conventional amorphous carbon have been investigated and silicon-based films show promise in terms of longevity.

In close collaboration with the Max Planck Institute of Biochemistry in Martinsried, FEI have developed a new type of phase plate with properties that make it very suitable for implementing it as a user friendly device in our TEMs. It produces high-contrast images, providing excellent contrast transfer in the low resolution range which is particularly relevant for cryo-electron tomography and may provide benefits for single particle analysis in the case of small and heterogeneous particles. No fringing effects around high-contrast features are observed and CTF oscillations can be avoided up to better than 10Å while maintaining contrast transfer at low spatial frequencies. Transmission losses by the phase plate are very modest. Moreover, the phase plate shows consistent performance for at least half a year of usage.

To facilitate routine phase plate usage we have added extra alignments and control panels to the microscope software. In particular, accurate adjustment of beam deflection pivot points is included to ensure a stable beam position at the plane of the phase plate. Also, software has been developed to easily navigate the phase plate in the back focal plane. We are developing detailed phase plate workflows for our applications software that will provide a seamless integration of the phase plate in the (automated) applications. In this talk a selection of results will be shown from cryo electron tomography.

[1] R. Danev, K. Nagayama, Ultramicroscopy 88, 243-252 (2001)

Fig. 1: Tecnai F20 results from cryo electron tomography on doxorubicin using conventional TEM at 4 μm defocus. Experimental conditions: total dose of 85 e-/Å2, tilt range +/-60º. 

Fig. 2: Tecnai F20 results from cryo electron tomography on doxorubicin using phase plate TEM at 0.5 μm defocus. Experimental conditions: total dose of 85 e-/Å2, tilt range +/-60º.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-1-O-2071 Sculpturing the electron wave function using nanoscale phase masks

Shiloh R.1, Lereah Y.1, Lilach Y.1, Arie A.1
1Department of Physical Electronics, Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv, Israel
royshilo@post.tau.ac.il

Electron beams are extensively used in lithography, microscopy, material studies and electronic chip inspection. Today, beams are mainly shaped using magnetic or electric forces, enabling only simple shaping tasks such as focusing or scanning. Recently, binary amplitude gratings achieved complex shapes. These, however, generate multiple diffraction orders, hence the desired shape, appearing only in one order, retains little of the beam energy. Here we demonstrate a method in electron-optics for arbitrarily shaping electron beams into a single desired shape, by precise patterning of a thin-membrane. It is conceptually similar to shaping light beams using refractive or diffractive glass elements such as lenses or holograms - rather than applying electromagnetic forces, the beam is controlled by spatially modulating its wavefront. Our method allows for nearly-maximal energy transference to the designed shape, and may avoid physical damage and charging effects that are the scorn of commonly-used (e.g. Zernike and Hilbert) phase-plates. The experimental demonstrations presented here – two solutions to the free-space wave equation: on-axis Hermite-Gauss and Laguerre-Gauss (vortex) beams, and computer-generated holograms – are a first example of nearly-arbitrary manipulation of electron beams. Our results herald exciting prospects for microscopic material studies, research in electron-matter interaction, enables electron lithography with fixed sample and beam and high resolution electronic chip inspection by structured electron illumination.

The work was supported by the Israel Science Foundation, grant no. 1310/13 and the German-Israeli Project cooperation.

Fig. 1: On-axis generation free-space modes: images taken at different effective distances near the diffraction plane. (A) Unmodulated beam passing through the membrane, (B) Hermite-Gauss11-like, (C) Laguerre-Gauss01-like (vortex), (D) Bragg diffraction pattern used as metric, (E) Bragg grating, (F) HG11-generating mask, (G) vortex-generating mask.

Fig. 2: On-axis holograms: (A) “TAU” hologram produced by the mask in (B); inset: magnification showing ~60nm holes composing the pixels. (C) Electrons orbiting a nucleus hologram produced by the mask in (D); inset: magnification showing the centre of the mask. Note: contrast and brightness levels in (C) were altered for visibility.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-1-O-2914 Tuning and Operation of a sub-20 meV Monochromator

Dellby N.1, Lovejoy T. C.1, Křivánek L. O.1
1Nion Co., 1102 Eighth St., Kirkland, WA 98033, USA
dellby@nion.com

When aiming for simultaneous high energy resolution and high spatial resolution in a monochromated scanning transmission electron microscope (STEM), three locations in the microscope are critical:
1) the monochromator’s (MC’s) energy-selecting slit, where the pass-band of energies admitted into the rest of the column is determined,
2) the sample, where the tuning determines the spatial resolution, and
3) the detector of the electron energy loss spectrometer (EELS).
To optimize the performance of the entire system, aberrations in all three locations must be accurately and repeatably tuned, so as to produce the smallest possible beam crossover at each place. In typical operation, all three crossovers are images of the field emission source, and upstream crossovers are re-imaged in subsequent stages. A mistuned monochromator can be largely compensated by a pre-sample aberration corrector that is mistuned in the opposite direction, or by a mistuned EELS.
The ideal method for monochromator tuning should therefore measure the actual aberrations at the plane of the energy selecting slit and not be affected by post-monochromator optics. We use a variation of the method developed by Foucault[1]: we image the far-field shadow of the energy-selecting slit near which the beam crossover is formed.
With a monochromatic beam coming into the monochromator, the aberrations would be tuned when the far-field image of the slit fades out uniformly as the slit is closed up. Non-zero focus and astigmatism would produce a stripe across the image of the beam-defining aperture, and one would focus and stigmate to make the stripe wider until it fills the aperture.
In practice, however, the slit is illuminated with an energy-dispersed beam some 300 meV wide, i.e. about 20 times larger than the energy width of our usual monochromated beam. This means that electrons with different incoming energies fill different parts of the aperture with stripes of different energies (Fig. 1), and the total beam after the slit is an incoherent superposition of a distribution of slit positions.
Fortunately, the tuning information is imprinted on the coherence properties of the beam exiting the MC slit, and we use this to determine the tuning at the slit to first and higher orders (Fig. 2). The end result is a repeatable tuning of the MC to 15 meV and better in Nion’s High Energy Resolution Monochromated EELS STEM (HERMESTM) (Fig. 3), as well as an ability to refocus the beam at the sample to sub-nm dimensions [2].
[1] L. Foucault, Comptes Rendus Academie des Sciences 47 (1858) 958-959.
[2] OL Krivanek et al, Microscopy 62 (2013) 3-21

Fig. 1:  Idealized far-field image (Ronchigram) for three electrons beams with slightly different energies, with astigmatism present at the MC slit.

Fig. 2:  Fourier transform of a Ronchigram obtained with the beam tuned at the MC slit.

Fig. 3:  Zero loss peaks before and after MC tuning
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-1486 Magnetic monopole like fields and electron vortices

Béché A.1, Van Boxem R.1, Van Tendeloo G.1, Verbeeck J.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
armand.beche@uantwerpen.be

The search for magnetic monopole particles has been in vain so far. However, an approximation to a magnetic monopole field can be obtained at the tip of a long, thin, nanoscopic magnetic needle [1,2]. We demonstrate that the interaction of an electron beam with such a field produces an electron vortex beam just like was predicted for a true magnetic monopole [3]. The total orbital angular momentum (OAM) produced by the magnetic needle can be precisely tuned by carefully selecting the amount of magnetic flux via the needle cross section.

The magnetic needle is extracted from a 60 nm thick nickel film using focused ion beam (FIB) milling. It is then deposited on top of a gold plated silicon-nitride grid with one end suspended over a pre-cut aperture hole (Fig.1 A). This aperture allows the impinging electron beam to interact with only one end of the needle. The magnetic field at the tip causes the fast electrons to obtain a spiral phase shift via the Aharanov-Bohm effect as revealed by holography in field free conditions in a transmission electron microscope (TEM) (Fig. 1B). The width of the needle is reduced in the FIB until the flux approaches one fluxon (total phase shift of to 2pi). Comparing the experimental results with simulations (Fig. 1C), an OAM of 0.8 was estimated.

In order to confirm the existence of a vortex after letting an electron beam interact with the magnetic needle aperture, a focal series was acquired in the far field plane of the needle (Fig.2 A). The presence of a dark center which does not disappear upon focusing is typical for a vortex beam, as demonstrated in simulated images (Fig. 2B). A second confirmation of the vortex character was made by cutting the slightly defocused far field images with the sharp edge of an objective aperture and noting the configuration of the Fresnel fringes [4]. Close to the vortex core, the phase dislocation pattern appears in the Fresnel fringes (Fig. 3A). The number of non-connected lines gives an approximation of the total OAM, close to 1 in the present case, confirming the holography result (Fig. 1B). The Fresnel fringes agree remarkably well with simulations (Fig. 3B).

An aperture containing such a monopole-like field provides a unique way of creating electron vortex beams with a pure OAM value, independent of the electron energy. As almost all the incoming electrons transforms into a specific OAM state, a high intensity vortex beam is created, greatly improving the potential for atomic scale magnetic measurements at much improved signal to noise ratios.

1. Béché A. et al., Nature Physics (2014), 10, p. 26-29.
2. Kasama T. et al., MRS Proc. (2004), 839, p. 107-118.
3. Aharonov Y. and Bohm D., Phys. Rev. (1959), 115, p. 485-491.
4. Verbeeck J. et al., Nature (2010), 467, p. 301-304.

This work was financially supported by the European Union: ERC grant 246791 COUNTATOMS, ERC Starting Grant 278510 VORTEX, Integrated Infrastructure Initiative grant 312483-ESTEEM2.

Fig. 1: A: Overview of needle surrounded by an aperture. B: Experimental phase map at the tip of the needle, figured by the dash square in B. The phase rosacea is scaled from 0 to 2pi. C: Simulated phase map for a total phase shift of 0.8x2pi over the full aperture.

Fig. 2: A: Experimental focus series of the aperture in far field conditions. The destructive interference center is typical of a vortex beam. B: Simulation of the focal series using the phase profile displayed in Fig. 1C.

Fig. 3: A: Cut of the defocused far field image of the needle aperture by a sharp edge, revealing a dislocation like feature in the Fresnel fringes close to the vortex center. As only one branch cannot connect, the total OAM is close to 1. B: Simulation of the cut aperture in the far field using the phase profile displayed in Fig 1C.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-1500 Measuring the Orbital Angular Momentum of Electron Vortex Beams in the TEM

Guzzinati G.1, Clark L.1, Béché A.1, Verbeeck J.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
giulio.guzzinati@uantwerpen.be

The exchange of orbital angular momentum (OAM) in the interaction between an electron beam and a sample is determined by the properties of the sample and the beam [1,2]. Studying this interaction could enable a new class of OAM based microscopy techniques if convenient measurement of OAM exchange would exist. These techniques could then be used to study in the TEM, among others, the magnetic state of atoms and the transfer of OAM nanoparticles.

Electron beams possessing intrinsic orbital angular momentum have recently risen to attention after the prediction and demonstration of electron vortex beams[3-5]. This discovery has led to the rapid development in the field of singular electron optics [1-7].
In order to employ electron vortices as a probe to study the OAM exchange between a beam and a sample, methods to manipulate or measure the OAM of the beams are fundamentally important. While several methods have been designed to produce vortex beams, there has not been an equal progress in the detection and measurement of intrinsic OAM in the electron microscope.

Aiming to bridge this gap, we have implemented several diffraction based OAM measurement methods: using a forked grating hologram, a triangular geometrical aperture, a knife-edge and an astigmatic phase plate. Fig.1 shows an overview of the experimental results of the different methods when different incoming vortex beams are used as input.
In particular the triangular aperture and the astigmatic phase allow to recognize high order vortex beams easily , but they require to record and analyze a full 2D diffraction pattern. Intentional astigmatic aberration is easier to implement but the OAM is revealed by observing the beam waist rather than the far field pattern which may be a disadvantage in scanned electron probe setups.
On the other hand the hologram and the knife-edge are only appropriate for the measurement of lower values of OAM, but they allow the measurement to be reduced to a simple electron counting process which makes them ideally suited for automated OAM measurement [7].

[1] P. Schattschneider et al., Phys. Rev. B 85, 134422 (2012).
[2] A. Béché et al., Nat. Phys.10/1 26 (2013).
[3] K. Bliokh et al., Phys. Rev. Lett. 99 190404 (2007).
[4] M. Uchida and A. Tonomura., Nature, 464/7289 737 (2010).
[5] J. Verbeeck et al. Nature 494, 331–335 (2013).
[6] V. Grillo et al., Phys. Rev. X 4, 011013 (2014).
[7] G. Guzzinati et al., Phys. Rev. A 89, 025803 (2014).

We acknowledge funding from the European Union under the FP7 program, ERC Starting Grant No. 278510 VORTEX and Integrated Infrastructure Initiative No. 312483 ESTEEM2.

Fig. 1: (a) Schematic representation of the experiment, depending on the OAM of the input beam and type of aperture used different patterns are produced. Experimental data are show for (b) forked hologram, (c) triangular aperture (d) knife-edge (e) astigmatic aberration.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-1541 Investigation of physical and chemical method to produce Möllenstedt electrostatic biprism for off-axis electron holography experiment

Cours R.1, Houdellier F.1
1CEMES-CNRS, Université de Toulouse, 29 Rue Jeanne Marvig, 31055 TOULOUSE FRANCE EU
robin.cours@cemes.fr

Denis Gabor has developed electron holography in 1948, as a method used to quantitatively retrieve the phase of the electron wave. D. Gabor proposed a configuration where the perturbed wave (object wave) and the reference unperturbed wave are observed in a common optical plane below the sample. In this plane a superimposition of the two waves can occur. This superimposition will induce an interference phenomenon and create the so-called in-line electron hologram, used to retrieve the phase difference between the two waves. In this configuration the sample is then out of focus. In 1955 G. Möllenstedt and H.Düker invented the biprism for electrons, a metallic wire biased relatively to the earth. The biprism effectively splits the electron beam into an object wave and a reference wave, which by electrostatic fields are brought to overlap onto one another. An interference pattern will be observed below the wire plane while the sample can still be in focus. This configuration, known as off-axis electron holography, is the one commonly used in all the major holography studies from dopant profiling to strain mapping through studies of nanomaterials magnetic configurations. Biprisms in common use today are constructed by coating ultrasmall quartz fibers with noble metals. The resulting biprisms, although they are quite small by most fabrication standards (approximately 700 nm in diameter), can have various mechanical, electrical, structural … properties. Depending on the quality of the biprism, the properties of the off axis hologram can be strongly modified. As an example, to avoid vibration, which drastically decrease the interference fringes contrast, the wire should be very taut; to minimize charge effect, which induce Fresnel fringes phenomena, the wire should be extremely clean; to increase the phase coherence of the beam across the biprism the wire should be the smaller possible, …

Regarding all these drastic requests that the wire should fulfilled to be a suitable biprism, the question of reproducibility become deeply problematic using standard biprism fabrication method. This question become even more crucial regarding our new microscope, the In situ interferometry TEM (I2TEM), a HF3300 TEM that fits with 4 biprisms wire used for various electron holography developments. In order to choose the most reproducible way which will give the best wire properties (size, vibrations, cleanliness, …), we have investigated several methods to produce them from chemical method to FIB (Focused Ion Beam) approach. The combination of these methods allowed us to make numbers of high quality biprism wire with a higher reproducibility rate.

This work has been supported by the French National Research Agency under the "Investissement d'Avenir" program reference No. ANR-10-EQPX-38-01.

Fig. 1: A: SEM image of a Wollaston wire thinned using a FEI Helios FIB B: The same wire installed inside a Hitachi HF2000 TEM

Fig. 2: C: Special and ultrafast chemical etching method using nanowetting of hot HNO3 onto a Wollaston wire
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-1672 Development of illumination system of a 1.2 MV Field Emission Transmission Electron Microscope

Kawasaki T.1, Kasuya K.1, Furutsu T.1, Ono S.1, Arai M.1, Moriya N.1
1Central Research Laboratory, Hitachi, Ltd., Hatoyama 2520,Saitama 350-03, Japan
takeshi.kawasaki.qb@hitachi.com

       In the FIRST Tonomura project, we have been developing a 1.2 MV field-emission transmission electron microscope (FE-TEM) for the atomic resolution three-dimensional reconstruction of electro-magnetic fields by electron holography. Here FIRST stands for funding program for world-leading innovative R&D on science and technology. In this paper we report its illumination system with the following requirements:
       (1) high brightness beam for electron holography
       (2) current fluctuation less than 10 % over 8 hours for stable observation
The requirement (1) is discussed in this presentation and the requirement (2) is discussed by Kasuya in this conference.
       Figure 1 shows schematic view of the illumination system and three ray paths. Separate valves are placed between the FE gun and the accelerator tube so that conditioning of emission and high-voltage can be performed separately. The pre-accelerating magnetic lens focuses the beam near the first electrode of the accelerator tube where the Butler lens is formed (Case A), and then the spherical aberration of the accelerator tube can be suppressed. When the magnetic lens excitation becomes stronger, the electron trajectory focuses twice in the accelerator tube (Case C). Between Case A and Case C, beams focus near the condenser lens and cannot focus on the specimen position (Case B). To obtain high brightness beam, total aberration of the illumination system has to be minimized. The optimum condition of the pre-accelerating magnetic lens was obtained by calculating mean brightness and probe current of the spot focused on the specimen position as a function of the lens excitation using WR5 software (MEBS Ltd.). The FE-cathode source diameter, the angular current density, and the energy spread are assumed to be 5 nm, 30 μA/sr, and 0.3 eV, respectively. Figure 2 shows the results. Two peaks of the brightness exist: The left peak corresponds to Case A, the right peak corresponds to Case C, and the bottom region D between two peaks corresponds to the Case B. Preliminary experimental results using the 1 MV FE-TEM showed the following:
       (1) existence of two brightness peaks
       (2) the maximum brightness of 1.8×1010 A/cm2sr [1]
This brightness value is almost the same as that calculated for the 1 MV FE-TEM. The calculated maximum brightness is 3.3 ×1010 A/cm2sr for the 1.2 MV FE-TEM. We expect it to reach 5×1010 A/cm2sr by increasing the angular current density of the cleaner FE-tip under ultra high vacuum condition (3.0×10-10 Pa) [2].

References
[1] T. Kawasaki et al.  J. Electron Microsc. 49 (2000) 711-718.
[2] K. Kasuya et al. submitted to  J. Vac. Sci. Technol. B.

 

This research was supported by the Japan Society for the Promotion of Science through the FIRST Program initiated by the Council for Science and Technology Policy.

Fig. 1: Schematic view of the illumination system and different ray paths A, B, and C.

Fig. 2: Calculated brightness and probe current as a function of the excitation of the magnetic lens in terms of IN(V1)-1/2 , where I is the lens current, N is the number of turns of the coil (1700), and V1 is the FE extraction voltage    (5 kV).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-1693 Measurement of current density distribution in shaped e-beam writers

Horáček M.1, Bok J.1, Kolařík V.1, Urbánek M.1, Matějka M.1, Krátký S.1
1Institute of Scientific Instruments AS CR, v.v.i., Brno, Czech Republic
mih@isibrno.cz

The ZrO W(100) Schottky cathode is used in our e-beam writing system working with a rectangular-shaped electron beam. The homogeneous angular current density distribution is crucial for quality of exposures of the shaped beam lithography systems. Two basic types of the angular emission distribution can be observed in dependence on the microscopic final end form shape of the emitter tip, with bright centre and more common dark centre [1]. The stable operation of the cathode thus stable end form shape requires a delicate balance of parameters inside the gun which however can slightly change during cathode life time. This implies the necessity of analysing and periodical monitoring the current density distribution in e-beam. Four methods enabling this measurement are presented.
First we implemented a method based on the modified knife-edge approach [2], when a part of the scanned element of the beam is blanked out and the current within the remaining "open" part is measured. The 2D information of the current distribution is obtained by stepwise opening of selected segments. The measurement error analysis was made and necessary measurement averaging in each segment were used in order to reduce the random error of the current [3]. The size of the scanned element was 6 × 6 µm2, a maximum usable segment for one shot in our lithography system (Fig. 1).
The current distribution obtained by the knife-edge method was compared with a method using a luminescent screen. The YAG:Ce single-crystal screen was irradiated by the e-beam stamp of the 6 × 6 µm2 and the areal light emission was recorded by a magnifying optical system with a CCD camera. The emitted light intensity is directly proportional to the e-beam current, thus the current density distribution can be compared with other measurements methods. However, the absolute measurement is hardly possible (Fig. 2).
Next the same e-beam stamp of the 6 × 6 µm2 was scanned over Faraday cup opening. The advantage of this method is uniform distribution of the measurement error instead of the modified knife-edge method. The absolute value of the current density is affected by the demagnification of the electron optics during measurement (Fig. 3).
Another method is based on evaluation of developed electron resist exposed by the 6 × 6 µm2 separate shaped e-beam stamp using atomic force microscope. The depth of the developed resist depends on the spread of the energy in the electron resist. The real current density distribution was obtained by the deconvolution of the developed resist with electron scattering model (Fig. 4).

References

[1] K-Liu et al., J. Vac. Sci. Technol. B 28, C6C26 (2010).
[2] M. Sakakibara et al., Jpn. J. Appl. Phys. 46, 6616 (2007).
[3] J. Bok et al., J. Vac. Sci. Technol. B 31, 31603-1 (2013).

The authors acknowledge the support from MEYS CR (LO1212) together with EC (ALISI No. CZ.1.05/2.1.00/01.0017), the TACR project No. TE01020118 and institutional support RVO:68081731.

Fig. 1: Modified knife-edge method.

Fig. 2: Luminescent screen method.

Fig. 3: Faraday cup method.

Fig. 4: Electron resist exposure method.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-1958 Quantitative measurement of the OAM spectra of electron vortex beams.

Clark L.1, Béché A.1, Guzzinati G.1, Verbeeck J.1
1EMAT, University of Antwerp, Antwerp, Belgium
laura.clark@uantwerp.be

Electron vortex beams have been subject to a great level of interest since their first demonstration only a few years ago [1]. Much of the interest in the field stems from their potential to measure magnetic transitions within a sample, at a previously unreachable scale. While much progress has been made, in producing electron vortices of high purity, high intensity and atomic scale, research into the required counterpart towards full experimental application, of orbital angular momentum (OAM) measurement, has not yet matured to its full potential [2-4].

In the last 12 months, the first methods to measure the OAM make-up of an electron vortex beam have been demonstrated [5-7]. However, the methods presented thus far, are limited to only those cases where the input beam is in a single vortex state, and do not allow measurement of the relative weightings of vortex states in a beam . Indeed, a generic electron wave can be seen as a superposition of multiple vortex modes and the weight of each of these modes can in principle be measured.

We introduce here an experimental technique able to measure the relative weightings of 5 or more OAM modes within an input beam, through the use of a multi-pinhole interferometer (MPI). This is a technique which has recently been used to measure the strength and location of optical vortices, but which is easily adaptable to practical implementation in a TEM, placing an MPI aperture in the SA plane, below the sample.

Experimental results are shown, having measured the OAM spectrum of pure l={-1,0,+1,+2} centred vortex beams, enabling the first quantitative discussion of their experimental purity. We further demonstrate the so-called mode broadening effect, by measuring the changes in OAM composition as a vortex beam is shifted away from the central axis of measurement.

This application of an MPI within a TEM has enabled measurement of an approximate OAM spectrum in the SA plane. We give experimental evidence alongside theoretical models, enabling rapid discrimination of different orders of vortex beams even if the electron beam consists of a superposition of different OAM modes. This capability serves as a promising tool to measure OAM exchanges in the interaction of electrons with a sample.

[1] Bliokh, KY, et al. PRL 99.19 (2007): 190404
[2] Verbeeck, J., et al. Nature 467.7313 (2010): 301-304
[3] Clark, L., et al. PRL 111.6 (2013): 064801
[4] Béché, A, et al, Nature Physics 10.1 (2014): 26-29
[5] Guzzinati, Giulio, et al. arXiv: 1401.7211 (2014)
[6] Saitoh, K, et al. PRL 111.7 (2013): 074801
[7] Shiloh, Roy, et al. arXiv: 1402.3133 (2014)

We acknowledge funding from the European Union under the FP7 program: ERC Starting Grant No. 278510-VORTEX and Integrated Infrastructure Initiative Reference No. 312483-ESTEEM2.

Fig. 1: Plot of idealised electron vortex beam – brightness represents intensity, and hue represents phase

Fig. 2: A five-pinhole multi-pinhole interferometer, enabling measurement of OAM modes in the set l={-2:+2}

Fig. 3: Experimental diffraction pattern from an l=+1 vortex centred on the MPI

Fig. 4: Autocorrelation function produced from the experimental diffraction pattern.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-1961 Development of Phase Contrast Scanning Transmission Electron Microscopy

Iijima H.1, Minoda M.2, Tamai T.2, Kondo Y.1, Hosokawa F.1
1EM Business Unit, JEOL Ltd., 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan, 2Department of Applied Physics, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan
hiiijima@jeol.co.jp

Phase contrast transmission electron microscopy (P-TEM) is a powerful tool to enhance the image contrast of transparent materials such as ice-embedded biological specimens and polymer materials. In P-TEM, a phase plate is placed at the back-focal plane (BFP) of the objective lens (OL). It gives a phase shift for scattered electron waves, resulting in a change of phase contrast transfer function (PCTF) from sine to cosine type. Eventually, phase variation of specimens is converted into intensity variation. Among various types of phase plates, a carbon film phase plate with a small central hole is the most practical1. However, there is a serious issue that high-density electron beam (cross-over) on the phase plate causes the charging and/or the alteration of the phase plate, resulting in decreasing the life time of the phase plate.

To overcome this issue, we are developing phase contrast scanning transmission electron microscopy (P-STEM). Figure 1 shows the schematics of P-TEM and P-STEM. According to the reciprocity theorem, the same contrast appears in the P-TEM and the P-STEM if a phase plate is placed at a front-focal plane (FFP) of an OL in P-STEM. In P-STEM, a cross-over is not formed on the phase plate, so that improvement of the phase plate life time is expected. In our experiments, we used a field emission electron microscope (JEM-2100F) equipped with a Schottky electron source, to obtain a coherent small probe on a specimen. Phase plate is placed on a condenser lens aperture plane conjugate to the FFP of the OL.

On the other hand, it is well known that the small detection angle is needed to obtain good phase contrast in STEM imaging. Figure 2 compares a conventional bright-field STEM and a P-STEM images of amorphous carbon film with different detection angle shown in Fig. 1. And Fourier transforms of the conventional bright-field STEM image and the P-STEM image with β = 4 mrad show the sine shape. By contrast, that of the P-STEM image at β = 0.3 mrad shows the cosine shape, which proves that the P-STEM can be achieved with small detection angle.

[1] R. Danev and K. Nagayama, J. Phys. Sci. Jpn. 70 (2001) 696.

This development was supported by the program for "Development of Systems and Technologies for Advanced Measurement and Analysis" under JST.

Fig. 1: Schematic of P-TEM (left) and P-STEM (right). The phase plate is placed at the BFP of the objective lens in P-TEM and the FFP of the objective lens in P-STEM.

Fig. 2: Conventional bright-field STEM and P-STEM images of amorphous carbon film. All images are taken close to focus. (a) Conventional bright-field STEM image. (b) P-STEM image with β = 4 mrad. (c) P-STEM image with β = 0.3 mrad. (d)-(f) Fourier transforms for images shown above. Scale bars; 10 nm in (a)-(c), 4 nm-1 in (d)-(f).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2090 Contrast enhancement of phase objects by using Phase Contrast Scanning Transmission Electron Microscopy

Minoda H.1, Tamai T.1, Iijima H.2, Hosokawa F.2, Kondo Y.2
1Department of Applied Physics, Tokyo University of Agriculture and Technology, 2EM Business Unit, JEOL Ltd
hminoda@cc.tuat.ac.jp

It is well known that an interaction between electron waves and molecules composed of light elements such as biological molecules is very weak. Therefore, it is very difficult to obtain their high contrast image in transmission electron microscopy (TEM). Contrast enhancement of the phase objects by using a phase plate was proposed at the middle of the 20th century [1], but it was realized at the beginning of 21st century [2]. In the pioneering work by Nagayama, a carbon thin film with a hole in its center is used as a phase plate (PP) and it was placed at a back focal plane (BFP) of the objective lens (OL). A role of the PP is giving a phase shift to scattered wave by means of the mean inner potential of the PP material. Electron waves having a phase shift interfere with electron waves without phase shift. Accordingly, phase image would be able to be visualized.

Applying the principle of reciprocity to scanning transmission electron microscopy (STEM), imaging optics of the STEM is equivalent to that of a conventional TEM. Therefore, a phase contrast scanning transmission electron microscopy (P-STEM) can be used to enhance phase contrast of the phase objects. In the present study, a PP can be set on the condenser lens aperture (CLA) plane that is optically equivalent to a front focal plane (FFP) of an OL. The P-STEM image which enhances image contrast could be obtained by getting an appropriate optical condition. Figure 1 show an example of the comparison of (a) the conventional STEM bright field image and (b) the P-STEM image. Ferritin molecules were used as a specimen. This comparison clearly shows contrast enhancement in P-STEM. In this paper, the results obtained by sung phase contrast microscopy to the STEM mode are introduced.

[1] F. Zernike, Physica 9 (1942) 686.

[2] R. Danev and K. Nagayama, J. Phys. Sci. Jpn. 70 (2001), 696.

This development was supported by SENTAN, JST.

Fig. 1: A comparison of (a) C-STEM and (b) P-STEM images of ferritin molecules.  The contrast enhancement in P-STEM is evident.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2325 AC- Voltage Operated Schottky Electron Source

Yada K.1, Saito Y.1
1Daiwa Techno Systems Co.,Ltd
yada@daiwatechno.co.jp

Introduction: Zr-O/W100 Schottky electron source has been widely used in electron beam instruments because of its favorable properties such as 1)vacuum technological tolerance at its operational condition, 2) rather long life time and high brightness. It is still required, however, that a vacuum must be better than 10 -8 Pa and stability of high voltage must be better than 10 -5 when DC high tension and electro-magnetic lens systems are used in the instrument. We tried to find promising materials. Among them, we selected BaZrO3 and SrZrO3 and tested their thermal field emission properties with both DC and AC high tension powers.

Results: Field emission tips of 110- and 100-oriented W wire were made by electrolytic method and powder of BrZrO3 or SrZrO3 was pasted near the apex as usual.　Thermal field emission patterns obtained by DC and AC voltage are very similar and crystal facets are indexed very easily. Fig.1 and Fig2 show emission patterns of BaZrO3/W(110) and BaZr3(W100) obtained by AC and DC operation ,where optimal working temperature is 800 degree C. Similar results are obtained with SrZrO3(W)100 cathode but its optimal temperature is little higher than the case of BrZrO3.

As advantages of AC operation of present schottky electron source, followings are concluded:

1)Schottky shield is not necessary because of low working temperature of the emission materials.

2)Emission beams can be focused, deflected and stigmated by using electrostatic lens,deflector and stigmtor, respectively.

3)Work function of newly adopted materials here is so small that working temperature is fairly low (800-850 degreeC). Consequently ,energy spread of the beam will be narrow.

4) As commercial AC electric supply can be used without any rectifire or stabilizer, factor cost will be fairly reduced.

5)We think that the present Schottky electron source is the best selection for a generation of strong and small X-Ray source of projection X-ray microscope.

Fig. 1: Fig.1

Fig. 2: Fig.2
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2198 The effect of Detector Thickness on Direct Detector Performance

Clough R. N.1, Moldovan G.2, Kim J. S.1, Kirkland A. I.1
1Department of Materials, University of Oxford, UK, 2Oxford Instruments NanoAnalysis, High Wycombe, UK
robert.clough@materials.ox.ac.uk

Direct detection refers to a detection system where signal is generated in the sensor chip directly by the imaging electrons; indirect systems generate photons in a scintillator from the imaging electron and it is these photon which are coupled to the sensor chip that generate signal. One of the key advantages of a direct detection system is the possibility of producing thin detectors; these are desirable as a thin detector has improved detection performance in terms of Modulation Transfer Function (MTF) and Detective Quantum Efficiency (DQE) [1]. This improvement arises from the fact that many electrons will pass all the way through the sensor and escape the detector system generating signal along the way, before large lateral scattering has occurred.

We have taken a prototype CMOS based direct detector featuring full frame resolution of 1024 by 1024 pixels, with a pixel size of 20µm and readout of 30fps [2]. Two different versions of the detection chip were produced. The first is a 20µm thick p- active layer on a p+ substrate mechanically thinned to 50µm. The second was made from silicon on insulator (SOI) wafer with a 20µm device layer with the handle wafer removed using a chemical etch. For each of these detectors the MTF and DQE were measured using standard techniques [3] at 80 and 200kV. Here we shall present the characterisation data along with images of gold particles on an amorphous substrate to show how thinner detectors lead to improved detector performance, allowing images taken at lower magnification to have improved resolution.

[1] G. McMullan, et al, Experimental observation of the improvement in MTF from backthinning a CMOS direct electron detector, Ultramicroscopy, 109 (2009).
[2] A.J. Wilkinson, et al, Direct Detection of Electron Backscatter Diffraction Patterns, Phys. Rev. Lett. 111 (2013).
[3] R. R. Meyer, et al, Experimental characterisation of CCD cameras for HREM at 300kV, Ultramicroscopy, 85 (2000).

We would like to acknowledge Dr T. Anaxagoras and Prof. N. Allinson from the University of Sheffield for provision of CMOS wafers, and C Wilburn of Micron Semiconductor Ltd. for chip packaging.

Fig. 1: MTF of a 20µm thick detector at 80 and 200kV.

Fig. 2: Au on amorphous Carbon at 80kV and 120,000x magnification taken with a 20µm thick sensor chip.

Fig. 3: Au on amorphous Ge at 200kV and 120,000x magnification taken with a 20µm thick sensor chip.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2263 Maximising Phase Contrast in Aberration-corrected STEM using Pixelated Detectors

Yang H.1, Pennycook T. J.1,2, Nellist P. D.1,2
1University of Oxford, Department of Materials. Parks Rd, Oxford, OX1 3PH, UK, 2EPSRC SuperSTEM Facility, Daresbury Laboratory, WA4 4AD, UK
hao.yang@materials.ox.ac.uk

For imaging weak phase biological specimens, phase contrast imaging using elastically scattered electrons provides the most information for a given amount of radiation damage as compared to electron inelastic scattering as well as X-ray and neutron scattering [1]. In scanning transmission electron microscopy (STEM), most phase information from weak scattering objects lies inside the bright field disc of the convergent beam electron diffraction pattern, which can be reconstructed using the method described by Rodenburg et al [2]. In this work we show that, compared to alternative modes including annular bright field (ABF) and differential phase contrast (DPC), phase contrast using a pixelated detector generates higher contrast in reconstructing the phase and therefore enjoys a higher dose efficiency in imaging weak phase objects.

With zero aberrations, any centrally symmetric detector will give no contrast for a weak phase object, as the two sides of disc overlapping regions in the convergence beam electron diffraction pattern are pi out of phase under weak phase approximation, and cancel each other when integrated using a central symmetrical detector geometry. Therefore, asymmetric detector geometries like DPC are expected to have higher phase contrast than ABF. In DPC, the quadrant detector can be divided into more segments with different collection angles, and the contrast transfer function is found to depend on the collection angles used, therefore the detector geometry of DPC can be further optimized to collect the maximum phase information per detected electrons. A pixelated detector provides even greater flexibility over where the information in the bright-field disc is retrieved from for each spatial frequency in the image.

Simulations have been done using an arbitrary weak phase specimen whose maximum atomic potential equals to that of a carbon atom, and has a Gaussian shape with a full width half maximum (FWHM) of 1nm. The artificially high width of the object is designed to test the lower spatial frequency transfer. The reconstructed phase with a dose as low as 50 electrons/Å2 and Nyquist resolution of 4.6Å still shows an interpretable feature (Figure 1). This dose is close to the critical dose of 5-50 electrons/Å2 for imaging biological specimen. In contrast to using a pixelated detector, neither ADF, ABF (Figure 2) nor DPC (Figure 3) show any recognizable structure feature under the same dose of 50 electrons/Å2. The formation of image contrast in ABF relies the presence of aberrations for a weak phase object, and here we are assuming an aberration-corrected microscope with zero residual aberrations.

[1] Henderson, R. Quarterly Reviews of Biophysics 28, 171-193 (1995).

[2] Rodenburg, J. M. et al. Ultramicroscopy 48, 304-314 (1993).

The authors would like to acknowledge financial support from the EPSRC (grant number EP/K032518/1) and the EU Seventh Framework Programme: ESTEEM2.

Fig. 1: Figure 1: Phase retrieval using a pixelated detector. (a) Schematic of a high speed pixelated detector. (b) A weak phase object with a maximum phase change of 0.15 radian. Reconstructed phase (c) assuming no noise, (d) with shot noise and a dose of 50 electrons/Å2. The scale bar is 5nm.

Fig. 2: Figure 2: Simulated (a,b) ADF and (c,d) ABF images of the weak phase object. The intensity is normalized to the number of incident electrons. (a)(c) assume no noise in image, and (b)(d) consider shot noise with the electron dose being 50 electrons/Å2. The scale bar is 5nm.

Fig. 3: Figure 3: Differential Phase Contrast (DPC) imaging using a 4-quadrants detector in (a). The simulated STEM DPC images using both (b,c) A-C quadrants, and (d,e) B-D quadrants, where (b)(d) assume noise free, and (c) (e) considers shot noise with the electron dose being 50 electrons/Å2. The scale bar is 5nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2346 Effects of dielectric substrate on localized surface plasmon in a silver nano-particle

Fujiyoshi Y.1, Nemoto T.1, Kurata H.1
1Institute for Chemical Research, Kyoto University, Kyoto, Japan
fujiyoshi@eels.kuicr.kyoto-u.ac.jp

Recently localized surface plasmons (LSPs) which are collective oscillation of conduction electrons of metallic nano-particles (NPs) attract researchers in nano-optics because of strong optical confinement and electric field enhancement, leading to many applications including biochemical sensors and surface-enhanced Raman spectroscopy (SERS) etc. Since the dielectric environment around the NP affects the property of LSPs, it is important to elucidate the effects of dielectric materials supporting NPs on LSPs.
In the present work, we examined special distributions of LSP excited on a silver NP supported by MgO substrate using electron energy loss spectroscopy (EELS) combined with scanning transmission electron microscopy (STEM). Spectral imaging (SI) data were acquired along the direction parallel to the MgO surface supporting a silver NP, which enabled us to observe the intensity distribution of LSP excitation as a function of the distance from the silver NP/MgO interface. The experiment was performed by an aberration corrected STEM (JEM-9980TKP1) equipped with a cold-FEG.
Figure 1 and 2 show a HAADF image of silver NP on MgO substrate and its LSP map extracted from SI data, respectively. From the HAADF image the NP can be regarded as a sphere. When a spherical metal particle is isolated in vacuum, the excitation probability of LSP should distribute isotropically around the particle. However, the LSP map in Fig. 2 shows anisotropic distribution, that is, the intensity at the top surface of silver NP is strong compared to that at other positions, which means that the effect of dielectric substrate is remarkable. In order to interpret such anisotropic distribution, we simulated the electromagnetic field induced in the silver NP on MgO substrate using finite-difference time-domain (FDTD) method.
Figure 3 shows the spatial distribution of field calculated by assuming the incident plane waves polarized perpendicular (a) and parallel (b) to the substrate surface. When the polarization of incident wave is perpendicular to the substrate, the field strength in the NP on MgO is enhanced compared to that in the isolated NP as shown in Fig. 3(a), which corresponds to the observed strong excitation at position A in Fig. 2. In case of the parallel polarization the field strength in the NP on MgO is weakened (Fig. 3(b)), corresponding to the observed intensity at position B in Fig. 2. Therefore, the anisotropic distribution of the LSP excitation in silver NP on MgO surface can be attributed to the direction of electric polarization induced in the NP depending on the electron positions.

Fig. 1: HAADF image of a silver NP supported on MgO surface.

Fig. 2: LSP map extracted from the energy range from 3.2 to 3.6 eV in the SI data.

Fig. 3: Spatial distribution of electromagnetic field calculated by FDTD simulations. Incident plane waves were assumed to be polarized parallel (a) and perpendicular (b) to the substrate surface. Solid and broken lines correspond to the intensity profiles for an isolated silver NP and the silver NP supported on MgO surface, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2410 Thermal Emission Properties of GdB6 Cathode

Saito Y.1, Yamagishi K.1, Yada K.1
1Daiwa Techno Systems Co.,Ltd.
saito@daiwatechno.co.jp

Introduction: Hexa boride of lanthanum (LaB6) has been widely used in electron beam
instruments because of its higher brightness than that of tungsten hairpin cathode. But it might
be that there are better materials than LaB6. Among many borides of lanthanide, hexa-borides
of Ce and Gd are promising from the existing data[1] based on Richardson-Dushman equation as
shown in Table 1. So we tested electron emission properties of GdB6.


Table 1
              A      φ(eV)       AT2         I(A/cm2)
LaB6      29      2.66     93960000   3.316363
GdB6    0.84     2.06     2721600     4.607963
GdB6     9.3      2.55     30132000   2.16234
GdB6      10      2.58     32400000   1.915988


R-D Eq. I=AT2EXP(-φ/kT), A:R-D constant, k:Boltsman cont, T:temperature(1800k), I:electron
density


Results: Fig.1 shows photograph of Ta wire covered with GdB6 powder where the central part is
slightly protruded. Fig.2 is beam pattern of GdB6 cathode at working temperature when the
cathode is installed in a scanning electron microscope. Fig.3(a) shows SEM image of ZnO
particles obtained with tungsten hairpin cathode and Fig.3(b) shows that obtained with GdB6
cathode. It is seen that image quality of (b) is superior to that of (a). It is also clear that emission
performance of present GdB6 powder cathode is nearly equal or little better than that of LaB6
single crystal. Sintered GdB6 cathode is now under examination to compare with the single
crystal LaB6 cathode.


Reference:[1]Japan-Soviet Communication “Emission Characteristics of Materials” pp.96-81 by
V.S.Fomenko, Published by Naukova Dunka, Kiev 1970

Fig. 1: Fig.1

Fig. 2: Fig.2

Fig. 3: Fig.3(a)

Fig. 4: Fig.3(b)
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2469 Electron differential phase microscopy with an A-B effect phase plate

Tanji T.1, Ikeda U.2, Niimi H.2, Usukura J.1
1EcoTopia Science Institute, Nagoya University, Nagoya, Japan 1, 2Graduate School of Nagoya University, Nagoya, Japan 2
tanji@esi.nagoya-u.ac.jp

    Observations of week phase objects, such as thin films of light elements, thin polymer films, biological sections etc., are available by electron phase microscopy[1]. Many of phase plates utilized are thin film types. Some electrostatic types have been developed, but they are not so general, because the fabrication of the filter with fine structures is very difficult. The mainstream of todays phase plate is the thin film type. This type of the phase plate, however, has some disadvantages, i.e. control of the film thickness, charging up, contamination and so on. We adopted the phase plate with a magnetic thin filament which generates the vector potential around itself by an Aharonov-Bohm (A-B) effect. The filament type phase plate with the A-B effect was proposed and constructed firstly by Nagayama. This type of the phase plate generates the differential phase contrast in the image, and has a longer life time than the thin film type. Any clear differential effect, however, has scarcely reported so far.
    We will report that the effect of a phase plate consisting of a Wollaston platinum filament of 1 µm in diameter covered with ferromagnetic material, Nd-Fe-B of 5 nm thick, deposited by Pules Laser Deposition. The filament with a clean surface selected by SEM is mounted on a single hole Cu grid. The phase difference in the both side spaces of the filament measured by electron holography shows 1.5 rad as shown in Fig.1. Being set on the aperture holder, the phase plate is inserted in the back focal plane of the objective. Figure 2 shows images of a colon bacillus stained with Pb. Fine structures can be observed clearer in the image using the phase plate than in the image taken ordinarily. The direction of the differentiation is shown by the arrowhead.

Refernce

[1]K. Nagayama, Another 60 years in electron microscopy: development of phase-plate electron microscopy and biological applications, Journal of Electron Microscopy, 60(2011) S43-S62.

Fig. 1: (a) Electron phase map reconstructed by electron holography. (b) A line profile along the arrow head in (a) which is averaged along the long side of the rectangle. The phase difference is about 1.5 rad. between both sides of the filament.

Fig. 2: mages of a colon bacillus stained with Pb taken at under-focus condition without the filament(a) , and in-focus with the filament(b).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2476 Effect of a phase plate on TEM imaging

Edgcombe C. J.1
1TFM Group, Dept of Physics, University of Cambridge
cje1@cam.ac.uk

The type of phase plate that has been most widely reported (eg [1]) consists of a plain disc of material such as carbon, of controlled thickness, with a central hole to pass the direct beam. Images made with this type of plate show bright outlines or halos around certain features [2, 3].  Analysis of geometrical imaging has shown how these halos occur.  It is necessary to consider the response to all spatial frequencies that are present in a typical object. In principle this can be done straightforwardly by Fourier transforming the object phase to find its spatial frequency distribution at the back focal plane (BFP), multiplying by the response of the phase plate and further transforming to find the image distribution.

The response has been found [4] for a weak phase object consisting of a circular disc of radius b, centred on the microscope axis. The phase plate is assumed to advance the phase of components with angular frequencies greater than a value q0 , defined as
q0 = 2 (pi) r2 ⁄ λf
where r2 is the radius of the central hole in the plate for the direct beam, λ is the electron wavelength and f is the focal length of the lens. The resulting image intensity is shown in figure 1 for a phase advance of (pi)/2 and a range of values of B = q0b. The object is imaged with little overshoot when B is less than about 1. Reported results [5] agree with this transition value for B.

The step changes at radius b are always imaged fully but as B increases, the low-frequency components are progressively lost from the image and for B > 1, the mean intensity across a step falls to the background value.  The full range of the step is maintained, so the intensity changes from +(half the range) at radii just less than b, to –(half the range) just outside the step. Thus a bright halo or outline is produced just outside the boundary r = b, for objects with B > ~1.  The darker central patch for B = 8 agrees with observation [3]. The maximum object diameter that corresponds to Bmax , the maximum B for accurate imaging, is
2bmax = 2Bmax ⁄ q0 = λ Bmax f ⁄ (pi) s2
where s2 is the radius of the hole needed to pass the direct beam.  To increase the size of object that can be imaged accurately, it will be necessary to reduce s2 or increase the focal length of the objective lens.

References
Danev R and Nagayama K 2001 Ultramicroscopy 88 243-52
Fukuda Y, Fukazawa Y, Danev R, Shigemoto R and Nagayama K 2009 J Struct Biol 168 476-84
Danev R and Nagayama K 2011 Ultramicroscopy 111 1305-15
Edgcombe C J 2014 Ultramicroscopy 136 154-9, http://dx.doi.org/10.1016/j.ultramic.2013.09.004
Hall R J, Nogales E and Glaeser R M 2011 J Struct Biol 174 468-75

Fig. 1: Figure 1. Image intensities produced by a (pi)/2 phase plate with fixed q0 for uniform disk objects with a range of diameters 2b.  Responses are shown for values of B = q0b (increasing from top left) of 0.2, 0.5, 1, 2, 5 and 8.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2510 Toward electron polarizators

Grillo V.1,2, Karimi E.3, Balboni R.4, Gazzadi G. C.1, Frabboni S.1,5, Mafakheri E.1,5, Tang W. X.6,7, Boyd R. W.3,8
1CNR-Istituto Nanoscienze, Centro S3, Via G Campi 213/a, I-41125 Modena, Italy, 2CNR-IMEM, Parco delle Scienze 37a, I-43100 Parma, Italy. , 3Department of Physics, University of Ottawa, 150 Louis Pasteur, Ottawa, Ontario K1N 6N5, Canada, 4CNR-IMM Bologna, Via P. Gobetti 101, 40129 Bologna, Italy, 5Dipartimento FIM, Universitá di Modena e Reggio Emilia, Via G. Campi 213/a, 41125 Modena, Italy, 6College of Materials Science and Engineering, Chongqing, 400044, China, 7School of Physics, Monash University, Clayton, VIC, 3800, Australia, 8Institute of Optics, University of Rochester, Rochester, New York 14627, USA
vincenzo.grillo@cnr.it

We describe the experimental and theoretical improvements toward the realization of an efficient electron spin polarizator. The initial proposed polarizator [1] was based on the spin-orbit conversion of a vortex beam [2] to a beam with a defined polarization. The conversion occurred within a compensated quadrupolar Wien Filter (WF).

The theoretical improvements are supported by simulations of the beam-field interaction through a new multislice for propagation including spin [3]. The experimental steps are based on the introduction of phase holograms to produce e-beams close to ideal Bessel beams [4]. To improve the flexibility and feasibility of the polarizer we have considered different possible alternative design: e.g. when the pitch fork hologram is positioned below the WF it is possible to obtain simultaneously the 2 polarized beams and switch between them [3]. Alternative design permit also to remove the electric fields. We have also studied the higher order corrections of the WF by magnetic multipoles of higher order and calculated the possible effects of the fringing fields: the efficiency in the selection of the polarized states increases with the order of the vortex and consequently of the multipoles in the WF.

Fig 1 is an example of simulation of the wavefunction after a WF for a beam at 15 KeV (e.g. for SPLEEM and low voltage TEM applications ) for 2 initial spin state. The brightness is proportional to the wave intensity, the phases encoded in the color. Due to the spin orbit coupling different spin are transformed, inside the WF, in different phase factors and orbital momentum. Only the center of the state |ℓ=0,↑> has stationary phase and therefore contributes to the intensity at the center of a pupil in far field diffraction.

For this simulation we corrected the asymmetric aberrations by multipolar elements but still obtained a strong phase oscillation beyond a radius dependent of the size of the field that must be further corrected to obtain maximal efficiency.

Fig 2 a,b is an example of phase hologram described in its thickness map and overall pattern. This pattern reaches an efficiency of 40%. In fig 2c an example of Bessel beam with ℓ=2 is shown. These beams, in the diffraction plane (see fig d), transform to narrow rings. This strongly reduce the demand of lateral stability of the fields and the problems of phase oscillations described in fig. 1

[1] E. Karimi et al. Phys Rev. Lett 108, 044801 (2012)
[2] J. Verbeeck et al Nature 467, 301 (2010).
[3] E. Karimi et al Ultramicroscopy 138, 22 (2014)
[4] V. Grillo et al. Phys. Rev. X 4, 011013 (2014)

Fig. 1: Wavefunction after a Wien filter for a beam at 15KeV. The initial beam had ℓ=1 and 2 spin states were considered. The final spin state are also separately plotted. The external phase oscillation are due to residual aberrations.

Fig. 2: Example of phase hologram: the thickness profile in Si3N4 a) and the full pattern b)are shown. Example of Bessel beam with ℓ=2 in the Fresnel c) and Fraunhofer d) regime
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2578 Design of a monochromator for aberration-corrected low-voltage (S)TEM

Mukai M.1, Omoto K.1, Sasaki T.1, Kohno Y.1, Morishita S.1, Kimura A.1, Ikeda A.1, Somehara K.1, Sawada H.1, Kimoto K.2, Suenaga K.3
1JEOL Ltd., Akishima, Tokyo, Japan, 2National Institute for Material Science (NIMS), Tsukuba, Ibaraki, Japan, 3National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
mmukai@jeol.co.jp

Low-voltage analytical electron microscope equipped with delta-type aberration correctors for image- and probe-forming lens system [1] was developed under a project “Triple-C phase-1” to study the atomic structures of carbon materials sensitive to the damage by irradiation of electrons. It enabled us to reveal the characters of graphenes by EELS [2] and to visualize and specify an encapsulated single metal atom in a fullerene [3]. However this microscope was equipped with a cold field emission gun to obtain high brightness therefore its energy resolution remains at approximately 0.3 eV.
For the next challenges, we have started to develop a new type of low-voltage aberration-corrected analytical electron microscope equipped with a monochromator working at 15-60 kV under a project “Triple-C phase-2”, whose targeted energy resolution is better than 25 meV. Fig. 1(a) and 1(b) show an appearance of the microscope and a configuration of components inside the cover.
The developed monochromator employs a double Wien-filter system, arranged between the extraction anode of Schottky source and the accelerator, which is similar configuration to previous design [4]. The electron trajectories from the electron source to the plane of the exit crossover of the monochromator are calculated as shown in Fig. 2. Electron trajectories are set to be symmetric to the plane of energy-selection slit so that the energy-dispersion formed by the first Wien-filter at a slit plane is cancelled by the second Wien-filter at an exit plane as a consequence of the double Wien-filter system. Thus, after the monochromator, the electron probe is achromatic and the energy spread is controllable by choosing the width of the slit, independently on the probe size. In addition, the setting of the monochromator and the electron trajectories inside the monochromator are independent of the change of the accelerating voltage since the accelerator of the electron gun is located after the monochromator and the potential along the optical axis inside the monochromator is kept constant.
We intend to evaluate the performances of the developed low-voltage monochromated electron optical system and the enhancement of spatial resolution arising from a small chromatic aberration in TEM at low accelerating voltage with large scattering cross-section and small specimen damage by reducing a primary electron energy.

References
[1] H. Sawada, et al.: J. Electron. Microsc. 58 (2009) 341.
[2] K. Suenaga and M. Koshino, Nature 468 (2010) 1088.
[3] K. Suenaga, et al.: Nature chemistry 1 (2009) 415.
[4] M. Mukai, et al.: Ultramicroscopy (2014) accepted.

This work is supported by Japan Science and Technology agency, Research Acceleration Program.

Fig. 1: Computer graphics of a low-voltage aberration-corrected analytical electron microscope equipped with the developed monochromator, (a) An appearance with the cover and (b) a column of the microscope inside the cover.

Fig. 2: (a) Calculated trajectories along optical axis from source to exit of monochromator, (b) beam shapes at slit plane with an energy-dispersed 1st focus and (c) beam shapes at exit plane with an achromatic 2nd focus. The red lines and the green lines show the trajectories having different energies of 1 eV inside of the monochromator.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2604 Holographic generation of Electron quasi-Bessel beams

Frabboni S.1,2, Grillo V.2,3, Karimi E.4, Balboni R.5, Gazzadi G. C.2, Mafakheri E.1,2, Boyd R. W.4,6
1Dipartimento FIM, Università di Modena e Reggio Emilia, Via G. Campi 213/A, 41125 Modena, Italy , 2CNR-Istituto Nanoscienze, Centro S3, Via G Campi 213/a, I-41125 Modena, Italy, 3CNR-IMEM, Parco delle Scienze 37a, I-43100 Parma, Italy. , 4Department of Physics, University of Ottawa, 150 Louis Pasteur, Ottawa, Ontario K1N 6N5, Canada, 5CNR-IMM Bologna, Via P. Gobetti 101, 40129 Bologna, Italy, 6Institute of Optics, University of Rochester, Rochester, New York 14627, USA
stefano.frabboni@unimore.it

Recently the attention of electron microscopy community has been attracted by the generation of electron beams by means of holographic element that allows to shape the electron wavefront through a modulation of the phase or amplitude transmittance. This new degree of freedom has already demonstrated huge potentialities in application with electron vortex beams [1]. In this contribution we discuss the case of the quasi-Bessel beams obtained as a coherent superposition of conical plane waves along a closed ring of finite angular aperture [2].
Fig 1a shows the simulated transverse distribution of the electron Bessel beam at the first order of diffraction propagating, in the Fresnel region, from the hologram shown in b). In Fig 1c is reported the scanning electron microscope image of the nanofabricated phase hologram with a zoom-in image of the central region shown in the upper inset. The hologram is obtained from of a FIB-milled silicon nitride membrane, which is almost transparent to the 200keV electron beam [3]. Different depths modify the local projected potential; thus, electrons see different effective paths at grooves. In Fig 1d the distribution of the diffracted electrons in the Fraunhofer region of propagation, is reported. In the first order of diffraction, the Bessel beam forms a ring in the far-field. Due to the limited number of grooves of the hologram, the ring, typical of the Bessel beam, is convoluted with the Airy function of the hologram aperture, thus forming a quasi-Bessel beam. In Fig 1e is shown the measured transverse intensity distribution of the quasi-Bessel beam of the zeroth order generated by the hologram shown in Fig 1c, in Fresnel regime. In Fig 1f the experimental radial intensity distribution of the Bessel beam, blue solid curve, is compared with simulations by varying the convergence of the beam incident on the hologram plane, thus showing the effect of the partial coherence on the Fresnel ring contrast.
Bessel beams have many interesting properties, namely resistance to diffraction and the smallest spot diameter compared to other ordinary type of beams that could be exploited in STEM tomography. In Fig 2 is reported the diffraction free range of the quasi Bessel beam shown in Fig 1c.


[1] J. Verbeeck, H. Tian, and P. Schattschneider, Nature 467, 301 (2010).
[2] V. Grillo, E. Karimi et al. Phys. Rev. X 4, 011013 (2014)
[3]V. Grillo, G.C. Gazzadi, E. Karimi et al. Appl.Phys. Lett. 104, 043109 (2014)

M.E. acknowledges  the support  of SPINNER 2013.

Fig. 1: Computer generated hologram and electron Bessel beams of the zeroth order.

Fig. 2: Propagation of Bessel beams of the zeroth order in the Fresnel regime.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2685 Low Voltage Mini TEM

Coufalová E.1, Mynář M.1, Štěpán P.1, Drštička M.1, Sintorn I. M.2, 3
1DELONG INSTRUMENTS a.s., Brno, Czech Republic, 2Centre for Image Analysis, Uppsala University, Sweden, 3Vironova AB, Stockholm, Sweden
michal.drsticka@dicomps.com

On the basis of experience with the low voltage transmission electron microscopy at 5 kV, which is intended for the study of samples with low contrast (organic matters), we tried to design a TEM optimized in many aspects:
1) Maintaining relatively low voltage to keep up high contrast.
2) The use of such energy, which would open the possibility to increase the resolution of the system to the area of atomic (molecular) resolution using the monochromatization of the primary beam and Cs correction in future.
3) Practical standpoints – reasonable dimensions, resistance to external influences.
4) Energy sufficient for the transmissivity of electrons through samples of "standard" thickness.
It turned out to be suitable to base such electron-optical system on the use of magnetostatic (the objective lens) and electrostatic (projection system) elements. For the above reasons, we have chosen a range of energy of 10-25 keV. This choice enables to maintain the concept of combination of electron-optical and light-optical magnification, which leads to a significant reduction of the dimensions of the unit and solving simultaneously the problem of TEM image digitalization. It emerged that the working energy of 25 keV is the highest possible energy, at which there is no degradation of the applicable high light-optical magnification due to scattering in the single crystal fluorescent screen.
Using light lenses with large numerical aperture (up to 0.95), we achieve a high collection efficiency of the light from the screen. Also, the level of the light signal is high enough at 25keV energy. We have verified that the electron-optical system can be operated in several modes:
1) TEM at 25 keV
2) STEM at 15, 10 keV
3) DIFF at 25keV
The first experimental results confirm the assumptions obtained by electron-optical simulations, in particular the expected resolution in various modes.
It is further confirmed that the contrast inevitably decreases at the energy of 25 keV compared to the lower energies, however, it is still significantly higher than in the energy area of above 50 keV. Even thin sections for which there is no significant increase of chromatic aberration provide sufficient contrast in the image at this working energy. This brings the opportunity to study both stained and unstained samples at low radiation damage.
This version has been optimized for identification of viruses – samples prepared with negative staining and fixation. It allows mobility of the device, and is equipped with user friendly control system with a simple concept that provides remote control resources to allow to be controlled by upper level image analysis software for automatic virus recognition (Kylberg and Sintorn EURASIP J. on Image and Video Processing 2013, 2013:17).

The work has been supported by Eurostars Programme of EUREKA and European Community.

Fig. 1: The body on MiniTEM on a standard desk

Fig. 2: Section of the column

Fig. 3: ATCC and rota viruses stained with 2%Uac in TEM mode at 25 keV

Fig. 4: ATCC and rota viruses stained with 2%Uac in STEM mode at 10 keV
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2688 Energy analyzer for point electron sources

Kolařík V.1, Coufalová E.1, Mynář M.1, Drštička M.1
1DELONG INSTRUMENTS a.s., Brno, Czech Republic
michal.drsticka@dicomps.com

We have built an energy analyzer for characterization of parameters of various types of point emitters, electron guns, and illumination blocks of electron columns. It can be also used for characterization of electron monochromators, and for studying the influence of electron – electron interaction on the beam energy spread.
The concept of the analyzer is very simple and physically straight, based on dispersion characteristics of magnetic prism: It is configured for measuring energetic spread of emitters with the virtual source size between 1 nm and 50 nm independently of the electron source distance, it means any design or type of electron gun can be measured.
The theoretic resolution of the analyzer is:
• < 15 mV for the virtual source size of 50 nm,
• < 3 mV for the virtual source size of 15 nm.
The image of virtual source is focused only in the dispersion direction (see Fig. 2). The dispersion of the magnetic prism in this plane is about 3 µm/V at the output edge of the prism. The optical set guarantees the resolution of electron spectrometer on the level of 10 mV or better, the use of slit aperture provides the capability of statistical evaluation of 2048 spectra (pixel columns).
Although the dispersion itself is relatively small (units of µm/V), the analysis is possible at the level of units of mV, because the source image size in the spectral plane is in units of nm. The dispersion plane can be enlarged electron-optically so that it is projected onto a screen with the size accessible for imaging by high-quality light optics (the dispersion and source image are magnified in the same proportion).
The significant input parameters that determine the resulting energy resolution are the virtual source size and used aperture angle. We illustrate on the chart that the effect of the virtual source size for cold field emission and Schottky cathode is in a significant range of aperture size under the resolution of a light objective lens with NA as high as 0.95 in this arrangement.
The high energy resolution of the electron-optical part can be used for very effective monochromatization of en electron beam.
Reference: V. Kolařík, M. Maňkoš, L. H. Veneklasen, Close packed prism arrays for electron microscopy, Optik 87, No.1(1991)

The work was supported by ”Electron Microscopy“ Competence Centre of Technology Agency of the Czech Republic

Fig. 1: Section along the optical axes

Fig. 2: Optical scheme

Fig. 3: The influence of the aperture on the energy resolution for CFE and Schottky emitter at dispersion of 2.8 µm/V in relation to the optical limit (at NA = 0.95, M=40×, pixel size = 7.4 µm)

Fig. 4: Examples: calibration of measurement, profiles - CFE energy spread – 0.34 eV, Schottky emitter energy spread – 0.58 eV
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2710 Spiral phase plates for electron vortices

Béché A.1, Winkler R.2, Planck H.3, Hofer F.3, Verbeeck J.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 2Center for Electron Microscopy, Steyrergasse 17, 8010 Graz, Austria, 3Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Steyrergasse 17, A-8010 Graz, Austria
armand.beche@uantwerpen.be

Vortex beams have been recently developed in electron optics and generate a lot of interest due to their potential ability of retrieving magnetic information down to the atomic scale [1, 2]. Several techniques are now available to produce such beams like the holographic mask [2] or the more recent magnetic needle [3]. In this work we propose to extend the idea of Uchida & Tonomura [1] by creating a spiral phase plate with smoothly increasing thickness.
The phase plate should be composed of a light material to prevent too much absorption from the plate itself and be ideally thicker than 100 nm at its highest point to allow a smooth increase of thickness. Focused electron beam induced deposition (FEBID) is an ideal tool to realize such structures as it can deposit functional materials with high spatial resolution. In the present case, ultrathin silicon nitride (SiN) was successfully used as substrate to fabricate SiO2 spiral phase plates as shown in Fig. 1. In order to prevent unwanted scattering from the central hole in the spiral, it was filled with a small amount of platinum via FEBID.
The phase plate was then introduced into the Qu-Ant-TEM, an FEI Titan3 transmission electron microscope, operated in Lorentz mode, to achieve a large field of view with extended spatial coherence conditions. Carefully illuminating the phase plate with a uniform electron beam and looking in the far field, typical features of vortex beams were recorded. Fig. 2 displays a through focus series of the resulting beam which reveals the presence of a doughnut like intensity pattern with the destructive interference centre of the vortex beam.
In order to quantify the orbital angular momentum (OAM) carried by the outgoing beam, electron holography was performed at the edge of the phase plate. By measuring the phase shift between the thickest and thinnest area, the total OAM was estimated to be 0.6 (Fig. 3).
Further tuning of this setup provides another method for creating atomic sized electron vortex beams with the advantage of providing a single vortex beam that is easy to obtain in a standard TEM.

[1] Uchida M. & Tonomura A., Nature Letters (2010), 464, p737-739.
[2] Verbeeck J. et al., Nature Letters (2010), 467, p.301-304.
[3] Béché A. et al., Nature Physics (2014), 10, p. 26-29.

This work was financially supported by the European Research Council under the 7th Framework Program (FP7), ERC grant 246791 COUNTATOMS, ERC Starting Grant 278510 VORTEX and Integrated Infrastructure Initiative No. 312483 ESTEEM2.

Fig. 1: (a) TEM view of the spiral phase plate with the central hole filled with platinum. (b) Atomic force microscope image revealing the thickness profile of the phase plate.

Fig. 2: Far field through focus series of an electron beam evenly illuminating the phase plate. The destructive interference area in the middle of the fully condensed beam (black hole) is typical of a vortex beam.

Fig. 3: (a) Phase image of the edge of the phase plate, between the thickest and thinnest area of the spiral, acquired by holography. (b) Phase profile taken along the dotted area displayed in (a) revealing a total phase shift of 4 rad corresponding to a total OAM of ~0.6.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2835 Projector lens and CCD camera distortions in a Hitachi HF-3300 TEM

Denneulin T.1, Gatel C.1, Houdellier F.1, Hÿtch M. J.1
1CEMES CNRS, 29 rue Jeanne Marvig, 31055 Toulouse, France.
hytch@cemes.fr

In a transmission electron microscope (TEM) the projector lenses are known to introduce large-scale distortions. The magnification and the rotation in the image can vary up to 5% and 2° across the field of view [1]. Therefore the accurate mapping of any physical field (strain, magnetic or electric field) using high resolution TEM or holography requires the calibration of those distortions. The method used here does not add noise to the phase image and alleviates the need for a reference hologram.
We have investigated the projector and the CCD camera distortions on a recently installed aberration-corrected HF-3300 Hitachi TEM (I2TEM-Toulouse). The distortions were measured using off-axis electron holograms acquired in the vacuum. A double biprisms setup was used to remove the Fresnel fringes [2]. The voltages of the biprisms were set so that the interference pattern fills entirely the 4k Gatan CCD camera. Two holograms with a different orientation of the biprisms were acquired in order to reconstruct the 2D strain field using geometrical phase analysis (GPA) [3]. Before GPA calculation, the reconstructed phase images were fitted using a 4th order polynomial to remove the noise.
The influence of the magnification and the values of P1 and P2 was investigated. It was found that the distortions are mainly dependent on the value of P2. Fig. 1 shows the strain field obtained for 4 different values of P2. Increasing P2 is equivalent to “zoom” into the distortion pattern. The variations across the image are then lower for high values of P2. At a nominal magnification of ×1.5M (P2 is equal to 5.3 A) the mean dilatation Δxz varies from 0 to 3% and the rigid body rotation ωxz varies from 0 to 1° from the center to the corner of the image.
According to the theory [1], Δxz and ωxz should be circular shaped. However it can be noted that the rotation image is slightly triangular shaped. After analysing the ronchigram of the camera provided by Gatan [4] we found that this is due to the low frequency distortions of the camera (see Fig. 2 after correction of the camera distortions). We then created an artificial ronchigram for correcting both the projector and the camera distortions. The procedure will be detailed during the presentation. Fig. 3(a) is an example of dark-field hologram acquired on a SiGe layer grown by epitaxy on a Si substrate. Without correction (Fig. 3(b)) the reconstructed phase image exhibits some variations in the substrate and the phase ramps in the layer are slightly distorted. Those artifacts are removed after correction (Fig. 3(c)).

[1] F Hüe et al, J. Electron. Microsc. 54(3) (2005), 181–190
[2] K Harada et al, Appl. Phys. Lett. 84(17) (2004), 3229–3231
[3] MJ Hÿtch et al, Ultramicrosopy 74 (1998), 131–146
[4] P Mooney, private communication

This work received financial support from the European Union under the Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative Reference 312483-ESTEEM2 and the European Metrology Research Programme (EMRP) Project IND54 Nanostrain. The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union.

Fig. 1: Distortions of the projector lenses as a function of the displayed value of P2. The strain field was calculated by geometrical phase analysis after fitting the phase images reconstructed from the holograms. From left to right are shown the horizontal εxx, the vertical εzz, the shear εxz strain, the mean dilatation Δxz and the rotation ωxz.

Fig. 2: Distortions (for P2 = 6.0 A) obtained after correcting the phase images with the CCD camera ronchigram.

Fig. 3: (a) (004) dark-field electron hologram of a SiGe layer epitaxially grown on a Si substrate. (b) Phase image reconstructed from the hologram without correction. (c) Phase image reconstructed with correction of the projector and camera distortions.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2975 Design and Characterization of a Single-Atom Electron Column

Lin C. Y.1,2, Chang W. T.1, Hsu W. H.1,3, Lai W. C.1,2, Chen Y. S.1, Hwang I. S.1
1Institute of Physics, Academia Sinica, Taipei, Taiwan, 2Department of Physics, National Taiwan University, Taipei, Taiwan , 3Department of Materials Science and Engineering, National Tsing Hua University, Hsinchu, Taiwan
cylin@phys.sinica.edu.tw

        It has been shown that noble-metal covered W(111) single atom tips (SATs) can be reliably prepared [1,2]. We have demonstrated full spatial coherence of electron beams emitted from the SATs [3]. Thus, single atom electron sources are suitable for phase retrieval imaging methods, such as holography and coherent diffractive imaging. We have proposed a SAT-based low-keV electron microscope that allows different imaging modes, as shown in Fig. 1. For this purpose, we plan to build an electron column with the capability to accelerate electron beams to 1~5 keV and a focused beam spot smaller than 100 nm. The column is composed of two parts: an electron gun and a condenser lens.

        The electron gun consists of a SAT, an extractor/suppressor, and an acceleration electrode. The tip is mounted on a holder that can be translated, tilted, and rotated in nanometer scale by piezo-positioners. Therefore, the tip-lens alignment can be done in vacuum without alignment coils. We have recorded the opening angles of the electron beams. As shown in the inset of Fig. 2, the emitter can be moved to different positions with the piezo-positioners and the corresponding beam profiles are recorded. Fig. 2 shows the half opening angles of the beams at an electron energy of 2.5 keV measured at different extraction voltage and different separations. Clearly the beam opening angle varies with the tip position. When the tip is positioned at about -2.5 mm, the half opening angle can be smaller than 1 mrad. We also find that the suppressor design that is often used in normal field emitters is not effective in reducing the beam divergence for the SAT emitter.

        The condenser lens consists of a limiting aperture, an einzel lens, and an octupole stigmator. We used Simion 8.1 software to simulate the lens parameters and determine the aperture diameter. In our simulations at the electron energy of 2.5 keV and the working distance of 2 mm, a spot size of 140 nm is obtained when the limiting aperture of 100 μm is used; a spot size of 20 nm is obtained when the limiting aperture of 20 μm is used. Fig. 3(a) shows the whole assembly of our instrument. As shown in Fig. 3(b), we have obtained a diffraction pattern on a small region of a suspended CVD graphene, which show two domains with different orientations. We are also designing a microcolumn based on the MEMS technique. Our ultimate goal is to determine the atomic structures of few-layer two-dimensional structures such as graphene and one-dimensional structures such as carbon nanotubes and bio-molecules.

References

[1] H. S. Kuo et al, Nano Lett. 4(12) (2004), p. 2379.

[2] H. S. Kuo et al, J. J. Appl. Phys. 45 (2006), p. 8972.

[3] C. C. Chang et al, Nanotech. 20 (2009), p. 115401.

This work is supported by National Science Council of ROC and Academia Sinica.

Fig. 1: Schematic of a multi-mode low-keV electron microscope

Fig. 2: Beam divergence, measured with the half opening angle at an electron energy of 2.5 keV, versus the extractor/suppressor voltage at different emitter positions. The inset is the schematic for characterization of the beam profile at different emitter positions.

Fig. 3: (a) Illustration and photo of a low-keV electron microscope (b) The diffraction pattern of a CVD graphene sample
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-2984 Wire corrector for aberration corrected electron optics

Nishi R.1, Ito H.2, Hoque S.2
1Osaka University, Osaka, Japan, 2Hitachi High-Technologies Corporation, Ibaraki, Japan
rnishi@uhvem.osaka-u.ac.jp

The wire corrector on the analogy of multipole correctors was proposed by H. Ito [1]. Two-parallel line current (Fig. 1) makes the magnetic field similar to that of a quadrupole as shown in Fig. 2. When using two-parallel line current, the filed cancels at the rotation symmetric axis and the two-parallel line current can generate quadrupole magnetic field because each magnetic field has opposite rotation direction of magnetic flux.
The wire corrector is only arranged by parallel line currents without using any magnetic materials, so it can be easily and simply fabricated and arranged in comparison to a conventional multipole. Adverse effect of hysteresis of magnetic material does not exist and homogeneity of magnet property is not needed. Magnetic field can be controlled by superimposition of parallel line currents. In actual layout, the wire corrector is configured to a coil shape in addition to the parallel currents with infinite length, but the effect of a coil shape can be reduced by consideration of its shape. Applying constant current to a main coil, fine adjustment of magnetic field can be performed by applying current to a sub coil. The wire corrector is valuable to the aberration corrected electron optics with high precision alignment and reproducibility.
When using the wire corrector of N=2, the magnetic field is similar to quadrupole field but the magnetic field is expanded in a series which also contains octapole field as a higher order term, as shown in Eq.(1) inset of Fig.1. Due to the wire corrector has octapole component, the wire corrector has possibility of simultaneous correction of spherical aberration in addition to chromatic aberration. Symmetric curved ray optical system constituted by combining both components of a deflector and the wire corrector of N=2, is expected that chromatic and spherical aberration is potentially corrected in such configuration.
The combination of the round lenses and the wire correctors of N=3 decreases the spherical aberration [2]. This shows the wire correctors of N=3 worked as a hexapole. The wire corrector has a potential of consisting an easy-to-use aberration corrector.

[1] Hiroyuki Ito et al, USP 7,872,240 B2 (date of patent: Jan. 18, 2011).
[2] H. Rose, Optik, 85 (1990) 19.

A part of this work of calculation was done by Dr. Eric Munro and Dr. John Rouse in Munro's Electron Beam Software Ltd.

Fig. 1: The wire corrector consisting of two parallel line currents (N=2).

Fig. 2: Magnetic flux in the wire corrector (N=2).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-3022 Measuring the Orbital Angular Momentum of Electron Vortex Beams by Forked Grating

Saitoh K.1, Hasegawa Y.2, Hirakawa K.2, Tanaka N.1, Uchida M.3
1EcoTopia Science Institute, Nagoya University, 2Department of Crystalline Materials Science, Nagoya University, 3Advanced Science Research Laboratory, Saitama Institute of Technology
saitoh@esi.nagoya-u.ac.jp

After the first report of the production of an electron vortex beam, an electron traveling in free space with orbital angular momentum (OAM) [1], electron vortex beams have been attracting a great attention owing to the unique physical property and application to a new microscopy in materials science [2]. In the present paper, we show the how the electron vortex beams are diffracted by forked gratings and how the OAMs of the electron vortex beams are transferred to each of the diffracted waves (Fig.1(a)). [3].

Figures 1(b) and 1(c) show a schematic diagram of the experimental setup of the present study. The binary masks of the spiral zone plates [Fig. 1(d)] and the forked gratings [Fig. 1(e)], fabricated from 200 nm thick PtPd films using a focused-ion-beam instrument (Hitachi FB-2100). The spiral zone plates and forked gratings were inserted into the condenser lens aperture position and selected-area aperture position, respectively, of a transmission electron microscope (JEOL JEM-2100F), which was operated at an acceleration voltage of 200 keV.

Figures 2(a) and 2(b) show electron vortex beams with OAMs of 10h and -10h, respectively, produced by the spiral zone plate. Each of the electron beams show a ring composed of 10 peaks at the center [4]. Figure 2(c) shows an electron diffraction pattern for an incident electron vortex beam with m = 10h. The diffraction pattern shows a series of diffracted rings, as indicated by the arrows. The central ring, composed of 10 peaks, is the transmitted beam with m = 10h. The 1st- and -1st-order diffracted electron beams show similar ringlike features, but have 11 and 9 peaks, respectively. This indicates that the electron OAMs of the 1st- and -1st-order diffracted beams are 11h and 9h, respectively. Figure 2(d) shows an electron diffraction pattern for an incident electron vortex beam with m = -10h. The pattern shows a series of diffracted rings as in Fig. 2(c), but is horizontally inverted from that shown in Fig. 2(c). The transmitted (0th-), 1st-, and -1st-order diffracted rings show 10, 9, and 11 peaks, respectively, indicating that the electron OAM of the 1st- and -1st-order beams are -9h and -11h, respectively. Our results indicate that the forked grating with a Burgers vector of b = 1 transfers not only linear momentum but also OAM, where the electron OAM transfer of the nth-order diffracted electron beam is nh. This diffraction property could be used as an electron OAM analyzer, as the nth-order diffracted beam shows a normal peak.

References

[1] M. Uchida and A. Tonomura, Nature 464, 737 (2010).

[2] J. Verbeeck, H. Tian, and P. Schattschneider, Nature 467, 301 (2010).

[3] K. Saitoh et al, Phys. Rev. Lett. 111, 074801 (2013).

[4] K. Saitoh et al., J. Electron Microsc. 61, 171 (2012).

The present work was partly supported by the Grant-in-Aid for Scientific Research (A) (No. 23241036), the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Mitsubishi Foundation.

Fig. 1: (a) Schematic drawing of the present experiment. (b),(c) Ray-path diagrams of the experimental setups for incident electron vortex beams of m = 10h (b) and m = 10h (c). (d) A spiral zone plate with a 20 μm diameter introduced to the condenser lens system (e) A forked grating with a 30 μm diameter introduced to the image-forming lens system.

Fig. 2: Incident electron vortex beams with m = 10h (a) and m = -10h (b), and diffraction patterns of the vortex beams with m = 10h (c) and m = -10h (d) generated by the forked grating shown in Fig. 1(e). 
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-3222 Contrast enhancement in TEM imaging by use of a central beam stop

Zandbergen H.1, Xu Q.1
1Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands
h.w.zandbergen@tudelft.nl

In TEM in life science, beam damage is the most important limitation. This is also the case for materials science samples like graphene, polymers and hybrid materials. On the imaging side an important boost is expected from the introduction of a phase plate. Phase plates have been researched over several decades and no easy to use system has emerged yet, indicating that is not easy task. Given the importance of efficient imaging, it is clearly necessary to explore other routes. We have explored [1] the possibility of dark-field imaging for contrast enhancement in which we have tried to block the central beam [2] and leave as many of the diffraction beams un-blocked.
Central beam block apertures (the abbreviation DF-000 is used in this abstract) in the shape of Mercedes star (see Figure 1) were made with a FIB. In our experiments we have observed no sign of charging, possibly due to the DF-000 shape. In central disk should preferably be smaller than the frequency, g, one wants to observe, which is of course much smaller for biological samples than for most materials samples. Our DF-000 removed frequencies corresponding to d-spacings of 8.7 Å and larger. In the presentation we will report how far we can decrease the size of central disk without charging problems and with still good blocking of the central beam.
For the drilling of holes in exfoliated graphene without contamination build-up we heated the graphene to 600°C. TEM experiments were done at 300keV and post-specimen aberration correction at 600°C. Figure 1c and 1d shows high-resolution and DF-000 images of multilayer graphene (4-5 layers). A hole was made in this sample using an e-beam. This hole can be seen very well in the DF-000 image and only faintly in the BF image. In both cases one can see that the graphene lattice continues up to the edge of the hole. The gradual decrease in thickness is clearly visible in the DF-000 image and not at all in the BF image. Thus we can obtain in the DF-000 image high-resolution information with a similar resolution limit as the BF image.
Figure 2 shows several images of graphene with three holes with varying size imaged at various focus values, showing that the bright field images in the range from -1500 to + 1500 nm show hardly any contrast and none at zero focus. On the contrary, the contrast in the DF-000 taken at 0 focus shows the largest contrast and in particular the smallest delocalization. In this case selecting ~ zero focus is easy by minimizing the blurring in the image.

1. Zhang C, Xu, Q, Peters PJ, and Zandbergen, H, Ultramicroscopy 134, 200 (2013)
2. Cowley, J., Acta Crystallographica Section A 1973, 29, 529-536

Fig. 1: (a) SEM image of the DF-000 objective aperture used to stop the central beam. (b) shows a typical DF-000 aperture in diffraction space. (c) and (d) show HREM images taken without an aperture and with the DF-000 image taken from the exactly the same area.

Fig. 2: Images from the same area of single layer graphene with three holes showing the effect of focus in BF and in DF-000 modes. No DF-000 at defocus values of -1000 and -3000 nm are given because these are too blurred. The two small holes are only barely visible in the BF image taken at -3000 nm and not at all in the other three BF images.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-6004 Determinationof geometrical form factor of emitter from Schottky plot

Emura Y.1, Murata H.1, Rokuta E.1, Shimoyama H.1, Yasuda H.2, Haraguchi T.2
1Faculty of Science and Technology, Meijo University, 2PARAM Corporation
hkmurata@meijo-u.ac.jp

In this paper we report preliminary experimental results on a LaB6 Schottky emission electron gun, which also includes our new findings that the electric field strength on the emitter surface can be estimated experimentally from the Schottky plot whose slope depends not on the work function but only on the reciprocal of the emitter temperature. According to the theoretical considerations on the Schottky emission, if the values of log10 j (j: emission current density) are plotted as a function of √F (F: field strength on the emitter surface), then the graph becomes a straight line with the slope of 1.913/T (T: emitter temperature), which is known as “the theoretical Schottky plot”. In experiment, on the other hand, the beam current I is measured as a function of the extraction voltage Va. Thus, the slope of “the experimental Schottky plot” is different from that of “the theoretical Schottky plot”. From I = j × ΔS (ΔS: emission area on emitter surface), the vertical axis of “the experimental Schottky plot” is expressed as log10 j + log10 ΔS, which means the graph is moved parallel to the vertical direction without changing the slope of the graph. We mark a new scale on the horizontal axis of “the experimental Schottky plot” in order that the slope may be equal to 1.913/T. Then, the new horizontal axis should be graduated in √F. This procedure makes it possible to relate the field strength F directly to the extraction voltage Va as F = β Va, where β is the geometrical form factor of the emitter.

The Schottky emission experiment has been done in the ultra-high vacuum chamber, using the experimental circuit shown in Fig. 1. A flat top LaB6 emitter is embedded into a rhenium conical sheath, and is heated by a tungsten hairpin filament, as shown in Fig. 1.

The beam current IF was measured as a function of the extraction voltage Va for a constant emitter temperature T = 1600 K by the Faraday cup placed behind the fluorescent screen. In Fig. 2, the values of log10 IF are plotted as a function of √Va. It can be seen that the plot is almost a straight line, which indicates that the emission is under Schottky emission mode. Figure 2 (a) and (b) also show emission patterns observed on the fluorescent screen at Va = 2 kV and 5 kV, respectively.

Figure 3 shows the above procedures, where the new horizontal axes scaled in √F are placed in addition to the original horizontal axes scaled in √Va. From the relation between F and Va, we have found that β = 99.6 [1/cm] for T = 1600 K. We have also performed the field calculation for the experimental system shown in Fig. 1. According to the calculation, the geometrical form factor β has been found to be β = 95.0 [1/cm], which is in good agreement with that estimated from “the experimental Schottky plot”.

Fig. 1: Experimental circuit for measuring the beam current by Faraday cup.

Fig. 2: Experimental Schottky plot for emitter temperature T = 1600 K. Emission patterns observed on the fluorescentscreen at Va = 2 kV (a) and 5 kV (b).

Fig. 3: Schottky plot for T = 1600 K for determination of geometrical formfactor β. A newhorizontal axis scaled in the square root of F is added so that the slope ofthe Schottky plot may be equal to 1.913/T.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-1-P-6020 Design and realisation of variable C shaped structured illumination

Mousley M.1, Thirunavukkarasu G.1, Babiker M.1, Yuan J.1
1Department of Physics, University of York, Heslington, York, YO10 5DD, United Kingdom
mgm514@york.ac.uk

Structured illumination is a new development in electron microscopy, with the advantage such as longer column channeling distances in crystals by donut-shaped illumination of atomic scale vortex electron beams [1]. In this paper, we introduce a controlled way to realize C shaped structured illumination. Analytical equations determining the parameters of the C shaped illumination pattern have been derived using phase gradient analysis, allowing independent control of the C-opening angle and radius of the C shape. Experimentally, we have used computer generated hologram (CGH) method to generate C shaped structured illumination in a 200 keV transmission electron microscope. Both amplitude and phase CGH masks have been used and comparisons with simulations show a strong match between the theoretical results and the experimentally recorded electron microscope images. C-shaped illumination has promises in potential applications such as electron beam lithography for production of metamaterials which utilise split ring resonance structures [2]. Physical dimensions of the artificial electromagnetic resonance structures as small as nanometres should now be possible. Furthermore the orientation of the C shape illumination can be readily identified, allowing the easy identification of the Faraday rotational effects of the vortex beams [3].

[1] H. Xin and H Zheng (2012) Microscopy and Microanalysis, Vol. 18, p711-9

[2] D. R. Smith, W. J. Padilla, D. C. Vier, S. C. Nemat-Nasser and S. Schultz (2000) Phys. Rev. Lett. 84, p4184-7.

[3] C. Greenshields, R. Stamps, S. Franke-Arnold (2012) New J Phys. 14, 103040

We wish to thank the UK Engineering and Physical Science Research Council (EPSRC) for financial support to this research by a grant (EP/J022098) and M. Ward of Leeds Electron Microscopy and Spectroscopy Centre, University of Leeds for the help with focused ion beam experiment.

↑ Go to the Table of content ↑

IT-2. High resolution TEM and STEM

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-2-IN-2458 Advanced scanning transmission electron microscopy with segmented annular all field detector

Shibata N.1,2
1The University of Tokyo, 2JST-PRESTO
shibata@sigma.t.u-tokyo.ac.jp

In this talk, I will review our recent and on-going findings from our exploration of new atomic-resolution imaging modes using an area detector which is capable of atomic-resolution STEM imaging [1]. One possibility is atomic-resolution differential phase contrast (DPC) imaging [2]. It has been reported that, to a good approximation, DPC STEM images represent the gradient of the object potential (= fields) taken in the direction of the diagonally opposed detector segments, provided the object scatters weakly [3-5]. Here, we show atomic-resolution DPC STEM images of SrTiO3 observed from the [001] direction [2]. Fig. 1(a) shows the orientation relationship between the SrTiO3 crystal and the detector segments used in this study. The probe-forming aperture angle was 23 mrad and the polar angle range of the detector segments was 15.3 to 30.6 mrad. Fig. 1(b) shows the experimental difference image and its intensity profile projected over the vertical direction in the image. The simultaneous ADF STEM image and its intensity profile are used for reference since the peaks in ADF image are a well-established indicator of the true atomic positions. Fig. 1(c) shows the results of corresponding image simulation. It is clear that the DPC STEM profile has a node (zero crossing) at the atom location. The profile is antisymmetric about this point, reflecting the reversal of the electric field direction across the atom along the direction of diagonal detector segments. Combined with detailed image simulations, atomic-resolution DPC STEM is found to provide information on the local electric field distribution in the vicinity of the atomic columns. Some application results of DPC STEM imaging for ferroelectics and their interfaces will be presented.
Another possibility is annular bright-field (ABF) imaging and its derivatives. Fig. 2(a) shows a schematic of the ABF detector geometry. We form “enhanced” (e)ABF images [6] by simply taking the difference between ABF images and the corresponding BF images using the area detector. As shown in Fig. 2(b), we find that light element imaging can be selectively enhanced by this process. We anticipate that the area detector will offer still further possibilities for new atomic-resolution STEM imaging modes useful for material characterization.

References
[1] N. Shibata et al., J. Electron Microscopy 59, 473 (2010).
[2] N. Shibata et al., Nature Phys., 8, 611 (2012).
[3] N.H. Dekkers and H. de Lang, Optik, 41, 452 (1974).
[4] H. Rose, Ultramicroscopy, 2, 251 (1977).
[5] W.C. Stewart, J. Opt. Soc. Am., 66, 813 (1976).
[6] S.D. Findlay et al., Ultramicroscopy, 136, 31 (2014).

I deeply thank S.D. Findlay and Y. Ikuhara for their collaboration in materials characterization and Y. Kohno, H. Sawada and Y. Kondo for their collaboration in the detector development. This work was supported by the PRESTO, JST. A part of this work was conducted in Research Hub for Advanced Nano Characterization, The University of Tokyo. 

Fig. 1: (a) Schematic illustration showing the relationship between the crystallographic orientation of SrTiO3 and the two detector segments. (b) The DPC STEM image formed by subtracting the signal in detector segment Y from that in detector segment X and its image intensity profile [2]. 

Fig. 2: (a)Schematic illustration of BF and ABF detector geometry. (b)ABF and eABF images of LaTiO3 observed from [001] direction [6].
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-2-IN-2576 Insights into Materials Properties with Quantitative STEM and EELS

Botton G. A.1, Bellido E. P.1, Bugnet M.1, Dudeck K. J.1, Gauquelin N.1, Liu H.1, Prabhudev S.1, Rossouw D.1, Scullion A.1, Stambula S.1, Woo S. Y.1, Zhu G. Z.1
1Department of Materials Science and Engineering, McMaster University, Hamilton, ON, Canada
gbotton@mcmaster.ca

The development of aberration correctors in scanning transmission electron microscopy (STEM) has dramatically improved the analytical “toolkit” of materials scientists. In particular, when combined with electron energy loss spectroscopy (EELS), STEM makes it possible to detect compositional and spectroscopic changes at the atomic level that can be used to understand the structure, and ultimately the performance of materials. Here we present some examples of quantitative STEM and EELS as applied to the study of graphene-based materials, complex nanoparticles used in electrocatalysts, and the defects generated in implanted Si and plasmonic structures.

An FEI Titan microscope was used for this work. With this system, we imaged Pt atoms on multilayer graphene nanosheets (GNS) and demonstrate that single Pt atoms are stabilized during atomic layer deposition on N-doped GNS. Quantitative analyses of images show that the single atoms are located at GNS edge steps and that the doping strongly suppresses the growth of Pt clusters (Figure 1a, b) [1]. Similarly, quantitative images have been used to detect atomic displacements on PtFe intermetallic core-shell nanoparticles that exhibit very high specific activity compared to pure Pt [2,3]. Not only is elemental mapping at the atomic scale possible, but the high beam current and fast spectrometers also allow the acquisition of maps with large sampling of the nanostructure. This is illustrated in the study of PtRu nanocatalysts used in fuel cells where Ru core-Pt shell structures are very clearly mapped (Figure 1c).

Beyond the “simple” deduction of the distribution of elements in nanostructures from maps, quantification is essential to understand the detailed structure of defects and correlate compositional measurement with the optical response of materials. The detailed quantification of the atomic position of a defect, in this case a so-called {311} defect [4] generated by the implantation of ions in Si [4,5] shows that an excellent agreement is obtained between the experimental atomic positions and molecular dynamics simulations (Figure 3) [4] with an accuracy of better than 0.05nm for more than 100 atomic columns. Similarly, quantitative analysis of SiGe alloys has allowed us to deduce compositional fluctuations and interdiffusion in proximity of interfaces [6].

[1] S. Stambula et al., J. Phys. Chem. C, on-line (2014), DOI: 10.1021/jp408979h
[2] S. Prabhudev et al., ACS Nano 7, 6103-6110, (2013)
[3] M.C.Y. Chan et al, Nanoscale 4 (22), 7273-7279, (2012)
[4] K.J. Dudeck et al., Physical Review Letters, 110, 166102 (2013)
[5] K.J. Dudeck et al., Semiconductor Science and Technology, 28, 125012, (2013)
[6] G. Radtke et al., Physical Review B 87, 205309, (2013)

The authors are grateful to NSERC for supporting this research. The microscopy was carried out at the Canadian Centre for Electron Microscopy, a National facility supported by NSERC and McMaster. We are grateful to Paolo Longo (Gatan Inc.) for the help in setting up the Quantum 966 spectrometer.

Fig. 1: HAADF STEM image of single Pt atoms and clusters stabilized on N-doped GNS. Raw signals (a), edges and atoms detected (b). Green arrows point to the few Pt atoms (pink) stabilized on GNS terraces (edges labeled in yellow) [1]. (c) elemental maps of PtRu core-shell nanoparticles

Fig. 2: HAADF STEM image of a {311} defect in Si (a) and the deduced atomic positions (crosses) in (b) with the expected atomic positions deduced by molecular dynamics calculations [4].
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-2-IN-2893 Extending the capabilities of high-resolution STEM:measuring depth dependent strain using optical sectioning and aberration-free phase contrast imaging of low-Z materials

Nellist P. D.1,2, Yang H.1, Lozano J. G.1, Hirsch P. B.1, Pennycook T. J.1,2
1Department of Materials, University of Oxford, UK, 2SuperSTEM Laboratory, Daresbury, UK
peter.nellist@materials.ox.ac.uk

The development of aberration correction in scanning transmission electron microscopy (STEM) has had a major impact on spatial resolution and analytical capability. Unsurprisingly, alongside these developments come further complications but also opportunities. The increased numerical aperture allowed by aberration correction leads to a reduced depth of focus (DOF), which in a modern instrument may be just a few nanometres, and typically less than the sample thickness. The increased numerical aperture of the probe converging optics also leads to a larger bright-field (BF) disc in the detector plane, and as a result much of the scattering by the sample remains in the BF disc. In this presentation we will two explore STEM imaging modes that make use of each of these effects to provide aberration-corrected STEM with new capabilities.


The reduced DOF means that in principle a three-dimensional (3D) data-set can be recorded as a focal series of images. In practice, a confocal configuration is generally required. At atomic resolution, however, nanometre-scale depth resolution is also available in the conventional STEM configuration [1]. For dislocations in GaN viewed end-on we show the detection of depth-dependent Eshelby twist displacements associated with screw dislocations. We also show that ADF STEM optical sectioning can be used to measure the screw displacements parallel to the dislocation line for dislocations lying in the plane of the TEM sample, and we use this effect to measure the dissociation reaction of mixed dislocations in GaN. Despite the channelling of the probe, the depth sensitivity persists, and Fig. 1 shows how a simple weighted potential model is a reasonable approximation to a full channelling simulation.


Use of a pixelated detector to record the entire BF disc in the detector plane as a function of probe position results in a 4D data set. A phase contrast image can be retrieved from this data set using a processing method proposed by Rodenburg et al [2]. Interference between the BF disc and a diffracted disc leads to intensity in the overlap region that oscillates with respect to probe position. Figure 2 shows the magnitude and phase of that oscillation for a bilayer graphene sample. From such data a full phase contrast image can be retrieved and we compare the sensitivity of this imaging mode with alternative techniques such as annular bright-field and differential phase contrast. The data is also an excellent instrument diagnostic, and effects such as aperture charging, residual aberrations and the effect of chromatic aberrations can also be observed.

[1] P.D. Nellist and P. Wang, Annual Review of Materials Research 42 (2012) 125-143.
[2] J.M. Rodenburg, B.C. McCallum and P.D. Nellist, Ultramicroscopy, 48 (1993) 303-314.

This research has received funding from the EPSRC and the EU 7th Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3) and was partly performed at the EPSRC National Facility for Aberration-Corrected STEM.

Fig. 1: Aberration-corrected ADF STEM simulated images along the a lattice direction for a 10 nm thick sample of GaN containing a screw dislocation lying parallel to [0001] in the mid-plane of the foil. For each defocus , the left panel shows a full frozen phonon calculation using the QSTEM code and the right panel a simple weighted potential approach.

Fig. 2: The (a) amplitude and (b) phase of the interference observed in the BF disc for one particular spatial frequency with respect to probe position in the 4D data set. The data was recorded from bilayer graphene at 60 kV using a Nion UltraSTEM 200 with a convergence angle of 30 mrad.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-1555 Imaging of light elements by annular dark-field Cs-corrected STEM

Lotnyk A.1, Poppitz D.1, Gerlach J. W.1, Rauschenbach B.1
1Leibniz Institute of Surface Modification (IOM), Leipzig, Germany
andriy.lotnyk@iom-leipzig.de

Nowadays, many crystalline lattices can be imaged directly at atomic resolution in Cs-corrected STEM. Recently, it was shown that light and heavy elements in crystalline lattices can be detected with an ABF method1 or with a double-detector STEM method.2 However, imaging of atomic columns of light elements by ADF method remains challenging. Particularly, the observation of light element columns at the interface between two different materials is still a difficult issue. In this work, we were able to detect directly and simultaneously the N and C atomic columns at the GaN-SiC interface and within the GaN and SiC materials. Additionally, the O atomic columns in a SrTiO3 single crystal were also observed by our method. We have studied the influence of imaging conditions on the appearance of N and C atomic columns in the GaN and SiC materials. The obtained results are discussed and are supported by image simulations.

       The GaN thin film for this study was grown on 6H-SiC(0001) substrate by ion-beam assisted molecular beam epitaxy. STEM experiments were performed on a probe Cs-corrected Titan3 G2 60-300 microscope operated at 300 kV. A probe forming aperture of 20 mrad was used. Cross-sectional samples for STEM work were prepared by FIB technique. To improve the surface quality of the TEM specimens and to reduce the samples thicknesses, a focused low-energy argon ion milling (NanoMill system) was applied.3 Ion energies from 900 eV down to 200 eV were applied to remove implanted Ga ions and amorphous regions caused by the FIB. Image simulations were performed with the xHREM/STEM software package.

       Figures 1 and 2 show the results of our work.4 We found that by adjusting the settings of  HAADF detector and defocus value in STEM, the light element columns at the GaN-SiC interface and within the w-GaN, 6H-SiC and SrTiO3 lattices can be imaged using only a single HAADF detector. We concluded that image simulations for interpretation of atomic-resolution STEM images are only necessary when the probe forming aperture angle overlaps the inner angle of an annular STEM detector or when a complex defect structure is observed in a studied TEM sample. Our method works well using either ADF or HAADF detector, because their angular ranges and defocus values can be easily adjusted on any Cs-corrected STEM. Thus, on TEM systems equipped with only one HAADF detector, the technique can be used without any doubt and upgrades to an ABF detector.

1. S.D. Findlay, N. Shibata, H. Sawada et al., Appl. Phys. Lett. 95, 191913 (2009).
2. Y. Kotaka, Appl. Phys. Lett. 101, 133107 (2012).
3. D. Poppitz, A. Lotnyk, J.W. Gerlach, B. Rauschenbach Acta Mater. 65, 98 (2014).
4. A. Lotnyk, D. Poppitz, J.W. Gerlach, B. Rauschenbach Appl. Phys. Lett. 104, 071908 (2014).

The financial support of the European Union and the Free State of Saxony (LenA project; Project No. 100074065) is greatly acknowledged.

Cs-corrected: aberration-corrected; STEM: scanning transmission electron microscopy; ABF: annular bright-field; ADF: annular dark-filed; HAADF: high-angle ADF; FIB: focused ion beam; w-GaN: wurtzite-type GaN; i: detector inner angle; o: detector outer angle.

Fig. 1: (a) Atomic-resolution STEM image of the GaN-SiC interface taken with a HAADF detector (i20.4-o124.6 mrad) and schematic representation of w-GaN and 6H-SiC lattices along the [2-1-10] zone axis. (b) and (c) Simulated images of w-GaN and 6H-SiC, respectively, at 5 nm underfocus. The TEM sample thickness is measured to be about 16 nm.

Fig. 2: High-resolution STEM images of SrTiO3 acquired with (a) ADF (i19.1-o106.5 mrad) and (b) ABF (i10.1-o19.1 mrad) detectors. The insets in (a) and (b) show the SrTiO3 structure viewed along the [001] zone axis. The TEM sample thickness is measured to be about 60 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-1645 Atomically Resolved 3D Shape Determination of an MgO Crystal from a Single HRTEM Image

Jia C. L.1,2,3, Mi S. B.1,4, Barthel J.3,5, Wang D.1, Dunin-Borkowski R. E.2,3, Urban K. W.2,3, Thust A.2,3
1International Center of Dielectric Research, The School of Electronic and Information Engineering, Xi'an Jiaotong University, Xi'an 710049, China, 2Peter Grünberg Institute, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany, 3Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany, 4Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016, China, 5Central Facility for Electron Microscopy, RWTH Aachen University, Ahornstr. 55, 52074 Aachen, Germany.
a.thust@fz-juelich.de

High-resolution transmission electron microscopy (HRTEM) allows one to investigate the structure of matter on an atomic level [1]. However, most atomic structure characterizations obtained by HRTEM were so far restricted to the determination of atomic column positions in the image plane perpendicular to the incident electron beam. Due to the fact that the depth resolution of the TEM technique along the beam direction is inferior to its lateral resolution, full 3D structure determinations on an atomic level remain highly challenging. The 3D structure retrieval problem can be solved with tomographic methods, where a multitude of images is acquired from different observation directions. Such multi-image approaches are very demanding at atomic resolution due to instrumental instabilities [2] and due to a possible radiation damage of the object. Alternatively, single-image approaches, where only one exposure is taken along a crystallographic zone axis, have been successfully used to count the number of atoms in crystalline columns running parallel to the beam direction. However, a full 3D determination of the crystal shape would additionally require a highly accurate determination of all column positions along the beam direction, which has not been achieved so far with the single-image approach.

We demonstrate that the full 3D shape of a thin MgO crystal can be determined in a nearly unique way from a single HRTEM image (Fig. 1). Our 3D determination of the crystal shape is based on refining an atomic structure model (Fig. 2) in such a way that a HRTEM image simulated on the basis of this model fits best to the experimental image. In contrast to the usual simplifying assumption of flat lower and upper object surfaces in conjunction with a single global defocus value [3], our structure refinement is executed now locally column-by-column, allowing also for atomically corrugated object surfaces. The comparison between simulation and experiment is made on the basis of absolute image intensity values [4]. A crucial part of our procedure is an extended statistical confidence test which yields detailed quantitative statements on the uniqueness and the reliability of the retrieved 3D crystal shape.

References:

[1] K.W. Urban, Science 321 (2008) 506.
[2] J. Barthel and A. Thust, Ultramicroscopy 134 (2013) 6.
[3] C.-L. Jia et al, Microsc. Microanal. 19 (2013) 310.
[4] A. Thust, Phys. Rev. Lett. 102 (2009) 220801.

Fig. 1: High-resolution image of the edge of an MgO crystal taken along the [001] zone axis with a CS-corrected FEI Titan 80-300 electron microscope at 300 kV accelerating voltage. The 3D shape reconstruction was performed at the area indicated by the dashed box.

Fig. 2: 3D structure model retrieved from the boxed area in Fig. 1. Red balls indicate Mg atoms, yellow balls O atoms, purple balls indicate formally half-occupied Mg positions, and green balls formally half-occupied O positions.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-1857 Towards a quantitative exit wave function: the influence of phonon scattering

Liberti E.1, Kim J. S.1, Kirkland A. I.1
1Department of Materials, University of Oxford, Parks Road, Oxford, OX1 3PH, UK
emanuela.liberti@materials.ox.ac.uk

To provide the utmost quantitative information about the atomic structure of the specimen is the ultimate challenge sought by modern high-resolution transmission electron microscopy. At the specimen exit surface, quantitative structural information is embedded in the object complex wave function, which can be recovered, with atomic resolution, from a focal (or tilt) series of aberration corrected HRTEM images [1]. Nonetheless, the quantitative information that is obtained from the exit wave is often in disagreement with imaging simulations. This disagreement is in effect a contrast mismatch, or Stobbs factor, which accounts for a reduction of the experimental image contrast by a factor of three with respect to the calculations [2]. The scattering of phonons following the electron beam-specimen interaction is amongst the possible causes of the Stobbs factor [3].
In this contribution, we discuss the role of phonon scattering in the quantification of the exit wave function of a single layer of graphene. For this idealized object, the contribution of the thermal phonon scattering to the total elastic scattering can be directly investigated by quantifying the exit wave function at different temperatures. For the imaging simulations, the influence of thermal motion upon modeling of the elastic scattering is studied quantitatively, using both the absorptive potential and frozen phonon approaches, addressing the role of the Debye-Waller factor in predicting the thermal displacement of graphene atoms. Experimentally, the exit wave function is recovered in the linear imaging approximation, in both heating and cooling conditions, as well as at room temperature.
To conclude, we present, and discuss, the comparison between the quantitative exit wave functions, obtained in both calculated and experimental approaches.

[1] A.I. Kirkland, S. J. Haigh, Jeol news, 44 (2009) 6 – 11.
[2] M.J. Hÿtch, W.M. Stobbs, Ultramicroscopy 53 (1994) 191 – 203.
[3] A. Howie, Ultramicroscopy 98 (2004) 73 – 79.

The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483-ESTEEM2 (Integrated Infrastructure Initiative–I3).

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-1921 Assessment of lower-voltage TEM performance using 3D Fourier transform of through-focus images

Kimoto K.1, Ishizuka K.2
1National Institute for Materials Science, Tsukuba, Japan, 2HREM Research Inc., Higashimatsuyama, Japan
kimoto.koji@nims.go.jp

The performance of an aberration-corrected TEM is determined by the information limit that is often demonstrated using Young's fringe method. However Young's fringe method could show unexpected high frequency information due to the non-linear terms as pointed out by several researchers [1,2]. The three-dimensional (3D) Fourier transform (FT) of through-focus TEM images allows us to discriminate between the linear and the non-linear imaging terms [3,4]. The linear imaging terms are observed on twin Ewald spheres in the 3D FT using an amorphous specimen. Here, we use the 3D FT of through-focus TEM images for the assessment of two low-voltage TEM systems.

Two spherical-aberration-corrected microscopes were assessed and compared. One was a Titan3 (FEI) equipped with a monochromator and a spherical aberration corrector for image forming (CEOS, CETCOR) operated at an acceleration voltage of 80 kV. The energy spread of the electron source was 0.1 eV under monochromated condition. The other microscope, the TripleC microscope, was equipped with a cold field-emission gun (CFEG) and the spherical aberration corrector developed for the TripleC project. This microscope was operated at 60 and 30 kV [5], and the energy spread was 0.3-0.4eV.

Figure 1 schematically shows various 3D data processed in this study [6]. Acquired through-focus TEM images are stacked as a function of the defocus z (Fig. 1a). The 3D Fourier transform Iuvw (Fig. 1c) of through-focus images shows two paraboloids called Ewald spheres, attached at the origin. The information limit can be estimated as an observable range of the Ewald spheres.

The signal of Ewald spheres depends on various factors, such as atomic scattering factors, a specimen structure, thickness, and the modulation transfer function of an imaging device; therefore, the quantitative evaluation of diverse TEM systems is not straightforward. Here we apply the tilted incidence in the 3D Fourier transform method (Fig. 2) to normalize those factors. We evaluate the spatial frequency at which information transfer decreases to 1/e2 (Fig. 3). It was found that the energy spread of the electron source is the major limiting factor even in a monochromated TEM [7].

[1] M. Haider et al., Microsc. Microanal. 16 (2010) 393. [2] J. Barthel, et al., Phys. Rev. Lett. 101 (2008) 200801. [3] Y. Taniguchi, et al., J. Electron Microsc. 40 (1991) 5. [4] M. Op. de Beeck et al., Ultramicrosc. 64 (1996) 167. [5] H. Sawada et al., Ultramicrosc. 110 (2010) 958. [6] K. Kimoto et al., Ultramicrosc. 121 (2012) 31. [7] K. Kimoto et al., Ultramicrosc. 134 (2013) 86.

We thank Drs. Nagai, Freitag, Sawada, Sasaki, Ohwada, Sato and Suenaga for invaluable discussions. This work is supported by Nanotechnology Platform of MEXT and Research Acceleration Program of JSPS.

Fig. 1: Schematics of (a) through-focus TEM images Ixyz, (b) stack of 2D FTs Iuvz, and (c) 3D FT of the through-focus images Iuvw. Since Iuvz and Iuvw are complex, their moduli are shown in gray scale. The cross section Ivz is similar to the Thon diagram. Two Ewald spheres attached at the origin are observed in the 3D Fourier space Iuvw.

Fig. 2: Cross sections of 3D FTs under on-axial and tilted incidence conditions. (a) Titan3 (80kV) and (b) TripleC (60kV).

Fig. 3: Information limit of (a) monochromated Titan3 (80kV), TripleC at 60kV (b) and 30kV (c).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2157 An alternative normalization method for quantitative STEM

Martinez G. T.1, Jones L.2, Béché A.1, Verbeeck J.1, Nellist P. D.2, Van Aert S.1
1Electron Microscopy for Materials Science (EMAT), University of Antwerp, Antwerp, Belgium, 2Department of Materials, University of Oxford, Oxford, United Kingdom
gerardo.martinez@uantwerpen.be

Techniques such as annular bright-field (ABF) or high-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) have become widely used in quantitative studies because of the possibility to directly compare experimental and simulated images when the experimental data is expressed in units of ‘fraction of incident probe’ [1]. This is achieved by subtracting by the amplifier’s ‘black-level’ normalizing the experimental image by the mean sensitivity of the annular detector. Since the detector response is spatially inhomogeneous [2], a ‘detector sensitivity’ profile needs to be included in image simulations in order to account for these irregularities. Unfortunately, the quantification procedure now becomes both experiment and instrument specific, with new simulations needing to be carried out for the specific response of each instrument’s detector. This not only impedes the comparison between different instruments but can also be computationally very time consuming.

In this work, we propose an alternative method for normalizing experimental data in order to compare these with simulations that consider a homogenous detector response. To achieve this, we determine the electron flux distribution reaching the detector by means of a camera length series, which is then used to determine the corresponding weighting of the detector response. Figure 1a) shows the detector scan and b) its corresponding active area. The electron flux reaching the active area of the detector is shown in Figure 1 c), which was determined using a camera length series (Figure 2). Next, after normalizing this flux profile to unity, it is multiplied pixel-wise with the experimental detector map, Figure 1d), in which the detector response inhomogeneity is clearly observed. By integrating Figure 1d), we obtain an overall ‘flux-weighted detector sensitivity’ value, which can be used for the experimental data normalization. To validate the proposed methodology, we simulated a [100] oriented Pt crystal using the StemSim software under the frozen lattice approach [3]. The simulations considered homogeneous and inhomogeneous detector sensitivities for 60 – 190 mrad detector acceptance angles. Figure 3 shows that the total intensity for a simulation considering inhomogeneous detector sensitivity followed by electron flux weighting (analogous to experimental conditions) is in perfect agreement with simulations performed with homogeneous detector sensitivity (the ideal case).

[1] J. M. Lebeau and S. Stemmer, Ultramicroscopy 108 (2008), p.1653-8
[2] K. MacArthur, L. Jones, and P. Nellist, Journal of Physics: Conference Series (2013)
[3] A. Rosenauer and M. Schowalter, Springer Proceedings in Physics, vol. 120 (2007), p. 169–172.

Funding from the FWO Flanders, the EU FP7 (312483 - ESTEEM2), and the UK Engineering and Physical Sciences Research Council (EP/K032518/1) is acknowledged.

Fig. 1: Proposed flux-weighted normalization steps: a) experimental detector map, b) detector active area, c) determined flux pattern using camera length series, and d) flux-weighted sensitivity resulting from product of plots a) and c).

Fig. 2: Measured electron flux distribution from simulated camera length series. Using this plot, Figure 1c) is computed for the detector active area.

Fig. 3: Total scattered intensity for homogeneous (blue) and inhomogeneous (black) detector sensitivity. Red circles correspond to the total scattered intensity of inhomogeneous detector sensitivity after electron flux weighted normalization.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2221 Channeling Effects on the Accuracy of HAADF STEM Quantification of Bimetallic Catalyst Nanoparticles

MacArthur K. E.1, Lozano-Perez S.1, Ozkaya D.2, Nellist P. D.1
1Department of Materials Science, University of Oxford, Oxford, UK , 2Johnson-Matthey Technical Centre, Reading, UK
katherine.macarthur@materials.ox.ac.uk

Quantification of high angle annular dark-field scanning transmission electron microscope (HAADF STEM) images uses atomic resolution images as data sets to extract sample composition and thickness information from. A new quantification method based on calculating the scattering cross-section (CS) of each atomic column has been shown to be more robust to microscope image parameters.1 Using an automated code,2 this analysis involves converting images to an absolute scale through detector normalisation3, integrating over each atomic column within an image and multiplying by pixel area.
Whilst mathematically robust to microscope imaging parameters there are many other factors which affect the accuracy of quantification results. Channelling occurs when the columns of atoms in a crystal are aligned parallel to the incident electron beam and they act like miniature lenses providing an extra focusing effect on the probe. The subsequent atoms in the atomic column see a more focused probe than the first atom; resulting in them supplying increased scattering out to the detector. Along the length of the column, oscillations in intensity are seen, much as though the electrons are propagating in a waveguide. The whole column may therefore have a different scattering CS than the sum of the individual CSs of its constituent atoms. The ordering of atom types within an atomic column also affects the overall CS. Comparably another process known as de-channelling provides cross-talk between neighbouring columns of atoms. Cross-talk occurs when part of the probe is scattered and becomes channelled by a neighbouring column of atoms and then scattered out to the detector, thereby contributing information to the signal from neighbouring columns.
Atomic resolution requires viewing a crystal down a low order zone axis; any sample mis-tilt away results in a reduction in the channelling contribution and therefore a loss in CS, Figure 1. By 4̊ of tilt the effects of channeling are almost completely lost, whilst some atomic resolution remains. Top-bottom effects in the bimetallic columns are also diminished by 4̊ mis-tilt. At small tilts, however, there is a plateau region where the CS is independent of tilt, the size of which is dependent on probe convergence angle size, Figure 1. We believe the robustness to tilt when imaging on axis is more beneficial than the potential composition information gain from tilting far off a zone axis. This is particularly the case for nanoparticles which tilt under the beam. Combining with spectroscopy techniques will be necessary for gaining compositional information.

1 H E et al, Ultramicroscopy 133 (2013), p109-19
2 The Absolute Integrator is free for academic use from www.lewyjones.com/software/
3 JM LeBeau et al, Nano Letters 10 (2010), p4405-8

The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3), and from the EPSRC (grant number EP/K032518/1).

Fig. 1: The CS of a column of 7 Pt atoms in a crystal plotted against tilt away from a <110> zone axis. (a) Pt, blue, with the top or bottom atom replaced with Co, red and green, show a plateau region before dropping with tilt. With no channelling the CS would be 7x1 Pt atom, dotted red. (b) Tilt plot with different probe convergence angles.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2283 Measuring surface atom structures in Pt and Au nanocatalysts with high precision STEM imaging

Yankovich A. B.1, Berkels B.2,3, Dahmen W.2,4, Binev P.2, Sanchez S. I.5, Bradley S. A.5, Voyles P. M.1
1University of Wisconsin – Madison, USA, 2University of South Carolina, USA, 3Universität Bonn, Germany, 4RWTH Aachen, Germany, 5UOP LLC a Honeywell Company, USA
ayankovich@wisc.edu

TEM and STEM aberration correctors make sub-Ang resolution imaging routine. Once atoms are resolved, the question is how precisely can their positions be measured? TEM and STEM regularly achieve precision smaller than the resolution, but STEM encounters practical limits, such as image distortions from instabilities, before reaching the signal to noise ratio (SNR) fundamental precision limit. Combining multiple frames improves SNR and precision. Rigid registration is a common approach, but it does not correct for all types of instabilities. We have developed a non-rigid (NR) registration scheme for STEM images that accounts for all types of image distortions caused by instabilities during acquisition[1, 2].

Fig. 1 shows the results of the NR registration and averaging of a series of 512 HAADF STEM images of GaN. We show that sub-pm precision is achieved by fitting each Ga column to a 2D Gaussian, calculating the interatomic separations as shown in the histograms in Fig. 1(c) and (d), and using the standard deviation as the precision. The sub-pm precision in the x and y directions (0.74 and 0.85 pm) is reproducible and is 5-7 better than rigid registration.

A multislice simulated HAADF STEM image of a Si [110] dislocation core, shown in Fig. 2(a) was used to create an image series that includes distortions representative of real experiments, including thermal drift, floor vibrations, acoustic noise, electromagnetic fields, and electronic instabilities. Fig. 2(b) shows the NR registered and averaged image of the distorted series, demonstrating that inhomogeneous strain is preserved by NR registration.

NR registering and averaging STEM images allows for pm-scale measurements of surface atom bond length variation in Pt nanoparticles, which are prototypical noble metal catalysts. NP’s surface structure is crucial to their chemical activity but measuring it is extremely challenging. Fig. 3(a) shows that a Pt nanocatalyst exhibits pm-scale contraction of atoms at a (1-11)/(-1-11) corner and expansion of a (1-11) facet, with very little lateral displacement. Standardless atom counting on the same NR registered STEM image shows that the Pt NP is between 1 and 8 atoms thick with <1 atom uncertainty, as shown in Fig. 3(b). High precision in both positions and thickness are enabled by the extremely high SNR after NR registration. In general, STEM imaging with pm-precision will aid in understanding atomic displacement fields important in catalysis, defects, interfaces, and ferroic materials.

[1] Berkels et al, Ultramic. 138, 46 (2014).

[2] Yankovich et al, “Picometer-Precision Analysis of STEM Images of Pt Nanocatalysts” under review (2014).

We acknowledge funding from the Department of Energy, Basic Energy Sciences (DE-FG02-08ER46547), NSF (DMS 1222390), USC’s Special Priority Program SPP 1324, and the Excellence Initiative of the German federal and state governments, and the UW Materials Research Science and Engineering Center (DMR-1121288).

Fig. 1: (a) The first of 512 HAADF STEM images of GaN [11-20]. (b) Average of 512 frames after NR registration. The red dots are the positions of the columns identified by fitting. (c) and (d) Histograms of the X and Y separation measurements from (b).

Fig. 2: (a) Simulated HAADF STEM image of a Si [110] dislocation core model displaying an inhomogeneous strain field. (b) NR registered and averaged HAADF STEM image after the simulated image in (a) was made into an image series with typical distortions representative of experimental series.

Fig. 3: (a) Average of 56 HAADF STEM images of a Pt [011] nanoparticle after NR registration. The red arrows show magnified displacement vectors of the surface atoms and the displacement magnitudes are labeled in white. (b) The number of atoms in each column determined by comparing the experimental absolute intensities to simulations.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2611 The detection of single dopant atoms by high-resolution off-axis electron holography

Cooper D.1, Mayall B.1, McLeod R.2, Haigh S.3, Dunin-Borkowski R. E.4
1CEA-LETI, Minatec, 17 rue des Martyrs, 38054 Grenoble, Cedex 9, France, 2CEA-INAC, Minatec, 17 rue des Martyrs, 38054 Grenoble, Cedex 9, France, 3School of Materials The University of Manchester, Manchester M13 9PL, U.K., 4Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, D-52425 Jülich, Germany
david.cooper@cea.fr

In 2003, a paper was published that discussed the need for a technique to detect single dopant atoms. It was stated that “single dopant atom detection is critical to device design, but it can also unravel complex and unexpected phenomena which may also open up new areas of materials exploration”. In 2014, it is still highly challenging to measure the locations, chemical identities and electrostatic potentials of single dopant atoms [1].

The technique of off-axis electron holography in the transmission electron microscope (TEM) involves the use of an electron biprism to interfere an electron wave that has passed through a thin specimen with a reference wave, in order to form an interference pattern that can be used to determine the phase shift of the electrons. Although electron holography has been used for many years to measure dopant potentials in semiconductors, soon devices will become so small that measurements of the electrostatic potentials of individual dopant atoms may be required.

Based on simulations, the expected step in phase shift across a single ionized P atom in Si is ~2π/1000 radians. This level of sensitivity can be reached easily in electron holographic measurements at low spatial resolution if long acquisition times are used. However, it is more of a challenge at atomic resolution. Here, we demonstrate progress towards the detection of single dopant atoms using electron holography. Figure 1(a) shows an electron hologram acquired at 80 kV using an aberration-corrected FEI Titan Ultimate TEM equipped with a high brightness gun, a monochromator and a single biprism. A careful choice of microscope lens settings allows holograms to be acquired with excellent interference fringe contrast and fine fringe spacing. Figure 1(b) shows an intensity profile extracted from the hologram, while Fig. 1(c) shows the experimentally measured phase resolution plotted as a function of interference fringe spacing, demonstrating that the conditions required to detect single dopant atoms are within reach if large numbers of phase images are added together.

Figure 2(a) shows part of an off-axis electron hologram of a thin MoS2 crystal recorded using an interference fringe spacing of 40 pm. The corresponding reconstructed phase image in Fig. 2(b) has a spatial resolution of 0.12 nm, while the line profile in Fig. 2(c) demonstrates that individual atomic columns with a spacing of 0.12 nm can be resolved. We are presently working towards the acquisition of signals from single dopant atoms in graphene and silicon and comparing our results with scanning TEM images. Great care is required to optimize specimen preparation and to minimize radiation damage, electron beam induced charging and contamination.

[1] Castell et al. Nature Materials 2, 129-131 (2003)

DC and RDB thank the ERC for the starting grant “Holoview” and the advanced grant “IMAGINE” respectively.

Fig. 1: (a) An off axis electron hologram acquired with a fringe spacing of 50 pm (b) profile of the fringe intensity showing a contrast of 33 %. (c) Experimentally measured phase resolution as a function of fringe spacing (spatial resolution is 2-3 times the fringe spacing).

Fig. 2: (a) Detail of an off-axis electron hologram of a MoS2 crystal with a fringe spacing of 40 pm (b) reconstructed map of the electrostatic potential profile and (c) profile showing that the 1.2 A spaced atoms have been resolved.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2414 A Method to Analyse the Chemical Composition in (InGa)(NAs) based on Evaluation of HAADF Intensity in STEM

Grieb T.1, Müller K.1, Mahr C.1, Cadel E.2, Beyer A.3, Talbot E.2, Schowalter M.1, Volz K.3, Rosenauer A.1
1Institute of Solid State Physics, University of Bremen, Otto-Hahn-Allee 1, 28359 Bremen, Germany, 2Groupe de Physique des Matériaux (GPM) UMR 6634, Normandie Université, Université et INSA de Rouen–CNRS, Av. de l’Université, BP 12, 76801 Saint Etienne du Rouvray, France, 3Materials Science Center and Faculty of Physics, Philipps University Marburg, Hans-Meerwein-Straße, 35032 Marburg, Germany
rosenauer@ifp.uni-bremen.de

InxGa1-xNyAs1-y is of technological interest for laser diodes in telecommunication and solar cells as both, In and N, lower the semiconductors band gap to emit or absorb in the infra-red spectral range. It was shown for ternary materials that an unknown chemical concentration (eg. of In in InGaN [1]) can be determined by high-angle annular dark field (HAADF) scanning transmission electron microscopy (STEM). For this purpose experimental HAADF intensities are compared with simulated ones. The experimental intensities are normalized to the total beam intensity which allows for determining the thickness in regions with known chemical composition. In this contribution this method is extended to evaluate the quaternary system InxGa1-xNyAs1-y. As a specific HAADF intensity cannot be allocated to a pair of concentrations (x,y) in a unique way, further information is needed. To this end, the local strain state is additionally determined from the high-resolution HAADF-STEM image.

The HAADF intensities were simulated with a frozen-lattice multislice approach implemented in the STEMsim software [2], considering thermal diffuse scattering (TDS). It was shown that for (In)GaNAs, besides TDS, Huang-scattering at static-atomic displacements (SADs) has to be taken into account [3]. SADs are distortions of the atomic lattice due to different covalent radii of In and Ga as well as As and N. The SADs were computed by relaxing the supercells using Keating's valence force field parametrization [4] in the LAMMPS code [5]. Fig. 1. shows the ratio of the simulated HAADF intensity of InGaNAs and GaAs versus specimen thickness for different In and N concentrations. For thicknesses above approx. 50 nm the intensity ratio increases not only with In but also with N concentration, although N has a smaller atomic number than As. This effect reveals the strong influence of additional scattering at SADs. An MOVPE grown InGaNAs/GaAs quantum-well sample is characterized by the outlined method. The mean concentrations of 32 % In and 2 % N (see concentration profiles in Fig. 2) are in good agreement with the results from XRD (marked by arrows). In addition, atom-probe tomography was applied to this sample, and the corresponding In profile is also shown in Fig. 2. Both, profile shape and mean concentration are in good agreement with the HAADF-STEM results.

[1] Rosenauer et al., Ultramicroscopy 111 (2011) 1316.

[2] A. Rosenauer and M. Schowalter, Springer Proc. Phys. 120 (2007), 169.

[3] Grillo et al., Phys. Rev. B 77 (2008), 054103.

[4] P. N. Keating, Phys. Rev. 145 (1966), 637.

[5] S. Plimpton, J. Comput. Phys. 117 (1995), 1.

We thank the DFG under contracts SCHO 1196/3-1, RO 2057/8-1 and GRK1782.

Fig. 1: Ratio of the simulated HAADF intensity for InGaNAs and GaAs (material contrast) as a function of specimen thickness for different indium concentrations (color) and nitrogen concentrations (line style). The HAADF intensity increases for specimen thicknesses above 50 nm with In and N concentration due to Z-contrast and scattering at SADs.

Fig. 2: Determination of the chemical composition of an InGaNAs layer embedded in GaAs. Concentration profiles from averaging concentration maps (HAADF analysis: indium and nitrogen) and from atom probe tomography (only indium). Concentrations derived from HRXRD are marked by arrows.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2538 Optimizing Phase Contrast Imaging in Aberration Corrected TEM

Kahl F.1, Hartel P.1, Linck M.1, Müller H.1, Haider M.1
1Corrected Electron Optical Systems GmbH, Heidelberg, Germany
frank.kahl@gmx.net

Since the realization of the first aberration corrected TEM [1], the number of corrected TEMs is still rapidly growing. Two key benefits are boosting the tremendous success of spherical aberration correction: vanishing delocalization and improved point resolution limit. The latter is achieved by using CS=C3 as additional optimization parameter to increase the aperture radius where the phase shift distribution (phase plate) of the elastically scattered electrons stays close to the optimum of +π/2 or −π/2 for dark or bright atom contrast, respectively. Various efforts have been made to optimize the phase plate, e.g. [2, 3], employing different measures for the distance of real and ideal phase plate as criterion for optimization. However, the different criteria lead to similar results.

Many users still use a π/4-limit for each aberration coefficient separately to assess the corrected state. However, advanced criteria such as minimizing the integrated mean quadratic deviation [3] or minimizing the largest deviation from the ideal phase over the aperture are much more sensible. While designing the SALVE II corrector [5] we used the latter criterion for optimizing the phase plate to assess imaging quality.

Fig. 1 shows the phase shifts generated by all axial aberration coefficients up to fifth order. Compensation schemes can be applied for aberrations of same multiplicity but different orders. In Fig. 1 potential partners are arranged within one column. The highest-order coefficient and the aperture size determine the optimum values for the lower-order coefficients (e.g. multiplicity 2: S5 given by corrector design; S3, A1 optimized during alignment). The procedure for optimizing coefficients of non-zero multiplicity is similar to optimize C3 and defocus C1 for a given C5, except that the deviation from zero instead of +π/2 or −π/2 is minimized.

The performance of the SALVE II corrector for 40 kV is shown in Fig. 2. The phase plate (a) corresponds to the output of the CEOS software after aberration correction. The π/4-circle is misleadingly small as it is largely determined by C3. In image (b) only the sum of all non-round contributions is shown. Optimizing C1 for given C3 and C5 yields passband (c). Only with a full compensation scheme (d, e) for all fourth- and fifth-order aberrations using all adjustable lower order aberrations, a passband of up to 50 mrad can be achieved.

References:

[1] M. Haider et al, Nature 392 (1998), 768-769.

[2] O. Scherzer, Ber. Bunsen-Gesellschaft phys. Chemie 74 (1970), 1154-1167.

[3] M. Lentzen, Microsc. Microanal. 14 (2008), 16-26.

[4] M. Born, E. Wolf, Principles of Optics, 6th edition (Cambridge university press, Cambridge), p. 468.

[5] SALVE II project, <http://www.salve-project.de>.

none

Fig. 1: Table of axial aberrations visualized as phase plates. At the aperture edge ±6π is adopted; the phase is wrapped to [-π (black); π (white)[. Aberrations of same multiplicity but different order can partly compensate each other.

Fig. 2: Left: Phase plates for measured aberration coefficients at the SALVE II microscope operated at 40 kV. The passband in (c) demonstrates the reduced contrast transfer due to non-round residual aberrations. Right: With a suitable compensation scheme a passband up to 50 mrad can be achieved. The aperture radius of all phase plates is 75 mrad.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2644 Low-voltage and energy-filtered chromatic aberration-corrected high-resolution TEM on the PICO instrument

Houben L.1,3, Luysberg M.1,3, Barthel J.2,3, Mayer J.2,3, Dunin-Borkowski R. E.1,3
1Peter Grünberg Institut 5, Forschungszentrum Jülich, Germany, 2Gemeinschaftslabor für Elektronenmikroskopie, RWTH Aachen, Germany, 3Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich, Germany
l.houben@fz-juelich.de

The advent of chromatic aberration correction in the transmission electron microscope (TEM) offers new prospects for high-resolution imaging at low accelerating voltages and for energy-filtered TEM (EFTEM). Examples of low voltage and energy-filtered images of complex oxides, thin layered materials, and nanoparticles will be presented, demonstrating the unique optical properties of an achroplanatic CEOS CCOR Cc/Cs corrector on Jülich’s chromatic aberration-corrected “PICO” microscope.
Atomic-resolution imaging at low voltages, currently down to 50 kV, allows high-resolution studies of radiation-damage-sensitive nanomaterials, such as CdSe/CdS nanostructures obtained from a cation exchange reaction, graphene and carbon nanotubes. It is also beneficial for the study of organic ligands, ligand-stabilised materials and materials that are functionalized with organo-metallic compounds.
The ability to acquire dose-efficient atomic-scale EFTEM elemental maps using the achroplanatic CCOR corrector on this microscope with a large field of view and large energy windows results from the fact that the chromatic focus spread is negligible after chromatic aberration correction. Figure 1 shows an example of an atomic-resolution elemental map of Ca obtained from a thin TEM foil of a calcium-titanate/strontium-titanate multilayer. Figure 2 shows a structural and compositional modulation in a (CeS)1.2CrS2 misfit-compound nanotube, which comprises alternating hexagonal CrS2 and rock-salt CeS sheets that have a repeat period of 11.2 Ångstrom.
The quantification of EFTEM elemental maps to provide atomic-resolution information about the local chemical composition of a specimen is complicated by the preservation of elastic contrast due to elastic scattering, which gives rise to thickness and defocus dependent contrast with fine details at all energy losses. Optical stability over minutes of collection time and careful image alignment and background subtraction are also required to obtain meaningful and reliable atomic-scale EFTEM elemental maps.

The authors thank J. Schubert (Forschungszentrum Jülich) and L. Penchakarla and R.Tenne (Weizmann Institute of Science) and M. Bar Sadan (Ben Gurion University) for kindly providing the materials used in this study

Fig. 1: High-resolution EFTEM images of a CaTiO3/SrTiO3 [001] multilayer sample taken at 300 kV. Ca L23 (a) pre-edge image, (b) post-edge image and (c) background-subtracted map. (d, e, f) Noise-reduced images obtained by averaging over 5x5 periods in the CaTiO3 layer, revealing Ca on the A sites of the pseudo-cubic perovskite lattice.

Fig. 2: Atomic-resolution micrographs and spectroscopic images of a CeCrS3 nanotube. (a) Schematic view of the CeCrS3 misfit lattice and a tubular structure. (b) HRTEM image and (c) magnified region of (b) with crystallographic projections superimposed. (d) EFTEM maps showing alternating Ce and Cr signals and (e) magnified region of (d).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2658 Atomic resolution secondary electron imaging and simulation of the SrTiO3 (001) c(6x2) surface reconstruction

Ciston J.1, Brown H. G.2, D'Alfonso A. J.2, Koirala P.3, Lin Y.3, Ophus C. L.1, Inada H.4, Zhu Y.5, Allen L. J.2, Marks L. D.3
1National Center for Electron Microscopy, Lawrence Berkeley National Lab., Berkeley, USA, 2School of Physics, University of Melbourne, Parkville, Victoria, Australia, 3Department of Materials Science and Engineering, Northwestern University, Evanston, USA, 4Hitachi High Technologies Corporation, Ibaraki, Japan, 5Condensed Matter Physics and Materials Science, Brookhaven National Laboratory, Upton, USA
jciston@lbl.gov

Aberration-corrected scanning transmission electron microscopes (STEM) enable simultaneous collection of atomically resolved signals relating to coherent scattering (bright field and annular bright field imaging), structural information based on thermal scattering to large angles known as high-angle annular dark-field (HAADF) imaging, bonding information using electron energy-loss spectroscopy (EELS), and element identification using both EELS and energy dispersive x-ray spectroscopy. Atomic resolution imaging based on secondary electron (SE) signals was demonstrated in 2009 [1], but the technique is only slowly growing in use. While these SE signals are highly surface sensitive due to the narrow escape depth of electrons with energies <50eV [2], atomic resolution SE imaging of surface structures that differ from a simple bulk crystal termination has not been previously demonstrated.


We have recently imaged the c(6×2) reconstruction on the (100) surface of single crystal SrTiO3 (Fig 1) through simultaneous atomic resolution SE and HAADF STEM with complementary HREM imaging. The ability to simultaneously record surface sensitive SE and bulk dominated HAADF signals at atomic resolution makes the problem of surface structure registration to the bulk lattice highly tractable, which is a distinct advantage over other scanning probe methods. By inspection it is clear that the registration of the previously reported structure, primarily refined from surface x-ray diffraction and scanning tunneling microscopy (STM) experiments [3], is incorrect. Interpretation of the experimental SE measurements from first principles is now possible using a recently developed quantum mechanical model to simulate SE images. This approach takes into account the probability and angular distribution of electrons that are ejected from atoms in the specimen when ionization of both core and semi-core electrons occurs [4]. Our preliminary simulations of a newly proposed structure of the SrTiO3-<100>-c(6×2) reconstruction are in good agreement with the bulk-subtracted experimental SE data (Fig 2), and consistent with previously reported data from STM, Auger spectroscopy, and x-ray diffraction measurements. The structure solved by SE imaging is also stable in density functional theory simulations, and is on the thermodynamic convex hull of known reconstructions on SrTiO3 <100>.


[1] Y Zhu et al., Nat. Mater. 8 (2009) p. 808
[2] A Howie, J. Microsc. 180 (1995) p.192
[3] CH Lanier, et al., Phys. Rev. B 76 (2007) 045421
[4] HG Brown et al., Phys. Rev. B 87 (2013) 054102

A portion of this work was performed at NCEM, supported by the Office of Science, Basic Energy Sciences of the U.S. Department of Energy under Contract No.: DE-AC02-05CH11231.

Fig. 1: (a) Weak beam dark field image of SrTiO3 001 c(6x2) single crystal with g=(200) showing atomically flat terraces (b) Transmission electron diffraction pattern of the c(6x2) reconstruction with c2mm symmetry acquired off-zone to minimize bulk dynamical diffraction

Fig. 2: (a) Bulk subtracted experimental secondary electron image of a 6×2 unit cell surface reconstruction on a <100> SrTiO3 substrate averaged over ~600 unit cells with c2mm symmetry enforced. The 200-keV probe had a convergence semi-angle of 25 mrad. (b) Prelimenary simulation of the result in (a) using the (projected) surface reconstruction indicated.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2674 Fast imaging with inelastically scattered electrons by off-axis chromatic confocal electron microscopy

Zheng C. L.1, Zhu Y.1, Lazar S.2, Etheridge J.1
1Monash University, Victoria, Australia, 2FEI Electron Optics, Eindhoven, The Netherlands
changlin.zheng@monash.edu

Imaging with inelastically scattered electrons is an important method for studying the composition and electronic properties of materials down to the atomic scale [1]. In this work, we describe an approach for fast mapping of inelastically scattered electrons using a scanning transmission electron microscope in a confocal mode, without using a spectrometer. We develop an off-axis scanning confocal electron microscope configuration using a double spherical-aberration corrected STEM/TEM. The electron probe is focused onto the sample at a significant angle to the optic axis of the imaging lens (Fig 1) and the probe-corrector retuned to form an atomic-scale electron probe in the specimen plane. Under the effect of the chromatic aberration of the imaging lens system, electrons with a chosen energy loss, the confocal energy, Ec, can be focused to a confocal point on the detector plane, while electrons of all other energies, including the zero loss electrons, will be chromatically defocused at that plane. In addition, the tilting of the incident beam laterally shifts the object exit wave in the back focal plane of the imaging lens, introducing an energy-related lateral displacement of the defocused probe. The inelastically scattered electrons are then chromatically dispersed both parallel and perpendicular to the optic axis, effectively separating electrons with different energies. In particular, electrons with the confocal energy can be detected selectively using an integrating detector. Using a synchronized set of scan-descan coils, these confocal electrons can remain focused on the detector as the electron probe is scanned across the specimen (Fig 1).

We illustrate the method with nanoscale core-loss chemical mapping of silver (M4,5) in an aluminium-silver alloy and atomic scale imaging of the low intensity core-loss La (M4,5@ 840eV) signal in LaB6 (Fig 2). The scan rates are up to 2 orders of magnitude faster than conventional STEM spectrum imaging methods recorded by CCD, enabling a corresponding reduction in radiation dose and improvement in the field of view [2]. Moreover, this off-axis chromatic confocal configuration offers the potential for fast nanoscale three-dimensional chemical mapping when coupled with the improved depth and lateral resolution of the incoherent confocal mode [3].

[1] R. F. Egerton, Electron energy-loss spectroscopy in the electron microscope (Plenum Press, New York, 1996), 2nd edn.

[2] C. Zheng, Y. Zhu, S. Lazar, J. Etheridge, Physical review letters, accepted (2014).

[3] T. Wilson and C. Sheppard, Theory and practice of scanning optical microscopy (Academic Press, London ; Orlando, 1984)

Funding is acknowledged from the Australian Research Council Grants DP110104734 and LE0454166.

Fig. 1: (a) Optical diagram of off-axis scanning confocal electron microscopy. (b) Chromatic defocused probe image of amorphous carbon. The refocused chromatic confocal energy is centered at energy loss of 300 eV. Chromatic confocal point is indicated by the arrow.

Fig. 2: Atomic resolution off-axis SCEM map of lanthanum M4,5 (~840 eV) core loss electrons in LaB6. The pixel dwell time is 1.5 ms with image size 256 x 256.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2881 Putting a New Spin on Scanning Transmission Electron Microscopy

LeBeau J. M.1, Sang X.1, Grimley E. D.1
1North Carolina State University, Raleigh, North Carolina, USA
jmlebeau@ncsu.edu

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-2885 Three-dimensional location of a single dopant with atomic precision by aberration-corrected ADF STEM

Ishikawa R.1,2, Lupini A. R.2, Findlay S. D.3, Taniguchi T.4, Pennycook S. J.5
1University of Tokyo, Japan, 2Oak Ridge National Laboratory, USA, 3Monash University, Australia, 4National Institute for Materials Science, Japan, 5The University of Tennessee, USA
ishikawa@sigma.t.u-tokyo.ac.jp

Impurity doping is the key technology for enhancing physical and chemical properties in semiconductors. These functional dopants usually take the form of isolated single atoms, and the materials properties have strong sensitivities to the doping concentration, spatial distribution and three-dimensional location of the dopants. The recent development of aberration-corrected electron microscopy has allowed the determination of the two-dimensional spatial distribution of single dopants with atomic spatial resolution. However, this resolution has been achieved only in the lateral directions, and the last dimension, depth, has not yet achieved atomic resolution.

Here we use quantitative annular-dark field scanning transmission electron microscopy (ADF STEM)[1] to directly visualize isolated single Ce dopants accommodated in bulk w-AlN single crystals[2], exhibiting strong visible-light photo-luminescence. Through combining with frozen phonon image simulations, we determine the three-dimensional location of the Ce dopant with single atomic-layer precision in depth[3].

On the basis of the mean signal value comparison between the experiment and the simulations, we estimate the number of atoms per column, and the atomic-resolution thickness map is shown in Figure 1a. During sequential acquisition, we observed a single Ce dopant jump from X to Y through the interstitial site (Figure 1b-d). For the two columns of X and Y, we performed image simulations of all the possible dopant configurations in depth. In the thicker specimen, it may be difficult to uniquely determine the depth location of a single dopant owing to strong dynamical intensity oscillation. To overcome this issue, we implemented multi-component analysis such as mean signal value, maximum peak intensity and profile fitting. As shown in Fig. 1e, the experimental profile at atom X is well matched with that of the simulation of dopant location to a 9 unit-cell depth. And similarly, atom Y is located to a depth of 8 unit-cells. To develop more general method, we also analyze the same data set with Bayesian statistical model, which does not require a priori knowledge of the number of atoms. And we obtained the same depth locations of Ce dopant. By tracking a single dopant, we could begin to determine the three-dimensional atom diffusion path within bulk materials.

References

[1] R. Ishikawa, A.R. Lupini, S.D. Findlay and S.J. Pennycook, Microsc. Microanal. 20, 99 (2014).

[2] R. Ishikawa, et al., Sci. Rep. 4 3778 (2014).

[3] R. Ishikawa, A.R. Lupini, S.D. Findlay, T. Taniguchi and S.J. Pennycook, Nano Lett., (2014) in press.

R.I. acknowledges support from JSPS Postdoctoral Fellowship. A.R.L. acknowledges support by the U.S. DOE. S.D.F. acknowledges support under the Discovery Projects funding scheme of the Australian Research Council (Project No. DP110101570). T.T. acknowledges support by a Grant-in-Aid for Scientific Research on Innovative Areas "Nano Informatics" (Grant No. 25106006) from JSPS.

Fig. 1: Figure 1. Sequentially acquired Z-contrast images of w-AlN viewed along the [11-20] direction, (a) atomic-resolution thickness map, (b) averaged over frames of 1-19, (c) frame 20, (d) frames of 21-40. (e) Z-contrast profiles obtained from atom X (exp.) and the simulations of Ce locations to 8, 9, 10-unit cells.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-3220 3D strain in HAADF – STEM images

Guerrero-Lebrero M. P.1, Bárcena-González G.1, Guerrero E.1, Liu Y.3, Kepaptsoglou D. M.4, Ramasse Q.4, Li L.3, Lazarov V. K.2, Galindo P. L.1
1Department of Computer Science and Engineering, Universidad de Cádiz, 11510 Puerto Real, Spain, 2Department of Physics, University of York, Heslington, York, United Kingdom, 3Department of Physics, University of Wisconsin, Milwaukee, WI 53211, USA, 4SuperSTEM Laboratory, STFC Daresbury Campus, Warrington, WA4 4AD, United Kingdom
maria.guerrero@uca.es

Strain mapping can be used to analyze materials at the atomic-column level, measuring local displacements and strain, and so revealing lattice translations, dislocations and/or rotations. Several methodologies have been developed to determine 2D strain field mapping from HRTEM images, either in real space (peak finding) [1, 2] or in Fourier space (geometrical phase analysis, GPA) [3]. Since 3-dimensional strain is independent of the image plane it might be ideal to gain insight into the behavior, shape and deformations of nanomaterials.

First, a HAADF focal series of 93 images (between 0nm and 14nm at steps of 0.15nm) of epitaxial Bi2Se3 (0001) thin films grown by atomic layer molecular beam epitaxy was taken with a Nion UltrastemTM 100 transmission electron microscope at 100 kV. Then, strain mapping of 23 images (from 19 to 41) was calculated using the Peak Pairs Analysis (PPA, [2]) plug-in for DigitalMicrograph available from HREM Research Inc.

Figure 1 shows one image of this focal series at a depth of 7nm, figure 2 shows the corresponding 2D strain map of this slice. It can observe that the screw dislocation position (red circle in figure 2) does not correspond to the apparent intensity change in HAADF STEM image (red circle in figure 1). The dark area in Figure 1 can be interpreted as a triangular spiral characterized by atomically smooth terraces, the way in which this material grows [4].

Figure 3 shows the 3D strain reconstruction where the screw dislocation movement, shape and tilt can be observed. Eshelby-Stroh twist [5] in the screw dislocation can be recognized, the upper part of the dislocation rotates in clockwise direction and the lower part turns in a counter – clockwise direction. The dislocation tilt has been estimated to be 6.3º with regard to the optical axis.

[1]Kret, S., Ruterana P., Rosenauer A., Gerthsen D. Extracting quantitative information from high resolution electron microscopy. Phys. Status Solidi (b) 227(1):247-295 (2001)

[2]Galindo, P. L, Sławomir, K., Sanchez, A.M., Laval, Y., Yañez A., Pizarro, J., Guerrero, E., Ben, T., Molina, S.I. The Peak Pairs algorithm for strain mapping from HRTEM images. Ultramicroscopy 107:1186-1193 (2007)

[3]Hÿtch, M. J., Snoeck, E., Kilaas, R. Quantitative measurement of displacement and strain fields from HREMicrographs. Ultramicroscopy 74:131–146 (1998)

[4]Liu.Y, Li, Y. Y., Rajput, S., Gilks, D., Lari, L., Galindo, P.L., Weinert, M., Lazarov V. K., Li, L.. Tuning Dirac states by strain in the topological insulator Bi2Se3. Nature Physics. (2014)

[5] Eshelby, J.D., Stroh, A.N. CXL. Dislocations in thin plates, Philosophical Magazines Series 7 42, 1401 (1951)

Fig. 1: HAADF Bi2Se3 slice at 7nm thickness. Apparent screw dislocation position is marked by the red circle.

Fig. 2: exx strain map that corresponds to the image in the Figure 1 calculated using PPA software [2].

Fig. 3: 3D strain reconstruction of a Bi2Se3 screw dislocation. Positive and negative strains are shown in red and blue respectively and the dashed red line represents the optical axis. The reconstruction makes it possible to collect a great deal of information about the dislocation motion.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-3292 Position resolved single electron response of the HAADF-STEM detector and improved method for intensity normalisation

Schowalter M.1, Krause F. F.1, Grieb T.1, Mehrtens T.1, Müller K.1, Rosenauer A.1
1Institut für Festkörperphysik, Universität Bremen, Bremen, Germany
schowalter@ifp.uni-bremen.de

Quantification of HAADF-STEM images as demonstrated in [1,2] is based on normalising the image intensity with respect to the incident electron beam and comparison with image simulations. For that the electron beam is scanned over the detector and the intensities Iout outside and Idet on the detector yield the normalized intensity Inorm=(I-Iout)/(Idet-Iout).

Recently, it has been shown that accidental electrons can hit the HAADF detector, although the electron beam is scanned in a specimen free area [3]. From such a “vacuum image” the number of counts caused by a single electron can be inferred and intensity can be scaled in units of electrons per pixels which enables an alternative way for STEM image quantification [3] and error estimation based on electron statistics.

In this contribution we show that accidentally impinging electrons cause artifacts in the normalization of image intensity using the detector scan technique (DST) [1,2]. We introduce an improved DST which is able to avoid such errors. In addition, we demonstrate a method for measuring single electron signals as a function of detector position.

The red line in Fig. 1 depicts a linescan through an HAADF image of an a-C wedge evaluated using the conventional DST. The normalised intensity exhibits a significant shift towards negative intensities in vacuum. This can be attributed to accidentally impinging electrons [3], whose dose is different for a detector scan (image mode) and a vacuum scan (diffraction mode). To account for this difference we suggest to replace Iout in the numerator by the intensity Ivac obtained from a vacuum scan. The result of this is shown by the blue line in Fig. 1, where the intensity in the vacuum region vanishes. Fig. 2 shows histograms of vacuum images for different dwell times, revealing a large peak at 9900 due to the background level of the detector as well as further peaks corresponding to one or more electrons per scan position. Different dwell times yield fundamentally different curves so that Ivac depends on dwell time. Therefore, vacuum scan and image scan must be performed with the same dwell time.

We also measured the spatially resolved response of the detector to a single electron by drastically decreasing the beam current and taking a series of 256 detector scans with 2048 by 2048 pixels. The position of the single-electron peak was measured in bins of 16 by 16 pixels and the position of the zero-electron peak was subtracted. Fig. 3 nicely depicts the position sensitive single-electron response.

[1] J. M. LeBeau and S. Stemmer, Ultramicroscopy, 108, 1653 (2008).
[2] A. Rosenauer et al., Ultramicroscopy, 109, 1171 (2009).
[3] R. Ishikawa, et al., Microscopy and Microanalysis, 20, 99 (2014).

Fig. 1: Normalized intensity along a linescan. Normalization was done using Iout and Idet as derived from a detector scan (red) as well as using the background level from the vacuum image (blue).

Fig. 2: Log. of the freq. of intensities in vacuum images for different dwell times. Each curve shows peaks corresponding to one or more electrons impinging on a certain pixel. The intensity is normalised with respect to the dwell time, so that the distance between e.g. one-electron peak and zero-electron peak is inversely proportional to the dwell time.

Fig. 3: Two-dimensional map showing the one-electron response as a function of position on the detector.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-2-O-3488 Linking Thickness, Channelling and Secondary X-ray signals in Atomic Resolution Scanning Transmission Electron Microscopy

Weyland M.1, Findlay S. D.2, D'Alfonso A. J.3, Allen L. J.3
1Monash Centre for Electron Microscopy, Monash University, Melbourne 3800, Australia, 2School of Physics, Monash University, Melbourne 3800, Australia, 3School of Physics, The University of Melbourne, Melbourne 3010, Australia
matthew.weyland@monash.edu

Quantification of EDX signals at atomic resolution can be treated by separation into two components; the scattering of electrons prior to ionisation and the subtleties of X-ray generation, emission and collection. Significant progress has recently been made concerning the first of these factors, with Forbes1 showing that consideration of both elastic and thermal scattering is required to explain anomalous contrast variations. However, true quantification requires a similarly detailed approach to the X-ray side of the system, with proportionality between signal and composition dependent on a multitude of factors including scattering cross-section, X-ray Fluorescence yield, Adsorption and detector geometry. Kotula has demonstrated a reference based approach2, scaling signals to averages from areas of known chemistry and Kothleitner recently showed the use of a ‘non-channelling’ (off-axis) approach to scale signals for quantification3. Both of these approaches offer a potential solution, but one of the main limitations is a lack of experimental data linking thickness, channelling and collected signal for a known specimen and well characterised instrument.

Results will be presented of a systematic study between thickness and EDX signal for known crystal structures and compositions. These will be matched with image simulation taking into account elastic and thermal scattering. The results presentenced will be carried out using a dual aberration corrected FEI Titan3, with well-defined probe illumination conditions, fitted with a standard 30mm2 ultra-thin window Si(Li) detector (0.13 sr) and a new 60mm2 windowless SSD detector (0.3 sr). Thickness will measured by position averaged convergent beam electron diffraction (PACBED), with EDX spectra acquired scanning over the same specimen area. Results will be presented from several specimens including Strontium titanate, GaAs/InGaAs radial nanowire heterostructures and Al-Cu alloys. By recording data from multiple areas with different thicknesses, trends between thickness, X-ray signal and channelling condition and its implications for quantitative high resolution EDX will be explored.

1. B. D. Forbes, A. J. D’Alfonso, R. E. A. Williams, R. Srinivasan, H. L. Fraser, D. W. McComb, B. Freitag, D. O. Klenov and L. J. Allen, PRB 86 024108, 2012
2. P. G. Kotula, D. O. Klenov and H. S. von Harrach, M&M 18(4), 2012
3. G. Kothleitner, M. J. Neish, N. R. Lugg, S. D. Findlay, W. Grogger, F. Hofer and L. J. Allen, PRL 112(8) 085501, 2014

The Australian research council is acknowledged for financial support through grants DP130102538 and LE0454166 (FEI Titan3).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-1522 Modification of an existing laboratory room to house a Cs corrected microscope.

Papworth A. J.1, Nellist P. D.1
1The Department of Materials, The University of Oxford, Oxford OX1 3PH UK.
adam.papworth@materials.ox.ac.uk

The first Cs corrected microscopes became generally available at the beginning of the 21st century. Cs corrected microscopes require very tight environmental conditions, which often means that when purchasing a Cs corrected microscope you also have to build a new building as well. An example of purpose built high resolution microscope laboratory is SuperSTEM in the UK. However, it is possible to meet the environmental conditions by converting existing rooms, removing the need for a new building, and therefore making ownership of a Cs corrected microscope more affordable.

The required environmental conditions fall into four groups; Electromagnetic force (EMF), Temperature, Acoustic and Vibration, where the biggest cause of instabilities can come from outside interferences, such as trains, power cables and general road traffic. In most cases these outside interferences can be mitigated, for example moving power cables; however trains and traffic cannot be relocated. This paper outlines measures that can be made to minimise the environment factors by careful design and choice of equipment.

The environmental targets set by the design team were as follows; EMF AC and DC <0.5mG, mechanical displacement (vertical and horizontal) <0.3µm, acoustic noise for all frequencies with a flat field response microphone <60db, room temperature and air movements targets were set as; temperature 20oC ±0.2 hr-1 fluctuation, air flow within the room was to be vertical with a minimum air flow of 100mm sec-1. These targets were considered as reasonable to obtain while also meeting the requirements of the Cs corrected microscopes, which were under consideration at that time of planning.

The final design of the rooms, equipment, anti-vibrational block and services gave the following results. The EMF measurement gave an AC X and Y of 0.05mG and Z 0.2mG with no significant DC component. The acoustics were compromised by noise coming from the floor above with a maximum of 55db at 120Hz. Room temperature was measured at 20oC ±0.08 over a five hour period with a 2.5kW load. The room remain within specification, even when the door was left open for two hours. The isolation block showed no external vibration being measured from the roads or surrounding buildings above the normal background. Vibration measurements were also taken during the night as wel

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-1525 Cs CORRECTED ATOMIC RESOLUTION TEM IMAGES OF THE HUMAN TOOTH ENAMEL CRYSTALS

REYES-GASGA J.1,3, TIZNADO-OROZCO G. E.2, BRÈS E. F.1
1Unité des Matériaux et Transformation (UMET). Université de Lille 1, Sciences et Technologies. Bâtiment C6. 59650 Villeneuve d’Ascq. Lille, France., 2Unidad Académica de Odontología. Universidad Autónoma de Nayarit. Edificio E7, Ciudad de la Cultura “Amado Nervo”, C.P. 63190 Tepic, Nayarit, Mexico., 3Permanet Address: Instituto de Física, UNAM. Circuito de la Investigación s/n. Cd. Universitaria, 04510 Coyoacán, Mexico D. F., México
jreyes@fisica.unam.mx

Aberration-corrected HR-STEM and HAADF images of the human-tooth-enamel crystallites are presented. These spatial and energy resolutions images have allowed to get information on the physical meaning of the central dark line (CDL) defect which leads to the anisotropic dissolution of the crystals.
Human tooth enamel is composed in 95% of hydroxyapatite crystals (HAP, Ca10(PO4)6(OH)2) which are elongated-plate-like of 30 to 60 nm wide and 100 to 200 nm long, approximately [1]. They are organized in microns-sized structures named “rods” or “prisms” that go from the enamel–dentin junction to the enamel surface (figure 1). The chemical analysis in the micron range of enamel by different analytical techniques, mainly EDS spectroscopy, has indicated the existence of trace elements. Thus, carbonated hydroxyapatite (c-HAP) with Na, Mg, Cl, as trace elements, has been stabled for these crystals [2].
When observed with the Transmission Electron Microscope (TEM), the enamel crystallites show a structural defect of 1 to 1.5 nm width in their central region approximately, the Central Dark Line (CDL) (figure 2), whose structure and role in the enamel structure itself is unknown yet [3, 4].
Several studies have shown that the CDL favors their anisotropic dissolution [4, 5]. During the carious process, for example, the enamel crystals are destroyed in a systematic fashion: first a series of hexagonal holes aligned along the [11-20] are observed, then the holes develop anistropically along the [0001] direction and cross the whole crystals [4, 5].
Enamel crystals are electron beam sensitive, other important parameter against the HRTEM observation (figure 3). Therefore, the use of low electron doses is critical during the study of the CDL. Therefore aberration corrected HR-STEM is the appropriated equipment for carrying out the chemical and structural analyses of the enamel crystallites.
Human tooth enamel samples were obtained from permanent non-carious human molar teeth, extracted for orthodontic or periodontal reasons. Samples were prepared in the FIB-FEI QUANTA 200 3D equipment using the two beams system.
JRG thanks to DGAPA-UNAM (contract IN106713), CONACYT and PASPA-DGAPA-UNAM for sabbatical support.

References
1. R.Z Le Geros, Calcium Phosphates in Oral Biology and Medicine ed H M Myers (San Francisco, CA: Karger). 1991.
2. G.E. Tiznado-Orozco et al., J. Phys. D: Appl. Phys. 42 (2009).
3. J. Reyes-Gasga et al., J. Mater. Sci. Mater. Med. 19, 877-882 (2008).
4. E. Brès et al., Journal de Physique, 51, C1-97-102, (1990).
5. E. Brès et al., Ultramicroscopy, 12, 367-372 (1984).

This research has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483-ESTEEM2 (Integrated Infrastructure Initiative–I3).

Fig. 1: TEM image of human tooth enamel crystals inside the micron-sized structure named “prism”.

Fig. 2: Magnification of one of the human tooth enamel crystals shown in figure 1. The row indicates the presence of the “central dark line”.

Fig. 3: HRTEM image of a human tooth enamel crystals aligned along the [0001] direction. The arrow indicates the electron beam damage.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-1600 Phase Contrast Transfer Function for Differential Phase Contrast in High Resolution Local Electric Field Measurements

Majert S.1, Kohl H.1
1Physikalisches Institut und Interdisziplinäres Centrum für Elektronenmikroskopie und Mikroanalyse (ICEM), Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Straße 10, 48149 Münster, Germany
s_maje03@uni-muenster.de

Differential Phase Contrast (DPC) is a contrast mechanism that can be utilized in the Scanning Transmission Electron Microscope (STEM). Since the advent of DPC, the technique has been used to image magnetic fields within a specimen [1]. To this end, a ring detector is divided into four quadrants and the direct electron beam is placed within the ring, only overlapping a small part of the detector. In a classical interpretation, the direct beam is slightly tilted by the magnetic fields in the specimen, so that subtraction of different detector segement signals yields DPC. Recently, this DPC geometry was also employed to investigate local electric fields with high resolution [2,3].

To determine whether this interpretation of DPC is still valid in high resolution, the wave nature of the electrons has to be taken into account. This can be done by calculating the Phase Contrast Transfer Function (PCTF) [4] to examine the contrast mechanism. For DPC, the PCTF should be proportional to the spatial frequency k=2π/λ whereas a PCTF constant as a function of the spatial frequency k would indicate conventional phase contrast.

Assuming an ideal lens, which is a good approximation for an aberration corrected STEM, the PCTF for a weak phase object can be calculated using elementary geometry. A cut through the two dimensional PCTF, evaluated for the parameters of a local electric field measurement, is shown in fig.1. It is striking that the area in which the PCTF is proportional to k is rather small (up to ca. 0.2 1/Å as seen in fig. 2), indicating that, for high spatial frequencies, DPC would not occur. While this is unproblematic at low resolutions (where the configuration described above leads to an improved signal to noise ratio [5]), it suggests that under these conditions the classical model is not valid for high spatial frequencies and the detector setup is therefore not suited for high resolution DPC applications.

The calculated PCTF shows that, for the given parameters, DPC is limited to spatial frequencies of about 0.2 1/Å. We are currently looking for possibilities to increase the resolution by optimizing the detector geometry.

[1] J. N. Chapman et al., Ultramicroscopy 3 (1978) 203
[2] M. Lohr et al., Ultramicroscopy 117 (2012) 7
[3] N. Shibata et al., Nature Physics 8 (2012) 611
[4] H. Rose, Ultramicroscopy 2 (1977) 251
[5] J. N. Chapman et al., IEEE trans. on magn. 26 (1990) 1506

Fig. 1: PCTF L(k) for an ideal microscope with an acceleration voltage of 300 kV, an aperture angle of 21.6 mrad, an inner detector angle of 21.0 mrad and an outer detector angle of 40.7 mrad, corresponding to the configuration in high resolution local electric fields measurements.

Fig. 2: Low spatial frequency region of the PCTF in fig.1, showing that DPC only occurs for spatial frequencies k smaller than ca. 0.2 1/Å.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-1633 Lifetime of the aberration-corrected optical state in HRTEM

Barthel J.1, Thust A.2
1Central Facility for Electron Microscopy, RWTH Aachen, Germany, 2Peter Grünberg Institute, Forschungszentrum Jülich GmbH, Germany
ju.barthel@fz-juelich.de

The technique of high-resolution transmission electron microscopy (HRTEM) experienced an unprecedented progress through the introduction of hardware aberration correctors, and by the improvement of the achievable resolution to the sub-Ångström level. As a consequence, the required precision level to measure and to adjust the optical properties of transmission electron microscopes has become increasingly demanding. A second consequence of this development, which has received little attention so far, is that aberration correction at a given resolution requires additionally a well-defined amount of optical stability. We investigate the qualification of a variety of high-resolution electron microscopes to maintain an aberration-corrected optical state in terms of a lifetime.

A comprehensive statistical framework is introduced for the estimation of the optical lifetime [1]. The temporal evolution of the twofold astigmatism is extracted from a series of images recorded over several minutes (Fig. 1). The twofold astigmatism serves as representative indicator for the optical stability since it is one of the most volatile image aberrations, has a strong influence on the image contrast, and can be measured rapidly in a simple experiment [2]. A model-based evaluation method was developed, which allows us to distinguish between two major components of astigmatism fluctuations, a random walk and a constant drift. A very useful output of the model-based evaluation is a probability curve (Fig. 2), which informs the operator about the chance to still work in an aberration-corrected state after a given timespan.

Optical stability evaluations for different high-resolution microscopes reveal surprisingly short lifetimes on the order of a few seconds up to a few minutes. The observed short lifetimes denote a critical limitation of the timespans between aberration measurement, aberration correction and the actual imaging. Therefore further investigations and technical developments are necessary in order to stabilize electron microscopes with respect to their sub-Ångström qualification. Since the topic of optical stability turns out to be of similar importance as the topic of resolution itself, we recommend to include a routine assessment of the optical stability in acceptance tests for high-resolution microscopes operating in the discussed resolution regime. For this purpose, the lifetime evaluation procedures developed in this work have been implemented in a user friendly and freely downloadable software [3].

References:

[1] J. Barthel, A. Thust, Ultramicroscopy 134 (2013), p. 6.
[2] J. Barthel, A. Thust, Ultramicroscopy 111 (2010), p. 27.
[3] J. Barthel, http://www.er-c.org/barthel/pantarhei/, (Feb 2014).

J.B. gratefully acknowledges funding within the core facilities initiative of the German Science Foundation (DFG) under the grant number MA 1280/40-1.

Fig. 1: Evolution of the twofold astigmatism extracted from images of amorphous carbon. Already after one minute the astigmatism fluctuations violate the π/4 limit for 300 keV electrons and for a microscope resolution of 0.8 Å.

Fig. 2: Decay of the probability to still work in an aberration-corrected state, evaluated from the astigmatism fluctuations shown in Fig. 1.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-1677 Preparation of thin film specimen by Cryo Ion Slicer for TEM cross-section (XTEM) observation 

Siddheswaran R.1, Medlín R.1
1New Technologies Research Centre, University of West Bohemia, Plzeň-30614, Czech Republic
rajendra@ntc.zcu.cz

An essential part of research in thin film fabrication is the microstructural analyses like morphology, grain distribution, texture, thickness of the layers and orientation of film structure. For such characterization, cross-sectional transmission electron microscopy (XTEM) is a very essential tool for the study of structure, phase, defects and interfaces. For such analyses, it is necessary to make the film electron transparent in a direction perpendicular to the interfaces. One of the methods is cryo ion slicing (from JEOL) with specific sample preparation procedure different from the PIPS from Gatan. The preparation of cross-sectional specimens with ion slicer are usually done by fabricating a sandwich structure (Thin film/Glue/Cover glass) and subsequently thinning it to transparent for electrons (thickness of the order of <50 nm for TEM and <10nm for HR-TEM). The cross-section specimen preparation is generally time consuming, specimen dependent and consequently a trial and error method. But the features of XTEM observations are in results more informative and necessary in addition with the other methods of the observations, i.e. XRD, optical studies or micrographs of scratched samples from thin films.
The present work describes the preparation of thin film specimen, includes mechanical (pre-preparation) and Ar+ ion slicing (milling). It was successfully used for the preparation of a-Si:H/a-SiO2, nc-Si/a-SiO2 and ZnO thin film specimens for transmission microscopic analyses. The pre-preparation of sample for ion milling consists of cutting samples by diamond disc using low speed saw cutter (Buehler IsoMet) and mechanically thinning using JEOL Handy Lap to get the specimen dimension 2.5mm×500µm×100µm with plan-parallel to the surfaces. The ion slicing was carried out using JEOL IB-09060CIS Cryo Ion Slicer using Ar gas of purity 99.9999%. Finally thin regions of range from 100nm to 10nm were achieved over the thin film layers. A very thin cross-section of ~10nm could be used to obtain high resolution TEM images. 

The result was developed within the CENTEM project, reg. no. CZ.1.05/2.1.00/03.0088. 

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-1739 Development of the on-line DigitalMicrograph scripts for TEM imaging using the “Virtual TEM”.

Potapov P.1
1temDM, Dresden, Germany
info@temdm.com

Practical high resolution imaging still depends strongly on skills and smartness of operators. DigitalMicrograph scripting [1] can facilitate the practice of high resolution imaging in numerous ways: the fine tuning of the aberrations; the automatic compensation for the specimen and lenses drift; easy navigation over the area of interest; minimizing the applied electron dose and therefore reducing the radiation damage; the rapid switching between the imaging and spectroscopy modes.
DigitalMicrograph scripting provides users with a set of commands controlling the TEM hardware. However these commands are available in the on-line version of DigitalMicrograph only, i.e. they require the physical connection with TEM. This hinders the progress of the on-line scripting - for TEM time is expensive and should be used for imaging, not programming.
The present work introduces a plugin “Virtual TEM” that simulates all the scripting commands for communication between TEM and DigitalMicrograph. With this plugin, a user is able to edit, debug and roughly test the on-line scripts with no actual connection to the TEM; when being in office, at home or during air travel. The plugin imitates a simple TEM interface with the basic control of magnification, focus, stage, beam and stigmators (Fig.1). Depending on the instrumental settings, the “Virtual TEM” generates the image of the model object that can be captured by DigitalMicrograph and used as a feedback for the communication commands. The simplest example scripts - “Auto Acquisition”, “Focal Series”, “Correct Stage Drift” et cet - are provided as a part of the “Virtual TEM” package. The example scripts are aimed to be a seed for the development of more sophisticated customized tools.
Beyond the simplest examples, the advanced on-line DigitalMicrograph scripts are presented. The “Batch Recording” allows a user to shoot the images by a single button touch and automatically put them into the image container optimized for easy resizing and sorting (Fig.2). The “Click Mover” provides the convenient navigation over the place of interest by simple mouse double-click on the live high resolution image (Fig.3).
The plugins can be free downloaded from [2].

References:
[1] http://www.gatan.com/resources/scripting/
[2] http://www.temDM.com/

Fig. 1: “Virtual TEM” interface including the virtual camera menu, virtual TEM and virtual Filter control panels. The image and spectrum of the generated model object are displayed.

Fig. 2: “Batch Recording” tool provides convenient live imaging and storing the recorded images in the image container.

Fig. 3: “Click Mover” tool moves the feature of interest (mouse double-clicked) to the center of the live image.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-1751 The atomic structure of epitaxially strained LaNiO3-LaGaO3 superlattices

Qi H. Y.1, Kinyanjui M. K.1, Biskupek J.1, Benckiser E.2, Habermeier H. U.2, Keimer B.2, Kaiser U.1
1University of Ulm, Central Facility of Electron Microscopy, Electron Microscopy Group of Materials Science, Albert Einstein Allee 11, D-89069 Ulm, Germany, 2Max Planck Institute for Solid State Research, Heisenbergstrasse 1, D-70579 Stuttgart, Germany
haoyuan.qi@uni-ulm.de

Many functional properties of ABO3 perovskite oxides are closely coupled to slight structural distortions in the perovskite lattice, thus defined symmetry constraints in oxide heterostructures can be used to access novel properties that are not found in bulk constituents [1].
Here, we present our study of an epitaxially strained [4 unit cell (u.c.)//4 u.c] х8 LaNiO3-LaGaO3 (LNO-LGO) superlattice grown on (001) SrTiO3 (STO) substrate (see the model shown in Figure 1). Due to the lattice mismatch, the superlattice is subject to tensile strain. We focus on the determination of the strain-induced distortions (changes in Ni-O bond length) and tilts (changes in Ni-O-Ni bond angle) of the corner-sharing octahedral network, as they may drastically influence the functionalities of the heterostructure. In order to discover the correlation between the NiO6 octahedra rearrangement and the functional properties of the material system, it is essential to study the interfacial structure with atomic-level accuracy. We studied the atomic structure of the octahedral network by means of aberration-corrected high-resolution transmission electron microscopy (AC-HRTEM). In order to enhance the image contrast, negative Cs imaging (NCSI) was applied [2].
Figure 2 is an experimental image of the LNO-LGO superlattice acquired in the vicinity of the top surface in [110] projection. It is clearly seen that the LNO and LGO layers manifest difference in both the zigzagness (out-of-plane corrugation) of BO2 layers and the image contrast. The smooth oscillation of the tilt angles indicates: 1) dissimilarity in tilt systems of each material, 2) proximity effect between adjacent layers. As a result of coherent epitaxial growth, the in-plane lattice parameter d220 remains constant while only the out-of-plane lattice parameter d001 varies. We will discuss further investigation at the substrate-layer interface and answer the question on the assignment of the layers. However, from merely HRTEM imaging, it is difficult to find out which of the layers, LNO or LGO shows the higher out-of-plane corrugation.

[1] H.Y. Hwang, Y. Iwasa, M.Kawasaki, B. Keimer, N.Nagaosa and Y. Tokura, Nat. Mater. 11, 103 (2012).

[2] C. Jia, M. Lentzen and K. Urban, Microsc. Microanal. 10, 174 (2004).

We are grateful to S. Grözinger for assistance with TEM specimen preparation and the German Research Foundation (DFG) for financial support (project DFG: KA 1295/17-1)

Fig. 1: Atomic structure model of a [4 unit cell (u.c.)//4 u.c] х8 LNO-LGO superlattice grown on STO substrate (viewed in [110] orientation). Unit cells are defined by pseudo-cubic symmetry axes.

Fig. 2: Experimental aberration corrected image of the LNO-LGO superlattice acquired in [110] projection in the vicinity of the top surface at 300kV. The oscillation of the tilt angles shown in the right diagram indicates non-identical tilt systems in LNO and LGO layers, which is clearly visible from the magnified areas (red and yellow rectangles).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-1996 Structural and spectroscopic analyses of exfoliated 2-D transition metal dichalcogenides nanosheets with special emphasis on TEM

Pokle A. S.1, Coelho J.2, Mendoza B.2, Nicolosi V.1, 2
1School of Physics, Advance Microscopy Lab, Trinity College Dublin, Ireland, 2School of Chemistry, CRANN, Trinity College Dublin, Ireland
poklea@tcd.ie

Having unique physical, chemical and structural properties, 2-D nanomaterials such as the Transition Metal Dichalcogenides (TMD’s) have attracted considerable attention. Similar to graphene, TMD’s are atomically thin two dimensional materials with electronic properties different from their bulk counterparts. Graphene’s vanishing band-gap for semiconductor application poses a major setback. As a result, it is not suitable for logic applications, because devices cannot be switched off. On the other hand, 2D TMDs (i.e. MoS2, NbSe2, MoSe2, etc.) are semiconductors with a variety of tunable bandgaps. This property makes them perfect contenders for replacing Silicon in the semiconducting industry. In addition, different classes of 2-D materials such as like Transition Metal Oxides (TMOs) have shown to exhibit excellent electrical, optical and electrochemical properties. In virtue of this properties they have become excellent candidates for applications in energy storage devices such as lithium-ion batteries and supercapacitors.

Few layered or single-layered TMDs and TMOs can be obtained either through exfoliation of bulk material or by a bottom-up synthetic approach. The approached used in our group is the synthesis of 2D materials by liquid-phase exfoliation. This method produces atomically-thin and few-layers sheets dispersed in a solvent media. In order to apply these materials to feasible applications it becomes crucial to analyse their structure and correlate that to the ultimate properties when these materials are used in devices.

In this work we present a structural and spectroscopic characterization of a range of liquid-phase exfoliated 2D materials. Major focus is given to the study of their crystallographic structure, presence of defects, possible oxidative processes, and edge-effects. For that we use a combined approach, where by X-ray diffraction (XRD), scanning electron microscopy (SEM), high-resolution (scanning) transmission electron microscopy - HR(S)TEM, energy dispersive X-ray spectroscopy (EDX), electron energy loss spectroscopy (EELS) and X-ray photoelectron spectroscopy (XPS) are all used to obtain a throughout characterization of the materials.

The authors gratefully acknowledge funding from the FP7 People Network – ITN: Initial Training Network and Science Foundation Ireland – European

Research Council: SFI - ERC Support Program

Fig. 1: TEM image of MoSe2 (LHS) and WSe2 with their respective SADP.

Fig. 2: HR-TEM image of MoSe2 (LHS) and WSe2 with FFT 
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2016 Surface Science of Metal Oxides by High-resolution TEM

Yu R.1, Zhan W.1, Lu S. R.1, Zhu J.1
1Tsinghua University, Beijing, China
ryu@tsinghua.edu.cn

Surfaces of metal oxides are of crucial importance for a variety of technological applications such as heterogeneous catalysis, thin film growth, gas sensing, and corrosion prevention [1]. Due to the complexities of oxides in crystal structure and electronic structure, however, the surface science of oxides lags far behind that of metals or semiconductors. Conventional surface-science techniques, typically scanning tunneling microscopy (STM) and low energy electron diffraction (LEED), are usually limited to surfaces of single crystals with relatively simple structures. Metal oxides are usually good insulators, either band insulators or Mott insulators, making them not suitable for STM, LEED, and most of spectroscopic methods using low energy electrons as probes. On the other hand, the complex atomic structures of oxides results in too many structural parameters to be determined by spectroscopy or diffraction methods. Recent developments in high-resolution transmission electron microscopy (TEM) provide us opportunities to overcome the above difficulties. With the realization of aberration-correction, the point resolution of TEM has been improved into the milestone 1 Angstrom scale. In addition, the correction of the spherical aberration has almost eliminated the contrast delocalization in high-resolution images. Therefore, high resolution TEM becomes an even more powerful tool than before for materials research at a truly atomic-scale. Here, we will present our recent works on atomic and electronic structure of oxide surfaces [2-4]. We will show that the structure and dynamics of oxide surfaces can be directly imaged and measured at the sub-angstrom scale with an accuracy of picometers, comparable to that obtained by conventional surface science techniques on single crystals. Special attention will be on line defects at the surfaces of MgO and Fe2O3.

References:

1. V. E. Henrich, and P. A. Cox, The Surface Science of Metal Oxides (Cambridge University Press, Cambridge, 1994).

2. R. Yu, L.H. Hu, Z.Y. Cheng, Y.D. Li, H.Q. Ye, J. Zhu, Phys. Rev. Lett., 105, 226101 (2010).

3. M.R. He, R. Yu, J. Zhu, Angew. Chem. Int. Ed., 124, 7864 (2012).

4. S.R. Lu, R. Yu, J. Zhu, Phys. Rev. B, 87, 165436 (2013).

This work was supported by National Basic Research Program of China (2011CB606406), NSFC (51071092, 51371102, 11374174, 51390471, 51390475), and Program for New Century Excellent Talents in University. This work used the resources of the Beijing National Center for Electron Microscopy and Shanghai Supercomputer Center.

Fig. 1: (a) HRTEM image of the α-Fe2O3 (-1102) surface defect viewed in the [1-101] direction. The inset shows the simulated image of the relaxed structure  by DFT calculations (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2042 Contrast Investigation of Annular Bright-Field Imaging in Scanning Transmission Electron Microscopy of LiFePO4

Zhou D.1, Sigle W.1, Müller K.2, Rosenauer A.2, Zhu C.3, Kelsch M.1, Maier J.3, van Aken P. A.1
1Max Planck Institute for Intelligent Systems, Stuttgart Center for Electron Microscopy, Heisenbergstraße 3, 70569 Stuttgart, Germany, 2Institute of Solid State Physics, University of Bremen, Otto-Hahn-Allee 1, D-28359 Bremen, Germany, 3Max Planck Institute for Solid State Research, Heisenbergstraße 1, 70569 Stuttgart, Germany
danzhou@is.mpg.de

Light elements, such as lithium, are difficult to detect using high-angle annular dark-field imaging (HAADF) in STEM because of their weak atomic scattering. Recently, a novel imaging mode in aberration-corrected STEM was presented which uses an annular detector spanning an angular range mainly within the illumination cone of the focused electron beam1. It was shown that due to the smaller dependence on atomic number Z, approximately Z1/3 compared to Z2 in HAADF, the resultant images enable one to visualize the light element columns2. This imaging mode has been called annular bright-field (ABF) imaging. The contrast differences are clearly visible in Figure 1 which compares HAADF (b) and ABF (c) images of LiFePO4 structure in [010] orientation (a).

In this work, we studied the contrast of ABF imaging in STEM on LiFePO4, correlating results of experiments from the newly installed probe-aberration-corrected JEM-ARM 200CF and simulations using the STEMsim program3. Previous work briefly demonstrated the possibility to acquire direct images of LiFePO4 and partially de-lithiated LiFePO4 at atomic resolution4. Figure 2 presents an experimental comparison of the visualization of lithium in LiFePO4 with HAADF and ABF. The present work aims at presenting a more detailed description of the contrast dynamics of ABF imaging of LiFePO4 with a view to its interpretation, and optimization. Thickness, defocus, angular range, and possible contamination introduced by sample preparation are taken into account to understand the image contrast. In particular, the probe and detector configurations in the microscope are taken into consideration to step from qualitative to quantitative contrast evaluation.

1. Okunishi, E.; Ishikawa, I.; Sawada, H.; Hosokawa, F.; Hori, M.; Kondo, Y., Visualization of Light Elements at Ultrahigh Resolution by STEM Annular Bright Field Microscopy. Microsc Microanal 2009, 15, 164-165.

2. Findlay, S. D.; Shibata, N.; Sawada, H.; Okunishi, E.; Kondo, Y.; Ikuhara, Y., Dynamics of annular bright field imaging in scanning transmission electron microscopy. Ultramicroscopy 2010, 110 (7), 903-923.

3. Rosenauer, A.; Schowalter, M., STEMSIM-a New Software Tool for Simulation of STEM HAADF Z-Contrast Imaging. Springer Proc Phys 2008, 120, 169-172.

4. Gu, L.; Zhu, C. B.; Li, H.; Yu, Y.; Li, C. L.; Tsukimoto, S.; Maier, J.; Ikuhara, Y., Direct Observation of Lithium Staging in Partially Delithiated LiFePO(4) at Atomic Resolution. J Am Chem Soc 2011, 133 (13), 4661-4663.

 

The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under grant agreement n°312483 (ESTEEM2).

Fig. 1: (a) Projection of the LiFePO4 crystal structure in [010] orientation. Simulated HAADF (b, angular range 40-100 mrad) and ABF (c, angular range 11-22 mrad) imaging using STEMsim. The parameters used for simulations are: high voltage 200 kV, convergence angle 22 mrad, spherical aberration 0 mm, defocus 0 nm, specimen thickness 30 nm.

Fig. 2: As acquired experimental results on the visualization of Li in LiFePO4 with HAADF (a, 90-370 mrad) and ABF (b, 11-22 mrad) imaging on the probe-aberration-corrected JEOL JEM-ARM 200CF microscope using a convergence angle of 22 mrad and a probe size of about 0.08 nm. (c) Assignment of atomic column in [010] orientation.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2046 HAADF STEM characterization of BST-MgO interface structure

Kuskova A. N.1, Zhigalina O. M.1, Khmelenin D. N.1
1Shubnikov Institute of Crystallography, Russian Academy of Sciences
xorrunn@gmail.com

The properties of thin perovskite ferroelectric films can be different from those of bulk materials, that is caused by the mechanical stress at the film–substrate interface [1]. Such stress is usually relaxed by the formation of misfit dislocations at the heterostructure interface.
It has been shown [2] that the degree of stress in epitaxial BST thin films is a function of thir thickness. In this study we have introduced a high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) investigation of this heterostructure interface combined with the modelling of the HAADF STEM images, a geometry phase analysis [3] and a statistical quantitative analysis [4].
The perovskite structure of BST (Ba0.8Sr0.2TiO3) allows two types of starting planes for growth on (100) cubic MgO substrate: the Ba(Sr)O or the TiO2 planes, which could enable different chemical bonding at the interface. Since the HAADF STEM image intensity is proportional to Z2 of the scanned crystal (Z is average atomic number of atomic columns), the Ba(Sr) atomic columns where are Z=52 observed as the brightest dots in the image (Fig.1). TiO columns with Z=15 have a lower brightness and MgO columns where are Z=10 demonstrated the least brightness in the images. The pure O columns (Z=8), located between the brightest Ba(Sr) columns, are not visible in the image. The misfit dislocations marked by arrows on Figure 1(a) were visualized by the geometric phase analysis [3]. Figure 1(b) illustrates the enlarged dislocation core and its Burgers vector identified as ½аBST[010].
It is obvious that to obtain the information about the chemical interface structure based only on the direct observation of changes in the image contrast is not correct. Model-based statistical quantitative analysis has quantified the chemical composition of ‘unknown’ atomic columns at the interface based on a comparison of their scattered intensities with ones of ‘known’ columns located far from the interface [4]. Figure 1d illustrates the estimated peak volumes for Figure 1c. This analysis of intensities of different types of planes has indicated that the first atomic layer of the film does not lie on top of the substrate, but is embedded into the upper layer of MgO.

1. Y.S. Kim, D.H. Kim, J.D. Kim, et al., Appl. Phys. Lett., 86 (2005), p.102907
2. O.M. Zhigalina, A.N. Kuskova, R.V. Gaynutdinov, et.al, Journal of Surface Investigation: X-Ray, Synchrotron and Neutron Techniques. 4 (2009). p. 542-547.
3. A.K. Gutakovskii, A.L. Chuvilin, Se Ahn Song, Izvestiya RAS, ser. phys., 71 (2007). p.1464-1470-
4. S. Van Aert, J.Verbeeck, R.Erni et al., Ultramicroscopy, 109 (2009) p. 1236-1244
5. J.W. Reiner, F.J. Walker, & C.H. Ahn. Science 323 (2009), p. 1018–1019.

This work was done using IC RAS Research Center equipment and supported by the Ministry of Education and Science of the Russian Federation and the grant RFBR №14-02-31223-mol_a.

Fig. 1: HAADF STEM images of the 120 nm BST film. Misfit dislocations marked by arrows and numbers of half-planes between them (a), an enlarged part of the interface with one misfit dislocation and its Burgers vector (b),an enlarged part of the interface and corresponding map with estimated scattered intensities (c) and (d), respectively
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2162 A channelling based approach for scattering cross sections of mixed columns in HAADF STEM images

van den Bos K.1, Van Aert S.1
1Electron Microscopy for Materials Science (EMAT), University of Antwerp, Antwerp, Belgium
Karel.vandenBos@uantwerpen.be

HAADF STEM is used to determine structure parameters of nanostructures, such as the number of atoms and the atomic column positions. In order to quantitatively evaluate HAADF STEM images different performance measures including peak intensities and scattering cross sections have been introduced [1-3]. Here, a channelling based approach is proposed to predict these measures for mixed columns.

In the analysis of experimental images performance measures which are sensitive for the parameter of interest are desirable. A comparison between scattering cross sections, determined using statistical parameter estimation theory [2], and peak intensities shows that peak intensities level off at a relatively low number of atoms whereas scattering cross sections increase nearly linearly up to relatively large thicknesses (Fig. 1). This is in agreement with the scattering cross sections computed by using the probe-position integrated cross sections [3]. For that reason, the number of atoms of monotype atomic columns has been successfully determined from experimental scattering cross sections [4]. However, in case of mixed columns the analysis is more complicated since more structure parameters are involved. Therefore, it is desirable to be able to predict performance measures as a function of composition and thickness. Often the assumption of longitudinal incoherence is considered where the scattering intensity of an atomic column is written as the sum of the scattering intensities of the individual atoms constituting this column. However, the non-linear behaviour of peak intensities as well as scattering cross sections makes it impossible to make a valid prediction using this assumption (Fig. 2). A more accurate prediction is obtained based on the channelling theory in which it is assumed that each atom acts as a lens focussing the electrons on the next atom [5]. In this approach the change in scattering intensity with thickness of monotype atomic columns is taken with respect to that of a single atom to estimate the scattering intensity of mixed columns. This approach leads to a significant improvement in the prediction of both performance measures (Fig. 2) and is especially accurate for scattering cross sections. This is an important step forward for the quantitative analysis of complex hetero-nanostructures.

In conclusion, scattering cross sections of mixed columns can be predicted more accurately using a channelling based approach as compared to assuming longitudinal incoherent modelling.

References

[1] Erni et al., Ultramicroscopy 94 (2003), p. 125
[2] Van Aert et al., Ultramicroscopy 109 (2009), p. 1236
[3] E et al., Ultramicroscopy 133 (2013), p. 109
[4] Van Aert et al., Nature 470 (2011), p. 374
[5] Van Aert et al., Ultramicroscopy 107 (2007), p. 551

The authors kindly acknowledge funding from the Fund for Scientific Research, Flanders (FWO).

Fig. 1: Simulations of the scattering cross sections and peak intensities of a single atomic column of (a) Al, (b) Ag, (c) Cd and (d) Pb with respect to thickness. Simulations were carried out using an aberration corrected system with a convergence angle of 21.78 mrad and a detector covering an area of 90-158 mrad.

Fig. 2: Prediction models of simulated scattering cross sections and peak intensities for 17 atom thick mixed columns. In (a) and (c) the centre of an Al column is replaced by Ag atoms keeping the thickness at 17 atoms. In (b) and (d) the centre of a Cd column is replaced by Pb atoms. The parameters for the simulations were the same as in Fig. 1.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2255 Probability of error for counting the number of atoms from high resolution HAADF STEM images

De Backer A.1, De wael A.1, Van Aert S.1
1Electron Microscopy for Materials Science (EMAT), University of Antwerp, Antwerp, Belgium
Annick.DeBacker@uantwerpen.be

During the last years, different quantification methods to count the number of atoms in an atomic column based on HAADF STEM images were developed [1-4]. These methods can be applied to a wide variety of experiments. However, to go beyond the current state-of-the-art, all sources contributing to errors in atom counting need to be understood. Therefore, we discuss a theoretical tool that can be used to quantitatively determine the probability of error for counting the number of atoms.
In principle, expressing the reliability in atom counts can be simplified to discussing the ability of distinguishing between n and n+1 atoms, i.e. the possibility to detect the difference of 1 atom in an atomic column. Using the principles of detection theory [5], this problem is written as a binary hypothesis test with the hypotheses corresponding to atomic columns having n (null hypothesis) and n+1 (alternative hypothesis) atoms. The goal is to minimise the probability of assigning the wrong hypothesis. This is illustrated in Fig. 1. For both hypotheses a so-called log likelihood ratio distribution can be defined. For a given atomic column, the log likelihood ratio then determines which of these hypotheses is decided. If this log likelihood ratio is larger than 0, the alternative hypothesis is decided; otherwise the null hypothesis is decided. From Fig. 1, it is clear that the probability of error is defined by the overlap of log likelihood distributions. This overlap can be calculated numerically.
As a preliminary example, the probability of error is calculated as a function of electron dose and number of atoms using a simple Gaussian model that linearly increases with the number of atoms. The analysis is shown in Fig. 2. As expected the probability of error increases for decreasing electron dose. Furthermore, it is shown that distinguishing between n and n+1 atoms in an atom column becomes more difficult for increasing n. One of the possible applications is to apply this method to realistic simulations in order to optimise the experiment design. This can be realised by minimising the probability of error as a function of a variety of parameters of interest, such as magnification, acceleration voltage, and inner and outer detector angle.
In conclusion, the method quantifies the error for counting the number of atoms as a function of the parameters of interest and enables us to understand the origin of miscounting the number of atoms.

References

[1] Erni et al., Ultramicroscopy 94, 125 (2003)
[2] LeBeau et al., Nanoletters 10, 4405 (2010)
[3] S Van Aert et al., PRB 87, 064107 (2013)
[4] A De Backer et al., Ultramicroscopy 134, p 23 (2013)
[5] den Dekker et al., Ultramicroscopy 134, p 34 (2013)

The authors kindly acknowledge funding from the Fund for Scientific Research, Flanders (FWO).

Fig. 1: The probability of error is depends on the electron dose D, the number of atoms n, the total scattered intensity of a column CS and the pixel size in the simulated HAADF STEM image dx; F denotes the cumulative distribution function of the normal distribution having mean μ and standard deviation σ.

Fig. 2: (a) Probability of error as a function of electron dose for choosing between 10 and 11 atoms in a column (b) Probability of error as a function of number of atoms for a constant electron dose (200 electrons per pixel) (c) Probability of error as a function of number of atoms and electron dose.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2256 STEM Optical Sectioning for Imaging Screw Displacements in Dislocation Core Structures

Yang H.1, Lozano J. G.1, Pennycook T. J.1,2, Hirsch P. B.1, Nellist P. D.1,2
1University of Oxford, Department of Materials, Oxford, UK, 2EPSRC SuperSTEM Facility, Daresbury Laboratory, Warrington, UK
hao.yang@materials.ox.ac.uk

Aberration corrected transmission electron microscopes have advanced our knowledge of the atomic structure of edge dislocations, which are viewed end-on with the tensile or compressive strain normal to the dislocation being clearly visible. Atomic displacements associated with screw dislocations however cannot be observed end-on because the helical screw displacements are parallel to the viewing direction. In this paper the helical displacements around a screw can be imaged with the dislocation lying transverse to the electron beam by “optical sectioning” in annular dark-field scanning transmission electron microscope imaging. In optical sectioning the few nanometer depth of focus is utilized to extract information along the beam direction by focusing the electron probe at specific depths within the sample. This novel technique is applied to the study of the c-component in the dissociation reaction of a mixed [c+a] dislocation in GaN that has previously been observed end-on [1].

Figure 1 shows atomic layers from different depths of a c-type screw dislocation aligned along the c axis [0001] in GaN. Each layer consists of a (2-1-10) plane, and is parallel to the dislocation line. In layers far from the screw dislocation, the displacements vary slowly across the field of view as expected from the lower shear strain that exists further from the dislocation core. Layers close to the screw dislocation core show displacements that very rapidly in the vicinity of the dislocation core, with a rapidly varying shear of the (0002) planes to given an apparent displacement of c/2 across the dislocation core (as expected for a total Burgers vector of c).

A focal series of experimental images were recorded using a Nion UltraSTEM100 aberration-corrected STEM operating at 100 kV (Figure 2). A 1μm thick sample of GaN, grown by metalorganic vapour phase epitaxy on a sapphire substrate, was thinned to be viewed along the a crystallographic axis. A dislocation was found lying in the plane of the sample, and characterized using weak-beam imaging to be of a mixed [c+a] type along [0001]. As the electron beam is focused closer to the dislocation from (a) to (e), the shearing of the (0002) planes becomes more localized in the image, and a more detailed observation of the screw displacements shows that the shearing occurs equally along two distinct lines along [0001], indicated by the arrows in Figure 2. It is therefore apparent that the screw component of the dislocation has dissociated according to the reaction c=[½ c + ½c] confirming the assumption made in previous end-on observations [1,2].

[1] P.B. Hirsch et al., The dissociation of the [a+c] dislocation in GaN, Philosophical Magazine, 93 (2013) 3925.
[2] H. Yang et al., manuscript in preparation.

The authors would like to acknowledge financial support from the EPSRC (grant number EP/K032518/1) and the EU Seventh Framework Programme: ESTEEM2.

Fig. 1: Figure 1. Structure model of a 10nm thick GaN c type screw dislocation viewed at different depths along direction, with the screw dislocation lying in the middle. From the top to the bottom panel, the distances from the dislocation core are +5, +1, +0.3, -0.3, -1 and -5nm, respectively.

Fig. 2: Figure 2. ADF STEM optical sectioning of a [c+a] dissociated screw dislocation viewed perpendicular to the dislocation line along the a direction. The focal step between (a),(c) and (e) is 4nm. (b,d,f) contain Fourier filtered versions of the corresponding images using just the (0002) Fourier components to highlight the shearing of the planes.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2265 Adding the Third Dimension to Atomic Resolution Spectrum Imaging

Pennycook T. J.1,2, Lewys J.1, Cabero M.3, Ribera-Calzada A.3, Leon C.3, Varela M.4,3, Santamaria J.3, Nellist P. D.1,2
1Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH, UK , 2SuperSTEM Laboratory, STFC Daresbury, Keckwick Lane, Warrington WA4 4AD, UK, 3Grupo de Fisica de Materiales Complejos, Universidad Complutense, 28040 Madrid, Spain , 4Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA
timothy.pennycook@materials.ox.ac.uk

Aberration correction made possible two-dimensional atomic resolution spectrum imaging in the scanning transmission electron microscope (STEM). It also led to a significantly reduced depth of field which has been utilised to perform optical sectioning with atomic number contrast annular dark field (ADF) imaging and determine the positions of individual dopant atoms in three dimensions. Here we combine these corollaries of aberration correction to demonstrate three dimensional elemental mapping with atomic resolution electron energy loss spectroscopy (EELS). Atomic lateral resolution is critical as the optical transfer function of the STEM has a large missing cone, leading to excessive depth elongation for laterally extended objects [1]. The longitudinal resolution varies with the lateral spatial frequency. In regions relevant to lateral resolutions achievable today, the missing cone causes the longitudinal resolution to vary as approximately d/α where d is the characteristic spacing of the object and α is the convergence angle. If the highest spatial frequency resolved is for example the width of a 3 nm nanoparticle, the depth resolution will be around 136 nm with a 22 mrad convergence angle. If instead we were able to resolve atomic columns with a spacing of 0.3 nm and use a 30 mrad convergence angle the depth resolution improves to 10 nm. This relationship between lateral spatial frequency transfer and depth resolution applies both to ADF and EELS imaging. However, although theoretical simulations suggested it was possible to perform optical sectioning with EELS, it had not previously been demonstrated experimentally.

Using a Nion UltraSTEM 100 operated at 100 kV with a 30 mrad convergence angle we acquired successive spectrum images from the same area of a sample, but with the probe focused to different depths. The EELS optical sectioning revealed the presence of a network of yttria-stabilized zirconia (YSZ) islands buried beneath strontium titanate (STO). These regions appear perovskite like from 2D imaging focused at the entrance surface, emphasising the importance of considering the possibility of three dimensional inhomogeneity. The results also highlight the unambiguous nature of EELS elemental mapping, revealing 3D compositional changes that cannot be determined through ADF optical sectioning with complete certainty.

[1] G. Behan et al, Phil. Trans. R. Soc. A 367 (2009), p. 3825.

SuperSTEM is the EPSRC UK National Facility for Aberration-Corrected STEM. Research at ORNL was sponsored by the U.S. DOE, Office of Science, Materials Sciences and Engineering Division (MV). The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3).

Fig. 1: Optical sectioning with atomic resolution spectrum imaging. The Sr (M-edge), Ti (L-edge) and Zr (M-edge) maps have been denoised with principle component analysis, and are shown alongside the simultaneously acquired high angle annular dark field (HAADF) images.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2307 Observing depth dependent strain via optical sectioning in the STEM

Lozano J. G.1, Yang H.1, Guerrero-Lebrero M. P.2, Yasuhara A.3, Okunishi E.3, Zhang S.4, Humphreys C. J.4, Galindo P. L.2, Hirsch P. B.1, Nellist P. D.1
1Department of Materials, University of Oxford, Oxford (UK), 2Departamento de Lenguajes y Sistemas Informaticos, CASEM, Universidad de Cadiz, Puerto Real (Cadiz), Spain, 3JEOL Ltd., Tokio (Japan), 4Department of Materials Science and Metallurgy, University of Cambridge, Cambridge (UK)
juan.lozano@materials.ox.ac.uk

The development of spherical aberration correctors for the scanning transmission electron microscopes (STEM) has led to a reduction in the depth of field, which can be of just a few nanometers in a modern instrument. Since this value is smaller than the typical sample thickness, it creates an opportunity to optically section the sample simply by imaging with the focal plane set to a specific depth within the sample [1]. By recording a series of images over a range of focus values, a full three-dimensional image can be obtained. Here we demonstrate that optical sectioning in the high angle annular dark field mode (HAADF)-STEM mode has a sufficiently small depth of field to detect depth-dependent atomic displacements associated with dislocations in GaN, in particular the so-called Eshelby twist [2]. The Eshelby twist is a consequence of the relaxation of the stresses on the free surfaces of the thin TEM sample in dislocations with a screw component of the Burgers vector normal to the foil. It can be seen as an apparent rotation of the lattice in one surface, with displacements that decrease with increasing depth below the surface until the mid-plane of the foil is reached where no displacements should be observed. A rotation in the opposite sense occurs at the opposite surface.
HAADF images of a GaN crystal containing the displacements associated with a right-handed screw dislocation and the Eshelby twist were simulated at different focus value using SICSTEM [3]. The results indicate that the displacements due to the Eshelby twist become larger as the distance from the core increases within the field of view of the simulations, leading to an apparent overall rotation of the lattice around the dislocation core (Figure 1). This allows the twist to be measured on the lattice planes that are parallel to the fast scan direction in the STEM image, providing an approach very robust to the effects of sample drift and scan distortion.
We show that the Eshelby twist can be experimentally measured, by recording focal series in dislocations with a screw component (either pure screw or mixed) imaged end-on. The rotation of the lattice with respect to the image of the crystal at the entrance, as a function of defocus, was measured using the Radon transform [4] (Figure 2), which allowed us to quantify the rotation rate in the different types of dislocations (Figure 3) and us to determine the sign of the screw component of their Burgers vector.
[1] G. Behan et al., Phil. Trans. R. Soc. A 367, 3825 (2009)
[2] J. D Eshelby and A. N. Stroh, Phil. Mag. Series 7 42, 1401 (1951).
[3] J. Pizarro et al. , Appl. Phys. Lett. 93, 153107 (2008).
[4] S. R. Deans, The Radon Transform and Some of Its Applications, New York: John Wiley & Sons (1983)

We gratefully acknowledge financial support from the EPSRC, the Spanish MINECO and the Junta de Andalucía.

Fig. 1: Displacements maps due to the Eshelby twist for focus values a) 0 nm (entrance surface), b) -10 nm (exit surface) for a GaN crystal with a screw dislocation normal to the foil with the Eshelby twist, and the atomic positions of the infinite crystal. For better visualization, a factor of 10 has been applied.

Fig. 2: (a) and (b) show two HR-STEM images of a screw dislocation taken at focus values 4 nm apart, and (c) and (d) their Radon transform maps .(e) and (f) represent the variance of each intensity profile in (c) and (d), where the maximum variance corresponds to the angle at which the horizontal a-planes are aligned with the Radon projection.

Fig. 3: Experimentally measured rotation angle as a function of defocus for three types of dislocations: a pure screw, a pure edge and two neighbouring mixed dislocations. The rotation is clockwise for the screw and one of the mixed dislocations and anticlockwise for the other. No significant rotation is observed for the pure edge dislocation.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2322 Comparison of intensity and absolute contrast of simulated and experimental high-resolution transmission electron microscopy images for different multislice simulation methods

Krause F. F.1, Müller K.1, Zillmann D.1, Jansen J.2, Schowalter M.1, Rosenauer A.1
1Institut für Festkörperphysik, Universität Bremen, Otto-Hahn-Allee 1, 28359 Bremen, Germany, 2National Centre for HREM, Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands
f.krause@ifp.uni-bremen.de

Discrepancies between experimental and simulated images were often reported for high resolution transmission electron microscopy (HRTEM). Simulated contrasts deviate by a factor of up to 3 from experimental ones [1,2]. This disagreement, termed Stobbs factor, has prevented evaluation of HRTEM contrast by comparison to simulations as successfully realised in Z-contrast scanning TEM [3]. The mismatch is caused mainly by improper consideration of the camera modulation-transfer function (MTF) [4]. It was further proposed that contrast-overestimation could be attributed to the use of absorptive potentials (AP) for thermal diffuse scattering (TDS) and to the treatment of incoherence by coherent envelopes [5]. A frozen lattice (FL) simulation with incoherent summation of intensities simulated for various Gaussian-distributed incident angles is more adequate.

The influence of each of these simulation methods on HRTEM contrast was examined by studies of simulated defocus series. Figure 1 shows the results for the simulation of 15 nm thick gold, the proper use of the MTF yields the largest contrast reduction by a factor of 140%. The consideration of TDS by FL instead of AP yields a small contrast decrease below 10%. Incoherent summation of different incident angles contributes a reduction of about 20%. Use of the coherent envelopes instead of the more accurate transmission cross coefficients (TCC) also causes overestimation of image contrast of 10%.

The mismatch of experiments and simulations, conducted with FL, incoherent summation and properly considered MTF, was investigated. Defocus series of a gold foil were acquired with a CS-corrected microscope. Specimen thickness and orientation were determined by diffraction pattern refinements and FL simulations were conducted for these parameters. With an aperture of 7 nm-1 radius, a very good agreement is achieved for image patterns and intensities quantitatively measured in units of incident intensity. The image contrast also coincides as shown in Fig. 2. The ratio of simulated and measured contrast is 0.98±0.07. For larger apertures a discrepancy of 20% is found and good agreement of intensities is observed. Without any aperture the difference amounts to a factor of 40%. Residual aberrations and drift as cause for this were ruled out.

The contrast mismatch between HRTEM simulations and experiments is definitely reduced by proper consideration of the camera MTF and FL simulations with incoherent summation but still remains observable with larger apertures.

[1] M.J. Hÿtch, W.M. Stobbs, Ultramicroscopy 53(1994) 191

[2] A. Howie, Ultramicroscopy 98(2004) 73

[3] J.M. LeBeau et al., Phys.Rev.Lett. 110(2008) 206101-1

[4] A. Thust, Phys.Rev.Lett. 102(2009) 22080-1

[5] D. V. Dyck, Ultramicroscopy 111(2011) 894

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) under contract № RO2057/4-2.

Fig. 1: Contrast of images of a defocus series simulated for 15 nm gold in [100] direction with different techniques using an objective aperture of radius 14 nm-1: The red curve is the result of conventional MS and incoherence treated by coherent envelopes. For the following curves, the image formation was successively simulated more accurately.

Fig. 2: Comparison of experimental and simulated contrast of a defocus series of [100] oriented gold of 12 nm thickness and an objective aperture of 7 nm-1 radius. Both the values and the periodicity agree well.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2407 Studying ω to α Phase Transformation in Ti-15Mo alloy by Combination of Aberration-corrected Scanning Transmission Electron Microscopy and Ab-initio Calculations

Sung Jin Kang 1 Sung-Hwan Kim 1 Heung Nam Han 1 Min-Ho Park 2 Cheol-Woong Yang 2 Hu-Chul Lee 3 Yoon-Uk Heo 3 Miyoung Kim 1
Department of Materials Science & Engineering, Seoul National University, South. Korea 1 Department of Materials Science & Engineering, Sungkyunkwan University, South Korea 2 High Temperature Energy Materials Research Center, Korea Institute of Science and Technology, South Korea 3
kang123@snu.ac.kr
Compared to α -Titanium alloys (Hexagonal), β-Titanium alloys (BCC) are inherently ductile and have promising potentials to substitute new technology materials in daily life1. Mo is commercially added as β-Ti stabilizer to precipitate finely dispersed round shaped α-Ti phase in the β-Ti matrix to enhance the hardness of the Titanium. Interestingly, this α-Ti precipitate is not nucleated directly from the β-Ti phase but from the nucleation sites provided by ω precipitates2. Though there have been intensive studies on the phase transformation of β → α, detailed atomistic dynamics, including the ω phase, have rarely been investigated. We study Ti-Mo(15 wt%) alloy for the phenomena of α-Ti phase formation from the ω precipitate using aberration corrected high annular angle dark field scanning electron transmission microscopy (HAADF-STEM) and electron energy loss spectroscopy for chemical information as well as atomic structural information. We present direct images of the early stage in ω → α transition state exhibiting a metastable state. In bright and dark Z-contrast regions of ω precipitates, the atomic arrangement as well as Z-contrast seems very different from each other. Bright Z-contrast regions show an atomically resolved projected image of ω precipitate crystal structure in the [112 ̅0] zone axis (Fig. 1B), while dark Z-contrast regions show a burry image which is not easy to be interpreted. The image of dark Z-contrast region has a layered periodic pattern and the atoms on each layer are not well resolved as presented in Fig. 1A. The Ti atomic layers could be considered as a metastable phase which will finally develop to a part of α precipitate. We speculate that the metastable structure is formed by distortions caused by local defects of the ω phase. Substantial amount of stacking faults and dislocations in an extremely early stage of ω→ α phase transition supports this hypothesis. Using systematic ab-initio calculations, we found that there is a reasonably stable defective ω-Ti structure which is relaxed to the structure similar to the Z-contrast in HAADF-STEM images (Fig. 2). It is also confirmed that the defective Ti structure is relaxed to the stable hexagonal α-Ti structure with additional Ti atoms on the Ti deficient sites, converting the [112 ̅0]ω phase orientation to [0001]α directly. This study demonstrates that ω → α transition in Ti-Mo alloy system is governed by defect mediated phase transformation. References 1. Ankem S, Green CA. Mater Sci Eng A 263 (1999) 127 2. S. Nag et.al, Acta Materialia 57 (2009), 2136-2147
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF 2013034238)
Fig. 1: Enlarged HAADF-STEM image of [11-20]ω//[110]β orientation in rapidly cooled Ti-15wt% Mo sample after aging at 400°C. The Z-contrast of region A is apparently different from the atomic array of ω phase in region B.
Fig. 2: Position of considered layered defects (left) and the Ab-initio calculation result of fully relaxed structure with defects (right).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2425 Sensitive X-ray analysis system on an automated aberration correction FE-STEM

Inada H.1, Hirayama Y.1, Tamura K.1, Terauchi D.1, Namekawa R.1, Shichiji T.1, Sato T.1, Suzuki Y.1, Konno M.1, Nakamura K.1, Hashimoto T.1
1Science & Medical Systems Design Div. Hitachi High-Technologies Corp.
inada-hiromi@naka.hitachi-hitec.com

In recent years the aberration-correction technique has brought a revolution in analytical microscopy by making atomic-resolution imaging and analysis routinely achievable in transmission and scanning transmission electron microscopy (TEM and STEM) 1). We have developed an aberration corrected STEM (Hitachi HD-2700) with an automated aberration correction function. The HD-2700 is equipped with a large solid angle Energy dispersive X-ray spectrometry (EDX) detector which enables an atomic spatial resolution and high sensitivity for EDX analysis.
In the new auto-aberration correction function, the aberration coefficients are measured from a Ronchigram image recorded on a CCD camera for an amorphous sample. Using the coefficients, the software determines the aberrations that need to be corrected and proceeds to correct them. The aberrations are measured and corrected repeatedly and automatically until they are settled under the thresholds. Experiment tests revealed that it took approximately 11 minutes to complete up to the 3rd order aberration2). Figure 1 shows an example of image comparison before and after auto correction for a silicon single crystal imaged along the <110> direction. The Si dumbbell structure is clearly observed after the correction.
To improve the X-ray detection efficiency of the STEM-EDX system, we adopted a design of a windowless3), large area (100mm2) silicon drift detector (SDD). The detector is located closely to the specimen to realize a large solid angle of 1.1sr. The detection sensitivity of the light element (Nitrogen) is more than 10 times higher than that of the 30 mm2 SDD. Figure 2 shows STEM-EDX mapping results for a Pd-Pt catalyst particle specimen using a 30mm2 Si(Li) conventional detector and the new 100mm2 SDD, respectively. The beam energy of 200kV, probe current of 800pA, and acquisition time of 3min. were used. Clearly the maps obtained using the new SDD show much better signal to noise ratio for both nano-particles and carbon support. The high-speed X-ray analysis with the new 100mm2 windowless SDD also largely reduces the beam irradiation damage to the specimen4).

1) H.Inada et al., J. Elec. Microsc., 58 (2009), 111.
2) Y. Hirayama et al., JSPS132 congress (2013).
3) S.Isakozawa et al., J. Elec. Microsc., 59 (2010), 469.
4) K. Tamura et al., Microsc. and Microanal., S2 19 (2013) 1192.

 

Fig. 1: Image comparison between before and after auto Cs correction of silicon dumbbells.

Fig. 2: EDX mapping comparison of Pt and Pd catalyst particles (a) Conventional 30mm2 Si(Li), (b) Newly developed 100mm2 SDD.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2453 3D-structural elucidation of highly ordered mesoporous TiO2 thin film by the method of electron crystallography

Xu B. B.1,2, Feng Z. D.1, Zhou H.1, Wang C.1
1College of Materials, Xiamen University, Xiamen, China, 2Center of Instrumental Analysis, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China
zdfeng@xmu.edu.cn

At present, transmission electron microscopy 3D reconstruction has become an important technique to elucidate the 3D structure of materials. Electron tomography, single particle analysis and electron crystallography are the common methods of 3D reconstruction. Thereinto, electron crystallography, comprising transmission electron microscopy and electron diffraction, determines the 3D structure of materials via their 2D structure information. This method is used to investigate the structure of crystal in general. In fact, due to the similar structure between highly ordered and crystal, highly ordered mesoporous structure can also form electron diffraction patterns, so the method of electron crystallography can be used to study the 3D structure of highly ordered mesoporous TiO2 thin film as well. In this experiment, we obtained TEM images and their corresponding slected-area electron diffraction (SAED) patterns from different zone axes. Especially, the corresponding SAED patterns were recorded at an instrument camera length of 200 cm, which is much longer than the average length. The top view TEM image of the film shows a highly ordered honeycomb arrangement with a nearly perfect hexagonal disposition. Its corresponding SAED pattern exhibits a 6-fold symmetry, which is compatible with the [001] zone axis of the hexagonal structure. Mainly, the diffraction patterns taken from three directions ([001], [1-10] and [121]) can be indexed in the P63/mmc space group. And the cross-section TEM image regarded as viewed from [100] zone axes shows an ABAB stacking sequence. Moreover, the diameter of the pores can be directly measured to be ~10nm. In summary, the method of electron crystallography implements an effective explanation of highly ordered mesoporous TiO2 thin film with 3D hexagonal structure.

Fig. 1: The TEM images along [001] with SAED

Fig. 2: The TEM images along [1-10] with SAED

Fig. 3: The TEM images along [121] with SAED

Fig. 4: The cross-section TEM images along [100]
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2571 Optimization of imaging conditions for atomic resolution in Titan TEM to minimize radiation damage and to study low angle boundaries in graphene-like materials

Lopatin S.1, Chuvilin A.2
1FEI Company, Eindhoven, Netherlands, 2CIC nanoGUNE, Donostia - San Sebastian, Spain
sergei.lopatin@fei.com

   Recent advances in spherical aberration (Cs) correction for TEMs in a combination with monochromated electron sources enabled imaging of single and bilayer graphene with atomic resolution [1]. Newly developed TEM techniques such as a single atom or single-atomic-column spectroscopy [2, 3] and atomic resolution electron tomography [4] drive the need for increased electron radiation doses applied to samples. The radiation damage started to be the key limitation factor for high-resolution TEM [5].

   For graphene-like (light element) materials [6] the radiation dose limitation is particularly severe. First, the knock-on damage cross section is higher for low atomic number elements [7]. Second, light elements produce less contrast than heavier elements, so even higher doses are needed to obtain a sufficient signal-to-noise ratio (SNR). Finally, the graphene-like materials appear in the form of low dimensional allotropes that have only one or a few atoms in a typical projection of a high-resolution image.

   To minimize the electron dose the optimization of acquisition parameters is needed. Here we present an extensive study of TEM tuning to obtain high quality HRTEM images of graphene. We used a Titan TEM (FEI Co) equipped with a Cs image corrector, a super-high brightness gun and a monochromator (energy spread better than 0.15eV). Tuning of the Cs corrector is based on measurement of images defocus (df) and astigmatism while recording so-called Zemlin tableau [8]. It was demonstrated that proper accounting for Cs of 3rd and 5th order (C3 and C5) and systematic error of C3 measurement results in more than 2 times increase of contrast, meaning more than 4 times decrease in dose needed for the same SNR (Fig.1).

   The optimal settings found were applied to study low angle boundaries (LAB) in graphene. LAB is a row of edge dislocations, separation of those defining the boundary angle. LABs are not visible directly on the image but can be identified by methods such as geometrical phase analysis (GPA), see Fig.2. Physically LAB may be interesting as they represent a perfect discontinuous layer with periodically spaced singularities.

[1] K. W. Urban, Nature Materials, 10 (2011) 165.
[2] P. E. Batson, Nature (London), 366 (1993) 727.
[3] D. A. Muller, et al, Science, 319 (2008) 1073.
[4] M. B. Sadan, L. Houben, S. G. Wolf, A. Enyashin, G.Seifert, R. Tenne, and K. Urban, Nano Lett., 8 (2008) 891.
[5] R. F. Egerton, P. Li, and M. Malac, Micron, 35 (2004) 399.
[6] K. S. Novoselov, A.K. Geim, S.V. Morozov, D. Jiang, Y. Zhang, S.V. Dubonos, I.V. Grigorieva, and A. A. Firsov, Science, 306 (2004) 666.
[7] F. Banhart, Rep. Prog. Phys., 62 (1999) 1181.
[8] F. Zemlin, K. Weiss, P.Schiske, W. Kunath, K. -H. Herrmann, Ultramicroscopy 3 (1978) 49.

Fig. 1: Simulation verification of the impact of optimum conditions for 0.1nm transfer: a) Scherzer conditions optimized; b) C5+C3+df optimized; c) C5+C3+df optimized and systematic error from Zemlin tableau is accounted; d) the intensity profiles across simulated images; e) an experimental image acquired at approximately optimum conditions.

Fig. 2: LAB in graphene: a) original HRTEM image; b) dislocations identification by GPA (rotation map).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2594 Aberration correction through auto-iteration system utilizing diffractogram analysis by profile fitting technique

Morishita S.1,3, Nakamichi T.1, Takano A.1, Satoh K.1, Hosokawa F.1, Suenaga K.2,3, Sawada H.1,3
1JEOL Ltd., 2National Institute of Advanced Industrial Science and Technology, 3Research acceleration program, Japan Science and Technology Agency
shmorish@jeol.co.jp

Spherical-aberration-corrected TEM/STEM has become widely used in the past decade. To automatically correct the aberrations in the correction system, a precise measurement of residual aberrations and an optimized correction procedure are crucial. Several methods have been reported for quantitative measurement of the aberrations [1-5]. We have developed corrector control software JEOL COSMO (Corrector System Module), in which aberrations are measured by diffractogram tableau method in TEM and SRAM method [6] in STEM. In the diffractogram tableau method, measurement precision for measurable defocus (df) and two-fold astigmatism (A2), at diffractograms with tilted illuminations, determines the final precision of the correction, since residual aberrations and the intrinsic A2 and df to be corrected are calculated from these measurable parameters. This paper reports a profile fitting technique to analyze the diffractograms incorporated into our developed auto-iteration system, which enables us to correct aberration with an improved precision.
In the diffractogram analysis, radial intensity profiles are used. Each of the radial profile is fitted with a phase contrast transfer function to pick up a parameter of the first-order components, that is, amount of defocus in particular azimuth. For searching the local minima in the profile that determine the parameters of the transfer function, profiles around local minima instead of simple detection of local minima are utilized to reduce affection of noise on the profile in our system. With thus obtained first-order components at many azimuths, the intrinsic df, A2 and other aberrations are calculated. Figure 2 compares the plots of A2 obtained using only the position of first zero and using the devised fitting method over 20 diffractograms. With this method, standard deviation of measured intrinsic A2 is improved from > 1 nm to a few angstroms.
Next, we developed the auto-iteration system for aberration correction using a script language integrated in the JEOL COSMO. The system automatically chooses the next targets of aberration to be corrected and corrects them. Our algorithm for correcting procedure preferentially corrects aberrations of lower-order or large higher-order to minimize the phase disturbance. Finally, we successfully performed the auto aberration correction in TEM with the improved procedure, which results in the residual third-order aberrations from about 10 μm to < 1 μm within 15 min.

[1] F. Zemlin, et al., Ultramicros. 3, 49 (1978).
[2] S. Uhlemann, et al., Ultramicrosc. 72, 109 (1998).
[3] M. Haider, et al., Ultramicrosc. 81, 163 (2000).
[4] J. Barthel, et al., Ultramicrosc. 111, 27 (2010).
[5] M. Vulovic, et al., Ultramicrosc. 116, 115 (2012).
[6] H. Sawada, et al., Ultramicrosc. 108, 1467 (2008).

This work is supported by Japan Science and Technology agency, Research Acceleration Program.

Fig. 1: Example of a diffractogram and its line profile. The experimental profile indicated by solid line is used for profile fitting.

Fig. 2: (a) Results of two fold astigmatism measurements by using positions of first zero (gray) and by using profile fitting (black). (b) Standard deviation of (a), which includes both measurement error and actual fluctuation.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2830 Quantitative Low-dose HRTEM Imaging and Analysis of Radiation-sensitive Materials

Huang C.1, Borisenko K. B.1, Kim J. S.1, Berkels B.2, Kirkland A. I.1
1University of Oxford, 2RWTH Aachen University
chen.huang@spc.ox.ac.uk

It is well-known that radiation damage caused by fast electrons in the electron microscopes is a main obstacle for high resolution transmission electron microscopy (HRTEM) characterisation of materials that are easily damaged by the exposure to electrons. Although extensive research has been carried out on damage mechanisms, critical doses, and low-dose imaging techniques for decades, the search for the ultimate resolution for radiation-sensitive materials imaging and the most optimal imaging conditions is still continuing.

Due to the usually simultaneous existence of more than one kind of damage mechanism and the innate complexity of each damaging process, theoretical predictions of the dose-limited resolution for many materials are still only qualitative. When it comes to the design of quantitative low-dose experiments, whether it is single-shot imaging or image series acquisition, a more accurate knowledge of the effects of dose rate, total dose, accelerating voltage and microscope aberrations on the resolution is needed to achieve the optimal resolution for a particular sample.

In this work we demonstrate an experimental approach to determining the dose-limited resolution of radiation-sensitive materials. Apart from the established low-dose imaging methods, we apply multiple related techniques, such as exit wave reconstruction and non-rigid image registration to improve the quantitative data analysis and interpretation. It is shown that with careful calibration, the suggested quantitative low-dose high resolution imaging and data processing procedures should be easily adaptable to any specific transmission electron microscope equipped with standard instrumentation.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2922 Real Space Characterization of the Finite Shape of the STEM Probe

Grimley E. D.1, Sang X.1, LeBeau J. M.1
1North Carolina State University, Department of Materials Science and Engineering, Raleigh, North Carolina, United States
edgrimle@ncsu.edu

The shape and size of the electron probe impact many aspects of scanning transmission electron microscopy (STEM), including resolution in imaging and related STEM spectroscopies. This work introduces the projective standard deviation (PSD) as a tool for examining the shape of the STEM probe from atomic resolution images of a reference material. The PSD possesses sensitivity to the shape and intensity of atom columns in a STEM image and has already proven pivotal in the recently developed Revolving-STEM (RevSTEM) technique for eliminating drift related image distortions [1].

The PSD utilizes the normalized Radon transformation mathematical construction that projects the normalized integrated intensities of an image onto an orthogonal vector; this transformation oriented along a lattice vector results in a profile with sharp, periodic oscillations while a projection away from a lattice vector forms a profile with nearly flat intensity. Fig. 1 (a) shows transformations over a 180° range for a simulated Si <100> image. Fig. 1 (b) displays the transformations at 45° (red oscillating line) and 60° (blue dashed line with comparatively flat intensity) from Fig. 1 (a) which are oriented along and away from lattice vectors, respectively. The standard deviation of a normalized Radon transformation over a desired angle range generates a PSD plot with standard deviation as a function of angle. Projection perpendicular to lattice vectors results in large standard deviations, while angles oriented away from lattice vectors result in small standard deviations, as exemplified by the peaks and flat regions of the PSD plot in Fig 2 (a).

We demonstrate herein that the comparison between the PSD of an experimental image and the PSDs of simulated images enables the identification of the probe shape for a given microscope and image. Fig. 2 (a) illustrates this principle as the PSD of a simulated Si <100> image convolved with a Gaussian function (FWHM 0.10 nm by 0.10 nm; red line) differs substantially from the PSD of the same simulated image convolved with an astigmatic Gaussian function (FWHM 0.10 nm by 0.07 nm; dashed blue line). Iterative least-squares fitting of PSD plots allows determination of a probable probe shape for given conditions. Fig. 2 (b) shows the fitting of a PSD of a background subtracted experimental Si <100> RevSTEM micrograph (red line) with the PSD of a simulated Si <100> image convolved with a Gaussian function of FHWM 0.102 nm by 0.092 nm and ~45° rotation (blue dashed line). Strikingly, the simulated Si <100> image with added noise (Fig. 2 (d)) is almost identical to the background noise subtracted experimental RevSTEM Si <100> image (Fig. 2 (c)).

Reference: [1] X Sang and JM LeBeau, Ultramicroscopy 138 (2014), p. 28

The authors acknowledge the use of the Analytical Instrumentation Facility (AIF) at North Carolina State University, which is supported by the State of North Carolina and the National Science Foundation, and they acknowledge use of the High Performance Computing (HPC) services at North Carolina State University.

Fig. 1: (a) Normalized Radon transformation spanning 180° of a simulated Si <100> image (~ 3 x 3 cells; 240 x 240 pixel) convolved with a Gaussian (FWHM 0.10 nm by 0.10 nm). (b) 45° (red) and 60° (dashed blue) projection angle line profiles from (a) showing oscillation at 45° and comparatively flat intensity at 60° due alignment with a lattice vector.

Fig. 2: (a) PSD of simulated Si <100> convolved with symmetric (red) and astigmatic (blue dashed) Gaussian functions. PSD (red trace in (b)) and image of (c) a background noise subtracted RevSTEM Si <100> image and PSD (dashed blue trace in (b)) and image of (d) simulated Si <100> image (noise added to (d)).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2932 Complementary Nature of Microscopy Techniques for Understanding Materials Phenomena

Ghosh C.1, Basu J.1, Divakar R.1, Mohandas E.1
1Materials Synthesis and Structural Characterisation Division, Physical Metallurgy Group, Metallurgy and Materials Group, Indira Gandhi Centre for Atomic Research, Kalpakkam-603102, Tamil Nadu, India
chanchal@igcar.gov.in

Advent of nanotechnology over last couple of decades has not only reduced the size of basic building block of materials by several orders of magnitude, complexity of materials architecture and chemistry has also increased enormously. In such a scenario, judicious application of suitable microscopy technique can only provide answer to a particular question. Even though, resolution limits, capabilities and instrumentation of microscopes have improved to a large extent, relatively older techniques even today are found to be equally relevant. In this paper, relative merits of several microscopy techniques will be compared with reference to materials problems encountered in our laboratory.
Understanding anti phase domain boundaries in polar crystals has been a major challenge. Till date, all the interface models for APB are based on (10-10) interfaces. However, anti phase domains apart from this interfaces and complex interaction of these interfaces with other defects are observed very frequently. It is almost impossible to suitably image such boundaries at atomic resolution as crystallography of the interfaces is not known. Diffraction contrast imaging is probably the starting point for understanding APB in polar crystals. In case of phase contrast microscopy the contrast is generated by the interaction of specimen potential with the incident e- wave which is further modified with the instrument CTF. Whereas, during STEM a very fine e- probe scans over the atomic columns and during scanning either electron energy-loss spectra or the X-ray signal is used to understand the chemistry. A number of complex oxides have been studied by this method. During incoherent imaging of complex oxides, heavier cations act as strong scattering centers while the relatively lighter oxygen anions scatter weakly. So the image contrast is mostly dominated by the scattering from the cations. The structural imaging of complex intermetallics e.g. V-doped TiCr2 Laves phase is quite different in nature from the complex oxides. The atomic numbers of V, Ti and Cr are pretty close and all of them will scatter almost equally. As a result differential contrast as is generated in a complex oxide will not happen for V-doped TiCr2 phase. Zero loss phase contrast microscopy with image simulation has been proven useful for structural imaging of Ti and Cr atomic columns and also providing V occupancy information.
Though all of these techniques fetch the materials information at the atomistic scale, still each one of them is unique by electro-optical configuration and interaction with the materials. In another words all of these techniques are complementary to each other and only a combination of all of these techniques can provide the complete solution of the materials related issues.

The authors would like to acknowledge UGC-DAE-CSR for providing the experimental support.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-2949 Development of highly stabilized water chiller for atomic resolution microscope

Hamochi M.1, Ishii T.1, Chisaka S.1, Okunishi E.1, Sawada H.1, Wakui S.2
1JEOL ltd., 2Tokyo University of Agriculture and Technology
hamochi@jeol.co.jp

With progress of high resolution imaging using an aberration corrected STEM/TEM, fluctuation less than 50 pm is detectable at a high magnification. As electrical and mechanical stability of the microscope has been improved, the disturbance, due to change of environmental conditions, becomes more crucial at the higher magnification. Among the conditions, a temperature of cooling water is extremely important because it directly affects the temperature of a lens or a column. Magnitude of the temperature fluctuation on a specimen can be roughly estimated as follows. Thermal expansion coefficients for typical metallic materials are in order of 10-5, so that the temperature fluctuation less than 0.1 degree C changes a length of nm-order for mm-size structures in a microscope. Thus, a highly stabilized water chiller system is desired for an atomic resolution electron microscope. The requirements for the microscope are temperature fluctuation of ± 0.05 degree C/min or less, and water temperature drift less than 0.2 degree C/h. We report a water chiller system developed by us with precise temperature control to realize very small fluctuation of water temperature.

Figure 1 (a) shows appearance of the developed water chiller. The size of the chiller is W550×L775×H1350 mm and the cooling power is 6 kW. The high stability of temperature for the cooling water was achieved by a developed temperature compensator with plural heat sources and an improved heat exchanger. Figure 1 (b) plots a temperature change of flowing water controlled by our system for an ultrahigh resolution Cs-corrected 300-kV microscope operated at 300 kV for an hour. The result shows that the fluctuation was less than ±0.01 degree C/min expressed as difference of maximum and minimum within a 60 sec time window, and the temperature drift was less than 0.006 degree C/h expressed as moving average deviations with 60 sec time window.

We recorded high-resolution images with long acquisition times to evaluate the performance of the chiller. Figures 2 (a) and (b) show HAADF STEM images of a Si[110] with an acquisition times of 10 s and 80 s. Fig. 3 (a) shows a high resolution STEM image of Si3N4 of 4k × 4k pixels with 160 s. The images with long acquisition times showed small distortion due to small sample drift, indicating that the temperature of flowing water was sufficiently stabilized for an atomic resolution imaging with long acquisition time. It should be noted that electrical and mechanical stabilities for this microscope were also devised against the other environmental disturbances such as external magnetic field, an external temperature change and so on. As we reported, we have successfully developed a highly stable water chiller that is compatible for an atomic resolution microscopy.

Fig. 1:  (a) Appearance of the developed precise water chiller, (b) Example of temperature measurement for a flowing water cooling an ultra high resolution Cs-corrected 300kV TEM/STEM

Fig. 2: HAADF STEM image of Si[110], (a) with acquisition time 10 s, (b) 80 s (512 x 512 pixels)

Fig. 3:  HAADF STEM image of Si3N4. Inset image is magnified from area indicated by dotted rectangle.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-3171 Quantitative study of defocus-dependent annular bright field images

Lee S.1,2, Oshima Y.2,3, Takayanagi K.1,2
1Tokyo Institute of Technology, Tokyo, Japan, 2JST-CREST, Tokyo, Japan, 3Osaka University, Ibaraki, Japan
slee@surface.phys.titech.ac.jp

Previously, we found that the lithium column intensity of an annular bright field (ABF) image varied by a step of a single lithium atom in correlation with the thickness change of the LiV2O4 crystal [1]. But, ABF imaging mechanism has not been investigated quantitatively. In this study, we observed ABF and high angle annular dark field (HAADF) images of very thin specimen simultaneously and investigated defocus dependency of visibility of atomic columns [2].
By using a spherical aberration corrected electron microscope (R005), both ABF and HAADF images were taken simultaneously for very thin lithium manganese oxide, LiMn2O4 specimen from the [001] view direction. The incident convergent semi-angle was 30 mrad, and the detector semi-angles were 15-30 mrad for ABF and 102-272 mrad for HAADF. Fig.1 shows the through focus series of ABF and HAADF images obtained from 10 nm over-focus to -10 nm under-focus condition. In the ABF images, the atomic column had dark contrast at over-focus, but the contrast reversed into bright one when the defocus condition was changed to under-defocus. While, the HAADF image showed the bright column contrast which did not reverse regardless the focus change. It indicates that ABF image is a kind of phase contrast image, and could be explained by weak-phase-object approximation (WPOA).
We measured visibility in the ABF and HAADF images in order to estimate the defocus for obtaining the maximum contrast and the depth of focal (DOF). The optimum defocus was different between both images: the maximum contrast of the atomic columns was obtained at a few nm over-focus in the ABF image, while it, at a few nm under-focus in the HAADF image. And, DOF was determined to be 4 and 8 nm in the ABF and HAADF image, respectively. DOF of ABF image is obviously narrower than one of HAADF image. ABF imaging which has a narrow DOF could be used for visualizing light elements three-dimensionally.

[1] S. Lee, et al., J. Appl. Phys. 109 (2011) 113530.
[2] S. Lee, et al., Ultramicroscopy 125 (0), 43-48 (2013).

Fig. 1: (a) A structure model of very thin lithium manganese oxide (LiMn2O4) specimen. Through focus (b) ABF and (c) HAADF images are shown from 10 nm (over-focus) to -10 nm (under-focus). The maximum contrast is obtained at the defocus indicated by red rectangle.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-3187 Quantitative analysis of CeO2 and Gd-doped CeO2 nanocrystals by HRTEM focal series restoration

Stroppa D. G.1 2, Dalmaschio C. J.3, Thust A.2, Lentzen M.2, Barthel J.2 4, Houben L.2
1International Iberian Nanotechnology Laboratory (INL), Braga, Portugal, 2Forschungszentrum Jülich, Jülich, Germany, 3Federal University of Espírito Santo, São Mateus, Brazil, 4Rheinisch-Westfälische Technische Hochschule Aachen (RWTH), Aachen, Germany
daniel.stroppa@inl.int

High resolution transmission electron microscopy (HRTEM) is a well-known characterization technique with atomic resolution imaging capability, and different approaches have been explored to extend its use with aim at the retrieval of quantitative information with high spatial resolution. A particular example is the restoration of the electron exit-plane wave-function (EPWF) from HRTEM focal series, as it ideally contains information on the atomic species and their position along individual atomic columns [1]. In this work, we explore the phase shifts on restored EPWFs from CeO2 and Gd-doped CeO2 nanocrystals aiming to extract their local chemical composition with atomic resolution.
HRTEM focal series from nanocrystals in <100> zone axis orientation were obtained using an aberration corrected TEM microscope at 300 kV under a negative Cs-imaging (NCSI) condition [2]. This procedure allowed the imaging of O and Ce-Gd atomic columns with enhanced contrast, as shown in Figures 1a and 2a. Electron EPWF from the two investigated samples were restored from the focal series taking into account the residual aberrations, the instrument instabilities and the detection system (CCD) modulation transfer function (MTF) [3]. Figures 1b and 2b show the respective electron EPWF phases reconstructed for CeO2 and Gd-doped CeO2 nanocrystals. Finally, the local EPWF phase shifts corresponding to the individual atomic columns centers were analyzed and compared to results of multislice image calculations.
The results show that the EPWF phase shift differences between columns containing heavy atoms (Ce or Ce-Gd) are significant with respect to the background, indicating that they can be used to map the atomic columns thicknesses and to infer nanocrystals 3D morphology [4]. Even though the phase shift differences between columns containing light atoms (O) are appreciable, they are on the same range of the background phase fluctuation. This indicates that spurious contributions to the EPWF, probably related to the carbon support film, the CCD read-out noise and the phase-shift tail from heavy elements, limit the direct extraction of quantitative information for the current experimental setup.

The authors would like to thank Dr. Antonio J. Ramirez and Prof. Edson R. Leite for the fruitful discussions that contributed to this project.

Fig. 1: Figure 1: a) HRTEM image and b) phase from the reconstructed EPWF after a HRTEM focal series from a CeO2 nanocrystal. While the direct identification of Ce and O atomic columns can be ambiguous from a HRTEM image, the reconstructed EPWF phase clearly shows Ce atomic columns with brighter intensity with respect to the O atomic columns.

Fig. 2: Figure 2: a) HRTEM image and b) phase from the reconstructed EPWF after a HRTEM focal series from a Gd-doped CeO2 nanocrystal.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-3197 Temperature dependence of Z-contrast in InGaN

Mehrtens T.1, Schowalter M.1, Tytko D.2, Choi P. P.2, Raabe D.2, Hoffmann L.3, Jönen H.3, Rossow U.3, Hangleiter A.3, Rosenauer A.1
1Institute of Solid State Physics, University of Bremen, Bremen, Germany, 2Max-Planck-Institut für Eisenforschung GmbH, Düsseldorf, Germany, 3Institute of Applied Physics, TU Braunschweig, Braunschweig, Germany
mehrtens@ifp.uni-bremen.de

Scanning transmission electron microscopy (STEM) combined with a high angle annular dark field detector (HAADF) gives rise to image contrast strongly depending on the nuclear charges of the scattering specimen atoms and is often referred to as Z-Contrast. The comparison of HAADF-STEM image intensities with simulated intensities from multislice calculations allows determining specimen thickness or material composition for each atomic column [1, 2] in a high resolution HAADF-STEM micrograph.

The main contribution to the measured signal in HAADF-STEM stems from thermal diffuse scattering (TDS) due to the thermal vibrations of the specimen atoms. An increase of temperature should result in a higher HAADF-signal due to the larger amount of generated TDS.

In this contribution we have studied the thermal dependence of Z-contrast for InGaN/GaN and specimen temperatures between 300K and 600K. Fig. 1 shows the intensity profile of an five-fold InGaN/GaN multi-quantum well structure measured at different temperatures. We observed an increase of the HAADF-STEM intensity with increasing temperature for GaN as well as for InGaN. However, we also noticed that the material contrast in the image (ratio between intensities for InGaN and GaN) decreased with temperature (Fig. 2).

In order to understand this effect, multislice simulations were carried out in the frozen phonon approach for different specimen temperatures using the STEMsim program [3]. A temperature dependent parameterization of Debye-Waller-Factors derived from density functional theory calculations [4] was used. The simulated material contrast in dependence of specimen thickness is shown in Fig. 3 for an indium concentration of 30% and for three different temperatures. The contrast decreases with temperature as is it was found in the experiment and is related to the effect of static atomic displacements (SAD). SADs occur, if atoms with different covalent radii share the same crystal lattice or sublattice., which is the case for In and Ga in InGaN. A rise of temperature increases the contribution of thermal diffuse intensity, whose material contrast is smaller than that of Huang-scattering [5] caused by SADs. Thus, the material contrast decreases with increasing temperature.

[1] Rosenauer et al., Ultramicroscopy 109, 1171-1182 (2009)
[2] Rosenauer et al., Ultramicroscopy 111, 1316-1327 (2011)
[3] Rosenauer and Schowalter, Springer Proc. in Phys. 120, 169-172 (2007)
[4] Schowalter et al., Acta Cryst. A 65, 227-231 (2009)
[5] Z. L. Wang, Acta Cryst. A 51, 569-585 (1995)

This work was supported by the Deutsche Forschungsgemeinschaft under Contract No. RO 2057/8-1 and the Bundesministerium für Bildung und Forschung (BMBF) in the frame of the “ERA-SPOT True Green (13N9634)” project.

Fig. 1: HAADF-STEM intensity profile of a five-fold InGaN/GaN multi-quantum well structure grown on GaN measured at specimen temperatures of 300K, 450K and 600K. The intensity is increasing with increasing temperature.

Fig. 2: Intensity profile of an InGaN quantum well normalized with respect to the intensity of the neighboring GaN for different specimen temperatures.

Fig. 3: Simulated material contrast (IInGaN/IGaN) of In0.3Ga0.7N for different specimen temperatures.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-3202 Sub-angstrom resolution realized with super high-resolution aberration corrected STEM at 300 kV

Sawada H.1, Okunishi E.1, Shimura N.1, Satoh K.1, Hosokawa F.1, Kaneyama T.1
1JEOL Ltd.
hsawada@jeol.co.jp

An aberration corrected scanning transmission electron microscopy (STEM) enables us to perform a structural analysis at sub-angstrom resolution [1-3]. By high angle annular dark field (HAADF) STEM method, a resolution of 47 pm was achieved using a Ge [114] [4,5]. For light elements, sub-angstrom distance between Si-N atomic columns in a β-Si3N4 was resolved by an annular bright filed (ABF) imaging technique [6]. Recently, we developed a 300-kV super high-resolution aberration corrected microscope. The stability of the electronic power supplies, mechanical stiffness, and optical parameters such as chromatic aberration coefficient were improved in the microscope. The microscope was equipped with a cold field emission gun to realize high brightness and smaller energy spread. In this paper, we report the results on observations of atomic dumbbells separated by sub-angstrom with the developed microscope.

We observed a GaN [211] [2,7]. Next, sub-50 pm imaging was performed using a Ge [114] and a Si [114] [7]. Figures 1(a, d) and 1(e, f) show HAADF and ABF images of GaN [211]. The Ga-Ga atomic columns separated by 63 pm was resolved by HAADF STEM in Fig. 1(a). Spatial information better than (63 pm)-1 was confirmed in the Fourier transform shown in Fig. 1(c). The intensity profile in Fig. 1(d) shows that 63-pm separated atomic dumbbell of the Ga-Ga was clearly resolved. The ABF was utilized for a light element imaging with sub-angstrom resolution. N-N atomic dumbbell with 63-pm separation was resolved as a gray contrast in Figs. 1(e) and 1(f). The intensity profiles in Figs. 1(e, f) show that the 63-pm is clearly resolved.

Figure 2(a) shows a HAADF STEM image of a Ge [114], which shows 47-pm separated Ge-Ge dumbbells. The Fourier transform in Fig. 2(d) shows -8-84 spots, which correspond to (47 pm)-1. The line profiles in Fig. 2(c) show separations of 47 pm. Next, we challenged a Si [114], which shows 45-pm separated Si-Si dumbbells. The resolution has never been reported. Fig. 2(f) shows HAADF image of the specimen. The -8-84 spots and the line profiles in Figs. 2(d) and 2(h) confirm the resolution of 45 pm.

In conclusion, we have successfully demonstrated that light element imaging with sub-angstrom resolution by ABF, and sub-50 pm resolution by HAADF with the developed super high-resolution microscope at 300kV with CFEG.

References:

[1] P. Nellist, et al., Science 305: 1741 (2004).

[2] H. Sawada et al., Jpn. J. Appl. Phys. 46: L568 (2007).

[3] C. Kisielowski et al., Microsc. & Microanal. 14: 469 (2008).

[4] H. Sawada et al., J. Electron Microsc. 58: 357 (2009).

[5] R. Erni, et al., Phys. Rev. Lett. 102: 096101(2009).

[6] E. Okunishi, et al., Micron 43: 538 (2012).

[7] M. O'Keefe et al., J. Electron Microsc. 54: 169 (2005).

The authors thank Professor Y. Ikuhara, and Associate Professor N. Shibata (The University of Tokyo) for collaboration and instrumental supports.

Fig. 1: (a) Raw HAADF STEM image of GaN [211] taken at 300 kV. The convergence semi-angle was 30 mrad. (b, c) Intensity histogram and Fourier transform of (a). (d) Raw HAADF image and intensity profile from dotted rectangle area. (e,f) Raw and filtered ABF images and their intensity profiles.

Fig. 2: (a, b, f, g) Raw and low-pass filtered dark field STEM images of Ge [114] and Si [114] taken at 300 kV with simulated images. (c, h) Intensity profiles from the dotted rectangles in (a, b) and (f, g). (d, e) Fourier transforms and intensity histograms from (a) and (f).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-3462 The mini-TEM: highquality imaging and analysis of biological specimen

Sintorn I.1,2, Kylberg G.2, Nordström R.2, Uppström M.2, Danielsson K.2, Fulin J.2, Åkesson J.2, Coufalova E.3, Drsticka M.3, Kolarik V.3, Stepan P.3
1Centre for Image Analysis, Uppsala University, Sweden, 2Vironova AB, Gävlegatan 22, Solna, Sweden, 3Delong Instruments, Brno, Czech Republic
ida.sintorn@it.uu.se

 

Traditional transmission electron microscopes are bulky and complex machines that are mostly operated by trained specialists. In this paper, we introduce the miniTEM, shown in Fig. 1, a desk-top instrument designed for imaging of biological as well as inorganic samples. The miniTEM has a high degree of automation in the microscope alignment, image acquisition, and analysis processes. The idea with the miniTEM is a small, cheap, robust, and easy to use system that requires no more training than any simple lab equipment, and can be hosted in any office or lab (even a mobile lab). Here we illustrate the imaging possibilities and show that it is good enough for quantitative and qualitative analysis.

The miniTEM microscope runs at 25 keV, which enables high-quality imaging of biological samples with a thickness up to at least 100 nm. The height of the microscope is only 70cm and it can sit and run on any desk in any lab or office space. It achieves a resolution sufficient for tasks such as virus identification in clinical samples, and morphological nanoparticle analysis. In addition to TEM functionality, the miniTEM can also run in STEM (scanning transmission electron microscopy) mode. We present here the achieved parameters of resolution, applicable sample thickness and image signal collection efficiency in both operating modes. One of the first images of inorganic nanoparticles acquired with a miniTEM microscope prototype in the TEM mode is shown in Fig. 2.

 

The graphical user interface is divided into three main views: live, edit, and analysis. It is developed for Windows 8, and designed for a touch screen, allowing convenient scrolling over the sample and zooming in (changing magnification). The live view, illustrated in Fig. 3, is used when manually investigating the sample by moving around, changing magnification and acquiring images. The edit view is for manually marking, drawing, measuring and annotating objects in the images. In the edit view the user can also manually correct analysis results, i.e., remove, add, and rename objects. The analysis view is where the user creates and applies automated image acquisition and/or image processing (GPU-accelerated) and analysis scripts. A graph-based interface is used to create scripts, which can be saved for future use and applied to multiple images.

This work is part of the miniTEM project funded by EU and EUREKA through the Eurostars programme. 

Fig. 1: The miniTEM microscope

Fig. 2: Image of inorganic nanoparticles acquired with the miniTEM, scalebar 200nm.

Fig. 3: The live-view in the miniTEM graphical user interface. In the default layout, thumbnails of acquired images, and the movement and position on the grid are shown in the left panel. The main window shows the current image. In the right panel the histogram and Fourier spectrum of the current view are shown.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-5735 Quantitative measurement of electron magnetic circular dichroism on polycrystalline iron film

Muto S.1, Rusz J.2, Tatsumi K.1, Adam R.3, Arai S.1, Kocevski V.2, Oppeneer P. M.2, Bürgler D. E.3, Schneider C. M.3
1Nagoya University, Nagoya, Japan, 2Uppsala University, Uppsala, Sweden, 3Peter Grünberg Institute, Jülich, Germany
jan.rusz@physics.uu.se

Electron magnetic circular dichroism (EMCD) is a measurement technique, which allows to measure element-specific spin and orbital magnetic moments using a transmission electron microscope. Since its discovery in 2006 [1] it went through a rapid development, bringing improvements in spatial resolution and signal strength. Yet, quantitative measurements remained difficult using the classical approach because of the low signal to noise ratio (SNR). The classical approach suggested to orient the sample into a 2-beam or 3-beam orientation and acquire core-level spectra (ELNES) at two distinct positions in between Bragg spots (so called Thales circle positions). Here we describe a different approach that overcomes these limitations and can be applied on a polycrystalline sample without setting any particular orientation [2]. We use a polycrystalline iron sample, Fig. 1, and acquire few hundreds of Fe L2,3 ELNES spectra, while scanning with the beam over the sample using a megaelectronvolt Jeol JEM-1000 K RS microscope. The scanning step is set to a value close to an average size of a crystalline grain in the sample, therefore it is very likely that every spectrum is taken from a different grain and thus at a different crystal orientation. The detector orientation is shifted by approximately G(110)/2 from the transmitted beam direction. In the next step the dataset is statistically processed and an averaged EMCD signal is accumulated, Fig. 2. The reliability of the method was checked on a NiO control sample, which shows no net magnetization. Finally, EMCD sum rules [3] have been applied to extract the ratio of the orbital to spin moment, Fig. 3. The obtained value 0.0429 +/- 0.0075 is in close agreement with x-ray magnetic circular dichroism measurements [4].

[1] P. Schattschneider, S. Rubino, C. Hebert, J. Rusz, J. Kunes, P. Novak, E. Carlino, M. Fabrizioli, G. Panaccione and G. Rossi, Nature 441, 486 (2006).

[2] S. Muto, J. Rusz, K. Tatsumi, R. Adam, S. Arai, V. Kocevski, P. M. Oppeneer, D. E. Burgler, and C. M. Schneider, Nature Comm. 5, 3138 (2014).

[3] J. Rusz, O. Eriksson, P. Novak, P. M. Oppeneer, Phys. Rev. B 76, 060408(R) (2007).

[4] C. T. Chen et al., Phys. Rev. Lett. 75, 152 (1995).

A portion of this work was supported by a Grant-in-Aid on Innovative Areas "Nano Informatics" (grant number 25106004) and on Young scientist A (24686070) from the Japan Society of the Promotion of Science. J.R. and P.M.O. acknowledge the support from the Swedish Research Council, J.R. acknowledges support from STINT and P.M.O. from the European Commission (grant No. 281043).

Fig. 1: TEM image of the polycrystalline iron sample. Scale bar corresponds to 50nm.

Fig. 2: Accumulated ELNES (blue and red curves) and EMCD (black line) spectra from the entire dataset of 225 individual ELNES spectra.

Fig. 3: Extrapolation of the ml/ms ratio as a function of the low-pass filter width. Low-pass filter was applied to individual ELNES spectra prior to statistical extraction of the EMCD spectrum. WIthout low-pass filter the statistical extraction was numerically unstable.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-5974 High quality FIB lamella preparation for wide area atomic resolution TEM investigations

Straubinger R.1, Beyer A.1, Gries K. I.1, Schneider C.2, Rohnke M.2, Mogwitz B.2, Janek J.2, Volz K.1
1Material Sciences Center and Faculty of Physics, Philipps-Universität Marburg, Germany, 2Physikalisch-Chemisches Institut, Justus-Liebig-Universität Gießen, Germany
rainer.straubinger@physik.uni-marburg.de

A large increase in research efforts on thermoelectric power generation is currently occurring because of the improved properties of various nano structured thermoelectric materials. NaXCoO2 is a thermoelectric material which for example makes the recovery of the waste heat emitted by vehicles and factories possible. In addition it can be used in electronic processors. The single phase NaXCoO2 crystals we are working with are grown by pulsed laser deposition on Al2O3 (001) or LaAlO3 (001). To improve this material transmission electron microscopy (TEM) investigations are indispensable. Especially for structures that reveal a lot of inhomogeneity it is necessary to have high quality focussed ion beam (FIB) TEM lamellas for wide area atomic resolution.
During our research we continuously improve the FIB preparation process. Because of the hardness of the sapphire substrate it is necessary to thin the lamella from the substrate side (shadow FIB). Especially when working with very ion beam sensitive structures this preparation technique is also very interesting for other materials, like even organic material.
In Fig. 1 a high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) image of a cross sectional TEM lamella is shown. This lamella consists of the Al2O3 substrate, the NaXCoO2 layer, a Pt protection layer to avoid oxidation and a W protection layer deposited in the FIB. The bright areas within the NaXCoO2 layer are CoO2 impurities.It gets obvious that the thickness of the lamella does not change significantly over the whole field of view with the width of approximately 4μm. Thus, a good overview of a big sample region showing different features can be created using FIB. In Fig 2 a high resolution HAADF STEM image of the same lamella is presented, showing the interface between the substrate and a CoO2 impurity. It can be seen, that FIB preparation is a useful method to obtain thin samples over a wide range. This enables in combination with (S)TEM the characterization of samples containing a lot of inhomogeneities. This presentation will summarize the necessary steps to optimize the FIB preparation to obtain optimal samples.

Fig. 1: HAADF STEM image of NaXCoO2 grown on Al2O3 with a Pt + W cap layer.

Fig. 2: High resolution HAADF STEM image of a CoO2 impurity on Al2O3 substrate.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-2-P-6055 Visualizing and correcting dynamic specimen processes in TEM using a large-format Direct Detection Device

Bammes B. E.1, Spilman M.1, Chen D. H.1, Jin L.1, Bilhorn R. B.1
1Direct Electron, San Diego, CA
bbammes@directelectron.com

Multiple factors reduce the resolution and signal-to-noise ratio (SNR) of transmission electron microscopy (TEM) images, including the microscope instrumentation, dynamic specimen processes (e.g., drift, beam-induced motion, charging, radiation damage, etc.), and inefficient electron detectors.

With the goal of overcoming many of these obstacles, Direct Electron introduced the first large-format Direct Detection Device (DDD®) in 2008, as the culmination of academic and industrial partnerships. Development has culminated in the DE-20 (5k x 4k) in 2012 and the new DE-64 (8k x 8k) this year. These DDD cameras deliver dramatically improved performance compared to traditional electron detectors such as film or CCD cameras.

In addition to improved efficiency and resolution, the architecture of DDD cameras allows for continuous streaming of unbinned full-frame images at ~30 frames per second, with no dead time between consecutive frames. Many TEM methods require a static specimen image, such as low-dose electron cryo-microscopy of biological specimens. In these methods, dynamic specimen processes are detrimental, causing either non-isotropic resolution loss (i.e., specimen drift) or overall degradation of the SNR in each image (e.g., beam-induced motion, charging, radiation damage, etc.). We have developed methods and algorithms for exploiting the “movie mode” output from DDD cameras to correct for these dynamic processes and maximize the isotropic resolution and SNR of each image. Briefly, a “movie” is acquired of a specimen at 2-3× the normal total electron exposure. To correct specimen drift (which is consistent across the entire image), the frames from the movie are iteratively aligned, and to correct beam-induced specimen motion and charging (which are local effects that vary across the image), sub-regions for each frame are iteratively aligned. To correct radiation damage, low-pass filters are applied to each frame based on expected damage rate of the specimen.

We have demonstrated the benefits of this method by using images of frozen-hydrated Brome mosaic virus (BMV). Images generated based on our method show improved isotropic high-frequency SNR along with significantly improved low-frequency contrast compared to conventional imaging (Fig. 1). We processed a data set of ~32,000 particles using both the conventional method and our new “damage compensation” method to generate de novo three-dimensional reconstructions of BMV. Our new method improved the resolution significantly from 4.4 Å to 3.8 Å resolution, thus demonstrating the power of damage compensation with a direct detection camera for high-resolution structural studies.

We sincerely thank the National Center for Macromolecular Imaging (Baylor College of Medicine, Houston, TX) for images and reconstructions of BMV. We also acknowledge funding from the National Institutes of Health (Grant #8R44GM103417-03).

Fig. 1: BMV imaging on a 300 kV TEM at 1 μm underfocus. (A) A particle with 20 e-/Å2 exposure, and (B) with the new method with a total exposure of 36 e-/Å2 with correction of dynamic specimen processes. (C) The Fourier transform of the image with the traditional method, and (D) the new method. (E) Comparison of the spectral SNR of (C) and (D).
↑ Go to the Table of content ↑

IT-3. Super-resolution light microscopy and nanoscopy imaging

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-3-IN-1768 Superresolution Light Microscopy of nuclear Genome Organization

Cremer C.1,2,3
1Institute of Molecular Biology (IMB), D-55128 Mainz/Germany, 2Institute for Pharmacy and Molecular Biotechnology (IPMB), University Heidelberg, D-69120 Heidelberg/Germany, 3Kirchhoff-Institute for Physics (KIP), University Heidelberg, D-69120 Heidelberg/Germany
c.cremer@imb-mainz.de

The spatial organization of the genome in the interphase nucleus has far reaching functional consequences for gene regulation. Recently, various methods of superresolution light microscopy have been developed which made possible to enhance the spatial analysis of nuclear structures far beyond the conventional limits of about 200 nm in the object plane and 600 nm along the optical axis. Here, we report on quantitative nuclear nanostructure analysis based on Structured Excitation Illumination/Structured Illumination Microscopy (SEI/SIM), and on Spectrally Assigned Localization Microscopy (SALM), respectively. Presently, these approaches realized with custom-built systems allow us to optically resolve nuclear structures down to the range of ca. 120 nm laterally/350 nm axially using structured illumination, and few tens of nanometer in 3D using a special variant of localization microscopy, Spectral Precision Distance/Position Determination Microscopy (SPDM). In addition, both SIM and SPDM techniques were combined in a  single microscope setup. Application examples will be presented on the use of such ‘nanoscopy‘ approaches to perform quantitative analyses of individual small chromatin domains labelled by Fluorescence-in situ Hybridization (FISH); fluorescence-labelled replication units; of repair foci induced by individual accelerated heavy ions; of Fluorescent-Protein (GFP/YFP/mRFP) tagged histones and chromatin remodeling proteins; or of immunolabelled transcription/splicing related nanostructures. In addition, we report on the direct high resolution SPDM of nuclear DNA distribution, localizing more than 1 million individual DNA sites in an optical section of various types of mammalian cell nuclei. Some perspectives of these novel, quantitative “superresolution” microscopy methods for deciphering the „4D Nucleome“ will be discussed.

The support of the Boehringer Ingelheim Foundation, and of Heidelberg University is gratefully acknowledged.

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-3-IN-2856 Smart NanoBioImaging: multimodal correlative nanoscopy.

Diaspro A.1, 2, 3
1Department of Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2Department of Physics, University of Genoa, Genoa, Italy, 3Nikon Imaging Center, NIC@IIT, Genoa, Italy
alberto.diaspro@iit.it

Nanoscopy and super resolution localization microscopy changed the paradigm in optical microscopy and increased the portfolio of applications along with new developments. Among biological applications, the demand for imaging cell aggregates (i.e., tumor spheroids) or tissues/organs and small organisms (i.e. zebrafish) and for performing multimodal investigations is challenging. Light scattering, polarization properties and other than light-based mechanisms of contrast can represent an important issue for further advances. Within such a framework, mixed technologies for investigating biological systems (and not only) at the nanoscale will be outlined. Specifically, the possibility of utilizing a Mueller matrix approach for scattering and polarization dependent data - also exploiting optically active biological structures, with particular interest in chiral objects - could lead to improve informative content of the formed images. For example, fluorescence and SHG data can be enriched by Mueller matrix signature and polarization considerations. As it was early demonstrated the possibility of getting ultrastructural information about chromatin-DNA organization by means of circular intensity differential light scattering makes the Mueller matrix integrated approach an effective good candidate projected to label free high-resolution imaging. To this end , a Mueller Matrix polarimetry integrated architecture will be outlined, based on photoelastic modulation. A Classical electrodynamics model can be the starting point to decipher high resolution information due to light scattering.
Moreover, although optical methods are a comparatively safe way to probe a biological system without substantial perturbation, scanning/surface probe microscopy had a relevant impact on biological imaging after the advent of atomic force microscopy (AFM). Force mapping and curves can be analyzed in order to obtain, for example, local elasticity information (Young’s modulus evaluation pixel by pixel) or performing molecular nanomanipulation, with a high specificity that generally lacks in atomic force microscopy. A hybrid modality, coupling super resolution methods based on individual molecule localization (IML, PALM, STORM) and on optical nanoscopy (STED, RESOLFT) with AFM will be critically discussed.
Multimodal and multidimensional correlative super-microscopy launches a new trend in microscopy. The focus is on asserting that the key elemental differences in the superresolution hyrbid approaches can be perceived as a modern overture for addressing old and new biological biological questions.

The auhor is indebted with members of the NanoBioImaging and NanoBIoPhotonics - LAMBS IIT research team. This work partially funded by  the Italian Programmi di Ricerca di Rilevante Interesse Nazionale PRIN 2010JFYFY2-002 grant.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-3-O-1457 Imaging of cleared biological samples with the Ultramicroscope

Dodt H. U.1, Becker K.1, Hahn C.1, Saghafi S.1
11Vienna University of Technology, Chair of Bioelectronics, 1040 Vienna, Austria
dodt@tuwien.ac.at

In the last years we have developed a special Ultramicroscope (light-sheet microscope) for visualizing neuronal networks in whole brains. In the Ultramicroscope whole cleared brains are illuminated with a sheet of light and the optical sections are used for 3D reconstructions. This approach allows one to employ also low power, wide field objectives for imaging of large samples.

By clearing neuronal tissue with organic solvents (BABB) after dehydration, we could visulalize GFP-labelled neuronal networks in the whole brain [1.[ Improving our clearing technology by using tetrahydrofuran for dehydration and dibenzylether (THF/DBE) for clearing we were able to image GFP-labelled axons even in heavily myelinated spinal cord [2,3]. Also nervous and muscle structures in drosophila melanogaster can be imaged [4]. Our and other clearing solutions have non standard refractive indices. Due to a heavy refractive index mismatch imaging in these solutions with e.g. air or water immersion objectives gives therefore suboptimal results. We thus developed special objective devices that allow refractive index matched imaging. We show that high resolution imaging through 10 mm clearing medium is possible (Fig.1).

Furthermore we substantially increased the axial resolution of our light-sheet microscope by developing completely new optics for light sheet generation. These optics create an extremely thin light sheet by the use of a Powell- and several aspheric lenses. As light sheet thickness determines the axial resolution it is of pivotal importance for the performance of the light-sheet microscope. Our light sheet is static and will thus in future allow combination with other microscopic techniques which need constant nonscanned illumination. Examples for the application of the ultramicroscope as imaging of mouse brain, spinal cord and whole drosophila are given.

References

1. H.U. Dodt, U. Leischner, A. Schierloh, N. Jährling, C.P. Mauch, K. Deininger, J.M. Deussing , M. Eder, W. Zieglgänsberger, and K. Becker, Nat. Meth., 2007, 4, 331-336.

2. A. Ertürk, C.P. Mauch, F. Hellal, F. Förstner, T. Keck, K. Becker, N. Jährling, H. Steffens, M. Richter, M. Hübener, E. Kramer, F. Kirchhoff, H.U. Dodt, and F. Bradke, Nat. Med., 2012, 18, 166-171.

3. A. Ertürk, K. Becker, N. Jährling, C.P. Mauch, C.D. Hojer, J.G. Egen, F. Hellal, F. Bradke, M. Sheng, and H.U. Dodt, Nat. Protoc., 2012, 7, 1993-95.

4. C. Schönbauer, J. Distler, N. Jährling, M. Radolf, H.U. Dodt, M. Frasch, F. Schnorrer, Nature, 2011, 479, 406-409.

FWF

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-3-O-1814 Large parallelization of STED nanoscopy using optical lattices

Yang B.1,2, Przybilla F.1,2, Mestre M.1,2, Trebbia J. B.1,2, Lounis B.1,2
1Univ Bordeaux, LP2N, F-33405 Talence, France , 2Institut d’Optique & CNRS, LP2N, F-33405 Talence, France
jean-baptiste.trebbia@institutoptique.fr

Recent developments in super-resolution microscopy techniques achieved nanometer scale resolution and showed great potential in live cell imaging. STED (Stimulated Emission Depletion) and more generally RESOLFT (REversible Saturable OpticaL Fluorescence Transitions) need parallelization in order to fully benefit from this spatial resolution for fast wide-field imaging. An approach for parallelization is based on structured illumination pattern. RESOLF parallelization has been proposed using 1D interference pattern, but the resolution improvement is only obtained along one direction. Recently methods for massive parallelization of RESOLFT with photo-switchable proteins and STED nanoscopy based on the use of 2D structured illumination have been reported. Larger field of view could be achieved for parallelized RESOLFT using photo-switchable fluorescent proteins, because it requires less intensity to switch. However, protein switching is a relatively slow on-off process (~10 ms), which sets a limit to the imaging acquisition rate. Moreover RESOLFT with photo-switchable fluorescent proteins is constrained in its versatility by the need for genetic modification and transfection.

We show how well-designed optical lattices, created by multi-beam interference can provide efficient depletion patterns with moderate laser power and can be used for large parallelization of STED, so far the most important and widely used RESOLFT technique. The stimulated emission depletion being an ultrafast on-off switching process (~1 ns), its imaging speed is therefore only limited by the number of detected photons and fast large field of view super-resolution imaging can be achieved. With optical lattices, acquisition of large field of view super-resolved images only requires scanning over a single unit cell of the optical lattice which can be as small as 290 nm x 290 nm. STED images of 2.9 µm x 2.9 µm with resolution down to 70 nm are obtained at a frame rate of 12.5 images/s (figure 1).

Photobleaching might be a constraint in STED nanoscopy when recording a large number of frames. We reduce the photobleaching (i.e. the probability of a molecule to get promoted to a highly excited and reactive levels), by structuring both excitation and depletion beams. In this case, we found a decay time two times longer than in the homogeneous excitation case. We demonstraste the high-speed capability of our STED microscope by imaging the movement of 20 nm fluorescence particles in a Carbopol gel [1] (figure 2). We clearly show that recording of fast relative movement of two particles separated by a distance well below the diffraction limit.

Reference :

[1] B. Yang et al.,“Large parallelization of STED nanoscopy using optical lattices”, Optics Express, In Press (2014).

We acknowledge financial support from the ANR, Région Aquitaine, the French Ministry of Education and Research, the ERC and FranceBioImaging (Grant N° ANR-10-INSB-04-01).

Fig. 1: Structured excitation (a) and depletion (b) patterns for OL-STED (Optical lattice STED). (c) Fluorescence signal decays in the case of structured excitation and homogeneous excitation. (d) diffraction limited image of 20 nm fluorescent beads, (e) Super-resolved OL-STED image. (f) Normalized fluorescence intensity profiles.

Fig. 2: Diffraction limited (left side) and OL-STED (right side) successive images of 20 nm fluorescent beads. Images are taken at the rate of 80 ms per image with PSTED = 280 mW, Pexc = 2mW. The squares indicate the region where one bead is moving around another one.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-3-O-2173 Effects of optical aberrations in single molecule and super resolution imaging

Coles B. C.1, Schwartz N.2, Rolfe D. J.1, Guastamacchia M.1,3, Lo Schiavo V.1, Martin-Fernandez M. L.1, Webb S. E.1
1Science & Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, UK, 2Science and Technology Facilities Council, Astronomy Technology Centre, Edinburgh, UK, 3Heriot-Watt University, Edinburgh, UK
benjamin.coles@stfc.ac.uk

The images created in a fluorescence microscope are imperfect because of aberrations caused by the various objects in the optical path. These aberrations may be due to both the microscope optics and the sample itself. More specifically, they may be due to imperfect optic design and manufacturing processes, imprecise alignment of optical components and mismatched and inhomogeneous refractive indices within the sample specimen. Aberrations caused by the sample are necessarily greater as we go deeper, particularly in heterogeneous biological samples. As a consequence, most single-molecule tracking and single-molecule based super-resolution imaging is performed in microscopes equipped with total internal reflection illumination, which limits fluorescence excitation to a thin layer a few hundred nanometres thick. The aberrations impose limitations on the resolving power of a fluorescence microscope, the localisation precision of super resolution imaging and the proportion of features detected in single molecule tracking analysis. Achieving the same high resolution throughout a 3D sample such as biological cells depends on correcting these aberrations and recovering a high signal-to-noise ratio in deeper layers, which can be achieved using adaptive optics. Correcting the aberrations is particularly important for measuring accurate distances within a 3D volume. Note that it is not, however, trivial to determine what the aberrations from each point in the sample are, particularly in widefield microscopes.

There are approximately ten significant low-order Zernike modes of aberration present in a standard fluorescence microscope. We have investigated the effects of these different aberrations on data analysis in single molecule imaging techniques such as tracking in live cells. In particular, we have studied the consequences of aberrations on single molecule detection rates and apparent intensities in the poor signal-to-noise ratio environment of biological cells. These have wide consequences for the accurate determination of, for example, stoichiometry, FRET and diffusion rates from single-molecule measurements.

We have also studied the impact of aberrations on data analysis in single molecule localisation super-resolution techniques, such as STORM/PALM imaging, with varying levels of noise. Aberrations directly affect the signal-to-noise ratio and hence the achievable localisation precision.

Lastly, both Gaussian and astigmatic point spread functions were considered in order to extend the improvements to three dimensional super resolution imaging and single molecule tracking.

This work was funded by the UK's Medical Research Council under grant number MR/K015591/1

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-3-O-2789 Identify and Localise: Algorithms for Single Molecule Localisation Microscopy

Best G.1 2 5, Prakash K.3 4 5, Hagmann M.1 2, Cremer C.1 3 4, Birk U.1 4
1Kirchhoff-Institute for Physics (KIP), University of Heidelberg, Heidelberg, Germany, 2University Hospital Heidelberg, University of Heidelberg, Heidelberg, Germany, 3Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Heidelberg, Germany, 4Institute of Molecular Biology (IMB), Mainz, Germany, 5equal contribution
K.Prakash@imb-mainz.de

Single Molecule Localisation Microscopy (SMLM) is increasingly viewed as one of the major tool for analysis of biological processes on a high resolution level in the range of 10 to 50 nm. The procedure relies on sequential detection of (a subset of) individual fluorophores. For dense regions (fluorophores with significant overlap), a compromise between fluorescence labelling density and the photoswitching behaviour of fluorophores is needed to have an optical isolation i.e. sparse distribution of molecules in each acquired frame.

Algorithms used to precisely identify the locations of these fluorophores can be broadly classified into two categories, namely fitting based and non-fitting based (usually Centroid) methods. While iterative fitting-based methods can usually provide fitted parameters equal or close to the maximum likelihood estimate, ad hoc centroid based methods are usually very quick. However, all localisation methods struggle if the underlying model poorly represents the observed data e.g. background level, out of focus signals, noise, etc. A particular challenge for the exact fluorophore determination is posed by spatially as well as temporally fluctuating background intensities arising from out of focus blinking fluorophores. This is to some degree always given if the structure is not per se 2-dimensional (e.g. PALM using TIRF illumination).

Here, we present a comparative analysis of a range of available localisation algorithms on complexity, applicability and performance by testing them on both synthetic and experimental data that cover examples of both sparse and dense regions, with both low and high background levels to determine, which method is suited for a given set of data.

We gratefully acknowledge the colleagues at IMB who supported us with reagents. In particular, we would like to thank Aleksander Szczurek and Hyun-Keun Lee for samples, reagents and many interesting discussions. This work is supported by the Boehringer Ingelheim Foundation. The support of University Hospital Heidelberg (Prof. S. Dithmar) to G.B. and M.H. is also gratefully acknowledged.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-1448 Nonlinear optical single-molecular image technique applying in nanostructure study of macromolecules from chicken egg

Wang X. M.1
1Hubei University of Chinese Medicine
foxglove@163.com

Nonlinear optical single-molecular image technique is a new technique which is not widely recognized in scientific community. It is our patent technique (Chinese patent 200910060951.7, PCT /CN2010/000138 ). It is a new innovative principle to get a profile image of tiny material noninvasively. by a series of lenses which diameter from small to large, adjusting each lens move back and forward carefully along a straight line ,the two different direction rays from the same point of out edge of objects can focus on a plate to form an image. This technique can magnify profile images of small samples. Its x-y resolution breakthrough the limit of Abbe’s diffraction law. Nowadays ,it resolution can reach 1-3nanometer . It can get single molecular image in water. It can be used to trace the trajectory of single molecule in living cell. The principle of technique will be broad application in many areas in near future. In this paper, we gave more supporting evidences that Nonlinear optical single-molecular image technique is a practical tool. The photos of our experimental results demonstrated that the principle of Nonlinear optical single-molecular image technique is correct. The photos showed the double helix structure of DNA extracted from chicken egg. This technique will bring human a lot of knowledge and information about molecules, especially about biological macromolecules in living cell. It will improve drug research and human understand micro world. With computer image reconstruct technique , Nonlinear optical single-molecular image technique will become more powerful tool for scientific research.

Gratful the Financial support of The nature science foundation of Hubei province 2006ABA060,The Education Department of Hubei province  D200516013

Fig. 1: macromolecules extracted from chicken egg white

Fig. 2: macromolecules deployed by double distilled water on glass slide 

Fig. 3: The photo showed double helix structure of macromolecules

Fig. 4: Diphenylamine assay demonstrated the macromolecules was DNA
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-2060 Sub resolution spectral discrimination of Lipufuscin-granules inside human RPE cells

Schock F.1,2, Best G.1,2, Celik N.2, Heintzmann R.4,5, Sel S.2, Birk U.3,6, Dithmar S.2, Cremer C.1,3,6
1Kirchhoff Institute for Physics, University of Heidelberg, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany, 2Department of Ophthalmology, Universityhospital of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany, 3Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany, 4Institute for Physical Chemistry, University of Jena, Lessingstr. 10, 07743 Jena, Germany, 5Institute of Photonic Technology, Albert-Einstein-Straße 9, 07745 Jena, Germany, 6Department of Physics, University of Mainz, Staudingerweg 7, 55128 Mainz, Germany
florian.schock@kip.uni-heidelberg.de

In the last decades, a variety of new microscopy methods has been developed to circumvent the classical resolution-limit[1]. Furthermore, combinations of confocal-microscopes and spectrometers have proven useful and are already commercially available. However, presently no existing device is used to combine spectrometry with superresolution.

Here we present a first attempt to analyze the emission spectrum of super resolution images in a clinically important field of application. We used a custom-made Structured Illumination Microscope (SIM) equipped for multicolor imaging[2]. At an excitation wavelength of 488 nm, this instrument provides an optical resolution down to about 120 nm in the object plane and 350 nm along the optical axis. To obtain spectral information we used different emission filters and calculated the resulting spectral bands.

We used this method on human retinal pigment epithelial (RPE) tissue sections and present first superresolution images on spectral information. We also present that this method is able to separate the intracellular autofluorescent Lipufuscin-granule-types that are connected to age related maculadegeneration. All work on human tissue was done according to the Declaration of Helsinki.

[1] Christoph Cremer and Barry R. Masters; Resolution enhancement techniques in microscopy; The European Physical Journal H; 2013
[2] Sabrina Rossberger, Thomas Ach, Gerrit Best, Christoph Cremer, Rainer Heintzmann, Stefan Dithmar; High-resolution imaging of autofluorescent particles within drusen using structured illumination microscopy; Br J Opthalmol 2013, 97, 518-523

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-2190 Super-resolution imaging of 3D-cultured cells by SAX microscopy

Yonemaru Y.1, Yamanaka M.1, Uegaki K.1, Smith N. I.1, Kawata S.1, Fujita K.1
1Osaka University, Osaka, Japan
fujita@ap.eng.osaka-u.ac.jp

We have proposed the use of saturated excitation (SAX) of fluorescence to improve the spatial resolution of confocal fluorescence microscopes [1]. SAX introduces the highly nonlinear relation between excitation and emission intensities. With using a focusing laser for fluorescence excitation, the nonlinear response is localized within the laser focus, therefore the extraction of fluorescence signal, which nonlinearly responds to the excitation intensity, can realize the spatial resolution beyond the diffraction limit [2]. Imaging of biological samples have been demonstrated firstly with a fixed sample [3] and recently applied to live cell observation with fluorescence proteins [4].

Since the spatial resolution in SAX microscopy is improved by the nonlinear relationship between the excitation and the emission, SAX microscopy has the imaging property similar to two-photon microscopy. The nonlinear relation between emission and excitation allows us to remove the background fluorescence signal generated at out-of-focus planes. With using this benefit, we have applied SAX microscopy to observed 3D-cultured HeLa cells [5, 6]. A cell cultured on a flat substrate expands its body and forms a thin and flat shape with approximately 10 µm thickness. On the other hand, a cell cultured in a 3D matrix (such as gel) has more freedom to expand in the 3D space and shape their bodies with a thickness of several tens of micrometers. Since many differences has already observed between cell functions in 2D and 3D cell culture and the 3D cell culture can provide a condition for cell growth closer to the nature, super-resolution imaging of 3D-cultured cell may become important in biological and medical researches in the near future.

Fig. 1a shows a SAX fluorescence image of actin in fixed HeLa cells cultured in 3D. The cells were cultured in gel and stained with ATTO Rho6G phalloidin. We scanned the entire cell cluster to obtain a 3D data set of fluorescence distribution in the sample to construct Fig. 1a as a projection of the data set. Fig. 1b shows the enlarged view of the dotted rectangle area in Fig. 1a. Fig. 1c shows the same area as Fig. 1b, but obtained by a typical confocal microscope without SAX. The comparison of Fig. 1b and 1c confirms the improvement of the spatial resolution and the image contrast by SAX.

Reference

[1] K. Fujita et al., Phys. Rev. Lett., 99, 228105 (2007).
[2] M. Yamanaka et al., Biomed. Opt. Express, 2, 1946 (2011).
[3] M. Yamanaka et al., J. Biomed. Opt., 13, 050507 (2008).
[4] M. Yamanakaet al. Interface FOCUS, 3, 2013007 (2013).
[5] M. Yamanaka  et al., J. Biomed. Opt., 18, 126002 (2013).
[6] Y. Yonemaru et al., ChemPhysChem, in press (2014).

This study was supported by Next-Generation World-Leading Researchers (NEXT program) of the Japan Society for the Promotion of Science (JSPS).

Fig. 1: a) SAX images of HeLa cells cultured in 3D matrix. Actin filaments were stained. b) the enlarged view of the area in the doted rectangle in a). c) the same area observed by a typical confocal microscope.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-2200 Structured Illumination Microscopy With Multifrequency Patterns and LED Light Sources

Švindrych Z.1, Křížek P.1, Ovesný M.1, Borkovec J.1, Smirnov E.1, Raska I.1, Hagen G. M.1
1First Faculty of Medicine, Charles University in Prague
deden@seznam.cz

Structured illumination microscopy (SIM) has grown into a family of methods which achieve optical sectioning, resolution beyond the diffraction limit, or a combination of both these effects in optical fluorescence microscopy. SIM techniques rely on illumination of a sample with patterns of light which must be shifted between each acquired image. The patterns are typically created with physical gratings or masks, and the final optically sectioned or high resolution image is obtained computationally after data acquisition. Here we used a high speed ferroelectric liquid crystal microdisplay together with incoherent LED illumination to generate the illumination patterns and a sCMOS camera for widefield image acquisition. The high precision and flexibility of the generated patterns allowed us to use advanced processing techniques relying on the precise knowledge of the display-camera mapping, such as scaled subtraction in the case of optical sectioning SIM [1] and precise determination of spectral parameters (modulation period, direction and phase) in the case of super-resolution SIM. The freedom in choosing the illumination patterns also allows to tune the spatial frequencies and orientations of the patterns. Here we demonstrate the use of multi-frequency one-dimensional patterns to achieve both increased lateral resolution and high contrast optical sectioning with incoherent illumination and two-dimensional data processing in the Fourier domain (see inset in Fig. 1 C). We have also evaluated the impact of incoherent illumination on the SNR (signal to noise ratio) of the recovered high-frequency image components [2].

[1] P. Křížek, I. Raška, and G. M. Hagen, Opt. Express 20 (2012), p. 24585.
[2] M. G. Somekh, K. Hsu, and M. C. Pitter, J. Opt. Soc. Am. A. Opt. Image Sci. Vis. 26 (2009), p. 1630.

This work was supported by the Grant Agency of the Czech Republic [P304/09/1047, P205/12/P392, P302/12/G157 and 14-15272P], by Charles University in Prague [Prvouk/1LF/1, UNCE 204022], and by European Union Funds for Regional Development [OPPK CZ.2.16/3.1.00/24010].

Fig. 1: Fixed HT-1080 cells labeled with EdU-Alexa647 (fluorescently tagged nucleotide that incorporates into newly replicated DNA, red) and fluorouridine (synthetic nucleotide which is incorporated into active transcription sites, green), maximum projections of 20 sections, 0.1 µm step. A – widefield, B – optical sectioning SIM, C – super-resolution SIM.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-2211 Flexible Structured Illumination Microscope with a Programmable Illumination Array

Švindrych Z.1, Křížek P.1, Ovesný M.1, Borkovec J.1, Raška I.1, Hagen G. M.1
1First Faculty of Medicine, Charles University in Prague
deden@seznam.cz

Structured illumination microscopy (SIM) has grown into a family of methods which achieve optical sectioning (OS-SIM), resolution beyond the Abbe limit (SR-SIM), or a combination of both effects in optical microscopy. SIM techniques rely on illumination of a sample with patterns of light which must be shifted and/or rotated between each acquired image. The patterns are typically created with physical gratings or masks, or by laser interference, and the final optically sectioned or high resolution image is obtained computationally after data acquisition. We used a flexible, high speed ferroelectric liquid crystal display for definition of the illumination pattern coupled with widefield detection and subsequent image processing. Focusing on optical sectioning, we developed a unique and highly accurate calibration approach which allowed us to determine a mathematical model describing the mapping of the illumination pattern from the microdisplay pixels to the camera sensor pixels. This is important for higher performance image processing methods such as scaled subtraction of the out of focus light, which require knowledge of the illumination pattern position in the acquired data. The calibration is also advantageous for SR-SIM reconstruction, as it provides precise information about reconstruction parameters (pattern period, orientation and phase) [1]. We evaluated the signal to noise ratio and the sectioning ability (see Fig. 1) of the OS-SIM reconstructed images for several data processing methods and illumination patterns with a wide range of spatial frequencies [2].

[1] M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc., vol. 198, pp. 82–87, 2000.
[2] P. Křížek, I. Raška, and G. M. Hagen, “Flexible structured illumination microscope with a programmable illumination array.,” Opt. Express, vol. 20, no. 22, pp. 24585–99, Oct. 2012.

This work was supported by the Grant Agency of the Czech Republic [P304/09/1047, P205/12/P392, P302/12/G157 and 14-15272P], by Charles University in Prague [Prvouk/1LF/1, UNCE 204022], and by European Union Funds for Regional Development [OPPK CZ.2.16/3.1.00/24010].

Fig. 1: Comparison of different optically sectioning microscopes on a pollen grain sample. The SIM system achieves an optical sectioning thickness of 300 nm, much better than is possible in CLSM (Confocal Laser Scanning Microscopy).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-3025 Measurement and simulation of PSF

Nahlik T.1, Stys D.1
1University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, Institute of Complex Systems, Nové Hrady, Czech Republic
nahlik@frov.jcu.cz

Goal of the microscopy is to observe nature and man-made products which are smaller than resolution of human eyes. Microscope should help us to see more details but it has its own limits. One of these limitation is PSF (Point Spread Function). Microscope is device with many lenses and each of the lens can bring some distortion to the final image. When the object is so small that it can be consider as a point the image of this point will be not only the point but set of points. This image is called PSF. It is important to know and describe behavior of the PSF because it helps us to understand how the microscope transfers and shows the image.
Many simulation and measurements were done on the fluorescent microscope, because it is much easier. The difference between bright field and fluorescent microscopy is in the light. In BF the light is scattered by the sample, in fluorescence the light is emitted out of the sample. We tried to use ENZ (Extended Nijboer-Zernike theory) simulation (See Fig. 1). This works for fluorescent microscopy but it fails in BF (See Fig. 2). We tried to measure the PSF in BF (See Fig. 3) under different condition; with different particles (15nm and 200nm) and under different light condition (Light intensity is expressed by current on the LED used for illumination). We came to conclusion that the shape and size of PSF depends not only on parameters described in ENZ like aberration, light wavelength, numerical aperture, size of the particle but also on light intensity.
The size and shape of PSF is connected with the real resolution of the microscope and terms like distinguishability and discriminability. We propose that measuring of PSF should be the basic experiment done with every microscope. Measured PSF should be used as a kernel for deconvolution function for improving microscopy images.

CENAKVA CZ.1.05/2.1.00/01.0024, and CENAKVA II (The results of the project LO1205 were obtained with a financial support from the MEYS under the NPU I program); GA JU 134/2013/Z;

Fig. 1: Different aberration in ENZ Simulation. Parameters of simulation were – wavelength = 200nm, NA (numerical aperture) was 0.5, diameter was 400nm; A – no aberration; B – Coma; C – Tilt; D – Astigmatism

Fig. 2: ferent Light condition – Upper row - 15nm gold particles, Bottom row – 200nm latex particle. Light is increasing from left to right (starts at 1000mA, step 500mA, end at 3000mA)

Fig. 3: Comparing of ENZ simulation and real PSF. Red line shows position of focus. Right column shows original images in different distance from focus and appropriate Z position in PSF.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-3035 Drift Correction Strategies for Single Molecule Localisation Microscopy

Hagmann M.1 2 6, Prakash K.3 4 6, Best G.1 2, Kaufmann R.5, Birk U.1 4, Cremer C.1 3 4
1Kirchhoff­-Institute for Physics (KIP), University of Heidelberg, Heidelberg, Germany, 2University Hospital Heidelberg, University of Heidelberg, Heidelberg, Germany, 3Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Heidelberg, Germany, 4Institute of Molecular Biology (IMB), Mainz, Germany, 5Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK, 6equal contributions
K.Prakash@imb-mainz.de

The correct position determination of fluorescent molecules is crucial for the interpretation of localisation microscopy data, e.g. of the biological structure investigated. The relative position of fluorophores with respect to the detector is highly sensitive to environmental disturbances (e.g. acoustic vibrations) and to mechanical instabilities of the microscope hardware (e.g. thermal expansion or mechanical relaxation). These disturbances cause distortion in the recorded image which pose a new natural limit to the localisation accuracy, especially for new acquisition protocols that allow acquisition times in the order of hours.

Here, we present two drift correction strategies based solely on data already acquired without any fiducial markers. We found that in some flavours of SMLM, many of the biological samples exhibit enough permanent (photostable) structure to reveal information about the sample location, or if this is not the case, reconstructions of a subset of the complete localisation data stack can be used at several time points to gain information about the sample drift. In both cases, the drift is found by determining the peak of the computed 2D-autocorrelation function. A polynomial or a set of Fourier functions is fitted through the data, based on which the dislocation of every localised fluorophore in a given frame of the acquired image stack is subtracted.

Using this approach, we successfully corrected localisation microscopy data down to a final drift less than 5 nm, which is comparable with fiducial markers based strategies. We demonstrate that with this procedure the resolution of the final reconstructions is substantially enhanced and the theoretical limit of localisation accuracy is almost restored.

We gratefully acknowledge the colleagues at IMB who supported us with reagents. In particular, we would like to thank Aleksander Szczurek and Hyun-Keun Lee for samples, reagents and many interesting discussions. This work is supported by the Boehringer Ingelheim Foundation. The support of University Hospital Heidelberg (Prof. S. Dithmar) to G.B. and M.H. is also gratefully acknowledged.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-5736 Three-Dimensional Surface Modelling Of Cellular Structures And Intracellular Proteins Expression In Urinary Bladder Cancer Cells.

ELKABLAWY M. A.2
1Pathology Department, Faculty of Medicine, Menoufyia University, Egypt; , 2Pathology Department, Faculty of Medicine, Taibah University, Almadinah Almonourah, Saudi Arabia.
elkablawy@hotmail.com

Introduction: Visualizing and describing structure and function at a subcellular level is of fundamental importance in the molecular pathological sciences. The majority of microscopic techniques give a single two-dimensional representation of often complex three-dimensional (3D) structures. In many cases it would be useful to be able to view objects seen under the microscope in three dimensions, an approach which has clear advantages for the user in terms of comprehension and interpretation.
The aim of this study: was to produce 3D models of data from confocal laser scanning microscopy (CLSM) with a user-friendly PC software package. The study would examine the spatial localization of cellular proteins p53 and Bcl2 in apoptotic human urinary bladder cancer cell lines.

Material and method: Various combinations of immunofluorescence for the proteins of interest and propidium iodide staining for nuclear delineation were carried out in urinary bladder l cancer cells using contrasting fluorochromes. Specimens were examined with CLSM and stacks of optical sections from each light channel saved to disk. Using a desktop computer, the volumes of data were merged and rendered using gradient shading ray tracing. Surfaces of nuclei and protein aggregations were made transparent and the subcellular structures could be viewed from any angle. Series of renderings could be tagged together to produce a 3-D video moving sequences (build-up, fly-by and clipping) for better studying the protein expression.

In conclusion: the rendered images and videos were of high quality and were extremely helpful in studying the intracellular proteins expression. The technique both aided the research process and improved the means of scientific interpretation of gene and protein localization at the subcellular level.

the Author Thank Sarah Elkablawy, Ayah Elkablawy and Mona Mawlana for technical help and support. Also Thank dr. Nashaat Shawky for revising the language  and grammer of the abstract.

Fig. 1: this Images show the conversion of 2D image into series of 3D images
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-5751 Light induced in situ observation technology in EM

Huang M. R.1, Lin C. L.1, Chen K. F.1, Liu S. Y.1, Haung T. W.1, Tseng F. G.1, Chen F. R.1
1Engineering and System Science Department/National Tsing Hua University, Hsinchu, Taiwan
sgps51238@yahoo.com.tw

Compare with optical microscope (OM), electron microscope (EM) has the advantage with higher resolution. Therefore, when Hans Busch developed the first electromagnetic lens in 1926 [1], the technique of electron microscope has attracted increasing attention. The first commercial transmission electron microscope (TEM) were produced in 1939 by Siemens. [2] In today’s development, EM is important for the use of observing smaller sample which like the microstructure of the materials, the device in MEMS manufacturing and microbiological analysis. On the other hand, since the rise of environmental awareness, green energy become more and more concerned recently and solar energy is one of the candidates. As a result, a growing number of researchers are studying on using catalysts to transfer light into chemical energy. However, the efficiency of commercial solar energy devices still lower than 30 %, we thick it may be limited by the comprehension and observation of reaction mechanisms. Due to the importance of solar energy, we designed an experiment combined the observation of light-induced reaction and electron microscope.
In our experiment, two kinds of light-induced system using in electron microscope are produced. They are named “LED-based system” and “fiber-based system”. Figure 1(a) and (b) showed the”LED-based system” and “fiber-based system” respectively. The ”LED-based system” contains a light-emission diode (LED) put under the sample holder and controlled by a power supply. This kind of light-induced system is used in Hitachi TM-1000. Another “fiber-based system” includes a UV light source with optical fiber to induce UV light through the EDS hole for JEM-2010. Then we use the wet-cell sealing technology [3] to fabricate the wet sample containing TiO2, H2O, CH3OH and H2PtCl6 solution. Finally, we successfully observe the in-situ light-induced deposition reaction in EM. Figure 2 showed the deposition process in scanning electron microscopy (SEM). When light Illuminate about 3 minutes, the Pt particles began to emerge in the upper right corner.

Reference:
[1] Mathys, Daniel, Zentrum für Mikroskopie, University of Basel: Die Entwicklung der Elektronenmikroskopie vom Bild über die Analyse zum Nanolabor, p. 8
[2] "James Hillier". Inventor of the Week: Archive. 2003-05-01. Retrieved 2010-01-31.
[3] Tsu-Wei Huang, Shih-Yi Liu, Yun-Ju Chuang, Hsin-Yi Hsieh, Chun-Ying Tsai, Yun-Tzu Huang, Utkur Mirsaidov, Paul Matsudaira, Fan-Gang Tseng, Chia-Shen Chang and Fu-Rong Chen, Lab Chip, 2012,12, 340-347.

Fig. 1: Figure 1 Two light-induced system development which named (a) the”LED-based system”, and (b) the“fiber-based system”.

Fig. 2: Figure 2 The deposition process in scanning electron microscopy (SEM).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-5777 ThunderSTORM: a comprehensive ImageJ plugin for PALM and STORM data analysis and super-resolution imaging

Křížek P.1, Ovesný M.1, Borkovec J.1, Švindrych Z.1, Hagen G. M.1
1Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague
pavel.krizek@lf1.cuni.cz

We present ThunderSTORM, an open-source, interactive, and modular software designed for automated processing, analysis, and visualization of data acquired by single molecule localization microscopy (SMLM) methods including STORM, dSTORM, SPDM, PALM, and FPALM. ThunderSTORM was developed using a home-built SMLM system, but the software has been tested, and works well with data acquired using commercially available Nikon N-STORM and Zeiss Elyra systems. Our philosophy in developing ThunderSTORM has been to offer an extensive collection of processing and post-processing methods which were developed based on extensive testing with both real and simulated data. We also provide a very detailed description of the implemented methods and algorithms as well as a detailed user’s guide. ThunderSTORM is written in Java and distributed as a plug-in for ImageJ. This enables users to run the software on computers with different operating systems, and to use all of the advantages of ImageJ including its rich collection of plug-ins. The latest version of ThunderSTORM and the source code are freely available at https://code.google.com/p/thunder-storm/.


ThunderSTORM can process data for both 2D and 3D SMLM imaging, including data with high spatial molecular density, which is known as the “crowded field" problem. The steps involved in SMLM data processing are shown in Figure 1. Several algorithms for each of the processing steps have been implemented so experienced users have many options to adapt the processing to their data. However, the default settings perform very well on many of the SMLM data sets we have experimented with.


ThunderSTORM is also capable of generating simulated SMLM data and of evaluation of the performance of localization algorithms based on the ground-truth positions of the molecules. This allows users to perform Monte Carlo simulations and to quantitatively evaluate the performance of the applied algorithms. Localization results, as well as the ground-truth positions of molecules, can be imported/exported to/from ThunderSTORM in a variety of data formats, allowing compatibility with other SMLM localization software.


We also present our preliminary data of replication and transcription processes in the cell nucleus of HeLa cells, see Figure 2. Our labeling strategy results in brightly stained cells revealing two distinct nuclear activities. Investigating how these processes are organized within the cell nucleus relates to the larger issue of understanding how DNA replication is regulated on a cellular level. The super-resolution data were processed by ThunderSTORM.

This work was supported by the Czech Science Foundation [P304/09/1047, P205/12/P392, P302/12/G157, 14-15272P]; by European Union Funds for Regional Development [OPPK CZ.2.16/3.1.00/24010]; and by Charles University in Prague [Prvouk/1LF/1, UNCE 204022].

Fig. 1: Data processing pipeline for single molecule super-resolution imaging.

Fig. 2: Replication and transcription, detail of nucleolus. HT-1080 cells labeled with EdU-Alexa647 (a fluorescently tagged nucleotide that incorporates into newly replicated DNA, red) and fluorouridine (a synthetic nucleotide which is incorporated into active transcription sites, green).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-5793 Nonlinear   optical single-molecular image technique

wang X. M.1
1Hubei University of Chinese medicine
foxglove@163.com

Nonlinear optical single-molecular image technique is a new technique which is not widely recognized in scientific community. It is our patent technique( Chinese patent 200910060951.7， PCT /CN2010/000138 ). It is a new innovative principle to get a profile image of tiny material noninvasively. by a series of lenses which diameter from small to large, adjusting each lens move back and forward carefully along a straight line ,the two different direction rays from the same point of the out edge of objects can focus on a plate to form an image. This technique can magnify profile images of small samples. Its x-y resolution breakthrough the limit of Abbe’s diffraction law.It can get single molecular image in water. It can be used to trace the trajectory of single molecule in living cell. The principle of technique will be broad application in many areas in near future. In this paper, we gave more supporting evidences that Nonlinear optical single-molecular image technique is a practical tool. The photos of our experimental results demonstrated that the principle of Nonlinear optical single-molecular image technique is correct. The photos showed the double helix structure of DNA extracted from chicken egg. This technique will bring human a lot of knowledge and information about molecules, especially about biological macromolecules in living cell. It will improve drug research and human understand micro world. With computer image reconstruct technique , Nonlinear optical single-molecular image technique will become more powerful tool for scientific research.

Fig. 1: single suger molecular image in double-distilled water

Fig. 2: starch chain  molecular image

Fig. 3: DNA double helix image

Fig. 4: our equitment of nonlinear single molecular image
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-5941 Development of the pump-probe nanoscopy architecture.

Korobchevskaya K.1, Bianchini P.1, Scotto M.1, Sheppard C.1, Diaspro A.1
1Nanophysics, Istituto Italiano di Tecnologia, 16136 Genova, Italy
ksenia.korobchevskaya@iit.it

Modern super-resolution microscopy techniques can provide high quality images with sub-diffraction resolution. However, a significant limitation for most of the methods is the use of fluorescence as readout, which results in photo-bleaching, and reduction of penetration depth, and significantly complicates the deep tissue imaging process due to strong scattering inside the sample. These issues can be avoided by using infra-red (IR) vibrational spectroscopy, but in that case, the image resolution is very low, due to the diffraction limit. As a new solution, the infrared absorption microscopy method was recently proposed [1, 2].

Strictly speaking the IR absorption microscopy method combines two well known techniques – transient absorption spectroscopy and stimulated emission depletion microscopy (STED) [3, 4]. In particular, it is based on a saturation effect, where the first laser pulse creates contrast within the sample, and the second pulse, at different frequency, detects the change. This allows label free transient images to be obtained. By introducing an additional doughnut-shaped depletion pulse, the excited area can be transiently saturated in the periphery of the focal spot, allowing collection of a signal from the central sub-diffraction area. By choosing the pumping and probing wavelengths, we can image different non-fluorescent species. The possibility to use IR light for sub-diffraction imaging is especially vital for deep tissue imaging, because the IR spectrum lies in the transparency window of biological tissues.


To verify the concept, we designed and assembled prototype of such a system presented in this work. The microscope combines two home-built setups – a pump-probe spectroscope and STED nanoscope. This configuration allows achieving high temporal and spatial resolution in the very same instrument. The setup is based on a femtosecond laser coupled with an OPA, which can generate laser pulses in a broad spectral range from visible to near IR, according to experimental needs. Therefore, explicit spatial and dynamical information about the sample can be obtained. This is very useful for cell biophysics and nanochemistry applications. We also present the possibility of implementing a 3D super-resolution capability.

The research leading to these results has received funding from the European Community’s Seventh Framework Programme: LANIR (FP7/20012-2015) under grant agreement n⁰ 280804.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-3-P-5986 Inherently coaligned dual color STED microscopy

Göttfert F.1, D'Este E.1, Lukinavičius G.2, Hell S. W.1
1Max-Planck-Institute for Biophysical Chemistry, Göttingen, 2Ecole Polytechnique Fédérale de Lausanne, Lausanne
fabian.goettfert@mpibpc.mpg.de

Stimulated Emission Depletion (STED) Microscopy provides a versatile tool for investigating cellular structures on the nanoscale: While preserving the advantages of fluorescence microscopy, such as easy sample preparation and live cell imaging, the resolution is not limited by diffraction.


Here, we demonstrate the capabilities of STED microscopy for protein mapping and colocalization analysis. Applying a two color, pulse interleaved excitation and detection scheme combined with a single STED beam, two spectrally distinct dyes can be imaged almost simultaneously at a resolution down to 20nm. As the fluorescent volume in the sample is defined by the STED laser, the described setup provides a colocalization accuracy well below its resolution, insensitive to moderate misalignment and drift.


We imaged various proteins of the nuclear pore complex (Figure 1). Following standard immunolabeling protocols the staining was done with primary antibodies targeting the protein and secondary antibodies, tagged with the dye, targeting the primary antibody. Although the spectra of the dyes used for two-color imaging are overlapping, the crosstalk between the channels is below 20% and no post processing is required. These properties open the possibility to map even closely neighboring proteins, as exemplified on gp210 and NUP133.


Until recently, the lack of a suitable dye in the red wavelength region confined live cell STED imaging to wavelengths below 600nm. Long wavelengths however have the tendency to be less phototoxic and reduce background by autofluorescence. Here, we demonstrate live cell imaging with a newly developed silicon-rhodamine dye. Using the STED system described above we achieve a resolution better than 40nm in living cells, imaging microtubules and actin (Figure 2).

Fig. 1: STED image of nuclear pores in Xenopus cells. The protein gp210 (red) is arranged symmetrically around the central pore channel. As the diameter of the ring is approximately 160nm, the structure would not be resolvable with conventional fluorescence microscopy. In green, various FG repeat nucleoporins were labeled with a pan-specific antibody.

Fig. 2: Live rat primary hippocampal neurons stained with the recently developed fluorogenic dye SiR-actin. Actin in axons can arrange in rings with a spacing of 180nm. The used silicon-rhodamine fluorophore is cell permeable and minimally toxic to the cell. Its high photostability makes it a versatile probe for superresolution microscopy.
↑ Go to the Table of content ↑

IT-4. Scanning electron microscopy

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-4-IN-1844 Ultra-low-energy STEM in SEM

Frank L.1, Nebesářová J.2, Müllerová I.1
1Institute of Scientific Instruments, Brno, Czech Republic, 2Biology Centre, České Budějovice, Czech Republic
ludek@isibrno.cz

Examination of thin samples in TEM or STEM has been performed at hundreds of keV. This energy range offered high resolution but low contrasts which meant that tissue sections had to be contrasted with heavy metal salts. Recent TEM with aberration correctors preserve an acceptable resolution down to 20 keV and provide enhanced contrasts [1]. The LVTEM device is operated at 5 keV on samples thinner than 20 nm [2]. STEM attachments to SEMs have become widespread [3] profiting from an image contrast substantially increasing even for light elements at tens or units of keV. The methods for the preparation of ultrathin sections of various substances are capable of producing layers at and even below 10 nm [4,5] which enables one to further decrease the energy of the electrons provided the image resolution is maintained. When using the STEM technique virtually all transmitted electrons can be utilised for imaging, while in TEM we are restricted to using electrons capable of forming the final image at acceptable quality. This forces us to narrow the ranges of the angular and energy spreads of electrons that enter the image-forming lenses. Consequently, the STEM technique promises higher contrasts at comparable resolutions. Unlimited reduction of the energy of the illuminating electrons is possible by employing the cathode lens principle [6]. This consists of biasing the sample together with its holder (made flat on both sides) to a high negative potential that retards the incident electrons before they land on the sample surface and accelerates backscattered and transmitted electrons to their respective detectors above and below the sample (Fig. 1). Calculations have shown a final spot size only moderately extended even at units of eV so that quality-consistent micrographs can be recorded over the full energy scale [7].

Ultra-low-energy STEM at hundreds of eV can be successfully applied to the examination of ultrathin tissue sections free of any heavy metal salts (Fig. 2) [8] or to 2D crystals. Single atomic steps are revealed at high contrast on multilayer graphene samples and transmittance of electrons at tens or units of eV can serve as a tool for “counting” the graphene layers (Fig. 3).

[1] Kaiser, U. et al., Ultramicroscopy 111 (2011) 1239.

[2] Drummy, L.F., Yang, J., Martin, D.C., Ultramicroscopy 99 (2004) 247.

[3] Morandi, V., Merli, P.G., Journal of Applied Physics 101 (2007) 114917.

[4] Riedl, T. et al., Microscopy Research and Technique 75 (2012) 711.

[5] Nebesářová, J., Vancová, M., Proceedings of IMC16, Sapporo 2006, Vol. 1, 500.

[6] Müllerová, I., Frank, L., Advances in Imaging and Electron Physics 128 (2003) 309.

[7] Müllerová, I., Hovorka, M., Frank L., Ultramicroscopy 119 (2012) 78.

[8] Frank, L. et al., Ultramicroscopy, submitted.

Support by the Technology Agency of the Czech Republic under no. TE01020118 and the institutional support RVO:68081731 are acknowledged.

Fig. 1: Trajectories of signal electrons toward transmitted (TE) and backscattered (BSE) electron detectors and through-the-lens detector (TLD) with the specimen immersed in the field of the open objective lens (a), with the biased sample retarding the beam 11 times (b), and with a combination of both (c).

Fig. 2: Section of mouse heart muscle, free of osmium tetroxide post-fixation and any staining, estimated thickness 5 nm, micrograph taken at 500 eV (a), electron energy dependence of the average edge resolution (b), and electron energy dependence of the relative variance contrast (c).

Fig. 3: Commercial CVD multilayer graphene imaged at 220 eV (a), total transmittance of extremely slow electrons through varying number of graphene layers (b).
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-4-IN-5709 A review of SE and BSE imaging in SEM and Variable Pressure SEM

Griffin B. J.1, Joy D. C.2, Michael J. R.3, Gauvin R.4
1Centres for Forensic Science, and for Microscopy, Characterisation and Analysis, The University of Western Australia, Perth, Australia , 2Centre for Nanophase Materials Science, Oak Ridge National Laboratory, Oak Ridge, USA, 3Sandia National Laboratories, PO Box 5800, Albuquerque, USA, 4Department of Materials Engineering, McGill University, Montreal, Quebec, Canada
brendan.griffin@uwa.edu.au

The SEM has evolved to a complex platform with multiple in-column and chamber-mounted SE and BSE detectors. With monochromated electron sources and biased stages the SEM can routinely provide sub-nanometre (0.4-7 nm) SE images and high voltages (15-30 kV), and in some cases low voltages (1 kV), on suitable samples. Live FFT analysis also now available on some instruments and it is particularly useful for high resolution operation. Another interesting and important development is the ability to collect images from the different detectors simultaneously, allowing consideration of the full range of sample information [figure 1], a consequence of higher resolution digital displays. The information content of the SE image is also much better understood, the in-column SED avoiding the well-documented but often forgotten “swamping” of the SE1 by SE2, SE3 and BSE through filtering and physical placement. Figure 1 also provides one example where surface contamination is most evident in the image from the in-column SED relative those from the BSED images and chamber-mounted SED image, even at high accelerating voltage (20 kV) and with C-coating. Stage tilt, reflecting angular selection, remains useful to enhance surface effects (such as relief in polished samples [figure 1b]). Electrostatic imaging of the chamber is a useful tool to illustrate the contribution of BSE generating SE3 on the pole piece and chamber walls in the vicinity of the E-T SED [figure 2b]. The angular sensitivity of BSE imaging has also been explored recently, particularly through the research of the late Heiner Jaksch in exploring the low angle BSE signals with ‘unconventionally’ short working distances. Two technologies have emerged, the use of in-column BSE detectors and selectable, segmented annular BSED. For example, the latter approach using a ‘tiled’, annularly-segmented single crystal Si diode detector [figure 2a] allows rapid switching through a range of collection angles, depending on the sample working distance. SE images in the variable pressure mode of SEM operation are strongly filtered by the positive ion cloud near and above the sample surface. The consequence is that such images lose the high resolution, near-surface component of the emitted SE. The collected signal is consequently dominated by higher energy SE and BSE and the deeper, delocalised ‘material’ contrast from the sample, even at the lower beam energies. It is currently difficult to envisage conditions in variable pressure mode where SE images will be directly comparable to those collected in high vacuum mode. The presence of a thin metal coating will assist. This filtering is not present where localized gas leakage is used for surface charge cancellation. 

FEI, TESCAN, Hitachi HT, and JEOL have all generously provided time on instruments.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-4-O-1411 Influence of the work-function changes on the contrast of images in SEM.

Cazaux J.1, Sato K.2, Kuwano M.3, Ikatura N.4
1Physics Department, Faculty of Sciences, BP 1039, 51687 Reims Cedex 2, France, 2. JFE Steel Corp., 1 Kawasaki-cho, Chuo-ku Chiba Japan, 3MJIIT, Universiti Teknologi Malaysia, Jalan Semarak, 54100 Kuala Lumpur, Malaysi, 4Kyushu-University, Kasuga, Fukuoka 816-8580, Japan
jacques.cazaux@orange.fr

In the present contribution the role of the local change of the work function on some contrasts in SEM is suggested and illustrated. When electrons, BSEs or SEs, escape from the sample they are partly refracted at the sample/vacuum interface. The refraction effect is given by [1]: √ES sin β= √Ek sin α (1) with ES: inner kinetic energy (referred to the bottom of the conduction band); β: inner incident angle to the normal; Ek: kinetic energy into the vacuum (referred to the vacuum level); α: emission angle into the vacuum. The relationship between ES and Ek obeys to ES = Ek+EF+φ (2a) for metals of  work function φ and  Fermi energy EF and to ES = Ek+χ (2b) for semiconductors or insulators of affinity χ. In addition, the transmission probability of the escaping electrons, T(α), differs from 100%. Then a local change of φ or χ with the crystalline orientation or oxidation or contamination will change the SE or BSE yields δ or η.

Fig. 1a shows the calculated change of T(α) when χ changes from 4.05 eV (Si; Ge; SiC) to 4.55 eV for SEs of an initial inner kinetic energy of ES= 5.05 eV. Fig. 1b shows the corresponding distortion of the spectral distribution of the emitted SEs, ∂δ/∂Ek, when χ changes by steps of 0.2 eV and the corresponding change in δ, from 100% to 69%, is indicated in caption. Performed for the calculated dependence of few-layer graphene on SiC [1] this type of evaluation applies also  to contamination effects on synthetic diamond:Fig. 2a.

Fig. 2b shows the contrast of dendritic SiGe crystals embedded in a SiGe amorphous matrix[2]. Such a contrast may be explained from the local change of χ between the two crystalline forms of SiGe.

The same analysis applies to the angular selective detection of the BSEs where the refraction effects increase with the detection angle α –Eq. (1)-.This point should be considered for the interpretation of Fig. 3 for two different Fe grains, A and B [3].

The same analysis may be transposed to the reflectivity of Very Low Energy Electrons, R(α)=1-T(α) [4] , a reflectivity changing rapidly when the incident beam energy, E°, varies from 1 to 10 eV: a point fairly illustrated by Frank et al.[5].

In conclusion, some material and crystalline contrasts reported in SEM using SE or BSE detection as well as in LVSEM may be explained from the local change of the work function or the electron affinity.

1. J. Cazaux Appl. Phys. Lett. 98 (2011) p 013109 1

2. M. Itakura, N. Kuwano, K. Sato and S. Tachibana, Journal of Electron Microscopy 5 (2010) pS165

3. J. Cazaux , N. Kuwano and K. Sato, Ultramicroscopy, 135 (2013) 43

4. J. Cazaux J. Appl. Phys. 111 (2012) DOI: .1063/1.3691956

5. L. Frank, S. Mikmekova, M. Hovarka, Z Pokorna and I. Müllerova; Proceed. 15 th European Microscopy Congress, Manchester, (2012) PS2.2

Fig. 1:  a: Refraction effects of SE’s at vacuum/sample interface. b: Distortion of the spectral distribution of the emitted SE’s as a fonction of the change of χ from 4.05 to 4.65 eV [1].

Fig. 2:  a: Contamination contrast on diamond. b: SE Crystalline contrast of SiGe[2].

Fig. 3: BSE Crystalline contrast for E°=5 keV [3]. From top left to bottom right, the angle of detection with respect to the normal, α, changes: 58.5°±4.5; 46°±5; 35°±5; 14.5°±5; 0°.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-4-O-1437 Advancements in Integrated Micro-XRF in the SEM

Witherspoon K. C.1, Cross B. J.2, Lamb R. D.1, Sjoman P. O.1, Hellested M. D.1
1IXRF Systems, Inc., 3019 Alvin De Vane Blvd, Suite 130, Austin, Texas, 78741, USA, 2CrossRoads Scientific, P.O. Box 1823, El Granada, CA 94018
mandih@ixrfsystems.com

In recent years, small x-ray tubes have been modified for mounting on Scanning Electron Microscopes. There have been two main types: low-power miniature tubes mounted re-entrantly within the SEM [1], and higher-power tubes with integrated x-ray optics to produce smaller beam spots at the sample, yet with intensities high enough for routine analytical work [1,2]. This addition allows samples to be analyzed both by X-Ray Fluorescence (XRF), and by the electron beam (SEM-EDS), as illustrated by the two spectra in FIG. 1.

Both techniques can be used independently or together by taking sequential e-beam and x-ray excited spectra. Quantitative analysis using this combined approach was first shown at the IMC16 conference in Sapporo [3]. This approach uses the advantage of e-beam excitation for lighter elements below 2.0 keV, and the more-efficient XRF excitation for x-ray lines above 2.0 keV. Micro-XRF with X-Y stage scanning can be used to collect x-ray elemental maps similar to those collected with e-beams, except the stage is moved versus scanning of the beam. This Micro-XRF mapping method has been proposed for some time [e.g.4], and was first commercially demonstrated in 1986 [5]. It is possible to collect e-beam and x-ray excited maps simultaneously for combined qualitative x-ray elemental mapping.

Currently 40µm and 10µm x-ray beam spot sizes are available inside the SEM. The 10µm beam has shown count rates that exceed 2000cps on steel. Future expectations are of even smaller excitation areas, with “useful” x-ray count rates. To create a smaller spot the polycapillary optic needs to be more tightly focussed. This means that the Focal (working) Distance (FD) of the XRF source must be shorter. For example, for a 40µm spot, an FD of 11 mm is typical. With a 10µm excitation spot, an FD of about 4.5 mm is required, making the integration of the x-ray beam a bit more of a challenge (FIG. 2).

It is now possible to use primary filters (thin foils) in front of the x-ray source. Using an automated filter wheel, allows in situ tuning of the x-ray source spectrum [e.g. 4], with improved elemental detection limits. An automated filter wheel between the x-ray source and sample provides comparable capabilities to those in benchtop XRF. FIG. 3 shows a comparison of unfiltered and filtered spectra, showing how the overall “shape” can be varied to optimize sensitivities and peak-to-background ratios.

Fig. 1: EDS (top) and Micro-XRF (bottom) spectra of NIST SRM 610

Fig. 2: Illustrates Focal Point and Working Distance

Fig. 3: Filtered Micro-XRF in SEM Spectrum
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-4-O-1674 Innovation possibilities of scintillation electron detector for SEM

Schauer P.1, Bok J.1
1Institute of Scientific Instruments of the AS CR, v.v.i., Brno, Czech Republic
petr@isibrno.cz

To evaluate performance of a scintillation detection system for SEM, it is necessary to consider many scintillator parameters. Various attributes of the scintillator for the SEM electron detector are listed in Fig. 1. The very important parameters are those affecting the detective quantum efficiency (DQE) which is primarily a measure of image noise. Not a less important indicator of image quality is the modulation transfer function (MTF) which describes the ability to show fine image details. Therefore, using a scanning imaging system, the detector bandwidth, which is given especially by the scintillator decay time, is the key to the good MTF. Currently, the YAG:Ce single crystal scintillator (introduced already in 1978 [1]) having somewhat limiting decay characteristic is the most frequently used scintillator in the SEM. The aim of this paper is to outline possibilities of scintillator innovation to get the improved MTF and DQE.

A database containing scintillation properties of various materials excited by hard x-rays and/or g-rays, taken from the literature, was established and is maintained at our laboratory. Among collected scintillators is only very limited selection of those that meet requirements for the SEM scintillation detector. For example, all hygroscopic materials must be excluded. Excluded must be also materials that have a low light yield and/or high luminescence decay. Thus the only suitable scintillators are those based on Ce-activated oxides characterized by a very fast 4f-5d emission as selected in Fig. 2.

Current research carried out in our laboratory tries to get faster scintillators by applying substitution of Y and/or Al in the garnet structure on the one hand and by increasing Ce-activator concentration on the other hand. Unfortunately, the Ce concentration increase is not an easy task for the Czochralski grown single crystals because of a sharp decrease of the distribution coefficient at crystal growth. But the development of optical ceramics is promising technology to get a more activated scintillator [2]. Our recent research includes the cathodoluminescence (CL) study of the commercial single crystal scintillators such as CRYTUR CRY18 and CRY19 as well as promising multicomponent garnet films grown by liquid phase epitaxy, for example GdGaLuYAG:Ce (formula (Gd,Lu,Y)3(Al,Ga)5O12:Ce3+). The new studied scintillators are quite fast as shown in Fig. 3. Their CL emission spectra show acceptable PMT matching as seen in Fig. 4.

References

[1] Autrata R., Schauer P., Kvapil Jos., Kvapil Ji.: A single crystal of YAG:Ce - new fast scintillator in SEM., J. Phys E: Sci. Instrum., 11 (1978), 707.
[2] Miyata T., Iwata T., Nakayama S. and Araki T., Meas. Sci. Technol. 23 (2012), Article No: 035501, DOI: 10.1088/0957-0233/23/3/035501.

The authors thank CRYTUR comp. for the supply with single crystal scintillators. They also thank Charles University, Faculty of Math. & Phys., for the supply with film scintillators. The work was supported by the Technology Agency of the Czech Republic (TE01020118). It was also supported by the European Commission and Ministry of Education, Youth, and Sports of the Czech Republic (EE.2.3.20.0103).

Fig. 1: Influence of various scintillator attributes on the choice of the best scintillator for the SEM electron detector.

Fig. 2: Compilation of x-ray and/or g-ray excited rare-earth activated oxides having the light yield ≥ 10 photons/keV and the decay time (τ1/e) ≤ 100 ns.

Fig. 3: CL intensity decay characteristics of the new scintillators: CRY18 single crystal and GdGaLuYAG:Ce garnet film. For comparison the decay of the improved YAP:Ce single crystal scintillator is also shown.

Fig. 4: Normalized CL intensity spectra of the new scintillators: CRY18 single crystal and GdGaLuYAG:Ce garnet film. For comparison the spectrum of the improved YAP:Ce single crystal scintillator is also shown.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-4-O-1791 Quantitative interpretation for angle selective backscattering image of iron oxide on steel

Aoyama T.1, Nagoshi M.2, Sato K.2
1JFE Steel Corporation, Fukuyama, Japan, 2JFE Steel Corporation, Chiba, Japan
to-aoyama@jfe-steel.co.jp

The contrasts in backscattered electron (BSE) images were studied from the cross section of a heat-treated steel sheet using a scanning electron microscope (SEM) equipped with a conventional annular BSE detector (Σigma, Carl Zeiss NTS GmbH). The specimen used was heat-treated low carbon steel with an oxide layer mainly composed of magnetite (Fe3O4). A cross-sectional specimen was prepared by argon ion irradiation (IB-09010CP, JEOL Ltd.) after polishing with diamond suspension. BSE images were observed at primary electron energies (Eps) of 2 keV, 5 keV, 10 keV and 15 keV at various working distance from 2 to 15 mm for an identical area of the specimen (cross section). The take-off angles (θ; measured from the specimen surface) of the detector were estimated to be 35-45°, 39-53°, 50-63°, 66-75° and 73-79° (except 2 keV) from the geometry of the detector and the specimen. The variation of BSE intensities between crystal grains was calculated from the images. According to the results, high Ep enhances bulk information and Z contrast, whereas low Ep improves surface information and channeling contrast. High θ also enhances bulk information and Z contrast, whereas low θ improves surface information and channeling contrast. In the case of the lowest θ, topographic information was enhanced by shadowing effect on BSEs, in addition to the amplification of channeling contrast. These results regarding channeling contrast and Z contrast can be understood by the ratio of low-loss electrons (LLEs) to the inelastic BSE components detected; LLEs contribute to channeling contrast, and their ratio increases with decreasing Ep and θ. The systematic results obtained in this study are useful for controlling SEM conditions in order to select Z and crystallographic information separately in BSE images for practical materials of interest.

Authors appreciate Dr. Š. Mikmeková of JFE Steel Corporation for her detailed advice on quantitative analyses of the image contrasts. And we would also like to thank Mr. M. Yamashita and Ms. K. Takase of JFE Steel Corporation for their technical supports.

Fig. 1: Schematic diagram showing dependencies of the BSE contrast on the θ and Ep. The areas where channeling contrast and Z contrast are enhanced in the BSE images are indicated by shaded and unshaded areas, respectively. The area where topographic information and channeling contrast are enhanced is indicated by dotted area.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1456 Photonic Crystal Structure of Butterfly Wing Scales Exhibiting Selective Wavelength Iridescence

Matějková-Plšková J.1, Mika F.1, Jiwajinda S.2, Dechkrong P.2, Svidenská S.3, Shiojiri M.4
1Institute of Scientific Instruments of the ASCR, v.v.i., Královopolská 147, Brno 612 64, Czech Republic, 2Bioresources and Biodiversity Section, Central Laboratory and Greenhouse Complex, Kasetsart University, Kamphaengsaen Campus, Nakhonpathom 73140, Thailand, 3Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Albertov 4, 12801 Praha 2, Czech Republic, 4Professor Emeritus of Kyoto Institute of Technology, 1-297 Wakiyama, Kyoto 618-0091, Japan.
filip.mika@isibrno.cz

Characteristic patterns and the vivid coloration of the wing scales of butterflies have lately attracted considerable attention as natural photonic crystals. The coloration of butterflies that exhibit human visible iridescence from violet to green has been elucidated. A Sasakia charonda (S. charonda) or ‘great purple emperor’ butterfly (Fig. 1a) was sampled in a woodland in Japan, and an Euploea mulciber (E. mulciber) or 'striped blue crow’ butterfly (Fig. 1b) was reared from an egg at the Environmental Entomology Research and Development Center, Kasetsart University. SEM observations, with the aid of the optical reflectance measurement, revealed that highly tilted multilayers of cuticle on the ridges in their iridescent scales (Figs.1e-1h and Fig. 2a-h) cause a dark zone where no reflection occurs (Fig. 2i)1-3 and produce a limited-view, selective wavelength iridescence (ultraviolet (UV)~green) as a result of multiple interference between the cuticle-air layers (Fig. 2j).3,4 TEM observation of S. charonda’s iridescent scales, sectioned with an ultramicrotome confirmed these results (Fig.2j and 2k). The iridescence from Chrysozephyrus ataxus (C. ataxus) or Thermozephyrus ataxus butterflies (Fig. 1c), which were sampled in Japan, originates from multilayers in the groove plates between the ridges and ribs (Fig. 3a-3f).3,5 The interference takes place between the top and bottom surfaces of each layer and incoherently between different layers. Consequently, the male with the layers that are ~270 nm thick reflects light of UV~560 nm (green) and the female with the layers that are ~191 nm thick reflects light of UV~400 nm (violet). A Troides aeacus (T. aeacus) or ‘golden birdwing’ butterfly (Fig. 1d) also grew in Kasetsart University, The butterfly does not produce any iridescent sheen which Troides magellanus does.3,4 No iridescent sheen is ascribed to microrib layers, which are perpendicular to the scale plane (Fig. 3g-3j), so that they cannot reflect any backscattering. The structures of these butterflies would provide us helpful hints to manipulate light in photoelectric devices, such as blue or UV LEDs.

1J. Matějková-Plšková et al., J. Micros. 236, (2009) 88.

2J. Matějková-Plšková et al., Mater. Trans. 51, (2010) 202.

3F. Mika et al., Materials 5, (2012) 754.

4P. Dechkrong et al., J. Struct. Biol. 176, (2011) 75.

5J. Matějková-Plšková et al., Mater. Trans. 52, (2011) 297.

Present address of J. Matějková-Plšková: Sadovského 14, Brno 612 00, Czech Republic.

Presenting author acknowledges the support from MEYS CR (LO1212) together with EC (ALISI No. CZ.1.05/2.1.00/01.0017).

Fig. 1: (a) S. charonda. (b) E. mulciber. (c) C. ataxus. (d) T. aeacus. (e-g) SEM images of iridescent white scales of the S. charonda. (h) Multiple cuticle-air gap arrangement on the rides of the scales.

Fig. 2: OM (a) and SEM images of iridescent white and blue scales of the E. mulciber (b-h). (i) Dark zone appearing on the S. charonda wing. (j) Iridescent reflection. TEM images of cross- sections of white (k) and blue scales of the S. charonda (l).

Fig. 3: (a-e) SEM images of iridescent scales of the male C. ataxus. (f) Rides and groves. (g-i) SEM images of yellow scales of the T. aeacus, which has not cuticle layers on the ridges but has microribs on the side of ridges. The microribs normal to the wing plane do not cause any backreflection as indicated by blue arrows.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1423 Development of high-efficiency DF-STEM detector

Kaneko T.1, Saitow A.1, Fujino T.1, Okunishi E.1, Sawada H.1
1JEOL Ltd. 1-2 Musashino 3-Chome, Akishima, Tokyo 196-8558, Japan
takekane@jeol.co.jp

Most recently, observations at low accelerating voltages have been increasingly popular for carbon-based materials such as carbon nanotubes or graphenes, to reduce knock-on damage due to irradiation of an electron beam. High-resolution dark-field (DF) imaging in a scanning transmission electron microscope (STEM) has been widely used for structural analysis in materials science. In conventional system, a STEM signal is detected as light intensity emitted from a scintillator which is hit by electrons. The conventional detector showed low signal conversion efficiency from an electron to a STEM image signal at low accelerating voltages, since the STEM detector is optimized for high energy electrons. Thus, a detector with good efficiency from low to high accelerating voltages is sought after. The STEM detector is consisted of a scintillator, a glass light guide and a photomultiplier tube. Many materials for scintillator were tested to improve the efficiency. The scintillator of the STEM detector is selectable either a powder scintillator or a single crystal scintillator. However, the good efficiencies from low to high voltages were not found yet so far. We measured the efficiency for powder and single crystal scintillators whose chemical compositions were the same, depending on the accelerating voltages. The measured results showed that the scintillation efficiency for the single crystal becomes higher than that of powder at accelerating voltage greater than 100 kV. Combining these features, we have developed a hybrid type scintillator, which consisted of powder deposited layer and a single crystal substrate. The luminescent quantum efficiency of the hybrid scintillator was measured to be twice as large as that of the single crystal at 60 kV and was about 8 times higher than that of the powder at 300 kV, and covers the observation at the accelerating voltages from low to high voltages. Especially, it is useful for low voltage observations of carbon-based materials consisted of few atomic layers that produces weak scattering of electron.

This work was supported by JST under the Research Accelerating Program (2012–2016).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1467 Energy Low Loss Backscattered Electrons Imaging in Material Characterization and Analysis

Liu X.1
1Carl Zeiss Microscopy GmbH
xiong.liu@zeiss.com

With the continuous size and structure shrinkage in semiconductor and electronic devices, the final performance and properties of the materials are dominated by the surface and interface layers. This requires scanning electron microscope (SEM) as a most conventional technical method in material characterization and analysis not only to be able to visualize and image such nanostructures with the secondary electron imaging under a low energy beam but also to analyze the tiny compositional differences like doping contrast, oxidation states of elements, small phases of hybrids or function group in polymers etc., which are not available via the classical backscattered electron imaging or other Energy-dispersive X-ray spectroscopy methods. Although the classical backscattered electron (BSE) imaging are from the multiple inelastic scattering process which could provide density related contrast like channeling contrast at high energy beam, the backscattering coefficient shows non-linear behavior and get very complicated.
In the classical backscattering process (Rutherford scattering), the backscattered electrons are mainly from the scattering of the high energy primary electrons with the nucleus charge or inner electron shells of the material. In such a case the contrast or brightness of the BSE imaging scale with material density, atomic number (Z). However the scattering between the primary beam with the outer electron shells of the materials at low impact energy (below 3 kV) region is not any more negligible which even becomes more dominant where the surface plasma resonance and ionization loss could happen and contribute to in the total contrast mechanism.
The unique design of the Gemini® lens integrated with a beam booster in the beam path not only maintains the brightness of the downward primary electron beam at low energies but also has a dispersion function for the generated reverse electron signals backward into the column. It means that the secondary electrons and backscattered electrons with a small energy and angle differences could be amplified and separated by the Gemini® lens in real time and space without converting the signals or by applying any additional stage bias. The separated backscattered electrons could be further filtered with an energy filtering grid and projected back into the corresponding detector. Backscattered electrons with a specific energy low loss could be picked out for imaging by setting an appropriate threshold potential to the filtering grid. After the grid filter the multiple inelastic scattered electrons could be cut away and the signal is consisted of the so-called energy low loss backscattered electrons which reveals some characteristic resonance of the materials.

Fig. 1: The SE1 image (left) and corresponding LL-BSE image (right) of the Ceincorporated into mesoporous silica as catalyst where the Ce ions andnanoclusters give high brightness.

Fig. 2: The SE1 image (left) and corresponding LL-BSE image (right) of the ZnSxO1-x thin film on Al2O3 substrate where the LL-BSE image is from the low loss BSEs with an energy between 700 eV and 800 eV.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1503 Using an EBSD Detector as a Microstructural Imaging Device

Wright S. I.1, Nowell M. M.1, de Kloe R.3, Camus P. P.2
1EDAX, Draper, Utah, United States, 2EDAX, Mahwah, New Jersey, United States, 3EDAX, Tilburg, The Netherlands
stuart.wright@ametek.com

Electron Backscatter Diffraction (EBSD) has proven to be a useful tool for characterizing the crystallographic orientation aspects of microstructures at length scales ranging from tens of nanometers to millimetres in the the scanning electron microscope (SEM). With the advent of high-speed digital cameras for EBSD use, it has become practical to the EBSD detector as an imaging device similar to a backscatter (or forward-scatter) detector [1-3]. When the EBSD detector is used in this manner, images exhibiting topographic, atomic density and orientation contrast can be obtained at rates similar to slow scanning in the conventional SEM manner. The same high-tilt (~70°) sample geometry is used and the camera is binned considerably – to a 5x5 “super-pixel” image - in order to get extremely fast acquisition rates. At such high binning, the captured patterns are not suitable for indexing. However, no indexing is required to for using the detector as an imaging device. Rather, a 5x5 array of images is formed by essentially using each super-pixel as an individual scattered electron detector as shown in Figure 1. The images formed in this way can then be combined in a variety of ways to form composite images of the microstructure as shown in Figure 2. The flexibility to combine these images together allows different contrast mechanisms to be emphasized in the composite images. While images formed in this manner lack the quantitative nature of the maps formed by using EBSD in the traditional manner, they still provide a wealth of information that can be obtained at rates much faster than the quantitative EBSD maps and with much less EBSD expertise required by the operator.

References

[1] S. I. Wright & M. M. Nowell (2006) “Microstructure Characterization Using EBSD Image Quality Mapping”, Presentation at THERMEC, Vancouver, Canada.
[2] R. Schwarzer, J. Sukkau & J. Hjelen (2011) “Imaging of topography and phase distributions with an EBSD detector in the SEM”, Poster presentation at Microscopy Conference, Kiel, Germany.
[3] E. J. Payton, L. Agudo Jácome & G. Nolze (2013) “Phase Identification by Image Processing of EBSD Patterns” Presentation at Microscopy & Microanalysis, Indianapolis, USA.

Scott Lindeman of EDAX is gratefully acknowledged for his assitance.

Fig. 1: A five by five array of images formed using a heavily binned EBSD detector as an array of twenty-five individual scattered electron detectors from a Mylonite sample.

Fig. 2: A composite image formed by combining the individual images shown in Figure 1together in the manner shown in the accompanying schematic. The circular outline in the schematic shows the position of the phosphor screen relative to the 5x5 pixel array.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1551 A simple way to obtain BSE image in STEM

Tsuruta H.1, Tanaka S.2, Tanji T.2, Morita C.3
1Department of Electronics, Nagoya University, 2EcoTopia Science Institute, Nagoya University, 3Meijo University
s-tanaka@esi.nagoya-u.ac.jp

Backscattered electron (BSE) signal has been used to image small objects in a liquid phase. A thin film such as silicon nitride (SiN) film was used to seal the liquid solution, and imaging electron beam was incident through the film, and BSE signals were detected for imaging. Such a technique is usually based on SEM, thus the accelerating voltage available is up to about 30 kV. In this paper, we introduce a simple BSE detector that is easily incorporated into a scanning transmission electron microscope (STEM) sample holder, and present some results for BSE imaging using STEM electron beam up to 200 kV.

Fig. 1 is a schematic representation of our BSE detector. The BSE detector is consisted of p-type silicon (Si) and Schottky contact. A dimple was made from one side, and a through-hole with a diameter of about 200μm was created at the bottom of the dimple. A thin Schottky electrode was made on this side. On the other side, an ohmic electrode was made. TEM grids were used to hold particle objects, and the grid was placed just below the detector. This was conveniently done with a silver paste. Observation experiments were performed using Hitachi H-8000 STEM (accelerating voltage 75 - 200 kV). The beam current was about 1.5 nA.

We used two types of samples. One was latex (Φ90 nm) and Au (Φ60 nm) particles on a carbon film coated grid. The other was Au (Φ60 nm) particles confined between two SiN membrane window grids (fig.2). The Au particles of this sample were in air atmosphere.

Figs. 3(a) and 3(b) are dark-field (DF) STEM and BSE images, respectively, of the latex and Au sample taken at 75 kV. Both latex and Au particles are visible in the BSE image, and they are distinguishable according to their intensity. Au particles appear brighter than latex particle. The detector current at bright Au particles was about 130 nA. On the other hand, for the STEM image, the difference of the intensities is not so noticeable, and it is difficult to distinguish latex and Au particles. Fig. 4 shows a BSE image of Au particles confined between the two SiN membrane window grids, taken at 200 kV. These particles were located on the upper membrane. In spite of the presence of 100 nm thick membrane, we can see each particle clearly, owing to the usage of a high accelerating voltage of 200 kV. The detector current at bright Au particles was about 8 nA. The low current is mostly due to the low backscattering probability as compared with 75 kV. And partially because of the fact that the BSEs at 200 kV are distributed higher angle than at 75 kV.

Our BSE detector was conveniently fixed to the sample grid with a silver paste. But it was able to remove the detector from the sample grid with tweezers. So, the detector was reusable until breakage which may happen by mistake.

Fig. 1: Schematic representation of the BSE detector.

Fig. 2: Au (Φ60 nm) particles were confined between two SiN membrane window grids. Au particles were in air atmosphere. This was fixed to the detector with a silver paste for BSE observation of Au particles.

Fig. 3: Latex and Au sample taken at 75 kV. (a) Dark-field (DF) STEM and (b) BSE images, respectively.

Fig. 4: BSE image of Au particles confined between the two SiN membrane window grids taken at 200 kV.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1565 Imaging of nanoparticles in cells with backscattered electrons in a scanning electron microscope

Müller E.1, Seiter J.1, Blank H.1, Gehrke H.2, Marko D.2, Gerthsen D.1
1Laboratory for Electron Microscopy, Karlsruhe Institute of Technology, Karlsruhe, Germany, 2Department of Food Chemistry and Toxicology, University of Vienna, Vienna, Austria
erich.mueller@kit.edu

Scanning electron microscopy (SEM) is an established technique for ultrastructure imaging of cells. Backscattered electrons (BSEs) yield subsurface information and atomic-number contrast [1] and are used in this work to image cellular structures and NPs incubated in cells. Specifically, optimum primary electron energies E0 for BSE imaging were determined for thin cell sections with thicknesses 100 nm ≤ t ≤ 1000 nm deposited on indium-tin-oxide (ITO-)covered glass slides which are interesting substrates for correlative light and electron microscopy imaging [2]. We also developed a technique to determine the information depth (ID) which denotes the maximum subsurface depth at which an object can be imaged.
Thin cell sections of HT29 colon carcinoma cells incubated with SiO2 nanoparticles (NPs) of 40 nm size were studied (see [3] for sample preparation). Poststaining was omitted to avoid artifacts. SEM was performed with an FEI Quanta 650 FEG with a low-voltage high-contrast detector (vCD).
Small E0 were selected to limit the escape depth of BSEs because electrons from large sample depths degrade image resolution and contrast. Fig. 1a shows a 2.5 keV BSE image of a 200 nm section. The SiO2 NPs, typically contained in vesicles, can be easily detected due to their bright contrast. Cell organelles display high contrast despite the lack of poststaining in Fig. 1b.
Fig. 2a shows a 1 µm section where E0 up to 7.5 keV can be applied without sample charging. In addition to the incubated SiO2 NPs, Au NPs with a size of 40 nm are present on the surface and can be distinguished due to their higher BSE intensity. BSE images were taken at different E0 between 1.5 and 7.5 keV for depth-dependent detection of SiO2 NPs. With increasing E0 more NPs become visible corresponding to the increasing ID. The depth of NPs from the surface was determined by tilting the sample and applying a triangulation method. In Fig. 2b the experimentally determined particle depths (dots) are plotted as a function of E0 and are compared with calculated ID values obtained by Monte-Carlo simulations (triangles). Based on the escape depth T = f·A·E01.67/(ρ·Z0.89) (Z: average atomic number, A: average atomic weight, ρ: density) proposed in [4], an analytical expression for the ID was obtained by fitting the experimental data with a modified factor f. This expression allows the determination of the ID of BSEs in biological samples. Experiments with entire cells grown on ITO-coated glass are promising with respect to NP detection and are subject of further work.

References
[1] H Niedrig, J. Appl. Phys. 53 (1982), p. 15.
[2] H Pluk et al., Journal of Microscopy 233 (2009), p. 353.
[3] J Seiter et al., J. Microscopy, accepted.
[4] K Kanaya and S Okayama, J. Phys. D: Appl. Phys. 5 (1972), p. 43.

Fig. 1: BSE SEM images of a 200 nm section of an HT29 cell deposited on an ITO-covered glass substrate. (a) Overview image taken at E0 = 2.5 keV. SiO2 NPs and organelles are visible in the cell. (b) High-magnification image taken at E0 = 3.5 keV. NPs and membranes can be well resolved.

Fig. 2: (a) 7 keV BSE SEM image of a 1000 nm section of an HT29 cell. SiO2 and Au NPs show different contrast compared to the cell matrix. (b) Plot of the information depth as a function of E0 with experimentally determined values (dots) and Monte Carlo simulations (triangles). Fit curves are based on the modified Kanaya-Okayama equation [4].
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1607 Application for Low Energy STEM with the In-lens Cold FE-SEM

Sunaoshi T.1, Orai Y.1, Ito H.1, Okada S.1, Ogashiwa T.1, Konno M.1
1Hitachi High-Technologies Corporation, Ibaraki, Japan
sunaoshi-takeshi@naka.hitachi-hitec.com

Inorganic carbon materials (primary carbon nanotubes and graphene) and organic polymeric materials are being developed more actively. The demands for fine structural, elemental, and chemical characterization of these materials by electron microscopes are rapidly increasing. These requirements have increased the demand to achieve high resolution STEM imaging at low accelerating voltages. It is necessary to determine methods to improve the contrast intensity at low accelerating voltage operation without loss of resolution. In order to respond to such demands, we have developed the Hitachi SU9000 (Figure 1), a cold FE-SEM (CFE-SEM) with an in-lens type of objective lens, capable of high resolution phase contrast STEM imaging. Through using this technique on this microscope, it is possible to routinely achieve lattice resolution of the graphite {002} planes with a spacing of 0.34 nm. In this study, we improve the observation conditions for obtaining enhanced lattice resolution in STEM imaging at an accelerating voltage of 30 kV. Additionally, we have shown the effectiveness of this method for imaging inorganic carbon based materials. Figure 2 shows a simplified lens diagram of the SU9000. By using newly optimized lens parameters and a specialized sample stage which reduces the distance between the objective lens and sample, the Cs was lowered from approximately 2 mm to 1 mm. Figure 3 shows a high resolution BF-STEM image with its inset Fourier transform (FFT) image, observed along the Si<110> zone axis at an accelerating voltage of 30 kV. The sample was prepared using the NB5000 FIB-SEM equipped with a unique micro-sampling system. The specimen was thinned down to approximately 30 nm thickness. (a) is the standard condition (WD: 3 mm,Cs:2 mm) and (b) is the optimized condition (WD: 1.8 mm, Cs: 1 mm). Both (a) and (b) imaged the Si {111} plane, which has a spacing of 0.314 nm, and reflection spots corresponding to 0.314nm were confirmed from FFT images. However, in the optimized condition, not only Si {111} planes (corresponding to 0.314 nm) but also the {002} planes (corresponding to 0.272 nm) are detected from FFT. This confirms that the image resolution is improved by reduction of Cs. Next we applied the optimized condition to a graphene sample. Figure 4 shows a high resolution BF-STEM image with its inset FFT image, the multi-layer graphene membrane was clearly observed at an accelerating voltage of 30 kV. Lattice fringes were easily observed and the reflection spots corresponding to 0.213 nm were successfully confirmed. These results reveal the potential for high contrast visualization without loss of resolution for any carbon-based materials and the latest semiconductor devices with minimal radiation beam damage.

The multi-layer graphene membrane specimen was kindly supplied by Dr. Tsuyohiko Fujigaya of the Department of Applied Chemistry Graduate School of Engineering, Kyushu University.

Fig. 1: General view of SU9000 In-lens FE-SEM.

Fig. 2: Configuration of SU9000.

Fig. 3: BF-STEM images and FFT images of Si <011> single crystal. (Accelerating voltage is 30 kV )

Fig. 4: BF-STEM image and FFT image of graphene. (Accelerating voltage is 30 kV )
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2405 Application of Multi-Tilt Specimen Stage for Advanced Electron Channeling Contrast Technique

Dluhoš J.1, Sedláček L.1, Ižák T.1, Hrnčíř T.1, Jiruše J.1
1TESCAN Brno, s.r.o., Brno, Czech Republic
jiri.dluhos@tescan.cz

New developments for a selected area electron channeling pattern (SACP) acquisition and electron channeling contrast imaging (ECCI) in the scanning electron microscope (SEM) are presented. Novel approaches for electron channeling contrast formation are introduced using multi-axial multi-tilt specimen stage.

The multi axial piezo-driven specimen stage allows high precision movement with all six degrees of freedom. Among other applications, it allows bi-axial tilting around any selected point on the sample. Such a technology enables precise selection of beam to surface angle which is the core principle of electron channeling contrast formation, as shown in Fig. 1.

The well-known "rocking beam" technique for electron channeling pattern (ECP) described e.g. in [1] is based on a special mode of scanning in the SEM. Limitation of this technique is given by spherical aberration of the objective lens, which restricts its use mainly to single crystals. A dedicated Cs corrected rocking beam mode was developed for TESCAN field emission microscopes for acquisition of SACP from a very small area as shown in Fig. 2. The practical use of this correction was demonstrated on polycrystalline samples in [2]. Further extension of the rocking angle by the use of stage tilt was also tested.

Specific properties of the ECCI technique in SEM for the observation of near surface defects are explained. The relation of ECP to the formation of ECCI is crucial for understanding the whole ECCI phenomenon. The oriented ECCI technique for reaching suitable diffraction condition as described in [3] was applied. Advantage of combination of Cs corrected SACP for oriented ECCI technique is shown. Newly, the use of precise bi-axial specimen tilt for oriented ECCI is demonstrated in Fig. 3.

Furthermore, new techniques for ECCI contrast improvement, such as color coded multi-axial specimen tilt, are introduced. The sample tilt angle is coded according to HSV color or RGB model to improve the informational depth of the micrograph (see Fig. 4).

References:

[1] A J Wilkinson et al, Micron 28 No. 4 (1997) p. 279.

[2] J Dluhoš et al, METAL Conference Proceedings (2012) p.453.

[3] B A Simkin et al, Ultramicroscopy 77(1-2) (1999) p. 65.

The research has been supported by the Technological Agency of Czech Republic TE 01020233(AmiSpec)

Fig. 1: Schematic diagram of forming the channeling contrast in relation to deviation from the Bragg condition. Image by Wilkinson et al. [1].

Fig. 2: Comparison of ordinary rocking beam mode without correction of spherical aberration (left) and a Cs corrected ECP mode (right). Images taken on polycrystalline stainless steel with grain size about 20 µm.

Fig. 3: ECCI imaging with the use of SACP a) navigation to diffraction condition on SACP (edge of the band, using a multi axial stage tilt. b) ECCI image of crystal defects

Fig. 4: Composite ECCI micrograph of copper sample with randomly oriented grains using the color coded tilt of the stage. R,G,B – images acquired with specimen tilt from -5° to +5°.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1688 High Sensitivity and Minimum Acquisition Time with the Annular EDX Pole Piece Silicon Drift Detector “Rococo2”

Liebel A.1, Eckhardt R.1, Bornschlegl M.1, Bechteler A.1, Niculae A.1, Soltau H.1
1PNDetector GmbH, Munich, Germany
andreas.liebel@pndetector.de

The operation conditions of Scanning Electron Microscopes (SEM) have changed a lot over the last years and many applications have to deal with very low primary beam energies and currents. Modern Energy Dispersive X-ray (EDX) detectors have to accomplish these tasks i.e. they need to support a large geometric collection efficiency (solid angle) in order to enable fast measurements even at weak X-ray intensities. This demands not only for large area detectors but also for intelligent detector designs.
The annular Silicon Drift Detector (SDD) “Rococo2” uses a highly optimized geometry which covers a very large solid angle. It consists of 4 cloverleaf shaped SDD cells combined on one monolithic chip with a total sensitive area of 60 mm² and a center hole. The detector is shown in Figure 1a. It can be positioned right underneath the pole piece extremely close to the sample which results in a very large solid angle up to 1.4 sr. [1] Comparing this number with the solid angle of a conventional 10 mm² SDD detector of typically 0.01 sr it is obvious that the Rococo2 detector can deliver 100 times larger signal intensities at the same measurement time and conditions. Figure 2 shows EDX mappings of a duplex brass sample which illustrate this benefit.
High energetic electrons which are backscattered from the sample are typically filtered by using magnetic electron traps in front of the EDX detector. In case of the Rococo2 detector this is not possible because the magnetic field would disturb the electron beam. In this case the Backscattered Electrons (BSE) are filtered by hardware filter foils which stop the electrons while transmitting the X-ray photons. We will present measurements with a combination of different filter foils made of 2 µm thick Beryllium and 2 µm Mylar for each two detector cells (see Figure 1b). With the used combination of foils a continuous undisturbed X-ray sensitivity down to carbon and boron can be achieved.
We will further present concepts for a combined annular detector for measuring backscattered electrons and X-Rays simultaneously. By increasing the central hole of the EDX detector it is possible to detect backscattered electrons at high take off angles in the central part of the detector. This enables the detection of X-ray and BSE signals at the same time with relatively high collection efficiency and just one single detector head. Figure 3 shows two 2 concepts for such a detector with the BSE detector positioned either above or at the same level as the annular EDX detector. We will show calculations of the solid angle and the collection efficiency of different EDX and BSE detector combinations and evaluate the results by comparing images or spectra.

[1] A Niculae et al, Microscopy & Microanalysis, vol. 18 S2 (2012) p. 1202-1203

Fig. 1: a) The annular pole piece EDX detector “Rococo2” inside the SEM and b) a view at the top of the detector showing the collimator with a filter combination of 2 µm Be and 2 µm Mylar.

Fig. 2: EDX Mappings of a duplex brass sample showing α and β phases with different concentration of copper and zinc.The left image was obtained with a 10 mm² single cell SDD with approx. 0.01 sr solid angle, the right one with the Rococo2 detector with a solid angle of more than 1 sr at the same conditions and acquisition time.

Fig. 3: Schematic drawings of a combined EDX and BSE detector setup with a) the BSE detector positioned above the annular EDX detector and b) the two different detectors positioned on the same level.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1795 SEM images using an energy/angle selective electron detector.

Otsuka T.1, Nakamura M.1, Yamashita K.1, Hara M.1, Timischl F.2, Honda K.1, Kudo M.2, Kitamura S.1
1JEOL Ltd., Tokyo, Japan, 2JEOL Technics Ltd., Tokyo, Japan
tootsuka@jeol.co.jp

Scanning electron microscopes (SEMs) are usually equipped with two types of detectors: secondary and backscattered electron detectors. The former produces secondary electron images (SEI) rich in topographic information[1, 2], whereas the latter produces backscattered electron images (BEI) rich in composition information[3]. Recently, however, a few other detectors have been installed in addition to these two types of conventional detectors[4]. In these practical detectors, however, it is difficult to see directly the effect of energy and take-off angle of the emitted electrons on the image contrast. In this study, an electron detector was designed and experimentally manufactured to detect electrons emitted in a definite, variable range of energy and take-off angle.


Figure 1 shows a schematic diagram of the newly designed electron detector, the E-θ detector, which can detect electrons emitted from a sample with a selected range of energy and take-off angle. The E-θ detector consists of a slit plate, inner and outer electrodes in a cage, and electron detectors. The slit plate is placed at a lower part of the E-θ detector. It serves as a selector of take-off angle. The range of take-off angle is selected mechanically by sliding the slit plate as shown in Fig. 1 and is measured from the horizontal direction parallel to the sample surface. The selection of electron energy is made by applying voltage to the inner and outer electrodes in the cage in accordance with electron take-off angles. In the case of the low angle range, positive and negative voltages are applied to the inner and outer electrode, respectively, as shown in Fig. 1(A), so that electrons are deflected towards the inside with the increasing amount of deflection with decreasing energy. In the case of the high angle range, the polarity of the applied voltage is reversed as shown in Fig. 1(B), so that electrons are deflected towards the outside. To enhance the electron detection, a metal mesh is placed in front of the electron detectors and a voltage of 2 keV is applied between the detectors and the metal mesh. When a series of concentric ring electron detectors with different diameters are placed at the upper part of the cage, each ring detector can collect electrons with a specific range of energy determined by the voltage applied to the two electrodes. As a preliminary study, we acquired images using commercial Si-photodiode (SiPD) as the electron detectors, as shown in Fig. 2.

[1] M. Kotera et al., Scanning Microscopy Supplement 4 (1990) p. 111.
[2] Y. Lin, D. C. Joy, Surface and Interface Analysis 37 (2005) p. 895.
[3] M. D. Ball, D. G. McCartney, Journal of Microscopy 124 (1981) p. 57.
[4] S. Asahina et al., Microscopy and Analysis, Nanotechnology supplement November (2012)

Fig. 1: Schematic diagram of the E-θ detector.

Fig. 2: SEM images acquired with the E-θ detector. The sample is a spherical single crystal of tungsten.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1825 The study of extreme low landing voltage scanning electron microscopy

Sakuda Y.1, Asahina S.1, Kazumori H.1, Kawauchi K.1, Nokuo T.1, Charles F.2
1JEOL.ltd, 3-1-2 Musashino, Akisima, Tokyo 196-8558 JAPAN, 2JEOL SAS, Espace Claude Monet-1, allee de Giverny, Croissy-Sur-Seine 78290 FRANCE
ysakuda@jeol.co.jp

The development of low voltage (LV) FE-SEMs have been in progress, and spatial resolution better than 1 nm can now be achieved even at 1 kV. General LV FE-SEMs are available for a sample surface observation and low voltage EDS analysis. Furthermore, by choosing appropriate observation conditions, we can selectively obtain different information such as material topography and composition. In this report, authors focused on observations at extreme low impact electron-energy in order to obtain information from surface. For example, the length of electron mean free path at 100 eV in a solid sample is smaller than 1 nm [1]. So that it is expected to observe clearly surface morphology. Moreover, we can expect what the low impact electron energy show less electron beam damages in general [2].

Recently, we can use combined lens both electrostatic and magnetic to minimize Cc as well as field emission type emitter with high brightness [3]. So-called Super Hybrid Lens (SHL) is equipped on JSM-7800F. In addition, negative surface potential can be applied on the specimen surface by so-called Gentle Beam mode (GB). Therefore, the impact electron energy to the specimen surface can be reduced down on 10 eV keeping the probe size small with high coherency.

Figs.1 (a) and (b) show carbon nanotubes observed at 80 eV and 500 eV. Those images clearly show their shapes even at 80 eV. Fig.1 (a) shows less edge effect compared with (b) due to small penetration depth of electrons at lower impact electron energy. The intensity profiles along the lines shown in Figs.1 (a) and (b) are shown in Fig.1 (c). The 80 eV image shows higher contrast than the 500 eV one. We assume that is due to higher efficiency of interaction with carbon materials at lower impact electron energy.

Fig.2 (a) shows a low magnification image of meso-zeolite (LTA) at 80 eV. Figs.2 (b) and (c) show high magnification images at 80 eV and 500 eV. All images clearly show topological information at meso-LTA. Especially, the image of 80 eV shows less edge effect due to small interaction volume. That is a useful feature of LV FE-SEM because it can reveal fine edges and give high accurate measurement of nano porous materials. One other useful feature is the reduction of specimen damage due to electron irradiation. The gap shown between two arrows in a circle in Figs. 2 (b) and (c) is observed to be wider in the latter than in the former. The observation indicates less electron damage at 80 eV.

References:

[1] C. R. Brundle, J. Vac. Sci. Technol., 11, 212 (1974)

[2] L. Reimer, Scanning Electron Microscopy: Physics of Image Formation and Microanalysis, 2nd ed., Springer,

Berlin, New York, (1998)

[3] J. Frosien, J. Vac. Sci. Technol. B 7 (6), Nov/Dec (1989)

[4] O. Terasaki, JEOL News, (2013)

Fig. 1: Low impact electron energy on Carbon nanotubes

Fig. 2: Images of meso-zeolite (LTA) at two impact electron energy of 80 eV (a,b) and 500 eV (c).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1829 Determination of the second critical energy of primary electrons in relation to dielectric thickness and angle of incidence

Evstaf'eva E. N.1, Rau E. I.1, 2, Tatarintsev A. A.2
1Faculty of Physics, M. V. Lomonosov Moscow State University , 2Institute of microelectronics technology and high purity materials RAS
rau@phys.msu.ru

At the second crossover energy E2C of incident electrons the equilibrium is maintained between the electron probe currents I0, the second electron emission (SEE) currents I0δ, the backscattered electron (BSE) currents I0η, as well as the leakage currents IL and the displacement currents Id that are responsible for the accumulated charge Q. At the equilibrium state the equality I0=I0(δ+η)+IL+Id is fulfilled, while for the target remaining uncharged the condition δ+η=1, VS=0 is valid, where δ and η are the emission coefficients of SE and BSE.

Experimental results for dielectrics, in the case when incident electrons impinge on the sample surface at the angle α, can be described by the following semi-empirical expression: E2C=E2C(0)exp[(ln(R2C/2λ))(1-cosα)], (1)

where λ is the effective emission depth of SE, R2C=76E01,67 ρ is the depth of penetration of primary electrons with the energy E0, ρ is the specific density of the dielectric material. As an example, fig.1(a) shows the experimental dependence and the dependence calculated by formula (1) of the second critical electron energy E0=E2C on the angle of incidence α, for the target potential VS=0, i.e. when the target remains uncharged. Fig.1(b) shows the dependence of the energy E2C on the angle of incidence α for ungrounded metals.

Consider the dependences of the VS of PММА films with the thickness d on a silicon substrate at the electron energy E0.

The experimental results are in qualitative agreement with the calculated results as shown in fig.2(a), presenting the dependences VS(d) for MICA plates 2 to 30 μm thick and for PММА films 0.4, 1.4, 2.7, 4 μm thick on the Si-substrate.

At the radiation energies E0 in the range of 0.5–1.0 keV the negative charging begins (note that according to previous views, positive charging was expected because E0<E2C). At this energy the sign polarity of VS changes, i.e. at the point where VS=0 V. For PММА this value lies in the range E0=0.4–0.6 keV, with the thicknesses of the layers of positive and negative charges and the values of these charges are approximately equal (λ≈R0, Q+=Q-), which is responsible for the total absence of charging. In the region of 1 keV <E0<Ecr2 one can clearly observe negative charging, and the greater d, the higher the value of -VS. This range corresponds to the condition λ<R0<d. The value of -VS at first increases and reaches the maximum, then as E0 and R0 grow, it starts decreasing slowly in the absolute value and reaches zero at the points of Ecr2, that are different for each film thickness d. These points can be used in high-voltage lithography, because it is at these values of R0≥2d that the conduction current IT is generated and it carries excessive negative charges (electrons) onto the substrate, hence VS=0 V.

Fig. 1: Characteristics of the value of the second critical electron energy E2C as a function of angle of incidence α for dielectrics (a) and ungrounded metals (b).

Fig. 2: (a) - Dependence of surface potential of dielectric films on their thicknesses: (1) VS(d) for mica (plot 1) and for PММА (plot 2). (b) - Dependences of surface potential VS on incident electron energy E0 for PММА films of different thickness d on Si-substrate.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1852 New possibilities of SEM for two-channel detection of energetically filtered secondary and backscattered electrons

Rau E. I.1, Kupreenko S. U.1, Tatarintsev A. A.1, Zaitsev S. V.1
1Faculty of Physics , M. V. Lomonosov Moscow State University
rau@phys.msu.ru

In this paper we present a preliminary study of new potentialities of SEM – microtomograph equipped with a toroidal spectrometer of electrons and two detector systems based on microchannel plates (MCP). A new modification of the instrument is shown in fig.1. Electron probe 1 scans across the surface of the sample under investigation 3. Toroidal spectrometer in a case 4 is mounted under SEM objective lens 2. SE an BSE emitted from specimen 3 pass through annular inlet slit 6 and are energy-separated in toroidal capacitor 5 and pass through outlet annular apertures 7 and 8. The energy filtered SE and BSE are detected by two MCP 9 (A and B) placed opposite each other. The signals A and B from these detectors can be sent either to PC 11 to record the spectra or to the SEM display. Using block 10 one can do the operations of addition (A+B) and subtraction (A-B) of signals. It is known that such operations allow us to obtain contrast from either the chemical composition of the specimen (Z-contrast) or the surface topography. In our case the contrast is enhanced and allows unique interpretation owing to filtration of detected electrons in a narrow energy window. The electrons with small energy losses escape mostly from the subsurface region and are modulated in escape angles, which favors domination of topographic contrast. The electrons, which lost considerable amounts of their energy, are emitted from much deeper regions and therefore mostly produce Z-contrast. Addition and subtraction of signals from the two oppositely oriented detectors enhances this effect considerably.

The examples presented in fig.2 and fig.3 show fragments of the sample having heterogeneous composition, consisting of the alloy of different materials, in particular, Cr, Si, Cu, W.

The images shown in fig.2 are obtained at the primary electron beam energy E0=10 keV, the current I0=1nА, the energy of filtered BSE forming the image EBSE=8 keV ((a) and (b)), and at the SE energy ESE=4 eV ((c) and (d)). One can see that the image contrast (obtained from signal addition) differs greatly in element composition and in surface topography (obtained from signal subtraction) for both BSE and SE regimes.

Fig.3 presents the images of another region of the sample taken at different energies of E0. The general image of this region demonstrated in fig.3a is taken in the standard SE mode in SEM at E0=5 keV. Fig.3b shows the image taken in the BSE added signal at E0=5 keV, in fig.3c – at E0=15 keV, and in fig.3d – in the subtracted signal. The fact that, contrast in filtered BSE and SE is higher and more informative than that obtained with standard signals and standard detectors in SEM makes it possible to more accurately visualize and reconstruct the sample 3D surface profile both in BSE and SE.

Fig. 1: Scheme of the spectrometer-microtomograph in SEM: 1–electron probe, 2–objective lens in SEM, 3–sample, 4–shielding case, 5–toroidal electrodes, 6,7,8–input and output annular slit, 9–MCP, 10–signal addition/subtraction block, 11–PC or SEM monitor, 12 – high-voltage power unit of the spectrometer, 13 – semispherical grid for SE potential contrast.

Fig. 2: Images of the sample of complex composition taken in the BSE mode with signal addition A+B (a) and subtraction A-B (b). Images in SE with signal addition A+B (c) and subtraction A-B (d).

Fig. 3: Images of complex sample in standard SE-mode (a) and BSE - filtred mode (b, c, d).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1865 Low-Damage SEM Imaging of Organosilicate Glass Thin Films in Semiconductor Industry

Garitagoitia Cid A.1,2,3, Muehle U.2, Rosenkranz R.2, Zschech E.1,2,3
1Technische Universität Dresden, Dresden, Germany , 2Fraunhofer Institute for Ceramic Technologies and Systems IKTS, Dresden, Germany, 3Dresden Center for Nanoanalysis (DCN), Dresden, Germany
aranzazu.garitagoitia@ikts-md.fraunhofer.de

On-chip interconnect stacks of high-performance microelectronic products like microprocessors consist of Cu interconnects and insulating organosilicate glass (OSG). Dielectrics with extremely low dielectric permittivity (k value) are needed to reduce signal delay time and cross-talk in on-chip interconnects systems. The OSG thin films are either dense (so-called low-k materials) or porous (so-called ultra-low-k materials).

Imaging of dense and porous OSG thin films with Scanning Electron Microscopy (SEM) is necessary in semiconductor industry for process monitoring and physical failure analysis. Due to weak chemical bonding in the glass network, these materials show strong degradation effects when observed in SEM, caused by electron-material interaction. Particularly, the glass network is densified during the electron beam application to the sample, which phenomenologically causes a significant shrinkage of the material. This shrinkage avoids e. g. a quantitative determination of geometric features in semiconductor structures, which is required for process monitoring. Imaging with reduced primary beam energy mitigates the materials damage; however, the spatial resolution is usually reduced at lower accelerating voltages. In this study, spatial resolution and OSG thin film degradation during SEM imaging are studied systematically as a function of the primary beam energy. A Carl Zeiss SEM/FIB system NVision40 with Gemini column and three types of detectors is used, the conventional Everhart-Thornley detector, the inlense detector and the energy selective backscattering (EsB) detector. The optimum parameters for SEM imaging of OSG thin films are provided for several types of materials.

We kindly thank Carl Zeiss Microscopy GmbH for funding the investigations in the framework of the project “Untersuchungen zur Charakteristik und Applikation des EsB Detektors”.

Fig. 1: Schematic of the Zeiss Gemini Column with three different electron detectors

Fig. 2: Crack in low-k dielectric after one single scan in SEM
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1912 Ionic Liquid Preparation for SEM Observation of Minute Crustacean

Shiono M.1, Sakaue M.1, Konomi M.1, Tomizawa J.2, Nakazawa E.1, Kawai K.3, Kuwabata S.4
1Tokyo Solution Lab., Hitachi High-Technologies Corp., Kawasaki, Kanagawa, Japan, 2Hitachi High-Technologies Corp.,hitachinaka, Ibaraki, Japan , 3Miyoshi Oil & Fat CO.,LTD. Katsushika, Tokyo, Japan , 4Graduate School of Engineering, Osaka University, Yamadaoka, Suita, Osaka, Japan
sakaue-mari@naka.hitachi-hitec.com

     Electron microscopic observation is often possible along the surface of an arthropod, however applying common fixative media present difficulties when penetrating beyond the exoskeleton. Ionic liquids (ILs) are unique in that they are incombustible, non-volatile, and have high ionic conductivity. The application of ILs as part of sample preparation for EM were conducted with some particles dispersed in ILs(1) and observed by TEM, including IL wetted seaweed with observation by SEM(2). Recently, we have developed a new ionic liquid (IL) HILEM, IL1000 for EM observation. The ionic liquid, IL1000 has been designed for EM preparation, having both a high level of safety and high solubility, resulting in a high suitability for biological tissue preparation. In this experiment, minute crustaceans were immersed in 10 % IL1000 diluted with distilled water for a period of 60 minutes to 3 hours. For surface observation retention, any extra IL covering samples were removed by an absorbent cloth. The samples were observed by the Hitachi SU3500 at an acceleration voltage of 5 kV in high vacuum condition.
     Figure 1 shows the secondary electron images of the minute crustaceans Gammaridea. Figure 1(a) is an image of the entire body of the Gammaridea, whose individual appendages are not easily distinguishable due to the overlapping of appendages. The imaged sample was removed from the SEM and tweezers were used with the aid of a binocular to separate its appendages from the body. Figure 1(b) is the separated appendage (the second thoracic appendage) of the Gammaridea and the attached organ shown by arrow in Figure 1(b). The organ shown functioned in protecting the egg, therefore it was determined that this specimen is female. The results show that the IL can deeply penetrate the specimen which aids electron conductivity inside the specimen. Figure 2 is the secondary electron images of the Tanaidacea. Figure 2(a) is the whole image of the Tanaidacea orientated to observe the ventral side. The female of the Tanaidacea has the brood chamber in the thorax (arrow) region. As aforementioned above, the observed specimen was removed from the SEM to separate its brood chamber. Figure 2(b) is a SEM image of the eggs that were removed from the brood chamber. Since the sample was soaked by IL, the sample is resistant to rapid dehydration while under vacuum, including soft materialsuch as eggs can be preserved by this technique. We emphasize that the IL1000 is a useful media for SEM observation of some soft and delicate biological material.

References
[1] E. Nakazawa. et al, Proceedings of the sixty-fourth Annual Meeting of The Japan Society of Microscopy. p 136 (2008)
[2] S. Kuwabata. et al, Kenbikyo, 44: p 61-63. (2009)

 

Fig. 1: Figure 1. Secondary electron image of Gammaridea treated by the ionic liquid. (a) the whole image of the Gammaridea, where a lot of appendages overlap. (b) The separated second thoracic appendage (arrow) functioned to protect the egg. Instrument: SU3500, Acc. Volt. 5 kV, Magnification: x 32(a), x75(b).

Fig. 2: Figure 2. Secondary electron images of Tanaidacea treated by the ionic liquid. (a) the inclined whole image holding the brood chamber (arrow). (b) the eggs scraped out of the brood chamber. Instrument: S-3400N, Acc. Volt. 5 kV, Magnification: x 35(a), x200(b).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-1917 New Preparation Method using Ionic Liquid for Fast and Reliable SEM Observation of Biological Specimens

Sakaue M.1, Shiono M.1, Tomizawa J.2, Nakazawa E.1, Kawai K.3, Kuwabata S.4
1Tokyo Solution Lab., Hitachi High-Technologies Corp., Kawasaki, Kanagawa, Japan , 2Hitachi High-Technologies Corp., Hitachinaka, Ibaraki, Japan, 3Miyoshi Oil & Fat CO., LTD., Tokyo, Japan, 4Graduate School of Engineering, Osaka University, Osaka, Japan
sakaue-mari@naka.hitachi-hitec.com

    For SEM observation, it is necessary for biological specimens to be treated with several types of preparation media to preserve their shape under vacuum. Ionic liquids are unique materials because of their natural incombustibility, non-volatility, and high ionic conductivity. Here, we used an ionic liquid to prepare samples for EM observation [1] [2]. The ionic liquid, IL1000 has been designed for use in EM sample preparation with a high level of safety and high solubility [3].
     Figure 1 shows the SEM images of a Helicobactor Bilis sample prepared by conventional procedures. To preserve the flagella structure, the sample was immobilized on the cover slip coated with poly-L-lysine, and freeze-dried after fixation with 2 % glutaraldehyde (GA) in 0.1 M phosphate buffer and dehydrated with acetone in descending concentrations. The conventional sample preparation method takes approximately 8 hours. The surface of the cell body and some flagella are clearly observed (fig.1). On the other hand, in the ionic liquid (IL) method, the sample fixed by the buffered 2 % GA is immersed in 10 % IL1000 solution for 15 minutes and dropped onto filter paper. The sample is then directly transferred into the SEM without further drying. The resulting SEM image of this sample clearly shows the helical shape of the bacteria and flagella (fig. 2). Figure 3 shows the SEM images of mold growing on a rice cake. The square cut sample x5mm2, was immersed in the 10 % IL1000 solution for approximately 4 hours, and then directly transferred into the SEM. Fine threads protruding from the rice cake are clearly observed (fig.3) and the higher magnification image shows the clear and smooth surface of the spores.
    These results indicate that the IL method for biological sample preparation greatly reduces preparation time, and is additionally better at preserving the sample’s original shape in the SEM.

References
[1] S. Kuwabata. et al, Chem. Lett., 35, p600-601. (2006)
[2] E.Nakazawa.et al, Proceeding of the Fifty-sixth Symposium of the Japanese Society of Microscope., 47-2, p92-95. (2013)
[3] K.Nimura. et al, Hitachi Hyoron., Vol.95, 9, p26-31. (2013)

 

Fig. 1: SEM images of Helicobactor Bilis prepared by conventional procedures　 (Frozen dried sample)　                     Instrument: SU6600, Acc. Volt. 1 kV, Magnification: x 30,000    Sample: Courtesy of Prof. Yoshiki Kawamura, Aichigakuin University

Fig. 2: SEM images of Helicobactor Bilis treated by the ionic liquid IL1000                                                               Instrument: SU6600, Acc. Volt. 1 kV, Magnification: x 25,000    Sample: Courtesy of Prof. Yoshiki Kawamura, Aichigakuin University

Fig. 3: SEM images of mold growing in the rice cake treated by the 10 % ionic liquid IL1000                                   Instrument: SU3500, Acc. Volt. 3 kV, Magnification: x 3,000

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2707 Low-Voltage Imaging of Non-Conducting Samples

Beránek J.1, Havelka M.1, Jiruše J.1
1TESCAN Brno, s.r.o., Libušina třída 1, Brno, Czech Republic
miloslav.havelka@tescan.cz

In the past years, considerable attention has been drawn to imaging of non-conducting samples without prior application of conductive coating. Conditions of low voltage microscopy allow such observation with its main benefits: sensitivity to the surface details and possibility to reach charge balance under which the charging of the sample is diminished [1, 2].

Uncoated non-conducting samples often exhibit undesirable charging that prevents the observation of finer details. This effect can be suppressed by using specific landing energy for which the total flow of electrons from the sample equals the charge coming into the sample. An example of the charge balance for nylon fibers is shown in Figure 1: a) exhibits positive charging effects, b) is an illustration of charge balance at 1200 V in agreement with [2], whereas c) has visible signs of accumulation of negative charge.

Conditions for charge flow equilibrium for the non-conducting materials generally lie in low voltage region [2]. To maintain the quality of imaging, preserving high resolution at low acceleration voltages is crucial. In Figure 2, we present images taken in low voltage regime. In Figure 2 a) uncoated polystyrene balls are shown. At 4.2 kV we can see fine details of their surface roughened by etching. The resolution at low voltages can be enhanced in the Beam Deceleration Mode (BDM) [3]. Figure 2 b) shows the structure of TiO2 imaged with the BDM at 800 V. In this mode, the electrons are maintained at higher energy during their path through the column and they are decelerated just after they leave the objective lens. BDM supports further lowering of landing energy, automatically to 50 eV and manually to 0 eV. Figure 2 c) shows para-hexaphenyl imaged at 20 eV. These images were taken by an ultra-high resolution microscope MAIA [4] by TESCAN, which has guaranteed resolution 1.4 nm at 1 kV.

Secondary electrons reveal sometimes surprising amount of details when primary beam interacts only with surface layers of material [1]. In Figure 3, the comparison of cracked oxidized copper imaged at acceleration voltages a) 20 kV, b) 10 kV and c) 2 kV is given. As can be seen in Figures 3 a) and 3 b), the shapes and edges of larger structures are well distinguishable and coarse surface is visible. Figure 3 c), taken at 2 kV, shows detailed structure of the studied object. In comparison with Figures 3 a) and 3 b), in Figure 3 c) the edges lose brightness and the contrast of surface cracks and contours is predominant.

References:

[1] I Müllerová et al, Adv. in imaging and electron physics 128 (2003) p. 310.

[2] D C Joy, Micron 27.3 (1996) p. 247.

[3] J Jiruše et al, 15th Eur. Microsc. Congress Proceedings (2012) p. 165.

[4] J Jiruše et al, Microsc. Microanal. 19 (Suppl 2) (2013) p. 1302.

The support from FR-TI2/736 (MOREMIT) funded by the Ministry of Industry and Trade of the Czech Republic is acknowledged.

Fig. 1: Charging artifacts of nylon fibers at acceleration voltages a) 900 V, b) 1200 V and c) 1500 V. Dark areas and lines in a) are an evidence of positive charging while localized brighter areas in c) are due to negative charging. Image b) at critical voltage shows least charging artifacts.

Fig. 2: a) Uncoated polystyrene balls at 4200 V, b) TiO2 with BDM at 800 V and c) Fiber-like structure of Para-hexaphenyl imaged at 20 V with BDM. Lowering the acceleration voltage makes fine surface details of the presented non-conducting samples clearly visible. Such details are frequently obscured when high acceleration voltages are used.

Fig. 3: Oxidized surface of copper imaged at different acceleration voltages, a) 20 kV, b) 10 kV and c) 2 kV, thus shrinking the interaction volume. The contrast is gradually changed, especially at the edges of surface cracks and contours.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2152 Purification of FEBID gold nanostructures using oxygen plasma

Shawrav M. M.1, Wanzenboeck H. D.1, Belić D.1, Gavagnin M.1, Wachter S.1, Bertagnolli E.1
1Institute of Solid State Electronics, Vienna University of Technology
mostafa.shawrav@tuwien.ac.at

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2277 Physical background of multiple detection possibilities of the Hitachi SU8030 SEM

Gernert U.1, Berger D.1
1Technical University Berlin, Center for Electron Microscopy (ZELMI), Straße des 17. Juni 135, 10623 Berlin, Germany
ulrich.gernert@tu-berlin.de

The spatial resolution of state-of-the-art high resolution scanning electron microscopes has nearly reached the physical limit of around 1 nm. On the one hand, this has been achieved by optimised electron sources and improved low kV electron optics. On the other hand, advanced and multifunctional detectors have been developed to detect separately energy- and angular-selective secondary electron (SE) as well as backscattered electron (BSE) signals with reduced interaction volumes. While early SEMs used one SE- and one BSE-signal only, a fully equipped modern HRSEM offers the possibility to record up to 20 different signals. Therefore, the skilled operator will find the most suitable detection method to optimise the contrast of the nm-structures under investigations, no matter if they show up due to potential-, edge-, binding energy-, working function-, Z-contrast or anything else.
The aim of this work is the quantitative analysis of nm-structured samples, i.e. all possible signals should be linked to the analysed sample properties and to the physical background of the detection process. Due to the large variety of parameters, this was performed by analysing known samples with the different detection methods of a modern Hitachi SU8030 SEM.
As an example, fig.1 shows a Si-chip with conductive Si-tracks and isolating SiO2-areas around. On top, there is a PMMA polymer layer with cracks. On sample areas, consisting of polymer coating on Si substrate, both detectors, the lower one (mounted at the specimen chamber) and the upper detector (in-lens), show the same image contrast, which in this case is a pure element contrast. On SiO2 substrate, however, using the lower detector to display the cracked polymer leads to the same image contrast as before, whereas the upper detector additionally shows the potential contrast since it records a pure SE1-signal.
Fig.2 shows a Si wafer with square Au pads with 130 nm height. The images are taken with the top detector, which is located well above the upper detector. In non-deceleration mode, a high angle BSE signal is detected with orientation- and Z-contrast (fig. 2b). In deceleration mode, a negative voltage is supplied to the sample decelerating primary electrons as well as accelerating escaping secondary electrons, which subsequently will then be detected with the top detector. In the latter case, the pure SE-signal shows a fine topographic contrast (edges of Au-pads and grain boundaries) and it is sensitive to the surface potential (bright lines in the grooves, fig. 2a).
Further sample properties might be analysed, if primary electron energy, deceleration voltage, detected energy and WD are varied in addition. Consequently, a state-of-the-art SEM provides many complementary imaging modes utilizing its high flexibility.

We kindly acknowledge financial support from the Deutsche Forschungsgemeinschaft (DFG).

Fig. 1: Si-chip analysed with Hitachi SU8030 SEM.  a: Lower detector (SE[L]); accelerating voltage 900 V; working distance 10 mm  b: Upper detector (SE[U]); accelerating voltage 900 V; working distance 10 mm

Fig. 2: Au-pads on silicon analysed with Hitachi SU8030 SEM.  a: Top detector (SE[T]); landing energy 2 keV (accelerating voltage 3 kV; deceleration voltage 1 kV); working distance 1.9 mm  b: Top detector (HA100[T]); landing energy 2 keV (no deceleration); working distance 1.9 mm
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3088 STEM in SEM imaging of gold nanoparticles in tissular ecotoxicity experiments

García-Negrete C. A.1, Jiménez de Haro M. C.1, Blasco J.2, Soto M.3, Fernández A.1
1Materials Science Institute of Seville (CSIC - Univ. Seville), Seville, Spain, 2Institute of Marine Sciences of Andalusia (ICMAN-CSIC), Puerto Real (Cadiz), Spain, 3Zoology and Cell Biology Dept., University of the Basque Country, Leioa (Bizkaia), Spain
cjimenez@icmse.csic.es

Because of their prospective widespread use, gold nanoparticles (AuNPs) will certainly account for a considerable and persistent nanomaterial input to environmental systems. Therefore ecotoxicological risks in non target organisms associated with AuNPs are showing increasing consideration. Location of AuNPs has been previously studied in our laboratory analyzing slices of gills and digestive gland tissues of the bivalbe Ruditapes philippinarum after “in vivo” exposure experiments. Analysis was carried out by TEM of ultrathin tissue’s slices (80 nm) operating at 80 kV [1].
In this communication we present the results of investigating the use of an “in vitro” methodology associated to the optimization of the STEM-in-SEM technique for the use of a scanning electron microscope (SEM-FEG) in transmission mode and operated at 20-30 kV.
The advantages of STEM-in-SEM over TEM are discussed [2, 3]. The localization of high Z nanoparticles in low Z tissue matrices is presented here by using the STEM-in-SEM coupled to EDX analysis as a powerful technique. In addition we have optimized the measurements with the goal of working with thicker slices. The work with thick samples also avoid the NPs displacement during cutting and increase the possibility of finding NPs when working with low NPs doses (environmental relevant concentrations).
For the optimization of measurements conditions, the resolution in our SEM-FEG has been estimated using Fast Fourier Transform (FFT) algorithms on specific images of our tissue slices. We have used the SMART macro running inside the “SCION Image” program under windows [4, 5]. Working at magnifications over 100 kx, for slices thicknesses of 200-300 nm and operating voltages of 20-30 kV, leads to resolutions below 10 nm (an adequate value for analyzing AuNPs of 23 nm average diameter).
Figure 1 shows a representative image of AuNPs accumulated into the gill tissues after “in vitro” exposures. From the obtained images it was possible to localize AuNPs (see also Figure 2) associated with vesicles (it can be a large phagosome or also exocytosis). Nanoparticles were also found in residual bodies (exocytosis).
In summary this communications presents new results for “in vitro” fast testing and STEM-in-SEM imaging of engineered AuNPs in a tissular ecotoxicity model.

References
[1] CA García-Negrete, J Blasco, M Volland, TC Rojas, M Hampel, A Lapresta-Fernández, MC Jiménez de Haro, M Soto, A Fernández. Environmental Pollution 174 (2013), 134-141.
[2] O Guise, C Strom, N Preschilla. Microsc Microanal 14 (Suppl2) (2008), 678.
[3] A Bogner, PH Jouneau, G Thollet, D Basset, C Gauthier. Micron 38 (2007), 390-401.
[4] C Probst, R Gauvin, RAL Drew. Micron 38 (2007), 402-408.
[5] DC Joy. J Microsc 208 (2002), 24-34.

The authors gratefully acknowledge financial support from the Junta de Andalucia and EU FEDER (project PE2009-FQM-4554 and TEP-217) and the EU FP7 AL-NANOFUNC project (CT-REGPOT2011-1-285895).

Fig. 1: SEM-FEG image (transmission mode) of a 200 nm slice of gills tissue

Fig. 2: EDX spectrum from the area containing the AuNPs
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2413 Hair Cuticular Characteristics: Potential of Animal Identification in Primates Order

HING L. H.1, TEO H. C.1, FOONG M. J.1, HUKIL S.1, SAHALAN A. Z.1, WAN NUR SYAWANI W.1, KASWANDI M. A.2, NORMALAWATI S.3
1School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, 2Institute of Medical Science Technology, Universiti Kuala Lumpur, Taman Kajang Sentral, 43000 Kajang, Selangor., 3Electron Microscope Unit, Faculty of Science & Technology, Universiti Kebangsaan Malaysiaa
hing61@yahoo.com

Hair is one of the common trace evidences in a crime investigation due to its easy shedding nature and could be easily transferred between surfaces or left behind at the crime scenes (1,2). Therefore forensic examination of hair sample plays a significant role in investigation of illegal wildlife-related crime cases (3,4). Four species of animals from Primates order were selected with their hair samples examined under scanning electron microscope. Species of Cercopithecidae family include blue monkey (Cercopithecus mitis), banded leaf monkey (Presbytis femoralis) and vervet monkey (Chlorocebus pygerythrus) (Fig 1) while chimpanzee (Pan troglodytes)(Fig 2) is from Homonidae family. Nol chemical or mechanical cleaning of hair examination was done. In Primates order, all species have  same regular wave cuticular pattern but variation was seen in other features. Blue monkey and vervet monkey have smooth cuticular dorsal margin whereas banded leaf monkey and chimpanzee have rippled structure. Banded leaf monkey  showed intermediate hair cuticular orientation and  is the only species having this characteristic. Statistics analysis proved that average scale layer difference could  be one of the criteria in examination of hair samples.  Comparison showed average scale layer difference of chimpanzee is significant lower than blue monkey and vervet monkey. In Primates order, cuticular dorsal margin, scale position and scale layer difference could be employed to differentiate all four species of animal successfully (4). This study also proved that analysis of cuticular scales pattern and other related characteristics was not affected when conventional cleaning procedure was not employed. Present study indicates that it is possible that positive identification of animal species through hair samples examination using various measurements and examination of hair cuticular characteristics.

References

B. J. Teerink, Atlas and Identification Key: Hair of West-European Mammals, Cambridge: Cambridge University Press (1991).

M.S. Dahiya, S.K. Yadav, Elemental Composition of Hair and its Role in Forensic Identification, Open Access Scientific Reports, 2(4): 10.4172, 721 (2013).

M.S. Dahiya, S.K. Yadav, Scanning electron microscope characterization and elemental analysis of hair: a tool in identification of felidae animal, J Forensic Research, 4(1), 178, 4172/2157-7145.1000178 (2013).

H. Brunner, B.J. Coman, The Identification of Mammalian Hair, Melbourne: Inkata Press (1974).

The author acknowledged the contribution of hairs by the National Zoo of Malaysia

Fig. 1: Hair of vervet monkey

Fig. 2: Hair of chimpanzee
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2468 Recent achievements of elemental analysis with low acceleration voltage FE-SEM

Kikuchi N.1, Morita H.2, Ikarashi M.2, Kawauchi K.1, Nokuo T.1, Charles F.3
1JEOL Ltd., 3-1-2 Musashino, Akisima, Tokyo 196-8558 Japan, 2Oxford Instruments KK, IS Building, 3-32-42 Higashi-Shinagawa, Shinagawa-Ku, Tokyo 140-0002 Japan, 3JEOL SAS, Espace Claude Monet-1, allee de Giverny, Croissy-Sur-Seine 78290 France
nkikuchi@jeol.co.jp

Field Emission Scanning Electron Microscopes (FE-SEMs) are widely used for high spatial resolution imaging and analysis. Energy dispersive X-ray spectroscopy (EDS) is a technique used for elemental analysis of a sample, through the detection of characteristic X-rays generated from the sample irradiated by an electron beam.

To obtain the best spatial resolution of imaging, as a matter of course, the accelerating voltage and the beam current of the electron probe should be optimized. In the case of JSM-7800F, the beam current under the optimum condition, especially in a lower accelerating voltage range, was too small to detect characteristic X-rays for the elemental analysis with a reasonably good signal to noise (S/N) ratio. As to the accelerating voltage, landing energy of the electron beam, in fact, has to be changed to be optimized, depending on the excitation potential of the characteristic X-rays of specific elements to be analyzed. In the case of analysis, we have developed a new gun, an inlens Schottky plus FE gun, which produces about two orders of magnitude larger probe current in that range of the landing energy with almost the same spatial resolution of imaging as the one under the optimum condition of JSM-7800F. JSM-7800F equipped with this new gun is named JSM-7800F Prime, which has one other key feature installed; a beam deceleration mode, named gentle beam for super high resolution (GBSH), in which negative potential can be applied to the sample surface to decelerate incoming probe electrons on to the sample. GBSH facilitates high spatial resolution of imaging in low landing energy [1]. Fig.1 shows gold particles on carbon images taken with JSM-7800F and JSM-7800F Prime respectively at the same accelerating voltage of 1kV under the same probe current. The amount of the current was almost two ordered of magnitude larger than the one under the optimum condition in JSM-7800F. The spatial resolution of the latter image is far better than that of the former, of the order of nanometers.

As to the detector of characteristic X-rays, their counting rate is desired to be as large as possible to obtain a better S/N ratio [2]. For this purpose, an improvement has been made to establish an ultrahigh solid angle double EDS detector system from Oxford Instruments.

Fig.2 shows image and their corresponding elemental map of a nanometer size Pt particles on carbon substrates. They were taken with JSM-7800F Prime. A particle with a size of 7nm is clearly observed in the elemental map in Fig.2 taken for 15min in the GBSH mode in 5keV landing energy.

References:

1. L. Reimer, Scanning Electron Microscopy: Physics of Image Formation and Microanalysis, 2nd ed., Springer, Berlin, New York, (1998)

2. Y. Nakajima et.al., Submitted to M&M2014

Fig. 1: Gold particles on carbon images taken with JSM-7800F and JSM-7800F Prime at 1kV under the same probe current.

Fig. 2: Pt particles on carbon substrates. Imaging and EDS analysis (The two EDS detectors of Oxford Instruments, each of which has the area of 150mm2, are installed to form a double detector system with the area of 300mm2 in total ) were made with JSM-7800F Prime in the GBSH mode in 5keV landing energy.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2766 A novel SEM triple in-lens detection system

Wall D. C.1, Vystavel T.2, Tuma L.2, Skalicky J.2, Sasam F.1, Wandrol P.2
1FEI Company, Eindhoven, The Netherlands, 2FEI Company, Brno, Czech Republic
David.Wall@fei.com

The advent of new materials and new techniques in SEM and DualBeam has driven the need for better detection in recent years. Traditionally, in-lens detection systems have focussed on energy selection of signal, due to the small opening angle of signal that can be detected, while modern, below-lens detectors have the benefit of separating angular differences in signal. This has typically meant that the in-lens detection system has had strong benefits for resolving materials with fine difference in the composition, while below-the-lens has been more suited to channelling contrast.
In this abstract, a new type of electron column design is introduced which broadens the spectrum of BSE’s and SE’s that can be detected with the in-lens detectors. The newly introduced NICol SEM column positions the in-lens BSE detector at the lowest point of the column, so that the opening angle of BSE that can reach the detector is far higher than those typically positioned higher up. The benefit of this can be seen in the images of figure 1. where strong channelling contrast is now possible with in-lens detection. This enables the collection of strong grain orientation images even while tilted or in 3D data collection in DualBeam configurations, where previously below-lens detectors is more difficult to use due to possible collisions. Additionally, by segmenting the annular design of this BSE detector into left and right segments, two separate signals can be detected and processed. Adding these these segments delivers material or orientation contrast, while a differential image generates strong topographical contrast. Where topographical images are necessary on charging material, this technique can avoid the charge.

By fully utilizing the experiment geometry, clear separation between high and low energy secondary electrons can also be enabled with the further two in-lens detectors in the SEM. Very low energy, surface sensitive signal will be affected most by the electrostatic field and travels closest to the beam axis. Higher energy secondary electrons less affected by the electrostatic lens are projected onto the middle detector. These effects can be seen in Fig. 2 (a, b) where the lower energy SE image shows excellent surface information while the higher energy SE image shows the best edge contrast. This signal can then be detected on the upper detector. Simultaneous collection of all three of these signals enables the collection of all information in a single scan, reducing charge up effects, and preventing beam damage or contamination to the sample.

 

Fig. 1: SEM image of FIB cross-section through steel sample acquired at 1.8 keV exhibiting strong channeling contrast. This enables clear identification of austenite and ferrite regions.

Fig. 2: Simultaneous SEM images acquired using high energy (left) and low energy (right) secondary electrons using a primary beam energy of 2kV revealing edge and surface sensitivity.

Fig. 3: Simultaneous SEM images acquired using high energy (left) and low energy (right) secondary electrons using a primary beam energy of 2kV revealing edge and surface sensitivity.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2553 Nanostructure Imaging with Topographic and Compositional Contrasts in a Cold-Field Emission Scanning Electron Microscope at Low Accelerating Voltage and with Energy-Filtration

Gauvin R.1, Brodusch N.1, Demers H.1, Woo P.2
1Department of Mining and Materials Engineering, McGill University, Montreal, Quebec, Canada, 2Hitachi High-Technologies Canada Inc., Toronto, Canada
raynald.gauvin@mcgill.ca

For developing new technologies, it is important to characterize the microstructure of materials with high spatial resolution at the nanoscale. To achieve high resolution, field emission scanning electron microscopes (FE-SEM) were developed. These microscopes allow working at low accelerating voltage, below 5 kV, to take advantage of the reduction of the interaction volume with accelerating voltage (from 1 μm in Al at 10 kV to 10 nm at 1 kV). Furthermore, their higher gun brightness compared to conventional thermo-electronic emitters, allow a probe size at the nanoscale. However, technical problems arise when SEM operates at low kV, i.e., the source brightness decreases and the chromatic aberration increases, all SEM parameters being equal. Using deceleration mode minimizes these problems and further improvement is achieved by using a cold-field emitter, which has a smaller energy spread and providing the highest brightness and the smallest source size of a FE-SEM. At low accelerating voltage, the emission volume of backscatter (BSE) and secondary (SEII, emitted by BSEs) electrons signals approach that of SEI (emitted by the primary electrons) signals. However it is not enough to reach the highest resolution. A magnetic field above the sample improves the spatial resolution by collecting mostly high-resolution signals. In addition, the energy-filtration of the electron signals allows selecting the type of contrast detected: topographic, compositional, or crystallographic.

Examples of high spatial resolution imaging are shown in Figure 1. Topographic imaging at a very low accelerating voltage of 50 V is possible with the deceleration mode with still an excellent spatial resolution of 2.8 nm as calculated with SMART-J (Figure 1A). The energy-filtration allows the observation of small compositional contrast as shown in Figure 1B where Al3Li precipitates (δ’) were observed in an AA2099 Al-Li-Cu alloy. A resolution of 2.2 nm was obtained for a combination of SE and BSE signals with a mix of topographic and compositional contrasts (Figure 2A). Simultaneously, an energy-filtered BSE signal was acquired with a resolution of 2.7 nm and a compositional contrast was observed (Figure 2B). Furthermore, Monte Carlo simulations were used to understand and to optimize the SEM parameters of these different imaging modes.

The HITACHI SU-8230 CFE-SEM provides low accelerating voltage, deceleration mode and energy-filtration of the electron signals and thus allows the characterization of the microstructure of materials with high spatial resolution at the nanoscale with various types of contrasts. The development of these new technologies permits to extend the imaging capabilities of the SEM towards new nanoscale applications.

Fig. 1: High resolution micrograph obtained with a CFE-SEM. (A) SE micrograph of a CNT decorated with Pt nanoparticles was acquired at 50 V in deceleration mode with the top detector. (B) Energy-filtered BSE micrograph of an AA2099 Al-Li alloy acquired at low energy with the upper detector.

Fig. 2: High resolution micrograph obtained with a CFE-SEM. CNTs decorated with Pt nanoparticles micrographs were acquired at 1 kV with: (A) combination of secondary and backscattered electron (SE+BSE) signals by the upper detector; (B) energy-filtered BSE signal by the top detector.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2654 POROSITY DETERMINATION ON IRON ORE PELLETS USING OPTICAL MICROSCOPE AND ELECTRON MICROSCOPE

Graça L. M.1, Lagoeiro L. E.2, Vicente T. M.3
1Geology Department, School of Mines, Federal University of Ouro Preto, 2Geology Department, School of Mines, Federal University of Ouro Preto, 3Mining Department, School of Mines, Federal University of Ouro Preto.
vicente_thais@hotmail.com

New procedures have been developed with the aim of improving the iron ore characterization and its agglomerated product, the pellets. The pelletizing plants have a considerable importance worldwide, as it becomes feasibly economic to use the fine particles (P90 of 0.045 mm). One of the main physical characteristics of pellets is its porosity, which directly interferes on its geometallurgical quality. Facing the lack of specific equipment to determine the porosity in indurated pellet; two methodologies were evaluated in order to determine the values for this variable: the reflected light optical microscope (OM) with and the scanning electron microscopy with the electron backscatter diffraction (SEM-EBSD). The same areas of the pellets (edge and center) were analyzed in both techniques, and with equal magnification (100X), in order to compare the results. Considering the MO results, mosaics of each examined area were created for subsequent imaging treatment that consisted of manual outline of regions that corresponds to the pore areas. The marked areas were subsequently quantified by specific software and it represented the pore percentages for each evaluated area. Considering the SEM-EBSD results, indexing maps of the crystal lattices of hematite, magnetite, wusthite and quartz phases were produced. In addition to the percentage of each mineral phase, the generated map determines the zero solution, which represents the regions of no indexing and therefore with no mineral phase. The percentage of zero solution in this technique represents the existing pores on the investigated area. It was collected three pellet samples, in each one the center and the edge were investigated. The percentages determined by EBSD were higher than those found by MO, both on border and center areas. The outline of the pores from MO was manual, which depends on the observer judgment and that may influence the final results. On the other hand, the SEM-EBSD does not index the amorphous phases, which is generated by the induration process. Although the amorphous material occurs in low percentages, it increases the zero solution. The determined porosity in both methods was higher on the central region of the pellet, as it was expected, since this region settles the nucleating particles. As in the MO, the average of the results obtained in edge regions of the pellet was 48.8% and in the central region of the pellet 51.8%. From the SEM, the average results obtained at the edge of the pellets was 53.66% and at the center 55.63%. Regions with higher porosity showed more differences in results between the methodologies. The analyzes made by the OM and EBSD showed coherent and consistent data when two methods were compared.

This study was partially supported by the FAPEMIG and the CNPq. All the analyses were performed in the MICROLAB at the Federal University of Ouro Preto.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2700 Volume reconstruction of biological samples by alternate physical and virtual slicing

Hovorka M.1, Boughorbel F.2, Potocek P.2, Cernohorsky P.1, van den Boogaard R.2, Hekking L.2, Korkmaz E.2, Langhorst M.2, Lich B.2
1FEI, Podnikatelska 6, 635 00 Brno, Czech Republic, 2FEI, Achtseweg Noord 5, 5651 GG Eindhoven, Netherlands
milos.hovorka@fei.com

The ability of scanning electron microscopy (SEM) to image large volumes with high spatial resolution, throughput and reliability goes hand in hand with the development of new approaches for data acquisition. Resin embedded tissues stained with heavy metals pose typical challenges with generally low electrical conductivity and charging. One way of imaging them in SEM is based on alternate slicing and imaging of the block-face. SBFSEM (Serial Block-Face SEM) utilizes an ultramicrotome inside the SEM chamber to cut slices of defined thickness. The revealed block-face is scanned and backscattered electrons are collected [1]. The depth resolution is determined by the achievable slice thickness and imposes a limit on voxel isotropy and on the quality of the reconstructed 3D information. The acquisition parameters and hence data throughput depend not only on the sample properties but significantly on the detection part of the microscope as well. Sample charging can be suppressed by working in low vacuum mode, in-situ coating of the surface with a very thin metal layer, by the usage of the accelerating voltage in the low kV range or introducing more metals during sample preparation.

 

We introduce the new integrated solution for SEM volume data acquisition based on a refined SBFSEM technique. It combines physical and virtual slicing which allows for extending the current resolution limit. Virtual slicing is enabled by using the MED-SEM (multi-energy deconvolution SEM) which is a non-destructive technique capable of high-resolution reconstruction of the top layers of the sample [2]. Following each cut, the exposed block-face is imaged and not only one image but a series of images is acquired using different accelerating voltages. Collected images serve as the input for a deconvolution algorithm that computes several subsurface layers. Subsequently, a given thickness of the tissue is removed mechanically using a diamond knife, a fresh block-face is exposed and the whole process is repeated for the needed number of iterations. While in the case of physical slicing the minimal slice thickness, and thus the depth resolution, is limited; virtual slicing is capable of extending it towards nanometer range and hence high-resolution isotropic datasets can be generated. To allow automatic data acquisition the whole workflow was integrated into a hardware and software solution that combines an SEM, an in-situ microtome and a reconstruction software. Increased ease of use is further facilitated through newly developed advanced auto-functions for electron column alignment.

 

References

[1] B. Titze & W. Denk, Journal of Microscopy, vol. 250(2), pp. 101–110 (2013).

[2] F. Boughorbel et al., SEM Imaging Method, Patent US 8,232,523 B2, 31st July 2012.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2793 Monte Carlo simulations of electron trajectories for the study of betavoltaic battery configurations

Napchan E.1
1DLM Enterprises, London NW6 1QH, UK
eli@napchan.com

Battery development goals are to produce small, light, safe, high power and very long lasting batteries. Betavoltaics batteries use semiconductors to convert beta particles (electrons) emitted from a radioactive source, much like photovoltaic panels convert sunlight to electricity. For betavoltaic devices the source can be within the devices themselves, while the radiant sun energy comes from outside the photovoltaic devices. A further difference is that betavoltaic cells can be stacked up.
The simplest structure for a betavoltaic battery consists of the beta layer on top of a pn junction producing electron-hole pairs, which are collected on both sides of the junction. Beta emission in the layer is isotropic within the layer, with randomization of the emission location and the emission characteristics. Each electron emission is isotropic in a sphere, calculated using direction cosines from the random localized emission point.
The Monte Carlo simulation program used is called MC–SET and deals with deposited beam energy calculations and with multi-layers. The simulation tracks each electron in its trajectory inside the specimen, and at each step calculates the energy lost by the electron. The energy deposited from all the electrons in the simulation is stored in a 3-D energy matrix. Other parts of the electrons energy, such as backscattered, transmitted and out to the device electrons are also recorded during the simulation.
The purpose of this investigation is to describe a methodology for simulating beta voltaic batteries, with different geometric configurations. The relationship between the nuclear radiation emission and the energy obtainable is evaluated.
Figure 1 presents the electron depth dose for a bulk Ni specimen, with a normal beam direction. The two selected energies correspond to the average beta emission energy and the maximum beta energy for the Ni-63 isotope. For the high value absorption in the Ni layer occurs at depths of up to about 10 um, while the curve for 17 keV indicates that all the average beta particle energy is absorbed within 1 um of Ni-63. Figure 1 inset shows the depth dose for a layered structure of Si-Ni-63-Si, for 2 um Ni-63 layer. This curve shows the relative amounts of energy deposited in the Ni-63 and the Si layers, the latter being the effective maximum energy available for conversion.
Figure 2 gives the energy deposited in one Si layer, for increasing values of Ni-63 thickness. The left hand curve corresponds to same activity for all layers, i.e. same number of beta emissions, while the right hand curve corresponds to all layers having the same specific activity (beta emissions per gram), corresponding to a typical Ni-63 isotope specific activity of 15 Ci/gr.

Fig. 1: Ni electron depth dose for 2 energies based on Ni-63 emission data and (inset) electron depth dose for Si-Ni-63-Si device

Fig. 2: Relative amount of beta energy emission from Ni-63 layer deposited in Si layer
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-2865 Estimation of the resolution in 2D Wet-STEM and Wet-STEM tomography by Monte Carlo simulations

Xiao J.1, Perret A.1, Foray G.1, Masenelli-Varlot K.1
1Université de Lyon, INSA-Lyon, Villeurbanne, France
juan.xiao@insa-lyon.fr

The microstructural characterization of water-containing materials in conditions closer to their native state is possible through Environmental scanning electron microscopy (ESEM) experiments. Among the possible ESEM imaging modes, Wet-STEM permits to observe nano-objects in suspension in a liquid with a nanometer resolution [1]. This technique is based on STEM (Scanning Transmission Electron Microscopy) configuration in ESEM. In parallel, a device has been developed for the characterization of the 3D structure of non-conductive and low-contrast materials, and it gives a compromise between the resolution level of a few tens of nm and the large tomogram size due to the large thickness of transparency [2]. Very recently, the implementation of a Peltier stage in the tomographic sample holder has enabled the acquisition of image series in wet samples (wet-STEM tomography) [3].
During Wet-STEM experiments, the contrast is influenced by water thickness and the particle size and composition. Furthermore, the thickness of water varies with the tilt angles, which can lead to contrast inversions. When performing Wet-STEM tomography, contrast inversions have to be avoided when tilting the sample since they may lead to reconstruction artifacts.
In the first part of this study, Monte Carlo simulations will be used to calculate the contrast which can be obtained when observing nanoparticles in suspension in water. We will present how the contrast is affected by the position of a Carbon particle, and its dimension compared to the thickness of the water film (see Figure 1). Then, the contrasts in an experimental Wet-STEM image (see Figure 2) and those calculated from Monte Carlo simulations will be compared.
In the second part, the Monte Carlo simulations will be used to define the best suited sample geometry for Wet-STEM tomography experiments. In particular, the conditions to avoid contrast inversion will be defined, and the resolution will be discussed in function of the nanoparticle composition.

[1] A. Bogner et al., Ultramicroscopy, 104 (2005), 290-301.
[2] Russias J, J. Am. Ceram. Soc., 91,(2008), 2337-2342. P. Jornsanoh et al., Ultramicroscopy, 111 (2011), 1247-1254.
[3] K. Masenelli-Varlot et al., Microscopy and Microanalysis, 2014. doi:10.1017/S1431927614000105

The authors acknowledge the Centre Lyonnais de Microscopie (CLYM) for the access to the microscope, the CSC and Institut Universitaire de France for financial support.

Fig. 1: Numbers of collected electrons for several Carbon particles with different thicknesses of water

Fig. 2: Contrast variation for several Carbon particles with different thicknesses of water

Fig. 3: Experimental Wet-STEM image of a SBA latex suspension – scale bar 500 nm
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3005 Microstructural characterization of metallic materials using advanced SEM techniques

Piňos J.1, Konvalina I.1, Kasl J.2, Jandová D.2, Mikmeková Š.1
11. Institute of Scientific Instruments of the ASCR, v.v.i., Czech Republic, 22. VZU Plzen- research and Testing Institute Plzen, Czech Republic
pinos@isibrno.cz

The development of advanced materials is inseparably connected with detailed knowledge of the relationship between microstructure and mechanical properties. Traditional high-voltage scanning electron microscopy (SEM) is one of the most commonly used techniques for microstructure analysis, though it may be insufficient particularly for the characterization of advanced materials exhibiting a complex microstructure.

The benefits of using slow electrons have been described in several articles [e.g. 1,2]. Experiments have been performed with a XHR SEM Magellan 400L (FEI Company) equipped with two detectors for secondary electrons (SE), an Everhart Thornley detector and an in-lens TLD detector, and solid-state BSE detector (CBS) located below the pole piece. This microscope can also be operated in the beam deceleration (BD) mode [3]. The field of the BD not only decelerates the primary electrons, but also accelerates the emitted (signal) electrons towards the detector. Furthermore, high-angle backscattered electrons (BSE) are also collimated towards the optical axis and are detected. These electrons carry, first and foremost, crystal orientation contrast. SE and low-angle BSE can be detected by the TLD detector located inside the objective lens. Angle-resolved detection of BSE is performed using a CBS detector divided into four concentric segments.

Fig. 1 shows the dependence of material contrast between BN precipitates, Laves phase and matrix on the landing energy and increasing of contrast between differently oriented areas in advanced creep resistant steel COST CB2. The presence of BN precipitates and Laves phase has been verified by EDX analysis. The prospect of angular separation of BSE is shown in Fig. 2. The trajectories of signal electrons were simulated in EOD software [4]. It is clearly visible that the low-angle BSE detected by means of the segments closest to the optical axis (S1 and S2) provide information about the chemical composition of the specimen. Segment S3 offers high crystallographic contrast, and material contrast between Laves phase and matrix is entirely suppressed. The final segment S4 exhibits topographic contrast due to the detection of electrons emitted under very high angles from the optical axis, which are products of the interaction of the primary electrons with surface irregularities.

[1] L. Raimer: Image formation in low-voltage SEM. SPIE Press (1993)

[2] I. Mullerova and L. Frank: Scanning low-energy electron microscopy. Adv. Imag. Elect. Phys., Vol. 128 (2003)

[3] Product specification. XHR SEM Magellan 400L. FEI Company

[4] J. Zlamal and B. Lencova: Nucl. Instr. Meth. Phys. Res. A, Vol. 645 (2011)

This work was supported by project no. TE0120118 (Competence Center: Electron Microscopy). The author Šárka Mikmeková is sponsored by an FEI Company Scholarship.

Fig. 1: Fig. 1 The same field of view imaged at 4 keV, 1 keV and 0.44 keV landing energy using the DB mode (specimen bias – 4 kV in all cases), together with corresponding EDX maps of N and Mo distribution.

Fig. 2: Fig. 2 Micrographs obtained at 440 eV landing energy (specimen bias – 4 kV) by means of separated parts of the CBS detector, together with information about detected angles by each segment.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3064 An approach to study antigenotoxicity assay in plants using Confocal Laser Scannig Microscopy and Scanning Electron Microscopy.

Walia A.1, Sharma M.2, Kumar K.1, Bhardwaj R.2, Thukral A. K.2
1Centre for Emerging Life Sciences, Guru Nanak Dev University, Amritsar, Punjab, India, 2Department of Botanical & Environmental Sciences, Guru Nanak Dev University, Amritsar, Punjab, India
adwalia@gmail.com

A new and rapid procedure has been followed using Confocal Laser Scannig Microscopy and Scanning Electron Microscopy for use in the determination of genotoxicity and antigenotoxicity of compounds in plants.

In the present study the above two techniques were used to analyse the genotoxicity and antigenotoxicity effects of compounds on plant system. Certain fluorescent dyes are more reliable indicators of cell viability than the commonly used colored dyes. DNA intercalating dyes like propidium iodide are known to pass only through the membranes of dead or dying cells. Staining with propidium iodide (PI) can be used for the determination of non viable cells. In this study we have evaluated the genotoxic effect of Cr(VI) at different concentrations along with ascorbic acid as a reducing agent in the plant roots. The results showed that the metal ions have a significant effect on the viability of root cells in a dose dependent manner. Also the reducing agent has its effect on reversing the negative effect of these metal ions.

The metal ions are not only genotoxic to plants but they also affect their root growth. To study the pattern of root growth using the same compounds we have scanned the roots of these plants using Scanning electron microscope. The results have shown significant changes in the features of the root tips in different binary combinations of Cr(VI) and ascorbic acid. The study suggests that these techniques can be effectively used for the study of physiological toxicity and antigenotoxicity assays in plants.

We are thankful to University Grants Commission for providing financial assistance to Dr. A.K. Thukral to conduct this work .

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3090 Low-voltage STEM tomography: an alternative for soft polymers and hydrated samples

Masenelli-Varlot K.1, Roiban L.1, Malchère A.1, Dhungana D. S.1, Xiao J.1, Jomaa M. H.1, 2, Ferreira J.1, Cavaillé J. Y.1, Seveyrat L.2, Lebrun L.2
1Université de Lyon, INSA-Lyon, CNRS, MATEIS, 7 avenue J. Capelle, 69621 Villeurbanne cedex, France., 2Université de Lyon, INSA-Lyon, LGEF, 8 rue de la physique, 69621 Villeurbanne cedex, France.
Karine.Masenelli-Varlot@insa-lyon.fr

Tomography has become a key characterization tool in materials science as well as in biology. The principle of tomography is based on the acquisition of a series of projections images at different tilt angles, computation of the volume using dedicated algorithms and data segmentation and three-dimensional (3D) quantification. Several tomography techniques are available, using different types of radiations, depending on the observation scale. X-rays are currently used for the 0.5 µm–1 mm resolution level and the three-dimensional characterization of nanoscaled structures requires transmission electron microscopy (TEM) tomography or an atom-probe approach. At the mesoscopic scale, corresponding to a resolution level between 10 nm and 500 nm, Scanning Electron Microscopy (SEM)-based techniques – such as Focused ion Beam (FIB) or serial block face SEM – use a slice-and-view method to directly obtain slices of the materials volume.
Moreover, in Environmental SEM (ESEM), the presence of the gaseous environment and the control of the sample temperature have also permitted the imaging of nanoparticles in liquid with a nanometer resolution, through STEM-in-SEM observations [1]. Its main advantage lays in the fact that water condensation or evaporation can be finely tuned by varying the environmental pressure, which enables in situ hydration / dehydration experiments.

In the first part of this presentation, we will briefly present an alternative tomography technique for the 3D characterization of materials at the mesoscopic scale. This method, called low-voltage STEM tomography, consists in performing tilted tomography in a SEM (in the transmission mode, the so-called STEM-in-SEM mode), see Figure 1 [2]. The potentialities of low-voltage STEM tomography will be compared to that of other 3D techniques through the study of polyurethane films containing two different kinds of carbon nanotubes (see Figure 2).
In the second part of this presentation, we will present the possibility of observing the 3D structure of hydrated materials [3]. In particular, we will discuss the role of different experimental parameters such as the temperature and the electron dose received by the sample. Two examples will be used: a porous material and a latex suspension. Monte Carlo simulations will also be used to estimate the resolution which can be expected in both cases.

[1] A. Bogner et al., Ultramicroscopy 104 (2005), 290-301.
[2] P. Jornsanoh et al., Ultramicroscopy 111 (2011), 1247-1254.
[3] K. Masenelli-Varlot et al., Microscopy and Microanalysis, http://dx.doi.org/ 10.1017/S1431927614000105

The authors acknowledge the CLYM (Centre Lyonnais de Microscopie) for the access to the ESEM XL30FEG microscope, the Agence Nationale de la Recherche and the Institut Universitaire de France for financial support.

Fig. 1: Device for low-voltage STEM tomography, composed of a) and b) piezoelectric elements; c) sample holder and d) STEM detector. The dashed lines represent the position of the Peltier stage.

Fig. 2: Low-voltage STEM tomography on polyurethane thin films containing 2 vol.% of carbon nanotubes (CNT): orthogonal slices extracted from the volume. a) grafted CNTs and b) ungrafted CNTs.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3106 SEM observation of several biological samples using a hydrophilic asymmetrical tetraammonium-type room temperature ionic liquid as a visualizing agent

ABE S.1, KAWAI K.2, YOSHIDA Y.1
1Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan, 2Miyoshi Oil & Fat Co., Ltd., Tokyo, Japan
sabe@den.hokudai.ac.jp

A room temperature ionic liquid (RTIL) is an organic salt that is liquid at room temperature and has specific physical properties such as noncombustibility, no vapor pressure, high heat resistance, and high ionic conductivity. These unique properties have led many researchers to study the application of ionic liquids in various fields including electronics and chemistry. Kuwabata et al reported that they had succeeded in using RTILs for electroconductive-pretreatment of some samples for scanning electron microscopy (SEM). Because RTILs have electrical conductivity and very low vapor pressure, they can maintain a liquid state even in vacuum such as in an SEM sample chamber. Thus, they can act as visualizing agent for SEM observation. Some types of RTILs, such as imidazolium salts, pyrimidinium salts and ammonium-type salts, have been investigated for the electroconductive pretreatment.

To apply this technique for wet biological samples, we used a novel asymmetrical tetraammonium-type RTIL (HILEM IL1000, Hitachi High-Technologies Corp., Tokyo, Japan). It has chemical structure similar to a choline, which is a bioactive compound. Its properties such as high fluidity, hydrophilicity and biocompatibility can allow using as the agent for SEM observation of biological samples. To elucidate usefullness of RTIL pretreatment, we investigated the conductivity pretreatment for SEM observation of the novel tetraammonium-type RTIL (IL1000). By immersion in an IL1000 solution, clear SEM images of several types of biological samples were successfully observed. We also succeeded in visualization of some bio samples, such as protozoans, red blood cells and bacteria, using IL1000 without dilution. In particular, the size of red blood cells pretreated with IL1000 was in good agreement with that of optical microscopic (OM) observation. When they were treated with traditional method, the obtained SEM images were shrunken compared with those in OM observation. Thus, these results suggested that the tetraammonium-type RTIL used in t his study (IL1000) was suitable for visualizing of biological samples for SEM observation as a "living" morphology. In addition, treatment without the need for dilution can obviate the need for adjusting the RTIL concentration and provide for a rapid and easy conductivity treatment for wet biological samples.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3134 Topotactic transformations of Goethite to Hematite during low metamorphic conditions

Souza D. S.1, Lagoeiro L. E.1, Barbosa P. F.2
1Federal University of Ouro Preto,Departament of Geology,Minas Gerais, Brazil, 2Federal University of Minas Gerais, Microscopy Center,Minas Gerais,Brazil
dansilvasouza@gmail.com

Crystallographic similarities between hematite and goethite allow us to model some topotactic transformation between these two minerals. For natural occurrence of goethite and hematite, such process is scarcely known. The aim of this contribution is to investigate the transformation that occurs in response to a change in deformation and metamorphic conditions of iron-formation rocks deformed at a very low temperature. We applied the EBSD technique to investigate the transformation between ferric oxides and oxyhydroxides combined with the observation of microstructures related to that process.
The samples came from iron-formation rocks in southeast of Brazil, in a region called Iron Quadrangle. Their mineralogy consists basically of quartz, iron oxides and oxyhydroxides. On the optical microscope magnetite is almost completely oxidized to hematite. Several magnetite core grains are filled with goethite and rimmed by hematite grains. The inner goethite and surrounding hematite grains occur in aggregates of irregularly shaped grains of varied sizes.
The EBSD analyzes of the clasts show a close relationship between different crystallographic axes of hematite and goethite crystals. The poles to the basal planes of hematite {001} match those of goethite crystals {001}.
Hematite and goethite, although belonging to different space group symmetries, have similar close packing structures. The structures of hematite and goethite can be described as a slightly distorted hexagonally close-packed of anions (O2- and OH-) stacked along their [c] axes. In these conditions, atom displacements are reduced, so that clear vectorial relations can be established between crystal parameters of the two structures. It is known that the transformation to goethite does not modify significantly the layers of anions in the structure of hematite. Therefore, the expected crystallographic orientation relationship can be described between these two phases.
We proposed that the transformation described in the studied iron formation rocks was performed in two different stages. Initially the original magnetite crystals were hydrated and transformed by oxidation into goethite. This might have been caused by a percolation of low temperature aqueous fluids in the early stages of the deformation. Subsequently, as the deformation proceeds and the temperature increases with the progressive metamorphism of the iron-formations, the newly formed goethite crystals dehydrate and transform into hematite. This can be described as a topotactic transformation because both hematite and goethite show coincidence of orientation in planes {0001} and {001}, and directions <a> and [010], respectively.

Fig. 1: Figure 1 –porphyroclast of magnetite completely transformed to goethite (dark gray) goethite in the magnetite crystal edges is transformed to hematite (light gray); matrix of elongated crystals of goethite (dark gray) occurring as aggregates with quartz ribbons.

Fig. 2: Figure 2- Pole figures for Goethite (a) and Hematite (b). Lower hemisphere, equal area projection. Note a coincidence between the poles to the basal planes of Goethite and Hematite, {001} and {0001}, respectively. The Y0 direction of the microscope system corresponds to the Z0 direction of the sample reference system.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3145 Application of EBSD technique to analyze the microstructure and texture in Peridotites from Archipelago of São Pedro and São Paulo

Pinto S. O.1, Lagoeiro L. E.1, Barbosa P. F.2, Simões L. A.3
1Federal University of Ouro Preto, 2Federal University of Minas Gerais, 3State University of Rio Claro
suellen_olivia@yahoo.com.br

The Archipelago of São Pedro and São Paulo consists in a set of island where rocks from the Upper Mantle outcrops above the sea level [1]. They are a rare example in the world with such this feature. Samples from their rocks were collected for microstructural and crystallographic texture analysis with the aim to get insight into the mechanisms involved in the formation of these rocks, as well as, to infer the deformation mechanisms that developed the observed structures. To achieve that, we use a combination of optical microscopy, to see the whole picture of the microstructure alongside with the powerful of the EBSD analysis. The rocks are Ultramylonite of Peridotite composition. Porphiroclasts of Olivine are deformed by dislocation creep and show subgrains, sweeping undolose extinction, and tails of recrystallized grains of delta type indicate a sinistral sense of shear [2]. The new recrystallized grains adjacent to the clast show crystallographic preferred orientation (CPO) compatible with recrystallization mechanisms of subgrain rotation with some grain boundary migration. In contrast, moving towards the matrix the Olivine grains are much smaller than those close to the clast and there is a weak to random crystallographic texture. A mechanism involving grain boundary sliding assisted by diffusional creep is proposed for the accommodation of the deformation in the matrix. The main challenge is that, it is also not completely ruled out some reaction between minerals in the matrix. Since some grains do not match any minerals loaded in the EBSD acquisition software (CHANNEL 5, in Flamenco mode), and if the nonindexing problem is a matter of phase reaction or the resolution SEM used in the analysis.


References:
[1] www.mar.mil.br/secirm/publicacao/arquipe.pdf
[2] Ron H. Vernon, A Practical Guide to Rock Microstructure, Cambridge University Press, Oct 7, 2004

Fig. 1: Optical image from a characteristic clast from the ASPSP.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3156 Microflow and Thermal Control System Design for Wet Cell in the Scanning Electron Microscope

Lee H. H.1, Lee C. Y.1, Chiang C. L.1, Tsai K. C.1, Lin W. T.1, Wang H. W.1, Fang J. M.2, Huang T. W.3, Liu S. Y.3, Tsai C. Y.3, Chen F. R.3
1Center for Measurement Standards, Industrial Technology Research Institute, Taiwan R.O.C., 2Taiwan Electron Microscope Instrument Corporation, Taiwan R.O.C., 3Engineering and System Science Department, National Tsing Hua University, Taiwan R.O.C.
kylelee@itri.org.tw

Scanning Electron Microscope has been widely used in different scientific areas such as material analysis, biology and life science. SEM integrated with a wet cell was proposed in recent years to satisfy the needs from live cell imaging. In the SEM, it requires a vacuum chamber to allow the operation of the electron beam and to minimize the scattering from other sources. To extend the ability of live cell observation in the SEM, the Si3N4 thin film supported by silicon microchip was developed in order to cultivate biological materials in the wet cell. Therefore, the wet cell can be used to visualize live tissues in fully hydrated conditions and to maintain the culture environment. It would be particularly valuable when applying to the analysis of lipid membranes in cells as they are difficult to preserve during dehydration and washing steps. The processes of sample preparation can be more efficient by using the wet cell. Furthermore, with the continuous flow control and real-time monitoring, the long-term operation can be achieved and also expand the applications of the wet cell.

In Fig. 1, it shows the meshes of simulation model in the wet cell. The pressure is critical because the flowrate needs to be well maintained in order not to break the thin film. To increase the flowrate and reduce the costs of the fluid mechanics, the geometry and flow conditions were optimized based on the skills of Design Of Experiments. The resulting pressure on the 50 nm thin film was simulated by CFD software (ANSYS Fluent v14) and the results are shown in Fig. 2. It demonstrates that the inlet flowrate still could be raised and the size of the wet cell can be reduced. With regard to the thermal control system, a preheating system of buffer liquid was developed by using a thermal control module. In addition to preheating system for the buffer liquid, an embedded micro thermal control element was also designed inside the wet cell with the capability of fine-tuning so as to achieve accurate and rapid temperature control. Furthermore, several design parameters including noiseless, non-vibration and long working life were also considered for the thermal control system. After preliminary experiments, the results are shown in Fig. 3. and the heating rate inside the wet cell can be achieved to 3.7 ℃ per minute. Eventually, the microflow and thermal control systems were integrated and the system architecture is shown as Fig. 4. With the microflow and thermal control modules integrated in the SEM, the flowrate and fluid temperature can be adjusted by users and the flow conditions including temperature, pressure and even the fluid properties can be simultaneously monitored as well.

Thanks for the technical assistance and suggestions from R&D team of Taiwan Electron Microscope Instrument Corporation (TEMIC) during system integration.

Fig. 1: Meshes of the liquid volume inside the wet cell

Fig. 2: The pressure on the thin film inside the wet cell

Fig. 3: The temperature variation in the wet cell during heating process

Fig. 4: System architecture of microflow and thermal control system

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3166 Combined EDS and WDS analysis of thin specimens with high spatial and energy resolution in the scanning electron microscope

Mitsche S.1, Poelt P.1
1Graz University of Technology, Institute for Electron Microscopy and Nanoanalysis, Steyrergasse 17, 8010 Graz, Austria
stefan.mitsche@felmi-zfe.at

X-ray analysis of bulk samples by energy-dispersive x-ray spectrometry (EDS) in a scanning electron microscope (SEM) is widely used to gain chemical information of materials. The combination of EDS and bulk samples is limited by the low energy resolution of EDS, the associated high detection limit and the low spatial resolution due to the interaction volume of the electrons which is correlated to the beam energy used. A wavelength-dispersive spectrometer (WDS) can increase the energy resolution dramatically. An improvement of the spatial resolution can be obtained by use of very thin samples (less than 50 nm). The combination of EDS, WDS and thin specimens improves the spatial and energy resolution and detection limits and keeps the analysis time down.

All investigations were performed on a Zeiss Ultra 55 equipped with a 10 mm2 Si(Li)–EDS detector and a parallel beam WDS detector with a multi capillary optics from EDAX. Thin specimens were prepared by ion milling with a FIB.

In order to minimize shadowing effects a special sample holder was designed (see Fig. 1). With this sample holder a linescan across a FIB-lamella from a semiconductor device (see Figure 2a) was performed by EDS and WDS. The resulting intensities of the Ti-K peak recorded with both EDS (grey) and WDS (dark grey) are shown in Figure 2b (dwell time 1000ms for EDS and 2000ms for WDS). This Figure demonstrates that the x-ray intensities for both EDS and WDS are sufficiently high for the x-ray analysis of thin specimens. Figures 2c and 2d demonstrate the benefit of the high energy resolution of WDS. Whereas the signal of the W-M line recorded with EDS runs quite similar to that of the Si-K line, WDS clearly proves that in the region with high Si content no W is present.

A FIB-lamella of a circuit board with a 40 nm Au layer followed by a 14 nm Pd layer was analyzed by EDS to validate the improvement of the spatial resolution of x-ray analysis by investigating thin instead of bulk specimens (see Figure 3a). The linescan across these two layers is plotted in Figure 3b. This Figure proves that both layers can be detected separately by x-ray analysis. As a Lorentz distribution was the best fit to the scan of the Pd intensity its half width was used as a measure for the thickness of the Pd layer. It gave a value of 15 nm which is close to the value obtained by the electron image

I would like to thank the FFG for the financial support (Project number: 825165)

Fig. 1: Modified specimen holder, a) side view b) top view c) bottom view of the optimized holder; 1: optimized holder, 2: original Zeiss STEM holder, 3: FIB-lamella

Fig. 2: X-ray linescans across a semiconductor structure, a) BSE image with linescan marked (image width: 800 nm) , b) EDS (grey) and WDS (dark grey) signal of the Ti-K line c) EDS and WDS signal of the Si-K line, d) EDS and WDS signal of the W-M line; abscissa: distance in microns, ordinate: number of x-rays.

Fig. 3: X-ray linescan across a circuit board, a) BSE image with linescan marked (image width: 380 nm), b) EDS signal of Au-L line (dark grey) and Pd-L line (dashed line grey), light grey the Lorentz fit to the Pd scan; abscissa: distance in nanometre, ordinate: number of x-rays.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3232 PERFORMANCE OF YAG:Ce SCINTILLATORS FOR LOW-ENERGY ELECTRON DETECTORS IN S(T)EM

Lalinsky O.1, Bok J.1, Schauer P.1, Frank L.1
1Institute of Scientific Instruments of the ASCR, Brno, Czech Republic
xodr@isibrno.cz

Cerium activated single crystals of yttrium aluminium garnet (YAG:Ce) Y3-xCexAl5O12 are widely used as scintillators in electron detectors for S(T)EM [1]. Nowadays, it is sometimes necessary to detect low-energy electrons without post-acceleration. In such cases, extremely sensitive detectors are required that are able to detect even electrons with energies of only hundreds of eV while avoiding charging of the scintillator surface. However, commonly used scintillators strongly lose their light yield with the decrease of the incident electron energy [2]. Moreover, a thinner conductive layer on the scintillator surface has to be used to allow low-energy electrons to pass through. Possible charging of the surface negatively affects its cathodoluminescence (CL) light yield. The low-energy electron excitation takes place closer to the scintillator surface where damage can be expected owing to its preparation, which also reduces the CL light yield. The aim was to study the influence of the scintillator and its conductive layer on the low-energy electron detection efficiency.
In general, the following demands are made of the conductive layer: it should have the highest possible optical reflectivity, conductivity and electron transparency. These demands are mostly met for metals with a low atomic number. However, if the layer is very thin, it can form “islands”, i.e. a non-continuous layer of drastically decreased conductivity. We decided to apply scandium as a possible option. The YAG:Ce single crystals were studied using both Monte Carlo simulation and CL measurement. The MC method (Fig. 1) used Mott cross-sections and the Bethe slowing-down approximation. Using the CL apparatus (Fig. 2) [3], incident electron energy can be changed from 0.8 to 10 keV. The detection dynamic range spans 5 orders of magnitude. The experimental results are shown in Fig. 3. The significant decrease of efficiency at lower energies may be caused by the layer which doesn’t allow more electrons to pass through, by the YAG:Ce single crystal that has a lower light yield near the surface, and finally, if the layer isn’t conductive enough, it can be charged and retard incident electrons. Even so, we have found that the YAG:Ce scintillator with a 3 nm scandium layer is applicable for the detection of electrons having an energy as low as 800 eV.

References

[1] P. Schauer, Nucl. Instrum. Methods Phys. Res. B 21 (2011) 2572–2577.
[2] G. F. J. Garlick, Brit. J. Appl. Phys. 13 (1962) 541–547.
[3] J. Bok, P. Schauer, Rev. Sci. Instrum. 82 (2011) 113109.

Thanks are due to CRYTUR, Ltd., for the supply of scintillators, to J. Sobota for the preparation of scandium layers, to the Technology Agency of the Czech Republic (TE01020118), to the European Commission and to the Ministry of Education, Youth and Sports of the Czech Republic (CZ.1.07/2.3.00/20.0103).

Fig. 1: Interaction volumes in the YAG:Ce single crystal simulated by the Monte Carlo method. Simulated without any conductive surface layer.

Fig. 2: Experimental equipment used for cathodoluminescence (CL) property measurements.

Fig. 3: The cathodoluminescence light yield of the YAG:Ce single crystal scintillator coated with a scandium layer of a thickness of 3 and 5 nm, respectively, as a function of the incident electron energy.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3358 Characterization of β-phase in Al-Mg-Si alloys by SLEEM and STLEEM techniques

Ligas A.1, Hida S.2, Matsuda K.3, Mikmeková Š.1
1Institute of Scientific Instruments of the ASCR, v.v.i., Czech Republic, 2Graduate School for Science & Engineering for Education, University of Toyama, Japan, 3Graduate School for Science & Engineering for Research, University of Toyama, Japan
ales.ligas@isibrno.cz

Knowledge of the distribution and morphology of the Mg2Si precipitates (i.e. β-phase) in Al-Mg-Si alloys are very important for many practical reasons [1,2] and the scanning electron microscopy (SEM) technique is widely used for their visualization. Unfortunately, in the standard SEM images these precipitates are barely visible and finding them can be very difficult. Using the cathode lens (CL) mode in the SEM (so called SLEEM [3]) these difficulties have been overcome and a very high contrast between the hexagonal-shaped β-phase and the matrix has been obtained. Moreover, it has been found that the SLEEM images offer the possibility to distinguish between the hexagonal-shaped and the conventional β-phase based on their different brightness, not only on their shape, which can be in some cases difficult or even impossible. Mg2Si precipitates have been also characterized by means of the scanning transmission low energy electron microscopy (STLEEM [4]) method based on the using of a STEM detector in the SEM operated in the CL mode.

[1] Laughlin DE, Miao WF. Automotive alloys II. TMS Warrendale PA. 1998; 63-79.
[2] Edwards GA, Stiller K, Dunlop GL, Couper MJ. The precipitation sequence in Al-Mg-Si alloys. Acta Materialia 1998; 46: 3893-3904
[3] Mullerova I, Frank L. Scanning low energy electron microscopy. Advances in imaging and electron physics 2003; 128: 304-443
[4] Mullerova I, Hovorka M, Hanzlikova R, Frank L. Very low energy scanning electron microscopy of free-standing ultrathin films. Materials Transactions 2010; 51: 265-270

The financial support of the project no. TE0120118 (Competence Centre: Electron Microscopy) from the Technology Agency of the Czech Republic is greatly acknowledged. The author (ŠM) is sponsored by FEI Company scholarship.

Fig. 1: Comparison between the SEM secondary electron image (a), backscattered electron image (b) obtained at 10keV and the SLEEM image (c) obtained at 2 keV landing energy of the hexagonal-shaped β phase.

Fig. 2: The same point of view imaged at 10 keV primary energy in the standard mode: by means of SE electrons (a) and BSE electrons (b) and in the CL mode: at 5 keV (c) and at 0.63 keV lading energy (d).

Fig. 3: The same point of view imaged at 10 keV primary energy in the standard mode: by means of SE electrons (a) and BSE electrons (b) and in the CL mode: at 5 keV (c) and at 0.63 keV lading energy (d).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-3491 Application of Ultra Low Voltage Secondary and Backscatter Imaging in FE-SEM for Imaging of Nanomaterials

Erdman N.1, Robertson V.1, Shibata M.1
1JEOL USA Inc
erdman@jeol.com

Significant advances in electron optics and detectors in field emission scanning electron microscope (FE-SEM) in the last decade have allowed the researchers to observe a variety of materials and biological specimens with ultra-high resolution and exceptional surface detail. In particular, low voltage imaging has been successfully employed as a key technique for charge control and reduction. Enhancements in electron column optics towards smaller chromatic and spherical aberration coefficients, with improved ability to deal with charging specimens via precise control of the landing energy of impact electrons and electron signal detection through in-column signal filtering or signal collection angle control have opened new avenues for specimen observation [1]. These new design improvements have significantly advanced the ability to image insulating specimens with previously unattainable nanometer scale resolution [2] at landing voltages as low as 10V (Fig. 1). In this paper we will discuss common approaches and challenges associated with ultra-low voltage imaging. The instrument employed for these studies is JSM-7800F ultra-high resolution FE-SEM that features a hybrid lens design and the ability to bias the specimen stage thus decelerating the primary beam (Gentle Beam). When beam deceleration is employed the accelerating voltage which along with lens aberrations determines the minimum probe size and thus the resolution limit is retarded by a negatively charged bias to a lower landing energy. The landing voltage can be varied to achieve the necessary charge balance as well as high resolution performance at ultra-low voltages. Beam deceleration also serves as a form of aberration correction [3]. The use of Gentle Beam function preserves all the advantages of high kV imaging (gun brightness, small probe size) with added advantages of reduced charging, reduced specimen contamination and improved surface detail. Moreover, through-the-lens detection system features an ability to precisely filter the detected signal, providing the user with an additional degree of control during the imaging. We will demonstrate our experiences with imaging a variety of specimens, such as zeolites, biological nanostructures, oxides, nano-structured metals and more. The advantages of low kV imaging for such techniques as cathodoluminescense imaging and voltage contrast will be highlighted. Additional methods for charge balance, such as reduced probe current and adjustment of scan speed will also be discussed. References: [1] D.C. Bell and N. Erdman. Low Voltage Electron Microscopy: Principles and Applications (2012) [2] S. Asahina et al., Microscopy and Analysis, (2012) p.S12. [3] L. Frank and I. Müllerová, Ultramicroscopy, 106 (2005) p. 28

Fig. 1: Anopore membrane filter imaged uncoated at 10V. Pore walls (14 and 21 nm) are clearly resolved.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-5734 Palynomorphology of Dianthus petraeus (Caryophyllaceae)

Mačukanović-Jocić M.1, Jarić S.2
1Faculty of Agriculture, University of Belgrade, 11080 Belgrade-Zemun, Serbia, 2Department of Ecology, Institute for Biological Research ‘Siniša Stanković’, University of Belgrade, 11060 Belgrade, Serbia
marmajo@agrif.bg.ac.rs

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-5765 E-BEAM CROSSLINKING AND THERMAL DEGRADATION OF HYDROGEL UNDER ELECTRON MICROSCOPE

Wong Y. H.1, Kuo C. H.1, Huang T. W.1, Liu S. Y.1, Hsieh H. Y.23, Chen F. R.1, Tseng F. G.14
1Department of Engineering and System Science, National Tsing Hua University, TAIWAN, 2Department of Mechanical Engineering, National Taiwan University, TAIWAN, 3Institute of NanoEngineering and MicroSystems, National Tsing Hua University, TAIWAN, 4Division of Mechanics, Research Center for Applied Science, Academia Sinica, TAIWAN
brian791213@gmail.com

Introduction

In the past decades, hydrogel has been becoming an important medium for incorporating cells together to from 2D or 3D structures for tissue engineering applications.Electron beam can be used to pattern the resulting hydrogels on silicon or glass surfaces with nanoscale and microscale feature sizes by radiation crosslinking[1]. The water content plays a role to form the hydrogen and hydroxyl radicals which initialize the polymerization reaction. To visualize the dynamic evolution of hydrogel, we use a hermetic micro-device (wet-cell) to preserve the hydrated hydrogel in vacuum system under electron illumination. We has reported the innovation of self-aligned micro wet-cell and demonstrated the TEM examination of hydrated Deinococcusradioduransin our previous work[2]. In this paper, we furthermore introduce an advancing micro wet-cell with miniature heater integrated to achieve a temperature manipulating for the rapid recovering of hydrogel.

Chip fabrication and set up

The multiple-electrode wet-cell device is composed of two silicon chips with complementary structure, as shown in Fig.1.The “cover piece“, a 3mm x 3mm square-shaped device made of 250μm-thick Si wafer, has an observing window which is formed by the bulk micromachining and covered by a silicon nitride membrane; the “electrode piece”, a 3mm x 6mm rectangle-shaped device made of 250μm-thick Si wafer, consists of a similar observing window with additional Ni/Cr heater (200nm/50nm, 20.07 kΩ) as well as the extended metal pad for wire connecting.

Experiment Results

To measure the temperature rising with increasing applied voltage, we use an infrared-thermal microscope to visualize the distribution and variation of temperature. Several different applied voltages and their corresponding temperatures are shown in Fig.2 . We can control our heater temperature ranging from 30 to 70 °C, reversibly. We also used the Hitachi TM-1000 and made a circuit on the side wall of the SEM, so that we can directly applied tunable voltage in the vacuum system. We first applied the e-beam radiation onto the specific region of GelMa. In Fig.3a-b, electron radiation posses higher effect on GelMa as we zoom in the field of view.The cross-linked regions, as shown in Fig.3 c-d, can be approximately characterized as 1,973μm2. The degradation of the hydrogel was observed after heating, and the cross-linked regions has been fully dissolved at a temperature of 50.2°C.Finally ,the detail results will be discussed in detail in the conference.

REFERENCES:

1. Tsu-wei Huang and Fu-Rong Chen,Korean Journal of Microscopy, 38, 41 (2008)
2. Tsu-wei Huang and Fan-Gang Tseng, Proc. MicroTAS’09, Jeju (2009)
3. Hsin-Yi Hsieh and Fan-Gang Tseng, Lab on a Chip, 10.1039/c3lc50884f

This work was supported by National Science Council
(NSC102-2321-B-007-007 and NSC 102-2120-M-007-006-CC1).

Fig. 1: Figure 1.(a)Schematic of two parts of our device assembly (b)Schematic of E-beam and chip observing area

Fig. 2: Figure 2.IR microscope image of our device heatingby applied voltage from 50.2℃ to 70.1℃

Fig. 3: Figure 3.Orange frame is the electrode part . Blue frame is the unchanged part. (a)-(b)electron radiation affected GelMa as we zoom in (red frame region). Heating result is acquired after e-beam treatment. (c)-(d) Due to heating, the contrast of a pointed region is changed which can be observed in SEM but not in OM
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-5785 Time-Resolved Electron Beam Induced Current (TREBIC) Method for Spatiotemporal Analysis of GaN HFETs Structures.

Šatka A.1, Priesol J.1, Donoval D.1, Uherek F.1
1Institute of Electronics and Photonics, Slovak University of Technology, Ilkovičova 3, 812 19 Bratislava, Slovakia, 2International Laser Centre, Ilkovicova 3 841 04 Bratislava 4 Slovak Republic
alexander.satka@stuba.sk

GaN-based Heterostructure FETs are becoming preferable devices for high-speed and high-power applications in harsh environments. However, they suffer from the significantly deteriorated switching properties whose mechanisms are intensively investigated [1]. One of the possibilities to study spatiotemporal behavior of the device structures is to temporarily inject limited amount of charge using focused electron beam and to investigate the device’s time response. The lifetime in GaAs heterostructures was investigated by TREBIC in [2]. Combined time-resolved OBIC and TREBIC were used to map time response of InGaAsP/InP pin photodiode [3]. Dynamic behavior of HEMTs was investigated using backside infrared OBIC technique [4]. Stroboscopic TREBIC system was applied for analysis of dynamically biased power devices in [5].
In this contribution we report on the developed TREBIC system using sampling time-gated boxcar averaging techniques for spatiotemporal analysisof GaN based HFET structures. A field emission gun SEM equipped with beam-blanking system and multi-contact vacuum feedthrough has been used. Unpackaged HFETs soldered in a sample holder were connected to the feedthrough by 50 ohm coaxial cables. Induced current was detected by I/V converter (Figure 1). Alternatively, e-beam induced conductivity changes were detected, when a voltage on the resistor divider formed by resistor Rd and transistor channel Rch was preamplified by voltage amplifier. TREBIC signal in selected point or in selected area on sample was detected by digital oscilloscope with periodic signal used for beam-blanking. This technique offers excellent time resolution but it suffers from the huge amount of transferred data for each position of the e-beam [3]. Therefore, time-gated boxcar integration technique has been used to map the EBIC signal after selected delay Td from the rising edge of the e-beam (Figure 2). The gate width Tg determines the time resolution. Box car averaging of pulses was set as a compromise between the sensitivity and acquisition time. TREBIC signals measured from InAlN/GaN HFET device are shown in Figure 3, revealing slow response of the device to the charge injected in the G-D region of the transistor. From the series of TREBIC maps taken at various Td and Tg (e.g. in Figure 4) formation of a virtual gate in G-D region and inhomogeneous electric field build-up and recovery at the gate edge has been observed.

1. J.G.Tartarin et al., In: Proc. of the IWS 2013, IEEE, 2013
2. L.J. Balk et al., In: Proc. of the SEMAS, Part I, 447-456 (1975)
3. A. Šatka, J. Kováč, Microel. Eng. 24, 195-201 (1994)
4. D. Pogany et al., Microel. Reliab. 42, 1673-1677 (2002)
5. A. Pugatschow et al. In: Proc. of the 18th ISPSDIC, Naples, 2006, 4pp.

This work has been supported by the Slovak Research and Development Agency (contract No. APVV-0367-11) and by Slovak Grant Agency (project VEGA No. 1/0921/13).

Fig. 1: Schematic drawing of the TREBIC experimental set-up using time-gated boxcar integration and oscilloscope techniques.

Fig. 2: Time dependence of induced current and formation of TREBIC maps.

Fig. 3: Normalized TREBIC curves as a function of gate voltage VGS taken at VDS = 9.6V and e-beam energy Ebeam = 1 keV.

Fig. 4: TREBIC map taken from the top of HFET structure (see inset) immediately after the switching e-beam on (Td = 0 s) using gate width Tg = 3 ms. Pulse width Tp = 50 ms, VDS = 4 V, VGS = -4 V, Ebeam = 5 keV.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-5814 Development of "Adaptive SEM" Technology for in situ Genome/Proteome Expression Analysis in Single Cell Level

Kim H.1, Terazono H.1,2, Takei H.1,3, Yasuda K.1,2
1Kanagawa Academy of Science and Technology, Kanagawa, Japan, 2Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan, 3Department of Life Sciences, Faculty of Life Sciences, Toyo University, Gunma, Japan
ykp-kim@newkast.or.jp

Obtaining expression information in a cellular system is essential for understanding the mechanisms of living systems. One useful way is in situ measurement of expressed biomarkers in single cell level using a lot labels; however, production and identification of such labels are still challenging. We propose a new sensing technology based on the field emission scanning electron microscopy (FE-SEM), which is a comprehensive development of production and identification of nano-particle (NP) labels for simultaneous in situ measurements of expressed biomarkers in single cell (Fig.1).

For the fabrication of NPs, various sizes of polystyrene spheres were used as templates, and metals were deposited on the spheres by thermal evaporation. By using this method, more than 500 types of NPs were fabricated. Metal shell layers were formed by thermal evaporation; therefore, multi-layered NPs can be fabricated with sequential evaporation. We used double-layered NPs; outer is Au for easy immobilization of biomolecules to use these NPs as labeling probes, and inner layer is various to apply label varieties in FE-SEM observation. In this study, probe DNAs were immobilized onto the outer Au layers (referred to as "NP probe" hereafter), and target DNAs on a substrate were reacted with the NP probes as a model. For its detection, FE-SEM observations were performed to identify numbers and elements of hybridized NP probes on the substrate. Spatial distributions and diameters of NP probes were identified by secondary electron (SE) observation, and elements of NP probes were identified by backscattered electron (BE) observation as the difference of intensities in the BE image caused by the difference of atomic number of inner metal layer (Fig.2). In results, six different elements were simultaneously distinguished by BE observation [1], indicated that targets can be simultaneously labeled and identified with high spatial resolution by the combination of NP probe labeling with BE image analysis. In addition, detection sensitivity of target DNA in this method was femto-molar order [2] (Fig.3), which is 1,000 times higher than that in conventional fluorescent labeling and optical detection, indicated that our method is suitable for the detection of a few biomarkers in single cell. We call it "adaptive SEM" technology (i.e., NP identification is "adaptive" for various targets). These results indicate a possibility for quantitative in situ detection of expressed biomarkers in single cell level by the suggested technology based on NP probe labeling and FE-SEM identification.

References

[1] Kim, H., Negishi, T., Kudo, M., Takei, H., Yasuda, K., J. Electron Microsc. 59 (5), 379-385 (2009)

[2] Kim, H., Kira, A., Yasuda, K., Jpn. J. Appl. Phys. 49 (6), 06GK07, 1-7 (2010)

We thank Ms. M. Murakami and Ms. M. Naganuma for their technical assistance. This work was financially supported by the Japan Prize Foundation, JSPS, and KAST.

Fig. 1: Overview of "adaptive SEM" technology. Firstly, target biomarkers in single cell were simultaneously labeled with NP probes on which probe DNAs were coated, and next, the attached NP probes were identified with SE and BE observations of FE-SEM to identify expressed targets in the cell.

Fig. 2: Discrimination of NP probes. In this example, 120 nm of Au, Ag, and Ni NP probes were simultaneously observed with SE (a) and BE (b) detections using FE-SEM. As shown in (b), difference of elements can be distinguished as the difference of intensities in the BE image. Bars, 500 nm.

Fig. 3: Evaluation of detection sensitivity using DNA chip. (a) Overview of the evaluation. Target DNA on the chip was labeled with NP probe, and the result was observed using FE-SEM. Hybridized NP probes were detected as white dots in FE-SEM images. Bars, 1 μm. (b) Relationship between the number density of observed NP probes and target concentration.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-5889 Microflow and Thermal Control System Design for Wet Cell in the Scanning Electron Microscope

Lee H. H.1, Lee C. Y.1, Chiang C. L.1, Tsai K. C.1, Lin W. T.1, Wang H. W.1, Fang J. M.2, Huang T. W.3, Liu S. Y.3, Tsai C. Y.3, Chen F. R.3
1Center for Measurement Standards, Industrial Technology Research Institute, Taiwan R.O.C., 2Taiwan Electron Microscope Instrument Corporation, Taiwan R.O.C., 3Engineering and System Science Department, National Tsing Hua University, Taiwan R.O.C.
KyleLee@itri.org.tw

Scanning Electron Microscope has been widely used in different scientific areas such as material analysis, biology and life science. SEM integrated with a wet cell was proposed in recent years to satisfy the needs from live cell imaging. In the SEM, it requires a vacuum chamber to allow the operation of the electron beam and to minimize the scattering from other sources. To extend the ability of live cell observation in the SEM, the Si3N4 thin film supported by silicon microchip was developed in order to cultivate biological materials in the wet cell. Therefore, the wet cell can be used to visualize live tissues in fully hydrated conditions and to maintain the culture environment. It would be particularly valuable when applying to the analysis of lipid membranes in cells as they are difficult to preserve during dehydration and washing steps. The processes of sample preparation can be more efficient by using the wet cell. Furthermore, with the continuous flow control and real-time monitoring, the long-term operation can be achieved and also expand the applications of the wet cell. In Fig. 1, it shows the meshes of simulation model in the wet cell. The pressure is critical because the flowrate needs to be well maintained in order not to break the thin film. To increase the flowrate and reduce the costs of the fluid mechanics, the geometry and flow conditions were optimized based on the skills of Design Of Experiments. The resulting pressure on the 50 nm thin film was simulated by CFD software (ANSYS Fluent v14) and the results are shown in Fig. 2. It demonstrates that the inlet flowrate still could be raised and the size of the wet cell can be reduced. With regard to the thermal control system, a preheating system of buffer liquid was developed by using a thermal control module. In addition to preheating system for the buffer liquid, an embedded micro thermal control element was also designed inside the wet cell with the capability of fine-tuning so as to achieve accurate and rapid temperature control. Furthermore, several design parameters including noiseless, non-vibration and long working life were also considered for the thermal control system. After preliminary experiments, the results are shown in Fig. 3. and the heating rate inside the wet cell can be achieved to 3.7 ℃ per minute. Eventually, the microflow and thermal control systems were integrated and the system architecture is shown as Fig. 4. With the microflow and thermal control modules integrated in the SEM, the flowrate and fluid temperature can be adjusted by users and the flow conditions including temperature, pressure and even the fluid properties can be simultaneously monitored as well.

Thanks for the technical assistance and suggestions from R&D team of Taiwan Electron Microscope Instrument Corporation (TEMIC) during system integration.

Fig. 1: Meshes of the liquid volume inside the wet cell.

Fig. 2: The pressure on the thin film inside the wet cell.

Fig. 3: The temperature variation in the wet cell duringheating process.

Fig. 4: System architecture of microflow and thermalcontrol system.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-5937 New scintillation low-energy BSE detector

Kološová J.1, Beránek J.1, Jiruše J.1, Horodyský P.2
1TESCAN Brno, s.r.o., Brno, Czech Republic , 2CRYTUR, spol. s r.o., Turnov, Czech Republic
jolana.kolosova@tescan.cz

In the electron microscopy research of nanomaterials, biomaterials or semiconductors, low energy electron beam imaging is often necessary. Reducing the primary beam energy decreases the depth of specimen radiation damage, enables clear visualization of non-conductive samples and leads to enhanced specimen surface contrast.
Low accelerating backscattered electron (BSE) imaging with sufficiently high signal to noise level can be done with the new generation of solid state detectors. These detectors have good sensitivity in the low energy region and their speed approaches the speed of scintillation-type detectors. However, in dual beam systems the deposition of sputtered material on the detection surface can lead to deterioration of performance. Further drawback is the sensitivity of solid state detectors to light.
Scintillation detectors are fast and versatile, but their sensitivity drops rapidly in the low energy region thanks to the ‘dead layers’ on the detection surface (e.g. conductive coating), which are impenetrable for slow electrons. CRYTUR in cooperation with TESCAN has developed a new scintillation type BSE detector with special surface treatment, which guarantees enhanced sensitivity in the low energy region.
Detection limit of the new detector is less than 1 keV. It’s high performance in the field of energies under 3 keV makes it ideal for example for BSE imaging of surface details and contrast changing (see Figure 1), high resolution imaging of sensitive biological samples, or artifact free imaging of nonconductive samples (see Figure 2).

Fig. 1: Change of contrast in BSE images of CeO2 ceramics taken at 3 kV (left) and 1 kV (right) accelerating voltages. More surface details are resolved with lower primary beam energy.

Fig. 2: BSE image of Vitrina pellucida shell taken at 3 kV (left) and 2 kV (right) acceleration voltages. Charging artifacts are not visible at 2 kV.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-5966 Effect of hydroquinone treatment on OTO en bloc stained biological specimens.

Togo A.1, Higashi R.1, Ohta K.2, Nakamura K.2
1Kurume University School of Medicine EM LAB, 2Kurume University School of Medicine Department of Anatomy
togou_akinobu@med.kurume-u.ac.jp

INTRODUCTION
In recent three dimensional (3D) ultrastructural reconstruction techniques such as serial block face scanning electron microscopy (SBFSEM), TEM-like ultrastructural images of biological specimens are directly obtained from block surface of resin embedded specimens. Since this TEM-like block face images (BFI) is usually obtained using backscattered electrons (BSE) as a material contrast image, specimens are stained strongly by heavy metals prior to embedding into resin. In order to enhance the membrane contrast for BFI, we usually stain specimens by the method of Deerinck (2010). As a recent large volume reconstruction requires very long time to obtain image sets, we need a new staining method which provides much higher contrast that enable to acquire images in a shorter time. Takahashi et al. (1986) have reported that hydroquinone (HQ) treatment during the traditional electro-conductive staining increases specimen conductivity and drastically reduces charge problem for SEM observation. They concluded that HQ treatment might increase the efficient secondary electron (SE) generation. As BFI could be obtained not only by BSE but SE, we examined whether HQ treatment in en bloc staining protocol increased the contrast for BFI using SE in this study.

MATERIALS & METHODS
C57BL/6 mouse liver was used. The animals were deeply anesthetized with diethyl ether and sodium pentobarbital, and fixative of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4) was transcardially perfused through the left ventricle with heparin containing saline. After perfusion, liver tissues were removed and cut into small cubes about 1 mm3 in the fixation, and were further fixed in the same fixative for 2h at 4℃. After that, en bloc staining was performed as follows: The specimens were treated with reduced-OTO staining method (1.5% potassium ferrocyanide and 2% OsO4, 1% TCH, 2% OsO4). Subsequently specimens were treated with 1% HQ solution. Some specimens were skipped this step as a control. Then, they were further stained by 4% uranyl acetate and Walton’s lead aspartate solution. After staining, specimens were dehydrated in an ethanol series and were embedded in epoxy resin (EPON812, TAAB). The surface of resin block with specimens was observed by SEM (Quanta 3D FEG, FEI).

RESULTS AND DISCUSSION
The contrast of SE images was drastically increased by HQ treatment, although there is no effect for BSE images. This result suggests that HQ treatment effectively enhances SE generation from specimens. This enhancement may accelerate data acquisition speed for SBFSEM 3D reconstruction.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-6025 Two customized low-cost systems for STEM in SEM microscopy

Caciagli A.1,2, Tessarolo F.1,3, Piccoli F.2,4, Caola I.2,4, Nollo G.1,3, Caciagli P.4
11) Department of Industrial Engineering, University of Trento, via delle Regole 101, 38123 Mattarello Trento, Italy , 22) Section of Electron Microscopy, Azienda Provinciale per i Servizi Sanitari di Trento, via Degasperi 79, 38123 Trento, Italy, 33) Healthcare Research and Innovation Program, Bruno Kessler Foundation, Via Sommarive 18, 38123 Povo Trento, Italy , 44) Department of Medicine Laboratory, Azienda Provinciale per i Servizi Sanitari di Trento, via Degasperi 79, 38123 Trento, Italy
federico.piccoli@apss.tn.it

Scanning transmission electron microscopy (STEM) has become an established technique in microscopy, combining the transmission mode with the sample scanning (Bogner 2007). Although STEM can be performed in dedicated facilities, it is possible to implement it on existing SEM microscopes (STEM-in-SEM) in a relatively simple and cheap way (Vanderlinde 2004). Two customized low-cost stages for STEM-in-SEM microscopy are presented with some representative images obtained from material and life science.
The systems were optimized for a XL 30 FEI, but they can be easily adapted to other microscopes with minor modifications. STEM Signal is detected by the back-scattered electron detector (BSED) or the secondary electrons detector (SED). TEM grids fit in both systems. The first system (STEM-A) (Fig. 1a) was adapted from the configuration proposed by Merli and Morandi in 2005. An aluminium table is mounted on four columns screwed in the x-y stage. The grid is placed in a 2.5 mm hole at a working distance of 10 mm. An aluminium ring blocks the grid. The BSED is cemented on the original sample older allowing to adjust the BSED-grid distance by regulating the z stage axis. To obtain both dark-field (DF) or bright-field (BF) images, BSED should be centred respectively on or off the beam axis. The second system (STEM-B) (Fig. 1b) was derived from the configuration proposed by Golla at al. in 1994. The beam is focused on the sample via a graphite tunnel that attenuate SE signal (different tunnel heights allow choosing the optimal SE-TE signals ratio). Transmitted electrons are scattered back to the SED by two gold-sputterd coverglasses.
STEM-A proved better results with high beam voltage (>20 KV), guaranteeing a sufficient transmitted signal. A distance from 30 to 40 mm between sample and detector gave best results. Details down to 20 nm can be easily resolved (Fig. 2a ). BF and DF images provide complementary information. STEM-B with tunnels of different heights allowed various advantages. The shortest tunnel provides a better image quality, even though a SE signal was also present (Fig. 2b). Deeper tunnels allowed to collect pure TE signal. A less sensitivity to spot size was found in respect to the STEM-B (spot size of 2.0 or 3.0 can be used for magnification up to 100000x). It proved to work with both low and high voltages providing suitable electron transparent sample (Fig. 3) No DF images are available with this system.
Both STEM in SEM system are cheap and offer different configurations that can be adapted to sample characteristics, allowing to achieve good resolution and contrast with conventional SEM microscopes for sample screening before analysis with TEM or high-resolution dedicated STEM.

Fig. 1: STEM in SEM systems: STEM-A (a) and STEM-B (b) inside the closed (open in the insets) chamber of a XL 30 (FEI). Transmitted electrons signal is collected by BSE or SE detectors, respectively.

Fig. 2: Ag nano-particles on a holey carbon film coated grid imaged with the STEM-A system. Dark field image (a). Ag nano-particles on the surface of a CaCO3 micro-particle imaged by the STEM-B system equipped with the short graphite tunnel. Bright field image (b). Original magn. 204800x (a) and 51200x (b).

Fig. 3: Escherichia coli (a) and Staphylococcus epidermidis (b) cells imaged by the STEM-B system equipped with the short graphite tunnel. Samples were alcohol fixed, dehydrated, unstained. Microstructural details of cell surface (cell pili, black arrows), and inner structures (chromatin, white arrows) are visible. Original magn. 32000x (a) and 81000x (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-6037 BASE – a web based, open source solution for microscope reservation and accounting

Iwan H.1, Timmermann J.1, Ritter M.1
1Hamburg University of Technology
ritter@tuhh.de

Central electron microscopy facilities often are responsible for all microscopes and peripheral devices such as sample preparation and analysis equipment at a University. Usually, central facilities are also responsible for the distribution of microscope time to other scientists, institutes or departments and they generally charge an hourly fee for microscope usage and for providing other services. Several scheduling systems, which were available at the time this project was initiated, offered only reservation features or were lacking at least one or more important requirements for accounting, such as project management or cost separation. Therefore, a new system for booking and accounting for microscope time has been developed and introduced at Hamburg University of Technology (TUHH) to reduce administrative burden. This new system is called BASE (Booking and Accounting System for Electron Microscopy) and is based on PHP and MySQL. It offers the following main features: Project management, project-based booking of microscope time, redistribution of microscope time to one or more projects after usage, setting caps on expenses, customer accounts with balance history, timelines (versatile categorization and accessibility of microscope time), automatically expiring microscope access (to enforce regular safety briefings), download area, invoice management etc. Therefore, BASE should fit most needs of central electron microscopy facilities in academic environments. It soon will be available as a web-based, open source solution.

Fig. 1: Schematic principle of BASE: BASE is a project-based booking and accounting solution for electron microscopy facilities. Microscope users work for one or more projects that are billed to a customer, i.e. an institute, department or an external company. Within a project, microscopes and services that are provided by the EM facility can be used.

Fig. 2: BASE login screen for TUHH showing the status of the microscopes.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-4-P-6044 Energy filtered imaging in a scanning electron microscope

Boese M.1
1ZEISS Microscopy, Oberkochen, Germany
markus.boese@zeiss.com

Electron spectroscopy and spectroscopic imaging in a scanning electron microscope is currently used to separate secondary electrons (SE) and backscattered electrons (BSE) in SEMs. Due to limitations of the energy resolution the underlying spectral information of the emitted SE and BSE remains less investigated and mostly unused for imaging. I this study we will present some results of a retarding field detector with an improved energy resolution.

The ZEISS EsB (Energy selective Backscatter) detector was the first commercially available retarding field detector for a SEM. This detector has a very good surface sensitivity since it can work even at low Voltages. With energy filtered imaging it is possible to enhance material contrast even beyond the usual Z-contrast imaging.

Especially the energy filtering properties for low loss BSE imaging conditions were explored by Jaksch [1] and contrasts were shown which can not be explained with the common BSE contrast mechanism. The material dependent difference of the BSE spectral distributions is utilized here for energy filtered imaging to enhance the contrast between different phases.

In general the spectral distribution for BSE emitted from low Z materials are flat, whereas the spectra of high-Z elements have a pronounced maximum near the elastic peak. This behavior is due to the higher penetration depth and the resulting multiple scattering events of the BSEs. By use of energy windows a gain of contrast can be achieved which is superior to the commonly used Z contrast in conventional BSE imaging [2].

As an example for filtering BSEs we can achieve a strong contrast gain between Y2O3 and MoSi2 grains in a SiC matrix, by using only electrons with energy losses up to 100eV from a primary energy of 700V.
Furthermore energy filtered imaging of secondary electrons can enhance contrast for imaging conductors next to less conductive materials. Here the spectra of the secondary electrons are shifted towards each other for the different materials by several eV. As an example the contrast between multilayer graphene and a carbon support film will be presented.
The dependency of the BSE energy loss due to multiple scattering and sample depth can as well be used to gain 3D information from a sample for BSE tomography [3]. The selection of energy windows can be chosen from the elastic peak for pure surface information down to the maximum escape depth of the BSE with maximum energy losses. As an example the different thicknesses of a graphite sample can be imaged by using energy windows of different energy losses.


1. Jaksch, H.: Microsc. Microanal. 17,Suppl. 2 (2011), 902.
2. Cazaux, . J.: Electron Microsc. 61.5 (2012), 261.
3. Niedrig, H. and Rau, E. I.: Nucl. Instrum. Meth. Phys. Res. B 142(1998), 523.

↑ Go to the Table of content ↑

IT-5. Analytical electron microscopy

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-5-IN-1534 Can orbitals be mapped in the TEM?

Löffler S.1,2, Bugnet M.3, Gauquelin N.3, Hambach R.4, Lazar S.3,5, Pardini L.6, Draxl C.6, Kaiser U.4, Botton G. A.3, Schattschneider P.1,2
1Institute of Solid State Physics, Vienna University of Technology, Austria, 2University Service Centre for Transmission Electron Microscopy, Vienna University of Technology, Austria, 3Canadian Centre for Electron Microscopy, McMaster University, Canada, 4Electron Microscopy Group of Materials Science, Ulm University, Germany, 5FEI Electron Optics, Eindhoven, Netherlands, 6Department of Physics, Humboldt University Berlin, Germany
stefan.loeffler@tuwien.ac.at

The energy, position, and momentum distributions of electrons inside a material are decisive for most of the material's properties, ranging from optical, electrical and magnetic properties to hardness, durability, or the melting point. Therefore, the electron distribution is a key quantity in many fields of research. Unfortunately, it is also elusive and directly imaging electronic orbitals and bonds in the bulk has not been possible so far.

In the last few years, several authors reported measurements of orbital properties using electron energy loss spectrometry (EELS) [1-3]. While these are great advances, it would be even better to actually "see" the orbitals in an image. Recently, the possibility to record maps of transition probabilities – from which orbitals can be deduced – was predicted theoretically [4]. In this work, we investigate the requirements and the feasibility to realize that prediction in an actual experiment.

On the one hand, the point group symmetry of the sample atoms plays a crucial role. Intuitively, this is readily understandable. Taking an isolated atom, for example, one is faced with a spherically symmetric problem. Clearly, its solutions must produce rotationally symmetric images. Hence, the symmetry of the system has an important influence on orbital maps. Here, we investigate the requirements on the crystal structure in order to be able to see a directional dependence of transition probabilities, orbitals, and bonds (see Fig. 1).

On the other hand, experimental parameters such as the signal to noise ratio (where the signal is the difference from the average; see Fig. 2), as well as the stability of the specimen and of the microscope are vital for successfully recording high-resolution maps. Based on experimental energy filtered images recorded with very high spatial resolution, we evaluate the requirements on both the sample and the microscope to obtain reproducible and directly interpretable maps. This nurtures the hope that orbital mapping will become a reality in the near future and will become an invaluable tool for many fields of research.

[1] Löffler et al., Ultramicroscopy 111 (2011) 1163
[2] Neish et al., PRB 88 (2013) 115120
[3] Hetaba et al., Micron, in print
[4] Löffler et al., Ultramicroscopy 131 (2013) 39

The authors acknowledge financial support by the FWF (I543-N20), the DPG, and the MWK Baden-Württemberg.

Fig. 1: Comparison of the K-edge maps for an Oxygen atom with full O(3) point-group symmetry (left) and with C2v symmetry (right). For the simulations, an acceleration voltage of 80 kV, a collection semi-angle of 24 mrad and ideal imaging conditions (Cs=0, Cc=0, df=0) were assumed.

Fig. 2: Predicted maps of the Ti L edge for a thin Rutile sample in [001] zone axis for different signal-to-noise ratios (SNR) as indicated (a-d). Preliminary experimental map as acquired (e). EELS signal of 1 px (dots) and averaged over 14000 px (line) (f). An acceleration voltage of 80 kV and an energy-window of 4 eV on the L2 edge were used.
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-5-IN-1656 Single atom imaging and spectroscopy with aberration-corrected STEM

Zhou W.1, Lupini A. R.1, Kapetanakis M. D.2,1, Lee J.3, Oxley M. P.2,1, Prange M. P.2,1, Pantelides S. T.2,1, Idrobo J. C.3, Pennycook S. J.4
1Materials Science & Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA, 2Department of Physics and Astronomy, Vanderbilt University, Nashville, TN 37235, USA, 3Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA, 4Department of Materials Science and Engineering, University of Tennessee, Knoxville, TN 37996, USA
wu.zhou.stem@gmail.com

Aberration-corrected scanning transmission electron microscopy (STEM) at low voltage can now provide real space imaging and spectroscopy measurements at the atomic scale with single atom sensitivity. This opens new opportunities for quantitative study of structural defects in 2D materials. Such studies, especially when combined with first-principles calculations, serve as an important step to correlate the defect structure with local properties, and help to create new functionalities in 2D materials via controlled defect engineering.

Figure 1 shows an experimental annular dark field (ADF) image of Se-doped MoS2 monolayer. Quantitative image analysis allows us to identify the chemical nature of the dopant and map out their distribution in MoS2, one atomic-layer at a time, providing a feasible way to quantify the local composition and measure the local band gap at the 10 nm scale [1].

Combining the imaging and spectroscopy power on a STEM, the changes in local optical response and electronic structure can be directly measured at defect sites. We show that the presence of a single Si atom in the graphene lattice can enhance the low-energy interband transitions with sub-nm spatial confinement [2]. Furthermore, the fine structure in electron energy loss spectra acquired under optimized dose levels provides the sensitivity to determine the nature of the chemical bonding of single atoms. We show that three-dimensional and planar bonding configurations for individual Si atoms in graphene can be directly discriminated (Figure 2) [3].

[1] Y. Gong et al., Nano Lett., 14, 442-449 (2014).
[2] W. Zhou et al., Nat. Nanotech., 7, 161-165 (2012).
[3] W. Zhou et al., Phys. Rev. Lett., 109, 206803 (2012).

This research was supported by a Wigner Fellowship of Oak Ridge National Laboratory (ORNL), the U.S. DOE Basic Energy Sciences, and ORNL’s Center for Nanophase Materials Sciences.

Fig. 1: Atom-by-atom Se dopant analysis in monolayer MoS2 adapted from Ref. [1]. (left) ADF image of Se-doped MoS2. (right) Structure model obtained from histogram analysis showing the distribution of single- and double-Se substituted S2 sites.

Fig. 2: Direct determination of the chemical bonding of single Si atoms via combination of ADF and spectrum imaging with first-principles calculations. (Left) ADF images of 3- and 4-fold coordinated Si atoms in graphene, and their respective Si L-edge fine structure extracted from spectrum images (Right). [3].
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-5-IN-5756 Advancing the spectroscopic frontiers of STEM

Stephan O.1, Arenal R.2, Bocher L.1, Bourrellier R.1, Colliex C.1, Gloter A.1, Kociak M.1, Losquin A.1, March K.1, Marinova M.1,3, Tararan A.1, Tencé M.1, Tizei L.1,4, Zobelli A.1
1LPS, UMR8502 CNRS, Université Paris-Sud, Orsay, France, 2LMA, INA, Universidad de Zaragoza, Zaragoza, Spain, 3UMET, Université Lille1, Villeneuve d'Ascq, France, 4AIST, Tsukuba, Japan
odile.stephan@u-psud.fr

The field of electron energy-loss spectroscopy (EELS) has recently achieved a succession of impressive successes linked with the development of aberration correctors, enabling atomically-resolved spectroscopy, which are now spreading worldwide. In addition, a new generation of monochromators is emerging, providing improvements in energy resolution of at least one order of magnitude and giving unprecedented access to low energy-loss ranges. The possibilities in elemental analysis have become exceptional, especially as the progress in EELS has been accompanied by advances in energy-dispersive X-ray (EDX) analysis thanks to improvements in signal detection efficiency. Similarly, recent progress in the collection of visible-range photons emitted by a sample illuminated by a focused beam, has enabled novel cathodo-luminescence (CL) experiments in the scanning transmission electron microscope (STEM). In addition, new ways of exploiting fast electron beams, including combining them with beams of photons, have opened up the field of nano-optics, providing a high-spatial resolution alternative to more conventional optical techniques. Thus, STEM instruments are now extremely versatile, allowing for the simultaneous detection of an increasing variety of signals. Some of these new possibilities will be illustrated. For example, going beyond elemental mapping to measure fine structure (ELNES) variations at the scale of individual atomic columns or atoms will be described. Spectroscopic data acquired with EM or low-noise CCD cameras will be discussed in connection with the quantitative measurement of electron densities and the identification of charge ordering in oxide materials [1] or probing the chemical bonds of heteroatoms hosted in a carbon lattice [2]. Novel experiments combining EELS and CL, using a dedicated, home-made, high-efficiency nano-CL system will be presented, demonstrating how the usual macroscopic concepts such as extinction, absorption, and scattering cross-sections are no longer sufficient to describe optical phenomena at the nanoscale [3]. When used in combination with TEM structural investigations, nano-CL experiments have proved to be a unique way to explore the intimate link between a crystal structure (h-BN), its defects and its optical properties [4].
These experiments open stimulating perspectives for the development of further new spectroscopic techniques, combining photons and electrons in time-resolved applications for example, or entering the field of quantum optics.

[1] L. Bocher et al Phys.Rev. Lett. 111 (2013) 167202

[2] R. Arenal de la Concha et al, arXiv:1401.5007

[3] A. Losquin et al, submitted

[4] R. Bourrelier et al, arXiv:1401.1948

The work has received funding from the french CNRS-CEA METSA network and the European Programme ESTEEM2

Fig. 1: Schematics of the Orsay STEM set-up dedicated to nano-optics experiments. The HAADF image of a gold nanoprism is shown in combination with the EELS and CL signal intensity maps (not acquired simultaneously in that specific case) revealing the spatial variation of the « tip » plasmon mode of the particle.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-1510 Quantitative electron magnetic circular dichroic signals acquired by 1000 kV (S)TEM-EELS

Tatsumi K.1, Kudo T.2, Muto S.1, Rusz J.3
1EcoTopcia Science Institute, Nagoya University, Nagoya 464-8603, Japan, 2Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan, 3Department of Physics and Astronomy, Uppsala University, Box 516, SE-75120 Uppsala, Sweden
k-tatsumi@nucl.nagoya-u.ac.jp

Electron magnetic circular dichroism (EMCD) in EELS is an attractive technique for studying spin-related properties in a higher spatial resolution than the well-utilized x-ray magnetic circular dichroism (XMCD). Even though several effective experimental geometries of the EMCD measurement have been established, these are still substantially in the testing stage due to the intrinsic difficulties associated with quantitative analysis, such as a low signal-to-noise ratio (SNR) and complicated dynamical and plural inelastic scattering effects depending on the sample thickness/orientation. The present study shows the EMCD measurements using a (S)TEM-EELS with an acceleration voltage V of 1000 kV, toward more quantitative local magnetic analysis.
Figure 1 shows the example results of Co L2,3 EELS near edge structures (ELNES) with 200 and 1000 kV acceleration voltages in the intrinsic EMCD method [K. Tatsumi et al., Microcopy, 2014; doi: 10.1093/jmicro/dfu002]. The dichroic signals as the difference spectra showed a better SNR in the 1000 kV case, because of the larger fraction of the dichroic signals.
Figure 2 shows theoretical fractions of the dichroic signals, here represented by a quantity, fd = (IB - IA) / (IA + IB) at the L3 peak energy, as a function of sample thickness t, with several different collection angles φcol represented as EELS aperture radii in units of g shown in Fig. 1. The calculations were performed based on the dynamical diffraction and single core loss scattering. The desirable collection angle is less than 0.2 g because the larger φcol significantly decreases fd. fd at 1000 kV are significantly larger than 200 kV for relatively large thicknesses (t = 30 to 40 nm), because of the larger extinction distance and inelastic mean free path. The simulated and experimental ratios of fd at 1000 kV to fd at 200 kV for t= 35 nm are 2.5 and 3.0, respectively, showing reasonable consistency.
Finally, this advantage is utilized to statistically acquire quantitative EMCD signals distributed over the diffraction plane, demonstrating that quantitative magnetic information can be routinely obtained using electron beams of only a few nanometers in diameter without any restriction regarding the crystalline order of the specimen [S. Muto et al., Nature Commun., 2014; doi: 10.1038/ncomms4138].

This work was partly supported in Grant-in-Aids for Scientific Research of JSPS (Wakate A: 24686070 and Innovative areas: 25106004) and Swedish Research Council.

Fig. 1: Experimental Co-L2,3 ELNES collected at two different EELS aperture positions A and B. Two sets of results obtained by using different TEM-EELS systems with different acceleration voltages, 200 kV (a) and 1000 kV (b), are shown.

Fig. 2: Theoretical fd with V = 200 and 1000 kV. Numbers inset are φcol, represented by EELS aperture radii in g. Filled circles are results with the experimental φcol.
Fig. 3:
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-1718   Towards Quantitative EDX Results in 3 Dimensions.

Goris B.1, Freitag B.2, Zanaga D.1, Bladt E.1, Altantzis T.1, Sudfeld D.2, Bals S.1
1EMAT, University of Antwerp, Antwerp, Belgium , 2FEI Company, P.O. Box 80066, KA 5600 Eindhoven, The Netherlands
sara.bals@ua.ac.be

Over the last 10 years, electron tomography has evolved into a versatile tool to investigate (hetero)nanostructures [1]. Nevertheless, resolving their chemical composition in 3D remains challenging. In principle, energy dispersive X-ray (EDX) mapping can be combined with electron tomography since the number of generated X-rays increases with sample thickness. However, early attempts to perform 3D EDX experiments were complicated by the specimen-detector geometry [2]. Recent efforts therefore led to a novel EDX detection system, enabling the extension of EDX mapping to 3D [3]. An example of a 3D EDX reconstruction is shown in Figure 1, showing a Au@Ag nanocube of which the Au core yields an octahedral shape. This example clearly illustrates the potential of 3D EDX mapping, but one needs to be careful when extracting quantitative information from such reconstructions. In order to obtain quantitative 3D reconstructions using EDX, different steps in the experiment need to be optimized.

The Super-X detection system consists of 4 EDX detectors that are symmetrically arranged around the sample. As a result, it is expected that shadowing effects are minimized and that the total number of detected characteristic X-rays for a spherical nanoparticle is independent of tilt angle. Figure 2 presents the EDX counts that were acquired from a Au particle using a Model 2030 Fischione tomography holder. Using this dedicated holder, shadowing is kept at a strict minimum, but even in this case, an asymmetric collection efficiency of the detector is still observed. This problem, caused by remaining shadowing of the sample grid, can be overcome by combining EDX signals, unaffected by shadowing, that are collected by different detectors during the tilt series.

Quantification of the EDX maps is typically performed using the “Cliff-Lorimer” method, originally developed for the investigation of thin films. Here, we evaluate the use of the “ζ (zeta)-factor” method to obtain quantitative 3D chemical data using the following equation [4]:

ρt=ζ I ⁄ (CDe)

In this formula, ρ is the density of the material and t equals sample thickness, which can be obtained from 3D high angle annular dark field STEM (HAADF-STEM) reconstructions. First, the ζ-factor can be determined by measuring the intensity I and the electron dose for monometallic nanostructures. After estimation of the ζ-factors for different elements, quantitative 3D elemental analysis becomes possible for heteronanomaterials having unknown composition.

[1] PA Midgley, RE Dunin-Borkowski, Nature Materials 8 (2009), p.271

[2] G Möbus, RC Doole and BJ Inkson, Ultramicroscopy 96 (2003), p.433

[3] P Schlossmacher et al, Microscopy Today 18 (2010), p.14

[4] M Watanabe and DB Williams, Journal of Microscopy 221 (2006) p.89

The authors acknowledge support from the European Research Council (ERC Starting Grant -COLOURATOMS) and the FWO.

Fig. 1:  (a) 2D EDX map of a Au@Ag nanocube. Based on a tilt series of such 2D EDX maps, 3D reconstructions (b) could be obtained showing the 3D distribution of the different chemical elements.

Fig. 2: (a) Detected X-ray counts as function of tilt angle for each individual detector of the super-X system. At certain tilt angles, shadowing effects may block the X-rays preventing them to reach the detectors. (b) Total X-ray count when adding the signal from different detectors.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-1642 Surface-plasmon-polariton coupling between adjacent submicron slits in a Au-film investigated by STEM-EELS

Fritz S.1, Walther R.1, Schneider R.1, Gerthsen D.1, Matyssek C.2, Busch K.2,3, Maniv T.4, Cohen H.5
1Laboratorium für Elektronenmikroskopie, Karlsruher Institut für Technologie (KIT), Karlsruhe, Germany, 2Humboldt-Universität zu Berlin, Institut für Physik, AG Theoretische Optik & Photonik, Berlin, Germany, 3Max-Born-Institut, Berlin, Germany, 4Schulich Faculty of Chemistry, Technion – Israel Institute of Technology, Haifa, Israel, 5Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel
stefan.fritz@partner.kit.edu

Surface plasmon polaritons (SPP) and associated cavity modes (CM) were recently analyzed by STEM-EELS in submicron slits in thin metal films.1, 2 Moreover, a strong enhancement of the CM was observed upon introduction of neighboring slits.3 Such nanostructures exhibit extraordinary optical transmission, which is further enhanced due to SPP coupling between slits.4
In this work, EEL spectra were acquired in a monochromated FEI Titan3 80-300. Background subtraction was performed by a fit to the ZLP tail. Slits with a size of 180 x 900 nm2 were milled in a 200 nm Au-film by FIB milling. Numerical simulations were performed with the Discontinuous Galerkin Time Domain method adapted for EELS.5
Fig. 1a shows spectra acquired at 10 nm distance to the walls of a double slit (cf. dots in the HAADF STEM image). In addition to the Au surface plasmon (SP) at ~2.4 eV, signals at 0.5 and ~1.5 eV are resolved which correspond to the fundamental ω1 and 3rd harmonic ω3 of a CM hybridized with SPPs supported by the metal wall. A significant enhancement of ω1 and ω3 is found close to the inner wall. Also, ω3 is red-shifted and the SP is reduced in intensity. Fig. 1b shows corresponding simulated spectra which agree well with the experiments.
The coupling was studied in double slits with inter-slit distances (p) of 280, 450, 900, 1080, and 1980 nm. Fig. 2a shows spectra acquired at 10 nm distance from the inner walls. For increasing p values, ω1 is red-shifted from 0.5 to 0.4 eV and reduced in intensity, nearly vanishing at p=1080 nm. At an even larger p value, ω1 is observed again at ~0.5 eV. Fig. 2b shows corresponding simulations agreeing well with the experiments. For p > 880 nm, a second signal at higher energy is observed which red-shifts and increases in intensity up to p=1580 nm. These two signals correspond to symmetric and anti-symmetric coupling of the hybridized SPP CM between both slits. At p=1080 nm, two weak modes are observed in the simulation which corresponds to the almost vanishing loss intensity in the experimental spectrum. For further increasing p the symmetric mode increases in intensity which is also observed in the experimental spectra for p=1980 nm. The latter effect impressively demonstrates the effect of coherent interference between SPPs of adjacent slits across the metal bar.

1 I. Carmeli et al., Phys. Rev. B 85 (2012).
2 B. Ögüt et al., ACS Nano 5, 6701 (2011).
3 R. Walther et al., arXiv:1212.1987 (2013).
4 F. J. Garcia-Vidal et al., Rev. Mod. Phys. 82, 729 (2010).
5 C. Matyssek et al. Photonics Nanostruct. 9, 367 (2011).

Funding by the DFG Research Center for Functional Nanostructures, the Ministry of Science, Research and the Arts of BW, and DFG project Bu 1107/7-2 (KB and CM).

Fig. 1: a) Experimental and b) simulated EEL spectra at the outer (black line) and inner (red line) walls in a double slit system (cf. HAADF STEM image in inset). The scale bar corresponds to 500 nm.

Fig. 2: a) EEL spectra from double-slit systems with varying p detailing the evolution of ω1. The signal at 0.9 eV for p=1980 nm corresponds to the energy of ω2. This signal was excited due to SPP coupling between the two slits despite having a node at the measurement position (see red dot in Fig. 1a).b) Simulated EEL spectra as a function for increasing p
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-1653 New EM signals made accessible by sub-20 meV resolution EELS

Křivánek O. L.1, Lovejoy T. C.1, Aoki T.2, Crozier P. A.2, Rez P.3, Egerton R. F.4, Dellby N.1
1Nion Co., 1102 Eight St, Kirkland, WA 98033, USA, 2Center for Solid State Science, Arizona State University, Tempe, AZ 85287, USA, 3Department of Physics, Arizona State University, Tempe, AZ 85287, USA, 4Department of Physics, University of Alberta, Edmonton T6G 2E1, Canada
krivanek@nion.com

Nion’s High Energy Resolution Monochromated EELS-STEM (HERMES) instrument [1] is able to combine Scanning Transmission Electron Microscopy (STEM) spatial resolution of a 1-10 Å with 12-50 meV Electron Energy Loss Spectroscopy (EELS) energy resolution. These capabilities promise to make new signals available in analytical EM, and thereby revolutionize it even more than aberration correction has revolutionized EM imaging. Here we explore two new signals: spatially-resolved phonon spectroscopy, and the detection of very light elements by energy-filtered imaging of electrons scattered to high angles.

Fig. 1 illustrates how important signals have up to now been “hidden in plain sight” – obscured by a broad EELS zero loss peak (ZLP). The solid green spectrum was recorded with the beam passing through the monochromator but the energy-selecting slit retracted. The full-width at half-maximum (FWHM) of the ZLP is ~250 meV. The red (line) spectrum was recorded with the slit in, in 0.1 s, and shows FWHM of 14 meV.

The blue (x1000) spectrum in Fig. 1 was recorded from a ~2 nm Ø area of SiO2. The optical phonon peak visible at 140 meV energy loss is in good agreement with the energy of the strongest feature in infrared spectra of SiO2, at 1100 cm-1. (To convert cm-1 to meV, divide by 8.)

Fig. 2 demonstrates that some phonon signals can be spatially resolved with a resolution of a few nm, and hopefully better in the future. The phonon intensity decays close to zero within ~3 nm inside the Si and there is also an initial sharp intensity drop-off at the SiO2–vacuum interface. There is also a long tail stretching tens of nm outside the sample, which suggests that damage-free phonon spectroscopy may be possible with an aloof electron beam.

Imaging phonons in compounds containing light elements such as H should allow the spatial distribution of the compounds to be mapped. It may, however, also be possible to image the light elements in a more general way, by using the fact that electrons scattered incoherently by atomic nuclei to high angles (Rutherford scattering) transfer small amounts of energy to the recoiling nuclei, inversely proportional to their mass.

Fig. 3 shows proof-of-principle energy-filtered high-angle dark field (EFHADF) mapping of light vs. heavy atoms: 60 keV spectrum-image data from Au particles supported on an amorphous carbon foil ~20 nm thick, next to a hole in the foil. Energy window B is centred on the ZLP (±10 meV) and the corresponding image 3(b) shows mainly Au particles. Energy window C is placed over energy losses of 85±10 meV, and image 3(c) shows only carbon. The fact that we are able to image only the carbon shows that we now have sufficient energy discrimination to map very light elements such as H and Li [2].

[1] OL Krivanek et al., Microscopy 62 (2013) 3-21.
[2] TC Lovejoy et al., M&M meeting (2014, Hartford).
[3] We are grateful for the use of LeRoy Eyring Center facilities at ASU.

Fig. 1: EEL spectra recorded under various conditions by Nion HERMES at 60 keV. Solid (green) spectrum: monochromator slit out; red spectrum: slit in; blue (x1000) spectrum: slit in, electron probe on SiO2, acquisition time 10 s, beam current ~10 pA, probe convergence angle ±12 mrad, collection angle ±12 mrad

Fig. 2: a) HAADF image of a Si-SiO2 cross-section; b) profile of SiO2 phonon intensity and sample thickness along the red line in (a). The SiO2 phonon intensity was measured from a series of 100 spectra in 10 s each, normalized by the ZLP. The sample thickness was determined from spectra of all energy losses up to 180 eV, recorded separately.

Fig. 3: EFHADF recoil mapping of Au on am. carbon: a) EEL spectra from a Au particle and from carbon film; b) image formed with window B showing only Au; c) image formed with window C showing only carbon. The angles admitted into the spectrometer were 120±30 mrad, and scattering from carbon nuclei was expected to give a broad peak centered on 40 meV.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-1665 Atom-by-atom chemical imaging of topological insulator nanostructures by ChemiSTEM

Jiang Y.1, Wang Y.1, Zhang Z.1
1Center of Electron Microscopy and State Key Laboratory of Silicon Materials, Department of Materials Science and Engineering, Zhejiang University, Hangzhou, China
jiang0209@zju.edu.cn

Topological insulators (TIs) have attracted ever-increasing attention due to their exotic physical phenomena, however, the overwhelming majority of reported work was focused on the physical properties [1,2]. In contrast, limited effort has been made to gain an accurate picture for their chemical compositions at atomic level, although such information is of critical importance to comprehend their demonstrated properties. Here by employing a state-of-the-art atomic-mapping technology (ChemiSTEM), we present a direct atom-by-atom chemical identification of nanostructures and defects in TIs [3]. We first identify and explain the layer-chemistry evolution of Bi2Te3-xSex TIs (Fig. 1). Significantly, we reveal a long neglected but crucially important defect which is universally present in Bi2Te3 films, the seven-layer Bi3Te4 nano-lamella (Fig. 2). This nano-lamella may explain inconsistencies in measured conduction type as well as open up a new route to manipulate the bulk carrier concentration. This work may pave the way to thoroughly understand and tailor the nature of the bulk, and to secure controllable bulk states for their future dissipationless devices.

References
1. Xiu, F.; He, L.; Wang, Y.; Cheng, L.; Chang, L.-T.; Lang, M.; Huang, G.; Kou, X.; Zhou, Y.; Jiang, X.; Chen, Z.; Zou, J.; Shailos, A.; Wang, K. L. Nature Nanotechnology 6, 216 (2011).
2. Wang, Y.; Xiu, F.; Cheng, L.; He, L.; Lang, M.; Tang, J.; Kou, X.; Yu, X.; Jiang, X.; Chen, Z.; Zou, J.; Wang, K. L. Nano Letters 12, 1170 (2012).
3. Jiang, Y.; Wang, Y.; Sagendorf, J.; West, D.; Kou, X.; Wei, X.; He, L.; Wang, K. L.; Zhang, S. B.; Zhang, Z. Nano Letters 13, 2851 (2013)

We acknowledge the support of NSFC (No. 11174244), the National 973 Program of China (2013CB934600), Zhejiang Provincial NSFC (LR12A04002).

Fig. 1: Layer-chemistry evolution of Bi2Te3-xSex (x=0, 1, 2, 3).

Fig. 2: Structural and chemical identifications of the 7-layer lamellae in Bi2Te3.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-1860 Nanocharacterisation of vanadium and niobium carbide and carbonitrides precipitates in austenite high manganese steels with DualEELS and HAADF techniques

Bobynko J.1, MacLaren I.1, Craven A. J.1, McGrouther D.1, Paul G.2
1Kelvin Nanocharacterisation Centre, SUPA School of Physics and Astronomy, University of Glasgow, Glasgow G12 8QQ, UK, 2ThyssenKrupp Steel Europe AG, Research and Development, FuE-E Modelling and Simulation, Kaiser-Wilhelm-Straße 100, 47166 Duisburg, Germany
j.bobynko.1@research.gla.ac.uk

In order to fulfil the twin goals of safety enhancement and reduced CO2 emissions combined with fuel efficiency for automotive applications, steels combining high strength and high ductility are required for structural members. One alloy system of interest for this application is high-manganese steel microalloyed with either Nb or V to provide high densities of nanoscale carbide or carbonitride precipitates to provide additional strengthening by the dispersion hardening mechanism. We report significant progress in the understanding of the atomic structure of such precipitates, together with their nanoscale chemistry using aberration-corrected analytical STEM. Specifically, we report the details of the precipitate morphology, and absolute measurements of the chemical composition, and use this to make plausible reconstructions of the 3D morphology of these few nm precipitates. This is achieved using DualEELS spectrum imaging datasets recorded with a Gatan GIF Quantum on a JEOL ARM 200F cold FEG, probe-corrected STEM at pixel spacing of a few Ångströms.
We show that a processing approach consisting of subtracting all artefacts from the dataset, followed by the subtraction of the matrix components from the dataset allows the creation of a spectrum image just representing the precipitate. This can then be quantitatively analysed both to produce an accurate thickness map using the t/λ method, and also to produce chemical maps of both the metal cation and carbon contents. Excellent correlation is found between all the maps showing that all elements are uniformly distributed throughout the precipitate, and it is shown that absolute thicknesses can be calculated using crystallographic parameters together with standards for the cross sections of Nb, V, C and Ti (present as impurities). It is shown that there is little evidence for any significant content of N in the precipitates, as is expected for an Al containing steel, and consequently we see little evidence of TiN nanoclusters acting as nucleation points for the precipitates. Rather, Ti impurities have exactly the same spatial distribution in the precipitates as the V or Nb. Nevertheless, we also show that in at least one case, a tiny concentration of N could be detected and mapped within a precipitate, which seems in this case to have been distributed across the whole precipitate.

We are grateful to the European Commission for the funding of this work as part of a Research Fund for Coal and Steel Project (Precipitation in High Manganese Steels, RFSR-CT-2010-00018). This work was only possible because of the generous provision of the MagTEM facility by SUPA and the University of Glasgow.

Fig. 1: Qualitative maps processed from spectrum images of vanadium carbide and niobium carbide precipitates, both from steels isothermally treated at 900°C for a fixed time of 100s followed by quenching. The vanadium precipitate after the background subtraction reveals small traces of N.

Fig. 2: The background subtracted spectrum of the core loss EEL spectrum from the central region of the NbC precipitate, and profiles of the integrated counts in a horizontal line across the centre of the precipitate. Ti counts have been multiplied by a factor of 10 for the purpose of this graph.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-1775 Study of layer by layer graphitization of 4H-SiC, through atomic-EELS at low energy

Nicotra G.1, Deretzis I.1, Ramasse Q.2, Longo P.3, Scuderi M.1, Twesten R. D.3, La Magna A.1, Giannazzo F.1, Spinella C.1
1CNR-IMM, Strada VIII, 5, 95121 Catania, Italy, 2SuperSTEM Laboratory, STFC Daresbury Campus, Daresbury WA4 4AD, United Kingdom, 3Gatan, Inc., 5794 W Las Positas Blvd, Pleasanton, CA, 94588, USA
giuseppe.nicotra@imm.cnr.it

Epitaxial graphene (EG) grown on Si-polarized SiC, play a crucial role by the presence of a so-called carbon “buffer layer”. Such layer has been shown to present a certain degree of sp3 hybridization since it is partially bound to the outmost Si atoms of the SiC (0001) surface [1].
Our results indicate a layer by layer graphitisation of the SiC as the Si evaporates. Atomic resolution EELS measurements show that the relative Si concentration across the buffer layer [2]. Moreover, the presence of oxygen has been revealed across the buffer layer as shown in Figure 1b. The presence of oxygen could be responsible of the slower decomposition of the SiC into graphitic layers. This and other aspects will be discussed.
All the STEM and atomic EELS measurements were performed at 60k. This consists of a probe corrected STEM microscope, capable to deliver a probe size of 1.1 Å, and equipped with a C-FEG and a fully loaded GIF Quantum ER as EELS spectrometer. Low- and core-loss spectra were nearly simultaneously acquired using the DualEELS capability. In this way an accurate measurement of the π*/σ* peaks ratio that is proportional to the sp2 contribution can be carried out. Low- and core-loss EELS spectra were taken across the green box in the ADF STEM image in Figure 1a using a pixel step size of 0.6Å and an exposure time of 20 ms for each pixel. The spectrometer was set to 0.25eV dispersion yielding 0.75eV energy resolution. Such energy resolution is sufficient to reveal different features in the fine structure of the C K-edge and Si L2,3-edges. The ADF STEM image in Figure 1a shows the presence of the buffer layer between the SiC substrate and the 3 graphitic layers. EELS spectra of the O K-edge, Si L2,3-edges and C K-edge are shown in Figures 1b,c,d respectively and were extracted from the selected positions in the sample as shown in Figure 1a. In Figure 1b, the O K-edge peak shows up only in regions 4 - 6 and is particularly strong in region 6. There seems to be in this region of the buffer layer an increase of the oxygen concentration. No oxygen is detected in either the SiC substrate or the graphitic layers. Particularly interesting are the C K-edge spectra in Figure 1d. The spectra in positions 1-3 in the SiC substrate region show different π* peak, indicating chemistry changes. The spectra extracted from the graphene layers in positions 7,8,9 show much higher contribution in the π* peak that leads to the fully sp2 hybridization indicating transition to graphitic structure. [1] G Nicotra et al, ACS Nano 7 (4), (2013) p. 3045.2 [2] G Nicotra et al, to be published

This work was performed at Beyondnano CNR-IMM, which is supported by the Italian Ministry of Education and Research (MIUR) under project Beyond-Nano (PON a3_00363); The SuperSTEM  Laboratory is supported by the U.K. Engineering and Physical Sciences Research Council (EPSRC)

Fig. 1: a) ADTE STEM survey image. b-d) EELS spectra extracted from the selected positions in Figure 1a each spectrum was corrected for the effects of energy drift and plural scattering; b) O K at 532 eV slightly enlarged for better visualization; c) Si L2,3-edges at 99 eV; d) C K-edge at 284eV. Spectra from positions 7 – 9 are from the graphitic layers.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-1847 Atomic resolution mapping of localized excitations using STEM-EELS spectroscopy

Gauquelin N.1, Egoavil R.1, Martinez G. T.1, Van Aert S.1, Van Tendeloo G.1, Verbeeck J.1
1EMAT,University of Antwerp, B2020 Antwerp, Belgium
nicolas.gauquelin@uantwerpen.be

Atomically resolved EELS experiments are commonplace in modern aberration-corrected transmission electron microscopes. Energy resolution has also been increasing steadily with the continuous improvement of electron monochromators [1]. Improving the energy resolution further seems attractive in order to study phonon lattice vibrations which typically occur between a few meV and 1 eV, but with very large scattering angle (10-1000 mrad).
However these interactions are known to be strongly delocalized due to the long range interaction of the charged accelerated electrons with the electrons in a sample, and were found to scale approximately inversely proportional to the energy loss. This has made several scientists question the value of combined high spatial and energy resolution for mapping interband transitions and possibly phonon excitation in crystals.
For phonon excitations, the fast electron couples to a lattice vibration mode via Coulomb interaction, which might result in strong localization of the scattering. [2]
On the other hand, preservation of elastic contrast in low-loss EELS mapping has been reported by S. Lazar et al. [3], where the filtered image of the zero loss peak (ZLP) intensity shows the complementary nature of the high angle annular dark field (HAADF) intensity and the elastic contrast. Furthermore, atomically resolved signatures were observed at 3 eV using high collection angle (124 mrad) and addressed as being possibly related to phonon assisted losses.
In this work we demonstrate experimentally that atomic resolution information is indeed available at very low energy losses of a few hundreds meV expressed as a modulation of the broadening of the zero loss peak. [4] Careful data analysis allows us to get a glimpse of what are likely phonon excitations. On figure 1, one can note the strong presence of atomic resolution contrast hinting to localized inelastic phonon scattering. The contrast vanishes for higher energy losses where delocalized electronic excitations prevail. On Figure 2e and 2f important deviations from the average when changing probe positions within a unit cell can be observed. The spectra show both gain and loss contributions in region where multiple phonon losses are expected. These experiments confirm recent theoretical predictions on the strong localization of phonon excitations [2] as opposed to electronic excitations and show that a combination of atomic resolution and recent developments in increased energy resolution will offer great benefit for mapping phonon modes in real space.
[1] O. L. Krivanek et al., Philos. Trans. A367, 3683 (2009).
[2] C. Dwyer, http://arxiv.org/abs/1401.6305 (2014).
[3] S. Lazar et al., Microsc. Microanal. 16, 416 (2010).
[4] R. Egoavil et al., in preparation (2014).

This work was supported by funding from the ERC grant 246791 - COUNTATOMS and ERC Starting Grant 278510 VORTEX. R. E. acknowledges funding from the  FP7 program grant nr NMP3-LA-2010-246102 IFOX. All authors acknowledge support from the FP7 Program Reference No. 312483-ESTEEM2. The fund for scientific research Flanders is acknowledged for funding FWO project G.0044.13N and G.0064.10N.

Fig. 1: Fig.1: (a) ADF survey image of an EELS SI acquisition taken on [100] SrTiO3 at 120 kV and collection angle β = 129 mrad. (b,c) Corresponding integrated EELS signal in a small window above the ZLP obtained by dividing each spectrum by a scaled average spectrum (b) or by subtracting (c) a scaled averaged spectrum from each individual spectrum.

Fig. 2: Fig.2: Same data averaged over 16 unit cells (a) ADF image (b,c) subtracted and divided maps over the same energy range. Normalized EELS spectra can be extracted for the 3 different types of atomic columns Sr (green), TiO (red) and O (blue) (d) logarithmically scaled zero loss peaks and (e,f) spectra for both division and subtraction treatments.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-1900 Calculation and Measurements of XEDS Collection Solid Angle in the AEM

Zaluzec N. J.1
1Electron Microscopy Center, Argonne National Laboratory, Argonne IL, USA
zaluzec@microscopy.com

      

        One of the most often misquoted parameters of a solid state x-ray detector interfaced to the AEM is its collection solid angle (Ω). Closed form analytical equations have been developed to calculate the solid angle of six of common geometries of solid-state x-ray detectors[1]. These include cylindrical, rectangular and annular configurations, with the detector in either a tilted or untilted configuration as illustrated in figure 1. These formulae have been integrated into an on-line calculator which is freely accessible and only requires a Javascript compatible browser. It can be accessed at: http://tpm.amc.anl.gov/NJZTools. The use of this tool, removes the ambiguity which besets the community in assessing the relative merits of different manufacturer’s claims, by providing an independent procedure to assess the characteristics of difference detector sizes and their geometries.

       

        Due to the advances in the design and construction of modern Silicon Drift Detectors (SDD) the collection solid angle which in the past has hovered about 0.1-0.15 sR, is now routinely quoted in the 0.2-0.8 sR range. There is is a very little real data published which can be used as a direct measurement of the absolute solid angle or real comparisons of individual system other than anecdotal observations. To illustrate this variation we show measurements of the Ni Kα shell x-ray intensity on 4 different instruments all normalized to the same operating conditions (Figure 2). The performance of a series of 8 analytical electron microscopes, operated at 200 kV were tested independently and their collection solid angle determined. This was accomplished through the measurement of the absolute intensity/nA/nm of the x-ray signal emitted from an amorphous 10 nm thick Germanium specimen[2]. The results of these measurements for the different microscopes, with 18 different detectors configurations are summarized in Figure 3.  The largest individual detector tested had a nominal solid angle of ~ 0.24 sR, which agrees well with the calculated value obtain for it using the on-line calculator. For systems with multiple detectors each individual detector was measured, the net solid angle in such situations is the simple sum of from each independent detector value (i.e. instruments 4, 6, 9). The relative variation in the experimental measurements was very large (0.02 sR – 0.24 sR) illustrating the need to quantitatively assess detectors in terms of their real solid angle, rather than the often misinterpreted “Detector Area”. 

     

 References

[1] N.J. Zaluzec, Microsc Microanal, 20, in press (2014)

[2] N.J. Zaluzec, Microsc Microanal, 19 (Suppl 2) , 1262-1263, (2013)

doi: 10.1017/S1431927613008301

This work was supported by the U.S. DoE, Office of Basic Energy Sciences, Contract No. DE-AC02-06CH11357 at the Electron Microscopy Center at Argonne National Laboratory.

Fig. 1: Figure 1.) User interface to the on-line XEDS Solid Angle Calculator (http://tpm.amc.anl.gov/NJZTools)

Fig. 2: Figure 2.) Experimental variation of the performance of 4 different 30 mm2 detector systems for the same NiO specimen.

Fig. 3: Figure 3.) Experimental variation of the solid angle as a function of instrument for a 10nm thick amorphous Ge specimen.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-1937 In situ observation of symmetry and bond length induced ELNES changes of the CaCO3 to CaO phase transformation by in-column energy-filtered low-kV TEM

Golla-Schindler U.1, Klingl T.1, Kinyanju M. K.1, Benner G.2, Orchowski A.2, Kaiser U.1
1Group of Electron Microscopy of Material Science,Ulm , Germany, 2 Carl Zeiss Microscopy GmbH, Oberkochen, Germany
ute.golla-schindler@uni-ulm.de

Calcite (CaCO3) is an important system for biomineralization and of enormous importance in the construction industry. When used as material for the construction of buildings, calcite is transformed into CaO by the reaction: CaCO3→CaO+CO2↑ releasing CO2. The same phase transformation of calcite is initiated in the electron microscope. Low dose rates and the low accelerating voltages achievable with the SALVE prototype microscope, enable to monitor right from the start in situ the phase transformation and track the change of chemistry and electronic environment by EELS and ELNES. The coordination of the Ca atom in calcite is close to an octahedral coordination if the ligands were aligned along the x, y and z axis and the octahedron is stretched along the <111> direction. The angles between the Ca-O bonds are 87.25° and 92.75° and the bond length is 2.357 Ǻ in all directions. In cubic CaO the calcium atom is octahedral coordinated with a bond length between Ca and O atoms of 2.407 Ǻ. Induced by the octahedral coordination of calcium in calcite and CaO the degenerated energy levels of the 3d shell split up into t2g and eg energy levels with an energy difference of Δo. Figure 1 presents the ELNES of the Ca L2,3 edge for the 40 kV and 20 kV series. Four peaks can be separated, with two of them correlated to the Ca 2p3/2 →3d (t2g eg) and two of them to the Ca 2p1/2 →3d (t2g eg) transitions. For 40 and 20 kV, we obtained starting peak positions of the Ca-L2,3 edges-characteristic for calcite, indicated by blue dashed lines. The second end peak-position-characteristic for CaO are indicated by red dashed lines. In the mid of the phase transformation a superposition of the starting and end spectra with resolved peak positions is detectable. Solely these two peak positions exist in all spectra. This proved that the change in the peak position cannot be initiated by energy drift of the experimental setup but is caused by the change of the electronic environment of the Ca atom. All peaks shift to lower energies and additionally the energy level splitting Δo increases. During the phase transformation of calcite to CaO, the octahedral coordination of calcium with oxygen as binding partner is preserved, but the distortion is removed and the bond length changes by 5 pm. Calculation of theoretically fitted EELS spectra with the CTM4X4S and Wien code are started to separate both effects. Muller [1] showed that changes in bond lengths generate core level shifts and his findings show the same relationship as our results, but the bond length variation and following the energy level shift are reduced approximately by a factor of 10 [2].

[1] Muller D. (1999). Ultramicroscopy 78

[2] Golla-Schindler U., Benner G., Orchowski A., Kaiser U. Microsc. Microanal. accepted

This work was supported by the DFG (German Research Foundation) and the Ministry of Science, Research and the Arts (MWK) of Baden-Wuerttemberg in the frame of the SALVE (Sub Angstrom Low-Voltage Electron microscopy and spectroscopy project.

Fig. 1: Figure 1. 40 kV (a) and 20kV (b) EELS spectra (raw data) of the time dependent changes of the Ca-L2,3 edge . The blue dashed lines are aligned to the peak position of the Calcite ELNES and the red dashed lines to the peak positions of CaO ELNES.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2005 Electron energy losses and cathodoluminescence from complex plasmonic nanostructures : spectra, maps and CL radiation patterns from a generalized field propagator

Arbouet A.1, Mlayah A.1, Girard C.1, Colas des Francs G.2
1CEMES-CNRS, Université de Toulouse, 29 Rue Jeanne Marvig, 31055 TOULOUSE FRANCE EU, 2Laboratoire Interdisciplinaire Carnot de Bourgogne (ICB), UMR 6303 CNRS, Université de Bourgogne, 9 Avenue Savary, BP 47870, 21078 Dijon Cedex, France
arnaud.arbouet@cemes.fr

Stimulated by both instrumental (monochromators, detectors) and methodological (signal deconvolution and processing) advances, fast electron based spectroscopies have demonstrated their unique potential in probing surface plasmons (SP) of metallic nanostructures. Their nanometer spatial resolution and ability to probe so-called dark modes have given Electron Energy Loss Spectroscopy (EELS) and Cathodoluminescence spectroscopy (CL) a central role in experimental nano-optics. Today, these techniques are used to investigate nanostructures of increasing complexity in which the particle morphology, the substrate, or the interparticle interactions strongly influence their optical response[1]. Several examples of recent breakthroughs in combined electron/optical spectroscopy techniques such as Electron Energy Gain Spectroscopy demonstrated in ultrafast Transmission Electron Microscopes or surface plasmon three-dimensional imaging push forward the need and development of novel simulation techniques. In this context, we have developed a novel simulation technique allowing to describe thoroughly the interaction of fast electrons with metallic nanostructures. Building on the 3D Green Dyadic Method, our technique yields accurate predictions of the energy losses and CL photon emission consecutive to the interaction of a moving charge with a metallic nanostructure. It can be applied to nanostructures of arbitrary morphology, both penetrating and non-penetrating trajectories and rigorously takes into account the dielectric response of the substrate. Several examples will be presented which show an excellent agreement with recent experimental results. The influence of the substrate on the EELS spectra will be addressed. EELS spectra and maps (Fig. 1), CL spectra, maps and radiation patterns (Fig. 2) of several gold nanostructures from well-known textbook examples (nanoprisms, rods...) to more complex architectures (nanoporous films, particle aggregates, Fig. 3-4) will be presented. Finally, the potential of our technique will be illustrated on complex scenarii involving electron/photon interactions.

The authors acknowledge financial support from the European Union under the Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative Reference 312483-ESTEEM2 and the National Research Agency under the program ANR HYNNA (ANR-10-BLAN-1016)

Fig. 1: EELS spectra computed at 9 nm from a gold nanoprism edge (edge length a = 950 nm, thickness t = 15 nm lying on a Si3N4 substrate (εsub = 3.9). The electron kinetic energy is 200 keV. Inset: EELS maps computed at 1 eV. The EELS probability is per electron and per unit energy.

Fig. 2: Above: CL intensity per electron per unit energy range at 1.42 eV induced by a 200 kV electron incident on a gold nanowire (length L=700 nm, diameter D = 50 nm) deposited on a substrate with εsub = 4. The collecting mirror has a numerical aperture NA = 0.8. Below: CL radiation pattern for an electron incident at the center of the nanowire.

Fig. 3: Randomly generated nanostructure composed of 29 gold spheres (diameter D = 9 nm). The particles are on a glass substrate (εsub = 2.25).

Fig. 4: Corresponding computed EELS probability maps for a 200 kV electron incident computed at 2 eV.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2070 Electron impact investigation of void plasmon ring resonators

Talebi N.1, Ögüt B.1, Sigle W.1, Vogelgesang R.2, van Aken P. A.1
1Max Planck Institute for Intelligent Systems, Heisenbergstr. 3, 70569 Stuttgart, Germany, 2University of Oldenburg, Oldenburg, Ammerländer Heerstr. 114-118, 26129, Germany
vanaken@is.mpg.de

Electron energy-loss spectroscopy (EELS) provides a fast and accurate way of investigating the optical density of local states (ODLS), especially plasmons. By detecting the amount of loss of the electron energy, one can study plasmonic modes. Furthermore, energy-filtered transmission electron microscopy (EFTEM) performed in the Zeiss SESAM microscope [1] is an efficient detection tool for mapping the optical modes in two spatial dimensions.

A specific domain of the possible ODLS excitable by the electrons is the localized plasmon resonance (LPR) excitation. LPR is attracting particular attention due to its possibility to introduce optical key elements such as waveguides [2] and resonators [3] below the diffraction limit of light. Due to the small spatial dimensions of these optical systems, investigating their behaviour in a wide energy band is challenging for optical spectroscopy techniques.

Here, using EFTEM and EELS, we investigate the possible modes of void hexamer and heptamer plasmon resonators, with respect to their symmetries and topologies. The hexamer nanocavity is composed of 6 holes with a diameter of 70 nm and rim-to-rim spacing of 30 nm, drilled into a 100 nm thick silver film. The heptamer resonator is composed of 7 holes with one hole located at the center, and the other 6 holes located along the circumference of a ring. Each hole has a diameter of 60 nm and a rim-to-rim spacing of 50 nm.

The proposed structures sustain similar symmetry; however they differ according to the number of holes and topology. In order to investigate the spatial distribution of LPR modes, a peak-finding algorithm [4] has been utilized. Four distinguished modes could experimentally be observed (Figure 1a,b). These LPR modes can be classified into those modes related to topology, such as toroidal plasmonic modes [5, 6], and those only related to symmetries, such as radially and azimuthally polarized modes [6]. In order to investigate the symmetries more systematically, FDTD calculations have been performed, which provides modal decomposition analysis (Figure 2) with selective excitation of different resonances, and hence yielding their eigenenergies and spatial distributions. In this presentation the concept of symmetry- and topology-related classification of LPR modes is thoroughly discussed.

References:

[1] C.T. Koch et al., Microscopy and Microanalysis 12 (2006) 506

[2] S. M. Raeis Zadeh Bajestani, M. Shahabadi, and N. Talebi, J. Opt. Soc. Am. B. 28 (2011) 937

[3] N Talebi, A Mahjoubfar, and M. Shahabadi, J. Opt. Soc. Am. B 25 (2008) 2116

[4] N Talebi et al. Langmuir 28 (2012), 8867

[5] B Ögüt et al. Nano Lett. 12 (2012) 5239

[6] N. Talebi et al. Appl. Phys. A, accepted for publication (2014)

The research leading to these results has received funding from the European Union Seventh Framework Program [FP/2007-2013] under grant agreement no 312483 (ESTEEM2).

Fig. 1: Peak maps obtained from the acquired EFTEM images at energy losses depicted in the figure for (a) a heptamer resonator and (c) a hexamer resonator. Acquired EELS spectra at the depicted impacts are shown in (b) for heptamer and (c) hexamer nanocavities.

Fig. 2: The eigenmodes simulated with 3D-FDTD which depict the spatial field distribution for the magnitude of the z-component of the electric field for (a) the heptamer nanocavity system at energy loss values of 2.1, 2.5, 3.0, 3.5, and 3.7 eV, and (b) the hexamer nanocavity structure at energy loss values of 1.8, 2.7, 2.9, 3.4, and 3.8 eV.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2097 Characterization of hybrid plasmonic waveguide by dispersion measurement

Saito H.1, Kurata H.1
1Institute for Chemical Research, Kyoto University
saito@eels.kuicr.kyoto-u.ac.jp

 Dielectric waveguides combined with plasmonic waveguides, so called hybrid waveguides [1], have a great potential for providing subwavelength confinement and long propagation length, leading to highly integrated photonic circuits. The simplest example of the hybrid waveguide structures is a three-layered film consists of a high-permittivity semiconductor layer separated from a metal substrate with a thin low-permittivity insulator gap. We performed dispersion measurements on Si/SiO2/Al films using angle-resolved EELS (AREELS) combined with a TEM [2].

 Figure 1(a) and (b) show AREELS patterns taken from the Si(157 nm)/SiO2(6 nm) film and the Si(157 nm)/SiO2(6 nm)/Al(35 nm) film, respectively. The dispersion curves of the first and second order Si waveguide modes are observed in Fig. 1(a), while the dispersion curves in Fig. 1(b) shift to the low energy side compared to those of the Si waveguide modes. These curves are the dispersion curves of the hybrid waveguide modes. According to the coupled-mode theory [3], to a first approximation, the hybrid waveguide mode can be described as a superposition of the Si waveguide mode and the surface plasmon-polariton (SPP) mode excited on the Al/SiO2 interface. The amplitude of the Si waveguide mode ASi is determined by the wave vector of the Si waveguide (kSi), the SPP (kSPP) and the hybrid waveguide (khyb) modes as follow [1],

|ASi|2 = (khyb-kSPP)/(2khyb-kSi-kSPP).    (1)

 Using the experimental dispersion plots (Fig. 2(a)) and the calculated dispersion relation of the SPP mode excited on the interface Al/SiO2, the square norm of ASi can be determined by equation (1). Figure 2(b) shows the resultant mode character depending on the energy of coupling. The hybrid waveguide mode with high energy has large component of the Si waveguide mode, so the electromagnetic energy of waveguide is mainly stored inside the Si layer, while it should be transferred from the Si layer to the Al/SiO2 interface with the decrease of energy because of the increase of the SPP component as shown in Fig. 2(b).

[1] R. F. Oulton et al. Nat. Photon. 2, 496 (2008).

[2] H. Saito et al. J. Appl. Phys. 113, 113509 (2013).

[3] A. W. Snyder and J. D. Love, Optical Waveguide Theory (Chapman and Hall, London, New York, 1983).

Fig. 1: Figure 1. AREELS patterns of (a) Si/SiO2 film and (b) Si/SiO2/Al film. The white curves and line are the calculated dispersion relations of light in Si bulk, SiO2 bulk and vacuum. The red curve is the dispersion relation of Čerenkov radiation in Si bulk.

Fig. 2: Figure 2. (a) The dispersion plots of the first order modes of the Si waveguide obtained from Si/SiO2 film (blue) and the hybrid waveguide obtained from Si/SiO2/Al film (red). (b) The energy dependence of the square norm of ASi calculated using the equation (1).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2167 Electro-optical characterization of single InGaN/GaN core-shell LEDs inside an SEM

Ledig J.1, Scholz G.1, Popp M.1, Steib F.1, Fahl A.1, Wang X.1, Hartmann J.1, Mandl M.1,2, Schimpke T.1,2, Strassburg M.2, Wehmann H. H.1, Waag A.1
1Institut für Halbleitertechnik, Technische Universität Braunschweig, Hans-Sommer-Str. 66, 38106 Braunschweig, Germany, 2OSRAM Opto Semiconductors GmbH, Leibnizstr. 4, 93055 Regensburg, Germany
j.ledig@tu-bs.de

Three dimensional light emitting diodes (LEDs) with a shell geometry around a columnar GaN core are supposed to have substantial advantages over conventional planar LEDs. The active area along the sidewalls of the GaN pillars can considerably be increased by high aspect ratios - leading to a lower current density inside the InGaN multi quantum well (MQW) at the same operation current per substrate area. Due to the 3-dimensional (3D) shape, the electrical and optical characterization of such device structures is a substantial problem because most of the conventional characterization techniques (e.g. Hall effect, capacitance/voltage) cannot be used with 3D geometries.
A nano-manipulator setup inside a scanning electron microscope (SEM) has been used in combination with a cathodoluminescence (CL) system to characterize the electro-optical properties by directly contacting single facets of the 3D structure. The investigated core-shell LEDs are grown by selective area metal organic vapor phase epitaxy on templates consisting of a patterned SiOx mask layer on an n-type GaN layer on 2” sapphire wafers. The light extraction of optical emission from a small region is related to the structure shape, this is estimated using ray tracing simulation and observed by spatially resolved and angle resolved microscope images inside the CL system.
Electron beam induced current (EBIC) images obtained on 3D structures contacted inside the SEM via the substrate and a probe tip clearly prove that a conjunct p-type shell is wrapped around the entire n-type column with an aspect ratio of about 5 forming a depletion region. By comparing spatially resolved CL and EBIC, the rate of charge carrier generation, trapping and separation in different regions of the structure are discussed.
We will present results of electroluminescence (EL) of MQW as well as defect related emission from single core-shell LED structures obtained at different injection currents. A wavelength shift of the MQW emission by 60 nm is observed along the structure height for both excitation methods (CL and EL), indicating a gradient of the indium incorporation caused by changing local growth conditions. The spectra are corrected with respect to the spectral sensitivity of the optical system - including the collection optics, monochromator and the CCD parallel detector.
In addition, metal contacts have been fabricated in order to get a defined contact area. By evaluating the contact area and the EL spectra we gain an insight to the internal efficiency of a single structure versus current density and the average spatially resolved extraction properties.

We thank Dr. Uwe Jahn for support regarding optical characterization. The financial support of the European commission (SMASH and GECCO) as well as the endorsement of the NTH and the JOMC are acknowledged.

Fig. 1: SE image of a core-shell LED structure on the cleaved growth template contacted by a probe tip on the sidewall at a height of 2.6 µm at an FOV =11.4 µm, EHT = 15 kV, tilt = 30°. EBIC image (right) at a reverse bias of VR = 7 V obtained by contacting a core-shell LED with a probe tip, the core is contacted via the n-type GaN buffer layer.

Fig. 2: Photograph of the CL-SEM chamber showing the arrangement of the parabolic mirror (1) for light collection, the micromanipulator (2) and sample (3) tilted by 30°. The sample is a contacted inside the focal point of the optical collection system by a tungsten probe tip attached to the micromanipulator.

Fig. 3: EL spectra of the core-shell LED shown above obtained by different injection currents through a tip contact placed on the sidewall at a height of 4.4 µm and CL spectrum (upper curve) obtained by electron probe excitation at the same height. The spectra are captured with a spectral FWHM of about 7.5 nm using a CCD parallel detector.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2181 Probing Near Fields of Nanoparticle Surface Plasmons with Electron Energy Loss Spectroscopy

Collins S. M.1, Nicoletti O.1, Ostasevicius T.1, Rossouw D.2, Duchamp M.3, Botton G. A.2, Midgley P. A.1
1Department of Materials Science and Metallurgy, University of Cambridge, Cambridge, UK, 2Department of Materials Science and Engineering, McMaster University, Hamilton, Canada, 3Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich, Jülich, Germany
smc204@cam.ac.uk

Localized surface plasmon resonances (SPRs) of metal nanoparticles enable nanoscale manipulation of electromagnetic fields for a variety of sensing and light concentration applications. Electron energy loss spectroscopy in the scanning transmission electron microscope (STEM-EELS) is emerging as a key technique for near field characterization of SPRs. However, far field light and electron beam excitation produce distinct responses in plasmonic nanoparticles [1]. In recent work, three-dimensional imaging of nanocube resonances described modal responses consistent with light scattering near fields [2]. In this presentation, single nanoparticle resonances observed experimentally by STEM-EELS in lower symmetry systems (e.g., nanorods, right bipyramids) are compared using discrete dipole approximation (DDA) simulations for both light and electron excitation sources [3].

Silver nanorod monomers exhibit Fano-like resonances among longitudinal modes in far field light scattering studies [4]. Electron energy loss spectroscopy (EELS) experiments and EELS electrodynamics simulations, however, exhibit symmetric spectral line shapes. Experiments and simulations do display spatial amplitude modulation of the longitudinal nanorod mode, consistent with near field interference effects (Figure 1). In this presentation, key differences in the near field responses of silver nanorods to far field light and electron beam excitation will be examined (Figure 2). Interference effects among longitudinal nanorod modes will be discussed in terms of near field amplitude modulation as well as coupled oscillator modelling to explain experimental EELS mapping results as well as simulated EELS and cathodoluminescence signals.

[1] Collins, S. M.; Midgley, P. A. Phys. Rev. B 2013, 87, 235432.
[2] Nicoletti, O.; de la Peña, F.; Leary, R. K.; Holland, D. J.; Ducati, C.; Midgley, P. A. Nature 2013, 502, 80.
[3] Bigelow, N. W.; Vaschillo, A.; Iberi, V.; Camden, J. P.; Masiello, D. J. ACS Nano 2012, 6, 7497.
[4] López-Tejeira, F.; Paniagua-Domínguez, R.; Rodríguez-Oliveros, R.; Sánchez-Gil, J. A. New J. Phys. 2012, 14, 023035.

S.M.C. acknowledges the support of a Gates Cambridge Scholarship. This work has received funding from the European Research Council under the EU’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement 291522-3DIMAGE and a contract for an Integrated Infrastructure Initiative (Reference 312483-ESTEEM2). G.A.B. is grateful to NSERC for a Discovery Grant supporting part of this work.

Fig. 1: STEM-EELS maps and line profiles (4 nm from rod side) of modal components m = 4 – 5 for a 540 nm long Ag nanorod on a 30 nm silicon nitride substrate (processed by non-negative matrix factorization). Extracted line profiles are compared with simulated line profiles. The electron trajectory is along the z-axis.

Fig. 2: (a)-(b) Phase analysis of light scattering and EELS responses of Ag nanorod simulated by DDA. The respective light absorption (Qabs) and light scattering (Qsca) efficiencies and EELS probability are plotted for comparison. (c)-(d) Net induced dipole moment along the axis of the rod (y¬-axis) calculated for light scattering and EELS.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2490 Single Eu atom M shell spectroscopy and X-ray fluorescence yield

G Tizei L. H.1, Nakanishi R.2, Kitaura R.2, Shinohara H.2, Suenaga K.1
1Nanotube Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8565, Japan, 2Department of Chemistry, Nagoya University, Nagoya 468-8602, Japan
Luiz.tizei@aist.go.jp

Europium (Eu) has a half filled 4f shell and two valences (2+, 3+). The transition between these is induced by pressure or oxidation. We report single atom EELS and EDX spectroscopy of Eu M shell. This shell is ideal for such experiments as its energy falls in a range where EELS and EDX are feasible. Atomically resolved EELS experiments were performed in the JEOL-CREST double corrected microscope operated at 60 kV. EDX-EELS experiments have been performed in an ARM-JEOL 200 operated at 60 kV, equipped with a Centurio-JEOL silicon drifted detector (SDD, 0.80 sr collection angle).
Eu double atomic chains confined inside carbon nanotubes (Figure 1a) have been studied. EELS analysis shows that all chains contain 2+ atoms. The crystal structure is related to that of bulk Eu hcp.
Firstly, Eu M edge EELS analysis (spectrum in Figure 1d) demonstrates the possibility of measuring the spectral signature of single Eu atoms and possibly their valence. HAADF (Figure 1b) and EELS (Figure 1c) intensity maps show the atomic positions and the measured signal.
To compare the absorption and emission of single atoms (Figure 2) EDX and EELS spectrum images have been acquired. Figure 2 shows the maps of the HAADF, EELS M edge and EDX M lines (Figure 2a-c). Atomic positions can only be distinguished in the first two due to the low signal level for the EDX. Profiles along the second atomic pair (arrow in Figure 2a) are shown in Figure 2d. The M and the L emission lines and the M edge absorption signal have been measured. The M EDX and M EELS spectra for one atom are shown in Figure 2e and 2f (equivalent to 12.5 s exposure), respectively. An average EDX spectrum (198 s exposure) is shown in Figure 2g.
We have estimated the total X-ray emission and absorption events (red square on Figure 2b). The absorption signal has been corrected for the CCD efficiency and the finite convergence and collection angles. Events not counted due to the finite energy integration window (100 eV) have been estimated using power laws (largest source of uncertainty). The X-ray signal was corrected by a geometric factor due to the detector’s solid angle. The creation of M-shell vacancies due to L shell transitions was estimated from the EDX signal. Coster-Kronig M sub-shell transitions were not considered. Estimated fluorescence yield lies between 0.02 and 0.03 (the theoretical value is 0.0136). The uncertainty stems from the necessity to extrapolate the tail of the M edge.

This work is partially supported by a JST Research Acceleration programme. The authors would like to thank Niimi Yoshiko for her assistance during initial experiments.

Fig. 1: a) HAADF image of a double Eu chain. b-c) HAADF and integrated M intensity (100 eV window) of a double Eu chain. d) Spectrum from one atom integrated on the red square in b (total exposure time 9x50 = 450 ms). The inset shows the signal after background subtraction.

Fig. 2: a-c) HAADF, M edge EELS and M line EDX for a double Eu chain, respectively. d) Profiles of the Eu M and L EDX, M edge EELS and HAADF signals along the arrow in a). e-g) Single atom EDX and EELS spectra, respectively. f) Average EDX signal in the spectrum image. The exposure time was 198s. Not all peaks are marked but all are identified.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2552 Imaging and X-Ray Microanalysis at the Nanoscale with a Cold-Field Emission Scanning Electron Microscope

Gauvin R.1, Brodusch N.1, Demers H.1, Woo P.2
1Department of Mining and Materials Engineering, McGill University, Montreal, Quebec, Canada, 2Hitachi High-Technologies Canada Inc., Toronto, Canada
raynald.gauvin@mcgill.ca

The scanning electron microscope (SEM) was primary developed for imaging applications. With the introduction of the Si(Li) energy dispersive spectrometer (EDS), simultaneous imaging and x-ray microanalysis became possible. However, long working distance and high current were needed because the position and small solid angle of the EDS detector. SEM was initially and is still optimized for imaging applications, where the high spatial resolution is generally obtained at short working distance. This problem is still relevant today and unfortunately x-ray microanalysis is never performed in the best imaging conditions, i.e., not with the smallest probe size. With the introduction of an annular silicon drift detector (SDD) system, scanning electron microscopy is facing a revolution. This detector is inserted below the objective lens which gives a higher solid angle (up to 1.2 sr). In consequence, a lower working distance and probe current can be used. An improved spatial resolution becomes possible during x-ray microanalysis. At this point, the time required for x-ray imaging will be of the same order as for the atomic number contrast images achieved through backscattered electrons (BSE) imaging.

Carbon nanotubes (CNTs) decorated with platinum (Pt) nanoparticles are often used to evaluate the spatial resolution of cold-field emission scanning electron microscope (CFE-SEM). Figure 1 shows an example of high spatial resolution imaging and x-ray microanalysis of CNTs at low accelerating voltage (2.5 kV). A resolution of 19 nm and 24 nm were measured with SMART-J on the SE micrograph and the Pt x-ray map, respectively. Figure 2 shows another example of high spatial resolution imaging x-ray map obtained with an annular SDD of CNTs with low voltage scanning transmitted electron microscope (LVSTEM) mode at 20 kV. The dark-field micrograph had a spatial resolution of 6.5 nm and the Pt x-ray map had a spatial resolution of 8.9 nm. Currently, this system is limited to accelerating voltage below 20 kV and the shortest working distance is around 10 mm, which is shorter than the one used with a conventional SDD (15 mm on our system).

With this x-ray detector installed on a HITACHI SU-8230 cold-field emission scanning electron microscope, quantitative x-ray microanalysis with high spatial resolution at low beam energy and low current becomes possible with the possibility of using the various different type of imaging at the same time. Also, since the count rate can be as high as 1,500 kcps with our system, which lowers significantly the detection limit of elements as well as the minimum feature sizes of different phases that can be distinguished.

Fig. 1: Secondary electron micrograph of CNTs decorated with Pt nanoparticles was acquired at an accelerating voltage of 2.5 kV and a working distance of 9.4 mm. The Pt X-ray map was acquired with an annular silicon drift detector. The map acquisition time was 1433 s with a count rate of 81 kcps.

Fig. 2: Dark field micrograph of CNTs decorated with Pt nanoparticles was acquired in LV-STEM mode. The Pt X-ray map was acquired with an annular silicon drift detector. An accelerating voltage of 20 kV and a working distance of 10.5 mm were used. The map acquisition time was 412 s with a count rate of 7 kcps.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2673 Probing the electronic structure of functional oxides with high energy resolution EELS

Bugnet M.1, Rossouw D.1,2, Liu H.1, Radtke G.3, Botton G. A.1
1Materials Science and Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4L7, Canada, 2Department of Materials Science and Metallurgy, University of Cambridge, Cambridge, United-Kingdom., 3IMPMC-CNRS, UMR 7590, Université Pierre et Marie Curie-Paris 6, Campus Jussieu, 4 place Jussieu, F-75252 Paris Cedex 05, France
bugnetm@mcmaster.ca

The development of monochromators in the transmission electron microscope (TEM) has allowed improvements in energy resolution comparable to what is currently achieved in x-ray absorption spectroscopy. High-resolution electron energy loss spectroscopy (HREELS) in the TEM provides an invaluable tool to probe subtle changes in the electronic structure of materials at the nanoscale. Selected examples highlighting how HREELS has provided insight into the electronic properties of functional oxides will be persented. The perovskite BaTiO3 is of particular interest because of its intrinsic ferroelectricity at room temperature, and its applications as a piezoelectric material and in capacitors. The ferroelectricity of BaTiO3 at room temperature arises from the off-center position of the Ti atoms in the tetragonal lattice. From a fundamental point of view, the understanding of this structural characteristic, based on the investigation of BaTiO3 complex electronic structure, is essential [1]. In the present study [2], the O 1s excitation is probed by HREELS, and the interpretation of the spectral features is performed with ab initio calculations. The effect of the core-hole potential is investigated, and the correlation between its effect and the geometry of the excited atomic site, i.e. the relative position of the two nearest Ti atoms with respect to the excited O atom, is shown (Figure 1). The link between the core-hole effect and the off-center position of the Ti atom appears as a broadening in the near edge structures, which is resolved with monochromated EELS. This broadening effect is highlighted by probing the O 1s excitation during the phase transitions between the low and high temperature phases of BaTiO3. The effects of continuous light illumination on the structural and electronic modifications of TiO2, a prospective material used for photocatalysis and water splitting, will also be shown. By using a recently-built in situ laser-illumination setup in the TEM [3], we explore the exposure of titania to intense light irradiation. The electronic structure modifications are probed by HREELS and are interpreted in terms of local reversible changes in the material. Finally, HREELS was used to probe the valence changes upon cycling of Li(Mn,Co,Ni)O2 battery cathode materials. Using scanning transmission electron microscopy combined with HREELS, it is shown that valence maps provide exquisite spectroscopic information on local changes from the charge and discharge process in battery materials.

[1] B. Zalar et al. Physical Review B 71, 064107 (2005)

[2] M. Bugnet et al. Physical Review B 88, 201107(R) (2013)

[3] D. Rossouw et al. Physical Review B 87, 125403 (2013)

The Authors are grateful to NSERC for financial support. The experiments were carried out at the Canadian Centre for Electron Microscopy, a national facility supported by NSERC and McMaster University.

Fig. 1: (a) Evidence of the wider fine structure at low energy in the O-K edge for tetragonal BaTiO3, as compared to cubic SrTiO3. (b) Experimental O-K edge and calculated contributions of the two independent O positions in tetragonal BaTiO3 (O1 in red dashed line, O2 in black solid line). (c) Illustration of BaTiO3 off-centre position of Ti.

Fig. 2: The valence state of the transition metals in pristine Li(Mn,Co,Ni)O2 battery cathode material probed by HREELS: (a) Mn4+, Co3+, and (b) Ni2+. The evolution of the electronic structure of Ni upon charging is highlighted in (b), indicating a change in valence state.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2679 Challenges and Opportunities in Materials Science with Next Generation Monochromated EELS

Crozier P. A.1, Zhu J.1, Aoki T.2, Rez P.3, Bowman W. J.1, Carpenter R. W.2, Krivanek O. L.4, Dellby N.4, Lovejoy T. C.4, Egerton R. F.5
1School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ 85287, USA , 2LeRoy Erying Center for Solid State Science, Arizona State University, Tempe, AZ 85287, USA , 3Department of Physics, ASU, Tempe, AZ 85287, USA, 4Nion Co., 1102 8th St, Kirkland, WA 98033, USA, 5Department of Physics, University of Alberta, Edmonton T6G 2E1, Canada
crozier@asu.edu

The development of monochromated scanning transmission electron microscopes (STEM) offering energy resolutions of better than 20 meV and electron probes of 0.1 nm in size provides a new tool for materials characterization. Unique opportunities opened by access to ultra-high energy resolution low loss EELS include determination of optical properties in the IR, bandgap mapping, detection of defect interband states and localized vibrational spectroscopy. At ASU we are currently applying ultra-high energy resolution low-loss EELS to a variety of materials that are important in fields such as energy, environmental science and information technology. Here we show representative initial results acquired on a newly installed Nion UltraSTEM equipped with a probe corrector and monochromator [1].

The optical properties of carbonaceous atmospheric aerosols are an important contributor to radiative forcing for climate change. By applying Kramers-Kronig techniques to energy-loss spectra acquired from the Nion, Figure 1 shows that the refractive index can be determined out to photon wavelengths of 2500 nm, thus covering most of the incoming solar spectrum [2].

Local measurement of bandgaps and states within the gap is of great importance for opto-electronic materials. Figure 2 shows the low-loss spectra from ceria (CeO2) and a ceria co-doped with Gd and Pr (Ce0.85Gd0.11Pr0.04O2-δ). From EELS, local bandgaps were about 3.5 eV and in some regions additional peaks were detected within the bandgap (Figure 2b). Interestingly, all the ceria based samples showed significant uniform intensity within the bandgap which will be discussed in terms of Cerenkov radiation, defects, and surface layers.

At lower energy transfers, localized phonon spectroscopy becomes possible. We have been able to identify vibrational peaks in a variety of compounds like SiO2 which match the Raman spectrum [1]. Figure 3 shows two regions of the low-loss spectrum from TiH2. The peak at 150 meV is prominent under aloof beam conditions.

Ultrahigh energy resolution EELS is an exciting new tool for characterization of materials. However, to realize its full potential, considerable experimental and theoretical work must be undertaken to develop a fundamental understanding of this form of EELS.

[1] O.L. Krivanek et al, these proceedings (2014)

[2] J. Zhu et al, these proceedings (2014)

The authors acknowledge support of the NIST 60NANB10D022, NSF Graduate Research Fellowship Program (DGE-1311230), NSF DMR 1308085,NSF MRI-R2 959905 and DOE DE-SC0004954. The authors acknowledge the use of facilities in the John M. Cowley Center for High Resolution Microscopy at Arizona State University.

Fig. 1: (a) EELS from two forms of carbonaceous particles. (b) Complex refractive index derived from EELS covering photon wavelength range 200-2500 nm.

Fig. 2: Low-loss spectra from a) CeO2, b) Ce0.85Gd0.11Pr0.04O2-δ and c) hexagonal BN.

Fig. 3: EELS from TiH2 showing a) wide energy range on sample and b) very low energy-loss region in aloof beam mode (~5 nm off sample).

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2752 FIB-SEM Instrument with Integrated Raman Spectroscopy, Scanning Probe Microscopy and Secondary Ion Mass Spectroscopy

Jiruše J.1, Haničinec M.1, Havelka M.1, Hollricher O.2, Schaff O.3, Oestlund F.4, Whitby J.5, Michler J.5
1TESCAN Brno, s.r.o, Brno, Czech Republic , 2WITec GmbH, Ulm, Germany, 3SPECS Surface Nano Analysis GmbH, Berlin, Germany, 4TOFWERK AG, Thun, Switzerland, 5EMPA - Materials Science & Technology, Thun, Switzerland
jaroslav.jiruse@tescan.cz

Integration of a number of techniques in a single tool gives the possibility of correlating multiple measurements and analyses of the same sample area, all made in-situ. A multifunctional tool comprising an SEM-FIB, an SPM and a TOF-SIMS has been presented recently [1]. Newly, a Confocal Raman Microscope (CRM) has been added to yield information about molecular composition and chemical bonds. The CRM image complements the high resolution SEM image, topographic image from SPM, chemical map from TOF-SIMS and sample modification by FIB. Fig. 1 shows the arrangement of the presented apparatus.

State-of-the-art Raman analyzers in SEMs use a parabolic mirror for focusing and lateral resolution is usually no better than 2-5 µm. The presented system provides a resolution of 360 nm by integrating a full confocal light microscope. The important property is the capability of Raman imaging. When a spectrum from a single point is acquired, one can never be sure if the position calibration is correct. Fig. 2 shows overlaid Raman and SEM micrographs of diorite sample. Besides lateral scanning, vertical movement is supported, which allows non-destructive 3D tomography.

Integration is possible with two alternative electron optical columns, each with a Schottky field-emission gun: the LYRA with a conventional objective lens or the GAIA with an immersion lens. The immersion lens column [2] is recommended for non-conductive or fragile samples, because it offers better resolution at low energies (1 nm at 15 kV and 1.4 nm at 1 kV).

The FIB is used to modify the sample and it also enables 3D tomography techniques by sequential FIB slicing followed by imaging to create 3D datasets with analytical information such as elemental composition, crystallographic information, etc.

The FIB also acts as a primary ion beam for the TOF-SIMS analysis. It allows 2D as well as 3D spectral maps, carrying elemental, isotopic and chemical information about the investigated sample. Fig. 3 shows a TOF-SIMS 3D tomography of sodium contamination on solar cell sample. Lateral resolution of TOF-SIMS maps can be better than 50 nm.

The integrated Scanning Probe Microscope (SPM) supports work in STM and AFM modes. Its compact design allows it to sit on the SEM stage. Simultaneous use of SPM, SEM and FIB enables a true depth calibration of TOF-SIMS depth profile as well as the calibration of 3D tomography techniques. The SPM head is designed for a depth resolution of 0.1 nm and an imaging speed of up to 20 s per image. Fig. 4 shows the AFM topography map of gold particles on carbon and corresponding SEM micrograph.

References:

[1] J Jiruše et al, Microsc. Microanal. 18 (Suppl. 2) (2012) p. 638.

[2] J Jiruše et al, Microsc. Microanal. 19 (Suppl. 2) (2013) p. 1302.

The research leading to these results has received funding from the European Union Seventh Framework Program [FP7/2007-2013] under grant agreement No. 280566, project UnivSEM.

Fig. 1: Geometrical arrangement of the presented apparatus.

Fig. 2: Raman (in color) and SEM (in gray) overlaid micrographs of diorite sample. Different colors correspond to various phases in the sample.

Fig. 3: TOF-SIMS 3D tomography of sodium contamination on solar cell sample.

Fig. 4: AFM topography map of gold particles on carbon and corresponding SEM image. Field of view is 1 µm.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2917 Full Optical Properties of Carbonaceous Aerosols by Very High Energy Resolution Electron Energy-loss Spectroscopy

Zhu J.1, 2, Crozier P. A.1, Aoki T.2, Anderson J. R.1
1School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, USA, 2LeRoy Eyring Center for Solid State Science, Arizona State University, Tempe, AZ, USA
jiangtao.zhu@asu.edu

Carbonaceous aerosols have a strong impact on the global climate by direct radiative forcing via light absorption and scattering, and/or indirect radiative forcing via influencing cloud formation. It is critical to determine their optical properties to understand their contribution to direct radiative forcing. It is also important to understand their local chemical composition which would affect their role in cloud dynamics. By employing monochromated electron energy loss spectroscopy in a newly installed Nion UltraSTEM 100 at ASU with a sub 20 meV energy resolution, we can now determine the optical properties of carbonaceous aerosols over the full range of incoming solar radiation 200-2500 nm including the infrared. In addition, the compositional variation in aerosol spherules can also be studied.

Depending on the sources and the combustion conditions, different types of carbonaceous aerosols [1], can be present in the atmosphere. Here two types of aerosols, graphitic and amorphous carbon collected from East Asia, were investigated. Low loss spectra of these two types of aerosols were collected from Nion UltraSTEM 100 at 60 kV with an energy dispersion of 10 meV/channel giving a zero loss peak (ZLP) full width at half maximum (FWHM) of 40 meV. As shown in Fig. 1a, the EELS of graphitic and amorphous carbons are different, which is related to their different microstructure and chemical bonding. We found that the standard thin film formulation of Kramers-Kronig analysis can be employed to make accurate determination of the dielectric function for carbonaceous particles down to about 40 nm in size [2]. Figure 1b and c show the complex refractive indices (n-ik) of graphitic carbon spherules and amorphous carbon spheres over the photon wavelength range of 200-2500 nm. The absorption in the infrared range is obtained although it is smaller than that in the UV and visible range. The variances in the refractive indices of different particles were related to their variances in composition. We also found that on the surface of both graphitic spherule and amorphous carbon, there is a several nanometer thick layer rich in silicon and oxygen (Fig. 2). The nano-size coating could modify the chemical interactions of the carbonaceous aerosols, for example, by changing their ice nucleation properties.

References

[1] J. Zhu, P.A. Crozier, J.R. Anderson, Atmos Chem Phys. 13 (2013), p. 6359.

[2] J. Zhu, P.A. Crozier, P. Ercius, J.R. Anderson, accepted by Microsc Microanal.

The authors acknowledge support of the National Institute of Standards and Technology under Award 60NANB10D022 and the use of facilities in John M. Cowley Center for High Resolution Electron Microscopy at Arizona State University.

Fig. 1: (a) Low loss EELS of graphitic and amorphous carbon (a) and their refractive index (n-ik) in the range of 200-2500 nm in optical wavelength (b, c), respectively.

Fig. 2: (a) The angular dark field image of a graphitic carbonaceous aerosols.(b) Intensity distributions of C, O, S, K, Ca, Ga and Si along the line in the image (a).

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-2999 Direct observations of local electronic states in a quasicrystal by STEM-EELS

Seki T.1, Abe E.1
1University of Tokyo, Tokyo, Japan
seki@stem.t.u-tokyo.ac.jp

    Quasicrystals (QCs) have long-range ordered complex structure without periodicities. Stability of QCs has been discussed in terms of energetic gains in electron systems, because most QCs reveal pseudogaps in their density of states around Fermi level. In fact, many QCs have been discovered by tuning valence electron density based on Hume-Rothery rule. Therefore, understanding electronic structures in QCs may provide an important clue for their stabilization mechanism. Generally, it has been frequently discussed based on an interaction between Fermi surface and Brillouin zone boundary within the framework of nearly free electron model, providing an underlying physics of a Hume-Rothery’s empirical criteria. However, the electronic structures of QCs have not yet been fully understood, particularly being in microscopic-macroscopic relations. In the present work, we investigate local electronic states in Al-based QCs using electron energy loss spectroscopy (EELS) combined with scanning transmission electron microscopy (STEM).
    The AlCuIr decagonal phase was used for the present study [1]. A cluster with a diameter of ~2 nm emerges as a building unit for AlCuIr decagonal phase (Fig. 1, 2). Principal components analysis clearly showed up the atomic-site dependence of plasmon loss spectra in a two-dimensional map correlated with the cluster arrangement. Qualitatively, there seems to be certain correlations between the plasmon peaks and the core-loss edges, Al L1, Ir O23, Ir N67 and Cu L23, all of which reveal different behaviours at the cluster centers and the edges (Fig. 3). All results indicate the cluster centers have metallic states, while the cluster edges have covalent states. First-principles calculations confirm the unusual electronic state. We analyse a distribution of covalent electrons by Fourier transformation of electron localization function. The distribution seems like a 10-fold charge density wave with Fermi wave length. It suggests that the Hume-Rothery mechanism play a key role even when the hybridization effect mainly contributes to pseudogap formation. Along with a context of orbital hybridization, the covalent electrons might reduce their energy by mimicking 10-fold charge density wave. On the other hand, the metallic regions at the cluster centers may have no contributions to the Hume-Rothery mechanism, since there are no distinguished peaks appeared along the 10-fold charge density wave at the relevant regions.

[1] 1. P. Kuczera, J. Wolny and W. Steurer, Acta Crystallographica B68 (2012), 578.

This work was conducted in Research Hub for Advanced Nano Characterization, The University of Tokyo, supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. It is acknowledged that T. Seki is a research fellow of Japan Society for the Promotion of Science.

Fig. 1: HAADF-STEM image of AlCuIr decagonal quasicrystal. Yellow circles indicate clusters with a diameter of ~2 nm. The image shows only Cu and Ir atomic columns.

Fig. 2: Structure model of the cluster. Blue, green and red circles correspond to Al, Cu and Ir atoms, respectively.

Fig. 3: EELS spectra consisting Ir-O23, Ir-N67 and Al L23 obtained from the cluster centers and edges. Intensity ratio of Ir-O2 and N67 to Ir-O3 depends on atomic sites.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-3107 Luminescence of structural defects in opto-electronic materials studied by CL-STEM

Perillat-Merceroz G.1, Alexander D. T.2, Zamani R. R.3, Arbiol J.3, Stadelmann P.2, Grandjean N.1, Hébert C.2
1Laboratory of Advanced Semiconductors for Photonics and Electronics (LASPE), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland, 2Interdisciplinary Centre for Electron Microscopy (CIME), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland, 3Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Barcelona, CAT, Spain
duncan.alexander@epfl.ch

The cathodoluminescence (CL) signal from opto-electronic materials is a sensitive function of their structural properties. CL mapping in scanning TEM (STEM) mode offers a number of advantages for studying this relationship. The interaction volume of the electron beam has a diameter of a few nm within the TEM lamella or sample, giving the potential for high spatial resolution CL studies (e.g. [1]). Further, the CL signal can be acquired simultaneously with defect contrast images taken with bright-field (BF) or low angle dark-field (LDF) detectors, and atomic number contrast images from a high angle annular dark-field (HAADF) detector. This allows direct correlation of luminescence to structure and defects.
Here we present studies using CL-STEM to probe the effects of structural defects on optical properties. Data are taken using a JEOL 2200FS equipped with the Gatan XiClone CL spectrometer, at 80 keV beam energy to increase CL signal and reduce beam damage. The instrument has BF, LDF, and HAADF STEM detectors; sample holders allow specimen temperatures from 5 K to room temperature. Para-CL spectrum image data are acquired on a liquid nitrogen cooled CCD camera using Gatan Digiscan, typically using a 300 lines/mm grating and sub-pixel scanning for the simultaneously acquired STEM images.
To optimize experimental conditions and to interpret data it is important to characterize the sample-instrument system. Fig. 1 shows the effects of specimen thickness and temperature on the CL signal of a GaN epilayer prepared for TEM by mechanical wedge polishing. Specimen cooling increases CL signal intensity and also reduces non-radiative recombination at the polished surfaces, so giving luminescence even for 30 nm thick material. Fig. 2 then shows threading dislocations in an InGaN quantum well (QW) on a GaN substrate. Some dislocations demonstrate modulations in wavelength that could correlate to In enrichment and depletion in the strain fields around their core [2]. Fig. 3 instead shows data taken from a GaN nanowire that contains stacking faults (SFs) on the basal plane [3]. The spatial correlation of SFs to the near-bandgap (NBE) and sub-bandgap (SBE) emission is complex, demanding further investigation, while midgap states generate a diffuse background. The observation of Fabry-Pérot resonator effects for CL in the TEM lamella will also be discussed. Together these results illustrate the great potential of CL-STEM for investigating the structure-optical domain.
[1] Zagonel et al., Nano Lett. 11, 2011, 568
[2] Mouti et al., PRB 83, 2011, 195309
[3] Schuster et al., Nano Lett. 12, 2012, 2199

The Competence Centre for Materials Science and Technology (CCMX) is acknowledged for CL funding, and Anas Mouti of ORNL for earlier works with the CL-STEM system.

Fig. 1: CL intensity of the GaN peak as a function of thickness at different temperatures. Extracted from CL line scans taken on a GaN epilayer sample with a wedge shape. Thicknesses estimated by electron energy-loss spectroscopy.

Fig. 2: (a) BF STEM image of a single InGaN QW on GaN substrate in plan view with threading dislocations coming to the surface. (b) Map of the wavelength of the QW peak (colour scale shown below). A butterfly shape is visible around some of the dislocations with emission blue-shifted on one side and red-shifted on the other.

Fig. 3: (a) BF STEM image and (b) RGB CL map of different emission bands for a GaN nanowire (red: NBE; green: SBE; blue: midgap states). While the planar defects in the nanowire have an influence on CL emission, there is not a direct spatial correlation between the two.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-3165 3D EDX in SEM and TEM: common problems and common solutions

Burdet P.1, Croxall S. A.1, de la Peña F.1, Rossouw D.1, Midgley P. A.1
1Department of Materials Science & Metallurgy, University of Cambridge, Cambridge, UK
pb565@cam.ac.uk

Energy dispersive X-ray spectrometry (EDS) has recently seen a step forward with the introduction of silicon drift (SDD) detectors for both SEM and TEM. The improvements in speed and detector efficiency have allowed EDS, a traditionally 2D technique, to be extended to 3D. For SEM, focused ion beam (FIB) ‘slice-and-view’ methods can be used. For TEM, tilt series of 2D EDS maps are recorded for reconstruction by back-projection. 3D EDS mapping in both the SEM and TEM faces acquisition and processing challenges, such as partial detector shadowing, detection of spurious X-ray and high level of noise. In this paper we explore which problems are common to both techniques as well as proposing common solutions. To compare the two techniques, samples of Ni-based superalloy are investigated with complex structures ranging from microns to nanometers (see figure 1) and containing more than 10 elements (see figure 2).
For 3D experiments, the acquisition time per spectrum is often reduced, to cover a large volume or to acquire a sufficient number of tilt images, in a reasonable time. The set of data contains millions of noisy spectra characterizing only a limited set of chemical phases. This is a favorable case for a multivariate statistical approach, such as principal component analysis (PCA), and, as shown in figure 3, an important reduction of noise can be obtained with this technique. However, such statistical approaches need to be used with care, especially with data containing few counts per channel. Alternative PCA algorithms and pre-processing methods will be explored. Before quantification, the X-ray line intensities are extracted from each EDS spectrum. The involved processing steps are similar for SEM and TEM. Due to the relatively low energy resolution of the EDS detector, X-ray lines often overlap, as observed in figure 2 for Hf Mα and Ta Mα. Moreover, the background needs to be subtracted. Given the high level of noise in SEM and TEM datasets, accurate intensity extraction is challenging. Different processing strategies based on curve fitting will be discussed.
For 3D EDS in SEM and TEM, other signals may be recorded simultaneously, such as secondary electron (SE) for SEM or energy loss spectra for TEM. This opens the way for new processing methods benefiting from complementary signals. For instance, SE images can be used to improve the spatial resolution of the segmentation obtained with the EDS map [1]. In order to facilitate the interactive data analysis of these complex multi-dimensional datasets, HyperSpy [2] a free, open-source and open-development software package, has been extended to EDS data for SEM and TEM.
1. P. Burdet, J. Vannod, A. Hessler-Wyser, M. Rappaz, M. Cantoni, Acta Materialia 61 (2013) 3090–3098
2. http://hyperspy.org

The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) /ERC grant agreement 291522-3DIMAGE. SAC and PAM acknowledge financial support from Rolls-Royce plc.

Fig. 1: 3D SEM-EDS reconstruction of the sample of Ni-based superalloy. The green volume shows the Ni rich γ’ phase, red show the Hf rich phase and blue the Ta rich phase. The bounding box measures 12.8 µm x 11.2 µm x 6.3 µm.

Fig. 2: Characteristic SEM-EDS spectrum acquired from a sample of Ni-based superalloy. The main lines excited at 15 kV are indicated. The inset shows a magnified picture of the low energy lines.

Fig. 3: Denoising a SEM-EDS spectrum with PCA. A running sum is used prior to the PCA.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-3209 Oxygen Vacancies at Grain Boundaries in Doubly-Doped Ceria Determined using EELS

Bowman W. J.1, Zhu J.1, Hussaini Z.1, Crozier A. P.1
1Arizona State University
wjbowman@asu.edu

 In oxygen conducting ceramics like CeO2, O2- diffusion occurs via thermally-activated hopping through vacancies whose concentration can be modulated by doping with aliovalent cations such as Gd3+ or Pr3+. Sluggish ionic conductivity in these polycrystalline electrolytes has been attributed in part to highly resistive grain boundaries (GBs) which degrade total ionic conductivity. Here we use a combination of electrochemical impedance spectroscopy (EIS) and electron energy-loss spectroscopy (EELS) to characterize the electrical conductivity and vacancy concentration of GBs in Gd-doped CeO2 also containing Pr or Co.
Gd-doped (GDC), Gd/Pr doubly-doped (GPDC) and a series of Gd/Co doubly-doped CeO2 ceramics were prepared using a spray drying approach together with traditional ceramic processing techniques. EIS was performed using a Gamry Reference 3000 potentiostat, and EELS was performed using a Nion UltraSTEM100. To facilitate interpretation of the EELS data, FEFF codes [1] were employed to simulate the CeO2 O K-edge spectra as a function of oxygen vacancy concentration.
 Fig. 1a shows a simulation of the O K-edge in CeO2 as a function of O2- vacancy concentration. In this model, the vacancies were randomly distributed in the fluorite structure. These results indicate a decrease in the first peak in the O-K edge fine structure with increasing vacancy concentration. Figs. 1b and 1c show experimental Ce M45 and O-K near edge structure acquired at the edge and center of a CeO2 particle. The Ce white line intensity switch is characteristic of Ce4+ reduction to Ce3+, and in this case is accompanied by a drop in the first O K-edge peak similar to that in the calculated spectra (1a).
 Conductivity data (fig. 2a) shows that the GB conductivity is an order of magnitude higher in GPDC compared to GDC. Figs. 2b and 2c show Ce white lines and O K-edge fine structure acquired at a GB and grain interior in GPDC. In this case the drop in the first O K-edge peak is not accompanied by a Ce white line intensity switch.
 Here we probe and correlate the cation distribution, oxidation state and the O K-edge fine structure to elucidate the vacancy environment at GBs in various doped CeO2 electrolytes. These measurements coupled with macroscopic characterization of GB conductivity will be used to relate atomic-level GB structure and chemistry with bulk electrical properties. We also aim to refine our FEFF calculations to improve the quantitative robustness of our experimental approach to determining the distribution of O2- vacancies near GBs.

References
[1] Rehr, J.J, et al., Phy. Chem. Chem. Phy., 2010 12 5503-5513

We thank Kevin Jorrisen for FEFF help, NSF GRFP-1311230 & DMR-1308085, ASU NASA Space Grant & ASU Cowley HREM Center

Fig. 1: (a) Calculated EELS O K-edge near-edge structure as a function of O2- vacancy in CeO2. The intensity of the first peak drops with increasing vacancy concentration. (b & c) Experimental Ce M45 white lines and O K-edge near edge structure acquired at the edge and center of a CeO2 particle.

Fig. 2: (a) Conductivity data showing the much higher GPDC grain boundary conductivity, σgb (◊). (b & c) Experimental Ce M45 white lines and O K-edge near edge structure acquired at a grain boundary and grain interior in GPDC.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-5-O-3427 Elastic scattering of atomic-size electron probes carrying orbital angular momentum in aberration-corrected scanning transmission electron microscopy

Idrobo J. C.1
1Oak Ridge National Laboratory
jidrobo@gmail.com

The pioneering work by Uchida and Tonomura in 2010 [1] showed that electron beams carrying orbital angular momentum (OAM) can be produced in a transmission electron microscope. Since then, there has been a large interest in the microscopy community to produce atomic-size electron probes carrying OAM [2,3]. The interest arises because using those probes, in principle, one could study magnetic dichroism at the atomic scale through electron energy-loss spectroscopy (EELS) in aberration-corrected scanning transmission electron microscopy (STEM) [2].

In this work, we will present calculations that show how an atomic-size electron probe carrying OAM (vortex probe) channels through the sample and how its OAM character is affected by channeling. We will discuss the reasons why STEM images using vortex probes seem to show lower intensity contrast than images obtained with conventional aberration-corrected probes (as the example illustrated in Figure1). The STEM images simulations were obtained with a multislice algorithm scheme, using a recently developed code in Python (pySTEM) at Oak Ridge National Laboratory. The code calculates electron probes (up to C7 aberrations) with OAM implemented following the electron optics setup outlined in Refs. 4 and 5.

References:

[1] M. Uchida and A. Tonomura, Nature 464, 737 (2010).
[2] J. Verbeeck et al., Nature 467, 301 (2010).
[3] B. McMorran et al., Science 331, 192 (2011).
[4] J.C. Idrobo & S.J. Pennycook, J. of Electron Micros. 60, 295 (2011).
[5] O.L. Krivanek, et al., Micros. Microanal. in press (2014).

This research was supported by ORNL’s Center for Nanophase Materials Sciences (CNMS), which is sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy (JCI).

Fig. 1: (Left) Simulated ADF images of monolayer MoS2 with an electron probe with and without orbital angular momentum (OAM).  Simulations done at 100 kV, a converge semi-angle of 30 mrad, and ADF collection semi-angles of 81-200 mrad.  (Rigth) Intensity profile along the centers of a Mo atom and a S2 atomic column.  Intensity profile width of 0.16 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1533 Characterization of EDS Systems with respect to the Geometrical Collection Efficiency

Terborg R.1, Hodoroaba V. D.2, Falke M.1, Käppel A.1
1Bruker Nano GmbH, Am Studio 2D, 12489 Berlin, Germany, 2BAM Federal Institute for Materials Research and Testing, 12200 Berlin, Germany
ralf.terborg@bruker-nano.de

Characteristic parameters are needed to compare the performance of different energy dispersive X-ray spectrometers (EDS). The ISO 15632 standard defines parameters such as energy resolution as the FWHM for the K lines of C, F and Mn. Another crucial feature is the solid angle Ω available for photon collection (Ω=A/r², A: active area of detector, r: distance between radiation origin and center of active detector surface). Ω is not an intrinsic spectrometer property. It can only be defined for a specific detector in combination with a specific system (e.g. SEM), since the minimal possible distance r is determined by the particular detector/microscope geometry. An approach to obtain Ω it is to simply determine A and r but, this is difficult if respective information is not provided by the manufacturer.

For TEM/EDS, the solid angle can be estimated from the ratio of the measured to the theoretical X-ray net count number in a specific element line using a sample of well-known thickness at well-defined acquisition conditions [1]. Parameters such as the detector quantum efficiency, detector and sample geometry as well as electron beam current and quality need to be known for this approach to deliver results close to the real geometric solid angle. Otherwise the strategy can be used to determine just a performance parameter to compare to other TEM-EDS systems.

A similar approach for SEM/EDS systems is to acquire an X-ray spectrum under defined conditions, e.g. a spectrum of a pure Cu bulk sample at 20 keV and known beam current and measure the number of counts in the Cu-K peaks [2]. Using high energy lines reduces the influence of absorption effects, sample surface morphology and contamination. However, some SEMs don't provide the possibility to measure the beam current or don’t have a well calibrated ampere meter. Again, the quantum efficiency must be known. With a significant dead time the input count rate must be used.

A practical approach we suggest for the determination of the real detector-sample distance without need of knowledge of the beam current and detector efficiency is to measure the count rate in a defined energy region for various detector positions retracting the detector in known steps without altering the take-off angle. The count rate I should be proportional to 1/r² and therefore 1/sqrt(I) vs. r should be a straight line through the ordinate origin. This can be used to determine the absolute distance, see Fig. 1, but also to find possible problems with, e.g. shadowing and alignment, which can cause lower count rates, Fig. 2. For the active area A the nominal value can be used. An alternative (if possible) are measurements with apertures of known area placed onto the front of the EDS in a fixed measurement position [3].

[1] R F Egerton, S C Cheng, Ultramicroscopy, 55 (1994), p. 43.

[2] F Schamber, ISO/TC202, Boulder, CO, USA, 2013.

[3] M Procop, Microsc. Microanal, 10 (2004), p. 481.

Fig. 1: Count rate parameter (expressed as cps-1/2) in dependence on the relative position of the EDS for the calculation of the absolute distance from the radiation origin to the detector chip.

Fig. 2: Count rate parameter in dependence on the relative position of the EDS showing shadowing or misalignment leading to a non-linear dependence for small distances.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1543 Optimizing spatial and energy resolution in the TEM at low dose - Lateral Resolved EELS of organic bulk heterojunctions

Kast A. K.1,2, Oster M.1, Pfannmöller M.1,3, Benner G.4, Kowalsky W.2,5, Schröder R. R.1,2,6
1CryoEM, CellNetworks, Universitätsklinikum Heidelberg, Germany, 2InnovationLab GmbH, Heidelberg, Germany, 3EMAT, Antwerp, Belgium, 4Carl Zeiss Microscopy GmbH, Oberkochen, Germany, 5Institut für Hochfrequenztechnik, TU Braunschweig, Germany, 6Center for Advanced Materials, Universität Heidelberg, Germany
anne.kast@bioquant.uni-heidelberg.de

The morphology of organic bulk heterojunction (BHJ) solar cells is strongly correlated to the efficiency of the device [1]. To improve device performance, knowledge of the nanoscale morphology is thus essential. We implement a novel analytical method using Energy Filtered Transmission Electron Microscopy (EFTEM), which allows visualization of donor and acceptor materials in these thin films by analyzing electronic excitation features in the optical and plasmonic electron energy loss region.
Segmentation by Electron Energy Loss Spectroscopy (EELS) reveals that the carbon based organic photovoltaic materials show characteristic optical excitations in energy-loss spectra. However, the blend materials are very sensitive to radiation damage, which impedes also spatial spectral mapping using EELS in conventional scanning beam mode [2].
We introduce an automated scheme which exploits the inherent spatial resolution in the EEL spectrum as it is obtainable from aberration corrected imaging energy filters. It involves automatic scanning of the image of a slit aperture in the illumination beam path. To eliminate residual image distortion in the EEL spectrum we apply additional correction algorithms to facilitate quantitative spectrum interpretation.
Fig. (1a) shows a bright-field image of a polymer:PCBM BHJ and (1b) the spectroscopic image. The polymer-rich areas are represented in green while PCBM-rich areas are red. The slit position during LREEL spectrum acquisition is marked in (Fig. 1a). It was divided into eleven segments from each of which an averaged spectrum was extracted. The acquired spectrum is shown for three segments, in Fig. (2) (electrons are spread horizontally according to energy loss, the zero-loss peak is shown in red) and the averaged spectra from these areas are seen in Fig (3a), (3b) and (3c), respectively. Distinct differences in the spectral information from the different areas are obvious.
The application of such automated laterally resolved EEL spectroscopy (LREELS) as described here allows spatial mapping of high-resolution spectra in two dimensions at low-dose conditions. This is crucial for our understanding of organic BHJ solar cells.

[1] Pfannmöller, M. et al. Nano Lett. 11 (2011) 3099–3107
[2] Egerton, R. F. et al. Micron 43 (2012) 2–7

Financial support by the BMBF (FKZ 03EK3505K, FKZ 13N10794) is gratefully acknowledged.

Fig. 1: a) Bright-field image of a Polymer:PCBM BHJ and b) spectroscopic image depicting the polymer-rich areas in green and the PCBM-rich areas in red. LREELS data was acquired using the slit position marked in a).

Fig. 2: Recorded spectrum after correlation with the slit position. Of the eleven marked blocks (Fig. 1), three are shown representatively. The zero-loss peak is depicted in red. The energy loss is on the horizontal axis. The vertical direction corresponds to the slit length.

Fig. 3: The averaged spectra from areas 1 (a), 2 (b) and 3 (c) as seen in Fig.2. The spectra from the different areas show distinct differences typical for the materials studied here.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1605 Comparisons on Energy and Wavelength Dispersive X-Ray Spectrometry Microanalytical Results: First Approach Based on Iron Ore Sinter Phases

Magalhaes M. S.1,2, Figueiredo e Silva R. C.1,3, Balzuweit K.1,4, Moreira B. B.1, Garcia L. R.5, Persiano A. C.4,5
1Center of Microscopy – Federal University of Minas Gerais (UFMG), 2Consulting & Research , 3Institute of Geosciences - UFMG, 4Department of Physics - UFMG, 5Microanalyses Laboratory of Department of Physics – UFMG
marilias@uol.com.br

Microanalyses of iron ore sinter constituents — hematite, magnetite (Mag), silicoferrite of calcium and aluminium (SFCA) and silicates — are particularly important to handle their impurities. For this reason, these phases need to be well-studied in characterization researches and this had been done for sinters produced with iron ores from Quadrilatero Ferrifero Mineral Province (MG – Brazil). Prior researches had included many qualitative/semiquantitative microanalyses processed by an energy dispersive system (EDS) on a thermo-ionic scanning electron microscopy (SEM). In order to compare different microanalytical approaches and spectrometric devices, which is the main concern of this work, two of these sinter phases have been chosen: SFCA, a typical phase of these sinters, and Mag. At this time, a field emission gun SEM has been employed to acquire qualitative/semiquantitative analyses as much as quantitative ones. In the latter, standards were used. In the same way, a microprobe with wavelength dispersive spectrometers (WDS) was also applied, using similar standards. In a first remark, the relative behavior among the major elements constituents of these phases was retained in spite of the applied method. However, some differences can be highlighted. For SFCA (fig. 1), a similar trend has been observed for a same element, but each one has its proper behavior. Considering all applied spectrometry devices and elaborating the assessment of microanalyses from one sample, some aspects have been observed: Fe stated as Fe2O3 occurs close to 60wt% and inferior to 90wt%, as a rule between 70-85wt%; Ca usually varies near to 10-15wt% of CaO; Si ranges from 3 to 6wt% of SiO2, reaching either higher contents like 8wt% or lower ones as 2wt%; Al commonly achieves 2-4wt% of Al2O3; Mg as major element ranges between 1-2wt% of MgO; in minor quantities, it is close to 1wt% of MgO. For magnetite, a similar trend was also observed for a same element: Fe quantified as Fe2O3 vary from 87 to 97wt% (all spectra), for all devices. For the other elements: SiO2 extends from almost 0.0 to 1.2wt%; Al2O3 varies from 0.5 to 2.0wt%; CaO reaches values from 0.5 to 1.7wt% and MgO achieves contents between 2.5- 9.5wt%. In Mag, Si, Al, Ca and Mg are impurities. Even though diverse behaviors have been observed, it is possible to reproduce the composition of SFCA and Mag with a relative similar evaluation regardless of the applied method, revealing that all types of microanalyses (EDS and WDS) represent the behavior of the discussed elements. In the following steps, the number of microanalyses will be enlarged aiming the validation of this first assessment and to ensure the most appropriate system to understand the phases and related individual requests.

We are grateful for the infrastructure of the Center of Microscopy and of the Microanalyses Laboratory of Department of Physics, both institutions from UFMG.

Fig. 1: The diagram shows Fe2O3 mean distribution in some portions of SFCA, a typical iron ore sinter phase, considering EDS and WDS devices. Stdless: Standardless; STD-A: General Standard; STD-B: Particular Standard for SFCA; STD-C: Particular Standard for Magnetite; No-N: no-normalized.

Fig. 2: Other diagram showing Fe2O3 mean distribution in some portions of SFCA, a typical iron ore sinter phase, considering EDS and WDS devices.Jeol: JSM-5410 thermo-ionic SEM with a NORAN TN-M3055 spectrometer; FEI Company: Quanta-200 (Q200) FEG SEM with an EDAX Sapphire Si(Li) spectrometer; Jeol: JXA 8900R microprobe with four WDS spectrometers.

Fig. 3: The diagram shows the elements mean distribution in some portions of SFCA, a typical iron ore sinter phase, using EDS and WDS devices. The elements are stated as oxides - SiO2; Al2O3; CaO; MgO. See figure captions on figures 1 and 2.

Fig. 4: Other diagram showing the elements mean distribution in some portions of SFCA, a typical iron ore sinter phase, using EDS and WDS devices. The elements are stated as oxides - SiO2; Al2O3; CaO; MgO. See figure captions on figures 1 and 2.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1618 Magnified pseudo atomic column elemental maps realized by STEM moiré method

Okunishi E.1, Kondo Y.1
1EM Business Unit, JEOL Ltd., 3-1-2 Musashino Akishima Tokyo 196-8558, Japan.
kondo@jeol.co.jp

In recent years, modern technologies such as aberration correction realized an atomic column elemental mapping with analytical tools such as electron energy loss spectroscopy (EELS) and/or energy dispersive X-ray spectroscopy (EDS) [1,2], which is useful, since atomic species and positions in a crystalline specimen can be determined directly. A crucial issue to perform the mapping is specimen damage due to high electron dose onto a specimen, since an excitation probability for core electrons is small.
STEM moiré fringes for a periodic lattice arise when a pixel interval is close to a lattice spacing, due to the under-sampling effect [3]. With the proper pixel intervals in x and y, the moiré fringe shows the pseudo 2D magnified moiré lattice, which is homothetic to the original lattice [4]. The magnification (M) of moiré lattice to the original lattice is determined as  M = |1 – r|-1, where r is the ratio of the lattice spacing to the pixel interval. A magnified moiré lattice is formed with under-sampled signals picked from original lattices, resulting in reduced electron dose by M-2 on the specimen to form an atomic column with an equal pixel resolution. This paper reports a method to observe the atomic column elemental map with less electron dose and higher pixel resolution, utilizing the STEM-moiré method.

The specimen for our experiment was SrTiO3 [001] that has a square lattice. The microscope used was a Cs corrected microscope (JEM-ARM200F), equipped with a SDD type EDS. Figs. 1(a-f) show the high angle annular dark field (HAADF) and annular bright field (ABF) [5] images of magnified moiré lattice at various r. The magnification (M) increases as r approaches one. Figs. 2(a-i) show simultaneously obtained elemental maps of Sr, Ti and O, detected with an EDS. Each element was clearly separated on each magnified moiré elemental map. No beam damage on the specimen was observed during the experiment. Fig. 3 shows three line profiles along a (Ti+O)-(O) row of O-Kα map, Ti-Kα map and ABF image shown in Figs. 2(g,d) and 1(d). The profiles clearly show the peaks at oxygen sites.

In conclusion, the STEM moiré method was successfully applied to atomic column elemental mapping. The method can be applicable to measure detailed physical properties such as delocalization or channeling in crystalline specimens with higher pixel resolution, better signal-to-noise ratio and less electron dose than the direct atomic column mapping.

References:
[1] K Kimoto et al., Nature 450 (2007), p. 702.
[2] E Okunishi et al., Microsc. Microanal. 12 (Suppl. 2) (2006), p. 1150.
[3] N Endo and Y Kondo, Microsc. Microanal. 19 (Suppl. 2) (2013), p. 346.
[4] D Su and Y Zhu, Ultramicroscopy 110 3 (2010), p. 229.
[5] E Okunishi et al., Microsc. Microanal. 15 (Suppl. 2) (2009), p. 164.

Authors thank to Mr. Hosokawa of JEOL Ltd. for valuable discussion on a theoretical consideration.

Fig. 1: High angle annular dark field (HAADF) and annular bright field (ABF) images of magnified moiré lattice at various r.

Fig. 2: Elemental maps of magnified moiré lattice at various r, detected with the EDS.

Fig. 3: Line profiles along a (Ti+O)-(O) row of the O-Kα map, the Ti-Kα map and the ABF image shown in Figs. 2(g), 2(d) and 1(d).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1630 How to Avoid Unexpected Artifacts from Multivariate Statistical Analysis on STEM Spectrum-Imaging Datasets

Ishizuka K.1, Watanabe M.2
1HREM Research Inc., Higashimatsuyama, Japan, 2Dept of Materials Science and Engineering, Lehigh University, Bethlehem, USA
ishizuka@hremresearch.com

The latest aberration-corrected scanning transmission electron microscope (STEM) makes possible to perform routinely not only atomic-scale imaging but also chemical analysis via electron energy-loss spectrometry (EELS) and X-ray energy dispersive spectrometry (XEDS) [e.g. 1]. In combination with the latest hardware, the advances in the recent software developments allow us to acquire large-scale datasets such as multidimensional image series and spectrum images (SIs). Therefore, it is challenging to deal with the large-scale datasets, e.g. extraction of unknown features and estimation of dominant trends. If the datasets were relatively noisy, which is very common for atomic-resolution EELS/XEDS SIs, data analysis would be much harder tasks. Multivariate statistical analysis (MSA) is one of efficient approaches to analyse the large-scale datasets in terms of feature identification and extraction.
Principal component analysis (PCA) is one of the MSA techniques [2]. Since a use of PCA is relatively straightforward, PCA has been applied to SIs as data-mining and noise-reduction tools [e.g. 3]. The PCA tries to explain the data variation (variance) as much as possible using a small number of the components. Here, the signal itself of course contributes the data variation. However, a small amount of signal will be buried with the whole random noise. Therefore, despite that the PCA approach is very efficient and useful, it may create unexpected artifacts especially in higher noise conditions [4] (Figure 1). Since these artifacts might mislead results, it is essential to avoid such artifacts. There may be two approaches to improve the PCA sensitivity: (1) reduction of random noise and (2) enhancement of true variations. The former requires modifications in experimental conditions (higher currents and longer acquisitions). Conversely, the latter can be achieved by PCA analysis to divided small segments within a SI, which is called the local PCA approach (Figure 2). The division can be made spatially and spectrally. The spatially local PCA will be especially useful to detect segregated element in the matrix. The spectrally local PCA is useful to detect a weak signal, if the weak signal is spectrally separated from the strong signal. Especially the spectrally local PCA is useful for EELS Sis, since the background intensity varies significantly. In this study, advantages of the local PCA approach will be addressed.
[1] S.J. Pennycook & P.D. Nellist ed. Scanning Transmission Electron Microscopy: Imaging and Analysis, Springer, NY, (2011).
[2] E.R. Malinowski, Factor Analysis in Chemistry, 3rd ed., Wiley, New York, (2002).
[3] M Watanabe et al., Microscopy and Analysis, 23, Issue 7, (2009), 5-7.
[4] S. Lichtert & J. Verbeeck, Ultramicrosc., 125 (2013), 35-42

The authors acknowledge J. Verbeeck for providing the simulated BN test data. M.W. wishes to acknowledge financial support from the NSF through grants DMR-0804528 and DMR-1040229.

Fig. 1: Failure of PCA [4]. (a) BN model, where there are one excess N or B atom at the positions 1 and 2, respectively. (b) Untreated and weight PCA N element maps for low and high noise. Note that the N map of high noise shows higher intensity at 2 than 1.

Fig. 2: (a) and (b): Spatially local PCA and spectral local PCA, respectively. (c): N maps reconstructed by the spectrally local PCA shows higher intensity at the position 1 even for the high noise case contrary to the normal PCA.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1706 Formation of oxidatively stable M@Fe3O4 and MPt@Fe3O4 (M = Fe, Co) core@shell nanoparticles using a simple and versatile synthetic procedure

Knappett B. R.1, Gontard L. C.2, Ringe E.1, Tait E. W.1, Fernandéz A.2, Wheatley A. E.1
1University of Cambridge, 2Instituto de Ciencia de Materiales de Sevilla
bk324@cam.ac.uk

Core@shell nanoparticle synthesis offers the ability to create materials with dual characteristics, such as a magnetic core and a functionalisable or catalytically active shell. They are currently the subject of extensive research due to the tuneability of their structure and therefore properties.1 Our research focusses on the coating of magnetically interesting materials to protect against oxidation. Iron and cobalt nanoparticles are both strongly magnetic, but are highly susceptible to oxidation. Much work has been carried out to coat these materials in organic surfactant and polymer layers in an attempt to protect against core oxidation;1 however the coating procedure used in the current work utilises a 2-3 nm inorganic layer of Fe3O4, formed by the decomposition of Fe(CO)5 in solution, to stabilize the particle cores. The recently published Co@Fe3O4 system has been shown to have detectable levels of carbon present after oxidative plasma cleaning. This carbon must therefore be contained within the particle structure, suggesting that surfactant molecules that capped the Co seeds became trapped during shell formation.2 This has been verified using scanning transmission electron microscopy (STEM) to record electron energy loss spectroscopy (EELS) line scans and point scans with very high spatial resolution. Figure 1 shows the use of EELS point scans on a Co@Fe3O4 sample to confirm that, after plasma cleaning, no carbon can be detected at the outer surface of the particle shell, yet carbon remains detectable at the core-shell interface. It is also observable that in spite of the highly oxidative plasma cleaning process, the Co core remains metallic in nature. Figure 2 shows a line scan through an Fe@Fe3O4 nanoparticle, confirming a structure similar to that of Co@Fe3O4.

Recently, research has focused on utilizing the coating procedure to encapsulate FePt and CoPt alloys. These particles have interesting magnetic properties for applications in magnetic arrays for data storage. However, once synthesised, these particles require annealing, which often causes sintering. It has previously been established for FePt particles that a coating of iron oxide will prevent the particles from sintering during annealing.3 However, the coating procedure used was different to that employed in our work. We are seeking to prove that the same stabilization is granted the particles by our coating procedure, and to further extend the annealing studies to the system of CoPt, the coating of which has not before been reported.

References
[1] Ghosh Chaudhuri, R.; Paria, S., Chem. Rev., 112 (2012), 2373.
[2] Knappett, B. R. et al., Nanoscale 5 (2013), 5765.
[3] Liu, C. et al., Chem. Mater. 17 (2005), 620.

The authors would like to acknowledge financial support from The Junta de Andalucia (FEDER PE2009-FQM-4554, TEP-217) and EU FP7 AL-NANOFUNC project (CT-REGPOT2011-1- 285895). B. R. K. thanks the UK EPSRC, The University of Cambridge and Downing College for grants. E. R. acknowledges support from the Royal Society in the form of a Newton International Fellowship.

Fig. 1: Electron energy loss spectroscopy (EELS) point scans of the support (Si3N4, shown in blue) and the boundary of the core and shell of a particle. The signal clearly shows the presence of carbon within the structure of the particle after plasma cleaning.

Fig. 2: EELS line scan of an Fe@Fe3O4 particle, evidencing the presence of carbon within the structure of the particle. Again, the sample had been plasma cleaned, thus removing all carbon-containing ligand molecules external to the particle structure.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1713 Detection and quantification of phosphorus dopants in germanium by energy dispersive spectrometry using an annular detector with large solid angle

Robin E.1, Mollard N.1, Guilloy K.1, Pauc N.1
1CEA-INAC, Grenoble, France
eric.robin@cea.fr

Energy dispersive spectrometry (EDS) is generally not considered as a sufficiently sensitive and accurate technique for dopant detection and quantification. Indeed, the concentrations of traditional dopants are typically below ≈ 1020 at cm-3, which is very close to the detection limit of conventional EDS.

We report here the detection and quantification by EDS of phosphorus (P) dopant concentrations in germanium as low as 5 1018 at cm-3 with a precision and detection limit around 1018 at cm-3. This is achieved by using the Flat Quad 5060F annular detector recently developed by Bruker-AXS. This new generation of silicon drift detectors is composed of four bean-shaped silicon diodes, each of 15 mm2, arranged in a ring around a 1.6 mm central hole for the electron beam passage (Fig.1a). It is positioned a few millimeters above the sample (Fig.2b), a geometry which results in a much wider solid angle (up to 1.1 sr) compared to traditional detectors (<0.1 sr), thus allowing a higher counting rate at any operating conditions (up to 1000 kcps). The passage of the electron beam through the detector precludes the use of a conventional electron trap, which role is to protect the diodes against the backscattered electrons. To prevent detector damage, three mylar windows are mounted on the detector, the first (1 µm thick) being permanent to operate in the range 0-6 kV, the two others (2 and 6 µm thick) being retractable to operate in the range 6-12 kV and 12-20 kV, respectively. Although it was not their primary function, the two retractable windows may act as a high-pass X-ray energy filter allowing enhancement of the detection sensitivity for high energy X-rays. For instance, the insertion of window 2 enhances by a factor of 2 the counting rate in the P region (Fig. 2a). The Ge pile-up is also reduced due to the absorption of the Ge L lines, which also improves the detection of low concentrations of P dopants.

We tested five Ge 2D layers previously analyzed by Tof-SIMS and containing 0.66, 0.71, 0.98, 2.5 and 36 1019 at cm-3 of P dopants. Samples were analyzed at 4 different voltages (3, 4, 6 and 8 kV) with the window 2 inserted. All spectra were acquired for 2 hours at ≈ 500 kcps and normalized to pure Ge spectra (Fig. 2b). The reproducibility was tested by repeating the analyses at least three times. Results show a relatively good consistency with Tof-SIMS results, even for the lowest concentrations of P dopants (Fig. 2c). The reproducibility is within the analytical uncertainty of the counting statistic. The precision and the detection limit depend on the voltage and the total acquisition time (Fig. 2d). Typically at 4 kV on P doped Ge nanowires, it is around 2 1018 at cm-3 and 1018 at cm-3 for 30 minutes and 2 hours of acquisition time, respectively.

Fig. 1: The Flat Quad 5060F annular detector from Bruker-AXS: a) bottom view showing the four bean-shaped Si diodes (d), the central hole (h) and the first retractable mylar window (w); b) top view with the sample in place.

Fig. 2: a) 6 kV EDS-FQ spectra acquired on pure Ge using increasing thickness windows; b) 4 and 8 kV Ge-normalized EDS-FQ spectra acquired with window 2 on P doped Ge 2D layers; c) Comparison of P dopant concentrations between EDS-FQ and ToF-SIMS; d) precision/detection limit for P dopants by EDS-FQ.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1781 Effect of the asymmetry of dynamical electron diffraction on intensity of acquired EMCD signals

Song D. S.1, Wang Z. Q.1, Zhu J.1
1National Center for Electron Microscopy in Beijing, School of Material Science & Engineering, Tsinghua University, Beijing 100084, China
todongsheng@126.com

One of the most challenging issues to characterize magnetic materials in the transmission electron microscopy is to obtain quantitative magnetic parameters on the nanometer scale. By the technique of electron energy-loss magnetic chiral dichroism (EMCD) is proposed and applying the sum rules, it is possible to quantitatively extract the orbital to spin magnetic moment ratio mL/mS with high spatial resolution. Compared with the technique of XMCD, the detection source of EMCD technology are the transmission electrons rather than the X-ray based on the precious synchrotron radiation. Therefore, the dynamical diffraction effects of electrons are quite remarkable in the periodic crystal structures, making the quantitative EMCD technique more complicated. By establishing the quantitative relation between EMCD and dynamical diffraction effects, spin and orbital moment of different elements and nonequivalent crystallographic sites are quantitatively determined in a spinel structure NiFe2O4 [1].
However, the diffraction geometry in EMCD experiment is strict and conditions of symmetric detector positions should be fulfilled. It has been reported that the inherent asymmetry of the two-beam geometry can lead to systematic errors in quantitative EMCD measurements [2]. Besides, the asymmetry of dynamical coefficients in the three-beam geometry also exists and is neglected in the previous study. Here, we point out that the asymmetry of dynamical electron diffraction should be accounted and its impact on the quantitative measurements of the EMCD signal needs to be evaluated.
Reference:
[1] Z.Q. Wang, X.Y. Zhong, R Yu, Z.Y. Cheng, J Zhu. Quantitative experimental determination of site-specific magnetic structures by transmitted electrons. Nature communications, 2013, 4: 1395.
[2] J. Rusz, P.M. Oppeneer, H. Lidbaum, S. Rubino, K. Leifer. Asymmetry of the two‐beam geometry in EMCD experiments . Journal of microscopy, 2010, 237(3): 465-468.

This work is financially supported by National 973 Project of China (2009CB623701) and Chinese National Nature Science Foundation (11374174,51390471 ). This work made use of the resources of the Beijing National Center for Electron Microscopy and Tsinghua National Laboratory for Information Science and Technology.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1792 Spatial and Temporal Coherences in Spin-Polarized Transmission Electron Microscopy

Kuwahara M.1, Kusunoki S.1, Nambo Y.1, Saitoh K.2, Ujihara T.1, Asano H.1, Takeda Y.3, Tanaka N.2
1Graduate school of Engineering, Nagoya University Nagoya, 464-8603, Japan, 2EcoTopia Science Institute, Nagoya University, Nagoya 464-8603, Japan, 3Nagoya Industrial Science Research Institute, Nagoya 464-0819, Japan
kuwahara@esi.nagoya-u.ac.jp

Great advances have recently been made in magnetic recording technology and spintronic devices, which are promising for high-density storage devices. Such devices are expected to lead to the development of systems that can analyze magnetic and spin states with a nanometer-order spatial resolution.

We have commenced a development of a spin-polarized transmission electron microscope (SPTEM), which consists of a polarized electron source (PES) and a conventional TEM [1-3]. Figure 1 shows a photograph of the SP-TEM. Spin-polarized electrons can be generated using an optical orientation of III–V semiconductors and vacuum extraction that uses a negative electron affinity (NEA) surface. Several beam parameters of the PES are vastly superior to those of conventional thermal electron beams. In addition, it has the ability to generate a sub-picosecond multibunch beam[4]. A high ESP of 92% and a high QE of 0.5% have been realized using a GaAs–GaAsP strained superlattice photocathode[5].

We have already demonstrated that the SPTEM can provide both TEM images and the diffraction patterns [1]. The TEM images can be obtained in a spatial resolution of 1 nm in a 30-kV acceleration voltage. The apparatus has a below 240-meV energy width of electron beam in the TEM without any monochrometors (Fig. 2). The energy width indicates the temporal coherence is about 2.7 fs (longitudinal coherence of 2.7×10-7 m) at 30-keV beam energy. A brightness is directly measured by taking a spot size and a convergent angle on an image plane. The measured brightness is about 4×107 A/cm2sr in a 30-keV beam energy with a polarization of 82 % and the drive-laser power of 800 kW/cm2 on the photocathode [6]. The brightness for a 200-kV beam energy is 3×108 A/cm2sr which is converted by using a Lorentz factor. The order of the brightness is enough to do an interference experiment. We also demonstrated interference fringes of spin-polarized electron beam by using a newly installed biprism as shown in figure 3. These results indicate the SP-TEM can provide enough coherence in both lateral direction and longitudinal direction even if the semiconductor photocathode is used for an electron emitter.

[1] M. Kuwahara et al., Appl. Phys. Lett. 101 (2012) 03310

[2] M. Kuwahara et al., AMTC Letters 3 (2012) 180.

[3] M. Kuwahara et al., J. Phys.:Conf. Ser. 298 (2011) 012016.

[4] Y. Honda, et al., Jpn. J. Appl. Phys. 52, 086401-086407(2013).

[5] X.G. Jin et al., Appl. Phys. Express 1 (2008) 045002.

[6] M. Kuwahara et al., to be submitted (2014).

The authors thank Drs. H. Shinada, M. Koguchi and M. Tomita of Hitachi Central Research Laboratory for fruitful discussions and encouragement. This research was supported by MEXT KAKENHI Grant Number 51996964, 24651123, 25706031 and Kurata Research Grants from the Kurata Foundation.

Fig. 1: Photograph of the spin-polarized TEM.

Fig. 2: Energy spread of spin-polarized electron beam as a function electron energy.

Fig. 3: Interference fringe of spin-polarized electron beam extracted from a GaAs-GaAsP strained superlattice photocathode using a biprism.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1793 Measurement of liquid vibrational spectra using monochromated STEM-EELS

Miyata T.1,2, Fukuyama M.1,3, Hibara A.3, Ikuhara Y.2,4, Okunishi E.5, Mukai M.5, Mizoguchi T.1
1Institute of Industrial Science, the University of Tokyo, Tokyo, Japan, 2School of Engineering, the University of Tokyo, Tokyo, Japan, 3School of Science, Tokyo Institute of Technology, Tokyo, Japan, 4Japan Fine Ceramics Center, Aichi, Japan, 5JEOL Ltd., Tokyo, Japan
tomo-m@iis.u-tokyo.ac.jp

  Liquid is widely used in daily life and industrial activities. The dynamic behavior of the molecules in liquid is an important factor to determine the various liquid properties. The dynamic behavior of liquid molecules has been extensively investigated using infrared (IR) spectroscopy and Raman spectroscopy. However, these spectroscopy techniques allow us to obtain only averaged information of the entire sample. On the other hand, a specific location, such as solid-liquid interface, plays important role for reactions in electrochemistry and organic chemistry, in which liquid is treated as reactants and reaction media. That is, the methods for analyzing the dynamic behavior of liquid molecules at high spatial resolution have been desired.
  In this presentation, thus, we will report the results of the measurements of liquid vibrational spectra by monochromated STEM-EELS. For the analyses, I used an aberration corrected STEM with a monochromator (JEM-2400FCS, JEOL Ltd., 120keV). The energy resolution reached 0.065eV using the monochromator. As a liquid sample I chose a popular ionic liquid, 1-ethyl-3-methylimidazolium bis (trifluoromethyl-sulfonyl) imide (C2mim-TFSI). In order to verify the vibrational spectra by STEM-EELS, an IR spectrum was measured from the same sample. In addition, first principles calculations were performed to interpret the peaks in the vibrational spectrum. The plane-wave pseudopotential method (CASTEP code) was used in the calculations.
  From the STEM-EELS measurement, the HOMO-LUMO gap of C2min-TFSI was estimated to be 5.3eV, which is consistent with the results of the first-principles calculations and a separately measured ultraviolet-visible (UV-vis) spectrum. The peaks ascribed to the molecular vibration were measured in the vicinity of 0.4eV. These peaks were also observed in the IR spectrum and the one from the first-principles calculations. From those analyses, it was confirmed that the peaks at the 0.4eV correspond to the CH bonds stretching peaks. Based on this study, we have demonstrated that the vibrational peaks of the nano area in liquid are available by the monochromated STEM-EELS.

This study was supported by of MEXT and JSPS (22686059, 25106003). Some calculations were performed using a supercomputer at Institute of Solid State Physics of the University of Tokyo.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1811 Features and Applications of an Analysis System using Double Silicon Drift-type Detectors for Transmission Electron Microscopy

Ohnishi I.1, Kawai S.1, Ishikawa T.1, Yagi K.1, Iwama T.1, Miyatake K.1, Iwasawa Y.1, Matsushita M.1, Kaneyama T.1, Kondo Y.1
1JEOL Ltd., Tokyo, Japan
ionishi@jeol.co.jp

  The elemental analysis using energy dispersive X-ray spectroscopy (EDS) requires considerably long time due to a small ionization cross section for core electrons of a specimen and a small solid angle for a detector. Therefore, the realization of an efficient, short time analysis not only serves the increasing needs of users but also reduces specimen damage due to electron irradiation. For this purpose, we have developed a new analysis system, which is extremely sensitive than the one at present. We report the features and applications of the newly developed analysis system, which is composed of two silicon drift-type detectors (SDDs).

  Our newly developed analysis system consists of two SDDs (double-SDD) with a large sensor area (100 mm2 in area). Figure 1 shows the schematic configuration of the system for a field emission electron microscope (JEM-2800). A new TEM column has two ports for the detectors. And a special analytical specimen holder, which is thinner than the present one, has been newly developed to reduce the distance between the detector and specimen.

  The total X-ray intensity of the new system has increased, because the X-ray signals collected from two detectors are integrated. The new specimen holder also promotes higher X-ray collection efficiency. As a result, total sensitivity has been significantly improved. For example, the peak intensity of Al K line obtained with a double-SDD has increased to be approximately 1.7 times higher than that obtained with a single SDD as shown in Fig. 2.

  Since the new analysis system can provide high analytical sensitivity, an atomic resolution elemental map with a high S/N ratio can be acquired in combination with Cs-corrected TEM/STEM machines such as JEM-ARM200F. In the upper parts of Fig. 3 are shown atomic resolution elemental maps sized 128 x 128 pixels for SrTiO3<100>. These maps clearly show atomic columns of Sr, Ti and O. The profiles of elemental columns, displayed in the lower parts of Fig. 3, show a significantly improved signal to noise ratio for the double-SDD compared with the one for the single SDD.

  Our new system has significantly high X-ray collection ability. Therefore, it provides a shorter acquisition time than a single SDD system. It helps us, beyond all doubt, to analyze a beam sensitive specimen and to detect trace elements in a specimen.

Fig. 1: Fig. 1: A schematic configuration of an EDS analysis system for a field emission electron microscope (JEM-2800).

Fig. 2: Fig. 2: EDS spectra of an Al foil specimen, obtained by using JEM-2800 (200 kV) with SDD1+SDD2 (red) and SDD1 (blue), respectively. The vertical axis has been normalized by the peak intensity of Al K line, obtained with SDD1.

Fig. 3: Fig. 3: STEM-ADF images and atomic resolution elemental maps (O, Ti, Sr) for SrTiO3<100>, obtained with JEM-ARM200F (200 kV) with SDD1 (left) and SDD1+SDD2 (right). The mapping sizes are 128 x 128 pixels. Line profiles of O Kα, Ti Kα, and Sr Lα gross intensity extracted from yellow lines in ADF images are also shown below the maps.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1826 Orientational dependence of EMCD signals of hcp Co with strong magnetocrsytalline anisotropy

Kudo T.1, Tatsumi K.2, Leifer K.3, Rusz J.4, Muto S.2
1Graduate School of engineering, Nagoya University, 2Eco Topia Science Institute, Nagoya University, 3Department of Engineering Science, Uppsala University, 4Department of Physics and Astronomy, Uppsala University
kudou.tomohiro@h.mbox.nagoya-u.ac.jp

Electron magnetic circular dichroism (EMCD) is an electron spin-related property of ferromagnetic samples revealed as the difference of the EELS inner shell spectra measured at two specific positions on the diffraction plane [1]. EMCD at transmission geometry can be advantageous to the X-ray counterpart, XMCD, in spatial resolution and probing bulk properties since the L2,3/M4,5 white-lines of transition metals/rare earth elements, to which the sum rule is applicable, are located in the soft X-ray energy region. For these advantages, a number of experimental schemes have been proposed for better quantitative measurement and higher spatial resolution [2].
In the intrinsic EMCD experimental scheme, with magnetization of a crystalline sample aligned along the strong magnetic field of the objective lens, the symmetric two- or three-beam condition is required [1]. The dichroic signal, Δσ, is acquired as the difference between the two ELNES spectra measured at the two positions, A and B (Fig.1-(a) and (b)). According to the inversion sum-rule [3], the dichroic signal intensity is approximately proportional to (q×q')∙M , where q and q' are the inelastic scattering vectors respectively pointing from O and G to the two detector positions lying on the Thale circle (cf., Fig. 1), and M is the magnetization of the sample. The measured signal intensity is proportional to M·H (H: external magnetic field, parallel to the optic axis) if the direction of M is not fully saturated in the direction of the external magnetic field, which has not yet been experimentally exploited.
We measured EMCD signals of L2,3 in hcp Co, a hard magnet, exhibiting relatively larger magnetocrystalline anisotropy. A thin sample was prepared by electrochemical polishing. In Fig.2-(a) and (b) are shown spectra collected at the two different geometries of Fig.1-(a) and (b), where the optical axis is nearly parallel and perpendicular to the [001] easy magnetization direction of hcp Co with the low-order systematic row excitation conditions. The spectral intensities are normalized by the L3 peak collected at the detector position A. The magnetic dichroism is clearly enhanced in the geometry (a), compared to (b), confirming the unsaturated magnetization along the external magnetic field.
Moreover, a theoretical simulation [3] predicts that with the specific EELS detector positions on the three-beam condition the spin moment in the plane normal to the optical axis can be probed, the trial of which is also presented.
References
[1] P. Schattschneider et al. Nature 441, 486 (2006)
[2] S. Muto et al., Nature Commun., 5, 3138 (2014): doi:10.1038/ncomms4138
[3] J. Rusz et al., Phys. Rev. B 84, 064444 (2011)

A part of this work was supported by a Grant-in-Aid on Innovative Areas "Nano Informatics" (grant number 25106004) and on Young scientist A (24686070) from the Japan Society of the Promotion of Science. J.R. acknowledge support from the Swedish Research Council and STINT.

Fig. 1: Schematics of two experimental geometries in the present study. Optical axis is nearly parallel (a) and perpendicular (b) to easy magnetization axis, respectively.

Fig. 2: Experimental Co-L2,3 ELNES and difference spectra respectively corresponding to geometries (a) and (b) in Fig. 1.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1842 Advanced SEM/EDS analysis using an Annular Silicon Drift Detector (SDD): Applications in Nano, Life, Earth and Planetary Sciences below Micrometer Scale

Salge T.1, Terborg R.1, Ball A. D.2, Broad G. R.2, Kearsley A. T.2, Jones C. G.2, Smith C.2, Rades S.3, Hodoroaba V. D.3
1Bruker Nano GmbH, Berlin, Germany, 2Natural History Museum, London, United Kingdom, 3BAM Federal Institute for Materials Research and Testing, Division 6.8 Surface Analysis and Interfacial Chemistry, Berlin, Germany
tobias.salge@bruker-nano.de

Analysis of fine-scale structures requires low accelerating voltages. Consequently, only low to intermediate energy X-ray lines with many peak overlaps can be evaluated which requires deconvolution. Examination of nano-scale structures also requires low probe currents which would give low X-ray count rates with traditional EDX detectors. The additional time required to acquire sufficient data for deconvolution risks altering the specimen as a result of beam-sample interaction or sample contamination. The BRUKER XFlash 5060F SDD has allowed us to overcome these limitations and offers additional benefits as we demonstrate here.

The annular SDD is inserted between the pole piece and sample and has a large solid angle (1.1sr). It is ideally suited for the analysis of topographically complex, three-dimensional samples. X-rays are collected from 4 separate detector segments and signals are processed in parallel by 4 detection units allowing high count rates at low dead-time. Even at lowest beam current (<10pA), samples can be investigated under high vacuum in natural state. In VP mode, sufficient statistics can be collected with reduced acquisition times, consequently reducing the likelihood of sample contamination.

4 studies are presented here. Experimental impact foil craters (Fig. 1) used to develop methodologies for the examination of samples from NASA’s STARDUST mission were examined (6kV, 1,100kcps, 45% dead time, 4096 x 3072 pixels, 62nm pixels, 7 min). Residues of glass projectiles can be clearly distinguished from the aluminium target with no detector shadowing across the field of view. Fig. 2 shows a portion of the Martian meteorite “Tissint”. This analysis was carried out at low beam current (4kV, <10pA, 733x853 pixels, 55nm pixels, 13 h). The mapped area revealed a thin coating and local enrichment of carbon (and nitrogen). The third study (Fig. 3) was carried out under low vacuum (20Pa, 5kV, 1.8nA, 20kcps, 320x240 pixels, 460nm pixels, 33 min) and shows mineralization in the ovipositor of a parasitoid wasp. Sufficient data quality allows deconvolution of overlapping element lines (Zn-L, Na-K). The final study (Fig. 4) demonstrates that the analysis of core-shell nano particles (5kV, 520pA, 14-72kcps, 250x250 pixel, 2nm pixels, 6 min) on thin film supports has become possible.

It can be concluded that improvements in SDD technology will stimulate new approaches for various fields. To minimize acquisition time and increase spectrum statistics, the total solid angle is relevant, not the active detector area. Element analysis at low kV and low beam current in combination with multi-segment SDDs provides high spatial resolution and high detection sensitivity without the necessity of applying a conductive coating or working in low vacuum.

Fig. 1: Stardust analogue crater experiment; Composite EDX map overlain with SE micrograph reveals residues of glass projectiles (red) on an aluminium target (green).

Fig. 2: Tissint Martian meteorite; Composite EDX map of carbon and oxygen overlain with SE micrograph shows a thin coating and local enrichment of carbon.

Fig. 3: Biomineralization in a parasitoid wasp Monolexis fuscicornis. The ovipositor (sting and egg-layer) reveals ZnO reinforcement and contamination with NaCl.

Fig. 4: Composite EDX map of fluorescent silica nano particles. At the line scan (net intensities, 229 points, 467nm length, 30kcps, and 6.9 s), five adjacent pixel/spectra were binned for each point in order to improve impulse statistics.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1944 Planer defect structure analysis based on electron channeling phenomena

Ichikawa T.1, Ohtsuka M.1, Muto S.2
1Nagoya University, Nagoya, Japan, 2EcoTopia Science Institute, Nagoya, Japan
ichikawa.takahiro@a.mbox.nagoya-u.ac.jp

 High-angular resolution electron channeled X-ray spectroscopy (HARECXS) is based on the principle that the electron wavefunctions (Bloch waves) in a crystalline sample change their symmetry with the incident beam direction. HARECXS has been applied to the analysis of point defects, partly because ICSC [1] has been only the theoretical simulation code available to date for predicting the inelastic scattering cross sections depending on the diffraction condition, which does not allow us to include 2D/3D defects. In this study an attempt is made to apply HARECXS to a planner defect lying on the {111} plane in heavily Si-doped GaAs [2] to extend its applicability.

 Thin films were prepared by dimpling the sliced single crystalline wafers followed by Ar ion milling. HARECXS was carried out using a JEM-2100 S/TEM equipped with an EDX spectrometer, operated at the beam-rocking mode at 200kV. The illuminated area was about 1μm in diameter and the incident beam angle was rocked by ±1.5 degrees with a step of 0.05 degrees under the systematic row excitation condition.

 A bright-field TEM image of the planer defect nearly viewed end-on is shown Fig. 1(a) and the corresponding HARECXS profiles in (b), as functions of the incident beam direction in units of g1-11, tilted in the direction perpendicular to the projected defect plane. The HARECXS profiles of the Ga- and As-K lines have a different symmetry due to the polarity of the GaAs. The slight asymmetric profile of Si-K similar to that of As-K suggests that Si mainly occupies the As sites. The theoretical simulations based on the model where a Si atom substitutes for the As and Ga sites are shown in Fig. 2(a), using the Bloch wave [1] method with the dynamical inelastic scattering process incorporated. For comparison, more realistic models where Si occupies the single (111) Ga or As layer are also simulated, as shown in Fig. 2(b), in which a multislice method [3] is developed for including a planar defect in the simulation. The simulation result with Si occupying the As single layer seems to be relatively more consistent with the experimental Si-K HARECXS profile.

References
[1] M. P. Oxley and L.J. Allen, J. Appl. Cryst. 36 (2003) 940-943.
[2] S. Muto, S. Takeda, M. Hirata, K. Fujii, and K. Ibe, Phil. Mag. A. 66 (1992) 257-268.
[3] M. Ohtsuka, et al., AMTC4 letters (2014) in publish

A part of this work was supported by a Grant-in-Aid on Innovative Areas "Nano Informatics" (Grant number 25106004) from the Japan Society of the Promotion of Science.

Fig. 1: (a)Bright-field TEM image of planer defect in Si-doped GaAs nearly viewed end-on. (b)HARECXS profiles of Ga-K, As-K, and Si-K characteristic X-ray peaks as functions of incident beam direction in units of g1-11.

Fig. 2: Theoretically simulated HARECXS profiles for models where Si atom substitute for Ga (red) and As (blue) sites (a) and Si occupies single (111) Ga (red) and As (blue) atom layers (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1976 EELS of Si-nanocrystals by hyperspectral segmentation and multivariate factorization.

Eljarrat A.1, López-Conesa L.1, López-Vidrier J.1, Hernández S.1, Estradé S.1, 2, Magén C.3,4, Garrido B.1, Peiró F.1
1MIND-IN2UB, Departament d'Electrònica, Universitat de Barcelona, c/ Martí i Franqués 1, 08028 Barcelona, Spain., 2TEM-MAT, Centres Científics i Tecnològics (CCiT), Universitat de Barcelona, Solís Sabarís 1, Barcelona, Spain., 3LMA-INA, Departamento de Física de la Materia Condensada, Universidad de Zaragoza, 50018 Zaragoza, Spain., 4Fundación ARAID, 50018 Zaragoza, Spain.
aeljarrat@el.ub.edu

This work is focused on advanced data analysis methods for the characterization of Si NCs by high angle annular dark field (HAADF) and electron energy loss spectroscopy (EELS) in the aberration corrected and monochromated scanning transmission electron microscope (STEM). These Si-NCs, of high interest for photovoltaic applications, are embedded in multilayer stacks where SiO2, SiC and Si3N4 are used as dielectric barriers.

A comparison will be made between different techniques that exploit the information within low-loss EELS-spectrum images (SI). In this sense, the generation of maps from measured properties on the spectrum, such as characterization of the plasmon peak and relative thickness from the measured spectra was complemented with segmentation of the EELS-SI using mathematical morphology (MM) and a detailed exploration of spectral factorization using multivariate analysis (MVA).

Plasmon energies determined at the EELS-SI reveal the approximate spatial distribution of the Si-NCs and barrier dielectric material (SiO2, SiC and Si3N4, depending on the case). This method is better suited than the examination of the HAADF images, because of the appearance of spurious features from the inhomogeneity of the sample, masking the Si-NC positions (see Fig. 1 and 2). Nevertheless, it was not possible to get a direct measurement of the pure contribution of the Si-NC to the spectra, as all measured data present at least a mixture of nanoparticle and substrate plasmon. Fitting these two peaks using a double plasmon model (DPM) is reliable only when they are well separated in energy and exhibit significant differences in FWHM, i.e. low energy narrow peak vs high energy wide peak (as in Si-NCs in a SiO2 substrate) [1]. However, for other non-favorable situations, segmentation of the EELS-SI by MM can be of help. Following this scheme, averages of the spectra in the particle and dielectric areas can be generated, along with slices of the EELS-SI. These slices are then analyzed using MVA algorithms (NMF and BLU) for a factorization of the EELS data (see Fig. 3).

The collection of computational tools enabling nanometric spatial resolution imaging of the Si-NCs using sub-eV energy resolution EELS will be presented. Maps of measured properties, such as mean free path to sample thickness ratio, will be plotted for the three studied systems with different dielectric barriers. Moreover, the extraction of particular features by segmentation and factorization of the EELS data will allow recovering the pure Si-NC plasmon in each sample. Finally, the possibility of extracting electro-optical properties by thickness-normalized Kramers-Kronig analysis of the spectra will be explored.

[1] A. Eljarrat et al. (2013) Nanoscale 5, 9963-9970

Fig. 1: HAADF (upper left panel) and EELS (blue dashed lines, lower left panel) simultaneously acquired of a SiC sample. The EELS is analyzed to form the plasmon energy map (central panel, with thresholded histogram at left). Si-NC and SiC regions are marked off in this map and the average EELS are overlayed to the raw EELS (black=Si-NC, red=SiC).

Fig. 2: Results from the SiO2 sample, showing the superior sensitivity of the plasmon energy map above the HAADF and relative thickness map. Si-NC and SiO2 positions are marked off in the map and in the histogram (lower panel) as thresholds, using the same color code as Fig. 1.

Fig. 3: MVA factorization results vs. average EELS from the same EELS-SI shown in Fig. 2. After segmentation of the upper Si-NCs region, factorization reveals two different nanoparticles, and their contribution to EELS (comp. 2) is separated from the background SiO2 spectra (comp. 1).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1978 Plasmon and dielectric function mapping of multiple InGaN QW sample by HAADF-EELS.

Eljarrat A.1, López-Conesa L.1, Magén C.2,3, García-Lepetit N.4, Gačević Ž.4, Calleja E.4, Estradé S.1,2, Peiró F.1
1LENS-MIND-IN2UB, Departament d'Electrònica, Universitat de Barcelona, c/ Martí i Franqués 1, 08028 Barcelona, Spain., 2LMA-INA, Departamento de Física de la Materia Condensada, Universidad de Zaragoza, 50018 Zaragoza, Spain., 3Fundación ARAID, 50018 Zaragoza, Spain., 4TEM-MAT, Centres Científics i Tecnològics (CCiT), Universitat de Barcelona, Solís Sabarís 1, Barcelona, Spain., 5ISOM, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
aeljarrat@el.ub.edu

A thorough examination of InGaN quantum wells (QWs) was carried out through high angle annular dark field (HAADF) and electron energy loss spectroscopy (EELS) by aberration corrected scanning electron microscopy (STEM). The considered nanostructure consists of ~1.25 nm QWs layers with 20% indium content, periodically distributed along ~6 nm InGaN barriers, with 5 % indium content.

High resolution HAADF images give structural information from the examined crystal lattice below the nanometer range. Z-contrast in these images reveals the position of the QWs, the occurrence of In diffusion or even the formation of In-rich islands. Moreover, this information can be exploited using geometric phase analysis (GPA) algorithms in order to obtain maps of the deformation along lattice directions. The resulting deformation maps reveal that the structure suffers a localized distortion along the growth axis related to the In-rich QW positions (see Fig. 1).

STEM-EELS spectrum images (SI) are used to gain insight into the material properties of the sample. For this purpose, maps of plasmon peak energy and width are obtained and compared with the HAADF images. Generally, for III-V materials, compositional information can be recovered from the analysis of the plasmon peak energy through Vegard law [1]. In the present case, the small size of the examined QW and plasmon delocalization make this approach difficult to apply. However, the analysis of the plasmon witdth reveals a consistent swelling of the peak related with the position of In-rich regions, along with some expected shift to higher energy (see Fig. 2).

Furthermore, Kramers-Kronig analysis (KKA) of the EELS allows recovering the complex dielectric function (CDF) which contains electro-optical information from the material. For instance, it is possible to calculate the electron effective mass (m*) from the recovered CDFs at each pixel of the EELS-SI. The obtained values of m*, ranging from 0.14·m0 to 0.18·m0 are among the expected for InGaN (m*GaN = 0.2·m0 m*InGaN=0.11·m0). Moreover, depression regions in which the m* values are consistenty lowered are found in regions related with the ones having wider plasmon peaks (see Fig. 3).

All the computational work has been performed using the Hyperspy toolbox. The collection of techniques that have been developed in order to perform these analyses, will be presented along with the obtained results.

[1] A. Eljarrat et al. (2012) Microsc. Microanal. 18, 1143-1154.

Fig. 1: Left panel, HAADF-STEM image of the structure, revealing the position and width of an In-rich QW. Right panel, deformation in the growth direction calculated from the previous image by GPA.

Fig. 2: HAADF image and results from the analysis of the simultaneously acquired EELS-SI on an In-rich QW region. The plasmon analysis reveals a shift towards higher energies (Ep) and a swelling (Γ) of the plasmon peak around the QW. The effective mass (m*) shows a characteristic depression around the same region as the swelling in Γ.

Fig. 3: The upper panel shows two average EELS in the narrow Γ (dashed line) and wide Γ (solid line) regimes. Also following this line code, the lower panel shows the real (red) and imaginary (black) parts of the average CDF from these same regions.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1986 Advantages of FEG-EPMA for Microphase Analysis in Nuclear Materials

BRACKX E.1, NONNET H.2, HOMBOURGER C.3, ALLEGRI P.1, DUGNE O.1
11 CEA, DEN, DTEC, SGCS, LMAC, Marcoule, 30207 Bagnols sur Cèze, France, 22 CEA, MAR, DTCD, SECM, LDMC, Marcoule, 30207 Bagnols sur Cèze, France, 33 CAMECA, 29 Quai des Grésillons, 92622 Gennevilliers, France
emmanuelle.brackx@cea.fr

Many nuclear materials include micrometer-scale particles or phases that require characterization to understand and improve material fabrication techniques and processes. The microphase inclusions can be characterized with better precision thanks to the new generations of Field Emission Gun Electron Probe MicroAnalyzers (FEG-EPMA) whose high-resolution electron beam operating at low voltage optimizes and reduce electron interaction volume [1] [2].
An example of characterization is presented in relation with a study of the crystallization of certain phases in a glass matrix used for nuclear waste applications. Depending on the glass composition and the melting and cooling conditions, crystals can form in the matrix. The characterization of these phases, often of micrometer size, and of the glass including them are primordial in basic studies to elucidate the mechanisms involved. The composition of the including glass can be characterized by means of a CAMECA SX 100 electron microprobe with a low-resolution electron beam. On the other hand, the microcrystals studied here, apatite containing rare earth elements and microparticles of platinum-group metals, require the use of FEG-EPMA to determine their chemical composition because of their small dimensions (less than 10 µm: Figure 1). The analyses were carried out with the CAMECA SX 100 and SXFiveFE at 12 keV and 10 nA. The improved analytical resolution obtained with the CAMECA SXFiveFE made it possible to optimize the analysis of the micro particles, and to determine their chemical formulas.
A second example of characterization is presented in the context of coating materials used in nuclear processes. The coatings must have satisfactory homogeneity to ensure material adhesion and sealing. Any chemical homogeneity defects must then be characterized in order to optimize the manufacturing process. In this context a sealing material containing impurities in the form of micrometer-scale layered inclusions was characterized by EPMA (Figure 2). The analyses were carried out with a CAMECA SX 100 and CAMECA SXFiveFE at 15 keV and 10 nA. A comparison of the results shows the optimization obtained with the CAMECA SXFiveFE due to the high resolution of the electron beam.
REFERENCES
[1] X. Llovet, E. Heikinheimo, A. Núñez Galindo, C. Merlet, J. F. Almagro Bello, S. Richter, J. Fournelle, J. G. van Hoek. Materials Science and Engineering, 32 (2012).
[2] D. E. Newbury. Journal of Research of the National Institute of Standards and Technology, 107, 605-619 (2002).

Fig. 1: Microparticles in a nuclear glass matrix

Fig. 2: Stratified material with a thin vein
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-1995 EDS Element Analysis with High Count Rates

Eggert F.1
1EDAX Inc., AMETEK Materials Analysis Division, Mahwah NJ, USA
frank.eggert@ametek.de

One major benefit of Silicon Drift Detectors (SDD) with count rates of X-ray acquisition was already highlighted with a high speed 250 kcps map in first publication about application for Scanning Electron Microscopes [1] . The high count rates are usual with X-ray spectral imaging in each SEM lab more than 15 years later. The modern SDD spectrometers are able to process high count rates without significant deterioration of their basic spectrometric properties. It will be given a short overview about state of the art.

In the past, analysts have acquired single EDS spectra after selecting objects. This is now performed already with scanning the electron beam across entire specimen surface, which is usually very heterogeneous. A complete spectrum is acquired at each point. The count rates vary about a high range, depending from different specimen composition and topography. Reaching very high count rates of about 200 kcps and more are usual in daily praxis. Despite all SDD electronics are equipped with X-ray coincidence rejection logics, so called pile-ups will pass and are then in spectrum as artefacts. This is very fundamental, with all systems and does not depend from vendors, never possible to neglect. The artificial counts will produce mistakes in qualitative and quantitative analytical results if not considered. An outline will be given about the effects and how to process (Fig 1) [2]. It will be demonstrated the quantitative results are stable up to 200…300 kcps, if a pile-up consideration is applied (Fig 2) [3].

But correction comes with higher result uncertainties and detection limits. Also, the pile-up consideration is with limits, e.g. the entire region must be homogeneous, were all X-rays in a spectrum came from [5]. Otherwise fundamental assumption about pile-up consideration with random emission was violated. It is not satisfied if the electron beam excitation involves areas of different specimen constituent. Phase determination by independent Principle Component Analysis (PCA) is useful to identify homogeneous specimen regions to avoid qualitative and quantitative analytical errors. This would be if total spectrum was taken from inhomogeneous area (Fig 3) [5]. Specialized single pixel spectra evaluation strategies are required for full quantitative maps.

References

[1] Strüder L, Meidinger N, Stötter D, Kemmer J, Lechner P, Leutenegger P, Soltau H, Eggert F, Rohde M and Schülein T (1998) Microsc. Microanal. 4 622
[2] Eggert F, Elam T, Anderhalt R, Nicolosi J (2012) IOP Conf. Ser.: Mater. Sci. Eng. 32, 012008
[3] Eggert F, Anderhalt R and Nicolosi J (2012) Microsc Microanal 18 (Suppl.2)
[4] Eggert F (2010) IOP Conf. Ser.: Mater. Sci. Eng. 7 012007
[5] Eggert F, Schleifer M, Reinauer F (2014) IOP Conf. Ser.: Mater. Sci. (in publication)

Fig. 1: The results of automated qualitative analysis [4] with two spectra of same specimen (a low; b very high count rate) are similar due to internal pile-up consideration (pile-up distribution is not included in reconstruction, blue line). This is despite big differences are visible in both spectra caused by pile-up artefacts (example from [2]).

Fig. 2: The quantitative results vary with count rate, if pile-up was not considered (a). They are much more stable with using pile-up consideration method (b) (example from [3]).

Fig. 3: Phase map of Kiruna mineral with very high count rates. Different phase areas indicate from which pixel regions sum spectra are possible to gather without analytical evaluation issues. The spectrum is from an inhomogeneous area to demonstrate the qualitative analysis challenge, even if the pile-up consideration was applied (example from [5]).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2431 Experimental detection of the Čerenkov limit in Si, GaAs and GaP

Horák M.1, Stöger-Pollach M.2
1Institute of Physical Engineering, Brno University of Technology, Brno, Czech Republic, 2USTEM, TU Vienna, Vienna, Austria
horak.michal@seznam.cz

Since the advent of monochromated electron energy loss spectrometry (EELS) the experimental detection of band gaps in semiconducting materials is of great importance. But due to the fact that the swift electron probe excites relativistic energy losses, like Čerenkov losses [1] and the corresponding light guiding modes, the band gap is hidden below them. Therefore a technique was developed to excavate them mathematically [2]. Another possibility is to reduce the beam energy [3] such that the speed of the swift probe electron v does not exceed the speed of light inside the sample c0/n (with n as the refractive index).

The investigated specimens are Si, GaAs and GaP single crystals. Sample preparation was performed by grinding and ion milling using a low voltage ion mill for the final preparation step in order to remove surface damage from prior milling.

With the TECNAI G20 at TU Vienna we are able to record EELS spectra in the energy range of 6–100 keV. Consequently we can experimentally verify the Čerenkov limit of Si, GaAs and GaP, which is 13.3, 20.6 and 30.1 keV, respectively (Fig. 1). The probability of Čerenkov photon excitation per unit path length of the electron inside the specimen PCP is proportional to

PCP = 1/c0 – 1/(v2ε1),

with ε1 as the real part of the samples dielectric function [3]. The theoretical values are computed for PCP equals to zero. The shaded area in Fig. 1 represents the beam energy needed in order to excite 0.2 to 0.4 Čerenkov photons.

Above the limit Čerenkov losses and light guiding modes cause a red shift of the signal onset in low loss spectra. The signal onset is shifted to higher energies when reducing the electron beam energy and there is no shift below the limit (Fig. 2). It must be noticed, that only the direct gap at 3.6 eV of Silicon is measured.

 

[1] E. Kröger, Z. Physik 216 (1968) 115-135.
[2] M. Stöger-Pollach, A. Laister, P. Schattschneider, Ultramicroscopy 108 (2008) 439-444.
[3] M. Stöger-Pollach, Micron 39 (2008) 1092-1110.

M. H. acknowledges FEI Company for financial support.

Fig. 1: Theoretical Čerenkov-limit of beam energy for refractive index between 1.5 and 6.5. The shaded area represents the beam energy needed in order to excite 0.2 to 0.4 Čerenkov photons.

Fig. 2: Zero loss deconvolved low loss spectra of Silicon at various beam energies. Below the Čerenkov limit at 13 keV no shift of the signal onset can be observed. Above the Čerenkov losses and light guiding modes cause a red shift of the onset.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2062 Nanoscale Luminescence Mapping of InGaN/GaN Multiple Quantum Dot Doped Nanowire LEDs with Scanning Transmission Electron Microscopy

Woo S. Y.1, Kociak M.2, Nguyen H. P.3, Mi Z.3, Botton G. A.1
1Department of Materials Science & Engineering, Brockhouse Institute for Materials Research, and Canadian Centre for Electron Microscopy, McMaster University, Hamilton, ON, Canada, 2Laboratoire de Physique des Solides, Université Paris-Sud XI, Orsay, France, 3Department of Electrical & Computer Engineering, McGill University, Montreal, QC, Canada
woosy@mcmaster.ca

Ternary InGaN compounds show great promise for light-emitting diode (LED) applications because of bandgap energies (0.7–3.4 eV) that can be tailored to have emission wavelengths spanning the entire visible spectral range. Complex III-N device heterostructures have been incorporated into GaN nanowires (NWs) recently, but exhibit emission linewidths that are broader than expected for their corresponding planar counterparts, as measured with photoluminescence (PL) spectroscopy. It is thus critical to understand how the structural and optical properties interplay, using spectroscopic methods that can resolve localized signals at the nanoscale.
Multiple InGaN/GaN quantum dot (QD) embedded nanowire (NW) LED structures, grown on Si(111) substrates by molecular beam epitaxy, were characterized by STEM. Elemental mapping using EELS has shown a systematic non-uniformity of the In-content between the InGaN QDs that are centrally confined within the active region, embedded between n- and p-doped GaN in the NW LED structure (Fig. 2(c,d)). To correlate these observations to the inhomogeneous broadening observed in PL from an ensemble of NWs, nm-resolution STEM-cathodoluminescence (CL) spectral imaging on single NWs was performed using a custom-made system on a VG HB-501 STEM as described in [2]. Individual NWs examined showed diverse optical responses, but most NWs exhibit one main emission peak centered at 500–550 nm in the yellow-green. Spectral features consisting of multiple sharp peaks (25–50 nm at FWHM) spanning a wavelength range of ~100 nm arise from the active region (Fig. 1(b)), showing an apparent spatial dependence of the spectral shifts (Fig. 1(a)). This is consistent with the PL, indicating that the broad emission originates from within single NWs and is not an inhomogeneous broadening. However, typical wavelength-integrated CL mapping was too ambiguous in the spatial assignment of some peaks that have overlapping intensities. Improved spatial-spectral correlation was achieved by inspecting orthogonal spatial slices from the spectrum image singly along x and y (Fig. 2(a,b)) to define various combined position and wavelength maxima. Multiple optical signals of varying emission wavelengths arising from well-defined locations within the QD active region were identified, and can be attributed to the observed In-content variation between successive QDs. Lastly, the evidence of localized emission intensity in the QDs towards the p-GaN, likely due to the diffusion of charge carriers generated by the electron beam, could suggest the accumulation of carriers within the active region towards the p-GaN.
[1] H.P.T. Nguyen et al., Nano Lett., 12(3), 1317-1323 (2012)
[2] Zagonel et al., Nano Lett., 11(2), 568-573 (2011)

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).

Fig. 1: STEM-CL spectrum image (SI) of the NW structures. (a) HAADF and BF image acquired simultaneously with the CL, and spatial maps of spectral features centered about the wavelengths labeled. The three marked regions of interest (ROI) that exhibit unique emission spectra are shown in (b). (c) HAADF image to better resolve the same NW studied using CL.

Fig. 2: Spatial-spectral plots of the SI from Fig. 1, (a) across the SI in the y-axis, (b) along the SI in the x-axis with concurrent HAADF signal overlaid to show the structure; CL intensity is color-coded. (c, d) HAADF image and corresponding STEM-EELS In-map of the boxed area in another NW, showing the varying In-content in the 10 InGaN QDs.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2077 Application of Energy Filtering in STEM (EFSTEM) mode for Mapping of Elements and Chemical Bindings

Muehle U.1, Gluch J.1,2, Zschech E.1,3
1Fraunhofer Institute for Ceramic Technologies and Systems - Materials Diagnostics (IKTS-MD), Maria-Reiche-Str. 2, 01109 Dresden, Germany, 2Institute for Materials Science, TU Dresden, 01062 Dresden, Germany, 3Dresden Center for Nanoanalysis (DCN), TU Dresden, 01062 Dresden, Germany
uwe.muehle@ikts-md.fraunhofer.de

During the previous decade, Scanning Transmission Electron Microscopy (STEM) has gained a growing importance, enforced by the availability of TEMs with high-performance Cs correctors for the condenser system [1, 2]. One major benefit of STEM is that additional signals like X-ray emission (EDX) or energy loss of transmitted electrons (EELS) can be acquired with the same local resolution as the image of a sample.

The energy filtering in the STEM mode provides an improved time-to-data (or time-to-result) at a spatial resolution which is sufficient for a lot of application cases. Compared to the acquisition of complete EEL spectra at every pixel which leads to a long measuring time and a large data volume, , the proposed technique allows to obtain a highly resolved elemental distribution without leaving the STEM mode of the instrument [4].

The energy-filtered STEM data can be acquired using an in-column filter in combination with a BF/DF detector or a HAADF detector, positioned in the electron-optical path behind the filter (Fig. 1). After passing the energy selecting slit, the beam contains only electrons of the chosen energy range, and the acquired signal is comparable with that of the well-known EFTEM method [4].

One application is the improvement of the image quality by removing the inelastic scattered electrons. Shifting the energy of the primary beam allows to acquire STEM images using an energy window with a defined energy loss in the low loss or the core loss region. A combination of several images allows the application of the 3-windows method similarly to EFTEM or of the jump-ratio-method [2] (Fig. 2). The acquisition of a series of images with an energy window of about 2 eV and with stepwise increasing energy loss enables a detailed characterization of chemical bindings – either in the plasmon range of the EEL spectrum or above the ionization edge of an element.

Advantages of this technique are a better utilization of the available beam intensity, which is often weak for large energy losses. This approach enables to improve the focus of the image for large energy losses. In addition, some materials show less electron-beam damage in case of STEM imaging [5]. For these materials, the described technique is the technique of choice to avoid long illumination times as they are necessary for EFTEM. Finally, a sample drift does not influence the results as much as in the TEM mode.

Pennycook, S.J. Scanning Transmission Electron Microscopy Springer 2011

Brydson, R. Aberration-Corrected Analytical Transmission Electron Microscopy RMS 2011

Egerton, R.F. Electron energy-loss spectroscopy in the electron microscope Springer  2011

Muehle, U. et.al. Patent.; 10 2013 011 674.0 2013

Yeap, K.B. et al. IEEE IRPS 2013

Fig. 1: Schematic of an In-column STEM with HAADF-Detector behind the filter

Fig. 2: Elemental mapping of a semiconductor structure, acquired by energy filtered STEM and evaluated using the jump-ratio method for the elements nitrogen (a), oxygen (b) and titanium (c)

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2130 Plasmon Mapping in Au@Ag Nanocube and their assemblies

Goris B.1, Guzzinati G.1, Fernández-López C.2, Pérez-Juste J.2, Liz-Marzán L. M.2,3,4, Trügler A.5, Hohenester U.5, Verbeeck J.1, Bals S.1, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 2Departamento de Química Física, Universidade de Vigo, 36310 Vigo, Spain, 3BioNanoPlasmonics Laboratory, CIC biomaGUNE, Paseo de Miramón 182, 20009 Donostia , San Sebastián, Spain, 4Ikerbasque, Basque Foundation for Science, 48011 Bilbao, Spain, 5Institut für Physik, Karl-Franzens-Universität Graz, Universitätsplatz 5, 8010 Graz, Austria
bart.goris@uantwerpen.be

Plasmons are collective excitations of conduction electrons in metallic particles. For nanostructures, the resonant surface plasmon modes are highly sensitive to the geometry of the structure and can therefore be tuned by controlling their morphology and/or size. Here, we applied monochromated STEM-EELS to map the surface plasmon resonances in Au@Ag nanocubes and their assemblies. [1]

These assemblies reveal interesting plasmonic properties with an increased flexibility as compared to their single particle counterparts.
For the isolated nanocubes, EEL spectra were recorded at different locations, revealing the presence of three distinct plasmon resonances at energy values of 2.2 eV, 3.2 eV and 3.5 eV. As presented in Figure 1, the extracted plasmon maps indicate that the two modes with the lowest energy have the highest probability to be excited at the corners of the particles, whereas the third mode is best excited at the side faces, in agreement with previous reports. [2]

Interestingly, when the nanocubes are dispersed on a C support, they tend to orient themselves side by side yielding regular assemblies. As a first example, 3 nanocubes may form an approximately triangular array as shown in Figure 2. It can be observed that the main plasmon modes are obtained at energy losses of 1.2 and 1.6 eV and are in qualitative agreement to the plasmon modes of a perfect nanotriangle with the same dimensions. [3] Plasmon modes that occur at higher energy losses originate from the deviation of the overall shape from a perfect triangle, resulting in multiple regions of high intensity that are mainly located at the corners of the individual cubes. A nanotriangle constructed by the random ordering of multiple nanocubes could possibly act as the first half of a bow-tie antenna. This antenna has great potential due to the field enhancement in the central region between the two triangles caused by plasmon coupling. [4] As illustrated in figure 3, even with the simplified geometry for a bow-tie antenna, the field enhancement due to the coupling can be clearly visualized in the centre of both the experimental and the simulated plasmon maps. The enhanced field occurs at an energy loss of 1.3 eV which can also be observed as an increased probability for the energy loss in the acquired EEL spectra.


[1] S. Gómez-Graña, J. Phys. Chem. Lett. 4 (2013) p.2209
[2] O. Nicoletti et al., Nature 502 (2013) p.80
[3] J. Nelayah et al., Nature Physics 3 (2007) p.348
[4] A. Koh et al., Nano Letter 11 (2011) p.1323

The authors acknowledge support from the European Research Council and the FWO.

Fig. 1: Low loss EEL spectra of a Au@Ag nanocube showing three distinct major plasmon modes (a-c). The first two modes have the highest possibility to be excited at the corners of the cubes whereas the third one is best excited at the edges.

Fig. 2: Surface plasmon modes of a triangle comprising three Au@Ag core-shell nanocubes. (A and B) The EELS spectra were acquired at the positions indicated by the dots of the corresponding colors. When inspecting these spectra, several modes are observed, which are in agreement with BEM simulations of a perfect triangular shape.

Fig. 3: Surface plasmon maps of a bow-tie antenna created by a specific order of self-assembled nanocubes. Both the acquired EELS spectra (A) and the experimental and simulated near field maps (B-G) show a large field enhancement in the region between both nanocube structures.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2151 Non-stoichiometry and order-disorder in the SbxV1-xO2 (0<x<0.5) solid solution

Landa-Cánovas A. R.1, Vilanova-Martínez P.1, Agulló-Rueda F.1, Hernández-Velasco J.1
1Instituto de Ciencia de Materiales de Madrid, ICMM-CSIC. Sor Juana Inés de la Cruz, 3; 28049 Madrid, Spain
landa@icmm.csic.es

~SbVO4 plays a key role in the catalyst for the ammoxidation of propane to acrylonitrile. Besides, it exhibits an amazing structural flexibility involving cation vacancies, changes in oxidation states and different degrees of order-disorder, ranging from Short Range Order (SRO) to periodic superstructures and structural modulations [1,2]. In this work we have studied the solid solution that ranges from VO2 to SbVO4 according to the following reaction stoichiometry:

[Sb2O3 + V2O5] + VO2 ---> SbxV1-xO2

 During the reaction all Sb3+ cations are oxidized to Sb5+ while all V5+ cations are reduced to V3+. This implies the following substitution in the basic rutile-type VO2 matrix: Sb5+ + V3+ <---> 2V4+. In this way we have been able to synthesize a whole solid solution SbxV1-xO2 ranging from SbVO4 (x=0.5) to Sb0.1V0.9O2 (x=0.1) by heating the stoichiometric amounts of Sb2O3, V2O5 and VO2 at 800ºC under argon atmosphere. In the Sb-richest phase, SbVO4, most of the vanadium is V3+, as confirmed by EELS spectroscopy, magnetic susceptibility and neutron diffraction, showing the latter magnetic ordering at TN < 50K. Electron diffraction shows the presence of intense SRO in the form of wavy two-dimensional sheets of diffuse intensity in the reciprocal space, see Fig. 1. HRTEM demonstrates that the SRO is due to low correlation between ...Sb-V-Sb-V... chains running along c. This SRO disappears at Sb0.33V0.67O2 and magnetic ordering happens at lower temperatures (TN~6K). For Sb0.25V0.75O2 compositions, a different type of SRO appears and its intensity increases as the amount of Sb decreases, Fig. 2. At Sb0.1V0.9O2 this SRO can also be observed by electron diffraction as very intense two-dimensional sheets of diffuse intensity forming a three-dimensional net of edge-sharing octahedra in reciprocal space. This phase presents a structural transition similar to that of VO2 but at lower temperature (51ºC). A phase transition can be observed by electron diffraction (Fig. 3) when heating with the electron beam from Short Range Order (SRO, diffuse lines) (a) to a two-fold superlattice (b) and back to SRO (c) as the temperature is lowered. During the whole series we range from SbVO4 with V3+ to VO2 with V<4+ and that big change in stoichiometry has been accommodated in a "soft way" through SRO mechanisms.

[1] A.R. Landa-Cánovas, J. Nilsson, S. Hansen, K. Staahl and A. Andersson. J. Solid State Chem.116, 369-377 (1995); [2] A. R. Landa-Cánovas, F. J. García-García, S. Hansen. Catalysis Today 158, (2010) 156.

Authors thank Spanish Government (project FLEXOCAT-MAT2011-27192) for financial support.

Fig. 1: SAED patterns of a crystal of Sb1.0V1.0O4 showing sharp rutile maxima and wavy diffuse planes indicating Short Range Order between the cations.

Fig. 2: SAED patterns of Sb0.25V0.75O2 showing diffuse scattering produced by Short Range Order of the cations. Note that the shape of the diffuse scattering is very different to the one observed in Sb1.0V1.0O4, see Fig. 1.

Fig. 3: SAED patterns of the phase transition observed at the Sb0.1V0.9O2 sample, changing as the crystal temperature is raised with the electron beam over 51ºC from SRO (diffuse lines) (a) to a two-fold superlattice (b) and back to SRO as the temperature is lowered. SAED patterns are misaligned to increase the intensity of the SRO diffuse layers.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2159 Developments in FESTEM & EDS for the Characterisation of Dopant Distributions within Advanced Semiconductors

Dijkstra H.1, Thompson K.1, Stephens C. J.1
1ThermoFisher Scientific
chris.stephens1@thermofisher.com

Recent years have seen an acceleration in the pace of semiconductor development, driven by an ever increasing demand for high-performance, low-cost electronic devices. The ability to mass produce complex structures on a nanometre scale is critical to this process, requiring reliable characterisation techniques to measure and control key parameters of interest. The concentration and distribution of dopant atoms within semiconductors directly affects device performance, requiring analytical electron microscopes capable of accurate elemental quantification on a nanometre scale. This work focuses on the challenges in analysing advanced semiconductor structures, and the developments in microscope technology, EDS detectors and post-acquisition analysis routines which make this possible.

Figure 1 left shows a HAADF STEM image of an As/ P dopant distribution within a NMOS transistor, characterised using a JEM-2800 transmission electron microscope (TEM), a JEOL 100 mm2 (solid angle = 0.95 Sr) silicon drift detector (SDD) in conjunction with the NORAN System 7 microanalysis platform. The concentration of the dopant regions is low (<0.1%), within small regions (<5% area), as shown in the cumulative spectra in Figure 1 right. Whilst quantitative elemental mapping (peak deconvolution and background subtraction) eliminates many of the problems associated with traditional elemental analysis (Figure 2), such as overlapping peaks, many hours can be required to acquire statistically significant data. Furthermore, determining phases from such ‘Quant’ maps often results in end-user bias and the misidentification of chemically unique phases.

COMPASS is an ideal tool for EDS analysis under such extreme conditions, utilising multivariate statistical analysis (MSA) in order to extract the principle components of the spectra at each pixel and group statistically similar phases. Figure 3 left shows the composite phase map using COMPASS, Figure 3 centre shows the principle component map of As Doped Si and figure 3 right overlays spectra of each component. COMPASS extracts spectra relevant only to the specific phase of interest, enhancing the signal-to-noise ratio in comparison to the quant maps and assists in the detection of trace elements within a phase of interest. The end result is the significant reduction in acquisition time and the detection of physically significant phases Figure 4, otherwise missed by conventional spectrum-based phase approaches.

This work is set in the wider context of developments in analytical electron microscopy and the role this plays in improving advanced manufacturing processes.

Fig. 1: Figure 1 Left HAADF image of analysed area of a NMOS transistor. Right Cumulative spectrum for all pixels in the data set.

Fig. 2: Figure 2 Left Conventional peak count. Right quantitative elemental map of selected elements within the NMOS device. Ta is incorrectly displayed on a conventional elemental map due to overlaps with Ha

Fig. 3: Figure 3 Left Composite COMPASS element map of Si regions. Centre Principle component map of As doped Si Right selected area specta of Si phases.

Fig. 4: Figure 4 Left Composite map of non-Si phases. Right Spectra of principal components 10 and 12. Component 12 was not uniquely identified during spectrum based phase analysis
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2324 Band gap measurements of ultra thin buried films using conical darkfield EFTEM and low voltage EFTEM

Stöger-Pollach M.1, Biedermann K.2, Beyer V.2
1USTEM, TU Vienna, Vienna Austria, 2Fraunhofer IPMS-CNT, Dresden, Germany
stoeger@ustem.tuwien.ac.at

New electronic devices require new techniques for characterization. We investigate a c-Si/a-SiOx/a-SiON/a-SiOx/pc-Si (SONOS) stack as used in flash memory devices by using valence electron energy loss spectrometry (VEELS) and energy filtered transmission electron microscopy (EFTEM).
In the present work we discuss the given physical limitations, which include relativistic energy losses – like Čerenkov losses – and the wide range Coulomb interaction. Whereas the first effect can alter the VEELS spectrum and can be easily avoided by reducing the beam energy, the latter affects the spatial resolution of the inelastically scattered electrons.
Although the range of the Coulomb interaction is smaller for slower electrons, the spatial resolution of the low voltage (LV-) EFTEM method will still be limited by this effect. In the case of conical dark field (conDF-) EFTEM we are going to locate the collection aperture in the reciprocal plane such, that we do not collect the small angle dispersed Čerenkov losses and such that we probe the indirect gap of Silicon. Still this method is critical, because its results do not give the optical properties. This is because the measurement is performed at q ≠ 0.
The EFTEM data cubes are recorded with a TECNAI G20. For the respective experiments we chose 40 keV for the LV-EFTEM experiment and 200 keV for the conDF-EFTEM experiment. In conDF the incoming electron beam is deflected by the Bragg angle of Si(111) and conically rotated during the EFTEM acquisitions. Therefore only dark field signal is used for the data cube.
Although the 40 keV experiment does not show Čerenkov losses inside the oxide-oxynitride-oxide (ONO) stack, it still shows some intensity in the direct gap of Silicon. Anyhow, the inelastic delocalization hinders an extraction of an SiO2 EELS signal. The major components of the spectrum extracted from the SiO2 layer positions are due to Si and SiON. The measured band gaps are 3.8 eV in SiON, 3.8 eV in SiO2, and 1.8 eV in Si (although the direct gap at 3.4 eV should be probed). They are all wrong due to delocalization and Čerenkov loss excitation.
In the case of the 200 keV conDF-EFTEM experiment, the spectra can be extracted quite well, although the SiO2 spectrum still suffers slightly from inelastic delocalization. The measured band gaps are 4.9 eV in SiON, 6.3 eV in SiO2, and 1.3 eV in Si (which is the indirect gap being probed under conDF conditions). The value for SiO2 still suffers from delocalization.
We demonstrate that the determination of optical properties of low is a problem with EELS as soon as the layer thickness is smaller than the inelastic delocalization. Probing the band gap can be done under the restrictions of a dark field experiment measuring q ≠ 0.

The authors acknowledge the USTEM facilities for providing the low-kV TEM.

Fig. 1: Figure 1: (a) Defocused bright field image of the area of interest. The ONO stack of the SONOS-transistor is located between the substrate and the pc-Si gate electrode. (b) elemental map of Silicon (green), Oxygen (blue) and Nitrogen (red). (c) HRTEM image of the ONO stack.

Fig. 2: Figure 2: (a) 40 keV spectrum image across the ONO stack extracted from the LV-EFTEM data cube. (b) 200 keV spectrum image across the ONO stack extracted from the conDF-EFTEM data cube. (c) Spectra of Si, SiO2 and SiON extracted from the corresponding data sets.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2163 An ELNES study of anisotropic materials using variable beam energies

Stöger-Pollach M.1, Hetaba W.1, Rodemeier R.2
1USTEM, TU Vienna, Vienna, Austria, 2GATAN GmbH, München, Germany
stoeger@ustem.tuwien.ac.at

The development of (S)TEMs in recent years is pointing towards a higher variability of beam energies. The reasons are manifold, as there are amongst others less beam damage [1], larger elastic and inelastic scattering cross sections, and less or even no excitation of Cerenkov losses for the analysis of optical properties [2,3]. Simultaneously the development of electron detectors being able to handle slower electrons efficiently allows detecting low signals with little noise. This is important, because low voltage electron beams have usually little beam current.
In the present work we demonstrate the effect of the varying beam energy on the ELNES of the B-K edge caused by the change in momentum transfer with respect to the forward (qz) and perpendicular (qperp) directions. With decreasing beam energy qperp is stronger decreasing than qz. For the experiment we orient the c-axes of a hexagonal BN crystal parallel to the electron beam. For low beam energies the van der Waals bonds contribute to the EELS spectrum stronger as compared to high beam energies showing a small qz. This can be seen in Figure 1, because the π* peak decreases with increasing beam energy. When orienting the beam axes perpendicular to the c-axes of h-BN the effect is inverted and the sp2 orbitals are contributing stronger with decreasing beam energy (see Figure 2).
We compare the experiments with ab initio calculations using the Wien2k code. Using the TELNES.3 routine, the effects of orientation dependence [4] as well as the influence of the beam energies on the fine structure of the Boron K-edge can be simulated. One can investigate orbital dependent properties by comparing these simulations to the experimentally acquired spectra. As changing the beam energy is in some cases a much easier task to perform than conducting ELCE experiments [5], this technique can be an alternative or a complementary procedure.

[1] U. Kaiser et al., Ultramicroscopy 111 (2011), 1239 - 1246
[2] M. Stöger-Pollach, Micron 39 (2008), 1092 - 1110
[3] M. Stöger-Pollach, Micron 41 (2010), 577 – 584
[4] C. Hebert-Souche et al., Ultramicroscopy 83 (2000), 9 – 16
[5] W. Hetaba et al., Micron, in press.

The authors aknowledge the USTEM facility for providing the low-KV TEM.

Fig. 1: Figure 1: a) Boron-K edge of BN recorded with various incident beam energies (normalized to the 195.8 eV peak). b) Bright field image of the BN specimen. c) Diffraction pattern of h-BN showing the [0001]-orientation. Consequently the c-axes of the h-BN is parallel to the beam axes.

Fig. 2: Figure 2: a) Boron-K edge of BN recorded with various incident beam energies (normalized to the 195.8 eV peak). b) Bright field image of the BN specimen. The circle indicates the position of the EELS experiments. c) Diffraction pattern of h-BN showing the (0002) spots only. Consequently the c-axes of the h-BN is perpendicular to the beam axes.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2169 QW emission shift along single InGaN/GaN core-shell LEDs evaluated by monochromatic cathodoluminescence image series

Ledig J.1, Fahl A.1, Popp M.1, Scholz G.1, Steib F.1, Wang X.1, Hartmann J.1, Mandl M.1,2, Schimpke T.1,2, Strassburg M.2, Wehmann H. H.1, Waag A.1
1Institut für Halbleitertechnik, Technische Universität Braunschweig, Hans-Sommer-Str. 66, 38106 Braunschweig, Germany, 2OSRAM Opto Semiconductors GmbH, Leibnizstr. 4, 93055 Regensburg, Germany
j.ledig@tu-bs.de

Three dimensional light emitting diodes (LEDs) with a shell geometry of p-GaN and InGaN multi quantum well (MQW) around a columnar n-GaN core are supposed to have distinct advantages over conventional planar LEDs. The active area along the sidewalls of the GaN pillars can substantially be increased by high aspect ratios - leading to a lower current density inside the MQW at the same operating current per substrate area.
The investigated core-shell LED structures are grown by selective area metal organic vapor phase epitaxy on templates consisting of a patterned SiOx mask layer on an n-type GaN layer on 2” sapphire wafers. Due to the 3-dimensional shape, the optical properties of the QWs in each structure show significant gradients along the height and variations between different facets on a micrometer scale.
The automatized capturing of monochromatic CL image series is realized by combining a scan generator (controlling the electron probe position on the sample) and monochromator control (grating rotation angle). Hyperspectral imaging as well as spectra from selected areas are generated by post processing of such image series with respect to the spectral sensitivity of the optical system - including the collection optics, monochromator and detector. The spatial 2-dimensional (2D)-resolution of the presented method is higher than that of using a parallel detector for capturing CL spectra for distinct points of excitation. In parallel, the flexibility of the wavelength range gives a benefit for hyperspectral investigation in different regions of emission from the 3D InGaN/GaN LEDs.
The CL shows near band edge emission (NBE), signals from the QWs and defect related yellow luminescence (YL) which proves that the InGaN is present on all sidewall facets. The properties of neighbor structures are similar – although their diameters might be different. By contacting a single facet with a tungsten probe tip electroluminescence (EL) spectra are obtained at different injection currents.
Investigation of those structures by electron beam induced current (EBIC) proves the geometry of a p-shell around an n-core. Similar to the CL intensity the spatially resolved EBIC analysis indicates how the generation rate is affected by topography (edges) and that the top facets show different properties. A wavelength shift of the MQW emission of 60 nm is observed along the structure height for both excitations by the electron probe (CL) and by a local current injection (EL). This shift is assigned to a gradient of the indium incorporation caused by diffusion mechanisms during growth.

We thank Dr. Uwe Jahn for support regarding optical characterization. The financial support of the European commission (SMASH and GECCO) and the endorsement of the NTH and the JOMC are acknowledged.

Fig. 1: SE image and monochromatic CL images of InGaN/GaN core-shell LED structures on the cleaved growth template using a spectral FWHM of about 7.5 nm at an FOV = 11.4 µm, EHT = 15 kV, tilt = 30°.

Fig. 2: Logarithmic contour plot of CL spectra obtained by exciting small areas at different structure height (17 positions on the sidewall and 6 positions on the top facets), captured with a spectral FWHM of about 7.5 nm using a CCD parallel detector.

Fig. 3: CL and EL spectra captured with a spectral FWHM of about 7.5 nm using a CCD parallel detector. The single core-shell LED was excited at different heights on the sidewall by an electron probe of EHT = 15 keV and a current injection of I = 1.5 µA via a probe tip contact, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2232 Distinguishing overlapping EMCD signals on oxidized metals in the TEM

Thersleff T.1, Rusz J.2, Rubino S.5, Hjörvarsson B.2, Ito Y.3, Zaluzec N. J.4, Leifer K.1
1Department of Engineering Sciences, Uppsala University, Uppsala, Sweden, 2Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden, 3Department of Physics, Northern Illinois University, DeKalb, IL, USA, 4Electron Microscopy Center, Argonne National Laboratory, Argonne, IL, USA, 5Department of Physics, University of Oslo, Oslo, Norway
thth@angstrom.uu.se

Energy-loss Magnetic Circular Dichroism (EMCD) is a powerful electron microscopy technique capable of extracting quantitative magnetic information from nano-sized features [1–3]. This technique has potential for high impact in research and industry where understanding interfacial magnetism is crucial to the design of nanostructured magnetic materials yet hampered by a lack of reliable small-volume characterization techniques. A key challenge facing further development of the EMCD technique is to extract reliable data from very small volumes of materials with sufficient quality for quantitative analysis. This is especially difficult for metals, as a thin oxide typically forms on the surfaces of the as-prepared lamella during transfer into the transmission electron microscope. In the case of iron, this surface oxide layer may be itself magnetic, potentially complicating the quantification of an EMCD signal from thin regions.

In this study, we investigate variations in the EMCD signal on an iron thin film with a surface oxide layer. The experimental design is depicted in figure 1 and yields a fully convergent beam with a diameter that can be reduced to approximately 1 nm. Under these conditions, we are able to provide a detailed structural and chemical assessment of the surface oxide. Subsequently we explore the consequences of its magnetization as well as how this modifies the detected EMCD signal (see figure 2). We conclude by proposing a method to quantify this effect and distinguish between the EMCD signals from either the underlying metallic film or its surface layer.
References
[1] P. Schattschneider, S. Rubino, C. Hébert, J. Rusz, J. Kuneš, P. Novák, et al., Detection of magnetic circular dichroism using a transmission electron microscope, Nature. 441 (2006) 486–488.
[2] P. Schattschneider, M. Stöger-Pollach, S. Rubino, M. Sperl, C. Hurm, J. Zweck, et al., Detection of magnetic circular dichroism on the two-nanometer scale, Phys. Rev. B. 78 (2008).
[3] H. Lidbaum, J. Rusz, A. Liebig, B. Hjörvarsson, P. Oppeneer, E. Coronel, et al., Quantitative Magnetic Information from Reciprocal Space Maps in Transmission Electron Microscopy, Phys. Rev. Lett. 102 (2009).

The authors acknowledge STINT research grant (1G2009-2017) and the Electron Microscopy Center at Argonne National Laboratory, a U.S. Department of Energy Office of Science Laboratory operated under Contract No. DE-AC02-06CH11357 by UChicago Argonne, LLC. J. R. acknowledges the Swedish Research Council, Göran Gustafsson's Foundation and Swedish National Infrastructure for Computing (NSC center).

Fig. 1: Figure 1 - Illumination conditions for the EMCD experiment. The probe size is limited by the C2 aperture and can be reduced to approximately 1 nm in diameter.

Fig. 2: Figure 2 – ELNES spectra acquired at two detector positions using a probe with a diameter of approximately 1.5 nm.  Their difference is an EMCD spectrum.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2235 The Use ofVery Large Area Detectors for fast Light Element Mapping and Data Acquisition in STEM

Rowlands N.1, Phillips P.2, Bhadare S.3, Klie R.2, Nicholls A.2
1Oxford Instruments NanoAnalysis, Concord, USA, 2University of Illinois, Chicago, USA, 3Oxford Instruments NanoAnalysis, High Wycombe, UK
neil.rowlands@oxinst.com

In recent years silicon drift detectors have become the logical choice for characteristic EDS X-ray analysis in both SEM and TEM. Large sensor sizes offer increased solid angle, allow data to be acquired faster in low signal regimes and with only Peltier cooling required, the need for liquid nitrogen is removed. High count rate situations are also handled more easily due to lower noise and excellent energy resolution.
Now ultra-large non circular SDD detectors have been designed for (S)TEMs.  With their non-traditional geometries, they minimize the detector to sample distance and give very high solid angles. These large EDS detectors are able to detect and map elements down to the atomic level whilst minimizing analysis times and thus limiting the effects of beam drift, beam damage and sample contamination.
By taking further advantage of the clean, high vacuum regimes present in modern field emission (S)TEMs and the fact that these new detectors may be operated at relatively high temperatures, windowless SDD detectors can also now be easily and safely incorporated as part of the analytical system. 
By dispensing with the window and support grid, not only is true solid angle increased, but the increased collection efficiency dramatically improves signal to noise ratios for lower energy X-rays such as NKα and OKα. Figure1 shows this huge improvement in light element performance for the new Oxford Instruments X-MaxN 100TLE detector compared to an 80mm2 SDD detector.
This improved performance is especially useful for TEM instruments without EELS capabilities, where light element analysis can be problematic. Sensitivity can be further increased by mounting more than one detector on suitable instruments – this can maximize solid angle up to 2.0sr.  Not only is collection efficiency for un-tilted samples increased, but it also gives the ability to tilt the sample in a negative direction which can be useful when collecting simultaneous diffraction data.
Figure 2 shows an example of elemental distribution in an LED nanowire taken with a single X-Max 100 TLE in 10 minutes and structures as small as 2nm can be identified. The Al and Ga distribution is well defined and N concentrations are evenly distributed throughout the wire. In addition a thin out coating of oxide only a few nanometers thick is clearly seen in the mapping data. High resolution maps such as these can be collected in just 5 -10 minutes using a single detector.

Conclusions:
Large solid angle windowless detectors extend the capabilities of EDS analysis to application areas which were formerly regarded as being limited to EELS only. Higher collection efficiencies, especially at low energy now makes EDS a truly viable option for light element analysis on the nanoscale.

Fig. 1: Comparison of X-Max 80T (yellow - 80mm2 window) and Maxᶰ 100TLE (red - 100mm2 very large solid angle windowless) SDDs. Spectrum has been normalized on the Ni Kα peak to show the sensitivity enhancement of the windowless operation for low energy X-rays.

Fig. 2: Mapping of LED Al-Ga-N nanowires showing the elemental distribution throughout the wire. Each map has a resolution of 266x113 pixels.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2281 Spatially resolved EELS with an in-column Omega filter - characterization of energy filter aberrations and their correction by image processing

Entrup M.1, Kohl H.1
1Physikalisches Institut und Interdisziplinäres Centrum für Elektronenmikroskopie und Mikroanalyse (ICEM), WWU Münster, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany
michael.entrup@wwu.de

Spatially resolved EELS (SR-EELS) [1] is a technique to preserve spatial information when recording EEL spectra. Essentially, many EEL spectra are recorded in parallel as a function of one spatial coordinate, perpendicular to the energy dispersive direction. This method is useful for investigating specimens like interfaces and layer systems. We apply SR-EELS in a TEM with an in-column Omega filter [2]. Remaining aberrations can be corrected by processing the recorded SR-EELS dataset, using the results of a previous characterisation measurement.
The characterization measurement is performed using the small filter entrance aperture - 100µm instead of 500µm used for the final SR-EELS measurement. The aperture is shifted along the lateral axis. At several positions a SR-EELS dataset is recorded. For each energy channel we can extract the position of the aperture borders (yb). From this information we can calculate the width (w) and the position (y) of the aperture. To increase the signal to noise ratio, up to 64 energy channels are binned.
One aberration is directly visible when inspecting a SR-EELS datasets. The width of the aperture decreases with increasing energy loss. Figure 1a) shows a superposition of 3 datasets recorded using the described method. The borders of the apertures are plotted in figure 2. In addition to the change of the aperture width, the borders are curved. A two dimensional polynomial of 2nd order (Σij Aij ΔEi yb(ΔE=0)j) is used to describe this aberration, where yb(ΔE=0) is the position of the border at E=200keV, the energy of electrons that are not deflected by the energy filter. The correction of the aberration is done by image processing. Figure 1b) shows the correction of figure 1a) using the polynomial plotted in figure 2.
The change of the aperture width is best visible when plotting the width of the aperture as a function of the position, for only one energy channel (see figure 3). With increasing distance to the image centre, the width of the aperture decreases. A polynomial of 2nd order describes this well. This aberration is depended on the excitation ΔQSinK7 of the 7th corrector of the energy filter. For ΔQSinK7=-32% there is nearly no change in width of the aperture. Figure 4 shows the variation of the aperture width for all recorded energy channels. A two dimensional polynomial of 2nd order (Σij Aij ΔEi yj) is used for fitting. The dependency w(ΔE) is clearly visible in both graphs, while only ΔQSinK7=0% shows the dependency w(y).

[1] L. Reimer et al., Ultramicroscopy 24 (1988) 339-354.
[2] S. Lanio, PhD thesis (1986), TH Darmstadt.
[3] The code that has been used to perform the characterisation is available on GitHub: https://github.com/EFTEMj/EFTEMj/Scripts+Macros

Fig. 1: a) A superposition of 3 SR-EELS datasets recorded while the excitation of the 7th corrector was changed by ΔQSinK7=-32%. A amorphous carbon film has been used to guaranty a uniform signal which simplifies the processing. b) A corrected version of a).

Fig. 2: The aperture borders extracted from the datasets shown in figure 1a). Only every 2nd data point is displayed. A polynomial of 2nd order can be used to fit each border separately. Introducing the position of the polynomial at ΔE=0eV, a single two dimensional polynomial of 2nd order can be used to fit all borders simultaneously.

Fig. 3: The width of the filter entrance aperture is plotted as a function of the position on the lateral axis (only the energy channel ΔE=0eV is considered). The graphs differ by the excitation ΔQSinK7 of the 7th corrector. A second order polynomial has been used for fitting.

Fig. 4: The width of the filter entrance aperture is plotted as a function of the position on the lateral axis. In contrast to figure 3 all recorded energy channels are considered. The optimal excitation (ΔQSinK7=-32%) of the 7th corrector is compared to the default excitation (ΔQSinK7=0%). Only every 4th data point is displayed.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2311 Characterising severe plastic deformation: a quantitative assessment of TEM imaging using transmission Kikuchi diffraction in the SEM

Trimby P. W.1, Xia J. H.2, Sha G.2, Schmidt N. H.3, Sitzman S.3, Tort M.4, Xia K.4, Ringer S. P.1,2
1The Australian Centre for Microscopy & Microanalysis, The University of Sydney, NSW 2006, Australia , 2School of Aerospace, Mechanical & Mechatronic Engineering, ARC Centre of Excellence for Design in Light Metals, The University of Sydney, NSW 2006, Australia, 3Oxford Instruments Nanoanalysis, Halifax Road, High Wycombe, Bucks, HP12 3SE, United Kingdom , 4Department of Mechanical Engineering, The University of Melbourne, VIC 3010, Australia
patrick.trimby@sydney.edu.au

The enhanced physical properties attributed to nanocrystalline materials have resulted in significant recent research focus on grain size refinement using severe plastic deformation (SPD). SPD-processed materials typically have ultrafine or nanocrystalline grain sizes, often with very high dislocation densities. These attributes make them difficult to analyse using conventional scanning electron microscope (SEM) based techniques such as electron backscatter diffraction (EBSD), resulting in most researchers relying on the higher resolution capabilities of the transmission electron microscope (TEM).
The recent emergence of transmission Kikuchi diffraction (TKD) in the SEM enables routine characterisation of materials with mean grain sizes below 50 nm, and with high intragranular dislocation densities [1, 2]. However, TKD analyses of samples previously characterised using TEM have sometimes produced discrepancies in the final grain size estimates.
This is the first in-depth direct correlation between TEM imaging and TKD in the SEM: we used a variety of Al-alloys that have been deformed using SPD, firstly imaging using 200 kV TEM (JEOL 2100) and then analysing the same areas using TKD in the SEM (Carl Zeiss Ultra Plus with Oxford Instruments AZtec EBSD).
The results reveal some startling differences. In an Al-6060 alloy deformed by high pressure torsion (5 revolutions at 180 °C, under 6 GPa), the recovery of dislocations enables relatively clear TEM imaging of the grain structure, as shown in fig. 1. TKD mapping (fig. 2) confirms the location of the high angle boundaries and shows that the intragranular dislocations and precipitates visible in the brightfield image are associated with very low misorientations (<1°). However, a misorientation profile across one grain shows that the cumulative lattice distortion can be significantly higher, in this case nearly 4° (fig. 3).
In Al-Cu-Mg alloys that have been deformed by equal channel angular processing (ECAP) at room temperature, the correlation between TEM and TKD is more challenging. The increased dislocation density makes clear TEM imaging difficult, and TKD results indicate that many grain-like features imaged in the TEM are in fact part of larger grains with significant intragranular subgrain structure. We will present numerous correlative analyses between the two techniques; the results have important implications for the characterisation of SPD materials, and show the benefit of the TKD technique for the rigorous measurement of microstructural properties.

References:
[1] R.R. Keller and R.H. Geiss, J. Microscopy, 245 (2012), p. 245-251.
[2] P. W. Trimby et. al., Acta Materialia, 62 (2014), p. 69-80.

Fig. 1: Bright field TEM image of an HPT deformed Al-6060 alloy.

Fig. 2: TKD orientation map (IPF colouring) of the same area (measurement step size 8 nm), with high angle boundaries (>10°) in black, low angle boundaries (1-10°) in red.

Fig. 3: Detailed quantitative analysis of a single grain in Figs 1 & 2. Top left: bright field TEM image. Bottom left: TKD map showing the change in lattice orientation relative to the central spot. Right: graph showing the change in orientation across the 700 nm transect A-B, relative to point A.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2335 WDX-measurement of Ta-, W- and Re-concentration profiles in a Nickel/Superalloy diffusion couple using Lβ-X-ray-lines

Nissen J.1, Berger D.1, Epishin A.2, Link T.2
1Technical University Berlin, Center for Electron Microscopy (ZELMI), Straße des 17. Juni 135, 10623 Berlin, Germany, 2Technical University Berlin, Institute of Material Science, Ernst-Reuter- Platz 1, 10587 Berlin, Germany
joerg.nissen@tu-berlin.de

Ni-base superalloys are multicomponent alloys used at temperatures up to about 1100°C. At such high temperatures, diffusion plays the principal role for structural stability and mechanical behaviour. The material under investigation is CMSX-10, which consists of 11 elements (Al, Ti, Co, Cr, Ni-base, Nb, Mo, Hf, Ta, W, Re). CMSX-10 is diffusion welded with pure Ni under vacuum at 1050°C, 10 MPa, 1 h, then annealed at 1050°C for 128 days. In order to quantify the diffusion kinetics in such a multicomponent system, the diffusion profiles in Ni/CMSX-10 diffusion couples have to be measured. However, for the key strengthening elements Ta, W and Re this task is not trivial because they are neighbours in the periodic system (atomic numbers 73, 74, 75) and their concentrations are quite small, 1-3 at%. Therefore, X-ray peaks of these elements are small and they overlap. For these reasons, an optimised method is presented.
Measurement of the diffusion profile by EDX-microanalysis in a SEM is not quite reliable because the energy resolution of the detector is too large (127 eV @ 5.9 keV), as can be seen from the overlapping of the M-lines of Ta, W and Re in Figure 1 and the L-Lines in Figure 2, respectively. Thus, WDX-analysis with high energy resolution becomes essential, in our case with the Field Emission Gun Electron Probe Microanalyser (FEG-EPMA) JEOL JXA-8530F, having a resolution of about 15 eV @ 5.9 keV (LIF). However, figure 1 shows, that even now the Ta-Mβ- and W-Mα-lines cannot be separated (ΔE=9 eV) as well as the W-Mβ- and Re-Mα-lines (ΔE= 8 eV).
Energies of the L-lines are in general about 5 times higher than those of the M-lines, thus also the separation of the lines. In Figure 2 it can be seen, that the Lα-peaks of W and Re are isolated, however, the very close and strong Ni-Kβ-line falsifies their background. Therefore, the Lβ-lines are used. The energy differences between TaLβ2 and WLβ1 (ΔE=20 eV) as well as WLβ2 and ReLβ1 (ΔE=50 eV) are large enough to allow a reliable peak deconvolution. To excite the L-lines of Ta, W and Re (E≈10 keV), a 20 kV accelerating voltage is applied. Anyhow, the small Ta-, W- and Re-concentrations give only small peak/background ratios, making necessary a careful background subtraction. The method was checked by measuring the element concentrations in CMSX-10, which gives results very close to the nominal composition.
The diffusion profiles were measured 1.8 mm across the interface with a step size of 5 µm. Figure 3 shows the profile scan for Ta-, W-, Re-, Ni and Al. Comparison of the experimental concentration profiles with such modelled by the software DICTRA shows a very close match. Therefore, it is proved that Lβ-lines might be used for the quantitative element analysis in WDX.

Fig. 1: M-lines of Ta, W and Re in an EDX/WDX-spectrum of CMSX-10

Fig. 2: L-lines of Ta, W and Re in an EDX/WDX-spectrum of CMSX-10

Fig. 3: WDX-Profile scan of Ta, W, Re, Ni and Al in CMSX-10
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2350 Momentum-resolved electron energy-loss spectroscopy of MoS2 and graphene heterostructures

Mohn M.1, Hambach R.1, Wachsmuth P.1, Benner G.2, Kaiser U.1
1Electron Microscopy Group of Materials Science, Ulm University, Ulm, Germany, 2Carl Zeiss Microscopy GmbH, Oberkochen, Germany
michael.mohn@uni-ulm.de

By comparison of momentum-resolved electron energy-loss spectra and ab-initio calculations we analyze high-energy plasmons in 2D heterostructures made of graphene and few- or monolayer MoS2.
We are particularly interested in MoS2 monolayers covered by graphene (G/MoS2/G sandwiches) as it has been shown that such a configuration protects the MoS2 from beam damage [1].

Our experiments have been performed using a low-voltage (20–80 kV) transmission electron microscope with simultaneous acquisition of spectra for different momentum transfers. We have recorded energy-loss spectra in the range of 0–50 eV for momentum transfers along certain crystallographic axes within the Brillouin zone. For very high momentum and energy resolution, we have used a monochromated Zeiss Libra 200 based TEM in diffraction mode with an in-column Ω energy filter (SALVE I [2,3]).

The corresponding ab-initio calculations have been performed as follows: For the ground-state simulations, we have used the density-functional theory (DFT) software ABINIT [4] with pseudopotentials and local-density approximation (LDA). Energy-loss spectra have been calculated with the dp-code [5] within the random-phase approximation (RPA).

Eventually, deficiencies in both the experimental data and the simulations can be spotted by comparing the ab-initio calculations to the corresponding electron energy-loss spectra. These deficiencies include consequences of the approximations we made in the ab-initio calculations. Besides, our measurements are subject to the following experimental difficulties: First, despite the use of low acceleration voltages, the beam-sensitivity of the MoS2 monolayers limits the acquisition times of the spectra. During an exposure of only a few minutes, beam damage and contamination may lead to significant changes in the spectra. Second, the signal decays drastically with increasing momentum transfer, so that the background noise of the CCD plays a crucial role.

[1] G. Algara-Siller et al., Appl. Phys. Lett. 103, 203107 (2013)
[2] U. Kaiser et al., Ultramicroscopy 111, 1239-1246 (2011)
[3] P. Wachsmuth et al., Phys. Rev. B 88, 075433 (2013)
[4] X. Gonze et al., Comp. Mat. Sci. 25, 478 (2002)
[5] V. Olevano, L. Reining, F. Sottile, http://www.dp-code.org (1998)

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2395 Toward X-ray Quantitative Microanalysis Maps with an Annular Silicon Drift Detector

Demers H.1, Brodusch N.1, Woo P.2, Gauvin R.1
1Department of Mining and Materials Engineering, McGill University, Montreal, Quebec, Canada., 2Hitachi High-Technologies Canada Inc., Toronto, Canada.
hendrix.demers@mail.mcgill.ca

The scanning electron microscope (SEM) was primary developed for imaging applications. With the introduction of the Si(Li) energy dispersive spectrometer (EDS), simultaneous imaging and x-ray microanalysis became possible. However, long working distance and high current were needed because the position and small solid angle of the EDS detector. SEM was initially and is still optimized for imaging applications, where the high spatial resolution is generally obtained at short working distance. This problem is still relevant today and unfortunately x-ray microanalysis is never performed in the best imaging conditions, i.e., not with the smallest probe size. The annular silicon drift detector (SDD) system is inserted below the objective lens and has four segments which give a higher solid angle (up to 1.2 sr). Also, a lower working distance and probe current can be used. An improved spatial resolution becomes possible during x-ray microanalysis. However, the effect of the detector geometry and position on the quantification microanalysis is unknown.

Because of the position of the detector, Mylar windows are used to prevent the backscattered electrons (BSEs) to damage the SDD segments. Three window thicknesses are available for this detector and their effect on the x-ray spectra is shown in Figure 1. The shape of the background was strongly affected by the window absorption at low x-ray energy. For accurate quantitative analysis, the calculation of peak net intensity depends on the background subtraction method used. Different approaches are currently studied with this annular SDD. Another artefact created by the window is the generation of C and O peaks and bremsstrahlung x-rays in the window by the BSEs. Figure 2 shows the variation of the output count rate for the spectrum and the Cu Lα peak with the working distance for the three window thicknesses. An optimum working distance was observed for the Cu Lα peak as predicted by the calculation of the solid angle of this detector. However, no decrease of the output count rate was observed. The x-ray emission in the window negates the effect of the solid angle. This effect is more pronounce at high accelerating voltage. An example of x-ray elemental maps of a mineral ore sample acquired with annular silicon drift detector (SDD) at low accelerating voltage is shown in Figure 3.

The effect of this detector geometry and position on the correction model is currently studied to obtain quantitative maps from the elemental maps. With adapted correction model, the annular SDD with its larger solid angle will clearly revolution the quantification microanalysis by moving from point analysis to quantitative micrograph with simultaneous electron imaging.

Fig. 1: Copper sample spectra with three different window thicknesses for annular silicon drift detector (SDD). The gray line shows the expected Duane-Hunt limit at 5 keV. The C Lα peak intensity decrease by 3.2 times with the 3 µm-thick window and 30 times with the 7 µm-thick window.

Fig. 2: Variation of the experimental output count rate with working distance for three different window thicknesses for annular silicon drift detector (SDD). The gray line shows the detector bottom position at 7.5 mm.

Fig. 3: X-ray elemental maps of a mineral sample acquired with annular silicon drift detector (SDD) at low accelerating voltage of 4 kV and working distance of 11 mm. The image size was 11 x 9 µm2 (1280 x 960 pixels) with an acquisition time of 1162 s (input count rate of 117.6 kcps).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2428 Obtaining an accurate quantification of light elements by EDX: K-factors vs. Zeta-factors

Lopez-Haro M.1,3, Bayle-Guillemaud P.1, Mollard N.1, Saint-Antonin F.2, van Vilsteren C.3, Freitag B.3, Robin E.1
1CEA, INAC/UJF-Grenoble 1, UMR-E, SP2M, LEMMA, Minatec, 38054 Grenoble Cedex 9, France, 2CEA, LITE-DTNM, L2N, 38054 Grenoble Cedex 9, France, 3FEI Company, P.O. Box 80066, KA 5600 Eindhoven, The Netherlands.
miguel.lopez-haro@cea.fr

The new energy dispersive X-ray (EDX) technology based on four silicon drift detectors (SDD) with a windowless design provides new possibilities in the field of analytical characterization at nanometer scale. The four detectors are symmetrically arranged with respect to the sample and this unique configuration provides very high collection efficiency, allowing high counting statistics and rapid acquisition of X-ray spectra, line scans and maps. However, new methodologies for the precise quantitative assessment of the elemental composition at nanometer scale are still needed.
Classically, EDX quantification has been carried out using “Cliff-Lorimer” ratio method. This method requires the knowledge of the k-factors and their precise determination is a key point to obtain an accurate quantification. They can be determined theoretically or experimentally, nevertheless, several limitations are found: i) the theoretical k-factors present large uncertainties, ii) the experimental determination of k-factors required multi-element samples with known compositions and iii) X-ray absorption correction may be important for low energy X-ray emissions, especially for light elements, which require the prior knowledge of the specimen mass thickness. To overcome such limitations, a new procedure named “zeta (ζ)-factor” method has been proposed [1]. In this method, the composition and mass thickness are computed simultaneously for each analysis point enabling X-ray absorption correction.
In this work, we present an accurate EDX quantification of various samples containing light elements or elements with low energy X-ray lines using the ζ-factor method. In this regard, a Super-X Tecnai-OSIRIS installed at PFNC-CEA-Grenoble and operating at 200kV has been used. Fig. 1 shows a representative HAADF image of a thin foil of wollastonite (CaSiO3) prepared by FIB, together with the EDX maps of Ca (3.69 keV), Si (1.74 keV) and O (0.53 keV). Individual profiles of the net X-ray counts are extracted from a line scan (see arrow in Fig. 1a) and quantified using k- and ζ- factors (Fig. 2). Quantification using the k-factors gives wrong results as a consequence of the strong absorption of oxygen (Fig. 2b). Conversely, an excellent agreement between the computed and expected results is obtained using the ζ-factor method (Fig. 2d), due to the specimen thickness is determined for each analyzed point (Fig. 2c) and allowing therefore the X-ray absorption correction.
This example clearly illustrates the potential of the ζ-factor method using the new Super-X detector. By measuring composition and mass thickness simultaneously, the ζ -factor method is a very promising tool for quantitative 3D reconstructions.

[1] M Watanabe and DB Williams, Journal of Microscopy 221 (2006) p. 89

Fig. 1: HAADF-STEM image of wollastinite recorded on a Tecnai-OSIRIS (a) and EDX elemental maps of Ca (b), Si (c) and O (d) using the Super-X detector.

Fig. 2: EDX Line scan extracted from the site marked with an arrow (a). EDX quantification using the k-factor method (b). Thickness measurement obtained by EDX analysis (c) and EDX quantification in atomic percentage using the ζ (zeta)-factor (d).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2448 A simple EDXperformance test for transmission electron microscopy

Van Cappellen E.1, Porcu M.2, Delille D.2, Sudfeld D.2
1FEI Company, Hillsboro, USA, 2FEI Company, Acht, The Netherlands
eric.van.cappellen@fei.com

In the last 5 years solid angles increased dramatically (a factor 10 to about 1srad) and some systems are windowless further improving collection efficiency. In practice this means that for most elements a few percent (up to 10% for heavy elements) of the ionization events are now detected. Besides speeding-up conventional X-ray analysis (point analysis and 2D EDX elemental maps) large solid angle detectors have also enabled new EDX applications such as atomic resolution elemental mapping and 3D EDX tomography but this last application only on condition that X-ray collection is possible over a large sample tilt range (like for FEI’s Super-X™ detector).
Although the geometrical definition of a solid-angle is straightforward it is tedious to experimentally verify specified numbers and not all solid-angles yield the same detection efficiency. Some areas of the available real estate around the sample are better than others for X-ray detection and this leads to the concept of “quality of solid-angle” (see fig. 1). Here we propose to determine the quality of solid angle by measuring the output X-ray count-rate per nA of primary electron beam current on a very well-defined sample. This calibration sample needs to have an undisputable and stable composition over time, as well as a fixed and known thickness and must be easily and reliably produced in large quantities to allow for comparisons between systems.
In this study we propose to use 200nm thick Si3N4 windows made in 200μm thick silicon wafers and cut into 3mm discs to fit in regular low-background TEM holders (see fig. 2). Wafer processing technology ensures very good thickness uniformity and thickness reproducibility. Furthermore Si3N4 is stoichiometric and stable certainly when the membrane is 200nm thick and the electron beam is defocussed. Last but not least each wafer yields over 300 TEM samples which keeps the price down and guarantees easy access and supply. Actual measurements will be discussed.

Fig. 1: A single large solid angle EDX: The average take-off angle is NOT θ as the lower part of the detector (dark area) doesn’t contribute at all to the signal. Below the red line no X-rays are counted and above the signal will gradually increase as the take-off angle increases (blue curve).

Fig. 2: The proposed sample: a Si3N4 window in a 3mm disc of 200μm thick silicon wafer, completely flat on the top to avoid shadowing.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2506 Dynamic, Analytical EDX studies of B/Ni composite nanowires with MEMS heating holder

Sudfeld D.1, Lourie O.1, Mele L.1, Dona P.1, Konings S.1, Delille D.1, Van Cappellen E.2, Barton B.1, Jinschek J. R.1, Freitag B.1
1FEI Electron Optics B. V., 5651 GG Eindhoven, The Netherlands, 2FEI Company, 5350 NE Dawson Creek Drive, Hillsboro, OR 97124, USA
Daniela.Sudfeld@fei.com

EDX measurements at high temperatures were a challenge for many years due to the technology constrains of the past related to the limitations of commercial Si(Li) EDX (energy-dispersive X-ray spectroscopy) detectors. With a newly designed MEMS holder and state-of-the-art SDD technology nowadays analytical EDX studies at elevated temperatures can be performed on a regular basis in a Scanning Transmission Electron Microscope (S/TEM), FEI’s TalosTM with ChemiSTEMTM Technology [1].
Here we present fast chemical maps of B/Ni composite nanowires on nanometer scale by EDX which were done with the new VeloxTM software, see Figure 1. Successful synthesis of crystalline nanowires composed of the refractory light materials such as Boron can enable novel applications for nanoelectronics [2-5]. Boron/Nickel composite nanostructures were prepared by a CVD-based synthetic procedure with a Ni-based compound catalyst; naturally blended with high conductivity and refraction index. The properties of this binary nanomaterial at room temperature, Figure 2, are compared to those achieved from heating experiments with temperatures up to 1000 °C. 2D-3D EDX chemical mappings show clearly the core-shell structure of the wires: B in the shell and Ni in the core. This is amplified at elevated temperatures of ca. 500 °C, see Figure 3. At ca. 1,000 °C EDX maps reveal also that Ni vanishes from the core, leaving behind hollow B nanowire (nanotube) structures.
For the given in situ experiments FEI’s NanoExTM heating holder was used with a small, consumable semiconductor (MEMS) device as the heater and providing a direct read-out of the temperature value at all times during the dynamic experiment with a known and reproducible temperature distribution over the heated area. The NanoEx solution is optimized for ChemiSTEM EDX experiments to trace compositional changes correlated with temperature and electrical stimuli. The holder geometry is suitable for high tilt angles, also allowing its use for 3D experiments.

[1] P. Schlossmacher et al., Microscopy Today 18(4) (2010) 14.
[2] CJ Otten, et al., J Am Chem Soc. 2002 May 1;124(17):4564.
[3] D. Wang et al., APL 2003, 183(25):5280.
[4] W. Ding et al., Mech, Comp. Sci. and Techn. 2006, 66:1109.
[5] J. Tian, et al., “Boron nanowires for flexible electronics”,APL 2008 93:122105-7-5.

Fig. 1: EDX compositional maps of Boron nanowires at room temperature. The total acquisition time is ca. 10 minutes for the map sized 256 x 256 Px with a speed of 9.1 ms/Px and ~50cs/Px signal for the B-K edge.

Fig. 2: Dynamic EDX compositional analysis at the begin of the experiment at room temperature.

Fig. 3: Dynamic EDX compositional analysis comparing the same area at the heating temperature of 500 °C showing the Ni particles before they disappear when the heating temperature gets further raised and the specimen finally got cooled down.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2551 Quantitative X-Ray Microanalysis in the Scanning and Transmission Electron Microscopes with the Generalized f-Ratio Method

Demers H.1, Brodusch N.1, Trudeau M.2, Gauvin R.1
1Department of Mining and Materials Engineering, McGill University, Montreal, Quebec, Canada, 2Materials Science, Hydro-Québec Research Institute, Varennes, Québec, Canada
hendrix.demers@mail.mcgill.ca

Quantitative x-ray microanalysis of bulk samples is usually obtained by measuring the characteristic x-ray intensities of each element in a sample and in a corresponding standard. The k-ratio of the measured intensities from the unknown material over the standard is related to the concentration using the ZAF or φ(ρz) correction methods. Under optimal conditions, accuracies approaching 1% are possible. However, all the experimental conditions must remain the identical during the sample and standard measurements. This is not possible with a cold-field emission scanning electron microscope (CFE-SEM) where beam current can fluctuate by 5% in its stable regime. To address this issue, a new method was developed using a single spectrum measurement (Horny et al., 2010; Gauvin, 2012). It is similar in approach to the Cliff and Lorimer (1975) ratio method developed for the analytical transmission electron microscope. However, corrections are made for x-rays generated from thick specimens using the ratio of the characteristic x-ray intensities of two elements in the same material. The proposed method utilizes the ratio of the intensity of a characteristic x-ray normalized by the sum of x-ray intensities of all the elements measured for the sample. Uncertainties in the physical parameters of x-ray generation are corrected using a calibration factor that must be previously acquired or calculated. With this method, relative accuracies better than 5% were obtained in a CFE- SEM.

The f-ratio method was generalized to more than two elements. The correction factors are still acquired experimentally relatively to two elements and they do not change with composition. They are obtained from measurement of one known phase. The concentration curves versus f-ratio are obtained by Monte Carlo simulations and the unknown concentrations are calculated from these curves and the measured f-ratios by combination of multi-dimensional interpolation and iterative procedure. An example is shown in Figure 1, where quantitative x-ray maps of ternary Al-Mg-Zn diffusion couple sample were obtained with a CFE- SEM at 5 kV. The generalized f-ratio method was also applied to a thin specimen in the transmission electron microscope. An example is presented in Figure 2, trace element concentration of Fe in a Zr-Nb alloy was determined. The generalized f-ratio method allows the quantification of multi-elements sample in both SEM and TEM. Furthermore, in which condition this method can be applied to heterogeneous sample is currently explored.

References:

P. Horny, E. Lifshin, H. Campbell and R. Gauvin, Microscopy and Microanalysis, 16, 821-830 (2010).

R. Gauvin, Microscopy and Microanalysis, 18, 915-940 (2012).

G. Cliff and G. W. Lorimer, Journal of Micrsocopy, 103, 203-207 (1975).

Fig. 1: Quantitative x-ray maps of ternary Al-Mg-Zn sample obtained with the f-ratio method at 5 kV. The weight fractions of each element (A: aluminum, B: magnesium, C: Zinc) are represented by a gray scale: black 0% and white 100%. D A ternary phase diagram was obtained from the x-ray maps.

Fig. 2: X-ray quantification of Zr-Nb-Fe alloy obtained with the f-ratio method in a TEM at 200 kV. A Correction factors measured with 76 wt% Zr, 19 wt% Nb and 5 wt% Fe alloy. B Iron concentration in a Zr-Nb phase was calculated with the f-ratio method and Monte Carlo simulations.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2570 Orbital ordering of A-site ordered SmBaMn2O6 studied by inelastic scattering accampanied by Mn-L shell excitation

Saitoh K.1, Toake Y.2, Tanaka N.1, Takenaka K.3
1EcoTopia Science Institute, Nagoya University, 2Department of Crystalline Materials Science, Nagoya University, 3Department of Applied Physics, Nagoya University
saitoh@esi.nagoya-u.ac.jp

Manganite perovskites have been drawing a grate attention from the unique properties such as metal-insulator transition, colossal magneto-resistance, etc. Such unique properties are attributed to a charge and orbital ordering (COO) of the 3d electrons in the eg orbitals of Manganese by resonant X-ray scattering experiments. SmBaMn2O6 shows the A-site ordering of Sm and Ba at room temperature. The crystal structure has a 2√2ap × 2√2ap ×4ap supercell with ap the fundamental cubic perovskite unit cell reported. In the present study, we determine the orbital ordering of SmBaMn2O6 by the convergent-beam electron diffraction (CBED) and inelastic scattering [3] accompanied by Mn-L shell excitation.

Samples of SmBaMn2O6 were synthesized by a solid-state reaction using Sm2O3, BaCO3, and MnO2. CBED patterns were taken from an area of about 10 nm in diameter. Inelastic scattering patterns accompanied by the Mn-L shell excitation were taken using an energy-filtering system fitted to the bottom of the electron microscope. A series of inelastic scattering patterns at successive energy losses from 620 eV to 670 eV with an energy step of 1-2 eV were taken with an energy window of 1-2 eV.

Figures 1(a), 1(b) and 1(c) show CBED patterns of SmBaMn2O6 taken at incidences in the [0 0 1], [0 1 0] and [0 4 1] orientations, respectively. The [0 0 1] and [0 1 0] patterns show two types of mirror symmetries and twofold rotation symmetry. Thus, the point group is determined to be mmm. The patterns does not show any systematic extinction rules of reflections, indicating that the lattice type is primitive P. From the dynamical extinction lines in the h00 (h = odd) reflections in the [0 0 1] pattern and 0 -4 17 reflection in the [0 4 1] pattern, the space group of SmBaMn2O6 was determined to be Pnam.

Figure 2(a) shows an inelastic scattering pattern of SmBaMn2O6 accompanied by the Mn-L shell excitation taken at an incidence in the [0 1 0] orientation. The pattern clearly shows an elongation along the a* axis. Figures 2(b) and 2(c) show inelastic scattering patterns simulated from two kinds of the orbital ordering composed of the 3z2-r2 type orbitals [1] and the x2-y2 type orbitals [2], respectively. The anisotropic feature of the experimental inelastic scattering patterns agrees well with that of the x2-y2 type model. An orbital-ordering model was constructed from the CBED symmetry and a qualitative comparison between the experimental and simulated inelastic scattering anisotropy.

References

[1] M. Uchida et al., J. Phys. Soc. Jpn. 71, (2002) 2605.

[2] M. García-Fernández, et al, Phys. Rev. B 77, 060402(R) (2008).


[3] 
K. Saitoh et al., J. Electron Microsc. 55 (2006) 281.; K.Saitoh et al., J. Appl. Phys. 112 (2012) 113920.

The present work was partly supported by the Grant-in-Aid for Challenging Exploratory Research (No. 23654117), the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Fig. 1: Convergent-beam electron diffraction patterns of SmBaMn2O6 taken at incidences in the [0 0 1], [0 1 0] and orientations. The and patterns show two types of mirror symmetries and twofold rotation symmetry. Dynamical extinction lines in the [0 0 1] and [0 -4 17] patterns indicate that there exist a and n glide planes.

Fig. 2: Experimental inelastic scattering pattern of SmBaMn2O6 accompanied by Mn-L shell excitation taken at an incidence in the [010] orientation (a) and simulated patterns from the 3z2-r2 orbital model (b) and the x2-y2 orbital model (c). The elongation feature in the experimental pattern agrees well with the pattern simulated from the x2-y2 model.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2665 Electron energy-loss mapping of a perovskite-based solar cell

Divitini G.1, Peng X.1, de la Peña F.1, Saliba M.2, Snaith H. J.2, Ducati C.1
1University of Cambridge, 2University of Oxford
gd322@cam.ac.uk

The investigation of nanomaterials for solar cells, aimed at determining chemical structure and morphology, is a vital step in the development of novel materials to face the current energy crisis. Thin film solar cells have been evolving in the last decades, attaining interesting performance levels. In particular, highly efficient perovskite-based solar cells were demonstrated last year [Liu2013], and there is booming interest in the design of such systems. The addition of metal-oxide core-shell nanoparticles is also being investigated in several thin film devices for increasing light harvesting.
Morphology, both at the nano- and the micro-scale, plays a major role in several processes which affect the behaviour of thin film solar cells. Focused Ion Beam milling (FIB) processing is a powerful tool to extract samples from a full solar cell device for analysis with a transmission electron microscope (TEM). Characterisation in a TEM can shed light on the local elemental composition of the device, as well as spatially-resolved information on the electronic energy levels.
Recent developments in TEM analytical capabilities have enhanced the possibilities for investigation further: brighter electron guns and new detectors for energy-dispersed x-ray spectroscopy (EDX) and electron energy-loss spectroscopy (EELS) now allow large spectrum images to be acquired quickly. As a consequence, devices with organic components can be examined with a reduced electron irradiation, limiting beam damage artifacts.
In this work we apply FIB preparation to a perovskite-based solar cell and investigate the morphology of the photoanode using TEM. Elemental and EELS maps are acquired, and the effect of plasmonic nanoparticles is investigated. Information on the spatial distribution of metal nanoparticles is extracted to provide feedback on the device fabrication process.
Spectrum images are analysed using principal component analysis and blind source separation to optimise signal-to-noise ratio, thus obtaining high quality maps while limiting the electron dose on the specimen. This procedure also naturally separates different compounds without introducing operator bias, and is particularly effective in the presence of complex compounds, such as the perovskite active layer.

[Liu2013] M. Liu, M. B. Johnston and H. J. Snaith, Nature 501, 395–398, 2013

GD, XP, FDLP and CD thank ERC for funding.

Fig. 1: Cross-sectional view of a perovskite-based solar cell.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2705 Simultaneous panchromatic and color live cathodoluminescence imaging

Kološová J.1, Jiruše J.1
1TESCAN Brno, s.r.o., Brno, Czech Republic
jolana.kolosova@tescan.cz

Cathodoluminescence (CL) imaging is a standard non-destructive analytical technique. It provides information about composition and crystal structure of the studied material. Scanning electron microscope (SEM) equipped with a CL detection system allows panchromatic, monochromatic or color CL imaging, often in combination with other techniques (EDX, WDX, EBIC…). We can see growing needs for a seamless integration of multiple detection devices into one multi-analytical SEM system. In line with this trend, we have developed a new versatile “two in one” CL detector capable of simultaneous panchromatic and color live imaging.

Figure 1 illustrates the advantage of such simultaneous imaging. It shows a single scan image of a rhyolite sample. In the color image, two types of grains are clearly distinguishable – blue quartz and pinkish topaz. On the other hand, in the panchromatic image the zoning of quartz and topaz grains is more distinct, as the panchromatic channel of the detector collects the signal for the whole spectral range and maintains higher signal to noise ratio.

Versatility of the detector lies in its unique collection optics. Both sub-micrometer high resolution images (see Figure 2) and images with extra large field of view (FOV) can be done. FOV up to 35 mm can be achieved with a single scan, no stage scanning is needed. The collection efficiency of the detector is uniform over the full FOV even when topographic samples are imaged.

The new compact CL detector can be easily integrated into a modular multi-analytical SEM-based system. Simultaneous acquisition of CL together with other signals is straightforward, see Figure 3 for simultaneous CL and BSE. There is no need for complex sample preparation or precise setting of working distance and thus high quality and fast CL analyzes of various samples can be done easily.

The authors acknowledge funding from the European Union Seventh Framework Program [FP7/2007-2013] under grant agreement No. 280566, project UnivSEM.

Fig. 1: Color (left) and panchromatic (right) image of a rhyolite sample with quartz (blue) and topaz grains. Zoning typical for volcanic quartz is more contrasting in the panchromatic image.

Fig. 2: Sub-micrometer high resolution color image of GaN wires covered with InGaN quantum wells (view from above). Quantum wells (bluish) are deposited close to the sidewall surfaces. Sample courtesy MPI for the Science of Light, Erlangen.

Fig. 3: Color CL (left) and BSE (right) images acquired simultaneously on a ruby containing rock with baddeleyite (blue) grains.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2719 Development of an Analytical TEM with a Transition-Edge Sensor type Microcalorimeter EDS detector

Hara T.1, Tanaka K.2, Maehata K.3, Mitsuda K.4, Yamasaki Y. Y.4, Yamanaka Y.5
1National Institute for Materials Science, Tsukuba, Ibaraki, Japan, 2Hitachi High-tech Science, Corp., Hitachi High-Tech Science, Corp., Oyama-cho, Shizuoka, Japan, 3Kyushu University, Fukuoka, Fukuoka, Japan, 4Japan Aerospace Exploration Agency, Sagamihara, Kanagawa, Japan, 5Taiyo Nippon Sanso Corp., Tsukuba, Ibaraki, Japan
HARA.Toru@nims.go.jp

X-ray spectroscopy is widely used for compositional analysis in a TEM. However, the accuracy and sensitivity of this method has not been realized to the required level from recent advanced materials research. One of the main reasons preventing accurate analysis is the low energy resolution of the detector itself. The energy resolution of a standard SSD(Si(Li) type) detector is approximately 130eV, which results in considerable peak overlap. To solve this problem, we have attempted to use a superconductor transition-edge sensor(TES) type microcalorimeter with a TEM as an EDS detector to improve the quality of compositional analysis(1).

Figure 1 shows an outlook of the first prototype of the TES-EDS mounted on a TEM. The characteristic points of this system are as follows: (i) Cryogen-free cooling system, based on a combination of a mechanical (GM type) and a dilution refrigerator, is newly developed(2). (ii) An X-ray polycapillary is applied to increase detecting solid angle. Figure 2 is an example spectrum from silicon device (Si+W) taken for system confirmation. It is well-known that a standard Si(Li) detector (dotted line) cannot separate adjacent peaks; i.e., the Si Ka and W Ma lines are overlapped to each other. As shown in the figure, the developed TES detector (solid line) can separate them clearly. The FWHM of the silicon Ka peak is 7.8eV, which is more than tenfold higher than that obtained by the standard Si(Li) detector.

The spectrum shown in Fig.2 was taken with a single-pixel TES detector mounted on the TEM (not STEM) with LaB6 thermal emitter (Fig. 1). The acceptable count rate of this detector is very low, approximately 100 cps., From these reasons, an EDS map couldn’t be obtained. In order to obtain EDS map with sufficient count rate, we are now developing a multiple-pixels detector system mounted on a STEM. Figure 3 is a current situation of the developing new system; we have succeeded to obtain an X-ray map with a single pixel TES detector and confirmed the mapping function can correctly work. Multipixel detector system is now under developing to increase count-rate in order to obtain an EDS map effectively.

References:

(1) T.Hara, et al.; “Microcalorimeter-type energy dispersive X-ray spectrometer for a transmission electron microscope”, J. Electron Microsc., 59(1),(2010),17-26

(2) K.Maehata, et al.; “A dry 3He-4He dilution refrigerator for a transition edge sensor microcalorimeter spectrometer system mounted on a transmission electron microscope”, Cryogenics,(2014), in press.

This work has been financially supported by the MEXT Leading Project and JST-Sentan program. The authors acknowledge JEOL Ltd. and Hitachi High-Tech corp. for their cooperation.

Fig. 1: Figure 1. Transition-Edge Sensor type microcalorimeter EDS mounted on a TEM.

Fig. 2: Figure 2. Comparison between the TES (solid line) and the SSD(Si(Li)) (dotted line).

Fig. 3: Figure 1. Transition-Edge Sensor type microcalorimeter EDS mounted on a TEM.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2735 Quantitative Position-Averaged Core-Loss Scattering in STEM

Zhu Y.1, Dwyer C.2
1Monash Centre for Electron Microscopy (MCEM) and Department of Materials Engineering, Monash University, VIC 3800, Australia, 2Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, and Peter Grunberg Institute, Forschungszentrum Julich, D-52425 Julich, Germany
ye.zhu@monash.edu

With the rapid development in core-loss spectroscopic mapping in the scanning transmission electron microscope (STEM), it raises the need for the quantitative interpretation of core-loss intensity and map contrast. However, a quantitative interpretation of atomic-resolution chemical maps is not straightforward due to the dynamical scattering of the electron probe. The effect of dynamical scattering on core-loss maps bears similarities to its effect on annular-dark-field (ADF) images. Dynamical-scattering calculations are thus required to interpret both the core-loss map and ADF image intensities on a quantitative level.


Based on recent progresses on quantitative STEM imaging and inelastic multislice simulations, we have performed a quantitative comparison between experimental position-averaged core-loss scattering from K-, L- and M-shells of various elements and simulations based on a single-particle description of the core-loss process. The materials we studied include single-crystal Si, LaB6, SrTiO3, and LaAlO3. To facilitate a direct comparison free of adjustable or compensating parameters, we compare absolute scattering cross-sections for zone-axis-aligned crystals whose thicknesses have been measured independently using convergent electron beam diffraction (CBED). Our study of the position-averaged scattering avoids the complexity and any errors associated with evaluating the effects of aberrations and source size. Experimental results are compared with simulations that include an accurate description of multiple elastic and thermal-diffuse scattering both prior and subsequent to the core-loss events (double-channelling). In order to exclude any pronounced solid-state effects, which are not included in our simulations, we have considered discrete-continuum transitions that are at least 30 eV above edge onsets. The results show that the double-channelling simulations based on a single-particle model quantitatively predict the position-averaged scattering from K-shells, as well as that from L-shells in some cases (Si-L2,3). On the other hand, limitations of the single-particle picture are clearly revealed by the discrepancies in the case of M-shells (La-M4,5). Our results represent a critical step towards quantitatively predicting the absolute intensity and contrast in core-loss chemical maps with nano- or even atomic-resolution.

This work was supported by the Australian Research Council (ARC) grant DP110104734. The FEI Titan at Monash Centre for Electron Microscopy was funded by the ARC Grant LE0454166.

Fig. 1: (a) Experimental (left) and Bloch-wave simulated (right) PA-CBED pattern from 108 nm [110] Si. (b) PA-CBED determined thickness versus t/λ on [110] Si. (c-d) Experimental and simulated (c) ADF and (d) BF average intensities as a function of PA-CBED determined thickness on [110] Si.

Fig. 2: (a) Raw EEL spectra showing Si-L edge at different thickness. (b) Background-subtracted Si-L2,3 edge. The average intensity in the region highlighted in gray was compared to simulation. (c) Experimental (scattered) and simulated double-channelling (line) Si-L2,3 edge intensity at 45 eV above the edge onset.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2785 Implementation of the Zeta-factor method for quantitative EDS

Falke M.1, Kaeppel A.1, Nemeth I.1, Terborg R.1
1Bruker Nano GmbH, Berlin, Germany
meiken.falke@bruker-nano.de

Energy dispersive X-ray spectroscopy is well-established for composition analysis of electron transparent samples in STEM, TEM and SEM. This contribution reports on the implementation of the Zeta-factor method [1, 2] as an absolute EDS quantification method and opposed to the widely used relative Cliff-Lorimer method. The latter can provide quantitative data on the accuracy level of a few at% already and if using large solid and take-off angles even ppm. The quantitative results from the Cliff-Lorimer method are only valid relative to a standard though. Additionally, it has to be assumed that absorption and fluorescence effects can be neglected or it has to be realized that the thickness and composition of the sample of interest are close to the thickness and composition of the standard used, so that absorption and fluorescence cancel out.

An alternative quantification procedure, the Zeta-factor method, has been suggested and developed by M. Watanabe. It includes information on the beam current, sample thickness and density for the standard and can thus provide the absolute quantification of sample compositions while accounting for absorption and fluorescence effects. To obtain all this data, the just mentioned parameters must be well known for a standard sample and it must be possible to measure the beam current during the experiment with the sample of unknown composition and unknown thickness as well.

The Zeta-factor method is currently being implemented and tested in the Bruker ESPRIT software using various standards. For an initial test procedure a 30nm Si3N4 foil (commercially available from Agar) was used as a standard. The foil was punctured by the electron beam to produce folds of known thickness and composition. The spectrum from one of these areas (Fig 1) was processed to determine the net count number for individual X-ray lines and then to compute the respective ζ-factors for Si-K and N-K. Those ζ-factors were then tested on a Si3N4 sample region of a different well known thickness and vice versa.

The experimentally determined ζ-values can be used to calculate a proportionality factor to the respective Cliff-Lorimer factors, theoretically obtained from available atomic data and considering the detection geometry. Based on the experimentally specified Zeta/Cliff-Lorimer factor ratio the Zeta-factors for all element K-lines can then be calculated (Fig.2). Further tests on more complicated material systems including absorption and fluorescence effects are necessary.

[1] Watanabe M, Horita Z, and Nemoto M, Ultramicroscopy 65 (1996) 187–198
[2] Watanabe M. & Williams D.B, J. of Micr. Vol. 221. (2006) 89-109.

We gratefully acknowledge helpful discussions with W. Grogger and S. Fladischer from the ZELMI in Graz, Austria.

Fig. 1: Two areas of the Si3N4 foil and the respective spectra used for testing the Zeta-factor method implementation. The 30nm Si3N4 foil was folded after rupture in the electron beam, so that well-known sample parts of 30nm and 60nm thickness were available for test measurements.

Fig. 2: ζ-factors for K lines calculated based on the theoretically determined Cliff-Lorimer-factors and the N- and Si-ζ-factors obtained experimentally using Si3N4 as the standard.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2909 Advantages of Combining EDS and Energy Filtered STEM Diffraction at Atomic Level

Longo P.1, Aitouchen A.1, Rice P.2, Topuria T.2, Twesten R. D.1
1Gatan Inc., Pleasanton CA, USA, 2IBM Research Division, San Jose CA, USA
plongo@gatan.com

Very recently a technique called STEM Diffraction has been used to collect the entire diffraction pattern (DP) in STEM mode at each probe position and store it in a data-cube [1]. The advantage is that every feature in the DP can now be recorded at each probe position. In this way it is possible to take advantage of the wealth of information present in the DP and have the spatial resolution offered by the STEM probe. In addition, virtual STEM detectors can be created and images can be generated by integrating the intensity of the DP over an appropriate angular distribution that depends on the particular element being imaged. For instance, light elements show high contrast within a narrow angular distribution at low angle in the DP and this is analogous to medium annular bright field (MaBF) imaging (2).

 

Here we propose an alternative approach where EDS spectra and DPs are acquired simultaneously at atomic level. DPs are acquired using the camera attached to an energy filter in EFTEM mode with a 10eV slit inserted in order to remove the inelastic scattering effects that blur the contrast in the DP. EDS can be used to generate elemental distribution maps. However, even with the use of the latest generation of detectors, EDS is not sensitive towards light elements especially when imaged at atomic level. STEM Diffraction can be used to generate and deliver all the additional structural information present in the DP and also images of atomic columns containing only light elements by integrating over low angular distributions in the DP.

 

As example, a EDS/(energy filtered) STEM Diffraction dataset was taken at atomic level across a SrTiO3/LaFeO3 (STO)/(LFO) interface as shown in Figure 1. Figures 2 show DPs extracted from the Ti +O, Sr and pure O columns respectively. Features in the DP at every angular distribution are different across each atomic column and can be used to generate additional structural information. Figure 3 shows color maps of Sr in red, Ti in green, La in amber, Fe in light blue acquired using EDS while pure O columns in blue obtained by integrating the signal over 0 – 8mrad angular range in the DP. It is quite interesting to notice how the position of pure O columns is slightly asymmetric in the LFO region compared to the STO. This might be due to the presence of strain at the interface whose effects can create some crystal distortion in the LFO region. This paper will show other examples and discuss the advantages of this combined EDS/STEM Diffraction approach.

 

References:

1) Okunishi E. et al., Micron 43 (2012) 538-544

2)Findlay S.D. et al., Ultramicroscopy 136 (2014) 31-41

The authors would like to thank IBM for providing the TEM samples and access to their microscope installation 

Fig. 1: ADF STEM survey image. The green box is the area where the beam was scanned for the acquisition of the EDS/STEM Diffraction dataset. According to the image the interface STO/LFO seems quite nice and abrupt.  

Fig. 2: Series of Diffraction patterns from the STO region and extrcated from the Ti + O, Sr and pure O atomic columns. It is interesting to notice how features in the diffraction pattern at low and high angular range are different in each atomic column

Fig. 3: Elemental color maps of Sr in red, Ti in green, La in amber and Fe in light blue obtained using EDS and pure O atomic columns in blue obtained selecting a 0 - 8mrad angular distribution in the diffraction pattern. The position of the pure O atomic column appears to be slightly asymmetric in the LFO area compared to the STO.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2927 Evaluation of valence state in manganese oxide by transition edge x-ray sensor.

Tanaka K.1,5, Ohgaki M.2,5, Miyayama M.3,5, Matsumura S.4
1Hitachi High-Tech Science Corporation, Shizuoka, Japan, 2Hitachi High-Tech Science Corporation, Tokyo, Japan, 3The University of Tokyo, Tokyo, Japan, 4Kyushu-University, Fukuoka, Japan, 5Japan Science and Technology Agency, CREST, Tokyo, Japan
tanaka-keiichi@hhs.hitachi-hitec.com

Electrochemical capacitors are promising candidates for future energy storage devices because of their high power density, long cycle life, and relatively high energy density. There has been considerable interest in MnO2 as a cathode material for such capacitors because of its low toxicity, environmental safety, cost effectiveness, and large capacitance. In particular, two-electron redox reactions involving Mn2+ and Mn4+ are expected to yield a high energy density. Valence states in transition metals are often studied by determining the branching ratio of the L3 and L2 absorption edges using transmission electron microscopy together with electron energy loss spectroscopy (TEM-EELS). In the case of Mn, EELS can distinguish the L3 (640 eV) and L2 (651 eV) absorption edges, and an adequate signal can be obtained.
In the present study, an attempt was made to evaluate the valence state for manganese oxide particles using scanning electron microscope with a transition edge sensor (SEM-TES). A TES is a kind of energy dispersive X-ray spectrometer, but it has a very high energy resolution (typically 10 to 15 eV) and can separate extremely close X-ray peaks. The valence state was determined based on the branching ratio for the Lb and La X-ray emission lines. It was found to be possible to determine the valence state even in stacked structures with layer thicknesses of about 100 nm. Positive electrode powder samples with a composition Hx(Ni1/3Co1/3Mn1/3)O2 were evaluated using both SEM-TES and TEM-EELS. Three types of samples were examined: charged (as-prepared), discharged and recharged. Using SEM-TES, the branching ratios for the three samples were determined to be 0.76, 0.699 and 0.73, respectively. Using TEM-EELS, the values obtained were 2.3, 5.2 and 2.6, respectively. The discharge sample changed valence state from as-prepared one and discharged one. Thus, the SEM-TES shows the capability of identifying a different valance state in the case of the discharged sample.

This work was supported in part by the CREST program, JST, MEXT, Japan. The SEM-TES measurements were performed as part as a research program (A-12-KU-0018) of the Nanotechnology Platform Project conducted by MEXT.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2960 Advanced EELS Spectrum Imaging of RRAM Devices: Chemical State and Three-Dimensional Element Mapping

Chang M. T.1, Lo S. C.2, Hsieh C. Y.3
1Dept. of Electron Microscopy Development and Application, Material and Chemical Research Laboratories, Industrial Technology Research Institute (ITRI)
mtchang@itri.org.tw

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2963 TEM-EELS/SXES studies on electronic structures of p-type CaB6

Terauchi M.1, Saito T.1, Sato Y.1, Inayoshi K.2, Takeda M.2
1IMRAM, Tohoku University, Sendai, Japan., 2Nagaoka University of Technology, Nagaoka, Japan
terauchi@tagen.tohoku.ac.jp

Metal hexaboride MB6 is based on a network of B6-clusters located on each corner of cubic unit cell. M atom occupies at the body center position of the unit cell. When an M atom can supply two electrons to B6-network, the valence bands (VB) of this material is fully occupied and becomes a semiconductor. Those semiconductor materials have been investigated as a candidate for a high-temperature thermoelectric-power material. Seebeck coefficients of MB6 (M=Ba, Sr, Ca) synthesized by solid-state reaction method were reported to be negative, indicating those are n-type materials [1]. Recently, the p-type character for CaB6 synthesized from the mixture of CaCl2 and NaBH4 in eutectic LiCl-KCl molten salt was reported [2]. Thus, the electronic structure of this new material has been studied by using electron energy-loss spectroscopy (EELS) [3] and soft-X-ray emission spectroscopy (SXES) [4], which are methods for probing over and below the Fermi energy level, respectively.

EDS analysis of the p-type CaB6 showed an inclusion of a few % of Na. Electron diffraction patterns showed a good crystalline order. As one Na atom can transfer one electron to B6-network, Na-doping can be a hole-doping based on the rigid band structure scheme when doped Na atoms occupy Ca site. Valence electron excitation (from VB to conduction bands: CB) EELS spectra showed smaller bandgap energy of 1.5 eV than 2.5 eV of n-type CaB6 synthesized by solid-state reaction method. B K-shell excitation EELS spectra of p- and n-type materials showed almost the same onset energy, which energy position corresponds to the bottom of CB.

Figure 1 shows B K-emission SXES spectra of p-type and n-type CaB6. Those spectra show different intensity distribution especially at the top region of VB, which correspond to the right hand side end of the intensity distribution. The intensity distribution of p-type material apparently extends into the bandgap region of n-type CaB6. Since the bottom of CBs of the two materials were the same, this higher energy position of the top of VB of p-type should be the origin of the smaller bandgap energy of p-type material. This is consistent with the result of valence excitation EELS experiment stated above. This indicates that the doping of Na atoms into the Ca site of CaB6 causes not only the creation of holes in VB but also a change the energy state at the top region of VB, not a simple rigid band structure scheme.

[1] M.Takeda et al., J. Solid State Chemistry, 179, 2823-2826 (2006).

[2] K.Inayoshi and M. Takeda, IUMRS-ICEM (2012).

[3] Y.Sato et al., Ultramicroscopy 111, 1381-1387 (2011)

[4] M.Terauchi et al, Journal of Electron Microscopy 61, 1-8 (2012).

Fig. 1: B K-emission SXES spectra of p-type and n-type CaB6.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2969 Valence Electron States of Carbon Materials studied by TEM-SXES

Terauchi M.1
1IMRAM, Tohoku University, Sendai, Japan.
terauchi@tagen.tohoku.ac.jp

X-ray emission spectroscopy is widely used as a practical tool for compositional analysis of local specimen area and elemental mapping analysis in electron microscopes. X-rays originate form electronic transitions from valence bands (VB, bonding electron states) to inner-shell electron levels inform us energy states of bonding electrons. This X-ray energy ranges in ultrasoft or soft X-ray region form about 0.1 to a few keV. Thus, soft X-ray emission spectroscopy (SXES) based on electron microscopy (EM) can be a sensitive tool for elemental and chemical identifications. For that purpose, we have developed and tested SXES instruments by applying to TEM, EPMA, and SEM [1,2,3]. This SXES spectrometer informs us energy states of VB from specified specimen areas in electron microscopy, which is hardly obtained by EELS and EDS.

Figure 1 shows carbon K-emission (VB→K-shell) spectra of zeolite-Y template carbon (ZTC) [4]. Spectra of graphite and momomer-C60 (Mono.-C60) are also shown for comparison. As this ZTC has a huge surface area of 4000 m2/g, it is a candidate material for applying to fuel cell and electrode of rechargeable batteries. Electron diffraction pattern of ZTC shows amorphous like broad rings. However, C K-emission spectrum shows apparent structures. Those structure positions similar to those of Mono.-C60 than those of graphite. This result suggests that ZTC is mainly composed of curved grapheme, sp2, network with a similar curvature with that of a C60 cluster. Additional structures at the top end and on the lower energy part of VB as indicated by arrows suggest a presence of a certain amount of sp3 component in carbon network of ZTC examined.

When a crystal has anisotropic bonding nature, emission intensities originate from VB electrons should be anisotropic. TEM based SXES experiment can examine this anisotropic emission intensity by changing the crystal orientation. Analyses of anisotropic intensity of C K-emission of graphite by using TEM-SXES have already demonstrated [5]. However, this analysis did not include the effect of polarization on a reflectance of grating used. When the polarization effect was included, the resultant was improved (not shown here). This indicates that the polarization correction is presumably necessary for an accurate analysis of VB of anisotropic crystalline materials by using a grating spectrometer.

[1] M Terauchi et al, Journal of Electron Microscopy 61 (2012), 1.

[2] H Takahashi et al, Microscopy and Microanalysis 19(Suppl.2) (2013), 1258.

[3] M Terauchi et al, Microscopy and Microanalysis, accepted.

[4] K Nueangnoraj et al., CARBON 62 (2013), 455.

[5] M Terauchi in “Transmission Electron Microscopy Characterization of Nanomaterials”, ed. CSSR Kumar, (Springer-Verlag, Berlin Heidelberg) 284.

Fig. 1: C K-emission spectra of zeolite-Y template carbon (ZTC), monomer-C60 (Mono.-C60) and graphite. ZTC shows a similar structure with those of Mono-C60 than those of graphite.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-2998 STEM EELS Analysis of 2D Layered Inorganic Materials at Atomic Resolution

Nerl H. C.1, McGuire E. K.1, Backes C.2, Seral-Ascaso A.2, Ramasse Q.3, Houben L.4, Nicolosi V.1
1Centre for Research on Adaptive Nanostructures and Nanodevices (CRANN) and Advanced Materials and Bio-Engineering Research (AMBER), Trinity College, Dublin, D2 Dublin, Ireland. , 2School of Physics, Trinity College, Dublin, D2 Dublin, Ireland. , 3SuperSTEM Laboratory, STFC Daresbury, United Kingdom., 4Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich GmbH, Jülich, Germany., 5Advanced Materials and Bio-Engineering Research (AMBER), Trinity College, Dublin, D2 Dublin, Ireland.
nerlh@tcd.ie

In recent years, methods for dispersion and exfoliation of 2D nanostructures of a range of nanomaterials have been successfully developed [1-8], opening up numerous possibilities for a range of innovative technologies [4, 6-10]. Chemical and physical properties of materials can however change when going from bulk material to the 2D state. To make real applications feasible there is a need to fully characterize these nanostructures at an atomic scale. Due to the recent advances in transmission electron microscopy (TEM), scanning TEM (STEM) imaging and STEM electron energy-loss spectroscopy (EELS) can now be used to study the structure and composition of nanomaterials, atom by atom [11]. The focus of the study presented will be on characterization of inorganic 2D layered materials produced by liquid phase exfoliation [3,4], a high-yield method for producing sheets of few atomic layers thickness for a range of materials. Atomic resolution STEM EELS analysis of these sheets allows the determination of the atomic structure, structural defects as well as electronic properties of the material, giving insight into their fundamental physical and chemical properties.

[1] AK Geim and KS Novoselov. The rise of graphene. Nature Materials 6 183 (2007)

[2] SD Bergin et al. Towards Solutions of Single-Walled Carbon Nanotubes in Common Solvents. Advanced Materials 20, 10 1876 (2008)

[3] Y Hernandez et al. High-yield production of graphene by liquid-phase exfoliation of graphite. Nat. Nanotechnol. 3, 563 (2008)

[4] JN Coleman et al. Two-dimensional nanosheets produced by liquid exfoliation of layered materials. Science 331, 568–571 (2011)

[5] M Chhowalla et al. The chemistry of two-dimensional layered transition metal dichalcogenide nanosheets. Nat. Chem. 5, 263-275 (2013)

[6] V Nicolosi et al. Liquid Exfoliation of Layered Materials. Science 340, 1226419 (2013)

[7] AK Geim. Graphene: Status and Prospects. Science 324, 1530-1534 (2009)

[8] KS Novoselov et al. A roadmap for graphene. Nature 490, 192-200 (2012)

[9] QH Wang, K Kalantar-Zadeh, A Kis, JN Coleman & MS Strano. Electronics and optoelectronics of two-dimensional transition metal dichalcogenides. Nat. Nanotechnol. 7, 699-712, (2012)

[10] M Osada & T Sasaki. Exfoliated oxide nanosheets: New solution to nanoelectronics. J. Mater. Chem. 19, 2503 (2009)

[11] OL Krivanek et al. Atom-by-atom structural and chemical analysis by annular dark-field electron microscopy. Nature 464, 571–574 (2010)

Support from the Advanced Microscopy Laboratory (AML), Science Foundation Ireland, Enterprise Ireland and the European Research Council (ERC) is gratefully acknowledged.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-3086 On the usability of electron vortices as probes for atomic resolution EMCD experiments

Pohl D.1, Schneider S.1, 2, Rusz J.3, Schultz L.1,2, Rellinghaus B.1
1IFW Dresden, P.O. Box 270116, D-01171 Dresden, Germany, 2TU Dresden, Institute for Solid State Physics, D-01062 Dresden, Germany, 3Uppsala University, Department of Physics and Astronomy, SE-752 37 Uppsala, Sweden
d.pohl@ifw-dresden.de

Recently discovered electron vortex beams, which carry a discrete orbital angular momentum (OAM) L, are predicted to reveal dichroic signals comparable to classical electron magnetic circular dichroism experiments (EMCD) [1]. Since electron beams can be easily focused down to sub-nanometer diameters, this novel technique provides the possibility to quantitatively determine local magnetic properties with unrivalled lateral resolution. For this purpose, specially designed apertures are needed to generate such non-planar electron waves [2]. Dichroic signals on the L2- and L3- edges are expected to be of the order of around 5% [3,4].

We have prepared and successfully implemented a spiral aperture into the condenser lens system of a FEI Titan3 80-300 transmission electron microscope (TEM) equipped with an image CS corrector (cf. fig. 1a). This setup allows for the generation of focused electron vortex beams with user-selectable OAM that can be used as probes in scanning TEM (STEM). Since for such spiral apertures, the different OAM are dispersed along the beam direction (z direction), the selection of the OAM is obtained by defocussing the beam.

First investigations aimed at probing the presence of an EMCD signal with such vortex beams were conducted on a 20 nm thin polycrystalline Ni film prepared by RF sputtering. Fig. 1b) shows the resulting EEL spectra subsequently acquired with L = +1 and L = -1 vortex states, respectively. In order to improve the signal-to-noise ratio, the binned-gain acquisition technique was used [5].
As can be seen from fig. 1b), these first experiments do not provide any evidence for differences in the absorption edges in the two EELS spectra.
In addition, the generation and propagation of the vortex wave functions and the spatial distributions of the OAM were simulated. The results of these simulations show that the orbital momenta and the beam intensity are largely localized (in all three dimensions) symmetrically around the geometrical focal points which are paraxial to the vortex cores (cf. fig. 2). Despite this localization, the superposition of contributions of vortex states (e.g., L = 0 and L = -1) adjacent on the one selected by appropriate defocussing (e.g., L = +1) are large enough that the average OAM is close to zero h, if the defocused portions of the wave are not properly prevented from interacting with the sample (cf. fig. 2b) which explains the absence of a dichroic signal in the experiment.

[1] J. Verbeeck et al., Nature 467 (2010), p. 301-304.
[2] J. Verbeeck et al., Ultramicroscopy 113 (2012), p. 83-87.
[3] P. Schattschneider et al., Ultramicroscopy 136 (2013), p. 81-85.
[4] J. Rusz and S. Bhowmick, Phys. Rev. Lett. 111 (2013), 105504.
[5] M. Bosman and V. J. Keast, Ultramicroscopy 108 (2008), p. 837-846.

Fig. 1: SEM image of the spiral aperture installed in the C2 aperture plane of the electron microscope.

Fig. 2: Ni L3/L2 edges in the normalized EEL spectra acquired with electron probes of vortex states with orbital angular momenta L = +1 and L = -1, respectively. No significant EMCD signal (= difference between the two spectra) is observed.

Fig. 3: Intensity profile of the simulated electron vortex beam with L = +1 generated with a spiral aperture. Arrows indicate radial positions where contributions of the L = 0 and L = -1 states become dominant.

Fig. 4: Normalized expectation value of the OAM as obtained from radial integration from the vortex core to a given radius (1000 a.u. correspond to roughly 130nm).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-3124 Core/shell structure in magnetic nanoparticles from HRTEM and EELS

Bertoni G.1,2, López-Ortega A.3, Lottini E.3, Sangregorio C.3,4, Turner S.5, de Julián Fernández C.1,3, Salviati G.1
1CNR-IMEM, Parco Area delle Scienze 37/A, 43124 Parma, Italy, 2Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy, 3INSTM and Dipartimento di Chimica , 4CNR-ICCOM and INSTM Via Madonna del Piano 10, Sesto Fiorentino 50019 Firenze, Italy, 5EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium
giovanni.bertoni@imem.cnr.it

Magnetic properties on nanoscale materials are strongly affected by the possible different composition of the surface with respect to the bulk material.

In this study we investigate the composition and morphology of spherical d ≈ 10 nm magnetic particles, presenting a core/shell structure, synthesized through thermal decomposition of Fe-Co oleate under high boiling point solvents and posteriorly controlled surface oxidation. Core-shell structure has been assessed by X-ray diffraction analysis and corroborates the formation of two differentiated crystallographic phases: Co doped iron oxide spine (magnetite-like) and Co doped iron monoxide (wustite-like). From a careful inspection of HRTEM images (acquired on an aberration-corrected JEOL JEM-2200FS), an extra reflection from the shell region is indeed visible. This is compatible with a (220) reflection from magnetite. The (400) magnetite and (200) wustite reflections are indeed close (about 0.21 nm), indicating that some solubility or epitaxy of the two structures is possible.

HAADF images and spatially resolved EELS maps are acquired an aberration-corrected FEI Titan “cubed” microscope equipped with an electron monochromator and Gatan Enfinium. The core/shell structure of the particles is evident in HAADF. The reflection from magnetite is visible in the shell region. Moreover EELS maps, obtained by model-based fitting, reveals that there is a depletion of cobalt in the magnetite shell, together with the expected increasing in oxygen with respect to wustite. This is accompanied by the change in valence state of the transition metals from +2 to +3, as verified on Fe-L2,3 edge by fitting reference spectra. The energy position of the transition metal edge onset is indeed a valid parameter for determining its valence state.(1)

By further oxidation of the particles the core/shell structure seems to fade, the EELS maps revealing a more uniform distribution of Fe2+ and Fe3+ ions, indicating the magnetite phase extends towards the core. The final particles show higher policrystallinity with respect to the pristine particles. We can conclude that after further oxidation, the two phases are mixed in the whole volume. Further simulations from EELS profiles and/or EDS are in progress for quantifying the shell extension.

[1] H. Tan et al., Ultramicroscopy 116 (2012) 24–33

European Union FP7 Grant Agreement 312483 ESTEEM2 (Integrated Infrastructure Initiative–I3) and the European Union FP7 project 310516 NANOPYME

Fig. 1: a) HRTEM image from a Fe-Co oxide particle, showing an orientation close to [001] of a cubic structure. The FFT from the core can be addressed with wustite (W), while the shell region has clear reflections from (220) of magnetite (M). b) The core/shell structure as revealed in HAADF

Fig. 2: a) Color map from model-based quantification; b) Linescan profile of the particle, showing the concentration of Fe, Co, and O, respectively; c) Valence map for Fe after fitting of reference spectra for Fe2+ and Fe3+ (explained in e); d) After further oxidation, there is no evidence of a core/shell structure
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-3210 Mapping SPR of Au metallic nano-objects with complex morphologies and environments

Florea I.1,2, Arenal R.3,4, Tréguer-Delapierre M.5, Ihiawakrim D.1, Hirlimann C.1, Ersen O.1
1IPCMS,CNRS/UdS, 23 rue du Lœss, 67034, Strasbourg Cedex , 2LPICM,Ecole Polytechnique, Route de Saclay, 91128 Palaiseau Cedex, France, 3Fundacion ARAID, 50018 Zaragoza, Spain. , 4LMA, INA, Universidad de Zaragoza, 50018 Zaragoza, Spain., 5ICMCB, CNRS, Bordeaux University, 87 av. Dr. A. Schweitzer, Pessac F-33608, France
lenuta-ileana.florea@polytechnique.edu

The remarkable optical properties of the metal nanoparticles(NPs), governed by the excitation of localized surface plasmon resonances (LSPRs), whose character and resonant energy depend on their size, shape, composition and environment, take nowadays a prominent position in research and applications. Imaging the LSPRs can be possible using various techniques such as scanning near optical microscopy(SNOM), photon electron emission microscopy(PEEM), electron energy loss spectroscopy(EELS)and photochemical imaging. Specifically,the EELS technique developed in the scanning(STEM) imaging mode of an electron microscope with a nanometer spatial resolution was often used for probing the SPR of metallic nanoparticles presenting classical morphologies such as spheres, cubes, rods, triangles.[1-3] Along this line the key-issue addressed by this work is the assessment of the optical response of the Au metallic NPs presenting more complex morphologies in presence of particular environments. The most important aspect on which we focused over the entire study relates on accessing information regarding, first the presence and the localization of the “hot spots” or areas as well as coupling effects between the LSPRs modes, and second the influence of the environment of Au NPs on the SPR modes. Regarding the effect of the NPs environment two aspects were closely investigated: the circular nature of the surface supporting the NPs and its dielectric environment.For the STEM-EELS experiments different systems with complex morphologies(see Fig.1) were considered: Au individual BP, Au assembled-BPs forming 2D clustered-structures; silica coated Au BPs;Au NPs presenting a patch morphology and silica beads casted inside the Au patches.At first, a close analysis of the monochromated EELS spectra recorded on a single Au NPs(see Fig.2) allowed identifying two main SPR centered at 1.5 and 2eV.This result helped us later in the analysis of the EELS spectra recorded when the more complex morphologies were studied. More exactly for the systems consisting of enchained Au BP we found that the presence of a neighbor NP induces slight modifications in the SP mode with respect to their position. For the other systems, the silica coated Au BP, Au patch and silica bead casted inside the Au patch the analysis of the plasmon spectra taken at different areas on the NP enabled us observing that the presence of the circular support as well as the dielectric environment are inducing energy shifts of some of the LPSR modes.

[1] J. Nelayah; et al., Nat. Phys.(2007)

[2] R. Arenal, Microsc. and Microanal.(2011)

[3] S. Mazzucco et al., Nano Lett.(2012)

The work was supported by the ANR under Grant no. ANR-BLANSIMI10-LS-100617-15-01. The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2-I3.

Fig. 1: Au complex morphologies: (a) HAADF image of an agglomerate of Au bipyramids ;(b) BF-TEM image of a Silica coated Au bypiramid;(c) HAADF image of a Au NPs presenting a patch morphology; (d) HAADF image of a silica bead casted inside the Au patch NPs.

Fig. 2: STEM-EELS analysis of the SPRs of an Au BP: (a) HAADF image of a Au BP; (b) Map of the energy of the detected surface plasmon resonances; (c) EELS spectra extracted from the SI registered on the whole area of the Au NPs after ZLP subtraction.

Fig. 3: STEM-EELS analysis of the SPRs of Au patches NPs: HAADF images of: (a) pure Au and (b) with a silica bead casted inside the NP; (c) EELS spectra extracted from the SI registered on the analyzed area after ZLP subtraction.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-3251 Parallel acquisition Auger electron spectroscopy

Walker C. G.1, Zha X.2, El-Gomati M. M.1
11-Department of Electronics, University of York, Heslington, York, YO10 5DD., 2York Probe Sources Ltd, York, YO10 5DD
MOHAMED.ELGOMATI@YORK.AC.UK

Auger electron spectroscopy (AES), a surface analysis technique, has traditionally required the use of Ultra High Vacuum (UHV) conditions on account of its high surface sensitivity and the rapidity with which surface become coated with contaminants under High Vacuum (HV) conditions. Energy Dispersive X-ray Spectroscopy (EDS) has limited resolution due to its large excitation volume. Imaging AES, thus achieves much higher spatial resolution than EDS even when the recently developed Silicon Drift Detector technology is employed. The introduction of techniques that acquire an electron spectrum in parallel will allow a much faster acquisition of AES spectra and thus relax the vacuum conditions required.
Two such parallel acquisition electron energy analyser is the Hyperbolic Field Analyser (HFA) [1] and the Magnetic Electron Energy Spectrometer (MEES) [2]. Figure 1 shows a schematic of the experimental setup used for acquiring Electron Energy Loss (EELS) data and AES data using the MEES analyser. Figure 2 shows an image acquired on an Active Pixel Sensor CMOS detector of an elastic peak using the MEES. The image shows the output of the two dimensional detector when the incident electron beam energy is 900 eV and the magnetic field is 80 Gauss. The x and y axes corresponds to an energy interval of ~700 eV to~1000 eV. The image data can be converted into a spectrum by integrating the image data over specific regions (here between the 2 straight lines from top left to bottom right on Figure 2). The resultant spectrum is shown in Figure 3.
The HFA has been used in an SEM (operating at HV; 10-6mbar) to acquire AES spectra. The samples are cleaned using argon ion bombardment as practiced in surface analysis and then rapidly analysed by AES a few seconds after the ion cleaning is ceased. An example of an AES spectrum from Indium is given in Figure 4. The increase in the carbon Auger signal on the samples can also be monitored over the next minutes as the surface is contaminated. This provides a simple demonstration of how parallel acquisition can monitor rapid changes in surface composition in a way that no other technique can. In this article, we also explore in greater detail the theoretical basis of the MEES and its potential as a device for use in Scanning Electron Microscopes (SEMs) for the high speed inspection of objects on the nanometre scale and show further spectra collected using images acquired with the Active Pixel Sensor.
References
[1] M. Jacka, M. Kirk, M.M. El Gomati, M. Prutton, “A fast, parallel acquisition, electron energy analyzer: the hyperbolic field analyzer”, Rev. Sci. Instrum. 70, (1999), 2282-2287.
[2] X. Zha, “Magnetic Electron Energy Spectrometer”, Ph.D. Thesis University of York, York, UK, (2009).

Fig. 1: Schematic representation of the magnetic analyser, MEES. (a) Helmholtz coils shown semi-transparently (b) sample (c) slit (d) sensor (e) board containing electronics for sensor (f) metal plate (g) electron column.

Fig. 2: The locus of the elastic peak is shown as detected by the CMOS sensor. Curve A is the elastic peak and lines B and C mark the limits of integration to create the spectrum in Figure 3.

Fig. 3: A spectrum determined from Figure 2 after image processing.

Fig. 4: The Auger spectrum of indium acquired using the HFA in HV vacuum conditions of an ordinary SEM (JEOL 6400F) and shows the In MNN Auger peak taken immediately after ion cleaning. A carbon contamination layer took about 10 minutes to build up after Ar ion cleaning and the acquisition of the AES data.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-3289 Ultra-Fast, High-Resolution Silicon Drift Detectors for Accurate EDS Microanalysis in Electron Microscopes

Niculae A.1, Bornschlegl M.1, Eckhardt R.1, Herrmann J.1, Jeschke S.1, Krenz G.1, Liebel A.1, Lutz G.2, Soltau H.1, Strüder L.2
1PNDetector GmbH, 2PNSensor GmbH
adrian.niculae@pndetector.de

In the recent years significant advances have been done in electron microscopy instrumentation with respect to electron beam intensity and spot size, pushing for higher energy resolution and faster EDS detectors. High-resolution, ultra-fast EDS microanalysis applications require detectors with extremely low input capacitance, insuring optimum detector operation at very short processing times. A substantial development work has been done in the past years in this direction at PNDetector by remodeling the geometry of the anode and of the integrated FET with the goal of reducing all the parasitic capacitances related to the detector anode. This led to a new generation of Silicon Drift Detectors  – the so-called SDDplus series.
The low capacitance anode/FET can be adopted for all SDD types (round or droplet shape) and sizes (from 5 and 10 mm2 up to 100 mm2 or multichannel devices). Fig.1a and 1b show spectroscopic performances measured with the 30 mm2 and the 60 mm2 SDDplus detectors. Whereas energy resolution values of 126 eV are achieved with the round-shape SDDplus devices, when applied to the droplet-shaped SD3 devices, the low capacitance FET drives the energy resolution below 122 eV at shaping times as short as 1 us. With the detector operated at 0.5 us shaping time (maximum input count rate of 400 kcps) the energy resolution is still below 125eV. Further measurements with SDDplus devices of various sizes and shapes will follow.
The improved spectroscopic performance of the SDDplus devices becomes much more visible when it comes to detection of light elements. Combined with a high-performance, loss-free detector entrance window, the SDDplus devices demonstrate their excellent light element detection capabilities. Measured spectra from carbon samples in SEM are shown in Fig. 2a with an achieved energy resolution of 37 eV FWHM for a 10 mm2 SD3plus detector. Even energy lines well below 100 eV (Si-L, Al-l or Li-K) can still be well distinguished from the noise peak (see Fig2b).
When analyzing thin samples or biological probes with a low photon yield the measurement time is directly related to the detector collection angle. Another important development direction is moving toward smaller, more compact detector packages and therefore increasing the solid angle coverage of the detector with respect to the analyzed sample. An example here is the new large area 100 mm2 SDD detector which has been mounted onto a very compact package of 18.5 mm diameter only (see Fig. 3a). The spectroscopic performance is similar to that obtained with smaller size detectors (Fig. 3b) and this at moderate cooling temperature of -30°C. Selected measurements will be presented and the results will be discussed.

Fig. 1: Energy resolution vs. shaping time measured at -30°C with: (a) 30 mm2 SDDplus/SD3plus detectors; (b) 60 mm2 SDDplus/SDD detector

Fig. 2: Light element spectra of SDDplus devices: (a) C-K line (277 eV) and (b) Al-L line (70 eV)

Fig. 3: (a) 100 mm2 SDD in ultra-slim line package (b) energy resolution vs. shaping time at -30°C for the 100 mm2 SDDplus/SDD detectors
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-3319 Improvement of EFTEM acquisition and data processing using prior knowledge of camera DQE

Lucas G.1, Hébert C.1
1Interdisciplinary Center for Electron Microscopy (CIME), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
guillaume.lucas@epfl.ch

In transmission electron microscopy (TEM), electrons are traditionally detected using a camera with either indirect or indirect detection. In both cases the “true” image is degraded by the non-ideal point spread function (PSF) of the detector leading to a blurring of the signal and the addition of stochastic noise components such as dark-current noise or readout-noise.

Detector performances can be assessed by the measurement of the modulation transfer function (MTF), the noise power spectrum (NPS) and the detective quantum efficiency (DQE) [1,2]. First of all, it allows us to verify the specifications provided by the manufacturers, to compare the relative performance of different detectors and to estimate their degradation over time. Secondly it can help to optimize the acquisition strategy for a given problem. Finally this information can be used as prior knowledge for data processing algorithms.

Energy filtered transmission electron microscopy (EFTEM) has been used to illustrate the benefits of the knowledge of the characteristics of the detection system. Images corresponding to different energy losses are sequentially recorded on the camera device, resulting in a 3D dataset for which each image plane is convolved with the PSF of the camera and each spectrum with the resolution response function of the spectrometer.

This works aims in a first step to measure the DQE of the Gatan US1000 camera used in our JEOL 2200FS microscope in order to improve our EFTEM acquisitions. The recently developed silhouette method [2] is used for the determination of the MTF. In a seconds step this works tries to apply principal component analysis [3,4] in order to perform the denoising of the data as well as the improvement of its spatial or spectral resolution by deconvolution techniques. The prior knowledge of the noise model and the MTF of the camera are embedded in the deconvolution algorithms in order to perform the regularization of the solutions in a realistic way.

The data processing procedure is demonstrated on a simulated dataset providing a ground truth for exploring the applications, limits and eventual pitfalls of the algorithms under known noise levels and MTF. After the measurement of the camera characteristics, acquisition parameters required for a good signal-to-noise ratio are optimized. The algorithms are then applied to the real datasets.

References:

[1] M. Vulovic et al, Acta Crystallographica Section D: Biological Crystallography. 66 (2010) p. 97-109.
[2] W. Van den Broek et al, Microscopy and Microanalysis 18 (2011) p. 336-342.
[3] G. Lucas et al, Micron 52-53 (2013) p. 49-56.
[4] Multivariate Statistical Analysis plugin for Digital Micrograph™, lsme.epfl.ch/msa.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-3376 Comparison of the silicon/phosphorus ratio in natural and synthetic nagelschmidtite for possible use as standard for microanalysis based on X-ray lines of Si and P

Walther T.1
1University of Sheffield
t.walther@sheffield.ac.uk

Quantitative chemical microanalysis by energy-dispersive X-ray spectroscopy (EDXS) in a (scanning) transmission electron microscope (STEM) relies on the use of accurate k-factors. The most commonly used reference line is Si K.

For semiconductor research, standards for elemental semiconductors and for III/V compound semiconductors including elements from groups III and V of the periodic table are required. For the arsenides we have published results on X-ray quantification based on standards of InGaAs [1]. InAs and InP can be used to link arsenides and phosphides. However, the nominal k-factor for the P K-line in the ISIS software of k=1.000 indicates that this has probably not been measured at all.

Here, we use a natural and a synthetic sample of the mineral nagelschmidtite, a calcium silico-phosphate (ideal formula Ca7(SiO4)2(PO4)2 [2]), to evaluate the Si/P ratio from EDXS.

The natural mineral stems from the Hatrurim formation [3] and was cut from a thin section by a focused ion beam to produce an electron transparent specimen for TEM. Electron probe microanalysis (EPMA) of a larger inclusion of nagelschmidtite yielded an atomic ratio of Si/P=3.15. Results from TEM-EDXS are displayed in green.

The synthetic mineral was prepared in the laboratory of C Wu, Shanghai Institute of Ceramics [4]. Its chemical analysis using a Spectro Cirus Vision ICP-OES spectrometer gave a Si/P ratio of 0.36 (by at%), i.e. an almost inverted ratio. (S)TEM-EDXS results from these particles are displayed in dark blue.

Figure 1 shows that, for the detector setting used the deadtime of the detector is linearly related to the count rate up to a max of ~2500 counts/second or 50%, above which the detector runs into saturation.

Figures 2 and 3 plot atomic ratios as obtained from ISIS without absorption correction. The synthetic compound (blue) clearly reveals a higher P/Si ratio than the natural mineral (green) in Fig.2.

If the chemical concentration xn of an element n is proportional to the product of X-ray intensity In, k-factor kn,Si (for weight%) and absorption factor an, divided by the atomic weight An, then we can calculate an effective k-factor [5]:

keffP,Si = kP,Si aP,Si = (ISi xP AP) / (IP xSi ASi)

This is plotted in Fig.4. While the data scatter is rather large, a linear fit to the spectra that gave reasonable densities (≤3.5 gcm-3) as determined by ISIS allows us to determine the thin-film k-factor by extrapolation to zero count rate. The result is kP,Si=1.16±0.45 (R2=0.287). 

[1] T Walther, Proc EMAG2009, J Phys Conf Ser 241 (2010) 012016

[2] G Nagelschmidt, J Chem Soc 1 (1937) 865

[3] M Fleischer, LJ Cabri, GY Chao, A Pabst, Am Mineral 63 (1978) 424

[4] Y Zhou, C Wu, Y Xiao, Acta Biomaterialia 8 (2012) 2307

[5] Y Qiu et al, Proc EMC2008, 2 (2008) 643

Fig. 1:      

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-3430 The “equivalent sphere” approach to fitting surface plasmon energy loss spectra

Ostasevicius T.1, de la Peña F.1, Collins S. M.1, Ducati C.1, Midgley P. A.1
1Department of Materials Science and Metallurgy, University of Cambridge, Cambridge, UK
to266@cam.ac.uk

With the advent of modern cold field emission guns and commercial monochromators on Schottky sources, the remarkably high energy resolution available in the low loss part of the electron energy loss spectrum (EELS) has enabled detailed characterisation of low energy excitations such as localised surface plasmon resonances (LSPRs) in metallic nanoparticles [1]⁠. Analysis of the LSPRs has conventionally involved fitting characteristic peak shapes using a series of Gaussian or Lorentzian functions. Whilst this has proved to be successful in some cases, inherently the LSPR peaks are asymmetric, most evidently with larger retardation effects, and the number and energy of each Gaussian/Lorentzian is not always easy to determine.
Here we propose a new approach to fit the LSPRs based on the line shapes of plasmon modes excited by an electron passing close to a metal sphere. EELS for some impact parameter and sphere radius can be calculated analytically using a linear sum of basis functions and associated coefficients [2]⁠. Allowing the line-shape to scale in amplitude and shift in energy axis enables fitting such functions to simulated or experimental data.
As an example, we consider fitting EELS of a silver nanocube (with rounded corners and edges) in vacuo. Fig 1 is an e-DDA [3]⁠ simulated loss spectrum from a cube with 10 nm edge. The spectrum has been fitted using three separate line-shapes, each calculated from Mie theory for LSPRs. For spheres of this size, the dipolar mode is dominant. Fig 2 shows an equivalent spectrum to Fig 1, but for a silver cube of 100 nm size. The spectrum again has been fitted using this method, but with different radii for the three spheres. Fig 3 shows how the “equivalent sphere” diameter changes with cube size for the lowest order (dipolar) excitations. The smaller (10nm) cube can be fitted using the usual collection of Lorentzians, but it does not work well with the 100nm cube due to asymmetric peaks.
Work is ongoing to apply this approach in analysing experimental data and determine the limitations of it in terms of nanoparticle size and geometry. Whilst empirically the fits are very promising, we continue to work on the underlying basis for why the spectra can be described in terms of a series of spectra from “equivalent spheres” but note that, for example, in the case of cubes, cubic harmonic functions describing the lattice harmonics of a cubic crystal can be written in terms of spherical harmonics [4].
[1] J. Nelayah et al., Nat. Phys. 3, 348 (2007).
[2] F. J. Garcia de Abajo, Phys. Rev. B 59, (1999).
[3] N. W. Bigelow et al., ACS Nano 6, 7497 (2012).
[4] S. Altmann and A. Cracknell, Rev. Mod. Phys. 37, 19 (1965).

We acknowledge the support received from the European Union Seventh Framework Program under Grant Agreement 312483 – ESTEEM2 (Integrated Infrastructure Initiative – I3) and under Grant Agreement 291522-3DIMAGE. FDP and CD acknowledge funding from the ERC under grant number 259619 PHOTO EM

Fig. 1: Fitted EELS spectrum of 10nm silver cube with the electron crossing between opposite midpoints of edges 6 nm above the surface with a beam energy of 300 keV. Components from separate “effective spheres” have been highlighted.

Fig. 2: Fitted EELS spectrum of 100nm silver cube (same trajectory and beam energy as Fig 1). Components from separate “effective spheres” have been highlighted. Particle is big enough to see retardation effects: lowest energy component can only be approximated with at least two spherical modes and is now asymmetric.

Fig. 3: Diameter of “effective sphere” for lowest energy component versus the simulated rounded cube edge length. A straight line with gradient 1 and no offset is plotted to guide the eye.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-3513 Fabrication of High Energy Resolution Silicon Drift Detector for Energy Dispersive X-ray Spectrometer

Hsu C. C.1, Tseng F. G.1, Chen F. R.1, Lee C. H.1, Chuang Y. J.2
1Department of Engineering and System Science, National Tsing Hua University, Hsinchu, Taiwan, 2Department of Biomedical Engineering, Ming Chuan University, Taipei, Taiwan
ful60406@hotmail.com

Energy Dispersive X-ray Spectrometer (EDS) is the most common analytical equipment in SEM or TEM used for the elemental analysis or chemical composition of a sample. The most critical part in EDS system is the x-ray detector. The most common detectors are made of Si(Li) crystal. The major drawback of Si(Li) detector is that they require hours to cool down before use, and cannot be allowed to warm up during use. Besides, the increase of Si(Li) detection area will increase the capacitance that cause the increase of electronic noise. There is a trend towards a newer EDS detector over past decade, called the silicon drift detector (SDD). The key advantage of the SDD is the very lower anode capacitance compared with conventional silicon detectors of the same area. This unique feature reduces electronic noise and shortens processing shaping time to achieve higher energy resolution and counting rate. Due to the small anode in the SDD the leakage current is so low that the SDD can be operated with moderate cooling by Peltier cooler.
High resolution SDD have been designed, fabricated and tested.(Fig.2) The SDDs were fabricated on n-type<111> and a resistivity of more than 4kΩ.cm silicon substrates with a thickness of 400μm. The SDD consists of fully depleted silicon, in which an electric field with a strong component parallel to the surface drives electrons generated by the absorption of x-ray towards a small sized collecting anode. The electric field is generated by a number of increasingly reverse biased cathodes on one side surface of the device. The radiation entrance window on the opposite side is made by a non-structured shallow implanted junction giving a homogeneous sensitivity over the whole detection area.
SDD comprise homogeneous radiation entrance window and the anode guard ring for improving the energy resolution of detector.(Fig.1) First, if the p-n junction is located at deep depth, the thickness of dead layer will to thick. Therefore the homogeneous window is important to control the loss of entrance X-ray. Detailed studies showed that a 1100-1200 Å thick layer of aluminum sufficiently attenuates visible light so that it has a negligible impact on the SDD leakage current. Second, in the detector all electrons generated at the Si/SiO2 interface are collected on the anode guard ring rather than contributing to the detector leakage current.
SDD were characterized to extract critical I-V performance parameter like total leakage current at anode. The value of leakage current of ring_2 is 248 nA without anode guard ring, and reduces to 4 nA with anode guard ring.(Fig.3) And will test the response for detector exposed to the X-ray source in the future. The goal of the testing have shown a FWHM at MnKα line of a radioactive 55Fe source of 170 eV at -20℃.

Fig. 1: Cross section and operation scheme of a SDD detector with anode guard rings and non-structured homogeneous x-ray entrance window.

Fig. 2: Photo image of a fabricated silicon drift detectors.

Fig. 3: Leakage current of SDD with testing anode guard ring.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-5719 Major update of the EELS database: eelsdb.eu

Lajaunie L.1, Ewels P.2, Sikora T.3, Serin V.4
1Institut des Matériaux Jean Rouxel, (IMN) – Université de Nantes, CNRS, 2 rue de la Houssinère - BP 32229, 44322 Nantes Cedex 3, France, 2Babraham Institute, Cambridge, UK, 3SAVANTIC AB, Rosenlundsgatan 50, 118 63 Stockholm, Sweden, 4CEMES, Université Toulouse 29 rue Jeanne Marvig BP 94347 31055 Toulouse, France
luc.lajaunie@cnrs-imn.fr

Since its creation at the end of the 1990’s, the EELS database has gathered more than 200 spectra covering 35 elements of the periodic table, becoming the largest open-access electronic repository of spectra from Electron Energy-Loss Spectroscopy and X-ray Absorption Spectroscopy experiments.1 The EELS database is now a common tool used by spectroscopists, theoreticians, students and private firms as a reference catalog for fine structures and data-treatment analyzes2-4 and has been referenced by more than 30 papers.

Much of this success is due to the open-access nature of the database. The database depends on voluntary user contributions; to encourage these contributions, we have performed a major update of the website which is now accessible at http://eelsdb.eu/. The design of the website has been improved (Figure 1) and several new functions have been implemented, including a plotting function (Figure 2) which allows the online comparison of spectra before downloading. The new design gives greater emphasis on the original work of the contributors by strongly highlighting their papers. In addition of the database itself, users can post and manage job adverts and read the latest news and events regarding the EELS community. All these improvements will be discussed further in the poster details.

1. T. Sikora and V. Serin, EMC 2008 14th European Microscopy Congress, pp-439-440, Springer-Verlag Berlin (2008)
2. N. Bernier et al., Materials Characterization, 86, pp-116-126 (2013)
3. L. Zhang et al., Physical Review B, 81, 035102 (2010)
4. R. Núñez-González et al., Computational Materials Science, 49, pp-15-20 (2010)

The authors would like to thank the IMN and CEMES laboratories, the European microscopy network ESTEEM 2, the French microscopy network METSA and the French microscopy society Sfµ, for the funding. The authors warmly acknowledge everyone who has contributed to the database.

Fig. 1: Homepage of the EELS spectra database: http://eelsdb.eu/.

Fig. 2: The plotting page of the website allows the online comparison of spectra before downloading thanks to zoom-in and normalization functions.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-5813 Spectrum-based phase mapping of apatite and zoned monazite grains using principal component analysis

Seddio S. M.1
1Thermo Fisher Scientific, Madison, WI, USA.
stephen.seddio@thermofisher.com

EDS X-ray mapping requires trade-offs between interaction volume, collecting enough above-background counts, selecting appropriate elements, and avoiding sample damage. These trade-offs may produce confusing results, especially in samples containing multiple phases with similar compositions. Applying contrast enhancements and filters to X-ray maps fails to eliminate the confusion of interfering X-ray lines and phases with similar compositions. However, acquiring an image cube with an EDS spectrum at every pixel and comparing the mapped spectra using principal component analysis (PCA), phases can be readily distinguished.
A rock sample containing accessory monazite ([La,Ce,Pr,Nd,Th]PO4) was polished, carbon coated, and examined in an FESEM. EDS spectral imaging was done at 5 and 15 kV. Phases were identified using COMPASSTM spectral phase mapping, which identifies phases based on PCA of the EDS spectrum at each pixel [1,2].
In Figs. 1 and 2, an apatite (Ca5[PO4]3[F,Cl,OH]) grain is partially included in a monazite grain. In 15 kV BSE imaging (Fig. 1a), transmission through thin phases (e.g., “Silica;” Fig. 1a) is evident. 5 kV imaging (Fig. 2a) produces images and X-ray maps more representative of the sample surface. In the 5 kV O and P Kα maps (e.g., Fig. 2c), apatite and monazite are indistinguishable (2.5 hour acquisition). If this sample was mapped without a light REE in the setup, monazite could be misidentified as apatite. However, after 7.5 minutes of acquisition time, COMPASS distinguishes the phases (Fig. 2c). Additionally, spectral imaging of the monazite grain in Fig. 1b (15 kV) reveals a partial rim, < 1 μm wide, that contains higher Th. PCA is able to distinguish the Th-rich rim at 5 kV as well (Fig. 2b).
PCA is an important tool for clarifying confusing X-ray maps. Using spectral imaging with PCA can provide higher confidence identification of phases in less time than traditional elemental mapping.

References
[1] Keenan et al., Method of Multivariate Spectral Analysis. Patent 6,675,106 B1. 06 Jan. 2004.
[2] Keenan et al., Apparatus and System for Multivariate Spectral Analysis. Patent 6,675,106 B1. 06 Jan. 2004.

Fig. 1: Fig. 3. EDS spectra from apatite (red), high-Th monazite (blue), and monazite (green). The vertical axis is a square root scale.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-5816 Overcoming Quantitative Challenges Presented By X-Ray Line Interferences in EDS and WDS.

Seddio S. M.1
1Thermo Fisher Scientific
stephen.seddio@thermofisher.com

Quantitative analysis using EDS or WDS of phases containing elements with interfering X-ray lines presents challenges to the microanalyst. To illustrate some of these challenges, quantitative analysis of a two phase Ti-V-Al-Fe sample was done.
A sample of Ti-V-Al-Fe metal was examined in an FESEM. EDS and WDS data were collected using a Thermo Scientific™ UltraDry™ EDS detector and the Thermo Scientific™ MagnaRay™ WDS Spectrometer. EDS and WDS data were processed using the Thermo Scientific™ NORAN™ System 7. Quantitative analysis was done at 15 kV. EDS spectral imaging was done at 10 kV. Two phases (Figs. 1, 2) were identified using COMPASS™ spectral phase mapping, which identifies phases based on the principle component analysis of the EDS spectrum at each pixel [1].
 ~5 µm, V-rich (~13 wt% V) grains occur along the boundaries of larger, ~10 µm, V-poor (~3 wt% V) grains. Quantitative results are in Table 1.
Ti Kβ line is only separated from V Kα by 17 eV; these X-ray lines are indistinguishable by EDS and are poorly resolved by WDS (Fig. 3). The effect of this interference in the WDS quantitative analyses of these phases is the over-estimate of V.
There are three methods by which this shortcoming may be overcome. First, the V Kβ line is an appealing peak on which to count because there is no interfering energy line in this sample. However, greatly (>10×) extended acquisition times are required for counting the V Kβ line in the V-rich grains. In the V-poor grains, the V concentrations are low (~3 wt%) and the V Kβ line cannot be distinguished from the background. Second, a difference method can be utilized. This method subtracts the wt% of the other elements from 100% with the remainder representing the V concentration. This method requires that only one line is confounding and that the measurement of the remaining elements is done perfectly. The third method is to perform EDS quantitative analysis with standards. It is typically assumed that WDS is more accurate than EDS. However, EDS has peak deconvolution methodologies with both standards-based and standardless quantitative analysis, providing more accurate results than WDS in this case.
WDS is necessary technique for confirming the presence or absence of interfering elements, but unless the WDS spectrometer is able to completely resolve interfering X-ray lines, it cannot be used for accurate quantitative analysis. In addition, interfering energy lines confound the phase mapping. The peak deconvolution methods involved in modern EDS quantitative analysis provide accurate results when WDS is unable to do so. In addition, the utilization of EDS-based COMPASS discriminates phases with only subtle compositional differences.
References
[1] P. Camus, Thermo Scientific (2009) White Paper 51782.

Fig. 1: .
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-5821 Quantitative measurement of site-specific spin and orbital magnetic moments by electron energy-loss chiral magnetic dichroism

Xiaoyan Zhong 1, Ziqiang Wang 1, Jing Zhu 1, Rong Yu 1, Zhiying Cheng 1
Beijing National Center for Electron Microscopy, School of Materials Science and Engineering, The State Key Laboratory of New Ceramics and Fine Processing, Laboratory of Advanced Materials (MOE), Tsinghua University, Beijing 100084, People’s Republic of China 1
xyzhong@mail.tsinghua.edu.cn

In this work, we have developed the site-specific electron energy-loss magnetic chiral dichroism (EMCD) method for local magnetic moment determination in magnetic materials with non-equivalent crystallographic sites at a nanometer scale. It’s the first work to experimentally demonstrate that the fast electron as a new source can be used to determine magnetic structure, including quantitative magnetic moments, for a wide range of materials, which is generally considered to be accomplished by neutron diffraction. Compared with previous EMCD works in which EMCD was just used for detecting the ferromagnetic signals of materials, we fundamentally raise the EMCD technique to the new level of magnetic structure determination.

In the example of NiFe2O4, we achieve comprehensive magnetic structure information using the site-specific EMCD method under the assumption of no magnetic information known previously. The magnetic structure information we obtain includes site-specific total magnetic moment, site-specific orbital to spin magnetic moment (mL/mS) ratio and total magnetic moment of a unit cell. Our method is testified to be valid by comparing our results with those obtained by theoretical calculations and other experimental techniques such as X-ray magnetic circular dichroism and neutron diffraction. Using transmitted electron in site-specific EMCD method, we can reach a high spatial resolution, and get site-specific and element-specific magnetic information, as well as distinguish the orbital and spin magnetic moments.

In the technical aspects, the extremely strong EMCD signals have been achieved by using site-specific EMCD method, which allow us to do quantitative analysis. We first did the quantitative works on EMCD spectra to obtain total magnetic moments (the sum of spin and orbital magnetic moments) for atoms in different sites. For example, our work first reports the experimentally determined mL/mS ratios of Fe atoms in octahedral and tetrahedral sites.

In sum, our work opens the door of using fast electrons to determine magnetic structures for a wide range of magnetic materials in a nanometer scale. Site-specific EMCD may benefit much not only to the fundamental research of magnetic states and behavior in complex magnetic materials, but also to revealing the magnetic structure in nanostructures or interface of the composite magnetic films.

This work is financially supported by National 973 Project of China and Chinese National Nature Science Foundation. This work made use of the resources of the Beijing National Center for Electron Microscopy. The authors are grateful to Profs. Z.H. Zhang, J. Yuan, X.Q. Pan, D.S. Wang, Dr. L. Xie, Mr. Y. Xia, D.S. Song, Z.Y. Wang, Profs. S.P. Crane, R. Ramash, Q. Zhan and Dr. S. Löffler.

Fig. 1: Schematic image of site-specific electron energy-loss chiral magnetic dichroism
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-5868 Trends in X-ray Nano-Analysis by TEM/STEM

von Harrach H. S.1
1SvH Microscopy, East Sussex, UK
svhmicroscopy@gmail.com

The improvements of X-ray detectors gained momentum in recent years with the introduction of silicon drift detectors (SDD), and the increase in collection solid angle (SA) and speed of acquisition. The ability to produce larger detectors up to 100 mm2 without impairing the energy resolution means that detectors with 1 srad collection angle are now available (1). Since the SDDs are capable of higher throughput they have also improved the minimum detection limit of an element in a given analysis time.
Improvements in TEM/STEM resolution were brought about in recent years by the correction of spherical aberration (Cs) and the introduction of higher brightness sources, both Schottky and cold field-emission sources. This has resulted in sub-Angstrom image resolution for thin specimens, but only sub-nanometer probe sizes at the currents (~1nA) required for acquiring X-ray data with Si(Li) detectors at reasonable signal-to-noise ratios (S/N).
The combination of these developments has resulted in the ability to use lower probe currents to produce good quality analytical data at the atomic level within a few minutes (2). This is crucial for many materials that are damaged by exposure to high-energy electrons at high intensity.
So how should the technique be improved further in the future? On the TEM side, the need to operate at lower kV to minimise specimen damage calls for the use of higher brightness sources with lower energy spread (dE) or for chromatic aberration correction. Cold field-emitters are the brightest sources currently available (reduced brightness Br~1e8 A/m2/sr/V) and, either in combination with a monochromator or Cc corrector, could result in sub-Angstrom resolution at 40 -80kV (see Fig.1). For monochromated (dE=0.1eV) and Cs/Cc corrected instruments with cold field-emitters the probe size (d50) at 10pA is below 0.1nm at 40-50 kV and above.
On the detector side, there is room for improvement of collection angles approaching the theoretical limit of 4π steradians. This would reduce the dose required for good quality nano-analysis (S/N>5) by a factor of 10 or more. Fig.2 shows the relative dose at constant X-ray counts detected as a function of acceleration voltage (kV). As the kV is reduced the X-ray yield increases roughly as inverse square root of kV (3). Consequently, with a 10 sr detector the electron dose could be reduced by a factor of almost 20 by operating at 60 kV and sub-Angstrom probe size, compared to the current best instruments at 200kV with 1 sr detectors.
References
1. H.S. von Harrach, P. Dona, B. Freitag, H. Soltau, A. Niculae & M. Rohde (2009) Microsc.Microanal. 15 (Suppl.2), 208-9
2. A. J. D’Alfonso, B. Freitag, D. Klenov, and L. J. Allen (2010). Phys. Rev. B 81, 100101(R).
3. C.J. Powell (1976) Rev.Mod.Phys. 48, 33

The author was working at FEI Electron Optics B.V, The Netherlands until 2013.

Fig. 1: Fig.1 Probe size (d50) at 50% of constant 10pA probe current vs. acceleration voltage for cold FEG source (reduced brightness Br=1e8 A/m2/sr/V) with Cs corrector (Cs<2um), mono-chromated CFEG with dE=0.1eV, Br=3.3e7 and Cc corrected CFEG system with Br=1e8, Cc<0.1mm. [ref. P.Kruit et al. 2006 J. Appl. Phys. 99, 024315]

Fig. 2: Fig.2 Relative electron dose vs acceleration voltage at constant number of X-rays detected for X-ray detectors of collection solid angles SA = 1 and 10 sr.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-5871 Single atom Electron Energy Loss Spectroscopy at Low Primary Electron Energy in the Electron Microscope

G Tizei L. H.1, Iizumi Y.1, Okazaki T.1, Nakanishi R.2, Kitaura R.2, Shinohara H.2, Suenaga K.1
1Nanotube Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8565, Japan, 2Department of Chemistry, Nagoya University, Nagoya 468-8602, Japan
Luiz.tizei@aist.go.jp

In this poster we discuss single atom electron energy loss spectroscopy (EELS) of lanthanides (La, Ce, Er and Eu). In particular we analyze the different possibilities of high spatial resolution spectroscopy at low primary electron energy (30 keV and 60 keV) and high electron energy losses (above 800 eV). Atomically resolved EELS experiments were performed in the JEOL-CREST double corrected microscope operated at 30 keV and 60 keV. The samples analyzed were lanthanide atoms (La, Ce and Er) encaged in fullerenes stored in carbon nanotubes and Eu atomic chains inside carbon nanotubes.
The use of low primary electron energies is beneficial due to the decrease in energy loss delocalization and the minimization of one possible sample damage mechanism (knock-on). Examples of the experiments performed at 30 keV and 60 keV are shown in Figure 1. Evidently, one can see that the fullerenes structure is maintained in the experiment at 30 keV, while it is modified at 60 keV. All experiments have been performed with acquisition times ranging from 20 ms to 200 ms.
To observe the effect of energy loss on delocalization, we have performed the parallel acquisition of the loss signal of the N45 and M45 edges of La and Ce at 60 keV. These edges can be observed at 117 eV (121 eV) and 832 eV (881 eV) for La (Ce). To estimate delocalization, we have measured the full width at 50% intensity (L) of profiles for the annular dark field image (ADF), N45 and M45 edges. For a single La atom we have observed LADF = (0.15±0.02) nm, LN = (0.32±0.02) nm and L = (0.20±0.02) nm. The significant decrease is expected. However, the absolute values are smaller than those predicted by simple considerations.
Some effects of atomic movement on spectroscopy experiments will also be discussed.

This work is partially supported by a JST Research Acceleration programme.

Fig. 1: 2D EELS maps identifying the position of La (blue), Ce (green) and Er (red) at 60 kV (a-c) and 30 kV (d-f). The maps presented are not the first on a series of acquisitions. For this reason, electron beam damage can be observed at 60 kV and not at 30 kV.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-5918 Identifying suboxide grains at the metal-oxide interface of a corroded Zr-1.0Nb alloy using (S)TEM, transmission-EBSD and EELS

Hu J.1, Garner A.2, Ni N.3, Gholinia A.2, Nicholls R.1, Lozano-Perez S.1, Frankel P.1, Preuss M.2, Grovenor C.1
1Department of Materials, Oxford University, Parks Road, Oxford, UK, 2Materials Performance Centre, School of Materials, University of Manchester, Manchester, UK, 3Department of Materials, Imperial College London , Royal School of Mines, London, UK
jing.hu@materials.ox.ac.uk

Here we report a methodology combining Transmission Electron Microscopy (TEM), Scanning Transmission Electron Microscopy (STEM), Transmission-EBSD (t-EBSD) and Electron Energy Loss Spectroscopy (EELS) to analyse the structural and chemical properties of the metal-oxide interface of corroded Zr-1.0Nb alloys in unprecedented detail. The sample which has been under autoclave condition for 360 days shows no sign of transition, suggesting its excellent corrosion resistance1. TEM and STEM results reveal the complexity of the distribution of suboxide grains at the metal-oxide interface. Convergent beam electron diffraction (CBED) patterns were acquired from a region close to the metal/oxide interface which matches with the [3 2 -4] zone axis of the hexagonal ZrO phase with P-62m symmetry and lattice parameters a=5.31 Å and c=3.20 Å predicted by Nicholls et al2. EELS provided accurate quantitative analysis of the oxygen concentration across the interface, identifying the existence of local regions of stoichiometric ZrO and Zr3O2, with significant local variations in thicknesses from 20 nm to 326nm, much thicker than observed previously in other oxidised zirconium alloys3. T-EBSD confirmed that the suboxide grains can be indexed with the hexagonal ZrO structure. The t-EBSD analysis has also allowed us to map a relatively large region (~7μm) of the metal-oxide interface, revealing the location and size distribution of the suboxide grains. These observations will be compared to previous reports of less corrosion-resistant alloys studied by the same techniques.
1. Wei, J. et al. Autoclave study of zirconium alloys with and without hydride rim. Corros. Eng. Sci. Technol. 47, 516–528 (2012).
2. RJ Nicholls, N Ni, S Lozano-Perez, A London, DW McComb, PD Nellist, CRM Grovenor, CJ Pickard, J. Y. Crystal structure of the ZrO phase at zirconium / zirconium oxide interfaces. Adv. Eng. Mater.Accepted
3. Ni, N. et al. How the crystallography and nanoscale chemistry of the metal/oxide interface develops during the aqueous oxidation of zirconium cladding alloys. Acta Mater. 60, 7132–7149 (2012).


This research was funded by the MUZIC2 consortium and JH is supported by the China Scholarship Council.

Fig. 1: (a): Bright field image of the TEM sample. Figure 1(b): HAADF-STEM image of the TEM sample. The area used for EELS analysis is highlighted. The inset shows higher magnification images of this same area after further FIB thinning (which has created the hole under the left hand crack)

Fig. 2: (a) High magnification bright field image the metal-oxide interface chosen for EELS analysis. The suboxide grain which was diffracting is highlighted using arrows. (b) STEM dark field image of the suboxide grain area, the area where CBED pattern was taken is highlighted. (c) CBED pattern of the suboxide grain which matched with hexagonal ZrO phase.

Fig. 3: (a) Band contrast map, (b) Phase map from t-EBSD analysis of the TEM sample. The yellow part near the metal-oxide interface is matched with hexagonal ZrO phase with P-62m symmetry and lattice parameters a=5.31 Å and c=3.20 Å2. The area for the EELS and CBED analysis is also highlighted.

Fig. 4: Positions of EELS line scans from the suboxide region relative to (a) the t-EBSD map. (b) the HADDF image. Lines started from the oxide towards metal. (c) Zirconium and oxygen concentration of the EELS line scan showing both stoichiometric ZrO and Zr3O2, with significant local variations in thicknesses from 20 nm to 326nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-5962 Quantitative electron probe microanalysis of Ga-doped BiFeO3 and (Ca,Zr)-doped BaTiO3 thin films

LONGUET J. L.1, JABER N.2, DAUMONT C.2, WOLFMANN J.2, NEGULESCU B.2, RUYTER A.2, FEUILLARD G.2, BAVENCOFFE M.2, FORTINEAU J.2, SAUVAGE T.3, COURTOIS B.3, BOUYANIFIF H.4, AUTRET-LAMBERT C.2, GERVAIS F.2
1CEA, DAM, Le Ripault, BP16, F-37260 Monts, France, 2Laboratoire GREMAN, UMR7347 CNRS Université François Rabelais, faculté de sciences et techniques 37200 Tours, France, 3Laboratoire CEMHTI, UPR3079 CNRS, Site Cyclotron 45071 Orléans cedex 2, France, 4Laboratoire LPMC, Université Jules Vernes Picardie - Amiens, France
jean-louis.longuet@cea.fr

The most efficient multifonctional piezoelectric materials are lead-based, sush as Lead Zirconate Titanate LZT for example. Considering the need to use lead-free materials, an interest in bismuth ferrite (BiFeO3, also commonly referred to as BFO in Materials Science) has grown due to its ferroelectricity. The substitution of Bismuth in BiFeO3 modifies its properties and enhances the piezoelectric activity. Doped BaTiO3 perovskite has also great interest in this domain.

Linear lateral composition gradients were created by combinatorial Pulse Laser Deposition (PLD) using targets of doping material and host perovskite : Gallium is the doping element in the BGFO material (BiFeO3 + GaFeO3). Calcium and Zirconium are the doping elements in the BCTZ material (BaTiO3 + (Ba,Ca)(Ti,Zr)O3).

Here, we report quantitative thin films analysis (TFA) performed by wavelength dispersive spectrometry (WDX) with an electron microprobe (EPMA). Experimental procedure is described to achieve acceptable quantitative results regarding some analytical issues such as small doping content (less than 3 %wt), small top layer thickness (less than 100 µg/cm2) and lines interferences occurring in energy dispersive spectrometry (EDS) but not in WDS analysis (like Ba Lα – Ti Kα).

Mass thickness of BGFO, determined by TFA-EPMA, was confirmed by focus ion beam (FIB) cross section imaging. A short link is made with optimum doping concentration found on BGFO according to local piezoresponse measurements using an atomic force microscope (AFM) in piezoelectric mode (PFM) ) and by dual beam laser interferometry.

Fig. 1: BGFO sample description (10x10 mm²)BGFO (~100nm)/LSM(~40nm)/STO(substrate)

Fig. 2: Ga-doping content along X-Line profile n°2 (round circles are coincidence points with Y-line profiles n°3-4-5-6)

Fig. 3: BGFO mass thickness along X-Line profile n°2 (round circles are coincidence points with Y-line profiles n°3-4-5-6)

Fig. 4: FIB Cross Section
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-5964 On application of the Multivariate Statistical Analysis in spectrum-imaging

Potapov P.1
1temDM, Dresden, Germany
info@temdm.com

Availability of TEM instruments with fast spectrum-imaging EDX and EELS facilities made it possible to map the composition and structural properties with a high resolution. A typical spectrum-imaging data cube now routinely exceeds the size of 100x100x1000 pixels. The extraction of the chemical/structural information from such huge arrays of data can be significantly improved by using well established techniques of Multivariate Statistical Analysis.

Among the multivariate statistical methods, the most attention is paid to the Principal Component Analysis (PCA) which decomposes the observation set into the set of linearly uncorrelated variables. The components with the highest variance are assumed to have the highest significance and to correlate with the variation of the material parameters such as composition or structure features while the lower-variance components might be associated with the statistical noise and therefore ignored. As PCA is closely related to the eigenvector decomposition in linear algebra, the very efficient algorithms for its implementation are available. However, a caution should be taken when treating a spectrum-image from a system of several objects, for instance, an agglomeration of the particles of different nature. Fig.1 shows the score plot of the PCA components for such a system clearly indicating the separation of the data onto the two distinct clusters. In this situation, the PCA results represent the average eigenvectors for the two unrelated data sets and cannot bear any physical meaning. A more efficient strategy is to segment first the data onto the appropriated clusters and then apply PCA for each cluster individually.

Another approach is the reconstruction of a spectrum-image using a small number of the highest-variance components while cutting off the rest “noise” components [1]. Here PCA is used as a kind of noise filter and the physical meaning of the PCA components is unimportant. The problem appears when the variance of the minor components is comparable or beneath the typical variance due to noise. In this case the useful signal might partially “leak” to the “noise” components and be lost during the subsequent reconstruction [2]. The possible solution is to retrieve a relatively large number of the PCA components and then apply to them the Independent Component Analysis (ICA). Similar to PCA, ICA can be thought of as a rotation in the variable coordinates that maximizes the Curtosis of a given component. This way, the truly independent not just statistically uncorrelated components can be retrieved while the noise can be cut off.

References:

[1] M. Watanabe, E. Okunishi, K. Ishizuka, Microscopy and Analysis 23 (2009) 5-7.

[2] S. Lichtert, J. Verbeeck, Ultramicroscopy, 125 (2013) 35-42.

Fig. 1: Scatterplots of the first three PCA components for the system composed of two objects.PCA retrieves the “average” eigenvectors that cannot unmask the nature of theobjects. The fragmentation of the data and the application of PCA to eachcluster individually make the maximal variance in each object coinciding withthe direction of the eigenvectors.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-5988 Distributions of cations and inversion parameter in nonstoichiometric magnesium aluminate spinel characterized by electron energy-loss spectroscopy

Halabi M.1,2, Ezersky V.2, Kohn A.1,2, Hayun S.1,2
1Department of Materials Engineering, Ben-Gurion University of the Negev, 2Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev
akohn@bgu.ac.il

The effect of composition and heat treatments on the distribution of cations and on the inversion parameter in magnesium aluminate spinel was studied using Electron Energy Loss Spectroscopy (EELS). Powders of MgO•nAl2O3 (0.95<n<1.07) with nano-sized grains were synthesized by solution combustion and heat treated using Spark Plasma Sintering (SPS) and Pressure-less Sintering (PS) methods. EEL spectra were collected at varying distances perpendicular to grain boundaries from which the Mg to Al cation ratio and the inversion parameter, which is the fraction of tetrahedral sites occupied by Al cations, were calculated. The Mg to Al cation ratio was calculated from their core loss K-edges. To estimate the fraction of tetrahedral sites occupied by Al cations, the inversion parameter was calculated from the Al L-edge using the methodology suggested by Bruley et al., [1] which is based on the integral ratio between L3 to L2 peaks.
We report that cations which segregate to the grain boundaries are the excess component relative to the stoichiometric composition of the spinel (Fig. 1). Heat treatment does not change the type of segregate cation but does affect the degree of order of spinel. We find that spinel powders with nano-sized grains subjected to SPS treatment results in higher order compared to PS, namely lower inversion parameter. Finally, we discuss the experimental requirements for measuring reliable EEL spectra from these materials which are sensitive to damage from the electron beam.

[1] J. Bruley, M.-W. Tseng and D. B. Williams "Spectrum-Line Profile Analysis of a Magnesium Aluminate Spinel Sapphire Interface," Microsc. Microanal. Microstruct, pp. 1-18, 1995.

Fig. 1: (a) Schematic representation of the collection of EEL spectra perpendicular to grain boundaries. (b) Mg to Al cation ratio as a function of relative position perpendicular to the grain powders of spinel samples heat treated by SPS. (SCZ represents space charge zone)
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-5-P-6028 Addressing challenges in Electron Energy Loss Spectroscopy on individual atoms

March K.1, Brun N.1, Gloter A.1, Tencé M.1, Mory C.1, Stéphan O.1, Colliex C.1
1Laboratoire de Physique des Solides Université Paris-Sud - CNRS UMR-8502, Orsay, France
katia.march@u-psud.fr

The latest generation of STEM microscopes based on many instrumental developments (Cs corrector, lower primary voltages, EELS and EDX detector improvements...) offers the ability to track in a spectrum-image mode several signals generated simultaneously by individual atoms [1,2,3] and to rekindle the STEM-EELS spectro-microscopy of single atoms.

A Nion UltraSTEM microscope equipped with a Cs corrector and with a home-made fast EELS detector has been used to record a few typical cases illustrating the present situation in individual atom spectroscopy. With the new spectroscopic hardware, we can acquire EELS spectrum images of typically 100x100 pixels and covering a range of 1600 channels at an acquisition rate of 2300 spectra/s. Furthermore, the representation, exploitation and analysis of such data require some specific algorithms. The most widely used technique is the Principal Component Analysis (PCA) [4][5] and, as a filtering technique, offers an improvement of the signal to noise ratio. However, for high noise levels, a bias is introduced by PCA as signal bearing components are discarded with the removal of components considered as noise [6]. We have tested some algorithms based on non-local methods for denoising by exploiting the natural redundancy of patterns inside an image.

The first case is the determination of the position of Sm interstitial/substitutional dopants in ceria nanoparticles together with their valence changes in accordance with the variation of the ferromagnetic properties measured as a function of the nominal doping level [7]. The spectrum image has a high noise level and Sm doping could not be identified with usual PCA denoising. We have therefore tested Non-Local Sparse PCA [8] which produces interesting results: the filtered spectra display fine structures of edges and both spatial and spectral resolutions are preserved. The second example addresses the challenge of identifying the characteristic EELS signals from heavy (Tb, Th) atoms in rapid motion on a thin carbon layer which imposes a compromise between time acquisition and detection limit (see Figure).

This contribution emphasizes the possibilities currently offered by a tiny electron probe, a suitable efficient detector strategy and a well chosen signal analysis tool for single atom spectroscopy.

[1] K. Suenaga et al. Nature Chemistry 1 (2009) 415.

[2] O.L. Krivanek et al. Nature 464 (2010) 571.

[3] C. Colliex et al. Ultramicroscopy 121 (2012) 80.

[4] N. Bonnet et al. Ultramicroscopy 77 (1999) 97.

[5] F. de la Peña et al. Ultramicroscopy 111, 2 (2011) 169.

[6] S. Lichtert, J. Verbeeck, Ultramicroscopy 125 (2013) 35.

[7] S.-Y. Chen et al. Phys.Chem.Chem.Phys. 16 (2014) 3274.

[8] J. Salmon et al. J.Mathematical Imaging and Vision 48 (2014), 279.

Thanks are due to E. Delain et S. Baconnais (IGR, Villejuif, France) for tricky specimen preparation.

Fig. 1: Imaging and spectroscopy of Th and Tb atoms in rapid motion on a thin carbon foil under the electron beam (60 kV). HAADF images at 2 µs per pixel (a) and at 100 µs per pixel (b). Raw EELS spectra extracted from the SI at two different positions (blue and red) – acquisition time: 100 µs per spectrum (c).
↑ Go to the Table of content ↑

IT-6. Environmental electron microscopy

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-6-IN-1652 Advances in Atomic Resolution-Environmental (Scanning) Transmission Electron Microscopy

Gai P. L.1,2, Yoshida K.3, Lari L.1, Ward M. R.1, Martin T.1, Boyes E. D.1,4
1The York Nanocentre and Department of Physics, University of York, UK, 2Department of Chemistry, University of York , York, UK , 3Institute for Advanced Research, Nagoya University, Japan and York Nanocentre, University of York, 4Department of Electronics, University of York, UK
pratibha.gai@york.ac.uk

Dynamic chemical reactions catalysed by solid surfaces in heterogeneous catalysis play a major role in the development of energy sources, healthcare, environmental controls and industrial chemicals. Visualisation of the evolution of structural changes in catalysts in their working state under controlled gas and temperature conditions at the atomic level in real time is crucial in the development of efficient catalysts and processes but is extremely challenging.

Previously we reported the development of the first atomic resolution-Environmental transmission electron microscope (atomic resolution-ETEM) for in-situ studies of dynamic gas-solid reactions at operating temperatures under controlled conditions at the atomic level [1,2]. Highlights of this development include a novel ETEM design with the objective lens polepiece incorporating radial holes for differential pumping and the regular EM sample chamber as the controlled reaction environmental cell (reactor) [2]. This atomic resolution-ETEM development is now widely used.

Recently we have developed a double aberration corrected E(S)TEM (AC E(S)TEM) at York incorporating a large gap objective lens polepiece for in-situ studies under controlled gas and temperature reaction environments with single atom sensitivity, using a JEOL 2200 FS [3,4,5]. The new E(S)TEM capability enables the visualisation of single atom dynamics in real time (Fig. 1) [4,5]. Here we present E(S)TEM studies of working catalysts at the single atom level. Supported Au nanoparticles are of interest in hydrogenation, water-gas-shift and low temperature oxidation of carbon monoxide. Supported Pt nanocatalysts are used in fuel cells where reactions in hydrogen, oxygen, CO and water are important and in vehicle exhaust emission control measures [4-6]. The E(S)TEM with single atom sensitivity is playing a key role in the development of a heterogeneous process for sustainable biofuels from biomass (including vegetable plants, weeds and grass) and environmentally benign heterogeneous processes to produce medicines for human healthcare.

References
[1] Gai P.L. et al, Science 267 (1995) 661.
[2] Boyes E.D. and Gai P.L., Ultramicr 67 (1997) 219.
[3] Gai P.L. and Boyes E .D., Micros Res Tech. 72 (2009) 153.
[4] Boyes E.D., Ward M., Lari L. and Gai P.L., Ann. Phys. (Berlin) 525 (2013) 423.
[5] Gai P. L., Lari L., Ward M. and Boyes E. D., Chem.Phys.Lett. 592 (2014) 355.
[6] Yoshida K. et al, Nanotechnology 2014 (submitted).  

 We thank the EPSRC (UK) for Critical Mass grant EP/J018058/1.

Fig. 1: Top: single atoms and raft-like clusters of platinum on carbon using our in-house development of E(S)TEM at York. Single atoms are single white dots in the image. (Scale bar=2nm); bottom: intensity profile of a single atom in (a).
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-6-IN-2314 Atomic structure and reactivity in catalysis studied by electron microscopy

Helveg S.1
1Haldor Topsøe A/S, Nymøllevej 55, DK-2800 Kgs. Lyngby, Denmark
sth@topsoe.dk

Developing efficient technologies for the production of fuel and chemicals as well as for reducing environmental harmful emissions are among the largest challenges for our modern society. As their solutions depend on catalysis, research and innovation in this field is mandatory to realize the vision of a clean and sustainable society. In recent years, new opportunities for catalysis research have opened up with remarkable progress in transmission electron microscopy (TEM). On one hand, advancements in aberration-corrected electron optics and data acquisition schemes enable TEM delivering images of catalysts with sub-angstrom resolution and single-atom sensitivity [1,2]. On the other hand, parallel developments of differentially pumped electron microscopes and of gas cells enable time-resolved observations of catalysts in situ during the exposure to reactive gas environments at pressures of up to the one-atmosphere level and temperatures of up to several hundred centigrade [3-5]. In this contribution, I will outline how such instrumentation and methodologies can advance in situ studies of surface structures and reactivity in catalysis. Specifically, the concept of using low electron dose-rates in TEM, in conjunction with in-line holography and aberration-correction, is introduced to allow maintaining atomic resolution and sensitivity during non-invasive in situ observations of catalysts [3,6]. Moreover, a novel nanoreactor concept is demonstrated for directly correlating time-resolved, high-resolution TEM images of catalysts with concurrent measurements of their catalytic functionality under reaction conditions at the ambient pressure level [4-5,7]. These competences expand the applicability of TEM in catalysis and build a foundation for “live” observations of structure-sensitive functional behavior at the single–atom level and in catalytically meaningful environments. Extraordinary benefits are illustrated by in situ studies in e.g. water splitting, hydrotreating and automotive emission abatement catalysis [1-10].

References

[1] C.F. Kisielowski et al, Angew. Chemie. Int. Ed. 49, 2708 (2010)

[2] L.P. Hansen et al, Angew. Chem. Int. Ed. 50, 10153 (2011)

[3] J.R. Jinschek, S. Helveg, Micron 43, 1156 (2012)

[4] J.F. Creemer et al, Ultramicroscopy 108, 993 (2008)

[5] S.B. Vendelbo et al, Ultramicroscopy 133, 72 (2013)

[6] S.Helveg, C.F. Kisielowski, J.R. Jinschek, P. Specht, G. Yuan, H. Frei (2014)

[7] S.B. Vendelbo, C.F. Elkjær, H. Falsig, I. Puspitasari, P. Dona, L. Mele, B. Morana, B.J. Nelissen, R. van Rijn, J.F. Creemer, P.J. Kooyman, S. Helveg (2014)

[8] Z. Peng et al, J. Catal. 286, 22 (2012)

[9] S.B. Simonsen et al, J. Am. Chem. Soc. 132, 7968 (2010); J. Catal. 281, 147 (2011)

[10] L.P. Hansen, M. Brorson, E. Johnson, S. Helveg (2014)

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-6-O-1684 in situ Nanoscale Hyperspectral XEDS Elemental Mapping in Liquids

Lewis E. A.1, Haigh S. J.1, Kulzick M. A.2, Burke M. G.1, Zaluzec N. J.1,3
1Materials Performance Centre and Electron Microscopy Centre, School of Materials, University of Manchester, Manchester, U.K., 2BP Corporate Research Center, Naperville, Illinois, USA., 3Electron Microscopy Center, Argonne National Laboratory, Argonne, IL 60439 USA.
edward.lewis@postgrad.manchester.ac.uk

Recent years have seen an explosion of interest in in situ (scanning) transmission electron microscope (S/TEM) studies of solution-phase processes. Work employing silicon nitride windowed environmental cells (e-cells) to study nanostructures in liquid has yielded important insights into mechanisms of nanoparticle growth.[1,2] One of the great strengths of the S/TEM platform is the potential to combine high resolution imaging with local analytical information obtained using electron energy loss spectroscopy (EELS) and X-ray energy dispersive spectroscopy (XEDS). However, both EELS and XEDS face challenges when applied to specimens in liquid e-cells and elemental mapping has proved impossible until now.[3,4] In this work we show that by rational redesign of an e-cell holder we are able to dramatically increase the collection efficiency of characteristic X-rays,[5] in order to achieve elemental mapping of nanostructures in liquid. Improved X-ray detection is obtained using an analytical XEDS version of the Protochips Poseidon 200 holder in a FEI Titan ChemiSTEM operated at 200 kV. Wet specimens were encapsulated between a pair of 50 nm thick SiNx windows in a Si e-cell with 150 nm spacers separating the windows.
As a proof of principle we have studied a sample consisting of a mixture of pre-synthesised nanostructures immersed in a Cu containing aqueous solution. Beam-induced interactions with the solution result in dynamic Cu nanoparticle growth processes (Fig. 1).[2] Simultaneous XEDS spectrum imaging of nanostructures in liquid facilitates interpretation of the dynamic processes occurring in this complex multicomponent system (Fig. 2). A beam-induced copper plating reaction occurs in the liquid-phase, (fig 2a and 2b) while similar growth is not seen in dry reference samples. The resulting spectrum image (Fig. 2c) reveals that Cu ions from the surrounding liquid are plating the pre-synthesised silver nanowires and gold nanoparticles producing bimetallic, core-shell, structures.
We have shown that it is possible to use XEDS to simultaneously map multiple elements in liquid with a spatial resolution approaching 10 nm. This new technique allows direct observation of nanoscale changes in composition and elemental distribution during solution-phase processes and has great potential in the field of nanoscience, to provide insights to aid the synthesis of mixed metallic nanostructures, as well for corrosion and biological studies.

1. Zheng H et al 2009 Science 324 1309-1312.
2. Liao H et al 2013 Chem. Commun. 49 11720-11727.
3. Jungjohann K L et al 2012 Microsc. Microanal. 18 621-627
4. Holtz M E et al 2013 Microsc. Microanal. 19 1027-1035
5. Zaluzec N J et al 2014 Microsc. Microanal. (in press) 20

This work was supported by multiple grants including: EPSRC Grants # EP/G035954/1 and EP/J021172/1, DTRA grant HDTRA1-12-1-0013, and the BP 2013 DRL Innovation Fund.

Fig. 1: Selected HAADF STEM images from a video sequence showing the beam-induced growth of Cu nanoparticles from an aqueous solution containing Cu ions. Images taken at time = 0s (a), 6.3s (b), 13.1s (c), 19.9s (d), 26.2s (e), and 31.4s (f). Scale bar = 80 nm.

Fig. 2: Beam-induced growth of Cu nanostructures occurs during extended spectrum imaging, as observed by comparison of the HAADF images before and after (a and b). XEDS data facilitates simultaneous mapping of multiple elements in liquid (c) with a spatial resolution of the order of 10 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-6-O-2397 Raman Spectroscopy coupled with environmental scanning transmission electron microscope

Picher M.1,2, Lin P. A.1,2, Blakenship S.1, Winterstein J.1, Sharma R.1
11Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Gaithersburg, MD 20899-6203, 22Institute for Research in Electronics and Applied Physics, University of Maryland, College park, MD 20740
renu.sharma@nist.gov

In recent years the environmental transmission scanning electron microscope (ESTEM), has been successfully employed to reveal and understand the structural and chemical changes occurring in the nanoparticles under reactive environments [1,2]. The lack of statistical information available from TEM measurements is generally balanced by using other, ensemble measurement techniques such as x-ray or neutron diffraction, x-ray photoelectron spectroscopy, infrared spectroscopy, Raman spectroscopy etc.  However, it is almost impossible to create identical experimental conditions in two separate instruments to make measurements that can be directly compared. Moreover, ambiguities in ESTEM studies may arise from the unknown effects of the incident electron beam and uncertainty of the sample temperature.  Here, we present a unique platform that allows us to concurrently measure atomic-scale and micro-scale changes occurring in samples subjected to same reactive environmental conditions by incorporating a Raman Spectrometer on the ESTEM. 

We use a parabolic mirror, attached at the end of a hollow rod that can be inserted between the sample holder and the lower pole piece of the microscope (Fig. 1-2a). The mirror focuses the incoming laser on the sample and collects the scattered Raman photons. A set of optics then carries the Raman signal up to the spectrometer. Fig. 2.b,c show the Raman D and G band as well as the radial breathing modes of single walled carbon nanotubes (SWCNT) formed in the ETEM and an atomic-resolution still image extracted from a video sequence, respectively. We can monitor the growth rates using the G-band intensity under different growth conditions (Fig. 2d). This versatile optical setup can also be used i) to measure the temperature using Raman shifts, ii) to investigate light/matter interactions iii) as a heating source, iii) for general spectroscopy such as cathodoluminescence. Details of the design, function, and capabilities will be illustrated with results obtained from experiments on the in situ synthesis of carbon nanotubes.

Reference:

[1] Sharma, R., J. Mat. Res. 2005, 20, 1695

[2] Hansen et al., Science 2001, 294, 1508

Fig. 1: Schematic representation of the Raman data collection system: the laser passes through the hollow parabola holder, and is then focused on the sample by the parabola. The parabola collects the Raman signal and directs is back to the spectrometer.

Fig. 2: (a) Location of the parabolic mirror that collects the Raman signal, is located between the sample holder and the lower pole piece. (b) Raman spectrum collected from SWCNTs grown in the ESTEM. (c) Atomic Resolution image showing as grown SWCNTs (d) Time resolved evolution of the G band intensity (SWNT growth rate) at 650 °C under two C2H2 pressure.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-6-O-2933 Operando TEM of CO Oxidation Catalyst by Quantification of Gaseous Reaction Products

Miller B. K.1, Crozier P. A.1
1Arizona State University, Tempe AZ, USA
benmiller002@aol.com

In-situ transmission electron microscopy allows materials to be observed at the atomic scale while they are simultaneously subjected to stimuli relevant to some application. Operando TEM goes one step further, and additionally measures some performance metric of the material during the in-situ observation. We have developed a technique for performing operando TEM of a catalyst for CO oxidation, which allows us to quantitatively monitor the gas composition leaving the environmental cell in an FEI Tecnai F20 ETEM [1]. We have done this by two complimentary methods [2]. Electron energy loss spectroscopy (EELS) was used to quantitatively probe the gas composition directly in the sample chamber of the microscope at discreet times in the course of an experiment. Mass spectrometry was simultaneously used to measure the gas composition continuously via a residual gas analyzer attached to the vacuum system near the main turbo-pump which pumps the environmental cell. High resolution images can then be linked to the precise conditions in the cell at all times as seen in Figure 1. All of this was made possible by the introduction of a novel sample preparation technique in which a pellet with a 0.5mm hole in the center was formed from glass-wool fibers, and impregnated with a silica-sphere supported catalyst. This pellet was then placed into a Gatan heating holder along with a metallic grid, which was also covered in the silica-sphere supported catalyst. This approach is currently being adapted to an aberration corrected FEI Titan ETEM.
Some of the initial results of this technique are shown in Figure 3. High resolution images of individual particles are placed into context within the operando experiment using a plot of the mol fraction of CO2 determined using EELS. A hysteresis of the CO conversion is clearly seen as the temperature is ramped up and then back down over a period of several hours. The EELS data which was acquired in the core loss region of the spectrum was quantified using a linear combination method to fit the carbon k edge π* peaks from CO and CO2, as shown in Figure 2. Changes in the structure of the Ru-RuO2 catalyst are clearly seen, including transitions from an oxidized to a reduced state at temperatures above 400°C.

References:

[1] Chenna, S. and Crozier, P. A. ACS Catalysis 2, 2395-2402. (2012).
[2] Miller, B. K. and Crozier, P. A. Microscopy and Microanalysis, 2014 (in press)

Financial support from National Science Foundation CBET-1134464 and the Fulton Schools of Engineering at ASU, and the use of ETEM at John M. Cowley Center for HR Microscopy at Arizona State is gratefully acknowledged.

Fig. 1: Operando data from CO oxidation experiment. a) Mass spectrometry data from residual gas analyzer (RGA), showing a sudden increase in CO2 as the temperature is increased to 230°C. b) Core loss EELS data with peaks from both CO and CO2. c) High resolution image acquired at the same condition as the EELS data.

Fig. 2: Method for quantifying the CO2 conversion from core loss EELS data by a linear combination of reference spectra. After background subtraction of both the reference and mixture measured spectra, a least squares fitting method is implemented in MATLAB allowing precise determination of the ratio of CO to CO2 in the gas phase.

Fig. 3: Mole fraction of CO2 as a function of temperature determined by analysis of core-loss EELS spectra. The data show a clear hysteresis as the temperature is increased and then decreased. This is attributed, in part, to the reduction of the initially oxidized Ru. Images at three temperatures illustrate the changes in the catalyst structure.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-6-O-3407 Correlative Electron Microscopy and Photon Science Characterization of Working Catalysts

Stach E. A.1, Li Y.2, Zhao S.3, Zakharov D.1, Nuzzo R.3, Frenkel A.2
1Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, NY 11733 , 2Department of Physics, Yeshiva University, New York, NY 10016, 3Department of Chemistry, University of Illinois, Urbana-Champaign, Champaign, IL, 61801
estach@bnl.gov

Characterization of catalytic reactions is often hindered by the fact that the behavior the system is mesoscopic, while the materials involved are nanoscale, with features that can span a broad range of temporal and spatial scales and which involve a broad range of competitive interactions. As a result, the description of a catalytic system requires interrogation with a variety of techniques – involving imaging, diffraction and spectroscopy – to describe the dynamic changes in structure that can occur during reactions. Commonly, this is done by simple use of standard techniques, and inference of how the results relate to the working condition of the system. It is, however, preferable that multiple probes are used to characterize physical and electronic structure of the catalyst during reaction, over multiple time and length scales. To date it has not been possible to directly link the observations across these techniques in such a way as to confirm that the data (imaging, diffraction, spectroscopy) is obtained from the system in the exact same “working” state. Here we report an experimental approach that allows: (1) characterization – via x-ray absorption spectroscopy, extended x-ray absorption fine structure, x-ray fluorescence, Raman spectroscopy, transmission electron microscopy, scanning transmission electron microscopy, electron energy loss spectroscopy and energy dispersive electron microscopy – from the same sample, (2) characterization at atmospheric pressures in reactive environments, and (3) simultaneous, real-time and on-line analysis of the reaction products – i.e. “operando” experimentation.

We take advantage of recent developments in sample holders for transmission electron microscopy that allow catalysts to be confined between two, thin nitride membrane supports that are separated by a narrow gap, and that allow continuous flow of liquid or gas through the system. We exploit the simplicity of this system in such a way as to allow utilization in both synchrotron x-ray beamlines and transmission electron microscopes. We have chosen a simple, model catalyst reaction for the demonstration phase of this work, the catalyzed conversion of ethylene to ethane, though the use of Pd/SiO2 and Pt/SiO¬2 heterogenous catalysts. Extension to high-temperature experimentation will be reported, thereby demonstrating the extension of this approach to the full class of catalytic systems. 

Research carried out at the Center for Functional Nanomaterials, Brookhaven National Laboratory, supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. Y.L and A.F.F acknowledge additional support through the Synchrotron Catalysis Consortium, U.S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-FG02-03ER15476.

Fig. 1: Compendium of data. RGA data demonstrating that the catalysts are in the same operating environment in both the TEM and the XAS experiment. XANES, EXAFS, HR-TEM and EELS data from the Pt/SiO2 catalysts during the dehydrogenation reaction.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-1552 In situ and related TEM techniques for the characterization of chemically synthesized nanomaterials

Kamino T.1,2, Shimizu T.2, Yaguchi T.3
1Yamanashi University, Kofu, Japan , 2Japan Automobile Research Institute, Ibaraki, Japan, 3Hitachi High-Technologies, Ibaraki, Japan
tkamino@yamanashi.ac.jp

The wide range of techniques associated with an environmental transmission electron microscope (ETEM) are now applied to the studies of the nanomaterials used in the batteries and fuel cells. We have developed a specimen heating holder for in situ TEM observation of gas-solid reaction at elevated temperatures. The specimen heating holder has a nozzle for gas injection adjacent to the specimen. This unique design of the holder, together with the differential pumping system of the TEM, has made it possible to achieve a maximum gas pressure of about 100 Pa, at the specimen area, without affecting the vacuum of the electron gun chamber. The short gas path length, of only 50 cm, from a gas bottle to the nozzle, enabled us to replace the gas atmosphere in the specimen area, within several seconds. In recent years, the technique has been applied to the characterization of the degradation mechanism of the electrocatalysts of polymer electrolyte fuel cells as reported elsewhere. Most of the chemically synthesized metal-matrix nanoparticles contain organic elements as a residue, and if electron irradiation exceeds critical levels, the residue may be sputtered out from the particle, and the particle may crystallized. Therefore, careful control of illumination conditions and irradiation time are essential for the analysis of those materials. The results of our recent study revealed that the classic selected area diffraction (SAD) is one of the preferable techniques in the structural analysis of the composite. Figure 1 shows TEM images and SAD pattern of a Nafion coated Pt/GC electrocatalyst observed at 200kV with
the electron beam density of 25 mA/cm2 on the specimen. Except for a slight deformation of the GC layer, the morphology of the specimen remained unchanged during the electron beam irradiation, for 14 minutes. The microscope used for the observation was a Hitachi H-9500 environmental TEM equipped with a standard high resolution objective lens pole-piece. The size of the field limiting aperture, used for the SAD pattern observation, was 1.0 micrometer and the size of the selected area was smaller than 15nm. The result reveals that an intermediate voltage TEM can be applied to the study of beam sensitive composites if the observation condition is carefully controlled. However, the influence of the electron beam, in the phenomena observed, in situ, can not be completely avoided. From this point of view, we have developed a gas reaction device for ex situ TEM study ( Fig.2 ) . The chamber is designed for use with the TEM specimen heating holders so that comparison of the results of in situ experiments, with that of an ex situ experiments, or a combination of both experimental techniques, using same specimen heating holder, are possible.

Fig. 1:  TEM images(a,c) and SAD patterns (b,d) of a Nafion coated Pt/GC electrocatalyst observed at 200kV             with the electron beam density of  25 mA/cm2 . a,b : 0min, c,d : 14min.

Fig. 2: Desktopgas reaction device equipped with a TEM specimen heating holder
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-1598 Quasi in situ TEM characterization of Ni reduction in regenerated Ni/alumina catalysts

GAY A. S.1, DUBREUIL A. C.1, BROURI D.2, MASSIANI P.2
1IFP Energies Nouvelles - Rond point de l'échangeur de Solaize - BP 3 - 69360 Solaize (France), 2Laboratoire de Réactivité de Surface, CNRS-UMR 7197, UPMC - Site d’Ivry, 3 rue Galilée - 94200 Ivry-sur-Seine (France)
anne-sophie.gay@ifpen.fr

Ni/alumina are active and selective catalysts for the selective hydrogenation of pyrolyse gasoline produced by steam cracker. This reaction allows removing alkadiene and alkenyl aromatics from C5+ fraction, without hydrogenating the aromatic rings and forming saturated hydrocarbons. In order to extend catalyst life, regeneration of spent catalysts followed by a reduction step can be applied to obtain a metallic and redispersed catalyst with activity as close as possible to the one of the fresh catalyst.
In this work, such reduction of a regenerated Ni/alumina catalyst containing reoxidized Ni particles was studied. The morphological evolution of the nanoparticles during reduction was followed by “quasi in situ” TEM, using a special Gatan HHST4004 “Heating Environmental Cell Holder” equipped with an ex-situ reactor and allowing the observation of the same zone before and after thermal treatment, in controlled atmospheric conditions [1].
After regeneration, all Ni in the sample was in oxidic form, as shown by FFT and SAED analyses. Two kinds of Ni oxide particles were observed, namely (i) small well-dispersed particles with sizes centered around 11 nm and (ii) large hollow particles (up to 30 nm in diameter), probably formed by Kirkendall effect from the initial metallic particles, as previously reported for Co Fischer-Tropsch catalysts [2]. After 2 hours of reduction at 410°C in Ar-5% H2 (treatment performed in the sample holder reactor), a modification of the morphology of the large hollow particles was observed. Thus, the hollow spheres broke during the reduction into a group of smaller Ni° particles forming a ring-like aggregate whose size is the same as for the initial hollow particle (figures 1 and 2). Besides, small nanoparticles with sizes nearly as above were still present but smaller nanometric particles also appeared. Moreover, all nickel was fully reduced, whatever the type of the nanoparticle.
These observations are in good accordance with previous results obtained for Ni catalysts used in the partial oxidation of methane [3] and for cobalt FT catalyst [2]. In summary, the present study shows that regeneration and reduction of a spent Ni/alumina catalyst used in selective hydrogenation of pyrolyse gasoline leads to a reduced well-dispersed catalyst. Besides, this study highlights the interest of the "quasi in situ" TEM technique to follow morphological evolutions during activation and/or regeneration treatments of supported catalysts.

[1] E. Sayah, D. Brouri, P. Massiani Catalysis Today 218– 219 (2013) 10–17
[2] C. J. Weststrate and al., Top Catal (2011) 54: 811-816
[3] S. Chenna and al., ChemCatChem 3 (2011) 1051-1059

Fig. 1: Hollow NiO particle in the regenerated catalyst

Fig. 2: Same zone - Ring-like aggregate of Ni° particles after 2h of reduction at 410°C under Ar/5%H2
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-1815 Development of a TEM specimen holder system for catalytic materials

Hashimoto A.1, Takeguchi M.1
1National Institute for Materials Science, Tsukuba, Japan
Hashimoto.Ayako@nims.go.jp

   Catalytic materials are often used as particles dispersed on a support material. Reduction of their size to nanoparticles, clusters and atoms is crucial for improving their performance. Transmission electron microscopy (TEM) is an indispensable tool for characterization of catalyst nanoparticles. However, the observation environment in general TEM differs significantly from those in actual applications. In this study, we developed a TEM specimen holder system for in situ observation of catalytic materials.

   Figure 1 shows a schematic of the developed system. The specimen holder includes a heater, a gas nozzle for introducing gases to the specimen, and a gauge for measuring pressure near the specimen. Orifice plates are arranged above and below the specimen to create differential vacuum.

   Figure　2　shows a TEM image and a fast Fourier transform (FFT) pattern of Pt nanoparticles on an amorphous carbon film taken in 0.5 Pa vacuum at room temperature, i.e., under gas-phase TEM conditions. The used microscope was JEM-2100 (JEOL, Japan), operating at an accelerating voltage of 200 kV. Lattice fringes of the Pt nanoparticle were observed, as indicated by the arrow in the TEM image and by the satellite spots in the FFT pattern (inset). These results demonstrate that the developed specimen holder system is compatible with high-resolution imaging in low vacuum. We have applied this holder system to study the movement of Pt nanoparticles on carbon materials in a gas atmosphere at high temperatures.

This work was partly supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research and Center of Materials Research for Low Carbon Emission.

Fig. 1: A schematic of the developed specimen holder system.

Fig. 2: TEM image and FFT pattern of Pt nanoparticles on an amorphous carbon film taken under 0.5 Pa air at room temperature.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-1924 Dynamic Observation of Gold Particles in Water by Environmental-cell TEM

Kawasaki T.1, 2, 4, Imaeda N.1, Murase H.1, Yamasaki K.3, Matsutani T.3, Tanji T.1, 4
1Nagoya University, Nagoya, Japan, 2Japan Fine Ceramics Center, Nagoya, Japan, 3Kinki University, Osaka, Japan, 4Global Research Center for Environment and Energy based on Nanomaterials Science, Japan
kawasaki@nuee.nagoya-u.ac.jp

An environmental transmission electron microscope (ETEM) is very powerful technique enabling to in-situ observation of specimens immersed in gases, electric/magnetic fields, and even in liquids by using a closed-type ETEM. In the closed system, diaphragms which are set at the top and the bottom of a space for the specimen to isolate from the vacuum are one of the most important components. Although silicon nitride (SiN) films are generally used for this purpose [1], charging effect on the diaphragms is inevitable because of the insulating SiN. In order to solve this problem, we have developed a carbon/SiN hybrid diaphragm which consists of amorphous carbon film as conductive base material and amorphous SiN thin layer coated on it [2]. In this study, the diaphragms were applied to in-situ TEM observation of motion of gold colloidal particles in water.
Figure 1 shows a photograph of our developed environmental liquid-cell. This consists of three parts; upper/lower grids and a spacer ring. Each of the grids had seven holes of ?0.1mm in diameter as windows for the electron beam, which were covered by the carbon/SiN hybrid membrane. The fluid specimen was sandwiched by these two membranes with controlling distance between them by 240 nm with the spacer deposited on the upper grid. The liquid-cell containing the fluid specimen was set in an environmental-cell specimen holder. In the experiment, the fluid specimen was gold colloidal particles having the size of 80±5.7nm in diameter dispersed in water in 1.1×107/?l concentration, which were observed by using a conventional TEM H-8000 (200kV; Hitachi High Technologies).
Figures 2 show results of dynamic observations of motions of the gold particles in water. A gold particle in FIG. 2(a) migrated slowly at a speed of 10 ~ 30 nm/s, as shown in a quantitative analysis of variation of the moving speed in FIG. 2(b). This is not a Brownian motion because the gold particle drifted in a single direction. In contrast, a gold particle in FIG. 2(c) moved quickly just after starting the electron beam illumination at high speed of more than 1200 nm/s. Although a next short movement occurred after the first quick motion, the gold particle subsequently stopped, as shown in FIG. 2(d). These different types of the motion mean driving force of the movement of the gold particles is not unique. In the present cases, it is considered that an effect of water flow and coulomb force by charging due to the electron beam irradiation caused the motions of the particles in FIG. 2(a) and (c), respectively.


References
[1] U.Mirsaidov, C.D.Ohl, and P.Matsudaira, Soft Matter 8, 7108-7111(2012)
[2] T. Kawasaki et al., Proc. M&M2011. (2011) 465.

The authors acknowledge the financial support of this work by the Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (#25390078).

Fig. 1: Photograph of our developed environmental liquid-cell which consists of upper/lower grids and a spacer ring.

Fig. 2: TEM images of motion of gold particles ((a) slow and (c) quick movement) (b), (d) variation of speed of motions in (a) and (c), respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-1943 The Study of Ice Impurities Using the Environmental Scanning Electron Microscopy at Higher Pressures and Temperatures.

Neděla V.1, Runštuk J.1, Klán P.2, Heger D.2
1Institute of Scientific Instruments of ASCR, Královopolská 147, 61264 Brno, Czech Republic, 2Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic
vilem@isibrno.cz

Natural ice and snow accumulate and concentrate significant amounts of impurities that can be stored or chemically transformed, and eventually released to the environment. The location of impurities and their interactions with the water molecules of ice have not yet been sufficiently clarified. The aim of this work is to observe an uranyl-salt brine layer on the ice surface using a back scattered electron detection and the ice surface morphology using a secondary electron detection under equilibrium conditions in a specimen chamber of environmental scanning electron microscope (ESEM).

Our specially modified ESEM AQUASEM II equipped with the YAG:Ce3+ backscattered electron detector, an ionization detector of secondary electrons, a special hydration system and a Peltier cooled stage was used. The pressures between 400-700 Pa, 50% water-vapor saturation, and the temperatures above 250 K were utilized in our experiments. At these conditions, the phenomena of etching and subsequent stripping of impurities are largely suppressed.

Our samples were frozen under atmospheric pressure on a silicon plate cooled by the Peltier cooled stage. The initial sample holder temperature was above –1°C. A droplet of pure water or the uranyl nitrate solution was exposed to freezing. The uranyl nitrate solution (0.01 M) acidified by perchloric acid to pH = 1 were used in our second experiment because the hydrolysis of UO22+ is suppressed and only a single species (i.e., a hydrated uranyl ion) is present under these conditions.

Figure 1A shows an ESEM image of the ice sample prepared by freezing of pure water under atmospheric pressure inside the specimen chamber. Different shapes and sizes (30–200 µm) of the ice grains can be distinguished. Due to the detection of secondary electrons (SE), which are sensitive mostly to the surface topography, the ice grain boundaries are visible as black lines with a bright halo. At this temperature, the ice crystals are covered with a disordered interface (also called quasi liquid layer), however it is too thin to be identified by ESEM. Since the amount of backscattered electrons (BSE) is related to the atomic number of the present elements, 92U-rich regions appear brighter, whereas the regions consisting of water molecules remain dark, see Figure 1B. The difference between pure ice and the frozen uranyl solution is largely manifested in the channels and pools of concentrated UO22+ solutions (bright) along with the individual ice grains (black). Pools are usually the largest at the triple junctions, although some may also be present on the ice surface. A liquid layer containing UO22+ was expected to be considerably more concentrated than the parent solution due to the freezing concentration effect.

This work was supported by the Grant Agency of the Czech Republic: grant No. GA 14-22777S.

Fig. 1: Figure 1: An ESEM image of ice prepared by freezing of a droplet inside the specimen chamber: (a) frozen pure water; a SE detector mode; 270 K, 695 Pa (5.2 torr); (b) the frozen uranyl salt solution; c = 10–2 M; a BSE detector mode; 267 K, 525 Pa (3.9 torr). Bar 100 um
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-1959 The Atmospheric Scanning Electron Microscope (ASEM) Observes Axonal Segmentation and Platelet Generation in Solution.

Kinoshita T.1, Motohashi H.2, Hirano K.1, Maruyama Y.3, Kawata M.3, Ebihara T.3, Sato M.3, Nishiyama H.4, Suga M.4, Yamamoto M.5, Nishihara S.1, Sato C.3
1Faculty of Engineering, Soka University, Tokyo, Japan, 2Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan, 3National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan, 4JEOL Ltd., Tokyo, Japan, 5Tohoku University Graduate School of Medicine, Sendai, Japan
ti-sato@aist.go.jp

The new Atmospheric Scanning Electron Microscope (ASEM) is a Correlative Light-Electron Microscope (CLEM) [1]. Cell cultures of a few milliliters can be grown and differentiated directly in the removable ASEM dish, in a CO2 atmosphere if required. After fixation, the cells are imaged in situ, immersed in liquid and at atmospheric pressure, by optical microscopy (OM) and SEM in a fully correlative manner. Various dish coatings have been developed to increase the range of culturable cell types [2] allowing axonal segmentation and platelet generation to be investigated.

Axonal partitioning of neurons was correlated with specific cytoskeletal structures including microtubules. For this, isolated Drosophila primary neurons were grown on a poly-DL-ornithine-coated ASEM dish and immunolabeled for neuron markers HRP and BP102. Fluorescence microscopy demonstrated the localization of HRP in the whole axial fiber (Figure 1, B) and the specific localization of BP102 in the proximal region (A). ASEM revealed a hexagonal frame-like structure of BP102 at the boundaries of the most intra-axonal segments (E, arrow) [4], which has not been observed by OM. Two tubulin bundles running alongside one another make contact at the intra-axonal boundary and possibly elsewhere. Eight of the ten axons examined showed such contacts. In the other two axons, the immunolabeling was disconnected at the intra-axonal boundary [3]. This may mean that the two tubulin bundles are separated, although the possibility that labeling is prevented by proteins bound to the α-tubulins in this region cannot be excluded.

Mature megakaryocytes (MKs) generate beaded cell projections named proplatelets, and further shed off platelets, which are indispensable cellular components of blood for hemostasis. We cultured MKs on ASEM dish and fixed the cells at an appropriate timing captured with OM. The cells were stained with heavy metal solution and observed at high resolution with the inverted SEM. The pseudopodia extended beaded strings (Figure 2A-B), including vesicles (C). These vesicles are necessary for blood clot formation, which is related to cerebral or myocardial infarction under pathological conditions [4]. Immunolabeling of P-selectin indicates that the vesicles could be a-granules. Additional labeling of α-tubulin indicates their transportation on microtubules (D-E).

References

[1] H. Nishiyama et al, J Struct Biol 169 (2010), p. 438-449.

[2] Y. Maruyama et al, J Struct Biol 180 (2012), p. 259-270.

[3] Microsc. Microanal. in press, doi:10.1017/S1431927614000178

[4] Ultramicroscopy in press, (http://dx.doi.org/10.1016/j.ultramic.2013.10.010)

We thank Dr. Toshihiko Ogura at AIST for valuable discussions.

Fig. 1: Figure 1. Axonal segmentation. (A) Localization of HRP. (B) BP102 (red). (C)Merge. (D-E) ASEM. (D) BP102 accumulates forming a special hexagonal frame-like structure at the intra-axonal boundary(arrows). (Ultramicroscopy. in press).

Fig. 2: ASEM of platelet generation from MKs. Primary MKs with proplatelet formation cultured on an ASEM dish were fixed, stained with Ti-blue (A-B) or gold-tagged for P-selectin (C), and further for microtubule (D-E). (B) Arrowheads indicate beaded proplatelets. (C-E) Arrows indicate putative alpha-granules. (Ultramicroscopy in press).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-1960 Time-resolved Observations of Single Protein's Motions Using Diffracted Electron Tracking (DET) with Wet Cell SEM

Ishikawa A.1, Ogawa N.1,2, Hirohata Y.1, Yohda M.2, Sekiguchi H.3, Sasaki Y. C.4
1Nihon University, Tokyo, JAPAN, 2Tokyo University of Agriculture and Technology, Tokyo, JAPAN, 3SPring-8/JASRI, Hyogo, JAPAN, 4The University of Tokyo, Chiba, JAPAN
ishikawa@phys.chs.nihon-u.ac.jp

Diffracted electron tracking (DET) method has been developed for obtaining the information about the dynamics of a single protein molecule[1,2]. DET can be performed using a Scanning Electron Microscope (SEM) equipped with a highly sensitive detector for electron backscattered diffraction (EBSD). DET can trace the rotating motion of individual nanocrystals linked to the specific site in the molecule. Fig.1 shows the principle of the DET and the parameters to be measured. (a) When the electron beam irradiates a nanocrystal, inelastically scattered primary electrons form a band-like EBSD pattern (EBSP) and the 3D motion of nanocrystals can be traced from the shifts of the pattern. (b) shows the rotation angle ω around a single axis and the rotation angles α, β, and γ of the principal lattice vectors , a, b and c of the nanocrystal, respectively, between each time step. For tracing the motion of protein molecule, we have developed the wet cell sealed with the very thin carbon film for SEM observation[1,2]. The EBSP can be obtained from the colloidal gold linked to chaperonin protein in water under the carbon sealing film of the wet cell. In DET, radiation damage of the specimen is the biggest problem. To reduce the damage, specimen supporting was improved, as shown in Fig.2. (a) When the chaperonin protein is fixed to the carbon sealing film, although the motion of the colloidal gold could be traced, no directional motion could be observed in both conditions with and without adenosine tri-phosphate (ATP) which causes the rotation of the chaperonin protein. With this supporting, the chaperonin was irradiated by both the incident electron beam and EBSD electrons, and so damaged it could not move. Therefore the chaperonin supporting system was changed. The molecules are fixed to thin tri-acetyl-cellulose film to the opposite side of the sealing film as shown in (b). With this supporting, the chaperonin is covered with the “shadow” of the colloidal gold and hardly irradiated by the electrons. With this supporting system, the motion of the colloidal gold was traced by DET. The mean square of displacement (MSD) of the rotation angles of the colloidal gold particles, in both conditions with and without ATP, are shown in Fig.3. (a) Without ATP, each MSD of the α, β, and γ is almost same, and no directional motion is observed. (b) On the other hand, with ATP, the magnitude of the γ is clearly decreasing compared to other angles. This means that the chaperonin, linked to the colloidal gold, increases rotational motion around the ND axis as shown in (c). These results correspond with other single protein observations using other techniques.

References [1] N. Ogawa et al., Scientific Reports, 3, 2201 (2013) 1-7 [2] N. Ogawa et al., Ultramicroscopy, 140 (2014) 1-8

This research was supported by the Japan Science and Technology Agency under the Core Research for Evolutional Science and Technology (CREST) program.

Fig. 1: Principle of DET and parameters to be measured. (a) Inelastically scattered electrons in the crystal form a band pattern and crystal motion can be traced from the shifts of the EBSP. (b) The rotation angle ω around a single axis and the rotation angles α, β, and γ of the principal lattice vectors a, b and c are measured.

Fig. 2: Improvement of specimen supporting system for DET to reduce the radiation damage for chaperonin protein. (a) The chaperonin protein is fixed to the carbon sealing film of the wet cell. (b) The chaperonin protein is fixed to thin tri-acetyl-cellulose film opposed to the sealing film and is covered from the electron beam by the colloidal gold.

Fig. 3: MSD of the rotation angles of chaperonin molecules measured by DET. (a) Without ATP, almost no directional motion is observed. (b) With ATP, the g was clearly decreasing compared to other angular. This means that the many motions are the rotations around ND axis (γ = 0) corresponding to the motion of each chaperonin protein (c).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-1968 Investigation of hexagon shape nanoparticle growth mechanism using in-situ liquid Cell TEM

Ahn T.1, Kim Y.1, Hong P.1, Nam K.1, Kim Y.1
1Department of materials science and engineering, Seoul National University, Korea
an2027@snu.ac.kr

A number of efforts have been made on the synthesis of monodisperse nanoparticles with various morphologies to take advantage of their physical and chemical properties of nanoparticles attainable from the chemical composition and their dimensions. Growth of nanoparticle can be affected by many factors, such as temperature, surfactants, types of precursor, and its relative concentration. In order to understand the growth and the formation mechanism of nanoparticles, it was proven that the liquid cell TEM is one of the most powerful techniques because of its nanometer-level spatial resolution with transparency of the internal structure. Researchers were able to observe the growth procedures in real time using the liquid cell TEM, which made a great fore-step to observe nanoparticle growth using electron beam induced process.
Growth behaviors of spatially aligned, hexagon single crystals were investigated from the liquid cell with D.I water based solution. Formation sequence was observed from home-built liquid cell TEM stage in JEOL 2010F. No intentional heating was made during the crystallization. Streaming video was recorded from the Gatan ES500W camera. Figure1 shows the boundary regions with and without the nanoparticles formed by the electron beam induced growth. Inhomogeneous distribution of the particles might be come from exposure time difference for the particle formation. Figure2 shows the snapshots from the streaming video taken while shining electron beam onto the liquid layer. Nanoparticles were formed in spherical shape up to 123 second irradiation. However as irradiation time increased, nanoparticles were gradually changed to hexagonal shape. Orientation alignment and the growth rate were measured from the snapshots up to 260 second irradiation. Effect of precursor concentration and the electron current density on the formation and the growth of nanoparticles were examined.

This research was supported by the Nano-Material Technology Development Program(Green Nano Technology Development Program) through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2011-0019984)

Fig. 1: Low magnification image of the field of view. The regions in which electron induced growth occurred or not.

Fig. 2: Snapshots from the streaming video. As time increases hexagon shape nanoparticles growth were confirmed.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-2009 Development and application of environmental high voltage electron microscope

Wakasugi T.1, Isobe S.2, Wang Y.2, Hashimoto N.1, Ohnuki S.1
1Graduate School of Engineering, Hokkaido University, 2Creative Research Institution, Hokkaido University
wakatake@eng.hokudai.ac.jp

Introduction:

For the practical use of hydrogen storage materials, the improvement of their hydrogen storage properties has been required. In order to improve the properties, we should understand the mechanisms and the dynamics of the materials in nano scale. Recent research indicated that a strain field introduced in the materials affected on the properties [1]. For understanding the dynamics in nano scale not only for improving the hydrogen storage properties, but also for developing other functional materials, in-situ TEM is the best way. In this research, we developed a high pressure gas Environmental cell for High Voltage Electron Microscope (EHVEM) and applied to in-situ High-Resolution (HR) observation with increasing hydrogen gas pressure.

Experimental:

The sample was a Pd thin film (~10nm-thick) deposited on a Silicon Nitride (SiN) window film. Firstly, this film was observed in a vacuum condition at room temperature. And then, an in-situ observation was carried out with the hydrogen pressure up to 40 kPa. The microscope used in this study was the EHVEM based on the JEOL ARM-1300 and operated at an acceleration voltage of 1250 kV.

Result and discussion:

The EHVEM allowed an in-situ HR observation in a hydrogen pressure up to 40 kPa. As shown in the Figure.1, however, the lattice fringes of PdH0.6 (200) and (111) grain with a distance of 0.20 nm and 0.23 nm were barely visible at the pressure. This result suggested that there was a significant influence of the gas pressure on the image resolution despite the use of high voltage electron beam.

On the other hand, using the corresponding IFFT (Figure.2) allowed us to recognize some dislocation cores introduced around/in a hydride grain clearly. The result told us that the increase in the number and the distribution change of cores with the pressure.

Reference:

[1] N. Hanada et al. J. Phys. Chem. C 113 (2009) 13450-13455

Fig. 1: High resolution images and FFTs (the insets) corresponding to PdH0.6 (200) and (111) in the hydrogen pressure of 10, 20, 40 kPa.

Fig. 2: High resolution images (the inset) and IFFTs (from white square) corresponding to PdH0.6 (111) in the hydrogen pressure of 10, 20, 40 kPa.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-2220 TEM Imaging of CO Oxidation Catalyst of Gold Nanoparticle on TiO2 in CO and O2 Environments

Tanaka T.1, 3, Yamamoto N.2, 3, Takayanagi K.2, 3
1Meijo University, 2Tokyo Institute of Technology, 3JST, CREST
ttanaka@phys.titech.ac.jp

Gold nanoparticle on TiO2 (Au/TiO2) is promising for application to a low temperature CO oxidation (2CO+O2→2CO2) catalyst [1-4]. It is reported that the catalytic reaction proceeds at a peripheral region of the Au/TiO2 interface [1, 4]. It is proposed that the catalysis emerges from a negatively charged O2 molecule (O2–) [1, 4], which is generated by Au-Ti co-bonding [4] and/or interstitial Ti ion [5]. We have studied the structure and electronic states of Au/TiO2 by using advanced TEM techniques [2, 3].
Interstitial Ti ions in a TiO2 substrate with and without a gold nanoparticle were observed by aberration corrected TEM [3]. Interstitials of Iv sites were observed at TiO2(001) surface as shown in Fig. 1(a) (a). Interstitials were accumulated at a perimeter/interface of Au/TiO2, while interstitials were depressed in a peripheral area of the accumulated region. A specific phase, which seems an expansion of the interstitial-accumulated region, was observed at the edge of the Au/TiO2 interface [2]. The specific phase grew extensively by exposing to O2 gas at 100 Pa into a pillar which has a chemical composition of Ti1-xO2 (x > 0) in Fig.1 (g). We will discuss report the CO oxidation catalysis.

[1] M. Haruta, et al., J. Catal. 144 (1999) 175.
[2] T. Tanaka, et al., Surf. Sci. 604 (2010) L75.
[3] T. Tanaka et al., Surf. Sci. 619 (2014) 39.
[4] Z.-P. Liu, X.-Q. Gong, J. Kohanoff, C. Sanchez, and P. Hu, Phys. Rev. Lett. 91 (2003) 266102.
[5] S. Wendt et al., Science 320 (2008) 1755.

The presentworkwas supported by a Grant-in-Aid for Scientific Research (A) (No. 16201020) and Exploratory Research (No. 24656031) of Japan Society for the Promotion of Science (JSPS). We thank Associate Professor N. Yamamoto (Tokyo Institute of Technology) for his valuable comments and stimulating discussion.

Fig. 1: (a) 
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-2380 Controlled environment specimen transfer for investigation of catalysts by ETEM

Damsgaard C. D.1,2, Zandbergen H.3, Hansen T. W.1, Chorkendorff I.2, Wagner J. B.1
1DTU Cen, Lyngby, Denmark, 2DTU CINF, Lyngby, Denmark, 3Kavli, TU Delft, Delft, The Netherlands
cdda@cen.dtu.dk

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-2451 Aberration-Corrected, Environmental TEM Studies on Carbon Nanotube Oxidation and the Influence of the Imaging Electron Beam

Koh A. L.1, Gidcumb E.2, Zhou O.2, 3, Sinclair R.1, 4
1Stanford Nanocharacterization Laboratory, Stanford University, Stanford, California 94305, USA, 2Department of Applied Physical Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA, 3Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA, 4Department of Materials Science and Engineering, Stanford University, Stanford, California 94305, USA
alkoh@stanford.edu

One of the major applications for carbon nanotubes (CNTs) is as field emission electron sources, for example in image displays and high-intensity medical X-ray tubes [1-3]. The emission currents and lifetimes of CNTs are found to decrease under less stringent vacuum conditions [4, 5]. Earlier reports of carbon nanotube oxidation performed in an external laboratory setting, and surveyed a posteriori with a transmission electron microscope (TEM), suggested that the nanotube caps were selectively attacked during the oxidation process [6, 7].

Recently, we reported the direct study on the structural changes in CNTs as they were oxidized in-situ using an aberration-corrected environmental TEM (ETEM) [8]. Nanotubes were identified and tracked for structural changes as they were heated to increasing temperatures in a 1.5 mbar, high purity (99.9999%) oxygen environment. In order to investigate the effect of gaseous oxygen molecules on the nanotubes, rather than ionized gas species, we established a protocol whereby heating and oxidation were performed without an imaging beam, and the changes on identifiable nanotubes were documented after purging the gas from the chamber. Our studies showed that the oxidation of multiwall CNTs proceeds layer by layer, starting with the outermost wall, and not initiating at the nanotube cap, as reported previously. Nanotubes with a larger number of walls (greater than six) were found to be more resistant to oxidation, with all walls remaining intact during the ETEM experiments [8].

To simulate the highly ionized environment, which is expected during field emission, we repeated these observations at room temperature in the ETEM in the presence of the imaging beam. Under such conditions, we found that more rapid attack takes place, even at room temperature, and both the hemispherical cap and side walls of the CNTs are vulnerable. The influence of the imaging electron beam in the observation of this gas-solid reaction in the ETEM will be discussed.

References:

[1] Q. H. Wang et al., Appl. Phys. Lett. 72 (1998) pp. 2912–2913

[2] G. Cao et al., Med. Phys. 37 (2010), pp. 5306–5312

[3] X. Qian et al., Med. Phys. 39 (2012), pp. 2090–2099

[4] K. A. Dean and B. R. Chalamala, Appl. Phys. Lett. 75 (1999), pp. 3017–3019

[5] J.-M. Bonard, et al., Ultramicroscopy 73 (1998), pp. 7–15

[6] P. M. Ajayan et al., Nature 362 (1993), pp. 522–525

[7] S. C. Tsang, P. J. F. Harris and M. L. H. Green, Nature 362 (1993), pp. 520–522

[8] A. L. Koh et al., ACS Nano 7(3) (2013), pp. 2566–2572

Funding from the National Cancer Institute grants CCNE U54CA-119343 (O.Z.), R01CA134598 (O.Z.), and CCNE-T U54CA151459-02 (R.S.) is acknowledged. We thank Dr. Bo Gao of Xintek for providing the CNTs.

Fig. 1: Aberration-corrected TEM images of multiwall carbon nanotubes (MWNT) at (a) 400°C, (b) 400°C after exposure to 1.5 mbar oxygen for 15 min, and (c) 520°C after exposure to 1.5 mbar oxygen for 15 min. The electron beam is blanked during the oxidation process. The MWNT is found to resistant to oxidation under these conditions.

Fig. 2: Aberration-corrected TEM images of multiwall carbon nanotubes (MWNT) acquired at room temperature and 1.0 mbar oxygen, with the imaging electron beam on. The red arrow indicates attack on the MWNT cap and side wall, due to exposure to the ionized oxygen. The time elapsed between (a) and (b) is 32 sec.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-2499 Carbon gasification by silver nanoparticles followed in situ at atomic resolution under oxygen partial pressure in an Environmental TEM (ETEM)

EPICIER T.1,2, CADETE SANTOS AIRES F. J.2, AOUINE M.2, LANGLOIS C.1, BLANCHARD N.3
1University of Lyon, MATEIS, umr CNRS 5510, INSA de Lyon/Université Lyon I, 69621 Villeurbanne, FRANCE, 2University of Lyon, IRCELYON, umr CNRS 5256, Université Lyon I, 69626 Villeurbanne, FRANCE, 3University of Lyon, ILM, umr CNRS 5306, Université Lyon I, 69622 Villeurbanne, FRANCE
thierry.epicier@insa-lyon.fr

Generally, the gasification of hydrocarbon-based materials leads to the formation of syngas. Gasification can also be performed, at high temperature in oxidative environments, in presence of metal catalysts supported on solid carbon materials. Indeed, oxygen adsorbs and dissociates on the metal surface then interacts with the carbon at the interface with the metal leading to the formation of carbon dioxide; concurrently carbon is consumed and the metallic nanoparticle advances to maintain the interface with the carbon forming in this way a trench on the surface of the carbon. On structured materials such as graphite or graphene these trenches tend to be rather 2D [1,2] at the surface of the material. In this study we chose to study the gasification of a non-structured material (amorphous carbon) by silver-based nanoparticles in a Cs-corrected TITAN ETEM, 80-300 kV, recently installed at CLYM in Lyon. Samples were prepared according to a synthesis described in [4]; the silver based nanoparticles (NPs) hang on onto the supporting carbon films and gasification of this film is observed between 400 and 500°C under variable oxygen partial pressures (between 10-1 and 5 mbar). We could follow in real-time the dynamics of carbon gasification and catalyst evolution by high resolution imaging (Fig.1) unlike previous studies. In situ EELS yields complementary information (regarding oxygen) necessary to support the proposed mechanism deduced from our in situ study and schematically summarized in Fig.2: (i) at the beginning of the gasification experiment the NP have an hexagonal structure consistent with Ag2O or hexagonal-Ag (known to exist under the form of NPs or nanorods [5]) containing diluted oxygen (Fig.2 a-c); (ii) at a given moment during gasification the NP transforms to fcc-Ag, gasification slows down, the particle begins to shrink (which is consistent with the decomposition of a surface oxide) while a coating forms around it (Fig.2d); (iii) once the particle is completely coated gasification stops and the NP shrinking stops (Fig.2e).


[1] S.K. Shaikhutdinov, F.J. Cadete Santos Aires, Langmuir, 14 (1998) 3501.
[2] T.J. Booth et al., Nano Letters, 11 (2011) 2689.
[3] N. Severin et al., NanoLetters, 9(1) (2009) 457.
[4] S. Li et al., Chem. Comm. 49 (2013) 8507.
[5] I. Chakraborty et al., J. Phys.: Condens. Matter, 23 (2011) 325401.

Thanks are due to the CLYM (Centre Lyonnais de Microscopie, www.clym.fr) for its guidance in the ETEM project which was financially supported by the CNRS, the Région Rhône-Alpes, the ‘GrandLyon’ and the French Ministry of Research and Higher Education. The authors acknowledge S. Li, A. Tuel and D. Farrusseng for providing samples and L. Burel for her assistance in preparing them.

Fig. 1: video frames from an HREM sequence of about 6 minutes recorded in situ at 495°C under 0.6 mbar of oxygen (see text and fig. 2 for details).

Fig. 2: a) starting geometry; b) O2 dissociation at the silver surface; c) gasification of the carbon support and motion of the NP (arrow); d) oxygen diffusion inside the NP and decomposition of the formed oxide; e): reaction of the Ag, O species with the surrounding (irradiation damaged) carbon to form a protective shell around the silver core.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-2722 Development Aberration Corrected Wet-ETEM System and Its Application to Pt/Carbon Fuel Cell Catalysts in Moisturized Gases Environments

Yoshida K.1,2, Zhang X.2, Hiroyama T.2, Boyes E. D.3,4, Gai P. L.3,4
1Institute for advanced Research, Nagoya University, Japan, 2Nanostructures Research Laboratory, Japan Fine Ceramics Center, Japan, 3The York Nanocentre, University of York, UK, 4Department of Physics, Electronics and Chemistry, University of York, UK
ky512@esi.nagoya-u.ac.jp

Environmental transmission electron microscopy (ETEM), which was first reported in 1997, has proven to be one of the most efficient tools for in situ visualisation of the deactivation of heterogeneous catalysts in a reactive gas atmosphere at the nanometer scale. Development of wet environmental TEM (wet-ETEM) was also an essential for in situ studies of liquid-catalyst reactions [1].
Here we report a progressive gas injection system (Figure 1) for the latest spherical aberration corrected environmental transmission electron microscope [2-4], which enables real-time/atomic-sacale observation in moisturised gas atmospheres. The newly developed wet-ETEM system [5] is applied to platinum carbon electrode (Pt/Carbon) catalysts in proton exchange membrane fuel cells (PEMFC) to investigate the effect of water molecules on the Platinum/Carbon interface during deactivation processes such as sintering and corrosion.
Pt/Carbon is a typical electrode catalysts in PEMFC. But it is now well established that degradation of the carbon support at the cathode limits the lifetime of Pt/Carbon catalysts and thus the performance of the PEMFC. We evaluated the robustness of the Pt/Carbon electrode catalysts using the new Wet-ETEM system (Figure 1(b)-(d)). Humidity in the E-cell was accurately measured/controlled using the quadrupole mass spectrometer (Figure 1(e)). Sintering and migration of Pt nanoparticles observed in moisturized N2 atmosphere was extremely faster than ones in pure N2 atmosphere as shown in Selected Area Captured (SAC) images of Figure 2. Fig. 2(b) shows connected Pt nanoparticles, which were typically observed in wet condition. The damage and shrinking of carbon is not reason of such connection because granular pattern of carbon support is still surviving. White contrast surrounding the Pt connections (arrowed in Fig. 2(b)) also indicates that thickness of carbon films became thinner at Pt/Carbon interface because of the hydrocarbon desorption. We considered that physical adsorption of water and hydroxylation of the carbon surface is a main reason of the higher mobility of Pt nanoparticles observed in moisturized N2 atmosphere. The present in situ observation suggested we should induce much stronger trapping sites on the carbon supports for use on cathode in the PEMFC. In situ microscopy to show the dynamic behaviour of the fuel cell catalyst is thus very valuable to improve understanding of the degradation mechanisms and thus improve robustness.

[1] Gai P. L. et al., Microsc. Microanal. 8 (2002) 21.
[2] Yoshida K. et al., J Electron Microsc. 61 (2012) 99.
[3] Yoshida K. et al., Nanotech. 24 (2013) 065705.
[4] Yoshida K. et al., Microsc. 62 (2013) 571.
[5] Yoshida K. et al, Invited Paper, MMC 2014 Conf. Proceedings, and Nanotechnology (sub).

The authors thank the EPSRC (UK) for Critical mass grant EP/J018058/1 and The JSPS for a Grant-in-Aid for Young Scientific Researchers (B) (No. 24710110).

Fig. 1: Fig 1. Design schema of the new wet-ETEM system (a), optical micrographs of (b) Humidifier on the hot stirrer, (c) Thermostatic chamber, (d) gas injection and differential pumping line of the ETEM. (e) QMAS spectra corresponding 24% moisturized nitrogen.

Fig. 2: Fig 2. (a) SAC image at 230s of a movie obtained from the Pt/Carbon samples in pure Nitrogen environment. (b) SAC image at 40s of a movie obtained from the Pt/Carbon in 24% moisturized Nitrogen environment. (c), (d) and (f) SAC images from other rigion in 24% moisturized Nitrogen environment at 0, 5.5 and 14 s, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-2774 Experimental evaluation of Environmental Scanning Electron Microscopes at high chamber pressure [200 - 4000 Pascal]

Rattenberger J.1, Fitzek H.1,2, Schroettner H.1,2, Wagner J.1, Hofer F.1,2
1Graz Centre for Electron Microscopy (ZFE), Steyrergasse 17, 8010 Graz, Austria, 2Institute for Electron Microscopy and Nanoanalysis (FELMI), Steyrergasse 17, 8010 Graz, Austria, Graz University of Technology (TU Graz)
johannes.rattenberger@felmi-zfe.at

Environmental scanning electron microscopy (ESEM) is an established method to investigate uncoated insulators, organic or biological samples in their original state. The presence of the imaging gas inside the specimen chamber is responsible for the secondary electron (SE) detection caused by gas amplification and the generated positive gas ions suppress charging artefacts. Water vapour as imaging gas at high chamber pressure (800 Pascal at 4°C or 2809 Pascal at 23°C) enables the opportunity to investigate wet samples or by varying the pressure or temperature to do wetting experiments [1].
Nevertheless, at high chamber pressures (200 - 4000 Pa) the gas amplification of SEs decreases and the scattering of primary beam electrons inside the imaging gas increases, which degrades the signal to noise ratio (SNR) and prevents image acquisition. Especially for low acceleration voltages, which are typically used for biological samples, the increase in scattering strongly limits the area of applications.
To evaluate the high pressure performance of ESEM and to compare different electron microscopes, information about special resolution and detector type is not enough. The contrast in SE images vanishes at high pressure and the big advantages of elaborated and expensive field emission guns are wasted.
Therefore a key feature for ESEM manufactures and users should be the stagnation gas thickness (additional distance the electron beam travels inside the imaging gas above the pole piece) and the SNR in SE detection for high pressure application, a fact which is not taken into account at the moment [2].
By using a special designed faraday cup, the fraction of scattered and unscattered electrons can be determined and the stagnation gas thickness calculated (see figure 1) [3]. The SNR in SE images can be measured by analysing a single image displaying a copper wire on carbon tape.
Results are presented for different types of SE detectors and beam transfer conditions (see figure 2 and 3). All experiments were performed using a FEI ESEM Quanta 200 or 600 (field emission gun).
1. G.D. Danilatos, 1988. Foundations of Environmental Scanning Electron Microscopy. Adv. Electron Electron Phys. 71, 109–250.
2. G. D. Danilatos, J. Rattenberger, V. Dracopoulos, Journal of Microscopy, (2010), DOI: 10.1111/j.1365-2818.2010.03455.x
3. J. Rattenberger, J. Wagner, H. Schröttner, S. Mitsche, A. Zankel, Scanning 31, (2009), p. 107

The author wants to thank Gerry Danilatos (ESEM Laboratory, Sydney) for helpful discussions and the Austrian Research Promotion Agency (FFG) for financial support (PN 839958).

Fig. 1: Stagnation gas thickness (Θ) [mm] as a function of chamber pressure P [Pa] using the gaseous secondary electron detector (GSED) as pressure limiting aperture.

Fig. 2: Test images: copper wire on carbon tape (imaging gas: water vapor)

Fig. 3: Signal to noise ration SNR [dB] as a function of chamber pressure [Pa] using the gaseous secondary electron detector (GSED)
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-3009 Multi-slice simulations for in-situ HRTEM studies of nanostructured magnesium hydride at elevated hydrogen pressures of 1 bar

Surrey A.1,2, Schultz L.1,2, Rellinghaus B.1
1IFW Dresden, Dresden, Germany, 2TU Dresden, Institut für Festkörperphysik, Dresden, Germany
b.rellinghaus@ifw-dresden.de

Nanostructuring of many hydrides has been shown to reveal improved thermodynamic and kinetic properties, which are needed for both mobile or stationary applications of solid-state hydrogen storage materials. During structural characterization utilizing conventional (HR)TEM, however, hydrides such as MgH2 degrade fast upon the irradiation with the imaging electron beam due to radiolysis in vacuum and as a consequence, the hydride phase cannot be studied at highest resolution. This problem can be overcome using a novel nanoreactor recently developed by H. Zandbergen (TU Delft) that allows for in-situ TEM studies at elevated H2 pressures (up to 4.5 bar) and temperatures (up to 500°C) [1]. A point resolution of 0.18 nm has already been demonstrated experimentally for Cu nanocrystals [2].

We have studied the feasibility of HRTEM investigations of light weight metals such as Mg and its hydride phases with the nanoreactor by means of multi-slice HRTEM contrast simulations. Such a setup provides the general opportunity to fundamentally study the dehydrogenation and hydrogenation reactions at the nanoscale under realistic working conditions. We analyze the dependence of both the spatial resolution and the HRTEM image contrast on parameters such as the defocus, the metal/hydride thickness, the hydrogen pressure and the nanoreactor geometry in order to explore the possibilities and limitations of in-situ experiments with this reactor. Such simulations may be highly valuable to pre-evaluate future experimental studies.

Fig. 1 shows schematically the details of the nanoreactor as it was implemented in a super cell used for the multi-slice simulations. The hydrogen is encapsulated between two 20 nm thin α-Si3N4 windows with the metal/hydryde positioned on top of the the bottom window. First simulations were conducted for a metallic Mg film of varying thickness oriented with its [001] direction parallel to the electron beam. The slicing was chosen to account for the varying density of atomic scatterers along the beam direction. While the slice thickness was reduced to contain only a single layer of scatterers within the Mg layer, it was increased to 1 nm and 100 nm in Si3N4 and in the hydrogen containing volume, respectively. Fig. 2 shows as an example the simulated Weber contrast of a Mg column (averaged over 611 individual columns) with respect to the background due to the Si3N4 windows as a function of the Mg thickness and the defocus. (Simulation conditions: Linear imaging. gmax = 20/nm. Imaging parameters match a FEI Titan microscope at 300 kV for NCSI imaging. Absorption, the MTF of the CCD, and a noise level of 3% were included in the simulations.)

[1] T. Yokosawa, Ultramicroscopy 112 (2012) 47.

[2] J.F. Creemer et al., Ultramicroscopy 108 (2008) 993.

Fig. 1: Schematic illustration of the simulated super cell representing the nanoreactor used for in-situ HRTEM investigations of the (de)hydrogenation of Mg(H2).

Fig. 2: Mean Weber-type image contrast of a column of Mg atoms as obtained from averaging over 611 individual atomic rows along the [001] direction of a Mg film with varying thickness. Despite the two Si3N4 windows and some scattering from the hydrogen atoms the individual Mg columns can be clearly imaged for thicknesses below some 30 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-3318 Understanding catalytic properties of nanoalloys by using aberration corrected electron microscopy in gaseous environment

Ricolleau C.1, Nelayah J.1, Nguyen N.1, Prunier H.1, Wang G.1, Piccolo L.2, Alloyeau D.1
1Laboratoire Matériaux et Phénomènes Quantiques, CNRS-UMR 7162, Université Paris Diderot-Paris 7, Case 7021, 75205 Paris Cedex 13, France, 2Institut de Recherche sur la Catalyse et l’Environnement de Lyon, Université Lyon 1 – CNRS, Lyon, France
christian.ricolleau@univ-paris-diderot.fr

Catalysis is involved in most of industrial chemical processes for refining, pollution control and synthesis of chemicals. Heterogeneous catalysis has always used nanoparticles in order to maximize the surface/volume ratio of active particles. Therefore, “particle size effect” is a well-known concept of catalysis. Moreover, combining metals within catalysts can improve catalytic performances with respect to pure metals, e.g., increased selectivity or resistance to poisoning. The “alloying effect” is classically ascribed to either electronic structure or active site geometry. However, this phenomenon is poorly understood and controlled, due to the difficulty to elaborate homogeneous collections of multimetallic nanoparticles with imposed composition, and to the lack of structural characterization.
Our general objective is to get insights into the interplay between the structure of nanoalloys (Pd-Au, Au-Cu and Pd-Ir on oxide supports) with well-controlled size and composition and their catalytic properties. For that purpose, we have synthesized and characterized supported bimetallic nanoparticles, and analyzed their catalytic behavior by using a MEMS-based technology developed by Protochips Inc.. This MEMS gas cell allows to image and to follow the dynamics of nano-objects in an encapsulated gas environment as a function of the temperature. By combining this technology with our JEOL ARM 200F cold FEG aberration correction microscope, we can obtain images of nano-materials with an information limit better than 0.8 nm under 1 bar gas pressure and at 1000°C (Fig. 1).
From the study of the above systems, we want to address, by using this instrumentation, the following fundamental questions:
- How does the structure of supported nanoalloys depend on particle size and bulk phase diagrams (total miscibility for Pd-Au and Au-Cu vs. miscibility gap for Pd-Ir)?
- How does the chemical structure (ordering, random alloying, partial segregation, core-shell, etc.) of the nanoparticles influence their catalytic properties towards the series of selected prototypic catalytic reactions (oxidation, hydrogenation…)?
- How does the nature of the support drive the structure of the nanoalloys? What is the particle-support interface structure?
- How do temperature and gaseous environment affect the structure of the nanoalloys? Can we gain insights into the atomic mechanisms of sintering, redispersion and strong metal support interaction (SMSI effect)?
The concepts are not new but the methodology is novel and promising thanks to the recent development of gas cells technology that allows reproducing the real operando conditions.

Fig. 1: (a) TiO2 substrate classically used in catalytic reactions with nanoalloys imaged under 1 bar pressure of O2 and at 1000°C (with a JEOL ARM 200F cold FEG microscope equipped with an aberration corrector of the objective lens). (b) Enlargement of the rectangular area in dotted line of (a).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-3521 Beam skirt resolution in Gaseous Scanning Electron Microscopy

Khouchaf L.1
1Univ-Lille Nord de France, Ecole des Mines de Douai, Douai France
lahcen.khouchaf@mines-douai.fr

The electron beam scattering by gaseous environment is the fundamental parameter limiting the performance of the Gaseous Scanning Electron Microscopy (GSEM). The result is the enlargement of the primary beam characterized by the radius skirt Rs. The scattering phenomena require a much closer re-examination. In fact, depending on the localization of EDX detector and the particles shape to analyze, the collected signal after the beam skirt will be different and Rs also will be different. So, except for homogeneous materials, Rs cannot describe the scattering behavior.
In fact, Danilatos, introduced the radius Rs which represents the radius containing 90% of the incident beam) as below:

RS = (364*Z/E)*(P/T)1/2.GPL3/2

where rs is the skirt radius, Z the gas atomic number, E the incident beam energy, P the pressure, T the temperature and GPL the gas path length.

As given by equation above, the value of Rs depends on the gas introduced, the incident energy, the pressure, the temperature and the working distance but does not depend on the total or individual cross section. In Figure 1 we can notice a nonlinear beahvior of Rs versus the pressure. In order to take into account this approach we introduced a surface of the skirt Ss instead of the Rs. In the case Ss is given by the equation below:

Ss = ∏*Rs2

Ss = α*P

Unlike Rs, expression given above, the equation above shows that Ss is a linear function versus the pressure. Figure 2 shows the evolution of Ss versus the pressure for water vapor and helium.
In this study, the surface skirt Ss instead of the radius skirt is introduced. Unlike Rs, the results show that Ss is a linear function versus pressure. This may help to use Ss in different scattering regimes and for a best interpretation of the consequences of electron scattering beam by gaseous environment. Examples are given with two gases environment: helium and water vapor.
References
G.D Danilatos, Scanning Microscopy 4 (1990) P. 799.
D Stokes in “Royal Microscopical Society”, ed. Mark Rainforth, (Wiley,Chichester) P. 221.
J.F Mansfield, Microchimica Acta 132, (2000), P. 137.
L Khouchaf et al, Vacuum 81, (2007), P. 599.
L Khouchaf et al, J.De Phys. IV France 118, (2004), P. 237.
L Khouchaf et al, Vacuum 86, (2011), P. 62.

L. Khouchaf. (2012). V. Kazmiruk (Ed.),  978-953-51-0092-8, InTech, Croatia (2012)

L Khouchaf et al, Microscopy Research, 2013, 1, 29-32.

Fig. 1: Variation of Rs versus pressure at 20 keV and GPL= 2mm for (a) H2O vapor, (b) He.

Fig. 2: Variation of Ss versus pressure at 20 keV and WD= 2mm for (a) H2O vapor, (b) He.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-5740 Cultivation and Observation of HeLa Cells in the Microfluidic Environmental Electron Microscopy

Huang Y. C.1, Ma T. W.1, Huang T. W.1, Liu S. Y.1, Chen F. R.1, Tseng F. G.1, Chuang Y. J.2
1Department of Engineering and System Science, National Tsing Hua University, Taiwan, 2Department of BioMedical Engineering, Ming Chuan University, Taiwan
v800213@gmail.com

  Progress in the processing of wet tissues, without the need of fixation and other complex preparation, may facilitate the microscopic examination of tissues and cells. To solve the challenge that the moisture in the wet sample will be dried out by electron microscope’s vacuum system when observing the living cell, we attempt to develop an advanced MEMS wet-cell device with fluid-exchange to achieve the macromolecular dynamics observation accompanied with in-situ manipulating/monitoring under an electron microscope (EM), and then we report the observation of cells dynamics in solutions.

  In this study, we design a special wet chamber (liquid SEM capsule) for environmental SEM by MEMS technology[1], consisted of one in-frame and one out-frame fitting to each other with controllable gap between for cell incubation and EM observation. In the current SEM application, the environmental wet chamber, composed of a disposable out-frame and a capsule, is inverted in SEM after sealing for the sample to facing up toward the incident electrons for getting stronger signals. Then, we connect the PEEK tubes to the capsule and use a syringe pump to provide liquid circulation (Figure1).

  For living cell incubation inside wet cell, we immersed out-frame into culture dish to contain culture medium (DMEM with 10%FBS and 1%Penicillin/Streptomycin) , and then incubated HeLa cells for 8-12hr. To improve the image quality for the thick cell, we process cell permeabiliztion treatment (100％Methanol), immersing cell in the Milli-Q water in place of the cytoplasm. Comparing the image of different immersing time, we found that the two-hour immersion had a clearer view of cytoskeleton and nucleus (Figure2).Then we observed the living cell with our self-design component by fluid circulation way and recorded the cell division with slow flow rate(0.01ml/hr) under eight long hours OM observation (Figure3), which confirm the practicality of our design. Since the existence of liquid seriously influence the contrast, we replace the culture medium with glycerol, finding that the resolution is improved under long time SEM observation (Figure4). With the unique liquid circulating system incorporated with SEM, we can successfully incubate HeLa cells for a long period of time in the wet micro environment. The image resolution under a wet condition is characterized as 40-50 nm, suitable for observing interaction between virus and cells or subcell organelles.

1. ”Self-aligned wet-cell for hydrated microbiology observation in TEM.” T.W. Huang, S.Y. Liu, Y.J. Chuang, et al., Lab on a Chip.12:340-347(2012).
2. ”A Novel Method for Wet SEM,” Iris Barshack, Juri Kopolovic, Yehuda Chowers, Opher Gileadi, Anya Vainshtein, Ory Zik and Vered Behar, Ultrastructural Pathology, 28:29-31(2004).

This work was supported by National Science Council (NSC102-2321-B-007-007 and NSC 102-2120-M-007-006-CC1).

Fig. 1: Figure1. Liquid-SEM device for dynamical study on HeLa cells

Fig. 2: Figure2. Cell permeabilization and the Milli-Q water immersion for (a) 2 hr, (b) 4 hr. The image resolution under 2 hours immersion, which is 65 nm, is better than that under 4 hours immersion, which is 107nm.

Fig. 3: Figure3. Hela cell division growth under liquid circulation environment.

Fig. 4: Figure4. Dehydrated Hela cell with fluid circulation under environmental SEM. (a) Culture medium, (b) Glycerol.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-6-P-5803 ETEM observation of degradation of platinum and platinum-cobalt alloy nanoparticle electrocatalysts on carbon black

Nagashima S.1, 6, Kang Y.2, 3, Yoshida K.2, 4, Hiroyama T.2, 4, Liu K.2, 4, Ikai T.5, Kato H.5, Nagami T.5, Kishita K.6, Yoshida S.1
1Materials Research and Development Lab., Japan Fine Ceramics Center, Atsuta-ku, Nagoya, 456-8587, Japan, 2Nanostructures Research Lab., Japan Fine Ceramics Center, Atsuta-ku, Nagoya, 456-8587, Japan, 3Dept. of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya, 466-8555, Japan, 4Institute for Advanced Research, Nagoya University, Chikusa-ku, Nagoya, 464-8603, Japan, 5Catalyst Design Dept., Material Design Div., Toyota Motor Corporation, Toyota-cho, Toyota, Aichi, 471-8572, Japan, 6Material Analysis Dept., Material Development Div., Toyota Motor Corporation, Toyota-cho, Toyota, Aichi, 471-8572, Japan
s_nagashima@jfcc.or.jp

   The Proton Exchange Fuel Cell (PEFC) is expected as a promising energy source to use of Fuel Cell Vehicle. Platinum nanoparticles on carbon black (Pt/C) are a typical electrode catalyst used in the PEFC. For development of advanced electrode catalysts in the PEFC, reducing Pt usage and enhancement of the durability are still problems. In order to reduce the Pt usage, Pt-metal alloys were investigated in recent few years and previous researches have reported that Pt-Co alloy represented the high Oxygen Reduction Reaction (ORR) activity and improved stability on cathode condition of PEFC. For further design concept of the Pt-Co electrode catalysts, it can be essential to understand the degradation mechanism in real space about both Pt and Pt-Co alloy. Because actual Pt-Co catalysts consist of pure Pt nanoparticle, Co nanoparticle, the ordered Pt-Co alloy (L12 etc.) and disordered Pt-Co alloys. In this report, we evaluated structural changes of the Pt/C and the PtCo/C electrode catalysts during electrochemical degradation by using Environmental Transmission Electron Microscopy (ETEM). In addition to such ex-situ approach, we achieved the dynamic in-situ observation in controlled water (H2O) atmosphere which is known as product molecule of ORR in PEFC.

   For electrochemical degradation tests simulating the start and stop test of PEFC, potentio/galvanostat was used with a potential range from +1.00 V to +1.50 V, a scan rate of 500 mVs-1 in 0.1 M HClO4 at room temperature for 40,000 cycles. In our degradation tests, Electrochemical Surface Area (ECSA) of Pt/C decreased 57% and PtCo/C decreased 27% through 40,000 cycles (Fig. 1(e, f)). The size of nanoparticle and the surface area of nanoparticles were clearly increased in ex-situ TEM images obtained from both Pt/C and PtCo/C (Fig. 1(a-d), Fig. 2(a)). It indicated that Pt and PtCo nanoparticles grow and carbon black particles shrink during the electrochemical degradation test.

   Then, we achieved a dynamic observation to investigate the cause of the coalescence of Pt nanoparticles. In 10 Pa of water vapor (H2O), Pt nanoparticles rapidly diffused on the carbon surface and formed an interconnected structure (Fig. 2(b-e)). We considered that physical adsorption of H2O molecule and hydroxylation of the dangling bond on carbon surface were the main causes of the rapid mobility of Pt nanoparticles. Therefore, we consider that much stronger trapping sites on the carbon is needed to reduce the mobility of Pt and Pt-Co nanoparticles.

Fig. 1: (a-d), Ex-situ TEM images of Pt/C and PtCo/C before and after degradation. Size distribution of nanoparticles as an inset. (e, f), CV curves of Pt/C and PtCo/C obtained before and after degradation test over a potential range from +0.05 V to +1.20 V at a scan rate of 500 mVs-1 in 0.1 M HClO4 at room temperature.

Fig. 2: (a), Specific surface area of Pt and PtCo nanoparticles before and after degradation. It was calculated by presuming that volume of a carbon black particle is volume of a sphere with a diameter of 35 nm (the circles in Fig. 1(a-d)). (b-e), Selected area captured images of a movie obtained from Pt/C in 10 Pa of water atmosphere.

Fig. 3:
↑ Go to the Table of content ↑

IT-7. In-situ microscopic techniques and cryo-microscopy

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-7-IN-2863 The opportunities and challenges of liquid cell electron microscopy

Ross F. M.1
1IBM T. J. Watson Research Center, Yorktown Heights, NY 10598, USA
fmross@us.ibm.com

Liquid samples, particularly samples containing water, have traditionally been difficult to examine using transmission electron microscopy because of the incompatibility between the microscope vacuum and the high vapour pressure liquid. But in recent years, advances in sample design have allowed us to enclose liquids in a form that permits examination by TEM. Microfabricated devices are constructed in which two electron transparent membranes are spaced 100nm-1um apart. A liquid is introduced between the membranes, allowing imaging of structures and processes in situ. The technique of liquid cell electron microscopy has been adopted by many laboratories worldwide, and is of interest to the microscopy, materials and biology communities because it enables data to be obtained at a spatial and temporal resolution not accessible with other techniques.

In this presentation we focus on the use of liquid cell electron microscopy to examine the mechanisms of electrochemical processes in aqueous electrolytes. Liquid cell microscopy is well suited for electrochemistry because electron-transparent electrodes can be included during device fabrication. Images and movies of the transient structures that form during nucleation or dissolution can then be correlated with electrochemical parameters (voltage, current) controlled or measured by a potentiostat. We show measurements made during deposition and stripping of metals (Cu, Zn) on Au or Pt electrodes. After nucleation and coalescence, we measure the propagation of the growth front outwards from the electrode and into the liquid layer. We will show that an initially planar growth front roughens and becomes unstable, forming dendrites or ramified patterns. Such growth instabilities can affect the charging of batteries and the electrodeposition of thin films and multilayers. We will show how the development of diffusion fields works together with kinetic roughening to cause the onset of growth instabilities. Techniques have been developed to control the onset of instability, including pulse plating, electrolyte flow and the use of additives to alter interface parameters. We will examine these approaches using liquid cell microscopy.

In any liquid cell experiment, obtaining quantitative data that is suitable for matching with models involves understanding the pitfalls and artifacts that can occur during liquid cell EM. We therefore discuss electron beam effects, in particular the strong changes in solution chemistry that can be induced by the beam. Beam-induced radiolysis of water can lead to phenomena such as particle and bubble formation. These can be minimised with low-dose techniques, but may also be useful in forming patterned structures and in measuring the properties of nanoscale bubbles.

The results presented here have been funded, in part, by the US National Science Foundation under grants 1129722, 1225104 and 1066573.

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-7-IN-6082 Connectivity between imaging tools under controlled conditions: learning’s from 20 years experience with a variable cryo transfer system for the future

Wepf R.1
1ScopeM/EMEZ, ETH Zürich, Zürich, Switzerland
roger.wepf@emez.ethz.ch

Sample preparation has become more crucial with modern microscopy and compositional analysis. The most obvious requirement, is that the specimen is reduced in size and exposed without relocating, changing or exchanging atoms or part of the sample, so to say with minimal or no alteration. Once prepared the transfer to the microscope is the last potential destructive step prior to the final analysis. To reduce such influences we therefore first established a controlled connection between a high vacuum cryo preparation device and a cryo-SEM (FEG-XL30 & cc-corrected LVSEM) in 1994 to avoid contamination of freshly prepared samples at cryogenic conditions and enhanced sample preservation and throughput for high resolution SEM work.

Soon later this system was brought to market by Bal-Tec under the name VCT, including a high resolution cryo-stage, and adapted to a large number of SEM’s, FIB/SEM, cryo-AFM, cryo-IonTof and others. Later it was also extended to ESEM’s for “inert-gas” or controlled environment sample handling.

Proofing that connectivity under “inert gas” or high vacuum between sample preparation devices and analysis devices combines higher sample quality with higher sample throughput without the risk of loosing samples due to contamination, change of structure by oxidation, amorphisation, cracking or simple loss of sample by remounting.

This kind of connectivity between single devices is well established in semi conductor industry in so-called fabrication plant or “FAB’s”-lines, where the sample (wafer) is handled between production and analytical stations such as LM, Spectrometer, EM, Auger- and SIMS instruments without any remounting and interference of an operator and mostly under high vacuum conditions for on-line quality and process control.

In structure research we often face the problem on non-periodic (non crystalline) samples that several independently and comparative studies do not merge into a common picture. Understanding materials heterogeneity at various order of scale very much depends on imaging and analysis the same area/region of interest (AOI/ROI) without the risk of changing the sample properties and configuration between the investigations. This not only helps to reduce multiple experiments but also allows to zoom-in on pin-point selected ROI by a correlative combination of analytical imaging investigations (EELS, X-ray, SIMS, APT).

If we want to maintain highest possible quality of our carefully prepared samples for multimodal analysis we need to establish versatile transfer devices between the different analytical tools. In addition we need to standardize sample handling and interfaces to be able to investigate “close to native” samples at different resolution and sensitivity scales ideally on the same ROI. This will not only help to save time and number of samples but improving the output of multimodal analysis.

Fig. 1: For connectivity between various analytical modalities a kind of (cryo) innert gas exchange workstation for sample exchange and remounting under controlled environmental conditions is needed. This exchange station should avoid influcences affecting the sample native or virgin composition and ultrastructure (Δ critical interface steps for transfer)
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-1567 In situ Scanning Transmission Electron Microscopy study of CuO reduction

Martin T. E.1, Lari L.1, Gai P. L.2, Boyes E. D.3
1The York Nanocentre and Department of Physics, University of York, UK, 2The York Nanocentre and Departments of Chemistry and Physics, University of York, UK, 3The York Nanocentre and Departments of Physics and Electronics, University of York, UK
tm526@york.ac.uk

Methanol is one of the most important basic components in the chemical industry (worldwide production approx. 45 million tons 2010). Furthermore, it has potential as an in situ source of hydrogen for fuel cells [1, 2]. Cu is one component used to catalyse methanol synthesis and consequently, understanding of the activation and deactivation mechanisms of Cu is necessary to improve both catalyst activity and lifetime. Due to the scale of methanol production, small improvements in catalytic technology can lead to large economic impacts and make green technologies, such as fuel cells, more financially viable whilst also providing improved function. The activation process (in this case reduction) required to transform the precursor, CuO, into catalytically active Cu is very significant in determining the final size, structure and distribution of catalytic nanoparticles [1]. Subsequent to reduction, deactivation mechanisms (such as sintering) cause the catalyst activity to reduce with time. Single atom imaging under reaction conditions in ESTEM (Environmental Scanning Transmission Electron Microscope) can provide insights into activation and deactivation mechanisms, as well as the basis for improved catalyst designs.

The ESTEM at the York JEOL Nanocentre has recently been modified to provide the unique capability to directly visualise single atoms and the atomic structure of heterogeneous catalysts, such as Cu, in a gas environment [3]. This has allowed the in situ reduction of CuO in H2 (Figure 1) and investigation of the temperature-pressure parameter space to ascertain effects on particle morphology and size. As temperature is increased the Particle Size Distribution becomes bimodal with the particles divided into two distinct categories (facetted and unfacetted, Figure 2). Using ESTEM combined with EDXS, CuO particles and the more facetted Cu particles are seen to coexist. This suggests that reduction is dependent on the characteristics of the particle in question and thus that an atomic scale observation is required to fully understand the reduction process. Subsequent deactivation of the Cu particles is driven by reduction of the surface free energy and is shown to be primarily via the Ostwald Ripening (OR) mechanism (Figure 2). Understanding of the OR mechanism at the atomic level is currently lacking and the single atom resolution of the ESTEM, combined with Kinetic Monte Carlo simulations, provide a unique perspective on the factors affecting sintering such as particle size, temperature, activation energy and particle distribution.

The authors thank the EPSRC for support from critical mass grant
EP/J018058/1

References:

1. Hansen, P.L., et al., Science, 2002. 295(5562): p. 2053-2055.

2. Avgouropoulos, G., et al., Applied Catalysis B: Environmental, 2009. 90(3): p. 628-632.

3. Boyes, E.D., et al., Annalen der Physik, 2013. 525(6): p. 423-429.

Fig. 1: Reduction of CuO particles in situ using ESTEM at 3Pa Hydrogen, 361˚C to Cu. Diffraction patterns observed before and after reduction in UHV TEM.

Fig. 2: (a) Bimodal distribution suggests 2 groups of particles. These can be seen as grey (A) and white (B-more facetted) particles, (b) before reduction (c) after heating at 312˚C at 2Pa, (d) after heating at 361˚C at 3Pa. Particles become more facetted with reduced surface area as sintering process occurs.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-1894 In-situ (S)TEM redesigned: Concept and electron-holographic performance

Börrnert F.1,3, Riedel T.2, Müller H.2, Linck M.2, Büchner B.1,3, Lichte H.1
1Technische Universität Dresden, Germany, 2CEOS GmbH, Heidelberg, Germany, 3IFW Dresden, Germany
felix.boerrnert@tu-dresden.de

The progress in (scanning) transmission electron microscopy and electron holography has led to an unprecedented knowledge of the microscopic structure of functional materials at the atomic level. Nevertheless, in-situ studies inside a (scanning) transmission electron microscope ((S)TEM) are extremely challenging. Here, we introduce a concept for a dedicated in-situ (S)TEM with a large sample chamber for flexible multi-stimuli experimental setups.

In conventional (S)TEMs the sample space is restricted by the pole pieces of the objective lens to a few millimeters; additionally, the sample is immersed into a strong magnetic field forbidding the investigation of magnetic phenomena. The solution to this problem is a radical redesign of the sample chamber and thus an adaptation of the electron optical layout. A versatile in-situ sample chamber requires space and access ports to incorporate different devices for applying various stimuli. This can be achieved by the use of a spherical-aberration corrected Lorentz type objective lens [1]. The size of the sample chamber is not anymore restricted by the electron optics and can be easily adapted to emerging experimental demands. Also, for the large-area control of experimental setups in situ a scanning surface imaging mode, i. e. a secondary electron detector, is needed.

A fundamental drawback of TEM is that the imaging process acts like an edge filter, thus no large-area field variations could be detected, and the image contrast is largely non-quantitative. In electron microscopy, the fully quantifiable image wave can be recorded only by an interferometric technique, i. e. off-axis electron holography [2]. Crucial for in-situ experiments is a large field of view while maintaining a high spatial resolution [3].

Here, we report on the state of the conversion of a JEOL JEM-2010F retro-fitted with two Cs correctors [4] from a dedicated low-voltage high-resolution (S)TEM into a large-chamber in-situ microscope. Both correctors are aligned to act as a corrected Lorentz lens in conventional as well as in scanning mode. The complete column section originally housing the pole pieces of the conventional objective lens will be replaced by a sample chamber providing multiple large ports for accessing the sample. Special care has been taken to make the chamber design most flexible.

[1] B Freitag et al., Microscopy and Microanalysis (2009), 184.
[2] H Lichte et al., Ultramicroscopy 134 (2013), 126.
[3] M Linck et al., Microscopy and Microanalysis (2010), 94.
[4] F Börrnert et al., Journal of Microscopy 249 (2013), 87.

The authors acknowledge financial support from the European Union under the Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative (Reference 312483—ESTEEM2). We thank Prof. A. Kirkland (University of Oxford) for providing the SE detector.

Fig. 1: Scheme illustrating the conversion of the (S)TEM sample region. Green – electron beam, red – lens magnetic field, blue − sample.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-1998 In situ STEM studies of reversible electromigration in thin palladium–platinum nanobridges

Kozlova T.1, Rudneva M.1, Zandbergen H.1
1Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands
t.kozlova@tudelft.nl

Electromigration is a process in which a metallic contact line is thinned by passing a current through it, thus gradually displacing atoms and ultimately leading to its destruction [1]. The electromigration process in Pd–Pt nanobridges was investigated by in situ scanning transmission electron microscopy (STEM), using a FEI Titan operating at 300 keV. This technique together with a special electrical sample holder, built in-house, allows the nanobridge morphology transformations to be imaged down to the atomic scale during passage of electrical current [2]. Correspondent I–V curves are also recorded in real time. We focus in particular on the direction of material migration in relation to the electric current direction.
Polycrystalline Pd–Pt nanobridges with different lengths (500–1000 nm) and widths (200–500 nm), and a thickness of 15 nm were produced by e-beam evaporation from a metal alloy source onto a 100-nm-thick freestanding silicon nitride membrane [3] (Fig. 1a). The experiments were conducted in bias-ramping mode, i.e. a uniform increase in voltage from 0 V to a maximum of 350–600 mV (this was chosen in each separate experiment), followed by a decrease back to 0 V, sometimes a subsequent increase into the negative range (−350 to −600 mV) was done, followed by a decrease back to the original starting point of 0 V (Fig.1b).
Electromigration in Pd–Pt alloy [4] is quite different from the pure elements Pt and Pd. Material transport in Pt and Pd is very similar: after a recrystallization (which resembles that of the Pd–Pt alloy) the bridge gradually becomes narrower until a nanogap is formed, whereby grain boundary grooving is not a dominant feature. For the Pd–Pt alloy the dominant change is grain boundary grooving (which occurs near the cathode side), where the outer shape of the nanobridge is maintained. For high current densities (3 – 5×107 A/cm2), material transport in Pd–Pt alloy occurs from the cathode towards the anode side, indicating a negative effective charge. While polarity is changed, the voids formed near cathode side are refilled (Fig. 2). The reversal of material transport upon a change of the electric field direction could be the basis of a memristor.

[1] Ho, P. S.; Kwok, T. Rep Prog Phys 1989, 52, (3), 301-348.

[2] Rudneva, M.; Kozlova, T.; Zandbergen, H. Ultramicroscopy 2013, 134, 155-159.

[3] Gao, B.; Osorio, E. A.; Gaven, K. B.; van der Zant, H. S. J. Nanotechnology 2009, 20, (41), 415207.

[4] Kozlova, T.; Rudneva, M.; Zandbergen, H. Nanotechnology 2013, 24, 505708.

The authors gratefully acknowledge NIMIC and ERC project 267922 for support.

Fig. 1: (a) Typical TEM image of the initial configuration of the bridge. (b) Typical I–V curve for one loop in bias ramping mode.

Fig. 2: Snapshots from the STEM footage. (a) Initial view of the bridge. During electromigration, voids form on the cathode side (shown with wide arrow) and material accumulates on the anode size (b, d–e, g–h). When the current is reversed, the voids are refilled (c, f). White arrows indicate the direction of electrons.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-2861 A cryo high-vacuum shuttle for correlative cryogenic investigations

Tacke S.1, Krzyzanek V.2, Reichelt R.1,3, Klingauf J.1
1Institute of Medical Physics and Biophysics, Muenster, Germany, 2Institute of Scientific Instruments of the ASCR, Brno, Czech Republic, 3Rudolf Reichelt initiated the project but unfortunately he passed away too early to see the results
s.tacke@uni-muenster.de

The preservation of the native state is the key element in sample preparation. In the case of hydrated objects, embedding in vitreous (amorphous) ice and subsequent examination under cryogenic (cryo) conditions are the means of choice [1,2]. Over the last years, cryogenic techniques such as cryo-electron microscopy (cryo-EM) or soft X-ray cryo-microscopy have become increasingly popular, as they provide a direct, unaltered view on the specimen [3,4].

However, to provide a snapshot of the pristine architecture of the specimen, cryo techniques require constant cooling below the recrystallization temperature of 138°K [1] and avoidance of any contamination. This has been proven to be particularly challenging in the case of correlative cryo investigations [4,5], since these methods include several transfer steps due to their extensive post-processing [6] and complex workflow [7]. In the past, several transfer concepts were introduced and they are now commercially available. However, these systems are limited either by not offering a high-vacuum environment or constraining the applications to a restricted workflow.

Here, we introduce an improved cryo high-vacuum transfer system (CHVTS) that allows for the first time to combine all kinds of cryogenic experiments. Moreover, we provide a solution that offers the highest degree of freedom in terms of connectivity of experiments (Fig.1). As shown in the detailed scheme of the CHVTS, our system is composed of cartridge, storage unit and cryo high-vacuum shuttle (Fig. 2). Once vitrified and mounted to cryo-holder cartridges (CT3500, Gatan) up to eight samples can be transferred to the storage unit. Thereafter, the cartridges can be transferred to the electron microscope or any other system extended by our docking device. A constant vacuum level of 7 ± 2 x 10-7 mbar and a temperature well below 133°K guarantee a contamination free transfer (see Fig. 3). Taken together, the CHVTS introduced in this work streamlines the handling of the frozen-hydrated specimen while solving for all problems generally associated with cryogenic investigations.

[1] J. Dubochet et al, Q. Rev. Biophys. 21 (1988), p. 129.

[2] L. Fitting Kourkoutis, J. M. Plitzko, and W. Baumeister, Annu. Rev. Mater. Res. 42 (2012), p. 33.

[3] S. G. Wolf, L. Houben and M. Elbaum, Nat. Methods online publication (2014), p. 1.

[4] C. Hagen et al, J. Struct. Biol. 177 (2012), p. 193.

[5] A. Rigort et al, J. Struct. Biol. 172 (2010), p. 169.

[6] S. Rubino et al, J. Struct. Biol. 180 (2012), p. 572

[7] E. Villa et al, Curr. Opin. Struc. Biol. 5 (2013), p. 771.

This research was supported by the DFG Grants RE 782/11-1,-2. Vladislav Krzyzanek acknowledges the support by the grant 14-20012S (GACR). We kindly acknowledge the help of the precision mechanical workshop, especially Martin Wensing. Additionally, we would like to thank Ulrike Keller for providing the EM grids, Harald Nüsse and Roger Wepf for numerous discussions.

Fig. 1: Theoretical concept for the connection of different types of cryogenic experiments. Green: established techniques. Red: possible extensions of the workflow.

Fig. 2: Main parts of the cryo high-vacuum transfer system: a) Cartridges assembly: (*) universal cartridge (**) EM grid (***) Clip for fixing the grid. Scale bar: 3mm. b) Storage device: (*) Cooling stage (**) docking device. Scale bar: 18cm. c) Cryo high-vacuum shuttle and pressure measurement during uncoupling of the shuttle. Scale bar: 21cm.

Fig. 3: Temperature measurement during the transfer of the cartridge.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-2696 The Effect of Electron Beam on Aqueous Solution Composition during Liquid Cell Microscopy

Schneider N. M.1, Norton M. M.1, Mendel B. J.1, Grogan J. M.1, Ross F. M.2, Bau H. H.1
1Department of Mechanical Engineering and Applied Mechanics, University of Pennsylvania, Philadelphia, PA 19104, USA, 2IBM T. J. Watson Research Center, Yorktown Heights, NY 10598, USA
schnic@seas.upenn.edu

Liquid cell electron microscopy has emerged as a powerful tool for the real time imaging of objects suspended in liquids, and for characterizing processes that take place in liquids with the nanometer resolution of the electron microscope. However, as with all microscopy experiments, the electron beam interacts with the sample. Energy transferred from the fast-moving electrons to the irradiated medium causes excitation and ionization, resulting in the generation of radical and molecular species, which for water include eh (hydrated electrons), OH, H+, H2, O2, and H2O2. The hydrated electrons, oxidizing agents, and gaseous species can cause, respectively, reduction and precipitation of cations from solution, dissolution of metals, and nucleation and growth of bubbles. A quantitative understanding of electron beam-induced effects is critical to assessing whether the electron beam significantly affects the imaged phenomenon, so that we can correctly interpret experiments carried out with liquid cells; design experiments so as to minimize and mitigate unwanted effects; and take advantage of beam effects. We have developed a mathematical model to estimate radiolysis products during electron microscope imaging. The model includes the production of species by the electron beam, their destruction by reverse reactions, and their continued diffusion and reaction outside the irradiated region. We compute the concentrations of radiolysis products as functions of beam intensity, beam geometry, time, position, and solution initial composition (Fig. 1). We will describe this model and use its predictions to delineate various phenomena observed during liquid cell electron microscopy. For example, we predict that radiation chemistry causes large changes in pH within the irradiated region (Fig. 1a), and localized concentrations of reducing agents (Fig. 1b, c) and oxidizing agents. The pH of neat water can drop from 7 to 3.5 or lower within the imaged region under normal imaging conditions. Changes in pH can have significant effects on the phenomena under observation and may be the cause of aggregation of colloids (Fig. 2) that was observed during liquid cell imaging. We will compare the model with experiments carried out in a liquid cell, the nanoaquarium, at 300 kV in a Hitachi H9000 TEM and at 30 kV in an FEI Quanta FEG ESEM with a transmission detector, in each case imaging at 30 fps. The experiments and simulations suggest that liquid cell microscopy can provide a unique tool for studying radiolysis and for examining the behavior of materials subjected to high doses of radiation. We hope that the modeling tools described here will be useful for interpreting microscopy data obtained with liquid cells and for designing experiments that minimize unwanted effects.

The authors acknowledge funding, in part, from the National Science Foundation, grants 1129722 and 1066573.

Fig. 1: Heterogeneous model predictions of the concentrations of a) H+, b) e-, c) H, d) OH as functions of space and time. The beam (gray region) and liquid cell radii are, respectively, 1 µm and 50 µm. The beam current is 1 nA and the dose rate is 7.5x107 Gy/s. These values are typical for TEM imaging.

Fig. 2: Beam induced aggregation of 5 nm gold nanospheres in water. Dynamic imaging of large cluster-to-cluster aggregation (a-b) and early stage aggregation of small clusters (c-d).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-2818 Cold-field emission and charge measurements of a carbon cone nanotip studied by in situ electron holography

de Knoop L.1, Gatel C.1, Houdellier F.1, Masseboeuf A.1, Monthioux M.1, Snoeck E.1, Hÿtch M. J.1
1CEMES-CNRS, Toulouse, France
ludvig.deknoop@cemes.fr

The cold-field emission gun (C-FEG) is the brightest electron source available, and also exhibits the smallest energy spread [1]. This technology has been greatly improved over the years concerning the electron optics and the vacuum, but the same cathode materials are still in use [2]. We have recently developed a new C-FEG source using a carbon cone nanotip (CCnT) mounted on a standard tungsten cathode using a focused ion beam (FIB) [3]. This source exhibits very good spatial coherence properties, which could be useful for electron interferometry applications [4].

Here, we have inserted a CCnT inside an in situ biasing transmission electron microscope (TEM) sample holder (Nanofactory Instruments) incorporating a nanomanipulator, in order to approach the CCnT towards a Au-anode plate (Fig. 1). We then ramped up the voltage between the nanotip and the anode from 0 to 95 V until the electric field around the tip was strong enough to allow the electrons to tunnel through the barrier and a field emission current could be recorded.

We have previously reported of how quantitative information of the local electric field of the CCnT (Eloc = 2.55 V/nm at the onset of field emission at 80 V) could be obtained by using off-axis electron holography and finite element method (FEM) modeling (Fig. 3 b)). By combining this with the Fowler-Nordheim equation [5], also the work function of the CCnT (Φ = 4.8 ± 0.3 eV) could be found [6].

Knowing the local electric field and the work function, the study has been expanded further to focus on the accumulation of charges on the CCnT before, at and after the onset of field emission. This was done with a technique that we recently have developed [7], which quantitatively measures the number of charges by applying the elegance and power of Gauss’s Law to electron holograms (Fig. 2). It provides a direct measurement of the charge inside a contour integral, with a sensitivity of one unit of charge.

The number of accumulated charges and the charge density on different places on the tip has been determined. We will show quantitative charge measurements along the CCnT as a function of applied voltage (Fig. 3 a)). Particularly the charge density at the beginning and during the field emission process provides some remarkable results. We will then discuss the importance of these values.

[1] O. L. Krivanek et al., Advances in Imaging and Electron Physics 153 (2008)
[2] A. V. Crewe et al., Rev. Sci. Instrum. 39 (1968)
[3] F. Houdellier et al., Carbon 50 (2012)
[4] F. Houdellier and M. Monthioux, International Patent Number WO2012035277 (2012)
[5] R. H. Fowler and L. Nordheim, Proceedings of the Royal Society of London 119 (1928)
[6] L. de Knoop et al., Micron, accepted (2014)
[7] C. Gatel et al., Phys. Rev. Lett. 111 (2013)

The authors acknowledge the European Integrated Infrastructure Initiative reference 312483-ESTEEM2 and the French "Investissement d'Avenir" program reference No. ANR-10-EQPX-38-01.

Fig. 1: The front part of the in situ TEM sample holder with a nanomanipulator for coarse and fine motion and biasing functionality. The inset shows the CCnT mounted on a wire in the nanomanipulator opposite the Au anode.

Fig. 2: Unwrapped hologram at field emission onset voltage of 80 V on the anode. The integration contour is indicated by small arrows, and the integration direction by the big, dotted arrow (analogous to the black dotted arrow in Fig. 3 a)).

Fig. 3: a) Number of electrons along the CCnT and in the vacuum for different voltages. b) Profiles of phase shift maps from electron holography and finite element modeling. The bias on the anode was 80 V and the tip-anode separation distance 680 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-2947 In Situ Analytical Electron Microscopy: Imaging and Analysis of Steel in Liquid Water

Schilling S.1, Janssen A.1, Burke M. G.1, Zhong X. L.1, Haigh S. J.1, Kulzick M. A.2, Zaluzec N. J.3
1Materials Performance Centre, The University of Manchester, Manchester UK 1, 2BP Corporate Research Center, Naperville, IL USA 2, 3Electron Microscopy Center, Argonne National Laboratory, Argonne, IL USA 3
m.g.burke@manchester.ac.uk

In situ transmission electron microscopy has become an increasingly important and dynamic research area in materials science with the advent of unique microscope platforms and a range of specialized in situ specimen holders. In metals research, the ability to image and perform x-ray energy dispersive spectroscopy (XEDS) analyses of metals in liquids are particularly important for detailed study of the metal-environment interactions with specific microstructural features. We have recently demonstrated that both STEM imaging and XEDS data can be successfully obtained from nanoparticles in liquid in an aberration-corrected FEI Titan G2 S/TEM with Super EDX [1] [2].  Furthermore, a special hybrid specimen preparation technique involving electropolishing and FIB extraction has been developed to enable metal specimens to be studied in the liquid cell TEM specimen holder [3].  We have applied these techniques to examine austenitic stainless steel in distilled H2O.

Conventional Type 304 austenitic stainless steel was prepared for examination in a Protochips Poseidon P200 liquid cell specimen holder with a 500 nm gap between the amorphous SiN windows. This specimen holder had been modified to optimize it for XEDS microanalysis [1]. TEM/STEM examination was performed using an FEI Tecnai T20 analytical electron microscope operated at 200 kV, equipped with an Oxford Instruments Xmax80TLE windowless Silicon Drift Detector (SDD) for XEDS spectrum imaging and analysis.  Fig. 1 shows the Type 304 steel specimen imaged in distilled H2O. Fig. 2a shows several crystalline particles that were observed after 24 hours in H2O. Spectrum images (Fig. 2b,c) obtained from this area revealed that these particles were enriched in Fe and depleted in Cr, and were consistent with the formation of an Fe-rich oxide. An XED spectrum (Fig. 3) obtained from the coarse angular oxide demonstrated that the particle was Fe-rich but also contained low levels of Ni. These Fe-rich oxides can form because the thin Cr2O3 film formed on the Type 304 foil surfaces depletes the matrix of Cr. Thus, any defects in the passive film will enable the local Cr-depleted matrix to oxidise, thereby forming Fe-rich oxides. Further studies on the development of surface oxides and coarse oxide particles can aid in the study of passive film development in steels.

References

1. Zaluzec, N.J. et al., X-ray Energy-Dispersive Spectrometry During In Situ Liquid Cell Studies Using an Analytical Electron Microscope. Microsc. Microanal. 20, in press doi:10.1017/ S1431927614000154 (2014).  

2. Lewis, E.A. et al., Wet Chemistry goes Nano. Submitted to Nanotechnology Letters.

3. Zhong, X.L. et al., Novel Hybrid Sample Preparation Method for In Situ Liquid Cell TEM Analysis. Submitted to Microsc and Microanal 2014.

The authors thank the BP 2013 DRL Innovation Fund, US DoE, Office of Basic Energy Sciences, and Contract No. DE-AC02-06CH11357 at the EM Center of Argonne National Laboratory.

Fig. 1: TEM image of steel sample in distilled H2O; 50 micron wide window.                               

Fig. 2: (a) STEM image and corresponding spectrum images for (b) Fe Kα and (c) Cr Kα obtained after 24 h in H2O.

Fig. 3: XED spectrum obtained from angular Fe-rich oxide (circled) that formed in H2O.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-2952 In situ TEM observation of electrochemical deposition process

Oshima Y.1,2, Tsuda T.3, Kuwabata S.3, Yasuda H.1, Takayanagi K.2,4
1Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan, 2JST–CREST, Japan, 3Department of Applied Chemistry, Osaka University, Osaka, Japan, 4Department of Condensed Matter Physics, Tokyo Institute of Technology, Japan
oshima@uhvem.osaka-u.ac.jp

Recently, in situ transmission electron microscope (TEM) observations of lithiation and delithiation processes in lithium ion battery have been achieved in order to improve the performance. However, the electrolyte is liquid in conventional lithium ion battery. In order to observe the lithiation and delithiation processes in situ, it is necessary to develop an electrochemical cell, which keeps the liquid electrolyte in vacuum.

In this study, we developed an electrochemical cell with three terminals (working, reference, and counter electrodes) and demonstrated the process of electrochemical copper deposition on gold surfaces in-situ. Figure 1 shows a photograph of our home-made liquid cell. It is composed of two quartz glass pieces which are glued by a heat-curing epoxy with each other. The observation window is covered with a 50 nm silicon nitride film to keep the liquid inside. The advantage of our cell is that it is available to arrange suitable materials as cathode or anode. In this observation, the working, reference and counter electrodes were gold (Au), gold (Au) and copper (Cu), respectively. The electrolyte contained 0.2M CuSO4 and 0.05 M H2SO4. Cyclic voltammetry (CV) was obtained by using a VersaSTAT4 with a scan rate of 25 mV/ s.

Figure 2 shows a series of TEM images taken during CV measurement and the corresponding CV curve. Darker background corresponds to the deposited Au thin film of about 30 nm in thickness. We observed that Cu clusters were nucleated on the Au film when the bias voltage was negative, while they were desorbed when the voltage was positive. And also, during measuring CV repeatedly, we observed that Cu clusters were nucleated at the same position, corresponding to dots of slightly darker contrast as shown in the TEM image of (a). We consider that these dots correspond to the position where gold atoms were alloyed with copper atoms.

In conclusion, we have developed a new electrochemical cell for in situ TEM observation. Using the liquid cell, we have demonstrated electrochemical Cu deposition process on thin Au film simultaneously with measuring cyclic voltammetry.

This research was supported by Japan Science and Technology Agency (JST).

Fig. 1: A photograph of our developed electrochemical cell.

Fig. 2: (a)-(d) A series of TEM images taken during measuring cyclic voltammetry. Graph of cyclic voltammetry curves.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-3046 MAGNETIZATION REVERSAL PROCESS OF MAGNETIC SUPERDOMAIN STRUCTURES IN COBALT ANTIDOT ARRAYS

Rodríguez L. A.1,2, Magén C.1, Snoeck E.2, Gatel C.2, Castán-Guerrero C.3, Sesé J.1, García L. M.3, Herrero-Albillos J.4, Bartolomé J.3, Bartolomé F.3, Ibarra M. R.1
1LMA-INA, Universidad de Zaragoza, Zaragoza, Spain, 2CEMES-CNRS, Toulouse, France, 3ICMA, Universidad de Zaragoza-CSIC, Zaragoza, Spain, 4Centro Universitario de la Defensa, Zaragoza, Spain
luisaf85@unizar.es

Geometric confinement of the magnetization in magnetic thin films by patterning regular antidot (hole) arrays has been considered a potential method to fabricate storage media of ultrahigh capacity or magnonic devices for high frequency applications [1]. Reducing the distance between antidots modifies favorably the magnetic properties towards the creation of individual magnetic entities that could be used as magnetic bit of information [2]. For this reason, it is necessary to use magnetic imaging techniques that can provide information of the local magnetic states at submicron scales. In this work, high spatial resolution Lorentz Microscopy (LM) combined with the in situ application of magnetic fields has been used to perform quantitative studies of the magnetic states of cobalt square antidot arrays with periodicities (p) ranging between 524 and 95 nm. Antidot arrays were patterned by Focused Ion Beam (FIB) etching on a 10-nm-thick cobalt film deposited on Si3N4 membrane. The FIB etching process produced holes of 55 nm diameter. At remanence, defocused LM images revealed a periodicity dependence of the magnetic domains and a transition in the domain wall geometry around p ~ 300 nm, changing from 90° and 180° walls to superdomain (SD) walls for small periodicities (see Fig. 1). A Fourier filtered method has been implemented to improve the direct visualization of one-dimension SD (magnetic chains) for arrays with p > 95 nm (see Fig. 2), which has allowed to determine the magnetic configuration inside the antidot cells in both the SD and the SD walls. The magnetization reversal processes by means of hysteresis cycles upon magnetic fields parallel and diagonal to the antidot rows have been studied by in situ LM experiments. As illustrated in Fig. 3, we have found that the reversal magnetization process occurs by simultaneous (parallel hysteresis cycle) or sequential (diagonal hysteresis cycle) nucleation and propagation of horizontal and vertical superdomain walls, respectively.

[1] Xiao Z L, Han C Y, Welp U, Wang H H, Willing G A, Vlasko-Vlasov V K, Kwok W K, Miller D J, Hiller J M, Cook R E and Crabtree G W 2003 Nanotechnology 3 357.

[2] Torres L, Lopéz-Diaz L and Iñiguez J 1998 Appl. Phys. Lett. 73 3766.

This work was supported by the Spanish Ministry of Economy and Innovation (MINECO) through the projects MAT2011-28532-C03-02 and MAT2011-23791 including FEDER funding, by the Aragón Regional Government through Projects E26 (MAGNA), E34 (IMANA) and CTPP4/11, and by the European Union under the Seventh Framework Program under a contract for an Integrated Infrastructure Initiative Ref 312483-ESTEEM2.

Fig. 1: Defocused LM images for square antidot arrays with periodicities of (a) 524 nm, (b) 327 nm, (c) 160 nm and (d) 116 nm. In the latter, magnetic contrast has nearly disappeared.

Fig. 2: (a) Raw and (b) Fourier filtered defocused LM images of the antidot array with p = 160 nm. (c) Color-coded magnetization orientation map obtained by the Transport-of-Intensity Equation reconstruction to a small region marked with a yellow rectangle in (a) and (b).

Fig. 3: Sequence of filtered defocus LM images recorded during the in situ application of in-plane magnetic fields (a) parallel and (b) perpendicular to the antidot rows.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-3113 In-situ biasing and switching of electronic devices into a TEM.

Mongillo M.1, Garbin D.1, Navarro G.1, Vianello E.1, Coue M.1, Mayall B.1, Cooper D.1
1CEA-LETI Minatec, 17 rue des Martyrs 38054 Grenoble, FRANCE
massimo.mongillo@cea.fr

In order to understand the physics of new materials that are currently being developed for use in electronic memories it is now necessary to perform in situ switching inside a Transmission Electron Microscope (TEM).
In this talk we will present our approach towards the development of a robust integrated characterization system that enables in-situ biasing and/or switching of electronic devices inside a TEM. As microscope time is valuable, the basic idea is to be able to electrically test a device before and then after specimen preparation outside of a TEM such that the electrical properties are understood before in situ operation in the TEM. The goal is to correlate the electrical properties to modifications in the crystalline structure and composition measured using HAADF STEM and EELS and the dopant/vacancy distribution measured by electron holography [1]. For this task we have been using a dedicated specimen holder featuring six static electrical contacts and a piezo-actuated movable probe tip which can act as a local electrical lead.
Figure 1 shows a TEM image of the movable tip used to switch a SrTiO3 resistive memory [2-3] that has been prepared using focused ion beam milling. The TEM image shows that the probe has introduced stress into the membrane. The poor electrical contact can also cause local heating and can even cause the specimens to explode. Despite these problems, the external electrostatic potential applied to the probe can cause a reversible switching of the active layer between high and low-resistive states, however the experiment is difficult, stressful and time consuming.
Our approach is to use fixed contacts on both simple and complicated devices. An example is provided in Figure 2 which shows a resistive memory cell [4]. A thick slice of the wafer has been sawn and then a Xenon-Ion FIB has been used to remove a large volume of material to provide a site specific region of interest. This region is then thinned to electron transparency using a conventional Ga FIB. Metal deposition in the FIB has been used to rewire the electrical contacts inside the device such that the switching can be performing by wire bonding the top contacts.
In this presentation we will present the two different approaches of switching memory devices in situ in the TEM and compare the advantages of each.

References:
[1]Nature Materials, 8, 271 (2009)
[2]Nature Materials, 5, 312 (2006)
[3]Advanced Materials, 21, 2632 (2009)
[4]Nature Materials,6, 824 (2007)

This work has been performed on the nanocharacterisation platform (PFNC) at Minatec. The authors thank the European Research Council for the Starting Grant “Holoview” and LabEx Minos.

Fig. 1: In-situ biasing using a probe manipulated by a piezo-electric motor. A thin TEM lamella prepared using conventional FIB specimen preparation techniques is mounted onto a TEM grid. The movable metallic probe (a) approaches and makes contact (b)to the top of the specimen.

Fig. 2: Memory cell prepared using Plasma FIB Xenon milling. The milling rate of the Xe-Fib enables us to remove large quantities of material. The electron transparent region containing the memory cell is patterned starting from a “bulky” slab. The two bonding pads can be hard-wired and used in the TEM to allow for in-situ switching of the device.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-3133 In-situ Nano-compression tests on Shape Memory Alloys

San Juan J.1, Gómez-Cortés J. F.1, López G. A.2, Hernández J.3, Molina S.3, Nó M. L.2
1Dpt Física de la Materia Condensada, Univ. del País Vasco, Bilbao, Spain, 2Dpt Física Aplicada II, Univ. del País Vasco, Bilbao,Spain, 3Dpt Ciencia de los Materiales, Univ. de Cádiz, Puerto Real, Cádiz, Spain
jose.sanjuan@ehu.es

Recently, there has been growing interest in the potential use of shape memory alloys (SMA) in micro and nano-scale structures and devices, for example as sensors or actuators in micro electromechanical systems (MEMS). With a growing worldwide market in excess of hundred billion dollars, MEMS constitute a new paradigm of technological development for the present century, and smart materials are converging with miniaturization technologies, enabling a new generation of smart MEMS or SMEMS. Among the different smart materials targeted for use in SMEMS, shape memory alloys (SMA) have attracted considerable interest because they offer the highest work output density, about 107 J/m3, and exhibit specific desirable thermo-mechanical effects such as superelasticity and shape memory.

In previous works, completely recoverable superelastic strain and shape memory in micro and nano pillars was first reported for Cu-Al-Ni SMAs [1] showing the competitive advantage of these SMAs over the commercially used of Ti-Ni. In addition several size effects on superelastic behaviour were also demonstrated [2, 3] in Cu-Al-Ni SMAs. For practical applications the superelastic behavior must be reproducible in order to be functionally reliable, and first studies on cycling SMA micropillars by nano compression tests were recently published [4, 5].

In this work we present an In-situ characterization of the nano-compression superelastic behaviour of Cu-Al-Ni micro-pillars at the scanning electron microscope. Micro-pillars were milled by Focused Ion Beam technique on [100] oriented Cu-Al-Ni single crystals. All pillars were tested in an instrumented pico-indenter Hysitron PI-85, introduced inside the chamber of a JEOL-FEG 7500, by using a diamond flat indenter, as can be seen on Figure 1. The nano-compression stage was tilted in order to allow imaging by the SEM. Simultaneous video-image was taken during the nano-compression test acquisition data in order to correlate the mechanical behaviour with microstructure evolution. Fully recoverable and reproducible superelastic behaviour has been obtained and a picture of the screen containing both, image and mechanical test, is shown in Figure 2.

[1] J. San Juan, M. L. Nó, and C. A. Schuh, Advanced Materials 20 (2008), p. 272.

[2] J. San Juan, M. L. Nó, and C. A. Schuh, Nature Nanotechnology 4 (2009), p. 415.

[3] J. San Juan and M. L. Nó, J. Alloys & Compounds 577S (2013), p S25.

[4] J. San Juan, M. L. Nó, and C. A. Schuh, Acta Materialia 60 (2012), p. 4093.

[5] J. San Juan, J. F. Gómez-Cortés, G. A. López, C. Jiao, and M. L. Nó, Appl. Phys. Lett. 104 (2014), p.011901

The authors thank the Spanish Ministry of Economy and Competitiveness, MINECO, project MAT2012-36421 and the CONSOLIDER-INGENIO CSD2009-00013, and the Basque Government for Consolidated Research Group IT-10-310 and ETORTEK-ACTIMAT-2013. J. San Juan and M.L. Nó also thank EOARD Grant FA8655-10-1-3074. J.F. Gómez-Cortés thanks the Ph.D. Grant from MINECO.

Fig. 1: Figure 1. Sub micrometre pillar of Cu-Al-Ni SMA, milled by focused ion beam, just before the in-situ nano-compression test. On the lower side of the image it can be appreciated the flat tip of the diamond indenter in contact with the top of the pillar.

Fig. 2: Figure 2. In-situ superelastic nano-compression test performed on a sub-micrometre pillar of Cu-Al-Ni SMA. The video image allow to correlate the different points of the load-displacement curve with the corresponding images taken at the SEM. The above image corresponds to the final screen image just after finishing the in-situ test.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-7-O-3169 Temperature-induced sphere-to-tetrapod transformation of CdSe nanocrystals investigated by in-situ transmission electron microscopy

van Huis M. A.1,2, Fan Z.3, Li W. F.1, Yalcin A. O.2, Tichelaar F. D.2, Talgorn E.4, Houtepen A. J.4, van Blaaderen A.1, Vlugt T. J.3, Zandbergen H. W.2
1Condensed Matter and Interfaces, Debye Institute for Nanomaterials Science, Utrecht University, Princetonplein 5, 3584 CC Utrecht, The Netherlands, 2Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands, 3Process and Energy Laboratory, Delft University of Technology, Leeghwaterstraat 39, 2628 CB Delft, The Netherlands, 4Department of Chemical Engineering, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands
m.a.vanhuis@uu.nl

Colloidal CdSe nanocrystals (NCs) can be synthesized in a wide variety of (heterogeneous) nanostructures including sphere, rod, tetrapod, and octapod morphologies. Using a low-drift TEM heating holder [1] employing MEMS microheaters with 15 nm thick SiN windows, the thermal evolution of spherical CdSe NCs was followed in real time and with atomic resolution. With increasing temperature, the NCs were found to transform from spheres to multipods to rectangular single crystals. The thermal evolution is shown schematically in Figure 1. 

The as-synthesized CdSe NCs consist of multiple subcrystals, but are spherical in shape. Upon heating to a temperature of 80 °C, most NCs transform into bipods, tripods, or tetrapods, whereby the core exhibits the zinc blende (ZB) crystal structure while the pods have the wurtzite (WZ) crystal structure [2]. These multipods are remarkably stable, up to a temperature of 300 °C, as long as they remain isolated. Multipods that are close together, though, fuse into rectangular single crystals having the ZB structure at temperatures of 170-200 °C. This is an unexpected result, as the stable bulk phase of CdSe is WZ. The ZB NCs undergo multiple crystal fusion events by oriented attachment, which was recorded in real time and with atomic resolution. The fusion is followed by coalescence into larger agglomerates which eventually transform to the WZ crystal structure. The last step in the thermal evolution is sublimation which takes place at temperatures of 360‒400 °C. 

Force-field molecular dynamics (FF-MD) simulations [2] (Figure 2), and density functional theory (DFT) calculations were performed in order to investigate the driving forces inducing these most remarkable transformation phenomena. It is concluded that off-stoichiometry can slightly favor the bulk ZB phase with respect to the bulk WZ phase, but that temperature-dependent interface-related energies are most likely the cause of the rich thermal behavior. Furthermore, it becomes clear that the ZB-WZ transformations are mediated by vacancies on the {111}Cd or {0001}Cd atomic planes.

[1] M.A. van Huis, N.P. Young, G. Pandraud, J.F. Creemer, D. Vanmaekelbergh, A.I. Kirkland, H.W. Zandbergen, ‘Atomic imaging of phase transitions and morphology transformations in nanocrystals’, Adv. Mater. 21 (2009) 4992-4995.
[2] Z. Fan, A.O. Yalcin, F.D. Tichelaar, H.W. Zandbergen, E. Talgorn, A.J. Houtepen, T.J.H. Vlugt, M.A. van Huis, ‘From sphere to multipod: thermally induced transitions of CdSe nanocrystals studied by molecular dynamics simulations’, J. Am. Chem. Soc. 135 (2013) 5869-5876.

Fig. 1: Schematic showing the thermal evolution of the CdSe nanocrystals, as they transform from spheres to tetrapods to rectangular zinc blende nanostructures, whereby the potential energy decreases

Fig. 2: Left: Result of a force-field molecular dynamics (MD) simulation performed at a temperature of 800 K, whereby a spherical CdSe NC has transformed into a tetrapod (details in Ref. [2]). Right: HRTEM images of several CdSe multipods formed during in-situ heating.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-1394 An in-situ transmission electron microscopy study on room temperature ductility of TiAl alloys with fully lamellar microstructure

Kim S.1, Na Y.1, Yeom J.1, Kim S.1
1Light Metal Division, Korea Institute of Materials Science, Changwon 642-831, South Korea
mrbass@kims.re.kr

Gamma titanium aluminides (TiAl) have gained great interest for research on high-temperature applications due to their weight saving in combination with excellent high temperature properties such as creep and oxidation resistance. However, their poor room temperature ductility and machinability have hindered their application in areas such as aerospace and automobile products. In this study, mechanical properties of newly-developed TiAl alloys were investigated. The new TiAl alloys contain less aluminum compared with conventional gamma TiAl alloy to improve processibility and machinability. Especially, room temperature ductility of fully lamellar TiAl alloys was acquired without heat-treatment or TMP process.Adding beta stabilizers and lowering Al contents in conventional gamma-based TiAl alloys were found to be beneficial for room temperature ductility of TiAl alloys. An in-situ transmission electron microscopy study was conducted at room temperature in order to understand an underlying mechanism on room temperature ductility of TiAl alloys. From in-situ straining transmission electron microscopy experiments, it was revealed that the crack path is different between the TiAl alloys with/without room temperature ductility. The crack in TiAl alloys having room temperature ductility interacted with lamellae by forming bridging ligaments between the two alpha lamellae and the gamma lamellae (Fig. 1). In contrast, the cracks in TiAl alloys without room temperature ductility propagated along grain (colony) boundaries showing brittle intergranular fracture (Fig. 2). Finally, we proposed the important microstructural factors to have room temperature ductility of TiAl alloys.

This work was supported by the Fundamental R&D Program of the Korea Institute of Materials Science.

Fig. 1: Bright field images of alloy having room temperature ductilitytaken during in-situ TEM experiment.

Fig. 2: Bright field images of alloy having no room temperature ductility taken during in-situ TEM experiment.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-1582 Simultaneous in situ SEM/STEM observation of catalyst reaction under an air atmosphere using a Cold-FE environmental TEM

Sato T.1, Matsumoto H.1, Nagaoki I.2, Yaguchi T.2
1Application Development Department, Hitachi High-Technologies Corporation, 2Advanced Microscope System Design Department, Hitachi High-Technologies Corporation
sato-takeshi-3@naka.hitachi-hitec.com

In the development of catalysts and fuel cell materials, there is an increasing demand for fine structural characterization using Environmental TEM (E-TEM). Recently, we have developed an E-TEM based on a conventional analytical TEM combined with a gas injection-specimen heating holder[1-2]. To further clarify the mechanism of the degradation of electrode-catalyst, a simultaneous in situ SEM/STEM study was carried out under the accelerated degradation condition. With the surface information from the SEM detector, we have obtained information in three-dimensional, gained significant new understanding of the behavior of Pt/C catalysts.
Figure 1 shows an overview and a schematic diagram of the specially designed Hitachi HF-3300 Cold-FE in situ TEM equipped with STEM and SEM imaging capabilities. In order to maintain the electron gun area under ultrahigh vacuum of better than 10-8 Pa near the gun, yet introducing gas into the specimen chamber, an additional ion pump (IP3) and an extra orifice have been added between the gun valve and specimen chamber. In situ simultaneous SEM/STEM observation in a gaseous atmosphere is realized by using the gas injection specimen heating holder.
A picture and a schematic diagram of the gas injection specimen heating holder are shown in Figure 2(a) and 2(b), respectively. The reaction gas is introduced to the area around the specimen by means of a gas injection nozzle. Therefore, in situ observation in a gaseous atmosphere can be carried out using this Cold-FE TEM even if up to 10 Pa near the specimen. The specimen was used a commercially available Pt/C catalyst. To simulate an accelerated aging, the specimen was heated to 200˚C. The morphological changes of Pt/C catalyst operated at accelerating voltage of 300 kV.
Figure 3 shows the results of in situ SEM/STEM simultaneous observation. At the beginning of air with a measured specimen chamber pressure of 1 Pa after 270 sec., a cluster of Pt particles on the carbon support have grown and agglomerated, and the number of Pt particles appeared to decrease on the carbon support, as indicated in red circle. After 660 sec., most of the Pt particles have gradually started inserting into the carbon support, and the grain growth and agglomeration of Pt particles have occurred inside the carbon support, as shown in Figure 3. After 1080 sec., the behaviors of migration, coalescence and grain growth of Pt particles inside the carbon support were clearly observed by STEM image.

References
[1] T. Yaguchi et al., J. Electron Microsc., 61(4), 199-206 (2012)
[2] T. Kamino et al., J. Electron Microsc., 54(6), 497-503 (2005)

The authors gratefully acknowledge Professor Kazunari Sasaki and Associate Professor Akari Hayashi of Kyushu University for valuable discussions.

Fig. 1: External view and a schematic diagram of the Hitachi HF-3300 Cold-FE TEM

Fig. 2: External view (a) and schematic diagram (b) of the gas injection specimen heating holder

Fig. 3: The results of in situ SEM/STEM simultaneous observation
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-1610 Observation method of cross-sectioned cells by cryo-scanning electron microscopy

Nishino Y.1, Ito Y.1, 2, Miyazawa A.1
1Graduate School of Life Science, University of Hyogo, 2Leica Microsystems K. K.
ynishino@sci.u-hyogo.ac.jp

Protein and cellular structures have been visualized in a close-to-native state by cryo-transmission electron microscopy (cryo-TEM). In many cases for cryo-TEM cells are so thick that we have to prepare cryo-ultrathin sections. In such case compression of cryo-sections must be taken into consideration. The compression makes the image complicated because it occurs inhomogeneously. Cells and organelles are compressed whereas small rigid complexes such as ribosomes and microtubules have been reported to resist compression.

On the other hand freeze-fractured cells and tissues have been examined by cryo-scanning electron microscopy (cryo-SEM). However this method is limited because observation objects are only randomly fractured surface.

In order to visualize non-distorted cross-sectioned cells, we focused on the block surface after cryo-sectioning, and imaged it by cryo-SEM. Budding yeastwas pelleted, high-pressure frozen and cryo-sectioned. The sections were imaged by cryo-TEM while the block was imaged by cryo-SEM. As a result, ultrastructure such as ribosomes and invaginated plasma membranes as well as organelles were clearly visualized by cryo-TEM, however vesicle structure such as whole cells, nuclei and vacuoles were ellipsoidal in the same direction (Fig. 1a). They were obviously compressed along the cutting direction. Meanwhile the block was transferred to cryo-SEM and observed. We could image cells and organelles without any staining or coating. In the cryo-SEM images yeast was oval in shape, and nuclei and vacuoles were circle in shape (Fig. 1b). They are consistent with the fluorescently-labeled images by light microscopy.

Furthermore we showed an example of repetitive cryo-sectioning and observation by cryo-SEM. A piece of diaphragm was cryo-sectioned in the direction parallel to sheet-like structure of diaphragm and observed by cryo-SEM. On the sectioned face near the surface of isolated diaphragm connective tissue was clearly observed (Fig. 2a). In order to observe structure beneath the connective tissue, the block observed by cryo-SEM was returned to the cryo-ultra-microtome using a cryo-transfer system. After the block was cryo-sectioned again, sectioned surface was observed by cryo-SEM again. As shown in Fig. 2b, sectioned muscle cells appeared. Repetitive sectioning and observing would be helpful to find objects localized in a limited area.

In this study it was shown that non-compressed coss-sectioned hydrated cellular and tissue architectures are clearly visualized by cryo-SEM.

We would like to thanks Ms. Ishihara A. (Leica Microsystems K. K.) for technical support. This work was partially supported by JSPS KAKENHI Grant Number, 23570196.

Fig. 1: Comparative observation by cryo-SEM and cryo-TEM. (a) Cryo-section of budding yeast observed by cryo-TEM. (b) Cryo-sectioned surface of budding yeast observed by cryo-SEM. Arrows: cutting direction, CW: cell wall, N: nucleus, V: vacuole, Mt: mitochondrion, IM:invaginated pasma membrane, Bars=500 nm.

Fig. 2: Repetitive sectioning and observation of a block surface of diagram. A piece of diagram was cryo-sectioned and observed by cryo-SEM repeatedly. (a) Sectioned diagram near the surface of the frozen block. (b) Sectioned diagram inside the frozen block. CF: collagen fibers, N: nucleus, MF: muscle fibers, Bars=1 μm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-1655 Characterization of Melting and Crystallization Behavior in the Au-Ge Eutectic System Using Au-catalyzed Ge Nanowires

Marshall A. F.1, Thombare S. V.1, Chan G.1, McIntyre P. C.1
1Stanford Nanocharacterization Facility and Materials Science and Engineering Department, Stanford University, Stanford, CA
afm@stanford.edu

The catalyzed growth of nanowires (NWs) can provide us with a useful platform for studying nanoscale phase transformations that are readily observed using in situ transmission electron microscopy. For example, following vapor-liquid-solid (VLS) growth of Ge and Si NWs, the re-solidified catalyst, typically Au, remains at the end of the NW, with an abrupt, planar interface between the two materials. Fundamental behaviors of these nanoscale eutectic systems, such as melting and crystallization, as well as metastable phase formation, can be studied by heating and cooling the NWs in the TEM. The use of a MEMS based heating holder (Protochips AduroTM) allows for a large range of heating and cooling rates, including quench rates that are comparable to those used in more traditional rapid quench studies. Here we present details of the melting and crystallization behavior of the metastable hexagonal close-packed beta phase of the Au-Ge eutectic system.

We have previously shown that the metastable hcp phase, which occurs following NW growth [1], can also be formed by melting and rapid quenching of the Au nanocatalyst at the tip of a Ge nanowire [2]. Fig. 1 shows the melting behavior of the quenched-in metastable phase. Melting occurs over a timeframe of seconds; it begins at the edges of the Ge NW-catalyst interface (Fig. 1a and b). In Fig. 1b melting is also visible at the top of the catalyst indicating that initial melting continues along the surface. An abrupt formation of additional stacking faults (Fig. 1c), characterizes a transition to a large volume of melt regions that form parallel to the {0001} planes of the remaining crystal (Fig. 1d). As the melt volume grows, the crystal pulls away from the surface, adopting a spherical shape, and “floats” in the liquid, then moves to the interface (Fig 1e), before abruptly dissolving into the liquid volume (Fig. 1f). This last process is accompanied by a notable darkening of the liquid as a result of mass contrast induced by the dissolved Au. We note that this same sequence of events has been observed a number of times in the hcp quenched structure, e.g. Fig 2. These results suggest that orientation of the crystal, and diffusion along the {0001} planes of the metastable hcp phase influence details of the melting process. We will also present results of cooling studies, which indicate a correlation between the formation and orientation of the metastable phase, and show that the amount of Ge that remains in the hcp structure can be controlled by the cooling rate and minimized by subsequent annealing.

[1] A.F. Marshall, et al, Nano Lett. 10 (2010), 3302. [2] A.F. Marshall, et al, Microscopy and Microanalysis 2013, Phoenix, AZ.

Acknowledgement: Financial support is provided by National Science Foundation grant DMR-1206511. This work was performed at the Stanford Nanocharacterization Laboratory.

Fig. 1: Fig. 1: Selected timeframes from a melting video of a quenched nanocatalyst with the metastable phase. Melting begins at the edges of the interface in the first frame and proceeds through a series of morphological changes until final melting at about 11 seconds.

Fig. 2: Fig. 2: Still images from another quenched in metastable structure (a) shows the same preferred melting along the <0001> direction (b), and a detached crystal within the melt (c) prior to final melting.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-1721 Maestro: a Matlab-based centralized computer control system for an electron microscopy laboratory

Bergen M.1, Dalili N.1, Malac M.1,2, Hoyle D.3, Chen J.1, Taniguchi Y.4, Yotsuji T.4, Yaguchi T.4, Hayashida M.5, Howe J.3, Kupsta M.1
1National Institute for Nanotechnology, Edmonton, Canada, 2Univ. of Alberta, Edmonton, Canada, 3Hitachi High Tech Canada, Toronto, Canada, 4Hitachi High Tech, Naka, Japan, 5AIST, Tsukuba, Japan
marek.malac@gmail.com

A modern electron microscopy (EM) laboratory needs to integrate a number of auxiliary devices with a (transmission) electron microscope (TEM). Integration refers to controlled operation of all the hardware and the TEM, image and spectra recording at precise moments of the experiment and accurate logging of the status of the microscope and all connected devices. Here we report extensive development of a Matlab-based central computer system for an EM laboratory, referred to as Maestro [1].

Matlab has been successfully used to control TEMs [2]. The Maestro computer control system offers extensive functionality beyond the microscope control. At present, the Hitachi HF-3300 TEM / scanning TEM and H-9500 environmental TEM (ETEM) can be fully controlled. Additional devices, such as video recording software, gas handling system for ETEM, custom-built controllers for sample heating, electron biprisms, Gatan Image Filter, Gatan DigiScan, electron tomography holders are included in the Maestro system. The status of all active devices is recorded within each data set (image, diffraction pattern or spectra) and can be later reloaded to reproduce the exact instrument status. Control and logging of multiple data sources is possible.

Fig. 1 shows a generic layout of an EM laboratory controlled by Maestro. The communication between the central computer with Maestro and Matlab is typically over LAN, but non-LAN methods, such as RS232 are possible. Maestro allows operation using a control Matlab script that accurately executes an experiment with control of multiple parameters and efficiency far exceeding that of a manually operated microscope. In an ETEM, the possibility to control and log multiple experiment parameters leads to about five fold decrease of experimental time and the elimination of user errors in the experiment execution. Fig. 2 shows an example of low loss EELS trace of gas composition obtained in an H-9500 ETEM with 100 ms time resolution. Both the gas composition and data acquisition were controlled by Maestro. Maestro can be also operated through a graphical user interface (GUI) shown in Fig. 3. Often experiments are developed as a script and the GUI is implemented for frequently repeated experiments. The settings of all devices used in an experiment are saved either as tags in traditional Digital Micrograph (DM) files or as a Matlab mat file. The tags with device status can be accessed in DM, as shown in Fig. 4, as well as in Matlab. Maestro can execute existing DM scripts within a Matlab instrument control script. Maestro can be used both as point-and-click GUI tool and an accurate script based advanced instrument control.

[1] M. Bergen et. al. Micr.  Microanal. 19 S2 (2013), p. 1394

[2] TOM toolbox: www.biochem.mpg.de/278655/tom_e

The work was made possible by extensive support of Mr. I. Cotton and Hitachi High Tech, Canada and by funding from NRC/NINT and Alberta Innovates Tech. Futures.

Fig. 1: Layout of a generic laboratory controlled by Maestro

Fig. 2: EELS trace of gas composition in a Matlab-controlled environmental TEM

Fig. 3: Graphical user interface for one click connection to multiple devices

Fig. 4: Tags with device status during data acquisition viewed in Digital Micrograph
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-1777 In situ investigations of the thermodynamic behavior of dislocation loops in nanopillars and their impact on nanomechanical properties

Kiener D.1, Jeong J.2, Lee S.2, Zhang Z.3, Oh S. H.2
1Department Materials Physics, Montanuniversität Leoben, Austria, 2Department of Materials Science and Engineering, Pohang University of Science and Technology, Korea, 3Erich Schmid Institute of Materials Science, Austrian Academy of Sciences, Austria
daniel.kiener@unileoben.ac.at

Studying the nanomechanical behavior of miniaturized objects was enabled by the availability of focused ion beam (FIB) microscopes to create nanoscale structures, and boosted by unique deformation mechanisms encountered in nanoscale dimensions. Quantitative testing in situ in the TEM [1] was seminal in aiding understanding of underlying processes. However, a remaining issue concerns near surface crystal defects created by the FIB [2] and their influence on the properties of nanoscale samples [3].
Here we combine in situ heating and in situ nanomechanical TEM testing to study the thermodynamic behavior of FIB induced crystal defects in the confined volume of nanopillars, and their influence on mechanical properties on fcc, bcc, and hcp metals.
We show that during annealing, initially the FIB induced prismatic dislocation loops undergo an Oswald ripening process (Fig. 1). From this time resolved process we were able to determine the activation energy of lattice diffusion in Al, similar to recent pipe diffusion measurements along dislocations [4]. Upon further annealing to about 0.6 Tm, the remaining loops exit the sample due to image forces, leaving behind a pristine pillar as confirmed by HRTEM (Fig 2a, b). Loading these pristine samples in compression in situ in the TEM, we observed that dislocation plasticity initiates by surface nucleation of dislocations at very high stresses, much higher than required to plastically deform specimens that still contain FIB induced loops (Fig. 2c, d). This demonstrates that we can undo the FIB induced damage and restore pristine crystals and probe their intrinsic mechanical behavior. Finally, annealing the samples to even higher temperatures closer to the melting point, we could study the sublimation, melting, and evaporation processes of such confined metallic volumes. For the case of Mg, we show that the Ga from the FIB processing stimulates the sublimation process of the Mg crystal with a flat interface. This sublimation causes enrichment of Ga at the interface, leading to formation of a lower melting point Mg-Ga alloy. Upon melting the surface forms cusps, and evaporation of the molten Mg encapsulated in a MgO shell continues (Fig. 3). Notably, the rate of material loss during sublimation and evaporation did not change.
These observations underline the importance of direct in situ observation in the TEM when attempting to investigate nanoscale thermal or mechanical processes.

References:
[1] Dehm G, Howe JM, Zweck J. In-Situ Electron Microscopy. Weinheim: Wiley-VCH 2012
[2] Kiener D et al. Mater. Sci. Eng. A 2007;459:262
[3] Shim S, et al. Acta Mater. 2009;57:503
[4] Legros M, et al. Science 2008;319:1646

DK acknowledges support from the Austrian Science Fund (FWF), projects I 1020-N20 and P 25325.

Fig. 1: Images showing FIB prepared Al pillar (a) and during loop growth (b - d).

Fig. 2: HRTEM image of a Cu pillar after FIB fabrication (a) and subsequent annealing (b). Stress-strain curve of annealed (c) and FIB prepared (d) pillar.

Fig. 3: Sublimation of Mg nanopillar: (a-d) Straightening and continuous sublimation of the Mg pillar. (e) Evaporation of Mg. Note the lack of diffraction contrast and the cusps formed.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-1812 Dislocation mediated creep/relaxation in nanocrystalline palladium thin films revealed by on-chip high resolution TEM in-situ testing

Amin-Ahmadi B.1, Colla M. S.2, Idrissi H.1,2, Malet L.3, Godet S.3, Raskin J. P.2, Pardoen T.2, Schryvers D.1
1Electron Microscopy for Materials Science (EMAT), University of Antwerp, Belgium, 2Université catholique de Louvain, Institute of Mechanics, Materials and Civil Engineering, Louvain-la-Neuve, Belgium, 3Université Libre de Bruxelles, Matters and Materials Department, Belgium
behnam.amin-ahmadi@uantwerpen.be

The high rate sensitivity of nanostructured metallic materials demonstrated in recent literature is related to the predominance of thermally activated deformation mechanisms favoured by a high density of internal interfaces. In the present study, we report for the first time in-situ high resolution transmission electron microscopy (HRTEM) creep/relaxation tests on electron beam evaporated nanocrystalline (nc) palladium (Pd) thin films using an original on-chip nanotensile method resembling the technique used in [1]. Unexpectedly, large creep/relaxation rates have been observed at room temperature. Figure 1 shows the microstructure of the as-deposited Pd films characterized in both cross-sectional and plan-view thin foils prepared by focused ion beam (FIB). In this figure, columnar nanograins can be observed with 2 or 3 grains confined over the thickness of the films with an in-plane grain diameter of ~30 nm. Automated Crystallographic Orientation Mapping in TEM (ACOM-TEM) shows a clear [110] fibre texture parallel to the growth direction. The microstructure involves Σ3 60° {111} coherent twin boundaries (CTBs) in ~ 25% of the grains.
The in-situ HRTEM characterisation of the evolution of the microstructure shows that, despite the small grain size, the creep/relaxation mechanism is mainly mediated by the stress driven thermally activated nucleation and propagation of dislocations. Interestingly, the formation and the destruction of sessile Lomer-Cottrell dislocations have been observed in-situ. Furthermore, clear loss of the coherency of CTBs with time was observed as indicated by the progressive increase of the thickness of these boundaries as shown in Figure 2. Such feature is attributed to the interaction of CTBs with lattice dislocations. The impact of these elementary plasticity mechanisms on the creep/relaxation behaviour of the Pd films is discussed and compared to recent experimental and simulation works in the literature. This constitutes a key issue in the development of a variety of micro- and nanotechnologies, such as Pd membranes used in hydrogen applications.

References
1. H. Idrissi, B. Wang, M.S. Colla, J.P. Raskin, D. Schryvers and T. Pardoen, Adv. Mater. 23 (2011), P.2119.

Fig. 1: Figure 1. a) ACOM-TEM orientation mapping of as-deposited Pd film. Corresponding inverse pole figure along different directions are shown. b) Grain size distribution of (a). (c) Bright field micrograph obtained on cross-sectional as-deposited Pd film (d) HRTEM image obtained in as-deposited films showing a Σ3 60° {111} coherent TBs.

Fig. 2: Figure 2. HRTEM images showing Σ3 {111} TBs at (a) t=0 and (b) t=3 days, respectively. Note the increase of the TBs thickness from (a) to (b) in the filtered images at the upper right insets. Corresponding Fast Fourier Transform (FFT) showing the twin character is shown in the lower right inset of each image.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-1868 In situ mechanical testing in the transmission electron microscope and finite element method simulations on nanoscaled amorphous silica spheres – Densification, hardening and improved intrinsic properties on nanoscale

Mačković M.1, Niekiel F.1, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), FAU Erlangen-Nürnberg, Cauerstr. 6, 91058 Erlangen, Germany
mirza.mackovic@ww.uni-erlangen.de

As functional members in electronic devices and fiber-based telecommunication techniques oxide glasses have become subject of intense research. In addition to functionality, oxide glasses often have to fulfil mechanical reliability. Because silica is relevant in electronic and optical applications, it is chosen as a suitable model system towards a general understanding of factors which control intrinsic strength, deformation and elastic properties of non-crystalline materials. Since glasses lack of long-range periodicity, usual strengthening strategies, which comprise introduction of defects or grain boundaries with the aim to inhibit dislocation motion [1], are not working. Hence, novel approaches are appreciated to improve their mechanical properties. In the past, electron beam (EB) irradiation was used to tailor the properties of materials [2], but facing the problem of increased specimen temperature. EB irradiation is also known to densify amorphous silica (a-SiO2) on macroscopic scale [3]. Recent in situ transmission electron microscopy (TEM) studies have shown that moderate EB irradiation is very useful to induce enormous ductility in nanoscale a-SiO2 [4].

In the present study combined in situ mechanical testing in TEM and finite element method (FEM) simulations were used to characterize the mechanical properties of nanoscaled a-SiO2 spheres. First, the dose-dependent densification of a-SiO2 upon EB-irradiation was monitored in situ in TEM (Fig. 1). At low beam current doses (LD) a-SiO2 spheres densify clearly less compared to higher dose (HD) irradiation. In order to investigate the effect of EB irradiation on the mechanical properties, the spheres were irradiated with either LD or HD and then compressed under beam-off (Fig. 3a,b) or beam-on (Fig. 3c) conditions. We observe a pronounced hardening effect (Fig. 3), whereby higher loads are required to compress a-SiO2 spheres, which are treated with HD irradiation prior to compression [5,6]. FEM simulations based on an elastic / ideally plastic model (set-up in Fig. 2) reveal an increase in Young’s modulus upon HD irradiation (not shown) [6], as well as different plastic strains for beam-off and beam-on compression (Fig. 3). This clearly proves that the intrinsic glass properties can be tailored by EB irradiation [5]. Our approach is highly promising and opens opportunities for fundamental studies on structure-property relations of nanoscaled glass.

1. K. Lu et al., Science (2009) 324:349; 2. A. Krasheninnikov et al., Nature Mater. (2007) 6:723.
3. W. Primak et al., J. Appl. Phys. (1968) 39:5651.
4. K. Zheng et al., Nature Comm. (2010) DOI: 10.1038/ncomms1021
5. M. Mačković et al., Microscopy Congress MC2013, Regensburg, Proc. (Part 1), pp. 470-471.
6. M. Mačković et al., submitted.

Financial support by DFG via SPP1594 „Topological Engineering of Ultra-Strong Glasses” and the Cluster of Excellence (EXC 315) is acknowledged. The authors thank M. Hanisch and R.N. Klupp-Taylor for providing the amorphous silica spheres.

Fig. 1: Quantitative in situ observation of electron beam induced densification of nanoscaled a-SiO2 spheres.

Fig. 2: FEM simulation showing an a-SiO2 sphere compressed at maximum displacement.

Fig. 3: Electron beam hardening of nanoscaled a-SiO2. TEM images of a-SiO2 spheres after compression (top); experimental load-displacement curves and corresponding FEM simulations (center); plastic strain fields at maximum loads from FEM simulations (bottom).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-1941 Deformation modes of Au nanowires revealed by mechanical testing in TEM

Lee S.1, Im J.1, Bitzek E.2, Kiener D.3, Oh S.1
1POSTECH, Pohang, Republic of Korea, 2Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany, 3Montanuniversität Leoben, Leoben, Austria
shoh@postech.ac.kr

Shrinking the size of metallic structures not only leads to an increase of strength (i.e. the ‘smaller is stronger’ size effect), but also to a change in the deformation mechanism. In the case of uniaxial deformation of face-centered cubic (fcc) metal nanowires, the deformation mechanism can also change with the loading condition. According to recent molecular dynamics (MD) simulations, ultra-thin Au [110] nanowires (diameters of a few nm) deform predominantly by dislocation slip in compression, but in tension by deformation twinning. Here we report, by combination of in-situ transmission electron microscopy (TEM) and molecular dynamic simulation, the conditions under which particular deformation modes take place during the uniaxial loading of [110]-oriented Au nanowires [1].

In our deformation setup in TEM, a wedge-shaped top end of Au [110] nanowire was first compressed with a flat diamond punch (Fig. 1a), thus the initial deformation was localized near the contact region. Under such a strain gradient condition, the initial compressive deformation began with the emission of small prismatic loops from the top corner (white arrows in Fig. 1b). Initially, the loops appeared to replicate the perimeter of the contact line, but after a certain number of closed loops were punched out (typically less than ten), there was a clear transition in the nucleation mechanism; open loop dislocations started to bulge out and then released from the contact area (yellow arrows in Fig. 1b-c). As the contact area increased, ordinary dislocation slip along the inclined {111} slip planes dominated the compressive deformation (Fig. 1c and d).

The deformation mode of Au [110] nanowires changes from dislocation slip to deformation twinning as the loading condition is reversed from compression to tension. Moreover, once a Au nanowire has been twinned by the initial tensile loading, the subsequent compressive deformation was carried predominantly by detwinning instead of the expected dislocation slip (Fig. 2a and b). This twinning-detwinning behavior is capable of accommodating large plastic strains (> 30%) reversibly and repeatedly over many tension-compression cycles (Fig. 2c). Molecular dynamics simulations rationalize the observed behaviors in terms of the orientation-dependent resolved shear stress, i.e. Schmid factor, on the leading and trailing partial dislocations, their potential nucleation sites and energy barriers. The present in-situ TEM results demonstrate the primary role of the loading direction in determining the governing deformation mechanisms under uniaxial loading conditions.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2011-0029406).

Fig. 1: a. Deformation setup of Au nanowire in TEM. b. TEM DF image showing the initial compressive deformation by prismatic loops (white arrows) and half-loops (yellow arrows). c. TEM DF image showing ordinary dislocation slip (orange arrows). d. MD simulation showing the emission of half-loops (yellow arrow) and ordinary dislocation slip (orange arrows).

Fig. 2: a. A series of TEM DF images showing reversible twinning-detwinning deformation of Au nanowire during cyclic tension-compression. b. Schematics illustrating the corresponding loading condition for each TEM image in a. c. Plot of the axial strain versus the lateral displacement during the cyclic loading.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-1969 Observation of Strain Aging Behavior in Strain Based Line Pipe Steels using in-situ Heating and Straining TEM stage

Hong S.1, Ahn T.1, Kim S.1, Ro Y.2, Lee C.2, Kim Y.1
1Seoul National University, Seoul, Republic of Korea, 2Technical Research Laboratory, POSCO, Pohang, Republic of Korea
hong0133@snu.ac.kr

One of the new concepts for American Petroleum Institute (API) X100 grade line pipe steels was the strain-based design (SBD) approach. As demands increased for the harsh environmental applications such as the artic and seismic area, SBD line pipe steels were considered as a key solution. Even though the strength could be diminished by the processing or design, uniform elongation is the top-most property to attain in the line pipe steel. Many researchers have focused on the alloy design, combined with microstructure analysis and mechanical properties, to fabricate line pipe steel delivering both the transport efficiency and the performance. Full size X100 steel plate and pipe with 32mm thickness were selected and investigated in this study. The pipe shaping was achieved through UOE (U-ing, O-ing, and Expansion) piping process. The mechanical properties such as yield stress (YS), tensile stress (TS), and uniform elongation (uEl) were measured from the tensile test. Furthermore, microstructures were observed by scanning electron microscope (SEM) and transmission electron microscope (TEM). The dislocation structures of the plate and pipe were analyzed by selecting several layers through the thickness. Because the plastic deformation history of the surface is different from that of the center during the UOE piping process, it is expected that the dislocation density and structures were formed differently through the thickness. UOE process is typically followed by the anti-corrosion coating process, which requires heating the pipe up to 200 ~ 250°C. During the heating process for the anti-corrosion coating, the pipe reveals the strain aging phenomena giving yield drop in the stress-strain curves. To investigate both the strain and the thermal effect on the strain aging behavior of SBD X100 steels, in-situ heating and straining TEM stage was designed and applied to test the alloy. Each step of process conditions, such as applying stress and heat/cooling, was simulated in the TEM while observing the microstructural change. The analysis of strain aging behaviors was conducted.

This work was supported by the Development program(No.10040025) of the Korea Evaluation Institute of Industrial Technology grant funded by the Korea government the Ministry of Trade, Industry and Energy.

Fig. 1: TEM images of dislocation structures of the line pipe: (a) surface, (b) 1/4t, and (c) 1/2t

Fig. 2: in-situ heating and straining TEM stage: whole view (left), detail view (right)
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2081 In Situ 3D Studies of the Chlamydomonas Chloroplast Using Cryo-Focused Ion Beam Milling and Cryo-Electron Tomography

Schaffer M.1, Engel B. D.1, Cuellar L. K.1, Villa E.1, Plitzko J. M.1, Baumeister W.1
1Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
schaffer@biochem.mpg.de

A comprehensive understanding of eukaryotic photosynthesis, the process that converts light energy into biochemical energy, requires a molecular-resolution three-dimensional model of the chloroplast’s intricate structure. Although the first transmission electron microscopy (TEM) studies of this important organelle date back to the early days of TEM in the 1950s, these observations, and the subsequent studies in the following decades, were limited by artifact-inducing sample preparation techniques. While valuable knowledge has been gained by both freeze-fracture and conventional heavy-metal stained plastic section preparations, the three-dimensional native architecture of the chloroplast can only be visualized by cryo-electron tomography (cryo-ET) of vitreous samples. In situ cryo-ET of specific subsystems within larger eukaryotic specimens requires selected areas of vitreous material to be thinned to electron transparency (less than 500 nm). Until recently, cryo-sectioning with an ultramicrotome was the only method capable of achieving this goal. However, cryo-ultramicrotomy is a laborious and technically demanding technique, and furthermore, mechanical sectioning introduces inevitable artefacts such as compression deformations.
In this work, we show that cryo-focused ion beam (cryo-FIB) milling (1,2,3) provides an alternative method of sample preparation. As a compression-free technique for thinning vitreous material to any specified thickness, it can produce ideal artifact-free specimens for cryo-ET. We combined cryo-FIB with cryo-ET in a complete integrated cryo-workflow to obtain in situ 3D tomograms of the chloroplast within the unicellular green alga Chlamydomonas reinhardtii, the canonical algal model organism for studying photosynthesis.

References:
[1] M Marko et al., Nat Methods 4(3) (2007) p.215.
[2] A Rigort et al., PNAS 109(12) (2012) p. 4449.
[3] E Villa et al., COSTBI 23(5) (2013) p.771.

Fig. 1: Cryo-FIB preparation of Chlamydomonas reinhardtii cells. (a) An SEM image of a vitrified specimen on a TEM grid. (b) A FIB SE image of a lamella edge-on and (c) a top-down SEM image of a lamella. (d) A 2D slice from a tomographic reconstruction of a chloroplast within the intact cell, revealing the thylakoids and the chloroplast double membrane.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2095 In-situ Lorentz microscopy of high Bs and low core-loss Fe85Si2B8P4Cu1 nanocrystalline alloys

Akase Z.1,2, Shindo D.1,2, Sharma P.3, Makino A.3
1Institute of Multidisciplinary Research for Advanced Materials, Tohoku University. , Sendai, Japan, 2Center for Emergent Matter Science, RIKEN, Wako, Japan, 3Institute for Materials Research, Tohoku University, Sendai, Japan
akase@tagen.tohoku.ac.jp

The Fe-Si-B-P-Cu nanocrystalline alloys which exhibit excellent magnetic softness and relative high saturation magnetic flux density have been newly developed. This material has a homogeneous nanocrystalline structure composed of alpha-Fe grains with a size of about less than 20 nm which are realized by crystallizing the heterogeneous amorphous alloys. In this study, we observed the movements of the magnetic domain walls in the heat-treated Fe85Si12B6P4Cu1 amorphous-ribbons by in-situ Lorentz microscopy using a transmission electron microscope equipped with a magnetizing system, in order to understand the dependence of the magnetic properties on the microstructures.

Figure 1 shows a schematic illustration of the magnetizing system installed on a JEM-3000F instrument. The magnetizing specimen holder and two deflection coils are connected to an electric power source via three independent amplifiers. The two deflectors control the incident angle of the electron beam to avoid shifting of the image on the screen. When the amplifiers are connected to the DC source, a static external magnetic field is applied to the specimen. In order to observe the motion of the magnetic domain wall, the amplifiers are connected to an AC source.

The smooth movement of magnetic domain walls was observed in the specimen which was heat-treated at 430 °C, while the specimen which was heat-treated at 470 °C showed less-smoothness of the domain wall motions. Both of two specimens have the nanocrystalline structure in which the size of alpha-Fe crystallite is about 5 nm, but the electron diffraction pattern indicates that the latter specimen contains precipitates of boride. Figures 2a to 2d show Lorentz micrographs of the specimen which was heat-treated at 470 °C in a static external magnetic field of 3.8 kA/m, 4.3 kA/m, 4.6 kA/m and 4.7 kA/m, respectively. The direction of the external magnetic field is indicated by arrow at the top right of the Fig. 2. The positions of the magnetic domain wall are indicated by a dotted line, and previous positions of the domain wall were also plotted on the images. It is noted that a pinning of the motion of domain wall was observed at the position indicated by a white circle in Fig. 2c. It was considered that the precipitates caused the less-smoothness of the domain wall motions.

This work was supported by "Tohoku Innovative Materials Technology Initiatives for Reconstruction (TIMT)" funded by MEXT and Reconstruction Agency, Japan.

Fig. 1: A schematic illustration of the magnetizing system used.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2143 Observing impregnation dynamics at the liquid-solid interface using scanning electron microscopy: Charged-controlled SAPO-34 zeolite particle dispersion on SiC substrates

Tran C. M.1, Fordsmand H.1, Appel C. C.1
1Haldor Topsøe A/S, Nymøllevej 55, DK-2800 Kgs. Lyngby, Denmark
chmt@topsoe.dk

Insight into colloidal and interface processes benefits from observations made by electron microscopy. A wide palette of materials manufacturing techniques rely on drying of colloidal systems. The drying step plays a key role in distributing the suspended solid particles on a substrate and the density, clustering and packing of solid particles will intimately depend on the evaporation of the solvent. In general, drying is believed to depend on several parameters such as the surface tension of the liquid, the zeta potential of the particles and the state of the substrate. Up to now, observations of the dynamic processes during drying are scarce. Here, such investigations are reported for the dispersion of approximately 1 µm wide SAPO-34 zeolite particles suspended in liquid onto a porous outer surface of the SiC substrate, as a model system for automotive exhaust abatement catalysts. Using scanning electron microscopy (SEM) in combination with a differentially pumped vacuum system and a Peltier cooling stage, time-lapsed image series are acquired in situ during humidity variation at constant specimen temperature, whereby the dynamic arrangement of particles on the substrates is directly observed. Specifically, the effect of surface-modifications for cationic, anionic and neutrally charged particles in the suspension is shown to markedly affect the distribution on the SiC. Moreover, complementary SEM observations under cryo conditions of freeze-fractures of the fully hydrated samples are pursued to provide a snapshot of the particle distribution inside the porous SiC, Fig. 1. Differences in the arrangements of the zeolite particles in the liquid, indicate that electrostatic interactions between the charged particles and the substrate in the porous structure. These results can directly be explained by the electrostatic interaction between the SAPO-34 zeolite particles and the SiC substrate and proposes a method for guiding particle dispersions in porous support systems.

Fig. 1: SEM SE images for cationic, neutral and anionic washcoats. The zeolites are in a different arrangement depending on the charge. The color maps show where the particles (yellow) are situated compared to the SiC (black). The blue color is representing the ice.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2418 EBSD Measurements of the Twinning Process in an Mg-4wt%Li-Alloy with an in-situ Tensile / Compression Module in the SEM DSM 982

Fahrenson C.1, Driehorst I.1, Lentz M.2, Camin B.2, Berger D.1
1Technical University Berlin, Center for Electron Microscopy (ZELMI), Straße des 17. Juni 135, 10623 Berlin, Germany, 2Technical University Berlin, Chair Metallic Materials, Ernst-Reuter-Platz 1, 10587 Berlin, Germany
fahrenson@tu-berlin.de

The combination of a tensile / compression module in a scanning electron microscope (SEM) enables the in-situ analysis of the microstructure modifications as a function of the applied load and strain direction. To analyze the microstructure in a SEM quantitatively, an electron backscattered diffraction (EBSD) measurement is the most promising tool. Unfortunately, the information depth of the EBSD signal is very small; therefore, it needs first to be clarified first if an EBSD-signal might be recorded from stressed specimen surfaces. This investigation is the purpose of this paper.
These measurements require a large specimen chamber with the possibility containing the module and a sample stage whose loading capacity is large enough for the module. Furthermore, for EBSD measurements it is essential that the EBSD-detector can be positioned as close to the sample as possible to optimize the data collection. Additionally, shadows of the module on the detector should be minimized. We used a tensile / compression module from Kammrath & Weiss in the "Narrow version" and a Zeiss SEM DSM 982 with Gemini optic. The used EBSD-detector is a Pegasus system from EDAX with a high speed CCD camera (Hikari). All technical difficulties with the integration of the module, the shadowing and the sample alignment are described in [1].
An Mg-4wt%Li-1wt%Al alloy was investigated whose misorientation relations should be determined. The deformation behavior of magnesium alloys is significantly influenced by the activation of mechanical twinning. Hence, twin nucleation and growth will be observed through characteristic reorientations during several load steps. Figure 1 and 2 show an EBSD-measurement of an Mg-4wt%Li alloy of an unloaded sample with the initial grain orientation. Figure 3 shows the surface of the same specimenposition after a compression of 14% in horizontal direction. The surface of the specimen became quite rough indicating a strongly strained surface. Nevertheless, it is still possible to record meaningful EBSD-maps (fig. 4). Only some areas close to grain boundaries are strongly deformed. In addition, the EBSD-map reveals that the grains have a preferred orientation after compression. The change of the orientation through twinning might be observed in subsequently recorded EBSD-maps during compression. In figure 4 several grains are still twinned while others are already completely sheared.
The presented results confirm that EBSD-measurements are still possible on strongly compressed specimens and that the complete twinning process during the increasing deformation might be observed and analysed in-situ.

[1] Microscopy Conference MC2013, Regensburg

The authors would like to thank Prof. Reimers for providing access to the tensile / compression module.

Fig. 1: SEM-Image from the initial state; the marked grain is in all images the same

Fig. 2: EBSD-map from the initial state

Fig. 3: SEM-Image after 14% compression

Fig. 4: EBSD-map after 14% compression
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2483 High resolution cryo-CLEM: from cryo-light microscopy to cryo-TEM, through cryo-milling

de Marco A.1, Mayer T.1, Mahamid J.2, Arnold J.2, Plitzko J.2
1FEI Company, Munich, Germany, 2Max Plank Institute of Biochemistry, Munich, Germany
alex.demarco@fei.com

Correlative light and electron microscopy (CLEM) aims at combining the large field of view and chemical specificity of fluorescence microscopy with the high resolution ultra-structural details revealed by electron microscopy. CLEM can be extremely powerful in extending electron microscopy analysis to rare events that are impossible to target based on EM morphology alone. If CLEM is done on frozen hydrated samples there is also the opportunity to perform structural studies of complexes in situ.

Here is presented an innovative design for a cryo-light microscopy stage, developed to acquire data in cryo-light microscopy maximizing the resolution and minimizing the contaminations typically deposited on the sample during acquisition. The proposed design is extremely simple, where the stage is immobile and an inverted microscope is moved underneath. This allows the sample to be stored at cryogenic temperature, while the microscope and the objective are kept at room temperature in order to optimize the image quality.

Once the sample has been imaged in the light microscope, if suitable, it can directly go into the TEM for cryo electron tomography or single particle data acquisition. Considering the minimal amount of contamination accumulated during imaging it can easily be used for structural studies. In case the sample is too thick to be inspected in the TEM then a thinning procedure can be performed in a cryo dual-beam (Rigort A. et al JSB 2010; Rigort A. et al PNAS 2012). Relocation of a feature of interest identified in the light microscope and the dual beam is trivial thanks to the use of a cryo-shuttle which can be hosted in predefined orientation in both the light microscope and the dual-beam, as well as the use of a dedicated software framework.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2779 In-situ TEM studies of the electromigration process in a single InAs nanowire

Neklyudova M.1, Zandbergen H. W.1
1Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands
m.neklyudova@tudelft.nl

Electromigration (EM) is a phenomenon in which the electrical current flow of high density through a solid can lead to intensive atomic motion due to the high speed electrons transfer part of the momentum to the atoms (or ions) by collision. This phenomenon can lead to morphological and structural instabilities not only in metallic interconnections but also in semiconductor nanowires [1,2]. Since semiconductor nanowires are the subject of active study in virtue of their usage as low-dimensional systems, as building blocks for future nanoscale circuits [3], the EM becomes the key issue that controls the lifetime and stability of a nanoscale device.
In this work the process of EM in a single InAs nanowire was investigated by in situ TEM technique using a FEI Titan microscope operating at 300 keV. The EM experiments were carried out in a bias-ramping mode which allowed to perform accelerated experiments for EM process visualization in-situ TEM. The voltage applied for all cycles of EM experiments was set to 1200 mV. The resistivity calculated for the nanowire diameter 221 nm was 2*10-2Ω·cm. The current density for EM activation was about 3.6*104A/cm2. It was found that the EM in InAs nanowire starts at a position close to the cathode with formation of the cubic-shaped nanoparticles in the place of failure. The EDX analysis of the nanowire after EM experiments showed that the particle formed near anode part is indium. In the presentation all structural and chemical evaluations of the InAs nanowires during the electromigration will be discussed.

[1] D. Kang, T. Rim, C.-K. Baek, M. Meyyappan. Appl. Phys. Let. 103, 233504 (2013).

[2] C.-X. Zou, J. Xu, X.-Z. Zhang, X.-F. Song and D.-P. Yu. Journal of Appl. Phys. 105, 126102 (2009).
[3] Law, M., Goldberger, J., Yang, P. Annu. Rev. Mater. Res. 2004, 34, 83–122.

I would like to acknowledge ERC project 26792.

Fig. 1: Snapshots from the real-time TEM movie showing the first cycle of EM in InAs nanowire. (a) Initial configuration of InAs nanowire before EM experiments. (b)-(e) Images of the nanowire part pointed by blue square on (a) and taken at B- E times on I-V curve respectively. (f) Typical I–V curve. The red arrows indicate the bias-ramping direction.

Fig. 2: Snapshots from the real-time TEM movie showing the 2nd EM cycle (a) Nanowire configuration after 1st EM cycle (b)-(i) TEM snapshots taken at B-I times on I-V curve respectively (j) TEM image of the nanowire part marked by green square on (i) (k) Magnified image of the nanowire part marked by red square on (j). (l) I–V curve for the 2nd EM cycle
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2824 Understanding mechanisms of assisted sintering through dedicated in situ TEM experiments

van Benthem K.1
1University of California, Davis 1
benthem@ucdavis.edu

Sintering describes the densification of powder agglomerates through elimination of “empty space” between individual particles. [1] The application of electrical fields, currents and/or pressure in addition to heating can enable the accelerated consolidation of materials. While electric field assisted sintering, which includes spark plasma sintering and flash sintering, is already employed for the synthesis of a wide variety of microstructures with unique macroscopic properties, a fundamental understanding of the atomic-scale mechanisms that lead to enhanced densification is mostly absent from the literature. In this presentation, recent in situ transmission electron microscopy experiments will be reported that were designed to investigate specific densification mechanisms, including surface cleaning effects, i.e., dielectric breakdown of insulating surface oxides [2], mechanical properties of individual ceramic powder agglomerates [3], and electric field effects on the densification of yttrium-stabilized zirconia.

To quantitatively evaluate densification behavior we have developed an image processing tool to obtain three-dimensional densification curves from powder agglomerates. A variety of in situ TEM experiments was used to electrically contact individual nanoparticles (Figure 1a), apply mechanical pressure to particle agglomerates (Figure 1b), or expose particle agglomerates to electrical fields in non-contact mode. The results reveal that dielectric breakdown of insulating surface oxides on nanometric metal particles causes retardation of densification, while the morphology of ceramic powder agglomerates can limit densification through stabilization of pores. The application of electrical fields during in situ sintering experiments in the TEM reveals that the field strength in the absence of current has an appreciable influence on the densification behavior of Y-stabilized ZrO2. Moreover, the application of electrical fields promotes the formation of coincident site lattice grain boundaries and, hence, can accelerate grain growth in ceramic microstructures.

References

[1] Castro R, van Benthem K. Sintering: Mechanisms of Convention Nanodensification and Field Assisted Processes. Heidelberg: Springer, 2013.

[2] Bonifacio C, Holland TB, van Benthem K. Evidence of surface cleaning during electric field assisted sintering. Scripta materialia 2013;69:769.

[3] Rufner J, Holland TB, Castro R, van Benthem K. Mechanical properties of individual MgAl2O4 agglomerates and their effects on densification. Acta mater. 2014;69:187.

This work was supported by the University of California Laboratory Fee Program (12-LR-238313) and the Army Research Office (program manager: Dr. S. Mathaudu) under grant W911nf-12-1-0491-0.

Fig. 1: (a) Ni nanoparticles were contacted with an STM tip mounted on the TEM specimen holder to apply a local electrical bias [2]. (b) MgAl2O4 nanoparticle agglomerates were compressed by in situ nanoindentation [3]. Figures reproduced with permission.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2853 Correlating internal structure and mechanical properties of amorphous silica and gold micro-/nanoparticles using in situ mechanical testing in the scanning and transmission electron microscope

Herre P.1, Paul J.1, Romeis S.1, Niekiel F.2, Mačković M.2, Spiecker E.2, Peukert W.1
1Institute of Particle Technology (LFG), University of Erlangen-Nuremberg, Erlangen, Germany, 2Center for Nanoanalysis and Electron Microscopy (CENEM), University of Erlangen-Nuremberg, Erlangen, Germany
patrick.herre@fau.de

The ongoing miniaturization and reliability of functional materials and devices at micro- and nanoscale inevitably necessitates information on small scale mechanical properties. In the fields of e.g. optics and biomedical imaging, nanospheres of both, silica and gold, as well as hybrid core-shell structures made of these materials are of great relevance [1,2]. In order to elucidate deformation mechanisms and related mechanical properties on the nanoscale, compression of individual particles is particularly suitable due to the absence of strain gradient plasticity effects [3].

In the present work we use in situ mechanical testing in the scanning electron microscope (SEM) and transmission electron microscope (TEM) with the aim to characterize the mechanical properties of amorphous silica and gold micro-/nanoparticles. Thereby a custom built SEM supported manipulation device [4] and a TEM Picoindenter® (Hysitron, Inc.) are used.

Silica spheres are synthesized according to the Stöber-Fink-Bohn method [5] followed by thermal treatments at 400°C (S400), 800°C (S800) and 1000°C (S1000), respectively. Structural changes and mechanical properties are studied using in situ SEM indentation (Fig. 1b), combined with solid state nuclear resonance spectroscopy (NMR) and infrared spectroscopy. Reduced Young’s modulus and hardness of untreated silica particles (S70) (29.6±6.2 GPa and 6.0±0.6 GPa, respectively) increase after calcination at 1000°C (58.2±8.8 GPa and 13.4±0.9 GPa, respectively), as shown in Fig. 2b. The values achieved after calcination at 1000°C agree well with values known for bulk fused silica. In the course of heat treatment, infrared and NMR spectra (Fig. 3) reveal condensation of internal OH-groups and enhanced cross-linking of the silica network, resulting in particles with chemically and mechanically similar properties when compared to their bulk counterpart [6].

Gold nanoparticles (20 nm – 1 µm in size) are prepared by solid/liquid state dewetting of thin gold films. In situ SEM compression experiments show large strain bursts after elastic loading. Currently, cross-sections of as-prepared and deformed Au particles are investigated by ex situ TEM to get more insights into the internal particle structure. Nanomechanical testing in the TEM is focused on smaller Au particles with the aim to reveal deformation mechanisms in situ.

[1] Oldenburg et al., Chem. Phys. Lett. 288 (1998):243.
[2] Fuller et al., Biomaterials 29 (2008):1526.
[3] Gao et al., J. Mech. Phys. Solids 47 (1999):123.
[4] Romeis et al., Rev. Sci. Instrum. 83 (2012):095105.
[5] Stöber et al., J. Colloid. Interface Sci. 26 (1986):62.
[6] Romeis et al., Part. Part. Syst. Charact. (2014).

The German Science Foundation is gratefully acknowledged for financial support within the priority program “Particles in Contact” and the research training group 1896.

Fig. 1: a) Cumulative particle size distribution for untreated (S70) and heat treated (S400, S800, S1000) silica particles. Insets show representative SEM images for i) S70 and ii) S1000. b) Experimental setup for compression of single silica spheres between a diamond flat punch and a silicon substrate inside a SEM.

Fig. 2: a) Representative force-strain curves for silica particles from samples S70, S400, S800 and S1000. b) Reduced Young’s modulus, hardness and yield strength with respect to calcination temperature. At 1000°C, E* and HCEB approach the bulk values of fused silica.

Fig. 3: a) Infrared spectra of the heated silica particles. For higher temperatures densification and dehydroxylation occur. b) 29Si HPDEC and 29Si CP MAS NMR spectra of the samples. The mean number of siloxane (Si-O-Si) bonds per silicon atom (Qi, 1≤i≤4) increases from S70 to S1000, confirming enhanced cross-linking of the silica network.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2870 Revealing dislocation activities and deformation behavior in Nb2AlC using in situ nanoindentation in the transmission electron microscope

Schrenker N.1, Kabiri Y.1, Mueller J.1, Mackovic M.1, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM),Erlangen, Germany
nadine.schrenker@ww.stud.uni-erlangen.de

MAX phases are layered crystals with ternary or quaternary chemical composition. Due to their excellent electrical and thermal conductivity, as well as high oxidation resistance, they are in focus of intense research activities. Dislocation activities in MAX phases at room temperature (RT) are believed to be limited to slip along basal planes. In cyclic stress-strain curves, fully reversible, rate independent and closed hysteresis loops are observed. These features are attributed to the formation and annihilation of incipient kink bands (IKBs) and dislocation walls (DWs) [1]. Fig. 1 illustrates the formation of a KB. Initiated by elastic buckling above a critical maximum shear stress dislocation pairs of opposite sign form and move in opposite direction. It is believed that a nucleated KB immediately extends to the free surface. Hence, the attraction force between DWs disappears and a kink boundary is formed. An IKB does not dissociate into mobile DWs and thus is reversible, when the load is removed. KBs were observed ex situ by transmission electron microscopy (TEM) in single crystal Ti3SiC2, after nanoindentation perpendicular to basal planes (Fig. 1e) [2]. However, to date, the precise nucleation mechanism of IKBs and DWs is not known.
By means of in situ indentation in the TEM we reveal dislocation activities in Nb2AlC [3]. Undeformed Nb2AlC specimens exhibit basal plane dislocations with 1/3<11-20> type Burgers vectors. In situ indentation in dislocation-free regions and parallel to the basal planes (Fig. 3) reveals that basal plane dislocations nucleate and move in the same slip system without cross-slip or entanglement (Fig. 4). This confirms that these dislocations are mobile at RT, as proposed by Farber et al. in Ti3SiC2 [4]. The strain bursts in the load-displacement curve (Fig. 2) are assumed to be caused by dislocation nucleation. Indentation perpendicular to the basal planes results in an elastic deformation response, followed by fracture. Currently the formation mechanisms of IKBs and DWs are investigated in situ. Furthermore, plastic anisotropy is investigated by comparing pillar compression in the µm- and nm-range using scanning electron microscopy (SEM) and TEM, respectively. Prior to compression the pillar orientation is determined by electron backscatter diffraction with SEM and electron diffraction with TEM. For compression edge-on to the basal planes it is assumed that KB formation is more likely to occur than in pillar compression parallel to the c-axis.
[1] Barsoum et al., Nat. Mater. 2 (2003): 107
[2] Molina-Aldareguia et al., Scr. Mater. 49 (2003): 155
[3] Kabiri, Master Thesis, University Erlangen-Nuremberg (2013).
[4] Farber et al., J. Amer. Ceram. Soc. 81 (1998): 1677
[5] Barsoum et al., Metall. Mater. Trans. A 30 (1999): 1727

Financial support by the DFG via research training group GRK 1896 is gratefully acknowledged. The authors further thank Prof. Dr. Peter Greil for providing the samples.

Fig. 1: Schematic of kink band formation: (a) Elastic buckling, (b) corresponding shear diagram, (c) formation of dislocation pairs and (d) kink band [5], (e) Cross-sectional TEM image of an indent in a Ti3SiC2 (0001) single-crystal thin film with a maximum load of 40 mN [2].

Fig. 2: Load-displacement and time-displacement curve of an in situ indentation parallel to the basal planes. Start and finish of the indentation corresponds to points 1 and 2 or 3, respectively [3].

Fig. 3: TEM image showing the sample tilted in two beam condition around the <0001> zone axis, before an in situ indentation parallel to the basal planes [3].

Fig. 4: TEM image after an in situ indentation parallel to the basal planes revealing nucleation and propagation of basal plane dislocations [3].
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2887 In situ reduction of graphene oxide by Joule heating with TEM-STM system

Martín G.1, Claramunt S.1, Varea A.1, Yedra L.1,2, Rebled J. M.1,3, Sánchez-Hidalgo R.4, López-Díaz D.4, Velázquez M. M.4, Cirera A.1, Peiró F.1, Estradé S.1,2, Cornet A.1
1MIND/IN2UB, Departament d’Electrònica, Universitat de Barcelona, Marti i Franqués 1, 08028 Barcelona, Spain, 2CCiT, Scientific and Technological Centers, Universitat de Barcelona, C/Lluís Solé i Sabaris 1, 08028 Barcelona, Spain, 3Institut de Ciència de Materials de Barcelona-CSIC, Campus UAB, 08193 Bellaterra, Spain, 4Departamento de Química Física, Facultad de Ciencias Químicas. Universidad de Salamanca, E37008 Salamanca, Spain
gmartin@el.ub.es

Graphene has attracted a great deal of interest from scientists due to its intrinsic mechanical, thermal and electrical properties [1], [2]. Graphene, one-atom-thick layer of carbon, is a semiconductor with zero band gap [3] and high intrinsic mobility [4]. The excellent properties of graphene [5] have driven the search for methods for its large-scale production.

Graphene can be prepared by various methods [6] including micromechanical cleavage, epitaxial growth, chemical vapour deposition, exfoliation using graphite intercalation compounds and oxidation-reduction methods [7], [8]. These methods render high-quality graphene flakes although its low productivity makes them unsuitable for large-scale applications. The alternative strategy is the chemical oxidation of graphite or different carbon materials followed by chemical or thermal annealing.

Although the chemical oxidation of graphite is considered one of the most attractive methods to obtain graphene because it is cheaply, scalable and versatile, it presents the disadvantage that the O-containing groups produced by chemical oxidation, which make graphene oxide (GO) non-conducting [9], cannot be completely removed by the thermal annealing reduction. Thus, the level of reduction of GO is directly related to the conductivity, which can increase several orders of magnitude through the reduction process [10], [11].

In this work, GO, produced using a slight modification of the Hummers oxidation method from natural graphite flakes [12], has been in situ reduced by Joule heating in a TEM with a STM holder. The reduction of GO has been measured qualitatively from the comparison of conductivity of the sample before and after the reduction, all in the same experiment. Besides, with this technique it is possible to control the reduction from the measure of the conductivity of the sample and also characterize the sample during the experiment (both through TEM observation and through I-V characteristic). Indeed, the results show how GO has been reduced by observing a decrease of the resistance of more than four orders of magnitude.

[1] K.Novoselov et al, Science 306, 666-669(2004)
[2] A.K.Geim, Science. 324, 1530-1534(2009)
[3] Y.Zhang et al., Nature 459, 820-823(2009)
[4] K.I.Bolotina et al., Sol State Com 146, 351–355(2008)
[5] Y.Zhu et al., Adv. materials 22, 3903–3958(2010)
[6] D.Galpaya et al. Graphene 1, 30(2012)
[7] F.Bonaccorso et al. Materials today 15, 12(2012)
[8] Novoselov, K. S., et al. PNAS 102, 10451(2005)
[9] I.Jung et al. Nano Lett., 8 (12), 4283(2008)
[10] C.Gómez-Navarro et al. Nano Lett., 7 (11), 3499(2007)
[11] A.Bagri et al., nature chem. 2, 581(2010)
[12] B.Martín-García et al., ChemPhysChem 13, 3682(2012)

Fig. 1: TEM image of the tip contacting the GO during the experiment.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2942 Deformation Behavior of Silica Microparticles under Electron Beam Irradiation

Stauffer D.1, Bhowmick S.1, Major R.1, Asif S.1, Warren O.1
1Hysitron, Inc.
sanjit@hysitron.com

 

The studies of irradiation damage in silica are of significant interest because of its application in nuclear reactors, nuclear waste containers, optical fibers, and semiconductor devices. In this work, we investigate plastic flow and failure behavior of amorphous silica particles (1050±30 nm) under compressive stress inside a scanning electron microscopy (SEM). In situ quasistatic compression experiments were conducted using a PI 85 SEM PicoIndenter (Hysitron, Inc., Minneapolis, MN) with 2.5 mm flat punch diamond probe inside an SEM.

The deformation behavior of the particles before and after the experiments with beam on and beam off conditions can be seen in figures 1a-d. A large variation in the total plastic strain and tendency to fracture has been observed which varies with peak loads and beam condition. Here, plastic strain has been calculated as the ratio d/D, where D is the diameter of the particle and d is the amount of compression along the indentation axis. In quasistatic experiments with a 190s hold at 1 mN peak load, a particle deformed plastically to 55.5% strain when the beam was kept on during the test (fig. 1a). However, when the beam was turned off (fig. 1b), a similar diameter particle showed negligible strain (<0.05%). When the peak load is increased to 4 mN peak load with the beam on, a plastic strain of 57.8% strain was found with a crack that appeared on the surface as marked in fig 1c. In beam off condition, a similar sphere deformed plastically to 37% strain, occurring in conjunction with a large fracture which created a wedge-shaped missing segment as observed in figure 1d. The results in this study can be explained with the structural changes of the particles that has been reported in the literature. It has been observed that electron beam with sufficient intensity can change the pore structure of amorphous silica where small pores shrink and larger pores expand. The change in pore structure leads to softening of the particles which causes viscous fluid-type deformation. However, it should be emphasized that all the particles used in this study were exposed to electron beam before testing. So, it can be assumed that irradiation induced damage or defects in all the particles before loading were similar. This leads to a conclusion that the applied stress on the particles is playing a significant role in enhancing the structural changes and/or inducing more defects when electron beam is kept on. An important implication of this study is that electron irradiation under applied stress can induce significant instability and reduction in strength in silica resulting in lower lifetime in many devices where silica is an integral component.

Fig. 1: a-d: Images of deformation behavior of silica particles after quasistatic compression experiments with beam on and beam off conditions 1 mN and 4 mN. Fig e-f: Load-displacement plots at Pmax= 1 and 4 mN shows the effect of electron beam on plastic flow of the materials.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-2970 Light Irradiation of ETEM Samples for In-Situ Studies of Photocatalysts

Miller B. K.1, Crozier P. A.1, Zhang L.1
1Arizona State University, Tempe AZ, USA
benmiller002@aol.com

Inorganic photocatalysts are currently being intensely studied for their potential use for the production of fuels from H2O and CO2. Designing new efficient photocatalysts requires an increased understanding of the link between catalyst microstructure and activity. Environmental TEM (ETEM) is a promising technique for elucidating this link. However, while gaseous environments and variable temperatures are common to ETEM work, illumination of the sample by visible, ultraviolet, and infrared light is much less common.
We have installed a variable wavelength light source to irradiate the sample area in an FEI Tecnai F20 ETEM [1]. This will allow detailed analysis of the interaction between light and photocatalysts under reaction conditions. The current design, as seen in Figure 1, consists of a broadband light source with filters, optical fibers with a vacuum feedthrough, and a manipulator to precisely position the fiber tip with respect to the TEM sample in the microscope. The Energetiq® light source we use is a xenon lamp which is powered by an infrared laser, rather than the standard arc discharge, providing a smaller and brighter source. As seen in Figure 2, the broadband light source is capable of illuminating the sample with high intensity over a broad range of wavelengths. Optical filters may be used to specify smaller pass-bands. The measured intensity distribution at the sample position is sharply peaked at the center, reaching a maximum of about 1400 mW/cm2. This is more intense than the typical solar irradiance on the Earth’s surface which is only about 100 mW/cm2. The area of the sample illuminated with at least 90% of the peak intensity is an ellipse about 200x400 μm in size. This is large enough to cover 8 grid squares in a 200 mesh TEM grid. As shown in Figure 3, the fiber comes into the TEM at 90° to the sample rod, and the tip is cut at an angle, which refracts the light up toward the TEM sample. The angle chosen for this design was 30°, in order to sufficiently refract the light exiting the fiber while avoiding total internal reflection at the tip. This configuration, with the fiber independent of the sample rod, gives flexibility in the choice of sample holders, allowing other in-situ capabilities simultaneous to the light illumination. We are using this new capability to study the structure of titania-based nanostructured photocatalysts, and have observed changes in the surface of the titania when exposed to water and UV irradiation [2].

References:

[1] Miller, B. K.. and Crozier, P. A. Microscopy and Microanalysis 19, 461-469. (2013).
[2] Zhang, L. and Crozier, P. A. Nano Letters 13, 679-684. (2013).

The support from US Department of Energy (DE-SC0004954) and the use of ETEM at John M. Cowley Center for High Resolution Microscopy at Arizona State University is gratefully acknowledged.

Fig. 1: Overview of the light illumination system, showing the laser driven broadband light source, optics, and fiber manipulator. The monochromatic laser is used to power a xenon plasma which produces the broadband light.

Fig. 2: Light characterization, both spatial and spectral. The spatial distribution shows the intensity variation at the sample position. A faint dashed circle indicates the size of a 3mm TEM sample. The spectral distribution of the light is compared to the solar spectrum incident on the Earth’s surface.

Fig. 3: Cutaway view of the fiber as it extends into the pole piece gap. The fiber is supported at a 90° angle to the sample rod. The fiber tip is positioned close enough to the sample to provide maximum intensity without interfering with sample tilt. The tip is cut at an angle to refract light up toward the sample.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-3047 Reversible In-Situ TEM Electrochemical studies of Fluoride Ion Battery

Chakravadhanula K. V.1, Fawey M. H.2, Kübel C.1,2,3, Scherer T.2,3, Rongeat C.2, Munnangi A. R.1,2, Fichtner M.1,2, Hahn H.1,2
1Helmholtz Institute Ulm for Electrochemical Energy Storage (HIU), Albert-Einstein-Allee 11, 89081 Ulm, Germany, 2Institute of Nanotechnology (INT), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany, 3Karlsruhe Nano Micro Facility (KNMF), Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany
cvskiran@kit.edu

New research directions in Li-ion batteries are focusing on improvements of battery performance. Alternative technologies are investigated based on different chemistries using, e.g., sodium, magnesium or chloride as charge transfer ions in secondary batteries. Batteries based on a fluoride ion shuttle (fluoride ion battery) are an interesting alternative to Li-ion batteries as they can theoretically provide substantially higher volumetric energy densities compared to Li-ion batteries. Recently, the principle of a secondary battery based on a fluoride ion shuttle has been demonstrated [1]. Here, the electrolyte is one of the key components to obtain good cycling properties (e.g., resulting from fast F- conduction in fluoride ion batteries)[2].

For performing in-situ electrochemical studies, the stability of the components towards the electron beam (with beam energy and beam current being critical parameters) is essential to clearly interpret the results for the battery system in terms of the electrochemical performance. In the case of the F- batteries, the components besides being stable under the electron beam do not require an inert transfer, thus being suited as a good model system for in-situ electrochemical studies inside the TEM.

Ball milling of a mixture of (1−y)LaF3 and yBaF2 was employed to prepare La0.9Ba0.1F2.9. Initially, the electrolyte (La0.9Ba0.1F2.9) was studied for its structure, composition, porosity and stability under the electron beam. The cathode material based on a mixture of Bi (active material), La0.9Ba0.1F2.9 (ionic conductivity) and C (electronic conductivity) was prepared. Both materials were pressed to form a pellet. A lamellae of 60X35µm was prepared and electricaly contacted on the Aduro Electrochemical device (E-AEK11 from Protochips Inc.) inside the focused ion beam system (FEI Strata 400S). An Aduro sample holder from Protochips Inc. along with a Keithley 2611 sourcemeter in the FEI Titan 80-300 TEM were used in this work. SAED and HRTEM studies indicated the formation of a BiF3 phase in the cathode (reflections corresponding to d-values of 5.85Å(100)BiF3 and 3.37Å(111)BiF3, which were absent in the as-prepared state). The electrolyte structure at the interface to the cathode also changed during charging, where reflections corresponding to La were observed, indicating local reactions in the electrolyte leading to the formation of a La/LaBaF3/BiF3 cell. During discharging, most of the BiF3 was again reduced indicating the reversible behavior of the battery system in the TEM.

[1] M. Anji Reddy, M. Fichtner, J. Mater. Chem 21 (2011), p17059.

[2] C. Rongeat, M. Anji Reddy, R. Witter, et.al., ACS Applied Materials and Interfaces, 6 (2014) p2103.

Robby Prang is acknowledged for discussions towards sample preparation.

Fig. 1: (A)Thin lamella on MEMS device through FIB preparation. HRTEM micrograph and SAED pattern of cathode (B),(E) at 0V and (D),(G) at 3V respectively. (C)I-V curve during charging. (F)Line profiles of diffraction patterns showing intensive peaks of Bi and BaF3 cleaving at 3V, as evidence of structural change in the electrode, through formation of BiF3.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-3015 In situ applications of quantitative magnetic TEM imaging in magnetic nanostructures

Rodríguez L. A.1, Magén C.1, Snoeck E.2, Gatel C.2, Marín L.1, Serrano-Ramón L.1, Prieto J. L.5, Muñoz M.5, Ortolani L.6, Algarabel P. A.3, Morellón L.1, de Teresa J. M.3, Ibarra M. R.1
1LMA-INA, Universidad de Zaragoza, Zaragoza, Spain, 2CEMES-CNRS, Toulouse, France , 3ICMA, Universidad de Zaragoza-CSIC, Zaragoza, Spain, 4ISOM, Universidad Politécnica de Madrid, Madrid, Spain, 5IMM, CNM-CSIC, Madrid, Spain, 6IMM, CNR Bologna, Italy
cmagend@unizar.es

Magnetic imaging TEM techniques such as Lorentz Microscopy (LM) and Electron Holography (EH) are powerful tools to extract valuable quantitative information with nanometer-range resolution on the local magnetic states of nanomaterials. We usually study magnetic nanostructures at room temperature and at remanent state, but the use of special TEM specimen holders and/or set-ups opens the possibility to explore in situ the evolution of magnetization states upon the application of external stimuli such as temperature changes, magnetic fields or electric currents.

In this work, we present different applications of in situ LM and EH experiments under different scenarios: the in situ application of magnetic field with a calibrated objective lens is achieved by a smart control of the tilt angles of a double-tilt holder. A mathematical procedure has been developed to determine and/or quantify the in-plane component of the applied magnetic field. This capability is illustrated with two applications: the analysis of the domain conduit properties of magnetic nanowires by measuring the nucleation and propagation (depinning) fields [1], as shown in Figure 1(a); and the accurate determination of the magnetic hysteresis loops in nanoscaled magnetic tunnel junctions (MTJs) shown in Figure 2(b), where the different orientation of the magnetic induction component normal to the electron beam with respect to the induction in the sample’s plane can be quantified and corrected [2]. The potential of cryogenic conditions to study magnetism in nanostructures (down 100 K) is demonstrated by the investigation of the magnetic properties of La0.67Ca0.33MnO3 manganite thin films [2] and the strain effects on the suppressed ferromagnetism observed, an example of this characterization is displayed in Figure 2. Finally, the use of a dedicated two-contact TEM holder for the injection of spin-polarized currents in Py nanowires, illustrated in Figure 3, is applied to the investigation of current-induced domain wall manipulation phenomena.

[1] L. A. Rodríguez et al., Appl. Phys. Lett. 102, 022418 (2013).

[2] L. A. Rodríguez et al., Ultramicroscopy 134, 144-154, (2013).

This work was supported by the Spanish MINECO Projects MAT2009-08771, MAT2011-28532-C03-02 and MAT2011-28532-C03-03. The authors acknowledge the European Union under the Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative Reference 312483-ESTEEM2.

Fig. 1: (a) Depinning DW processes in CoFe nanowires by application of parallel and transversal magnetic fields (yellow arrows indicate the magnetic field directions), (b) Hysteresis loops in a Fe/MgO/FeV MTJ.

Fig. 2: Amplitude, magnetic phase shift (MAG) and magnetic flux (B) recorded by Electron Holography at low temperature (100 K) of (a) full magnetized and (b) with a superficial non-ferromagnetic layer (NFL) epitaxial La0.67Ca0.33MnO3 thin films grown on SrTiO3 substrates.

Fig. 3: Defocused LM images recorded before and after injecting an electrical current pulse of an amplitude of 1 mA and a duration of 100 μs. Red arrows point a domain wall which is propagated after the pulse.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-3115 In-situ heating using MEMS devices on FIB/SEM systems

Novák L.1, Vystavěl T.1, Faber P.2, Mele L.2, Šesták J.1
1FEI Company, Podnikatelská 6, 612 00 Brno, Czech Republic, 2FEI Company, Achtseweg Noord 5, 5600 KA Eindhoven, The Netherlands
libor.novak@fei.com

Introduction

Information on the kinetics of microstructural evolution is important in materials science fields like recrystallization, grain growth and phase changes. This requires reliable discrimination of differently oriented crystallites or different crystal phases, coupled with useful spatial resolution, temporal resolution and temperature change rate. Currently available SEMs have spatial resolution below 1 nm, temporal resolution below 10 ms (100 Hz frame rate), but existing heating holders only allow heating bulk samples up to 100°C per minute (~2°C/s). This prohibits experiments like quenching of metals and the long ramping time may cause the sample to change (oxidize, recrystallize) before the temperature range of interest is reached. In addition the backscatter (grain-, phase-) contrast is deteriorated because solid state detectors are blinded by the infrared radiation from the sample.

As a solution to these problems we present a MEMS heating holder [1], [2] in combination with in-situ sample preparation using a DualBeam FIB/SEM.

 

Sample preparation

A chunk of material is cut with the FIB and attached to the micromanipulator needle using beam-induced deposition (Figure 1a). After lift-out it can be further shaped using the FIB (1b). It is then placed on the MEMS heating holder, fixated with beam-induced deposition and cut loose from the needle (1c).

 

Ramping rates

The tiny thermal mass of the MEMS heater and sample allow temperature changes of 1000°C in just 50 ms (2·104°C/s) for a Cu sample of 20x50x50 um3 size (including settling to within 20°C, both for heating and cooling).

 

Imaging

The small heated area of the MEMS heater reduces the infrared radiation sufficiently that solid state detectors such as in-chamber BSE detectors and EBSD cameras can be used at elevated temperatures. Figure 2, for example, shows the melting of gold micro-particles at 1064°C imaged with the solid state BSE detector.

 

References:

[1] L. Mele et al. “A molybdenum MEMS microhotplate for high-temperature operation”, Sensors & Actuators: A. Physical, 2012 | 188 | 173-180

[2] B. Morana et al. “A silicon carbide MEMS microhotplate for nanomaterial characterization in TEM”, Micro Electro Mechanical Systems (MEMS), 2011 IEEE 24th International Conference on, 23-27 Jan. 2011, 380-383

This work was supported by Technology agency of the Czech Republic, project no. TE01020118 (Competence centre: Electron microscopy).

Fig. 1: Figure 1: Sample preparation from bulk sample: extraction using ion beam and manipulator (a); shaping of sample on manipulator needle (b); placement on MEMS heating holder (c). Horizontal field width is 50µm.

Fig. 2: Figure 2: Solid State Detector BSE imaging of gold particle solidification (a → b) and re-melting (b → c). Horizontal field width is 10µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-3273 Atomic Level In-situ Characterization of NiO-TiO2 Photocatalysts under Light Irradiation in Water Vapor

Zhang L.1, Crozier P. A.1
1Arizona State University, Tempe, USA
liuxian.zhang@asu.edu

Photocatalysts have potential applications for solar fuel generation either through water splitting. It is now recognized that atomic level in situ observations are critical for understanding the structure-reactivity in photocatalysts in the presence of reactant and product species and during in-situ light illumination. NiO loaded semiconductor photocatalysts with Ni first reduced and then partially re-oxidized at the surface has been reported to have good photocatalytic properties by forming a metallic Ni ohmic contact between NiO and the semiconductors [1]. TiO2 is a promising photocatalyst which has attracted intense research interest for decades since photo-decomposition of water by TiO2 was discovered. The TiO2 photocatalysts are either anatase or rutile which has been well known. Herein we use anatase as a model material to develop in situ photocatalytic experimental methodology and explore structure changes of NiO/semiconductor photocatalysts. In-situ heat treatment in H2 or O2 is applied to prepare initially Ni/TiO2, NiO/TiO2 or NiO-Ni-TiO2 materials in an environmental transmission electron microscope (ETEM). Then, without exposure to air, analysis can be performed in the same modified ETEM under in situ conditions in the presence of light and reactants to explore oxidation/reduction or interface changes under photocatalytic reactions.
NiO-Ni-TiO2 was prepared using Ni(NiO3)2 as the precursor following impregnation, calcination, reduction and partial re-oxidation. Ex-situ experiments were performed to achieve preliminary observations under exposure of xenon lamp with mirror reflecting light in the range 360nm to 460nm light. TEM images for ex-situ experiments were recorded with a FEI aberration corrected Titan TEM. Figure 1A&1B show initial Ni-NiO core-shell structures on anatase particles. The inside rounded darker particles are Ni metals with outside shells of polycrystal NiO. After 6 hrs exposure to light in liquid water the oxide shells become porous and the Ni metal is absent leaving a void (Figure 1C&1D). Ni may either be oxidized to NiO or dissolved into the solution during photocatalytic reactions.
In-situ heat treatments using a hot stage sample holder with H2 or O2 allows Ni or NiO to be prepared as the starting material for in situ photocatalytic experiments. A FEI Tecnai F20 ETEM was modified to allow samples to be illuminated with light from a broadband laser driven light source (EQ-99, Energetiq Inc.) with the intensity up to 10 suns [2]. Changes taking place in these Ni metal and NiO structures under in situ light exposure in presence of water vapor will also be discussed.
References:
[1]. Domen, K.; Kudo, A.; Onishi, T.; J. Catalysis, 1986. 102,92-98
[2]. Miller, B.K.; Crozier, P.A. Microscopy and Microanalysis 2013, 19, 461-469

The support from US Department of Energy (DE-SC0004954) and the use of ETEM at John M. Cowley Center for HR Microscopy at Arizona State University is gratefully acknowledged.

Fig. 1: a) Initial 5%wt NiO on anatase particles, b) zoom in of initial NiO-Ni-TiO2 structure, c) after ex-situ 6hrs exposure to 360nm-460nm light in liquid water, d) zoom in of NiO particle on anatase after exposure
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-3335 Mems-based heaters for ultrahigh temperature in situ TEM studies

Erdamar A. K.1, Zandbergen H. W.1
1Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands
a.k.erdamar@tudelft.nl

MEMS-based heaters are presently used in situ TEM studies for different purposes and applications such as morphological transformations of gold nanoparticles [1], sculpting of graphene [2], gas nanoreactors [3], thermal stability of nanoparticles [4]. An obvious question is how high we can go with such heaters. The heaters used in references 1-4 were made out of Pt embedded in SiN. Other heater materials like Mo and W can be applied to allow a higher temperature, in particular for temperatures above 1000 0C. Table 1 gives some thermal properties of various materials. Note that these are bulk properties and that for a thin film or in combination with another thin films they can be quite different. The applicability of the various metals depends strongly on the layer package of the membrane and the heater in the MEMS fabrication. For instance we use Pt heaters are embedded in ~500 nm thick SiN membrane, with ~6 µm wide viewing windows of 10-20 nm thick SiN (Figure 2). Since the embedding requires two SiN fabrication steps, Pt has to be stable in the gases used in the second SiN deposition. However, W is not stable in this process and thus embedding of W requires a different fabrication route.
Our MEMS heater contains four electrical connections that allow for temperature determination and heating. The big advantage of MEMS-based heater holder that the heat produced is low and thus little drift. It will be the thickness of the membrane, the size e.g. 1000 µ wide, and its thermal conductivity that determines the heat transfer to the holder. In this respect it is useful to consider the total system from specimen to holder as a set of thermal resistors (Figure 3). The temperature of the sample on a thin window will depend on the heat transfer (and thus the thermal resistances of the components between the sample and the heater. At low temperatures (up to 500°C) the irradiation is relatively small and one can assume that the temperature of the sample is about equal to that of the heater even if the thermal resistance between the thin window and the sample is high. But at high temperature various components of the heater will irradiate which add to the uncertainty of the temperature of the sample.
We are exploring in particular the use of W as heater material, with SiN, SiC and Al2O3 as membrane material. Recently we made heaters that are at least stable at 1250°C over 24 hours and are trying to push this up to 1400°C by a optimization of materials and process steps. We will report on these optimizations in the presentation.

[1] Young, N.P. et.al. Ultramicroscopy 2010, 110, 506-516.
[2] Song, B. et.al. Nano Letter 2011, 11, 2247-2250.
[3] Malladi, S. et.al. Chemical Communication 2013, 49, 10859-10861.
[4] Yalcin, A.O. et.al. Nanotechnology 2014, 25, 055601.

This work is part of the research programme of The European Research Council (ERC) NEMinTEM 267922.

Fig. 1: Table 1: (a) Thermal properties of support materials, (b) and heater materials.

Fig. 2: (a) Optical images of the center of the MEMS-based heater with an embedded Platinum wire for local heating with four connections, (b) electron-transparent windows with a diameter of 6 µm and 20 nm thick SiN.

Fig. 3: Schematic representation of the various thermal resistors (R1-R6) that determine the heat transfer of the heater to the outer tube of the TEM holder. At high T the specimen itself, its contact with the support of the heater and the support-heater contact introduce thermal gradients and thus the real situation is more complicated than indicated.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-3342 Applying of 6-carboxyfluorescein (6-FAM) to cytogenetics

Galkina S.1, Saifitdinova A.1, Bogomaz D.1, Radaev A.1, Gaginskaya E.1
1Saint-Petersburg State University, Saint-Petersburg, Russia
chromas.spbu@gmail.com

6-carboxyfluorescein (6-FAM) is one of the most commonly employed and simplest fluorescent reagents to use in oligonucleotide synthesis. 6-FAM is highly reactive, water-soluble single isomer of fluorescein, with absorbance/emission maxima in the visible region of the electromagnetic spectrum (492/517 nm respectively). 6-FAM plays a particularly important role in real-time PCR and SNP-analysis, being used in TaqMan probes, Scorpion primers and Molecular Beacons. Oligonucleotides labeled with 6-FAM at the 5’-end are widely adopted as PCR and DNA sequencing primers to generate fluorescently-labeled products for sequencing and genetic analysis. Per se 6-FAM-labeled oligonucleotides can be used as hybridization probes for fluorescent in situ hybridization (FISH), for example for a direct visualization of microorganisms in human and animal clinical samples (e.g. Behrens et al., 2004; Lin et al., 2011; Fontenete et al., 2013).

We used 6-FAM-labeled oligonucleotide probes specific for various chicken tandem repeats to detect RNA-transcripts and to localize them on giant transcriptionally active lampbrush chromosomes dissected from growing chicken oocytes. Lampbrush chromosomes have distinctive chromomere-loop patterns that enable high-resolution cytogenetic mapping of unique and repeat nucleotide sequences. We report that due to the high brightness and relatively long lifetime, the 6-FAM is found to be well suited for FISH proceeded accordingly with a DNA/(DNA+RNA) hybridization protocol.

The work is supported by SPbSU grant 1.37.153.2014 and grant for Leading Scientific Schools 3553.2014.4. The equipment used was provided by SPbSU Resource Research Centers “Chromas” and “Center for molecular and cell technologies”.

Fig. 1: FISH with oligonucleotide probe PO41 on chicken lampbrush microchromosomes. (a) Representative smallest microbivalents probed with PO41 labeled with 6-FAM (green signal); (b) microbivalents probed with PO41 labeled with Cy3 (red signal). Chromosomes are counterstained with DAPI. Left panels – phase contrast images. Scale bar 5 μm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-3408 In situ TEM deformation of a bulk metallic glass with a K2-IS detector

Gammer C.1, Rentenberger C.2, Karnthaler H. P.2, Czarnik C.3, Beitlschmidt D.4, Pauly S.4, Eckert J.4, Minor A. M.1
1National Center for Electron Microscopy, Lawrence Berkeley National Laboratory, Berkeley, USA, 2Physics of Nanostructured Materials, University of Vienna, Vienna, Austria, 3Gatan, Inc., Pleasanton, USA, 4IFW Dresden, Dresden, Germany
cgammer@lbl.gov

Bulk metallic glasses are an exciting new class of materials due to their unique mechanical properties, such as high strength and good wear resistance. However, potential applications are hindered by their low ductility caused by the formation of shear bands leading to catastrophic failure [1]. The origin of these shear bands remains unknown. In order to investigate the structural mechanisms of shear band formation, in-situ deformation was carried out inside a TEM using a Hysitron Picoindenter and a Gatan K2 high speed direct electron detector. Samples were made from a bulk CuZrAlAg rod produced by vacuum casting.

During the compression of nanopillars slipping events were observed but since the bands were not necessarily in projection they were difficult to analyze. To overcome this problem we used notched pillars to localize the deformation. Fig. 1 shows the results of a compression test carried out in dark-field mode. The video was recorded using a Gatan K2-IS direct detection camera at a frame rate of 400 f/s. The load displacement curve shows a long elastic regime followed by a sudden load drop. Two images were extracted, one right before and one right after the load drop (indicated with a and b, respectively). The images show the formation of a shear band as concluded from the shear offset that is indicated in (b). During further loading multiple small load drops can be observed followed by a larger loaddrop. The images taken before and after the large loaddrop (c and d) reveal that the load drop is caused by an abrupt slip event. The same shear band is reactivated (the trace of the shear band is indicated in (d)). Due to the fast frame rate of the camera it is possible to conclude that the time for the slip event was less than 2.5ms. The results indicate that after the initial formation of a shear band the pillar slips along this shear band in a stick-slip motion.

In addition to compression tests, tension tests of the metallic glass were carried out. Tension samples were made by transferring the sample to a Hysitron Push-to-Pull Device that allows using the Picoindenter as in-situ tensile apparatus [2]. Fig. 2 shows the result from an in-situ tensile test acquired in bright-field mode. The corresponding load displacement curve shows elastic deformation followed by an abrupt fracture with no indication of plasticity. In addition, digital image correlation from decorated samples was used to examine the origin of shear band formation. These results will be described in terms of the relationship between local shear transformation zones and eventual shear band formation and propagation.

[1] A.L. Greer. Science 267 (1995) 1947.
[2] H. Guo, et al. Nano Lett. 11 (2011) 3207.

The authors acknowledge support by the Austrian Science Fund (FWF):[I1309, P22440, J3397] and by the National Center for Electron Microscopy, Lawrence Berkeley Lab, supported by the U.S. Dept. of Energy under Contract # DE-AC02-05CH11231.

Fig. 1: Load displacement curve recorded during in-situ compression. After elastic deformation loaddrops can be observed. The initial one corresponds to the formation of a shear band (images from the dark-field video are shown in a+b). After some small loaddrops, a larger one is observed that results from an abrupt slip along the same shear band (cf. c+d).

Fig. 2: Stress strain curve recorded during an in-situ tensile test. The video acquired in TEM bright-field mode shows an elastic elongation of the tensile specimen. Two frames corresponding to the initial state and the elongated state are shown in (a) and (b). After elongation the sample fractures abruptly and shows no ductility (c).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-3442 Direct evidence for orbital angular momentum transfer from electron vortex beam

Thirunavukkarasu G.1, Yuan J.1, McKenna K.1, Babiker M.1
1Department of Physics, University of York, Heslington, York, YO10 5DD, United Kingdom.
t.gnanavel@york.ac.uk

Optical vortices have become well known for a vast range of applications such as optical sensors, tweezers, nanoparticle trapping and manipulation etc., since they were first reported by Allen et al [1]. Compared to optical vortices, electron vortices are relatively new. They were first predicted theoretically by Bliokh et al [2] and experimentally realised by several groups in the subsequent years utilising either a phase plate method [3] or the holographic mask method in a transmission electron microscope [4-6]. These electron vortex beams have the characteristics of orbital angular momentum.

The rotation of gold nanoparticles subject to electron vortex beams has been reported by our group [6] as well as by Verbeeck et al [7]. In this presentation, we focus on experimental evidence that such rotation is direct proof of the mechanical transfer of orbital angular momentum from the beam to the particles. The experiment has been conducted in a JEOL 2200FS double-aberration corrected TEM operating at an acceleration voltage 200kV which utilises a specially designed condenser mask aperture with a fork dislocation to produce the required electron vortex beams. The motion of the nanoparticle subject to the vortex beam illumination is examined by video microscopy and frame-by-frame image analyse. The time series of particle rotation can be obtained and detailed analysis allows the rate and sense of the rotation to be determined.

The chirality of the beam is deduced by comparing the through focus images of the hologram mask in the condenser aperture with the simulation. From the phase structure of the simulated beams, the sense of the rotation of the particle flux can be deduced unambiguously (Fig. 1). As can be seen in figure 2a-d and figure 2e-h a clear trend of opposite rotation in both l = ±1 beams was observed. This shows that the rotation induced on the nanoparticle is consistent with the chirality of the electron vortex beam, indicating direct transfer of orbital angular momentum. However, a detailed examination of the induced rotation shows signs of stochastic processes, indicating that the rotation is dissipative due to friction between the nanoparticle and the substrate.

[1] L Allen et al, Physical Review A 45 11 (1992), p. 8185.

[2] K Bliokh et al, Physical Review Letters 99 (2007), 190404.

[3] M Uchida and A Tonomura, Nature 464 (2010), p. 737.

[4] J Verbeeck et al, Nature 467 (2010), p. 301.

[5] BJ McMorran et al, Science 331 (2011), p. 192.

[6] T Gnanavel, J Yuan and M Babiker, in Proc. European Microscopy Congress, ii, edited by DJ Stokes and J Hutchison (Royal Microscopical Society, Oxford, 2012).

[7] J Verbeeck et al, Advanced Materials 25 (2013), p.1114.

The authors gratefully acknowledge funding from the EPSRC (Grant No. EP/J022098). Thanks are also due to the York JEOL Nanocentre for the provision of microscopy facilities and JEOL, U.K. for financial support.

Fig. 1: Determination of chirality of the electron vortex beams.

Fig. 2: Series of images showing (a-d) anticlockwise rotation under = +1 and (e-h) clockwise rotation under = -1 beams and corresponding FFTs. The arrows are pointing to the same diffraction peak to highlight its angular orientation.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-5752 Development of In-Situ Wet-Cell Electron Microscope Holder for Oxygen Nano-bubbles by Platinum

Zheng H. T.1, Liu S. Y.1, Tsai C. T.2, Haung T. W.1, Tseng F. G.1, Chen F. R.1
1Engineering and System Science Department/National Tsing Hua University, Hsinchu, Taiwan, 2Dept. of Material Science and Engineering, National Chung Hsing University, Tai-Chung, Taiwan
applebyapple1@gmail.com

Recently, wet-cell electron microscopy provides a new method for investigating crucial scientific issues within liquid which beyond the conventional electron microscopy. The progress in electron microscopy pushes the capability of viewing as close as the original phenomena occur and thus may open new scientific windows in multi-field due to the spatial resolution in sub-nanometer as well as tens of millisecond time-resolved power. Much more researches has been published and included various field such as electrochemistry [1], catalyst material [2, 3], and biophysics [4]. Heimei et al firstly visualize growth dynamics of Platinum nanocrystal with nanometer resolution in wet cell TEM [2]. The above discoveries provide the key to understanding toward whole mechanism for synthesizing more efficient catalyst materials. In this research, owing to the significance of catalyst, we put more focus on investigating catalytic process of Platinum. For this purpose and to approach real case, we built up the platform with function of liquid circulation to fit with currently TEM (JEOL, JEM-2010 LaB6 equipped with Gatan multi-scan CCD) observation respectively, that is in-situ wet-cell electron microscope holder. The wet-cell chip is made by micro electro mechanical systems (MEMS) process and the observing window is 50nm Si3N4 [5, 6].
For TEM observation, the platinum nanoparticles was carried by multi-wall carbon nanotube was dropped onto wet-cell chip with electron transparent Si3N4 membrane then sealing was completed by a set of o-rings. The solution contains 0.08wt% of H2O2 have been transported into the observation area by syringe pump; additionally, the flow rate was accurately controlled below 0.15 mL/hr that is the key to preventing membranes broken. The bubble formation is due to the well-known equation: 2H2O2→ 2H2O+O2. Platinum serves as catalyst to promote hydrogen peroxide decomposed into water and oxygen, which contribute to the source of bubble generation.
References:
[1] M. J. Williamson, R. M. Tromp, P. M. Vereecken, R. Hull and F. M. Ross, Nature Materials 2 (2003), p. 532.
[2] H. Zheng, R. K. Smith, Y. W. Jun, C. Kisielowski, U. Dahmen and A. P. Alivisatos, Science 324 (2009), p. 1309.
[3] J. M. Yuk, J. Park, P. Ercius, K. Kim, D. J. Hellebusch, M. F. Crommie, J. Y. Lee, A. Zettle and A. P. Alivisatos, Science 336 (2012), p. 61.
[4] M. J. Dukes, D. B. Peckys and N. D. Jonge, ACS Nano 4 (2010), p. 4110.
[5] T. W. Huang, S. Y. Liu, Y. J. Chuang, H. Y. Hsieh, C. Y. Tsai, Y. T. Huang, U. Mirsaidov, P. Matsudaira, F. G. Tseng, C. S. Chang, and F. R. Chen, Lab chip 12 (2012), p. 340
[6] T. W. Huang, S. Y. Liu, Y. J. Chuang, H. Y. Hsieh, C. Y. Tsai, W. J. Wu, C. T. Tsai, Utkur Mirsaidov, P. Matsudaira, F. G. Tseng and F. R. Chen, Soft Matter 9 (2013), p.8854

This work was supported by National Science Council (NSC102-2321-B-007-007 and NSC 102-2120-M-007-006-CC1).

Fig. 1: Fig. 1(a) The cross section view of TEM liquid holder tip: electron beam penetrate the liquid thickness which defined by metal spacer between chips and sets of o-rings are used for vacuum sealing. The nitride membrane is 50 nm for each piece and (b) assembling diagram of TEM liquid holder.

Fig. 2: Fig. 2, TEM images of nano-bubbles, phenomena of the bubbles merge as indicated in dash line region with time by injecting H2O2 solution (a) ~ (g).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-5919 Real-time observation of in-situ cation exchange in CdSe-PbSe nanodumbbells during epitaxial solid-solid-vapor growth

Yalcin A. O.1, Goris B.2, Bals S.2, Van Tendeloo G.2, Casavola M.3, Vanmaekelbergh D.3, Tichelaar F. D.1, Zandbergen H. W.1, van Huis M. A.3
1Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands, 2Electron Microscopy for Materials Science (EMAT), University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 3Debye Institute for Nanomaterials Science, Utrecht University, Princetonplein 5, 3584 CC Utrecht, The Netherlands
a.o.yalcin@tudelft.nl

Both the synthesis and design of hetero-nanocrystals (HNCs) have undergone a rapid development, whereby PbSe and CdSe NCs are key materials acting as functional building blocks within a wide variety of heterogeneous nanostructures.1,2 Heat treatment of HNCs can induce new interface designs, exemplified by the transformation of PbSe/CdSe core/shell systems into PbSe-CdSe bi-hemispheres.2 Here, we report an in-situ heating-induced epitaxial PbSe NC domain growth at the solid-solid PbSe-CdSe nano-interface through cation exchange. We show that Pb replaces Cd at the PbSe/CdSe interface, resulting in growth of the PbSe phase at the expense of the CdSe phase.3 In analogy with vapor-liquid-solid4 and vapor-solid-solid5 growth mechanisms, the currently observed process could be called solid-solid-vapor (SSV) growth as the Cd evaporates, either as neutrally charged Cd atoms or in a molecular complex such as Cd-oleate. Figure 1 shows the elemental maps of CdSe-PbSe HNCs at each stage of the cation exchange during epitaxial SSV growth mechanism. As a result of the cation exchange from CdSe to PbSe, the crystal structure transformed epitaxially from hexagonal wurtzite (WZ) to cubic rock-salt (RS). Figure 2 shows this transformation at atomic resolution. When the HNC was heated from 160 ⁰C (Figure 2a) to 180 ⁰C (Figure 2b), the brighter intensity corresponding to PbSe advanced into the CdSe region. The PbSe RS (200) lattice spacings started to appear along the nanorod domain instead of the CdSe WZ (0002) lattice spacings, as confirmed by the Fourier Transformation (FT) patterns shown in the insets. It is clear that the cation exchange takes place at the PbSe/CdSe interface and propagates epitaxially (layer by layer) along the WZ<0001> direction.

[1] Son, D. H. et al. Science 2004, 306, 1009-1012.
[2] Grodzińska, D. et al. J. Mater. Chem. 2011, 21, 11556-11565.
[3] Yalcin, A. O. et al. Nano Lett. 2014, 14, 3661–3667.
[4] Gudiksen, M. S. et al. Nature 2002, 415, 617-620.
[5] Persson, A. I. et al. Nat. Mater. 2004, 3, 677-681.

This work is part of the research programme of the Foundation for Fundamental Research on Matter (FOM), which is part of the Netherlands Organization for Scientific Research (NWO).

Fig. 1: HAADF-STEM images and chemical maps of CdSe-PbSe HNCs at (a-d) 100 ⁰C (initial configuration), (e-h) 170 ⁰C, and (i-l) 200 ⁰C. In (e-h), a partially transformed nanorod is present. In (i-l), two PbSe-CdSe HNCs became full PbSe domains. The Se remains in place during the transformation. Reprinted with permission from Ref. [3].

Fig. 2: HAADF-STEM images of CdSe-PbSe HNC. With heating from 160⁰C (a) to 180⁰C (b), WZ CdSe nanorod started to transform to RS PbSe. The spot depicted with an arrow in the inset FT in Fig. 2a corresponds to WZ CdSe(0002) spacing. It disappeared in the inset FT of Fig. 2b, confirming the WZ to RS transformation. Reprinted with permission from Ref. [3].
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-7-P-6054 In-situ observation of gold nanorod self-assembly

Novotný F.1, Wandrol P.2, Proška J.1, Šlouf M.3
1FNSPE, Czech Technical University, Břehová 7, 115 19 Prague, Czech Republic, 2FEI, Podnikatelská 6, 612 00, Brno, Czech Republic, 3Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
filip.novotny@fjfi.cvut.cz

Self-assembly organize gold nanorods (AuNRs) encapsulated by cetyltrimethylammonium bromide (CTAB) bilayer into an ordered material. The adsorbed molecules not only stabilize colloidal dispersion but also create “glue” among nanorods in the superlattice. Configurational entropy and depletion mediated interactions are commonly considered in the process of supracrystals growth. However, many questions arise about the dynamics of self-assembly in general and particularly about gold nanorods self-assembly in drying colloidal drop. Here we demonstrate the vizualization of the dynamic behaviour of the AuNRs (20 nm x 60 nm) in viscous CTAB/water environment using scanning transmission electron microscopy (STEM) in environmental conditions (STEM-in-ESEM). We observed several distinct stages of AuNRs self-assembly at the liquid-gas interface under space confinement, during controlled evaporation of solvent. The formation of free standing membrane of close-packed nanorods with vertical orientation around the inner edge of the carbon membrane hole, the formation of side-by-side AuNR chains and the sensitivity of the self-assembly process to the irradiation of the electron beam will be shown. Moreover, the viscous environment of the membrane enables to observe the dynamics of the self-assembly process on timescale of seconds. Particular events can be traced such as the nucleation and growth of the 2D crystals around the rim of the holey carbon membrane, the slowing down of the Brownian motion of loose tip-to-tip rod assemblies and convective flows in nano-environment revealed by their collective translation movement besides and the effects of the electron probe upon the prolonged exposure. A time lapse series of micrographs will used to demonstrate such capatibility.

This work was supported by the Czech Science Foundation project P205/13/20110S and P205/10/0348, Technology Agency of the Czech Republic project TE01020118, and also joint FEI & Czechoslovak Microscopy Society scholarship 2011.

Fig. 1: Scheme of in-situ observation of self-assembly of AuNRs. (a) AuNR solution is deposited on top of a holey carbon TEM grid. (b) Droplet undergoes rapid evaporation. (c) Collapsed drop concentrates the AuNR/CTAB volume to form a electron transparent hydrated viscous membrane. (below) Micrograph of AuNR array forming inside holey opening.
↑ Go to the Table of content ↑

IT-8. Ultrafast microscopies

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-8-IN-1690 Imaging surface plasmon polaritons by fs-transmission electron microscopy

Carbone F.1
1LUMES, ICMP, SB, Ecole Polytechnique Fédérale de Lausanne
fabrizio.carbone@epfl.ch

In this seminar, we will review the recent advances in fs-transmission electron microscopy. The design and implementation of a fs-resolved transmission electron microscope will be briefly introduced and its overall performance in terms of time, energy and spatial resolution will be presented. Thanks to this technology, the direct imaging of light-induced surface plasmon polaritons in nanostructures is enabled. When electrons and photons are overlapped spatially and temporally on a nanostructure, the evanescent field photoinduced at the edges of the latter interacts with the electrons allowing them to absorb and emit photons from the pump laser beam. This results in sideband peaks spaced by an energy corresponding to the pump photon energy on both the energy gain and loss sides of the elastic electrons peak. By selecting one or more of these sidebands via energy filtered imaging, snapshots of the surface plasmon polaritons can be taken. Such a technique is called Photon Induced Near Field Electron Microscopy (PINEM). By controlling the properties of light excitation, its energy, polarization and intensity, the distribution of the field around a given nanostructure can be controlled, providing a unique tool for the characterization and manipulation of optoelectronic circuits. The life-time of the surface plasmon polariton waves on metallic materials is found to be ultrafast, comparable to the laser excitation pulse duration (100 fs), and reveals information about the surface morphology and its electronic properties.

This work was supported by an ERC starting grant.

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-8-IN-5754 Time-Resolved Imaging of Surface Plasmon Polaritons by Photoemission Microscopy: The Next Generation

Meyer zu Heringdorf F. J.1
1Faculty of Physics and Center for Nanointegration Duisburg-Essen (CENIDE), University of Duisburg-Essen, 47048 Duisburg, Germany
meyerzh@uni-due.de

Observing surface plasmon polaritons (SPPs) in a photoemission electron microscope (PEEM) is possible via a two photon photoemission (2PPE) process, if ultra-short laser pulses of a suitable wavelength are directed onto a surface with plasmonic structures. In the past, we used a grazing incidence angle of 65-74° of the laser light relative to the surface normal for PEEM-based SPP imaging. The resulting SPP contrast was in this case described as a Moiré-pattern [1,2]. Properties of the SPP, however, can only be inferred indirectly from the Moiré pattern in grazing incidence geometry. For instance, SPPs propagating in different directions across the surface produce Moiré-patterns with a different fringe-spacing. A “normal incidence” geometry – harder to achieve due to the geometrical restrictions of the available PEEMs – is overall better suited for SPP imaging. The cylindrical symmetry caused by the incidence of the laser pulses normal to the surface results in the same imaging conditions for all SPPs, independent of their propagation direction. Also, the spacing of the Moiré fringes resembles the SPP wavelength, and in this respect normal incidence 2PPE PEEM provides a direct conceptual visualization of the SPP phase fronts in time and space. In time-resolved experiments under normal incidence conditions the direct observation of isolated SPP wave packets is then possible. Normal incidence 2PPE PEEM offers the possibility to study SPP reflection, transient SPP interference, and SPP focusing in time and space.

[1] L.I. Chelaru, F.-J. Meyer zu Heringdorf, Surface Science 601 (2007) 4541
[2] N.M. Buckanie, P. Kirschbaum, S. Sindermann, F.-J. Meyer zu Heringdorf, Ultramicroscopy 130 (2013) 49

Financial support by the German Research Foundation (DFG) through programs "SFB616: Energy Dissipation at surfaces" and "SPP1391: Ultrafast Nanooptics" is gratefully acknowledged.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-8-O-1703 Electron Pulse Properties and PINEM Aberrations in Ultrafast Transmission Electron Microscopy

Flannigan D. J.1, Plemmons D.1
1Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN, USA
flan0076@umn.edu

In ultrafast transmission electron microscopy (UTEM), extension of the static imaging and analytical capabilities of transmission electron microscopy to the ultrafast temporal domain relevant for many atomic and nanoscale processes allows for visualization of non-equilibrium structural phenomena. As in pump/probe spectroscopic techniques, the operating principle of UTEM requires – at some point during the experiment – temporal overlap of the femtosecond photon and electron pulses at the specimen. At time zero (i.e., precise overlap of the pulses), significant photon absorption by the freely-propagating electrons and population of virtual states occurs, and peaks occurring at integer multiples of the photon energy can be observed to the gain-side of the zero-loss peak in electron-energy spectra. Because this process, called photon-induced near-field electron microscopy (PINEM), is observed when the pulses are overlapped in space and time at the specimen, proposals for using this phenomenon to measure the total UTEM response function and the electron pulse shape and duration emerged during the initial experimental observations.

In this talk, we will discuss considerations for isolating the inherent artifacts of the highly non-linear near-field interactions from the actual pulse characteristics. Using theory developed to describe these interactions, we will discuss how temporal cross-sections of peaks in the electron-energy spectra corresponding to high-order transitions are expected to exhibit the true temporal behavior of the electron pulses. In general, the exceedingly small portion of the pump laser pulse capable of initiating such transitions results in the temporal widths converging to the electron packet duration. Additionally, population of quantized virtual states occurring for an electron beam focused on the edge of a nanostructure suggest that the resulting energy distribution may produce well-defined chromatic aberrations in images arising from the velocity-dependence of magnetic lens focusing (Fig. 1). As such, we will discuss the prospect for detecting such phenomena and its potential as a means of determining the UTEM instrument response without the need for a spectrometer. Appropriate interpretation of observed spectroscopic and image features should in principle enable systematic temporal and spatial deconvolution allowing for a more accurate depiction of intrinsic ultrafast dynamics, especially the critical initial excitation rising edge which, as advances continue, will drop below 50 fs.

This work is supported by 3M, and acknowledgment is made to the Donors of the American Chemical Society Petroleum Research Fund.

Fig. 1: (a) PINEM energy spectrum. Real-space annular point-spread functions due to (b) quantized chromatic aberration (i.e., PINEM aberration) and (c) spherical aberration. Convolution results in the point-spread function shown in (d). (e) Temporal variation of Fourier intensity for a discrete frequency arising from the convoluted aberrations.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-8-O-1901 Diffract-before-destroy with electrons?

Egerton R. F.1, Li R. K.2, Zhu Y.3
1Physics Department, University of Alberta, Edmonton, Canada T6G 2E1, 2Department of Physics & Astronomy, UCLA, Los Angeles, California, USA, 3Center for Functional Nanomaterials, Brookhaven Nat. Lab., Upton, NY11973, USA
regerton@ualberta.ca

Free-electron lasers provide x-ray pulses with short enough duration (< 100 fs) to record diffraction patterns from biological molecules (allowing their structure to be determined) before the molecules are destroyed by radiation damage [1]. Since fast electrons are elastically scattered more strongly than x-rays [2], it is reasonable to ask whether damage by radiolysis can be similarly outrun using electrons.


Electrons carry greater momentum than photons of the same energy, leading to additional knock-on damage, but in organic materials the knock-on damage is less severe than that caused by radiolysis [3]. More importantly, electrons carry electrostatic charge that can cause charging of insulating specimens (deflecting the beam or disrupting the specimen) and limit the incident-current density because of electrostatic repulsion between the electrons.


The Brookhaven ultrafast electron diffraction (UED) apparatus [4] produces 100fs pulses containing as many as 106 electrons at 2.8 MeV kinetic energy and this high energy helps to reduce Coulomb repulsion effects. To record ten diffracted electrons per pulse from a 10nm particle (e.g. macromolecule), the beam would need to be focused down to about 500 nm, giving a current density of almost 109 A/cm2. At these high current densities, space-charge effects are more important than statistical repulsion. Even so, it appears impossible to focus the beam to below a few micrometers diameter by means of a solenoid (Fig 1), due in part to the increased energy spread (approaching 0.3%) caused by Coulomb repulsion.


Lateral coherency of the beam is of concern for diffractive imaging, which nevertheless offers advantages over direct imaging since it avoids imaging lenses that degrade resolution because of aberrations and beam crossovers [5].


[1] JCH Spence, U Weierstall and HN Chapman, Rep. Prog. Phys. 75 (2012) 102601.
[2] R Henderson, Quarterly Rev. Biophys. 28 (1995) 171.
[3] RF Egerton, Microsc. Research & Technique 75 (2012) 1550.
[4] XJ Wang et al., J. Korean Phys. Soc. 48 (2006) 390.
[5] BW Reed et al., Microscopy and Microanalysis 15 (2009) 272.

Ray Egerton wishes to thank the Natural Sciences and Engineering research council of Canada for financial support. Renkai Li acknowledges DOE Grants No. DE-FG02-92ER40693 and No. DEFG02-07ER46272, and ONR Grant No. N000140711174. Work at BNL was supported by US DOE-BES under Contract no. DE-AC02-98CH10886

Fig. 1: Beam diameter versus distance along optic axis, calculated using the 3Dmesh method for a 0.16pC bunch of 2.5MeV electrons focused by a solenoid with four different values of maximum field strength B0.

Fig. 2: Pulse profile, calculated using the 3Dmesh method and assuming an initial energy spread of 0.001%. Colors represent electron energy, red being the highest.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-8-O-2020 Resolving Landau State Dynamics with Electron Vortex Beams

Schachinger T.1, Schattschneider P.1,2, Löffler S.1,2, Stöger-Pollach M.2, Steiger-Thirsfeld A.2, Bliokh K. Y.3, Nori F.4,5
1Institute of Solid State Physics, TU Vienna, Vienna, Austria, 2USTEM, TU Vienna, Vienna, Austria, 3iTHES Research Group, RIKEN, Wako-shi, Japan, 4Center for Emergent Matter Science, RIKEN, Wako-shi, Japan, 5Physics Department, University of Michigan, Michigan, USA
thomas.schachinger@tuwien.ac.at

Since the advent of electron vortex beams (EVB) [1] and techniques to routinely produce them in the TEM [2], some exciting applications have been proposed, such as particle manipulation and mapping magnetic moments on the atomic scale, owing to the fact that they carry quantized orbital angular momentum (OAM) of Lz=mħ as well as a quantized magnetic moment of M=µBm per electron. With m being the topological charge m=…,-1,0,+1,….

Recently, it has been argued quantum mechanically [3] and classically [4] that EVB in a homogeneous magnetic field resemble free electron Landau states (LS), exhibiting peculiar azimuthal dynamics that deviate from standard electron optics’ Larmor rotation (Ω), where M=0. Depending on the orientation of M (for M≠0) with respect to the magnetic lens field B, cyclotron (double-Larmor) rotation (2Ω) and no rotation (0Ω) have been predicted. In solid-state physics, these states are well known describing phenomena like the diamagnetism of metals [5]. Even though great advances in mapping LS in solid-state systems have been made, their azimuthal dynamics could not be resolved experimentally so far [6].

The application of EVB opens up the road for the observation of free electron LS in the quasi-homogenous objective lens field in the TEM. In breaking the rotational symmetry of the annular structure of an EVB with a Si-knife-edge, it is possible to resolve azimuthal variations by scanning the EVB along the propagation (z-) direction, see Fig. 1a. Due to the converging character of EVB in the TEM, see Fig. 1b, the free electron LS are only approximated well by the EVB in a specific z-shift region. Fig. 2 shows experimental images from that region. Indeed, m-dependent rotational speeds can be seen. Fig. 3 summarizes the experimental data of many measurements by giving the fitted slopes ‹ω›, which represent the electrons’ rotational speed. There, the comparison to the theoretical calculations indicates excellent agreement with the predicted dynamics of no rotation for m<0 states, Larmor rotation for m=0 and cyclotron rotation for m>0. It conclusively shows the OAM dependent Landau state behavior. Note that with the Larmor frequency being Ω~2π·19GHz, the discrimination between those three rotational states represents an energy resolution of ~100µeV. These findings provide new insight into the fundamental properties of LS and prepare the route towards detailed investigations of their otherwise hidden characteristics.

[1] K Y Bliokh et al, PRL 99 (2007), 190404, M Uchida et al, Nature 464 (2010), 08904
[2] J Verbeeck et al, Nature 467 (2010), 09366
[3] K Y Bliokh, et al, PRX 2 (2012), 014011
[4] T Schachinger, Master Thesis, TU Vienna (2013)
[5] L Landau, Z f Phys 64 (1930) , 629

[6] K Hashimoto et al, PRL 109 (2012), 116805

This work was supported by the Austrian Science Fund FWF (I543-N20) and RIKEN iTHES Project.

Fig. 1: (a) Sketch of the experimental setup. Half of the incoming beams (blue, green, red), immersed in the B-field of the lens, are blocked by a knife-edge, which is scanned in the z-direction, showing azimuthal dynamics in the observation plane. (b) Cross section of an |m|=1 beam showing the z-region corresponding to the lateral LS extension 2wB.

Fig. 2: Experimental images of the cut EVB with azimuthal angle measurements (in false colors), showing m-dependent azimuthal dynamics. Negative m-states show decreased angular variations, while the m=0 and the positive m-states show larger angular variations. The red line acts as a guide to the eye and represents a constant azimuthal angle.

Fig. 3: Scatter plot of the fitted slopes for the LS z-region validating the m-dependent rotational speeds. The solid lines stand for the theoretical slopes, e.g. no rotation (blue), Larmor rotation (green) and cyclotron rotation (red) and the dashed lines represent averaged slopes.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-8-O-3019 High Throughput Imaging in a Multibeam SEM

Ren Y.1, Hagen C. W.1, Kruit P.1
1Delft University of Technology, Delft, the Netherlands
y.ren-1@tudelft.nl

Nowadays there is a growing demand to increase the throughput of scanning electron microscopes, especially in biological research where 3-D images of organ’s structures are desired but too time-consuming. By adopting our Multi-Beam Scanning Electron Microscope (MBSEM) and proper stage moving strategy, the time for constructing a 3D image of 1mm3 brain can potentially be dramatically reduced from 200 days to 1 day. There is also a place for high throughput imaging in the semi-conductor industry where inspecting patterned wafers often asks the high resolution of the SEM, but the standard SEM is too slow.
The MBSEM which we have developed is based on a regular FEI Nova-Nano 200 SEM, but equipped with a novel multi-electron beam source module containing a MEMS fabricated aperture array that delivers a 14x14 array of focused beams with a resolution and current per beam comparable to a state of the art single beam SEM 1,2 .
A Secondary Electron (SE) imaging system, a Transmission Electron (TE) imaging system and a Backscatter Electron (BSE) imaging system have been designed for this MBSEM. The most challenging issue for these 3 imaging systems is how to separate and collect different signals belonging to corresponding beams, especially considering that the pitch of the primary beams on the sample is only 0.5~5 µm, and that the systems should work well for different landing energies and working distances.
Here we will present analysis and the simulation results of the SE and BSE detection systems, and recent experimental results of TE imaging. For the latter we have made use of the in-vacuum high resolution light microscope developed for in-situ correlative microscopy3 as shown in figure 1. We demonstrate that all beams arrive at the specimen in a regular grid (figure 2) and that each beam gives a focused image (figure 2). We will discuss the detection problems that arise when so much data can be detected simultaneously.

References:

1. A. Mohammadi-Gheidari, P.Kruit, Nuclear Instruments and Methods in Physics Research A 645 (2011) 60
2. A. Mohammadi-Gheidari, C. W. Hagen and P. Kruit, J. Vac. Sci. Technol. B28, (2010) 1071
3. A.C. Zonnevylle, R.F.C. van Tol, N. Liv, A.C. Narvaez, A.P.J. Effting, P. Kruit and J.P. Hogenboom; Journal of Microscopy, (2013) doi: 10.1111/jmi.12071.

Fig. 1: Multi-Beam SEM with transmission detection

Fig. 2: Top: Multi-beam probes on the YAG : There are 14*14 beams in the system and the 4 quarters are used to identify beams. Bottom: TE images, based on the beam identification indicated above, TE images of different beams are shown, with the same field of view 4.3 µm, collected simultaneously.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-8-P-1428 Analysis of image distortion on projection electron microscope image

Iida S.1, Hirano R.1, Amano T.1, Terasawa T.1, Watanabe H.1, Murakami T.2
1EUVL Infrastructure Development Center, Inc.(EIDEC), Tsukuba, Japan, 2EBARA CORPORATION, Fujisawa, Japan
susumu.iida@eidec.co.jp

We have been developing a high throughput projection electron microscope (PEM) for EUV (Extreme ultraviolet) patterned mask inspection system. The PEM provides a sample target with areal illumination at a throughput higher than that obtained from a conventional SEM as shown in Fig. 1. However, image distortion is one of the main issues to be fixed. In order to understand the mechanism behind this issue, simulated PEM images through the imaging electron optics (EO) were analyzed using an upgraded advanced Monte Carlo software CHARIOT. Fig. 2 shows a schematic illustration of a target sample using this approach. Near the pattern with 100 nm half-pitch lines and spaces (L/S), a metal contact with an applied 10 V was added to the substrate. This metal mimics a positively charged area. When a simulated L/S pattern image was obtained by an image sensor placed 30 nm above the target sample, the electrons forming this image could not pass through the imaging optics and remained unaffected by the local charge. On the other hand, when secondary electrons could pass through the imaging optics, the image from the detector placed at a focal plane 200 mm away from the target sample resulted in a distorted image as shown in Fig. 3. These results clearly show that image distortion can be reproduced not by the near-field image but by the focal plane image because the virtual source image is projected on the focal plane in PEM. In the case of focal plane, the L/S patterns bent away from the positively charged area in spite of the fact that secondary electrons should be attracted by the charged area. This phenomenon can be explained using Fig 4. If the SE was bent by the charged area, the virtual source, which SE generates, shifts away from the positively charged area. When the SE bends more, the source shifts farther. As a result, focused L/S patterns are bent away from the positively charged area.These simulation results of image formation including electron scattering and long-range effects of the charging help to understand the mechanism of image distortion and to overcome this issue. At higher energies of SE, the effects of bending become smaller. The energy of SE can be controlled by the extraction voltage. In a novel concept of PEM under development, we applied an extracting electrical field eight times stronger than in conventional PEMs in order to considerably reduce the charging distortion. This reduction effect of image distortion was confirmed by this simulator using the EO data of the novel PEM.

This work was supported by New Energy and Industrial Technology Development Organization (NEDO)

Fig. 1: Schematic illustration of PEM

Fig. 2: Schematic illustration of the test sample

Fig. 3: Simulated image from a detector placed behind the electron optical system

Fig. 4: Schematic illustration of distortion due to local charging
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-8-P-1696 New Operation Modes with the direct detecting pnCCD-camera in Transmission Electron Microscopy

Simson M.1, Hartmann R.2, Huth M.2, Ihle S.2, Müller K.3, Rosenauer A.3, Ryll H.2, Schmidt J.2, Soltau H.1, Strüder L.2
1PNDetector GmbH, Emil-Nolde-Str. 10, 81735 Munich, Germany, 2PNSensor GmbH, Römerstr. 28, 80803 Munich, Germany, 3Universität Bremen, Otto-Hahn-Allee 1, 28359 Bremen, Germany
martin.simson@pndetector.de

The pnCCD’s ability to directly detect and image single electrons in a transmission electron microscope (TEM) at energies from 300 to 80 keV has already been demonstrated [1,2] with the dedicated mechanical setup shown in Fig. 1. For these measurements a pnCCD with a physical pixel size of 48x48 μm² with 264x264 pixels was read out as fast as 1000 fps. At low dose conditions a sub-pixel resolution of 1320x1320 after processing of the raw data has been reached. Meanwhile the pnCCD-camera was used to detect TEM electrons with energies down to 20 keV.
A common limitation of CCD-based cameras is the charge handling capacity. Primary electrons from the TEM generate many signal electrons when they scatter in the bulk of the pnCCD. These electrons are stored in potential wells (the pixels) until the CCD is read out. If the number of signal electrons in one pixel exceeds its charge handling capacity, surplus electrons will spill over to neighboring pixels. This effect is usually called “blooming”.
In order to have an optimal behavior of the pnCCD-camera under many different experimental conditions, several new operation modes were developed. While the normal operation settings offer the best spectroscopic properties of the camera, especially at very low energies, the charge handling capacity is limited to about five 80 keV electrons per pixel and frame, i.e. 1 ms. In the high charge handling capacity (HCHC) mode this can be increased by a factor of 3 to 4 without noteworthy losses of other performance parameters. This means, for a primary electron energy of 80 keV and a readout rate of 1000 fps, up to 16,000 TEM electrons per pixel can be processed during one second.
To process even higher electron rates the anti-blooming (AB) mode is available. In this operation mode the number of electrons that can be stored in one pixel is reduced compared to the HCHC mode but excess charge does not overflow into neighboring pixels. Instead it is drained from the detector and does not contribute to the readout signal. Therefore it is possible to image spots of very high intensity without degrading spatial information. At the same time single electrons can be detected in other sections of the detector.
The XPLUS mode is a hybrid of HCHC and AB mode. The charge handling capacity is increased compared to the AB mode, while overflowing signal electrons are drained off.
In the presentation the advantages of the different operation modes will be explained and visualized by different measurements under varying TEM conditions and primary electron energies from 300 to 20 keV.

[1] H. Ryll et al., Microscopy and Microanalysis 19 (2013), p.1160-1161.
[2] K. Müller et al., Appl. Phys. Lett. 101 (2012), p. 2121101-2121104.

Fig. 1: The directly detecting pnCCD- camera for TEM applications.

Fig. 2: Images of 300 keV electrons taken in different operation modes. In normal operation mode (a) the high electron rate causes blooming. The HCHC mode (b) allows to store more charge in each pixel. In the AB mode (c) surplus charges are removed, suppressing all blooming effects. The XPLUS mode (d) is a hybrid of the HCHC and AB modes.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-8-P-1895 Design of the novel flange-on high lateral and energy resolution ultrashort electron pulse compression system for ultrafast microscopy

Grzelakowski K. P.1
1OPTICON Nanotechnology
k.grzelakowski@opticon-nanotechnology.com

An instrumental realization of the idea [1,2] for the ultrashort electron pulse source based on the newly developed imaging energy filter called α-SDA (Spherical Deflector Analyzer) [3] is reported. Its compact design enables the realization of the flange–on instrument concept. It consists of six independent subsystems: photocathode/immersion lens, primary electron column, pass energy tuning element, α-SDA as a central part, focusing/ compression column and detector/target with XY-stage, Fig1. The ultrashort photoelectron bunch created by an attosecond laser pulse propagates through the primary column towards the mirror plane of the α-SDA, where according to simulations, the focusing and temporal reversion occurs [2]. As a consequence, the time-divergent primary electrons at the mirror plane are transformed to a time-convergent pulse at the same plane after 2π deflection. It has been also previously shown, that the aberration free imaging properties of the α-SDA assure a very high lateral (<<1nm) and energy (ΔE/E<10-3) resolution.  In the symmetric case with the first time compression exactly at π, the shortest electron pulse behind the α-SDA analyzer is mirror symmetric to the original electron pulse at the photocathode [2]. As a consequence, an extremely dense: ultrashort (<<1fs) and perfectly focused (<<1nm) high energy (104-105 eV) electron bunch strikes the target.

1 K.P. Grzelakowski, US Patent Nr. 7,126,117
2 K. P. Grzelakowski, R. M. Tromp, Ultramicroscopy, 130 (2013) 36
3 K.P. Grzelakowski, Ultramicroscopy 116 (2012) 95

The author acknowledges the financial support by the NCBR (National Centre for Research and Technology) in Warsaw. I would like also to express my gratitude to Dr.Rudolf Tromp for stimulative discussions.

Fig. 1: General outlook of the electron pulse compression system
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-8-P-2148 Sub-picosencond electron beam and femtosecond optical pump system in spin-polarized TEM

Kuwahara M.1, Nambo Y.1, Saitoh K.2, Ujihara T.1, Asano H.1, Takeda Y.3, Tanaka N.2
1Graduate School of Engineering, Nagoya University Nagoya, 464-8603, Japan, 2EcoTopia Science Institute, Nagoya University, Nagoya 464-8603, Japan, 3Nagoya Industrial Science Research Institute, Nagoya 464-0819, Japan
kuwahara@esi.nagoya-u.ac.jp

Time-resolved measurements at nanometer spatial resolutions are very important for investigating relaxation processes, catalyzed reactions and phase-transition phenomena. It is possible to carry out such time-resolved analysis using transmission electron microscopy (TEM) by using a pulsed electron beam as a probe beam. Such an approach has been applied in dynamic TEM (DTEM) and ultra-fast electron microscopy (UEM), which use metals and LaB6 for a photoemission source driven by a pulsed laser. These methods have led to the possibility of four-dimensional electron microscopy with high spatial and temporal resolutions. Spin-polarized transmission electron microscopy (SP-TEM) can satisfy two abilities of spin-resolved imaging and pulsed electron gun operation simultaneously, because the instrument consists of a laser-driven polarized electron source (PES) and a conventional TEM system [1].

Spin-polarized electron can be generated by photoemission from III–V semiconductors with a negative electron affinity (NEA) surface. Several beam parameters of a PES are vastly superior to those for conventional thermal electron beams. A high spin-polarization of 92% and a high quantum efficiency of 0.5% have been simultaneously realized using a GaAs–GaAsP strained-layer-superlattice photocathode. In addition, such a photocathode has the ability to generate a sub-picosecond multibunch beam [2]. In order to realize a pump-probe method using the spin-polarized pulse electron beam, we have developed a synchronizing system and demonstrated a phase-locked TEM image of wobbling probe beam by using a pulse electron beam [3]. TEM images were already acquired with a pulsed electron beam with a 1.4-ns pulse duration. Now we have constructed a new optical system, which can provide a sub-picosecond pulse laser and femtosecond pulse simultaneously, to realize an ultrafast temporal resolution. The figure 1 (b) and (c) show the photograph and the schematic diagram, respectively. The sub-picosecond pulse laser is used to drive the electron gun. Another femtosecond laser is transferred to pump a specimen to create an excited state. The sub-picosecond pulse is generated by narrowing a bandwidth of a seed laser which is emitted from a mode-lock Ti:Sapphire laser. Figure 2 shows the femtosecond and picosecond pulse. The sub-picosecond pulse laser is necessary to keep the high polarization. These results suggest the possibility of pump-probe measurements in SP-TEM using the pulsed electron beam as a probe, allowing nanometer-scale time-resolved spin mapping.

[1] M. Kuwahara et al., Appl. Phys. Lett. 101 (2012) 03310

[2] Y. Honda, et al., Jpn. J. Appl. Phys. 52, 086401-086407(2013).

[3] M. Kuwahara et al., Microscopy 62, 607-614 (2013).

The authors thank Drs. H. Shinada, M. Koguchi and M. Tomita of Hitachi Central Research Laboratory for fruitful discussions and encouragement. This research was supported by MEXT KAKENHI Grant Number 51996964, 24651123, 25706031.

Fig. 1:  (a) Photograph of the spin-polarized TEM, (b) Photograph of pulse laser system and (c) the schematics.

Fig. 2: Auto-correlation amplitudes of pulse lasers as a function of correlation time. (a) a correlation amplitude of a femtosecond pulse laser for excitation of a specimen. (b) a correlation amplitude of picosecond pulse laser for driving an electron source.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-8-P-2668 Ultrafast measurement in spin- polarized pulse TEM

Nambo Y.1, Kuwahara M.1, Kusunoki S.1, Sameshima K.1, Saitoh K.1,2, Ujihara T.1, Asano H.1, Takeda Y.3,4, Tanaka N.1,2
1Graduate school of Engineering, Nagoya University, Nagoya, 464-8603, Japan, 2EcoTopia Science Institute, Nagoya University, Nagoya, 464-8603, Japan, 3Nagoya Industrial Science Research Institute, Nagoya, 464-0819, Japan, 4Aichi Synchrotron Radiation Center, Aichi Science and Technology Foundation, Seto, 489-0965, Japan
nambo.yoshito@f.mbox.nagoya-u.ac.jp

Investigations of ultra-high-speed phenomena in nanometer scale are important for analysis of dynamics in advanced nano-devices. Furthermore, measurement of a spin information is necessary for densification of magnetic recordings and developments of spintronics devices. It is expected that spin-polarized transmission electron microscopy (SP-TEM) can clarify the spin-dependent dynamics.
　　The SP-TEM consists of a laser-driven electron source and a conventional TEM (Hitachi H9000-UHV). The electron source is configured with GaAs-GaAsP strained superlattice photocathode using a negative electron affinity surface, which has high polarization of 92% and high quantum efficiency of 0.5% [1]. Moreover, the photocathode also has an ability of generating a sub-picosecond pulsed electron beam [2]. The Pulsed electron beam is emitted by illuminating a pulse laser to the photocathode.
Figure.1 (a) and (b) show the TEM images obtained by using a continual electron beam and pulsed electron beam, respectively. The image of Fig.1 (a) is wobbled TEM image by an alignment deflector coil. The wobbling amplitude is dramatically decreased by using the pulse beam as shown in Fig.1 (b) [3]. Fig.2 is an illustration of an optical bench for a pump-probe method. A femtosecond pulse laser is separated two beam line by a polarized beam splitter. One of the beam line is delayed by passing a variable delay-line. The other is irradiated to a dispersive grating as a pulsewidth stretcher and extracts sub-picosecond pulse laser. Each of lasers are transferred to the SP-TEM by using optical fibers. Fig.3 shows the pulse-duration of a picosecond pulse laser measured by an auto-correlator. The picosecond pulse lasers are generated by selecting a part of the wavelength of femtosecond pulse laser.
　　Consequently, a pump-probe method which picosecond and femtosecond pulse lasers are used for photocathode and specimen excitation respectively can be carried out in the SP-TEM. This instrument gives a possibility of investigations of spin dependent phenomena with a high temporal resolution such as a time-evolution of photo-induced magnetism on nanometer scale.

[1] X G Jin et al., Appl. Phys. Express 1. (2008) 045002
[2] Y. Honda et al., Jpn. J. Appl. Phys. 52 (2013) 086401.
[3] M. Kuwahara et al., Microscopy 62 (6) (2013) 607-614.

The authors thank Drs. H. Shinada, M. Koguchi and M. Tomita of Hitachi Central Research Laboratory for fruitful discussions and encouragement. This research was supported by MEXT KAKENHI Grant Number 51996964, 24651123, 25706031 and Kurata Research Grants from the Kurata Foundation.

Fig. 1: Wobbling TEM images acquired by illuminating (a) continual electron beam and (b) pulsed beam.

Fig. 2: A schematic of the optical bench for the pulse laser.

Fig. 3: Correlation time-structures of series of picosecond pulse lasers measured by an auto-correlator.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-8-P-2678 Implementing in situ experiments in liquids in the(scanning) transmission electron microscope and dynamic TEM

Abellan P.1, Woehl T. J.2, Russell G. T.1, Schroeder W. A.3, Evans J. E.1, Browning N. D.1
1Pacific Northwest National Laboratory, Richland, USA, 2U.S. DOE Ames Laboratory, Ames, USA, 3University of Illinois at Chicago, Chicago, USA
patricia.abellanbaeza@pnnl.gov

Developing a fundamental understanding of phenomena that take place in liquids, such as nanoparticle growth, protein conformational dynamics or the transformation of active materials during battery operation requires characterization tools able to provide in situ information with nanometer spatial resolution. In principle, this can be achieved using fluid stages in the (scanning) transmission electron microscope ((S)TEM). One of the main experimental challenges in the field is obtaining reproducible data free of beam-induced effects to enable quantitative analysis. Methods of calibration of the amount of radiation damage resulting from beam-induced reactions with the sample continue to be needed [1,2]. For instance, in situ growth of particles in solution by the electron beam is typically observed in (S)TEM experiments and has been used to calibrate the effect of electron dose in a Ag precursor solution in an in situ fluid stage [3] (Figure 1 (a) shows areas where Ag was grown under different experimental conditions). Custom image analysis algorithms can be applied to analyze movies of nanocrystal nucleation and growth and extract important information on growth dynamics and parameters such as the induction threshold below which no nucleation occurs [3] (see an example of image analysis in Figure 1(b)). Besides electron dose, factors such as accelerating voltage, imaging mode (e.g. TEM, STEM, SEM), liquid thickness, and solution composition are expected to affect the results of in situ experiments. Reproducing an experiment in a different instrument operating with different electron optical settings, introduces a large set of variables whose effect must be calibrated. Here, we present our recent developments in the design and implementation of calibration experiments using in situ fluid stages, including an identification of beam-sample interactions for changing imaging and experimental conditions. Since fluid stages are designed to fit in any transmission electron microscopy, the different capabilities of each instrument can be applied to the study of liquid phase reactions. When using fluid stages in combination with the dynamic TEM (DTEM), a combined temporal and spatial resolution of ~10-6 and ~10-10 m, could be achieved (see schematic of the DTEM design at the Pacific Northwest National Laboratory (PNNL) in Fig. 2). The unique qualities of the DTEM that benefit the in-situ experiments with fluid environmental cells will be also discussed.

[1] T.J. Woehl et al., Ultramicroscopy 127 (2013) 2927

[2]J.M. Grogan, Nano Letters 14 (2014) 359

[3] T.J. Woehl et al., ACS Nano 6 (2012), p. 8599; Nano Letters 14 (2014), p. 373.

[4] J.E. Evans et al., Microscopy 62 (2013) p. 147-156

This work was supported by the CII; under the LDRD Program at PNNL. PNNL is a multiprogram national laboratory operated by Battelle for the U.S. DOE under Contract DE-AC05-76RL01830. A portion of the research was performed using the EMSL, a national scientific user facility sponsored by the DOE's Office of BER and located at PNNL.

Fig. 1: (a) Low magnification BF STEM image showing a set of nanocrystals growth experiments using different electron dose rates. (b) Number of particles grown from solution as a function of time measured from an in situ dataset using 300kV, 7.1pA beamcurrent, 3 μs pixel-dwell time and M=40000x in STEM, to give a dose per frame of 39.1 e-/nm2f.

Fig. 2: Schematic of the DTEM showing theupgrades planed at PNNL. Modified from [4]. Copyright 2013 Oxford UniversityPress.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-8-P-3023 SPIM-Fluid: High-throughput platform based on Light-Sheet Microscopy

Gualda E. J.1, Pereira H.2,3, Pinto C.2,3, Simaõ D.2,3, Brito C.2,3
1Instituto Gulbenkian de Ciências, Portugal, 2Instituto de Biologia Experimental e Tecnológica, Portugal, 3Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal
emilio.gualda@gmail.com

Drug screens on complex cell models and organisms are a key factor to understand and treat human diseases. However, fast and effective conclusions have been hindered by the lack of robust and predictable models amenable to high-throughput (HT) analysis. Animal models can mimic s pathological features, however species-specific differences may occur and are prone to increase experimental costs. On the opposite side, adherent cell cultures have been used in drug screening and tumor modeling but they do not properly represent biological tissues. Recently, important advances have been made towards the development of 3D cellular models, using human immortalized cell lines, stem cells and other patient derived cells, which better recapitulate features of tissues. These advances bridge the gap between adherent cell culture and animal models, making 3D cellular aggregates an extremely powerful in vitro model for preclinical research.

A major hurdle, hampering the widespread utilization of complex in vitro models, is the lack of robust analytical tools. The development of innovative methodologies will allow more comprehensive readouts, generating more accurate and predictive human cell-based 3D models for drug and toxicity screenings. Imaging techniques like confocal microscopy are not optimal for thick samples, providing a short penetration and long imaging times. As an alternative approach, light sheet microscopy (LSM) has been proposed to overcome those limitations. Novel LSM configurations fusing its inherent capabilities with microfluidics will allow massive live 3D cell cultures studies in real-time and with a high spatio-temporal resolution, enabling sophisticated cell-based assays in 3D cell cultures (disease diagnosis and therapy; drug screening; cell differentiation; etc.). Using this approach we will be able to make HT quantitative analysis of the spatio-temporal organization of the different cell types in a spheroid, as well as the response to different environmental conditions with high resolution, high speed and minimal photo-damage.

We will present new designs and prototypes, and how the use of 3D-cell cultures and full system automation will contribute to measure a large set of biological parameters with statistical relevance to investigate drug response on the central nervous system (CNS), cancer therapy and cell differentiation. Also, it would facilitate the development of new typologies for 3D-cell cultures and optimize staining protocols. Those systems will be primarily devoted to 3D cell cultures studies, but the expansion to other biological systems, such as full brain imaging in zebrafish embryos with cellular resolution, will be also presented.

The authors acknowledge support from Fundação para a Ciência e Tecnologia, Portugal - grants SFRH/BD/80717/2011, SFRH/BD/78308/2011, EXPL/BBB-IMG/0363/2013 and PTDC/EBB-BIO/119243/2010; and from Innovative Medicines Initiative Joint Undertaking (EU), grant agreement n° 115188.

Fig. 1: Schematic of the Light Sheet Fluorescence Microscope at IGC (top). Detail of the SPIM-Fluid set-up (bottom).

Fig. 2: Viability of cells within differentiated neurospheres visualized with NucView 488 and MitoView 633 Apoptosis Kit (Biotium, Hayward, CA, USA) image during 15 hours. Tert-butyl hydroproxide (tBHP) (Sigma), an oxidative stress inducer, was used to trigger apoptosis at a concentration of 1mM in Hibernate medium (Invitrogen).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-8-P-3340 In-Situ Lorentz Microscopy with Femtosecond Optical Illumination

Gatzmann J.1, Eggebrecht T.2, Feist A.1, Zbarsky V.2, Münzenberg M.2, Ropers C.1, Schäfer S.1
1IV. Physical Institute, Georg-August-University, 37077 Göttingen, Germany, 2I. Physical Institute, Georg-August-University, 37077 Göttingen, Germany
schaefer@ph4.physik.uni-goettingen.de

Ultrafast electron microscopy as a laser-pump/ electron-probe technique allows for the investigation of structural and electronic dynamics occurring at sub-picosecond timescales and nanometer length-scales. However, current implementations necessitate compromises in electron source brightness compared to conventional electron microscopy techniques. In-situ transmission electron microscopy with temporally-structured optical sample excitation, i.e. by employing femtosecond laser pulse trains, offers a complementary approach to access ultrafast processes, without the need for customized pulsed electron sources. To this end, we implement free-space-coupled femtosecond sample excitation in a Schottky field-emission electron microscope and investigate the optical response of magnetic domain structures with Lorentz microscopy. Specifically, we study laser-induced domain rearrangements in polycrystalline iron thin films on silicon nitride membranes which are pumped with single sub-50-fs laser pulses. By inverting the observed image contrast at large defocus, we reconstruct the local in-plane sample magnetization based on a transport-of-intensity approach. Prior to laser-excitation, the iron thin films display the well-known magnetic ripple domain structure (cf. Fig. 1A). Upon optical excitation, at laser fluences below a sharp threshold of about 5 mJ/cm2, single laser pulses induce local magnetic domain wall. At laser fluences above the threshold, a single laser pulse generates a network of magnetic vortex/anti-vortex (V/AV) structures, as depicted in Fig 1B-D. Subsequent laser pulses lead to nearly complete rearrangement of the V/AV network (left panels in Fig 1C and D). While the network is stable without optical excitation and shows no discernible dynamics on timescales of minutes to hours, V/AV annihilation can be triggered by illuminating the sample with laser pulses below threshold. After several low fluence optical pulses, the equilibrium ripple domain structure is recovered. The generation of a V/AV-network is remarkable as it presumably is the result of a partially melted, non-equilibrium spin system which is quickly quenched to a metastable state. Possible processes leading to a V/AV network are discussed on the basis of micromagnetic simulations and with respect to ultrafast all-optical pump-probe experiments. The nature and dynamics of the laser-driven magnetic reorganization will be further experimentally investigated with temporally-structured illumination utilizing femtosecond pulse pairs separated by variable time delays. In conclusion, we report the optically-induced vortex/anti-vortex generation mapped by in-situ Lorentz microscopy and discuss possible pathways for their generation.

We gratefully acknowledge financial support by the DGF through SFB 1073 "Atomic Scale Control of Energy Conversion" and by the DFG and the State of Lower Saxony under grant Inst186/867-1FUGG.

Fig. 1: (A) Electron micrograph with Lorentz contrast prior to optical excitation. (B-D) After single-fs-pulse laser excitation a network of vortices and anti-vortices appears (C, left panel). The reconstructed in-plane magnetization is displayed in (C, right panel) and (B). Subsequent laser pulses lead to a rearrangement of this network (D).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-8-P-6038 Towards RF photo injector based dynamic transmission electron microscopy with REGAE

Manz S.1, Casandruc A.1, Keskin S.1, Zhang D. F.1, Bayesteh S.2, Hirscht J.1, Felber M.2, Gehrke T.4, Loch R. A.1, Marx A.1, Delsim-Hashemi H.2, Schlarb H.2, Hoffmann M.2, Hada M.1, Epp S. W.2, Floettmann K.1, Miller R. J.1,3,4
1Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany, 2DESY, Hamburg, Germany, 3University of Toronto, Toronto, Canada, 4University of Hamburg, Hamburg, Germany
stephanie.manz@mpsd.mpg.de

The relativistic electron gun for atomic exploration (REGAE) has been designed
to study structure and dynamics in a wide range of systems. Aiming for
a time resolution of far less than 100 fs, we plan to observe fast structural changes
in solid, solution and gas phase with single-shot femtosecond electron diffraction
in the energy range from 2 - 5 MeV.
As a prove of principle study, we investigated static electron diffraction of sample
thicknesses close to micrometer.
This poster will present latest feasibility studies of performing dynamic single shot
real space imaging with REGAE. The requirements for single shot imaging result in
bunch charges beyond pC. Both the electron’s high energy as well as space charge
in the electron bunches call for a special lens column pre- and post-sample.
The lenses need to be strong enough to diminish spherical and chromatic aberrations. In order to achieve nanometer resolution a focal length in the
millimeter to centimeter rage is necessary. For electromagnetic solenoid lenses
this means peak currents on the order of Tesla. Although the relativistic energy of
the electrons decreases space charge fields compared to a dc electron gun or
conventional electron microscopes, they come back in play when considering single
shot imaging. We study the effects of space charge on the resolution for our newly designed lens system. We find that the bunch
charge strongly affects both chromatic and spherical aberrations. Simulations show,
that space charge fields affect the resolution already from fC bunch charges on,
even though only the meanfield is considered yet. An optimized imaging system will be presented as well as
strategies to circumvent chromatic aberrations by temporal pulse shaping with an
additional RF cavity in order to achieve nanometer spatial resolution.

The project receives funding from the Centre for Ultrafast Imaging (CUI) at the University of Hamburg.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-8-P-6048 Ultrafast transmission electron microscopy with nanoscopic electron sources

Feist A.1, Bormann R.1, Schauss J.1, Gatzmann J. G.1, Rubiano da Silva N.1, Strauch S.1, Schäfer S.1, Ropers C.1
1IV. Physical Institute, Göttingen, Germany
feist@ph4.physik.uni-goettingen.de

Ultrafast transmission electron microscopy (UTEM) is a laser pump/electron probe technique which enables the investigation of ultrafast processes on nanometer length scales [1]. Here, the dynamics of an inhomogeneous system after ultrashort laser excitation are probed by stroboscopic illumination with sub-picosecond electron pulses. However, current implementations to create short electron pulses, employing a flat photocathode, are intrinsically limited by their low emittance.
We present the implementation of a pulsed electron source, based on localized laser-triggered emission from a needle-shaped tungsten emitter [2], which we employ in a commercial Schottky field emitter TEM. Within this setup, we experimentally characterize the minimum spot size, overall brightness and intrinsic emittance of the electron beam. To further study the emission properties of the electron gun, numerical finite element calculations are carried out. In addition, photon induced near-field electron microscopy (PINEM) [3] of metallic nanostructures is utilized to investigate the temporal structure of the electron pulses, currently yielding pulse durations of 700 fs.
These electron bunches will allow us to study structural dynamics of heterogeneous systems at and near interfaces, defects and structural inhomogeneities with a sub-ps temporal and nanometer spatial resolution.
[1] A.H. Zewail, Science, 328, 187 (2010).
[2] C. Ropers, D. R. Solli, C. P. Schulz, C. Lienau, T. Elsaesser, Phys. Rev. Lett. 98, 043907 (2007).
[3] B. Barwick, D. J. Flannigan, A. H. Zewail, Nature, 462, 902 (2009).

↑ Go to the Table of content ↑

IT-9. Electron and X-ray diffraction techniques

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-9-IN-1862 Multiple-scattering assisted electron crystallography

Koch C. T.1
1Institute for Experimental Physics, Ulm University, Ulm, Germany
christoph.koch@uni-ulm.de

The ab-initio determination of crystal structures typically requires highly complete single-crystal diffraction data, i.e. diffraction intensities should have been measured for almost all unique reflections. The reason for this is that, if many more reflections have been measured, than there exist atoms within the structure, the sparseness (peaked nature) of the real-space representation of the charge density (in the case of X-rays) or potential (in the case of electrons) can be utilized to solve the crystallographic phase problem (e.g. by direct methods, or charge flipping, or similar kinematic scattering based techniques). While electron diffraction has the great advantage over X-ray or neutron diffraction, that very small crystallites are already sufficient to produce such single crystal patterns, multiple scattering of electrons within the material generally prevents electron diffraction data to be used in as quantitatively a manner as X-ray or neutron data. This limits the application of electron diffraction tomography [1] to samples that are small along at least two dimensions (e.g. rods), and makes the investigation of other geometries (e.g. platelets) generally more difficult.

It is a well-established truth that, if electron diffraction data corresponding to a few different dynamical diffraction conditions is available, the relative phases of the structure factors that correspond to this data are uniquely determined. This fact is being exploited in structure-factor refinement by quantitative convergent-beam electron diffraction (QCBED) [2,3]. Applying the same real-space constraints as are used for solving the crystallographic phase problem from kinematical diffraction data, a lot less properly phased structure factors are necessary to find the corresponding arrangement of atoms than would be the case if the phases were not known.

In this talk I will show that by applying the recently developed large-angle rocking-beam electron diffraction (LARBED) technique [4], as implemented in the QED plugin [5] for DigitalMicrograph (Gatan), highly quantitative dynamical electron diffraction data sufficient to solve the structure can be acquired from nanocrystals even without tilting the specimen at all.

[1] U. Kolb, E. Mugnaioli, T. E. Gorelik, Cryst. Res. Technol. 46 (2011) 542 – 554

[2] C. Deininger, G. Necker, J. Mayer, Ultramicroscopy 54 (1994) 15-30

[3] J.-M. Zuo, M. Kim, M. O’Keefe, J.C.H. Spence, Nature 401 (1999) 49

[4] C.T. Koch, Ultramicroscopy 111 (2011) 828 – 840

[5] http://www.hremresearch.com

[6] C.T. Koch and J.C.H. Spence, Journal of Physics A: Mathematical and General 36 (2003) 803-816

Financial support by the Carl Zeiss Foundation as well as the German Research Foundation (DFG, Grant No. KO 2911/7-1) is acknowledged.

Fig. 1: Illustration of the recovery of structure factor phase triplets from a simulated 2D rocking curve (LACBED disc of radius 2°) for a single reflection of 3.5 nm thin GaAs by applying a recently developed scattering path expansion [6]. The structure factor phases can be further refined assuming sparseness of the potential in real-space.

Fig. 2: (001) LARBED pattern of SrTiO3. The range of beam tilts applied for acquiring this pattern spans the disc indicated by the red circle (diameter = 140 mrad). The beam tilt has been compensated by the diffraction shift coils to produce non-overlapping discs. Individual background-subtracted discs have been extracted and are shown as well.
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-9-IN-2477 Application of 3D EBSD: Growth of tin whiskers and hillocks

Michael J. R.1, Susan D. F.1, Rye M. J.1
1Materials Science Center, Sandia National Laboratories, Albuquerque, NM USA
jrmicha@sandia.gov

International agreements now require lead-free surface finishes for most electronic components. Pure tin finishes have been shown to form whiskers and hillocks. Tin whiskers can grow rapidly to long lengths that can cause faults in electronic devices and circuits. Long whiskers have been shown to be largely single crystals with specific crystallographic growth directions. Hillocks are smaller bump-like growths that can be made up of single grains or many grains and are less likely to cause electrical faults. Both whiskers and hillocks have been shown to grow from their bases.
The actual growth mechanisms for these features still remain elusive, although tin whiskers and hillocks are generally thought to grow as a response to stress in the plated tin films. In order to provide more information about the growth mechanism it is important to have a complete understanding of the crystallography of the growth substrate and the whiskers or hillocks that form. In this work we will apply the technique of 3D EBSD using the dual platform FIB/SEM to provide a more complete picture of whisker crystallography.

Shown in Figure 1 is a long thin whisker that contains low-angle grain boundaries. In this case, the whisker formed first followed by the formation of a hillock grain at the base, which caused subsequent whisker growth to stop. Note that the growth directions of the whisker segments are <001> and are aligned with the <001> direction in the hillock grain. The 3D crystallographic reconstruction shown in Figure 1 allows all of the grains at the base of the whisker to be visualized which is not possible with single 2D sections as are normally used for EBSD orientation maps.

Figure 2 is a 3D EBSD reconstruction of a hillock on the same Sn-film as the whisker in Figure 1. This reconstruction demonstrates that the hillock is polycrystalline with a grain size that is substantially larger than that of the electrodeposited Sn-film. When the area under the hillock is examined there is considerable grain growth associated with the film grains that continue into the hillock, indicating that grain growth and recrystallization may be contributing to hillock growth.

The capability to visualize whiskers and hillocks and the underlying grains in the plated films with 3D EBSD provides new insights into possible growth mechanisms and therefore may enable strategies to eliminate their occurrence on tin plated Cu surfaces

Sandia National Laboratories is a multi-program laboratory operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Company, for the U.S Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.

Fig. 1: 3D reconstruction of a straight tin whisker that has grown from a pure tin-plated surface on Cu.

Fig. 2: 3D reconstruction of a large tin hillock that grew on a pure tin-plated surface on Cu. Note that the hillock is polycrystalline as compared to the single crystal whisker in Figure 1.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-1603 Split-Illumination Convergent Beam Electron Diffraction (SICBED) of Strained Crystals

Houdellier F.1, Arbouet A.1, Hÿtch M. J.1, Snoeck E.1
1CEMES-CNRS, Université de Toulouse, 29 Rue Jeanne Marvig, 31055 TOULOUSE FRANCE EU
florent@cemes.fr

Convergent beam electron diffraction (CBED) is a well-established Transmission Electron microscopy (TEM) method mainly used to characterize crystal structural properties like space group, charge density distribution or strain. CBED became very popular with the advent of stress engineering in microelectronics where the carriers mobility can be enhanced by tuning the strain of a channel in transistors. It is then of major interest to measure the strain state of the channel in order to understand the electronic properties of transistors. To tackle this problem, various strain measurement methods have been developed by X-Rays analysis, wafer curvature measurements or TEM techniques. Among different TEM methods (HREM, dark-field electron holography, (nano)diffraction, …) CBED is the most sensitive, because of the strong influence of the crystal parameters on the position of the High Order Laue Zone (HOLZ) lines. Furthermore, it has been shown by dynamical simulation method, that the HOLZ rocking curve is extremely sensitive to the displacement field changes along the electron beam path. This explains the occurrence of HOLZ line broadening when the strain is not constant along electrons trajectories in thin sample where surface relaxation occurs. CBED is therefore extremely efficient for local strain measurement, however, like all other strain measurement methods in TEM, the absolute strain measurements necessitates the use of an unstrained reference area. Depending on the sample geometry this reference can be located far from the area of interest. As example, in an epitaxial layer, due to stress relaxation, the substrate generally used as reference can be strained over hundreds of nanometers below the interface. In order to have access in a single CBED pattern to both the area where the strain measurement has to be performed and the unstrained reference (the latter being located microns apart from the former), we have developed a new convergent beam diffraction method, which combines split illumination and CBED optical configuration. This method called split-illumination CBED (Fig. 1) has been developed on the I2TEM microscope. I2TEM is an Hitachi HF3300C TEM fitted with a 300kV cold FEG, an electrostatic biprism located above the three condensors illumination system, two stages capability, a multibrism set-up, a 4k X 4k camera and a Cs-corrector from CEOS. The biprism located above the condenser system and the adjustment of the three condensors allow to separate the convergent beam in two parts which can be shifted apart on the surface of the sample and be recombined in the focal plane of the objective lens. In a single CBED disk, this allows half of the disk to from the strained region and half from a reference region located far from it (Fig. 2).

The authors acknowledge financial support from the European Union under the Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative Reference 312483-ESTEEM2 and the National Research Agency under the program “Investissement d’Avenir” reference No. ANR-10-38-01-EQPX.

Fig. 1: A : SICBED optical configuration. The condenser biprism is used to split the beam convergent in two parallel half cones. B: Geometry of the FIB prepared SiGe/Si multilayers sample studied by SICBED

Fig. 2: SICBED patterns obtained when the condenser biprism voltage increase. Zone 2 (a=0V, b=3V, c=6V, d=9V), Zone 1 (1=0V, 2=3V, 3=6V, 4=9V)
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-2129 Mapping distortion and strain with EBSD in Cu single crystals

Kalácska S.1, Groma I.1, Ispánovity P. D.1
1Eötvös Loránd University, Budapest, Hungary
kalacska@metal.elte.hu

Cross-correlation based analysis of electron backscatter diffraction (EBSD) patterns is often carried out to map plastic strain variations in deformed polycrystalline samples [1]. In this work this method is applied to characterize the evolution of dislocation structures and corresponding distortion fields in Cu single crystals compressed to different levels. We aim at developing a statistical method that can be used to measure the total dislocation density in the specimen.

Firstly, the effects of sample surface preparation methods were investigated including Ar ion polishing and traditional electropolishing treatments. Then the distortion maps of the specimen are computed with the cross-correlation technique. This method is capable of detecting changes of the crystal orientation to higher accuracy than the commercial software provided for standard EBSD devices that analyse each EBSD pattern individually. The distribution of distortions shows broadening with increasing load and a slow decay. To give a more detailed evaluation of the microstructure the measurements are complemented with the analysis of broadened X-ray diffraction (XRD) peaks. The total dislocation density and its fluctuation within the sample are determined by the variance method [2,3]. The good qualitative agreement found between the two methods indicate that the cross-correlation method is capable of giving a statistical characterization of the dislocation structure.

In the last part of the talk EBSD measurements on thin foils are presented where the cellular dislocation structure can be directly observed by transmission electron microscopy. These results demonstrate the advantage of the EBSD method compared to XRD analysis, namely that the former is not only capable of determining the dislocation density but also yields the spatial distribution of dislocations.

References:

[1] T.B. Britton and A.J. Wilkinson, High resolution electron backscatter diffraction measurements of elastic strain variations in the presence of larger lattice rotations. Ultramicroscopy 114 (2012) 82-95.
[2] I. Groma, X-ray line broadening due to an inhomogeneous dislocation distribution. Phys.Rev.B 57 (1998) 7535-7542.
[3] F. Székely, I. Groma and J. Lendvai, Changes in the dislocation density fluctuations during plastic deformation. Scripta Mat. 45 (2001) 55-60.

Special thanks to Károly Havancsák, Zoltán Dankházi and Gábor Varga for consultation and valuable suggestions.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-2147 Using electron vortex beams to distinguish enantiomorphic space groups

Juchtmans R.1, Verbeeck J.1
1University of Antwerp, Antwerp, Belgium
roeland.juchtmans@uantwerpen.be

Twenty two truly chiral space groups exist which are characterized by a screw axis. They can be divided in eleven enantiomorphic pairs of two space groups being each others mirror image. Telling apart crystals belonging to enantiomorphic space groups appears to be a difficult task. A few methods have been developed making use of dynamical scattering in which experimental observations have to be compared with numerical simulations [1-4]. We propose a new method to distinguish enanthiomorphic space groups without the need for simulations, based on the use of electron vortex beams in the kinematical approximation allowing a direct interpretation of the handedness of a crystal.
Ever since their first creation [5,6], electron vortex beams (EVB) have been subject of intensive  research [7]. EVB are solutions of the free space Schrödinger equations of the form Ψ(r,φ,z)=exp(imφ)Ψ(r,z). Being eigenfunctions of the orbital angular momentum operator, they carry a well defined orbital angular momentum (OAM) of mħ per electron and a transverse current around the vortex core. In order for the wave function to be continuous, the intensity of the beam has to be zero in the center of the beam resulting in the well known donut shape of the beam, a bright ring with a dark hole in the middle. As can be seen in fig.1, the wave fronts of such a beam have an helical form. Based on a simple model we have derived a relationship between the symmetry of the higher order Laue zones in the diffraction pattern and the OAM of the vortex when scattered kinematically on helically arranged atoms, as is shown schematically in fig.1. For crystals having one heavy atom near a 3-fold screw axis this provides a simple way of measuring the chirality of the space groups without the need for simulations. We verify our conclusions with multislice simulations of the diffraction patterns shown in fig.2 and fig.3 and we discuss the feasibility with experimental results.


[1] Goodman, P. & Johnson, A. W. S. (1977). Acta Cryst. A33, 997–1001.
[2] Goodman, P. & Secomb, T. W. (1977). Acta Cryst. A33, 126–133.
[3] Haruyuki I. et al. (2003), Acta. Cryst. B59, 802-810.
[4] Johnson, A. W. S. (2007), Acta Cryst. B63, 511-520.
[5] Uchida M. & Tonomura A. (2010), Nature 464, 737.
[6] Verbeeck J., Tian H. & Schattschneider P. (2010), Nature 467, 301.
[7] Verbeeck J. et al. (2014), C. R. Phys., http://dx.doi.org/10.1016/j.crhy.2013.09.014".

This research was supported by an FWO PhD fellowship grant (Aspirant Fonds Wetenschappelijk Onderzoek - Vlaanderen). The authors acknowledge support from the EU under the 7h Framework Program (FP7) under a contract for an Integrated Infrastructure Initiative, Ref. No. 312483-ESTEEM2, the European Research Council under the FP7, ERC grant N246791 – COUNTATOMS and ERC Starting Grant 278510 VORTEX.

Fig. 1: A vortex beam scattered on helically arranged atoms in a crystal. In our setup the vortex core coincides with the screw-axis and the size with the radius of the helix.

Fig. 2: Multislice simulation of the zeroth and first order Laue zone of the diffraction pattern of a focused 300keV vortex beam with convergence angle 8mRad and OAM=+1, scattered on a 3-fold screw-axis in right handed Mn2Sb2O7. The sample thickness is 20nm.

Fig. 3: Same as fig.2 for the left handed enantiomorph. The lack of 2-fold symmetry in the first order Laue zone, in contrast to fig.2, allows a direct interpretation of the handedness of the crystal.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-2387 Retrieving nanoscale third-dimension information directly from TEM data using stacked-Bloch-wave simulations and artificial neural network tools

Pennington R. S.1, Van den Broek W.1, Koch C. T.1
1Institute for Experimental Physics, Albert-Einstein-Allee 11, Ulm University, 89081 Ulm, Germany
robert.pennington@uni-ulm.de

Transmission electron microscope (TEM) specimens are three-dimensional, but TEM images and diffraction patterns are two-dimensional. To retrieve the "third-dimensional" information, we have developed a direct-retrieval algorithm including dynamical diffraction that can use TEM data (such as a single convergent-beam electron diffraction [CBED] pattern) and retrieve variations of a range of nanoscale specimen parameters, including strain, crystal tilt, and chemical composition. The retrieval algorithm itself is detailed elsewhere [1], and uses the stacked-Bloch-wave algorithm [2-3] and artificial neural network optimization tools [4]. In this work, we show the effectiveness of our algorithm and discuss considerations for applying this algorithm to realistic experimental data.
A demonstration of this algorithm’s third-dimension (depth-dependent) retrieval ability is seen in Figures 1 & 2. Figures 1 and 2 show CBED patterns of a 100-nm-thick Si specimen at 80 kV at the [110] zone axis, simulated using the stacked-Bloch-wave [2-3] forward-simulation algorithm and 197 zero-order-Laue-zone reflections. Figure 1 has "asymmetric" diffraction features due to the third-dimension variation of crystal tilt. Figure 2 is a CBED pattern like that in Figure 1 but without third-dimension variation, and fails to reproduce the correct diffraction features. Figures 3 and 4 demonstrate our algorithm’s effective and accurate retrieval of third-dimension variation in crystal tilt (Δα, mean over all layers) from the specimen shown in Figure 1a, and decreasing mismatch between simulated and experimental CBED intensity (given by ΔE, mean over all reciprocal-space points). Figure 4 shows how well the unknown α is determined for a known E mismatch.
This algorithm can retrieve third-dimension material properties from a single CBED pattern; however, other techniques like dark-field image series or large-angle rocking-beam electron diffraction (LARBED) series can also be used. Each technique has its own advantages and challenges, especially for analysis of strain or compositional variations. Large lattice-parameter variations can also require a modification to the algorithm in [1].
In this work, we present practical considerations for using our third-dimension information-retrieval algorithm [1]. We demonstrate its effectiveness, discuss different acquisition techniques and consider how different parameters affect our algorithm.
[1]: R. S. Pennington, W. Van den Broek, C. T. Koch. (submitted)
[2]: R. S. Pennington, F. Wang, C. T. Koch. Ultramicroscopy, 2014. http://dx.doi.org/10.1016/j.ultramic.2014.03.003
[3]: D. J. Eaglesham, C. J. Kiely, D. Cherns, and M. Missous. Phil. Mag. A 60, 161 
(1989).
[4]: R. Rojas. Neural Networks: A Systematic Introduction (Springer Verlag, Berlin, 1993).

We acknowledge funding from the Carl Zeiss Foundation and Grant No. KO 2911/7-1 of the German Research Foundation (DFG).

Fig. 1: Simulated zero-loss-filtered convergent-beam electron diffraction (CBED) pattern, generated from a specimen with third-dimension crystal tilt variation. The specimen has ten 10 nm layers, tilted along the [001] direction {0.00, -0.04, -0.10, -0.20, -0.30, -0.30, -0.20, -0.14, -0.06, 0.00} degrees, respectively.

Fig. 2: A CBED pattern like Figure 1, but generated from a specimen with no layer-by-layer crystal tilt variation but with the same mean crystal tilt, which fails to reproduce the "asymmetric" diffraction features seen.

Fig. 3: Our algorithm [1] retrieves third-dimensional variation in crystal tilt (see text) using a (13x13) point reciprocal-space grid, each point 0.05 degrees apart, starting at the 000 point and moving in the [001] and [-110] directions. (This area does not correspond to the discs in Figure 1, but is from the same specimen.)

Fig. 4: The unknown third-dimension parameter mismatch (Δα, mean over all layers), plotted as a function of the known intensity mismatch (ΔE, mean over all points).

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-2763 Measuring strain with high precision and high spatial resolution using precession and convergent beam electron diffraction

Rouviere J.1, Martin Y.1, Beche A.2, Cooper D.3, Bernier N.3, Vigouroux M.3, Zuo J.4
1CEA, INAC/SP2M UJF-Grenoble Minatec campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France, 2FEI Electron Optics, Achtseweg Noord 5, 5651 GG Eindhoven, The Netherlands, 3CEA, LETI, Minatec campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France, 4Univ Illinois, Dept Mat Sci & Engn, 1304 W Green St, Urbana, IL 61801 USA
jean-luc.rouviere@cea.fr

Stimulated by the demand of the semiconductor industry, several new TEM based techniques have been recently proposed to measure strain with high sensitivity and high spatial resolution. In this presentation the interest of using diffraction techniques, either Convergent Beam Electron diffraction (CBED, fig. 1) or Nanobeam Precession Electron Diffraction (N_PED, Fig. 2 and 3) [1] will be shown. Off-axis CBED can give 3D maps of the complete 3D strain tensor ε or equivalently of the deformation gradient tensor F (Fig. 1b), but it is computationally and experimentally demanding. In constrast, N-PED is a straightforward and simple technique, although it is limited to the projected 2D strain tensor. Thanks to its robustness, great precision of about 2x10-4  and simplicity, N-PED should be the preferred tool for the microelectronics industry (Fig. 2).

Fig. 1 illustrates the principle of our strain measurement using off-axis CBED. The originality of our approach is to use both the deficient HOLZ lines of the transmitted beam and the excess HOLZ lines of the diffracted beams to measure the strain. Using Bloch wave calculated CBED patterns as tests, we could retrieve of 7 out of the 9 components of the deformation gradient tensor F (Fig. 1b); in particular the volume of the cells can be determined (Fig. 1c). By using two different directions which makes an angle of 22°, we show that it is possible to determines the whole tensor F. In addition, the method can also be extended to the analysis of split HOLZ lines that allow measuring the variations of the strain tensor along the electron beam.
For N-PED, best results were obtained on a FEI TITAN microscope using a 2kx2k CCD camera. Strain maps of 40x50 points can be acquired in about 20 minutes (Fig. 2). Precession can be used either with nearly parallel beam (NBED like condition, Fig. 3b) or with a convergent beam (on-axis CBED like condition, Fig. 3d). Slightly higher precision were obtained by using the CBED like condition. The main advantage of precession is to suppress the contrasts in the diffraction disks, which leads to improved strain precision.

A major advantage of diffraction based techniques is to be able to analyze samples of non-uniform thickness and non uniform composition along the electron beam. To demonstrate this, results on core shell nanowires (NWs) - Ge NWs embedded with SiN, or Si NWs with a surrounding polycrystalline gate - observed either parallel or perpendicular to the growth direction will be presented.


[1] J.L. Rouvière et al., Appl. Phys. Lett. 103 (2013) 241913.

This work was supported by several projects and contracts: the European catrene UTTERMOST project, the French ANR AMOS and the FEI-CEA common laboratory.

Fig. 1: (a) Simulated CBED pattern along a <651, 441,31> direction in Si. (b) Definition of the deformation gradient tensor F and its link to the strain tensor ε and rotation θ. (c)  Without using the excess HOLZ lines, fzz and (fxx+fyy) are correlated. The volume (fzz+fxx+fyy) can be determined only by using the excess lines.

Fig. 2: Maps of 2 transistors with SiGe source (S) and drain (D). As SiGe has a greater lattice parameter than Si the Si channel is compressed by the SiGe source and drain in the x-direction (εxx). (a) HAADF image computed from the series of N-PED patterns. (b-c-e-f) Strain and rotation maps. d) A typical N-PED pattern of the series.

Fig. 3: Diffraction patterns obtained for various semi-convergence angles α and beam diameters d. At the bottom right of each diffraction, an image of the associated electron probe passing through the [011] silicon crystal gives an estimation of d. In (a) and (b) α = 0.6 mrad (NBED like condition), in (c) and (d) α = 2.2 mrad (CBED like condition).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-2773 Combining real space and reciprocal space tomography in the TEM

Eggeman A. S.1, Krakow R.2, Midgley P. A.3
1Department of Materials Science and Metallurgy, University of Cambridge, Cambridge, CB3 0FS, UK
ase25@cam.ac.uk

Precession electron diffraction (PED) [1] is a valuable technique for investigating crystal structures and when combined with a well-defined raster is able to produce high quality virtual dark-field (VDF) images and orientation maps [2] with ca. nm resolution. Many microstructures vary in all 3 dimensions and tomographic techniques are needed to investigate such complex structures. Combining scanning PED (SPED) with electron tomography offers a way to study local orientation across a volume of interest in 3D.

In this study a tilt-series (from -60o to +70o with 5o steps) of SPED images were recorded from a Ni-base superalloy sample, the scanned maps were recorded with 140x140px of 7.5nm step with a 5nm probe and a precession angle was 0.5o. The image processing is shown in Fig. 1: a) a VDF image (at 5o) that shows a large (ca. 200nm) precipitate, b) shows a VDF image of a smaller (ca. 50nm) inclusion, c) shows the components in the microstructure after segmentation. Geometric tomography (shape-from-silhouette) [3] was used to recombine the VDF tilt-series into a tomogram, Fig. 1(d). The tomogram allowed the contributions to each diffraction pattern in the tilt-series to be determined. As such, the individual diffraction components could be isolated and combined to produce 3D reciprocal lattice reconstructions.

The smaller particle was found to have the ordered η-phase structure (sp. gr. P63/mmc, a=5.314Å and c=8.351Å), the larger precipitate had the MC carbide structure (sp. gr. Fm3m, a=4.32Å) and the γ-matrix has the disordered fcc structure (sp. gr. Fm3m, a=3.59Å). The correspondence between the orientation of the diffraction pattern and the tilt step allowed the orientation of the different phases to be examined. The (001) plane of η-phase has a coherent registry with (111) of the γ-matrix. A test of the reciprocal lattice alignment confirmed that this registry existed across the ‘top’ facet of the inclusion. In the literature there has been no reported registry between the MC and γ phases. However, the front facet of the precipitate (shown in Fig. 2a) was found to be parallel to the (111) plane, the projected reciprocal lattice from this component at the appropriate orientation is shown in Fig. 2(b). The corresponding reciprocal lattice for the matrix is shown in Fig. 2(c) and returned the (531) plane as the matrix surface. Since the entire reciprocal lattice is projected, for clarity the ZOLZ reflections are highlighted and indexed where appropriate. Analysis of the interface showed a semi-coherent registry with the inclusion of a small interface strain (ca. 4%).
[1] R. Vincent & P. A. Midgley, Ultram. 53 (1994), 271-282
[2] P. Moeck et al. Cryst. Res. Tech., 46 (2011), 589-606
[3] Z. Saghi et al. J. Phys. Conf. Ser. 126 (2008) 012063

The authors acknowledge funding from the ERC though grant 291522-3DIMAGE, the 7th Framework Programme of the EC: ESTEEM2 and Rolls Royce plc.

Fig. 1: Figure 1a) and b) virtual dark-field images of second phase particles in a nickel-base superalloy microstructure, c) segmented image of the two particles and d) representative surface render of the reconstructed tomogram.

Fig. 2: Figure 2a) Tomogram surface of a carbide precipitate normal to its largest facet. b) and c) projections of the reconstructed reciprocal lattices from the carbide and matrix, respectively, at the same orientation. The ZOLZ reflections are highlighted in each showing that the interface is composed of tye carbide (111) and the matrix (531) planes
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-2884 Orientation measurements in TEM foils using transmission EBSD

de Kloe R.1, Nowell M. M.2, Suzuki S.3, Wright S. I.2
1EDAX, Ringbaan Noord 103, 5046 AA Tilburg, The Netherlands, 2EDAX, 392 E. 12300 S., Suite H, Draper, UT 840201, USA, 3TSL Solutions KK, #SIC2-401, 5-4-30, Nishihashimoto, Midori-Ku, Kanagawa, Sagamihara 252-0131, Japan.
rene.de.kloe@ametek.nl

For a number of imaging modes in the TEM, knowledge of the orientation is critical. For example dislocation analysis by weak beam dark field imaging requires orienting the grain of interest along one of a limited number of orientations (fig 1). Obtaining this orientation can be done by diffraction pattern analysis of multiple zone-axes. Only when multiple zone axes are identified can it be determined if the zone axes required for the imaging of certain defects are within tilting range. This can be a time consuming process with often limited success, especially on low-symmetry materials. There are a number of automated orientation mapping methods available in the TEM that can assist in this orientation determination [1,2], but the available analysis area in the TEM is limited and it is difficult to obtain a complete overview of a sample.
Combining orientation measurements in the SEM with subsequent TEM analysis can bridge this gap. Standard EBSD measurements can be obtained from most electron transparent crystalline samples that have been prepared for the TEM. Such samples can be mounted in the traditional 70 degree tilt orientation to collect larger area EBSD maps. Recently high resolution EBSD results have also been collected using TEM foils in transmission mode in the SEM (fig 2) [3,4]. But in addition to high resolution orientation mapping, this transmission analysis mode also allows identification of the electron transparent areas in the sample. And in combination with orientation simulations the transmission EBSD orientation results can be used to identify grains that are suitable for specific diffraction analysis on the same sample.

 

[1] Rauch E. F., Véron M., Portillo J., Bultreys D., Maniette Y., Nicolopoulos S., Automatic Crystal Orientation and Phase Mapping in TEM by Precession Diffraction. Microsc. and Anal. 93 (2008) S5-S8
[2] Dingley, D. J. (2006). "Orientation imaging microscopy for the transmission electron microscope." Microchimica Acta 155(1-2): 19-29
[3] Trimby P.W. Orientation mapping of nanostructured materials using transmission Kikuchi diffraction in the scanning electron microscope. Ultramicroscopy. 2012 Sep;120:16-24.
[4] Suzuki S. Features of Transmission EBSD and its Application. J.Japan Inst. Met. Mater’ Vol.77(2013), p268-275

Fig. 1: Weak beam dark field images of a dislocation structure in olivine imaged along different g-vectors

Fig. 2: Images of same area of 8Cr tempered martensite steel specimen. top: TEM bright field image (200kV), middle: t-EBSD IQ map (25kV), bottom: t-EBSD IPF crystal direction map // sample normal. The (sub)grain boundary structure is clearly represented in the t-EBSD images [4].

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-2923 Study of nanoscale local structures of ferroelectric barium titanate using convergent-beam electron diffraction

Tsuda K.1, Sano R.1, Yasuhara A.2, Tanaka M.1
1Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan, 2JEOL Ltd., Tokyo, Japan
k_tsuda@tagen.tohoku.ac.jp

  Convergent-beam electron diffraction (CBED) is established as the most powerful technique to determine crystal point- and space-groups from nanometer-sized specimen areas.1) The CBED method was extended to quantitative crystal structure analysis by Tsuda and Tanaka,2, 3) which enables determinations of structural parameters such as atom positions, atomic displacement parameters (ADPs), as well as electrostatic potential and electron density distributions. In the present study, we applied the CBED method to examine nanometer-scale local structures of BaTiO3.

  It is well known that BaTiO3 undergoes successive phase transformations from the cubic paraelectric phase to three ferroelectric phases: tetragonal, orthorhombic and rhombohedral ones. Coexistence of the displacive and order-disorder characters in the phase transformations of BaTiO3 was pointed out from many experiments and theories. However, local structures related to the order-disorder character were discovered neither in crystal structure analyses using neutron and X-ray diffraction nor by TEM observations.

  Using the CBED method, rhombohedral nanostructures were observed in the orthorhombic and tetragonal phases of BaTiO3.4) It was found that the symmetry of the orthorhombic phase is formed as the average of two rhombohedral variants with different polarizations, and that of the tetragonal phase is formed as the average of four rhombohedral variants. These results indicate an order-disorder character in their phase transformations.4) Similar results were obtained in the ferroelectric orthorhombic phase of KNbO3,5) while it was found that the ferroelectric tetragonal phase of PbTiO3 does not have such rhombohedral nanostructures.6)

  We also proposed a combined use of STEM and CBED methods (STEM-CBED method7)) to observe the nanostructures of polarizations, which is schematically shown in Fig. 1. Using the STEM-CBED method, two-dimensional distributions of the rhombohedral nanostructures, or nanoscale fluctuations of the polarization clusters, were successfully visualized in the tetragonal phase of BaTiO3 as shown in Fig. 2.

References

1) M. Tanaka and K. Tsuda, J. Electron Microsc. 60(Suppl. 1), S245 (2011).

2) K. Tsuda and M. Tanaka, Acta Cryst. A 55, 939 (1999).

3) K. Tsuda et al., Acta Cryst. A 58, 514 (2002).

4) K. Tsuda, R. Sano and M. Tanaka, Phys. Rev. B 86, 214106 (2012).

5) K. Tsuda, R. Sano and M. Tanaka, Appl. Phys. Lett. 102, 051913 (2013).

6) K. Tsuda and M. Tanaka, Appl. Phys. Express 6, 101501 (2013).

7) K. Tsuda, A. Yasuhara and M. Tanaka, Appl. Phys. Lett. 102, 051913 (2013).

This study was supported by JSPS KAKENHI Grant Number 25287068.

Fig. 1: (a) Schematic diagram of the STEM-CBED method.7) (b) a STEM-CBED map of the tetragonal BaTiO3 and CBED patterns,7) which shows the intensity difference between the 020 and 0-20 reflections, (I020-I0-20)/I020. The CBED patterns obtained at positions A, B, and C are, respectively, shown in (c), (d), and (e).

Fig. 2:
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-2948 Direct determination of atomic structures from the observation of phase

Etheridge J.1,2, Nakashima P. N.2, Moodie A. F.1
1Monash Centre for Electron Microscopy, Monash University, VIC 3800, Australia, 2Department of Materials Engineering, Monash University, VIC 3800, Australia
joanne.etheridge@monash.edu

To determine a crystal structure, we need to determine the amplitude and phase of its structure factors from the intensity in its diffraction pattern. However, phase information is either missing or extremely difficult to extract from the diffracted intensities, the infamous “phase problem”. To compensate for this, conventional structure determination methods measure thousands of amplitudes and then deduce the missing phase information using computer-intensive statistical analysis. Although this is time-consuming and the solution is not unique, it has remained the only structure determination approach for a century because of the inability to measure phase.

Here we demonstrate the opposite approach. We show that a centrosymmetric structure can be determined purely from the observation of phase from 3-beam convergent beam electron diffraction (CBED) patterns [1], without the need to measure intensity or analyse it with computer simulations or statistical analysis.

The equations for three beam CBED patterns of centrosymmetric crystals can be inverted analytically, so that the crystal structure factors are described directly in terms of distances to specific features in the pattern [2,3]. This enables the direct measurement of the 3-phase invariant as well as the amplitudes of the structure factors, without recourse to pattern-matching routines [4,5]. Most notably, the sign of the 3-phase invariant can be determined directly by inspection, from the direction of deflection of the rocking curve near the 3-beam Bragg condition (Fig. 1) [4,5], and the individual phases can then be determined from the Bormann effect [6]. This then opens the possibility of solving a crystal structure starting from the observation of phases, rather than the measurement of amplitudes.

We illustrate the method with α-Al2O3, which has 30 atoms in its unit cell. We determine 9 of the structure factor phases, simply from observation of features in 3-beam CBED patterns [1,4,5]. Using these 9 phases only, we can determine the structure to better than 0.1Å precision with no a priori knowledge, except for its space group [1] (Fig. 2). In comparison, the determination of this structure using conventional X-ray diffraction required the measurement of over 2,000 structure facture magnitudes [7].

References

1. P.N.H. Nakashima, A.F. Moodie, J. Etheridge: Proc. National. Acad. Sci. 110 14144 2013.
2. A.F. Moodie, Chem. Scr. 14 21 1978.
3. A.F. Moodie, J. Etheridge, C.J. Humphreys Acta Cryst. A52 596 1996.
4. P.N.H. Nakashima, A.F. Moodie, J. Etheridge Acta Cryst A63 387 2007.
5. P.N.H. Nakashima, A.F. Moodie, J. Etheridge Ultramicroscopy 108 901 2008.
6. G. Borrmann Phys Z 42 157 1941.
7. E.N. Maslen, V.A. Streltsov, N.R. Streltsova, N. Ishizawa, Y. Satow, Acta Cryst B49 973 1993.

The data used in this work was obtained at the Monash Centre for Electron Microscopy. We are grateful to Prof. R. Withers for helpful discussions. This work was supported by the Australian Research Council (DP0346828 and FT110100427).

Fig. 1: An example of a 3 beam CBED pattern. The phase of a crystal structure factor can be determined by inspection of features in such patterns.

Fig. 2: The structure of α-Al2O3 was determined to <0.1Å resolution from the observation of just 9 structure factors phases from 3 beam CBED patterns. No intensities were measured, no computer simulations were required, no statistical analysis is used, no a priori information is needed, other than space group.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-3034 3D Electron Diffraction Tomography without limits: structure analysis of a hyper-complex approximant to icosahedral quasicrystal

Oleynikov P.1, Ma Y. H.1, Fujita N.2, Garcia-Garcia J.3, Yoon K. B.4, Tsai A. P.2, Terasaki O.1,5
1Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden, 2IMRAM, Tohoku University, Sendai, Japan, 3Facultad CC. Químicas, Universidad Complutense de Madrid, Madrid, Spain, 4Department of Chemistry, Sogang University, Seoul, Republic of Korea, 5Graduate School of EEWS, KAIST, Daejeong, Republic of Korea
peter.oleynikov@mmk.su.se

Analyzing the crystal structure of approximants is of vital importance in deriving structural information of building units (or basic clusters) and their arrangements toward icosahedral quasicrystals (IQCs). The acquired knowledge is essential in performing a hyper-space modeling, which is the only feasible way of today to elucidate the structural details of IQC’s. For approximants conventional single-crystal X-ray diffraction can in principle be applied to analyze their atomic structure. However, it becomes quite challenging for the case of approximants to Al-based F-type IQCs, Al-Pd-TM (TM = transition metal) [1]. These approximants often have very large unit cells with lattice constants of over a few tens of Ångströms [2]. A recent study also suggests that, except for the solved case of [2], it is often very difficult to grow single crystals having coherent crystallinity within the width of the incident X-ray beam. It is therefore desirable if the crystal structure can be assessed using electron diffraction from a sub-micron sized crystal domain.
The aim of this study is to assess the possibility of taking the advantage of 3D Electron Diffraction Tomography (3D EDT) [3] in order to solve the crystal structure of the Al-Pd-TM IQC approximant (cubic, s.g. Pa-3, a = 40.54Å). Automated 3D EDT is a fast and efficient technique that has been recently developed by us [3]. It can be used for fast 3D reciprocal space scanning with a given fine step (0.01° – 0.1°) using conventional transmission electron microscopes.
The crystal structure of the individual sub-micron single crystal was determined from the EDT data collected in conventional selected area electron diffraction (SAED) mode using EDT-COLLECT software package [3] on JEOL JEM-2100 FEG CTEM equipped with a single high tilt holder (+/–50°) and Gatan UltraScan 1000 CCD (2048*2048). The acquired data set contains ~2000 unique electron diffraction patterns (exposure 0.5 sec/frame). Reciprocal space coverage was ~90°. The recorded frames were processed using the EDT-PROCESS software package [3] and assembled into a corresponding 3D volumetric representation of reciprocal space (Fig. 1). The crystal structure was successfully determined (Fig. 2) using the direct methods software Sir2011 [4] from the integrated intensities extracted by EDT-PROCESS program.
In this work we show that 3D EDT as a very powerful technique which offers a facile and systematic way to study complex crystal structures.

[1] A.P. Tsai et al, Mater. Trans. JIM 31 (1990), pp. 98-103.
[2] N. Fujita et al, Acta Cryst. A, 69 (2013), pp. 322-340.
[3] M. Gemmi and P. Oleynikov, Z. Kristallogr. 228 (2013), pp. 51-58.
[4] M.C. Burla et al, J. Appl. Cryst. 45 (2012), pp. 357-361.

We kindly acknowledge Swedish Research Council (VR, 1486801), JEOL Ltd., Japan and BK21Plus, Republic of Korea.

Fig. 1: Reconstructed 3D reciprocal space along 001 direction.

Fig. 2: The potential map of the solved structure using direct methods.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-3230 Transmission Kikuchi Diffraction (TKD) in SEM

Palasse L.1
1Bruker Nano GmbH, Berlin, Germany
Laurie.Palasse@bruker-nano.de

It is well known that the study of ultrafine grained materials with grain/cell diameters smaller than ~100 nanometers is very difficult or impossible to characterise by Electron BackScatter Diffraction (EBSD) technique. The spatial resolution limitation of the EBSD technique is function of the electron probe diameter and energy as well as the backscattering coefficient of the analysed material. The incident angle between the beam and the specimen surface (~20º) is another critical parameter influencing the highly anisotropic character of the lateral spatial resolution of the EBSD technique.

As an alternative, the recently introduced Transmission Kikuchi Diffraction (TKD) technique is a SEM based method capable of delivering the same type of results as EBSD but with a spatial resolution improved by up to one order of magnitude [1, 2]. And it only requires a commercial EBSD system and a sample thin enough to be electron transparent, e.g. TEM thin lamellae.

The spatial resolution improvement of TKD compared to EBSD will be demonstrated using results obtained by both techniques. Examples on deformed sample as well as orientation contrast images acquired at unprecedented resolution will also be shown.

In addition, we aim to compare the grain size distribution results between the TKD and  the TEM based “Automated Crystal Orientation mapping” (ACOM) techniques in order to evaluate the feasibility of these advanced methods and discussed the parameters influencing the TKD analysis.

References:

[1] R.R. KELLER and R.H. GEISS, Journal of Microscopy, Vol. 245, Pt. 3, pp. 245–251, 2012.

[2] P. W. Trimby, Ultramicroscopy, 120, 16–24, 2012.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-9-O-3245 Pushing the boundaries of symmetry determination with ‘digital’ electron diffraction

Beanland R.1, Woodward D. I.1, Evans K.1, Römer R.1, Smith K.1, Thomas P. A.1
11Department of Physics, University of Warwick, Coventry, CV4 7AL, UK
r.beanland@warwick.ac.uk

The symmetries in convergent beam electron diffraction (CBED) patterns and their relationship to crystal space groups were first explained almost 40 years ago, and there have been many investigations which have used this to solve crystal structures. The utility of CBED lies in the ability to obtain patterns from regions only a few nm in size, well below that attainable by other methods, sampling perfect crystal that is unaffected by defects or domain structure. Nevertheless, the technique is restricted by small Bragg angles, making it difficult or impossible to apply to materials with closely-spaced spots in a diffraction pattern. Use of computer control to collect patterns at different incidence angles is now relatively straightforward and overcomes this limitation. Capture of many hundreds or thousands of CBED patterns allows reconstruction of ‘digital’ large-angle CBED (D-LACBED) patterns from regions only a few nm in size. The vast increase in information allows previously intractable problems of symmetry determination – particularly for materials with lattice parameters >1nm – to be solved with relative ease. We give several examples, including AgNb7O18, Ca2Mn3O7, polarity measurements in thin PZT films, and polar nanodomains in Na0.5Bi0.5TiO3.
Figure 1 shows [001] diffraction patterns from AgNb7O18. X-ray diffraction showed the material to be orthorhombic with lattice parameters a = 1.4331, b = 2.6151 and c= 0.3836 nm, but was unable to distinguish between four possible space groups: I222, I212121, Imm2 and Immm. Selected reflections from the corresponding D-LACBED pattern, a combination of 2600 CBED patterns, are shown in Fig. 1b. The whole pattern has a vertical mirror but not a horizontal mirror. Opposing dark field patterns with ±g vectors are not equivalent when translated onto each other, demonstrating that the crystal structure is acentric and eliminating the space group Immm. The projection diffraction group of the pattern is therefore m1R, which fixes the point group as mm2. This is consistent with dielectric permittivity measurements which show that is AgNb7O18 is an ergodic relaxor ferroelectric.
Data from, the Ruddlesden-Popper phase Ca2Mn3O7, is shown in Fig. 2. Occasional stacking faults are visible in the HREM image (Fig. 2a) and these were avoided in the collection of D-LACBED patterns. Again, X-ray diffraction is able to limit the possible space groups to a small number of possibilities, in this case Cmcm or Cmc21. The spacing between spots in the SAED pattern (Fig. 2b) is such that no detail is visible in CBED patterns (Fig. 2c). The D-LACBED pattern projection diffraction group is m1R, indicating the lack of a centre of symmetry and confirming the space group to be Cmc21.

Fig. 1: Fig 1. (a) SAED pattern from [001] AgNb7O18. (b) D-LACBED patterns showing a vertical mirror, no horizontal mirror, and acentricity (projection diffraction group m1R).

Fig. 2: Fig 2. [001] Ca2Mn3O7. (a) HREM image of stacking faults; (b) SAED and (c) CBED patterns, (d) selected D-LACBED patterns (projection diffraction group m1R)
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-1450 Sr25Fe30O77 : A complex layered and modulated structure solved by electron diffraction

Lepoittevin C.1
1Institut Néel, CNRS et Université Joseph Fourier, Grenoble, France.
christophe.lepoittevin@neel.cnrs.fr

These past few years, many new structures have been solved using electron diffraction methods. Zone axis precession electron diffraction (PED) and tomography in reciprocal space are two methods enable to reduce importantly the multiple scattering of the electron beam, so that the reflection intensities can be used for structure determination by direct methods.

The ferrite Sr25Fe30O77 belongs to a family of phases whose structures consist of an intergrowth of m perovskite layers with complex rocksalt type layers [1-2]. Our compound of interest is the member m = 4 of this family and its structure has been solved by combining both electron diffraction methods cited above. This oxide crystallizes in an orthorhombic system with the sub-cell parameters a ≈ b ≈ 5.4 Å and c ≈ 42 Å. The structure exhibits modulation along axis with a modulation vector . Due to the commensurate nature of the modulation, the structure can be described in a supercell with the parameters a ≈ 27 Å, b ≈ 5.4 Å and c ≈ 42 Å. PED patterns were recorded in zone axis with a Spinning Star unit using a precession angle of 2°. The intensities were extracted with CRISP software [3] in “shape fitting” or “integer” modes. The data were then implemented in SIR2008 software[4] and many trials were made with or without application of geometrical Lorentz correction to obtain the structure. The tomography data collection, recorded by tilting manually every 0.5 degree from -30 to +30 degrees, was inserted in EDT (Electron Diffraction Tomography) software [5], which reconstructs the 3D reciprocal space and integrates automatically the reflection intensities. The resulting intensity file was then used on SIR2008 for structure resolution. The solved structure, by combining both methods, consists of four consecutive layers with Fe in octahedral environment alternating with one complex layer containing Fe in three different environments. The oxygen atoms in this last layer are responsable of the modulated nature of the structure.

References:

[1]Pérez, O., Mellenne, B., Retoux, R., Raveau, B. & Hervieu, M. (2006). Solid State Sciences. 8, 431-443, [2]Grebille, D., Lepoittevin, C., Malo, S., Pérez, O., Nguyen, N. & Hervieu, M. (2006). J.Solid State Chem. 179, 3849-3859, [3]Hovmöller S., www.calidris-em.com, [4]Il milione II suite http://wwwba.ic.cnr.it/content/sir2011-v10, [5]Oleynikov P.www.edt3d.com.

Fig. 1: [010] electron diffraction pattern of Sr25Fe30O77

Fig. 2: solved structure of Sr25Fe30O77
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-1516 Substrate threading dislocations imaged by weak beam dark field TEM on samples with GaN nano-LEDs

Lenrick F.1, Bi Z.2, Ohlsson J.3, Ek M.1, Hetherington C.1, Samuelson L.2, Wallenberg L. R.1
1Centre for Analysis and Synthesis/nCHREM, Lund University, Box 124, S-221 00 Lund, Sweden, 2Solid State Lighting Center, Lund University, Box 118, S-221 00 Lund, Sweden, 3QuNano AB, Ideon Science Park, Sheelevägen 17 , S-223 70 Lund, Sweden
filip.lenrick@polymat.lth.se

The semiconductor material GaN is used in blue and white light emitting diodes (LEDs). It’s also a promising material high power and RF electronics Traditional planar epitaxial fabrication of GaN is, however, not adequate due to the large lattice mismatch between GaN and the available substrates, such as sapphire, Si and SiC. At the strained interfaces threading dislocations (TDs) are formed, degrading efficiency, reliability and lifetime of the devices.
Nano-sized structures show the potential to be free of TDs due to their small dimensions, and morphologies such as nano-wires and nano-pyramids (grown along <0001>) have additional benefits. For instance, the quantum confined Stark effect can be reduced since these morphologies can offer non-polar and semi-polar planes, respectively.
Truncated GaN pyramids were grown by selective area metal-organic vapour phase epitaxy on a GaN substrate with high TD density. A 30 nm thick layer of amorphous Si3N4 (grown by low-pressure chemical vapor deposition) with openings about 100 nm in diameter, patterned by electron-beam lithography and etched by reactive ion etching, was used as the selective area mask. The mask blocks most of the TDs in the substrate from entering the pyramids, but the ones that cross through the mask are interesting to study due to their degrading impact on the device.
To clearly observe the threading dislocations, weak beam dark field (WBDF) transmission electron microscopy (TEM) was applied on focused ion beam (FIB) prepared cross sections. The images facilitate tracing of TDs through the material and how they enter the nano-structures. The FIB lamella, which was about 100 nm thick, showed a TD density of about 10 TDs/µm in projection. Six adjacent pyramids were analyzed where two was found to have TDs from the substrate coming through the mask. The WBDF technique is challenging on a high acceleration voltage microscope due to the low curvature of the Ewald sphere. WBDF condition such as 3g(9g) was found to be more suitable than the standard g(3g) since many diffraction spots are excited. By slightly defocusing the diffraction pattern and using Kikuchi lines as guide lines WBDF conditions became easier to set up.

Fig. 1: 3g(9g) weak beam dark field (WBDF) TEM image of FIB prepared cross sections of truncated GaN nano-pyramid grown through small openings in Si3N4 mask on a GaN (0001) surface. Threading dislocations (TDs) are visible as bright lines. Two TDs marked by red arrows are blocked by the mask, while one TD marked by a green arrow enters the pyramid.

Fig. 2: The low curvature of the 300kV Ewald sphere causes a challenge to set up the WBDF conditions. Kikuchi lines, visible at slight defocus, are usable as guidelines.

Fig. 3: Schematic illustration of one truncated GaN pyramid (grown through openings in amorphous Si3N4 on a GaN substrate) as seen in TEM projection. Threading dislocations (TDs) are marked as red lines. The Si3N4 mask acts as a filter, keeping the TDs in the substrate, but occasionally a TD pass through the opening.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-1526 PRECESSED NANO-ELECTRON DIFFRACTION PATTERNS OF THE HUMAN TOOTH ENAMEL CRYSTALS

REYES-GASGA J.1,2, ADDAD A.1, BRÈS E. F.1
1Unité des Matériaux et Transformation (UMET). Université de Lille 1, Sciences et Technologies. Bâtiment C6. 59650 Villeneuve d’Ascq. Lille, France., 2Permanet Address: Instituto de Física, UNAM. Circuito de la Investigación s/n. Cd. Universitaria, 04510 Coyoacán, Mexico D. F., México
jreyes@fisica.unam.mx

In this work we present the precessed electron diffraction patterns of the nano-sized human-tooth-enamel crystallites. These diffraction patterns have allowed us to obtain crystallography information the enamel’s unit cell [1].
The intensity of selected area electron diffraction (SAED) and nano-electron diffraction (n-ED) patterns is difficult to interpret due to the multiple interactions which take place (dynamical diffraction, absorption, etc). However, when the electron beam is tilted and precessed at high frequency the dynamic effect is minimized [2]. The crystal is not moving but the Ewald’s sphere is precessing around the optical axis producing that the dynamical SAED patterns become close to kinematical conditions and they can be used to obtain information on crystal structures [3, 4].
We have obtained the precessed n-ED from human tooth enamel crystals along different zone axes. Human tooth enamel is composed in 95% of hydroxyapatite crystals (HAP, Ca10(PO4)6(OH)2). These crystals are elongated-plate-like of 30 to 60 nm wide and 100 to 200 nm long [5], approximately (figure 1).
The human tooth enamel samples were obtained from permanent non-carious human molar teeth, extracted for orthodontic or periodontal reasons. Samples were prepared in the FIB-FEI QUANTA 200 3D equipment using the two beams system. A Philips CM30 transmission electron microscope with LaB6 filament working at 300 KV was used for TEM observation, the n-ED and the precessed electron diffraction patterns obtaining using a double-tilt holder. The precession of electron diffraction patterns were obtained with the Nanomegas “Spinning Star” equipment. The patterns were recorded on a Gatan “ORIUS” CCD camera using the Digital Micrograph software. JEMS software (version 3.8431U2012) was used for electron diffraction simulation.
Therefore, we have obtained precessed nano-electron diffraction patterns from crystals in the range from 30 to 100 nm (figure 2).

References
1. See papers in Ultramicroscopy, vol.107, issue 6-7, July 2007.
2. R. Vincent, P.A. Midgley, Ultramicroscopy 53 (1994) 271-282.
3. J.P. Morniroli, A. Redjaımia, S. Nicolopoulos, Ultramicroscopy 107 (2007) 514-522.
4. H. Klein Acta Cryst. A67, (2011) 303-309.
5. J. Reyes-Gasga et al., Materials Sci. Eng. C 33 (2013) 4568-4574.

JRG thanks to DGAPA-UNAM (contract IN106713), CONACYT and PASPA-DGAPA-UNAM for sabbatical support.

Fig. 1: TEM bright field image of the human tooth enamel FIB sample used in this work. Note the nano-sized crystals.

Fig. 2: Nano-electron diffraction pattern along the [0001] zone axis (A) and the corresponding precessed electron diffraction pattern (B). C) Simulated [0001] electron diffraction pattern for a HAP sample with thickness sample of 15 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-1563 Nano-beam Diffraction of Pt/Al2O3 and Pd/Al2O3 Catalysts

Ward M. R.1, Boyes E. D.1,2, Gai P. L.1,3
1Department of Physics, University of York and the York Nanocentre, UK, 2Department of Electronics, University of York and the York Nanocentre, UK, 3Department of Chemistry, University of York and the York Nanocentre, UK
michael.ward@york.ac.uk

Pt-Al2O3 and Pd-Al2O3 catalysts are used in a wide range of applications including automobile emissions catalysts (1, 2). Although there have been many studies on this system, there are few studies which examine in detail the nanoparticle interface with the complicated nature of γ-Al2O3 and its associated polymorphs. Commercial Pt-Al2O3 and Pd-Al2O3 catalysts tend to be composed of small metal nanoparticles (< 10 nm) on high surface area Al2O3, which are often agglomerations of 10-30 nm crystallites. SAD analysis of such catalysts rarely provides quantitative crystallography of individual Al2O3 crystals but nanobeam diffraction (NDB) has been shown to be a useful tool for examining individual small crystals (3). The nano-sized probe, combined with nanosize crystals has been shown to produce well defined shape effects in the diffraction pattern which can provide further structural insights compared to images alone (4). Here, we have used nano-beam diffraction to investigate how this technique can provide useful insights into the structural relationships between the nanoparticles and the Al2O3 support.

A double aberration corrected JEOL 2200FS was used for this study. The catalysts were provided by Jonhson Matthey as powders. (S)TEM specimens were prepared by depositing an ethanol suspension of the powder onto a holey-C film Cu TEM grid.

Figure 1 shows a typical SAD pattern of an agglomeration of many Pd nanoparticle coated Al2O3 crystals. The bright rings are the {400} and {440} rings assuming γ-phase. The related θ and δ phases have similar bright spatial frequencies but are indexed differently. From the SAD pattern in Figure 1, no useful information can be extracted from it regarding the phase of individual Al2O3 crystals and the nanoparticles supported on them. Figure 2 shows NBD pattern of an individual Al2O3 crystal. The NBD pattern was taken with a probe of approximately 2 nm in diameter. The convergence angle of the probe in this case is sufficiently small to produce a diffraction pattern composed of well defined points rather than discs. The diffraction pattern shows well ordered spots of γ-Al2O3 in [110] orientation. Diffraction patterns such as this from the support and a nearby nanoparticle provide insights into the structural relationship between the two. Where it is applicable, for quantitative analysis the method is clearly superior to FFTs of images in terms of the effects of SNR, aberrations and drift etc and in not requiring an exact zone axis orientation.

References

1. A. Russell, W. S. Epling, Catal Rev 53, 337 (2011).

2. O. K. Ezekoye et al., J Catal 280, 125 (May 16, 2011).

3. M. R. Ward, T. Hyde, E. D. Boyes, P. L. Gai, Chemcatchem 4, 1622 (Oct, 2012).

4. F. Tao et al., Science 322, 932 (Nov 7, 2008).

The authors thank the EPSRC for support from critical mass grant EP/1018058/1.

Fig. 1: (a) TEM image and (b) SAD pattern of the Pd-Al2O3 catalyst. The SAD pattern rings are not useful for indentifying the phase of individual Al2O3 crystals.

Fig. 2: (a) TEM and (b) NBD pattern of a single crystal (indicated) in the Pd-Al2O3 catalyst with (b) being identified as γ-Al2O3
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-1573 Charge Density Determination for Transition Metals and Intermetallics by Convergent Beam Electron Diffraction and Density Functional Theory Validation

Sang X.1, Kulovits A. K.1, Wang G.1, Wiezorek J. M.1
1Department of Mechanical Engineering and Materials Science, University of Pittsburgh, Pittsburgh, PA, 15261, USA
wiezorek@pitt.edu

The electron charge density difference, ∆ρ(r), i.e., the difference between the crystal electron density and that of the equivalent independent atom model (IAM), represents quantum mechanical characteristics central for fundamental understanding of materials. Convergent beam electron diffraction (CBED) permits probing of nano-scale volumes of perfect crystal and can enable measurements of low-order structure factors, Fg, with sufficient accuracy to obtain ∆ρ(r) for transition metals and binary intermetallic phases [e.g. 1-3]. The high accuracy and precision of the CBED measurements warrants their use as additional metrics in validation of density functional theory (DFT) calculations for these d-electron system materials [2]. Here, sets of multiple Fg and the Debye Waller factors have been determined simultaneously by CBED for transition metals (e.g. Cr, Fe, Ni, Co, Cu, Ta) and chemically ordered intermetallic phases (e.g. NiAl, TiAl, FePd). Using the local density approximation (LDA), LDA + U, and different generalized gradient approximations (GGA) functionals implemented in WIEN2K low-order Fg and thus ∆ρ(r) have been calculated for comparison with the CBED measurements. While many of the different GGA calculations achieve good overall agreement with the experimentally determined low-order Fg for the elements, LDA and GGA functionals fail to predict accurately the low-order Fg for β-NiAl and γ1-FePd. For equiatomic γ-TiAl GGA based DFT achieved considerably improved agreement with experimentally determined ∆ρ(r), when compared with LDA calculations [2]. Fig. 1 shows the difference between the X-ray structure factors, Fg, determined by CBED for two different composition TiAl crystals (Ti-50at%Al and Ti-52at%Al) and the IAM based Fg. Select data from ∆ρ(r)-maps obtained from CBED measurements and GGA DFT calculations are compared in Fig. 2 for the equiatomic and slightly Al-rich TiAl phases. Effects from the small (2at.%) Al-excess in the intermetallic γ-TiAl have been detected by the CBED experiments and are discernible in the ∆ρ(r) most clearly for the (001)-sections (Fig. 2). The excess Al is incorporated substitutionally on Ti sites and appears to enhance delocalization of charge density between second nearest neighbor Ti atoms along <010>, while reducing it for nearest neighbor Ti atom bonds along <110> (Fig. 2).

References

[1] XH Sang, AK Kulovits, JMK Wiezorek, Acta Crystal. A66 (2010) p. 694

[2] XH Sang et al., J. Chem. Phys. 138 (2013) p.084504

[3] XH Sang, et al., Phil. Mag. 92 (2012) p.4408

The authors acknowledge support from the Office of Basic Energy Sciences, Division of Materials Science and Engineering (Grant No. DE-FG02-08ER46545).

Fig. 1: Fig. 1: Difference between the CBED determined X-ray structure factors and the IAM structure factors, ∆Fg, for equiatomic (TiAl) and Al-rich off-stoichiometric (Ti-52Al) γ-TiAl phase. The hkl are plotted along the abscissa. The [uvw] in the legend (inset) indicate the approximate incident beam direction in CBED experiments.

Fig. 2: Fig. 2: Example ∆ρ(r) sections in (001), all Ti plane for the equiatomic composition phase, of L1o-structure tP4 unit cell of TiAl. CBED derived for Ti-50Al (equiatomic) on the left, CBED derived for Ti-52Al (Al-rich) in the middle, and DFT calculated for Ti-50Al (equiatomic) on the right.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-1631 Axial transmission electron diffraction in a scanning electron microscope

Volkenandt T.1, Müller E.1, Gerthsen D.1
1Laboratory for Electron Microscopy, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany
erich.mueller@kit.edu

Scanning transmission electron microscopy (STEM) at low electron energies is a well suited technique to achieve sensitive material contrast in the high-angle annular dark-field (HAADF) mode where contrast is attributed to incoherently scattered electrons. HAADF STEM can be exploited for sample thickness determination and composition analysis [1]. Transmission electron backscattered diffraction (t-EBSD) patterns were recently recorded from a thin specimen by a detector placed laterally to the tilted sample [2]. In our study the detector was placed on-axis below the sample and coherent electron scattering at energies up to 30 keV was analysed which yields axial Bragg-diffraction patterns with Kikuchi lines.
A FEI Strata 400S scanning electron microscope equipped with a segmented semiconductor STEM detector was used. A conventional imaging plate (IP) was inserted below the sample as a detector. The sample consists of a GaN layer with 140 nm thickness on a 120 nm AlN layer epitaxially grown on a Si(111) substrate. A TEM sample with a thickness of 120 nm was prepared by focused-ion-beam milling.
Figure 1 shows a 25 keV HAADF STEM cross-section image of the sample. Dislocations and columnar regions (indicated by dashed lines) with slightly different intensities can be seen in the GaN layer. A tilt series was recorded which shows changes and even contrast inversion within the GaN layer which is a strong indication for coherent scattering.
Figure 2 shows a transmitted on-axis IP-image taken at 25 keV at the position marked by a cross in Figure 1. Figure 2a depicts the illuminated area with the STEM detector segments marked by circles. Kikuchi lines are visible on the whole detector area which can be identified by comparison with simulated EBSD patterns. Diffraction patterns from different positions along the GaN layer show a shift of Kikuchi lines due to orientation changes in the columnar layer. Figure 2b depicts the inner region of the diffraction pattern. A GaN [1-100] zone-axis pattern is identified by measuring the scattering angles for the Bragg reflections. This pattern also yields information on the first-order Laue zone and shows (0002) two-beam excitation condition.
Axial diffraction patterns recorded with IP reveal Bragg reflections and Kikuchi lines within the scattering range covered by the STEM detector. They provide information on the crystal structure of the sample and show that coherent scattering must be considered even at large scattering angles at low electron energies. Moreover, the diffraction pattern shows the local orientation and excitation condition of the sample.

References
[1] T. Volkenandt, E. Müller, D. Hu, D. Schaadt, D. Gerthsen, Microsc. Microanal. 16, 604 (2010)
[2] N. Brodusch, H. Demers, R. Gauvin, J. Microscopy 250, 1 (2013)

This work was funded by the Deutsche Forschungsgemeinschaft (DFG).

Fig. 1: 25 keV HAADF STEM cross-section image with dislocations marked by arrows and columnar regions separated by dashed lines. The cross indicates the position where the diffraction pattern in Figure 2 was taken. The sample was covered with a Pt/C-layer for protection during FIB milling.

Fig. 2: a) Diffraction pattern taken at marked position in Figure 1 at 25 keV. The layout of the STEM detector is indicated by dashed-line circles. b) Inner region of a) showing a diffraction pattern of GaN [1-100].
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-1694 Strain mapping at the nanoscale using precession electron diffraction in a non-corrected Transmission Electron Microscope

Vigouroux M. P.1, Delaye V.1, Lafond D.1, Bernier N.1, Rouvière J. L.2, Chenevier B.3, Bertin F.1
1CEA, LETI, MINATEC Campus, 17 rue des martyrs, 38054 GRENOBLE Cedex 9, France, 2CEA, INAC, MINATEC Campus, 17 rue des martyrs, 38054 GRENOBLE Cedex 9, France, 3LMGP, CNRS, 3, parvis Louis Néel, 38016 GRENOBLE Cedex 1, France
mathieu.vigouroux@cea.fr

The electron precession [1] technique is a recent innovation in electron crystallography. The advantage of this technique is to minimize the dynamical effect to such an extent that diffraction images can be analyzed using a kinematical approach with minimal user intervention. As a first step we have performed Precession Electron Diffraction (PED) strain measurement on a simple calibration sample paving the way to the strain analysis on more complex devices from micro-electronic.
PED measurements were made using a JEOL-JEM2010FEF non corrected microscope operating at 200 kV. Precession beam scan alignment is performed employing NanoMEGAS’s “DigiSTAR” add-on device. Precession semi-angle was set to 1.44° to take full advantage of PED kinematical behavior. With a probe size as small as 4.2 nm FWHM is obtained on the sample with a convergence of 1 mrad.
The sample we have used is prepared from materials grown by RPCVD on a [001] Si Substrate. It is composed of four 10 nm SiGe layers with different contents in Ge separated by 30 nm of Si and covered with 150 nm Si capping layer. A 8 kV FIB operating voltage was used to provide 50 nm thin parallel-sided lamellae with reduced surface damage. This sample was specifically designed to benchmark strain studies [2] as it is easy to simulate the strain expected in TEM.
PED are recorded every 2.7 nm in a 185 nm x 240 nm region indicated in Fig. 2. using a 512 x 512 pixels Camera deported from the microscope optical axis. Classical projective geometry was used to correct most of distortions in misaligned cameras. Figure 1 (a) illustrates typical diffractions patterns acquired during experiments. Beam probe images were made with a CCD camera (Fig. 1) able to deal with high brilliance scenes and dedicated software was designed to compute PED patterns for strain analysis. The algorithm used takes advantage of the whole “kinematic” region in reciprocal space. The basis of vectors inherent in that periodic region is found using Delaunay triangulation and introduced in a reciprocal matrix G. From this matrix, the distortion matrix D can be retrieved, giving access to the strain ε matrix and rotation Ω matrix.
Figure 2 shows εxx strain mapping obtained by analysing the acquired diffractions patterns set with this method. The noise in 800 contiguous εxx values far from SiGe layers is rather small so that an rms of 3 10-4 is obtained. Strain profiles (Fig. 3) reveal the strong repeatability in measures. Both of them agree very well with finite element COMSOL simulation of the strain averaged along the beam direction and convoluted with the measured electron beam shape.

[1] Vincent, R., et al. « Ultramicroscopy, 53, 3,1994

[2] Rouviere, et al. Applied Physics Letters 103, 24,2013

This study was made possible through funding provided by ANR LABEX MINOS and ANR AMOS programs. Experiments have been done within the Nanocharacterisation Platform of the CEA/Grenoble, MINATEC Campus.

Fig. 1: (a) [110] PED diffraction patterns obtained in Silicon with the probe displays in (b). (a) 4.2 nm full width at half maximum spot size in silicon measured on CCD camera with 1.44° Precession semi-angle.

Fig. 2: SiGe Strain mapping with Precession (semi-angle set to 1.44°)

Fig. 3: εxx SiGe Strain profile along y=70 profile shown Fig. 3; mean εxx strain profile all over y; εxx SiGe Strain profile obtained by finite element COMSOL simulation.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2034 Imaging of grain boundaries in polycrystalline samples by HRTEM

Kiss Á. K.1, 2, Pécz B.1, Rauch E. F.3, Nicolopoulos S.4, Lábár J. L.1
1Institute for Technical Physics and Materials Science, Research Centre for Natural Sciences of the Hungarian Academy of Sciences (MTA TTK MFA), Budapest, Hungary, 2University of Pannonia, Doctoral School of Molecular-and Nanotechnologies, Veszprém, Hungary, 3SIMaP, Grenoble INP/CNRS, France, 4NanoMEGAS Sprl, Brussels, Belgium
kiss.akos.koppany@ttk.mta.hu

Simultaneous imaging of neighboring grains and the grain boundary between them is tedious if polycrystalline samples are to be examined with random orientation distribution of submicron sized grains. The tilting range of HRTEMs is limited to about 20° and there is a low probability to find simultaneously resolved planes and especially low index zones for both grains within this tilting range by chance. Operation of a computer assisted method is demonstrated here that aids such imaging. The method is a combination of the commercial precession electron diffraction (PED) system [1] deployed on a JEOL 3010 with a new computer program that predicts tilt values needed for simultaneous HRTEM imaging of the grains selected from the orientation map.

The best scenario is when we are able to orient low index zones parallel to the electron beam in both grains and the grain boundary is also parallel to the beam simultaneously. A solution with compromise is if only one of the grains is seen from a low index zone while only one plane-set is resolved for the other.

Miller indices of the grain boundary plane in the coordinate systems of both grains are determined from its projection and the local thickness (or from projections at two tilt values as an alternative). The method also comprises the calibration of the directions of the tilt axes in the image.

The evaluation process can be applied to both cubic and non-cubic crystal systems and even to phase boundaries since the calculation of orientations and sample tilts is based on the general metric matrix formalism.

Application of the method is demonstrated here on hcp ZnO thin film with grain size of ca. 20‑40 nm deposited on Si substrate. Figure 1 shows the orientation map of the interested area. Different colors represent different orientations (blue area at bottom is the Si substrate) therefore individual grains can be recognized. The chosen boundary is marked by the white arrow. Figure 2 shows BF image of the layer while Figure 3 presents the HRTEM image of the observed boundary. The area marked by the dashed rectangle indicates a region where the two grains do not overlap, so the boundary is almost in the beam direction here. Fast Fourier transforms of the two grains, shown as inserts, corroborate that both grains are seen from the predicted orientations.

[1] J.Portillo, E.F.Rauch, S.Nicolopoulos, M.Gemmi, D.Bultreys: Precession Electron Diffraction assisted Orientation Mapping in the Transmission Electron Microscope, Materials Science Forum Vol. 644 (2010) pp 1-7 doi: 10.4028/www.scientific.net/MSF.644.1

Z. Baji is acknowledged for the preparation of the ZnO layer by ALD.

Fig. 1: Orientation map; probe size: 10 nm, step size: 5 nm. The observed boundary is marked by the white arrow.

Fig. 2: Bright field image taken at the area of interest. The observed boundary is marked by the white arrow.

Fig. 3: High resolution image of the neighboring grains showing the first grain from [011] i.e. [-1 2 -1 3] zone. The (011) i.e. (0 1 -1 1) planes are only resolved for the second grain. The selected area shows the best insight into the structure of the boundary.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2120 Statistical evaluation of 3D planes in polycrystalline materials from 2D Electron Backscatter Diffraction (EBSD) maps

Jäger A.1, Klinger M.1, Tesař K.1, Malachov M.1
1Institute of Physics AS CR, Na Slovance 2, Prague, Czech Republic
jager@fzu.cz

Scanning electron microscope (SEM) fitted with electron backscatter diffraction (EBSD) detector reached widespread popularity for gaining crystallographic information from a surface of crystalline materials. The main limitation of EBSD technique during two-dimensional mapping is missing depth information. However, in comparison with time-consuming 3D EBSD that require focused ion beam (FIB), 2D EBSD technique needs simpler equipment and easier post-processing.

In this work, uncomplicated statistical approach is presented to find dominant planes such as grain boundaries and fracture planes in bulk polycrystalline materials. The model is based on analysis of intersections of demanded planes with plane of EBSD mapping. Intersection of the two planes generates traces which are further evaluated. For experimental verification metals with hexagonal close packed (hcp) structure were selected; namely magnesium and titanium since they are very attractive for many industrial applications. Data were acquired on SEM FEI Quanta 3D FEG fitted with Hikari EBSD camera. It is shown that the approach combining 2D EBSD mapping with calculations in Matlab software can evaluate the results very well even with moderate amount of experimental data. With this technique dominant planes such as abundant {10-12} <11-20> 86° twin boundaries in wrought magnesium alloy AZ31 (nominally Mg-3wt%Al-1wt%Zn) and preferred fracture plane in duplex phase titanium grade 2 (nominally <0.3wt%Fe, <0.25wt%O, <0.015wt%H) submitted to uniaxial tension at room temperature were successfully analyzed. An example of statistical evaluation in wrought magnesium alloy AZ31 with a number of {10-12} <11-20> 86° twin boundaries is shown in Fig. 1.

The authors would like to appreciate financial support offered by GACR GBP108/12/G043 and MEYS LM2011026.

Fig. 1: Fig. 1: Normals to boundary planes found with the help of the approach. The results correctly show abundant {10-12} <11-20> 86° twin boundaries in wrought magnesium alloy AZ31.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2149 Precession Electron Diffraction Tomography study of new materials derived from Aurivillius phases.

Mouillard-Stéciuk G.1, Boullay P.1, Barrier N.1, Pautrat A.1
1Laboratoire CRISMAT, UMR CNRS 6508, ENSICAEN, 6 Bd Maréchal Juin, F-14050 Caen Cedex 4, France
gwladys.mouillard@ensicaen.fr

Oxides of the Aurivillius family (Bi2O2)2+(Am-1BmO3m+1)2- (A = Ca, Sr, Ba, Pb, … and B= Ti, Nb, W, …) have attracted constant interest in the solid state chemistry community considering both their complex layered structure and their wide range of potential applications. A large number of Aurivillius phases exhibit ferroelectric properties at room temperature and present structural distortions leading to predictable structures and space groups [1]. While their dielectric properties have been intensively studied over past decades, Aurivillius phases have recently proved to also present good potential as semi-conductor photocatalyst [2,3].

In the search for new ferroelectrics derived from Aurivillius phases, we recently found [4] a series of layered materials in the pseudo-binary system Bi5Nb3O15-ABi2Nb2O9 (A=Ca, Sr, Pb, Ba). Preliminary observations made by Transmission Electron Microscopy (Fig. 1) indicate that these compounds exhibit a complex incommensurately modulated structure. Following the procedure described in [5], a (3+1)D structural model was obtained using ab-initio phasing by charge flipping (Superflip) based on the analysis of Precession Electron Diffraction Tomography (PEDT) data (Fig. 2). The (3+1)D structure was further validated by a refinement against powder X-ray diffraction (PXRD) in JANA2006 (Fig. 3).

The new materials possess a layered Aurivillius-type structure with periodic crystallographic shear planes (CSP) leading to the formation of “collapsed” structures with discontinuous (Bi2O2)2+ slabs and perovskite blocks (Fig. 3b) quite similar to what is known in the high-Tc superconductors and related compounds [6]. It appears that the structural difference between the compounds of this series is the length of the collapsed layers, related to the evolution of the modulation vector with the cationic radius A.

Nevertheless, instead of “conventional” Aurivillius phases, where the possibility of non-stoichiometry is mostly limited to a partial substitution of A cations for Bi in the (Bi2O2)2+ slabs, the newly found compounds exhibit a wide compositional stability domain.

Our results define the contour of what appears as a new family of layered perovskite oxides and emphasizing the role of PEDT in the search for new materials.

[1] P. Boullay, G. Trolliard, D. Mercurio, J.M. Perez-Mato and L. Elcoro, J. Solid State Chem. 164 (2002) 252.
[2] H.H. Kim, D.W. Hwang and J.S. Lee, J. Am. Chem. Soc. 126 (2004) 8912.
[3] X. Chen, S. Shen, L. Guo and S.S. Mao, Chem. Rev. 110 (2010) 6503.
[4] G. Mouillard, Master 2 Recherche (2013) Université de Caen.
[5] P. Boullay, N. Barrier and L. Palatinus, Inorg. Chem. 52 (2013) 6127.
[6] M. Hervieu, M.T. Caldes, S. Cabrera, C. Michel, D. Pelloquin, B. Raveau, J. Solid State Chem. 119 (1995) 169.

Fig. 1: a) [0100] electron diffraction zone axis patterns of one representative member of the new layered compounds. b) Enlarged area of a) revealing the existence of a modulation of the form q = αa*+γc*. c) Corresponding HREM image.

Fig. 2: Results for BaBi7Nb5O24, [0100] projection of a 14ax14c: a) Electron density map as obtained from the charge-flipping structure solution procedure. b) Cationic structural model obtained after interpretation of theelectron density map and the addition of discontinuous functions (crenels) (Bismuth red and Niobium green).

Fig. 3: a) Final observed, calculated, and difference plots obtained for the PXRD Rietveld refinement of BaBi7Nb5O24. The black tick marks indicate the main reflections and the green set the satellite reflections. b) [0100] projection of a 14ax14c supercell as obtained from the PXRD refinement.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2158 Multiple scattering in amorphous structures

Mu X.1, Koch C. T.2, Sigle W.1, Neelamraju S.3, van Aken P. A.1
1Max Planck Institute for Intelligent Systems, Stuttgart, Germany, 2Institute for Experimental Physics, Ulm University, Ulm, Germany, 3Max Planck Institute for Solid State Research, Stuttgart, Germany
muxiaoke@gmail.com

Electron diffraction is a convenient technique to study the structure of materials with the advantage of high spatial resolution compared to X-ray diffraction. This fact has recently also increased interest in measuring the pair-distribution function (PDF) of amorphous materials by electron diffraction.[1] However, electrons are likely to scatter multiple times on their path through the sample, due to their strong interaction with matter. Thus, understanding the effect of multiple scattering (MS) on extracting PDFs from electron diffraction is crucial for the quantitative interpretation.

It is generally accepted that for materials possessing a 3-dimensionally isotropic structure subsequent scattering events along the electron path are independent from one another. It implies that MS can be accounted for by a simple convolution.[2] The single-scattering signal should thus be extractable from a diffraction pattern containing the contribution from MS electrons by deconvolution.[3] In our study of amorphous MgF2,[4] we found that the PDF extracted from the deconvolved diffraction pattern does not differ significantly from the PDF extracted from the original experimental data in peak shape and positions, even though there has been a significant amount of MS.

In order to investigate this similarity between the original and the deconvolved data, we used the QSTEM package [5] for simulating a dynamical diffraction pattern of an amorphous structure [6] and extracted the PDF from it. The first multislice simulation (figure 1c) was done to simulate a diffraction pattern from a small model (figure 1a) obtained by molecular dynamics simulation, mimicking single scattering because of the very thin specimen. Another simulation (figure 1d) was done to simulate the diffraction pattern from a supercell being constructed by vertically stacking the original model 20 times (figure 1b), mimicking a 20 times thicker specimen. Figure 2 shows that, except for a reduction in peak height at low frequencies, the diffraction pattern containing MS agrees rather well with the kinematical one. The PDFs (figure 2d) extracted from the MS data and the kinematic data also show no difference in peak shape or position. We finally conclude that, apart from a reduction in peak height, MS has no significant effect on the PDF. Therefore, deconvolution is not necessary in case that correct retrieval of coordination numbers is not important.

[1] D. J. H. Cockayne, Annu Rev Mater Res 2007, 37, 159-187.

[2] G. R. Anstis et al., Ultramicroscopy 1988, 26, 65-69.

[3] J. E. Ankele et al., Z Naturforsch A 2005, 60, 459-468.

[4] X. Mu, Ph.D thesis, TU Darmstadt 2013, 91-94.

[5] C. T. Koch, Ph.D. thesis, Arizona State University 2002.

[6] C. T. Koch et al., Ultramicroscopy 2006, 106, 383-388.

Acknowledgements: The research leading to these results has received funding from the European Union Seventh Framework Programme [FP/2007-2013] under grant agreement no312483 (ESTEEM2).

Fig. 1: Figure 1. (a) A MgF2 cell containing 6150 atoms. (b) Supercell constructed by stacking 20 randomly orientated single cells (shown in a) to mimic the thick material for the dynamical diffraction simulation. (c) Simulated diffraction pattern from the single cell of the model shown in a. (d) Simulated diffraction pattern from the supercell shown in b.

Fig. 2: Figure 2. (a) Profiles of simulated diffraction patterns; (b) structure factors extracted from a; the black dotted line is a 4th-order polynomial function fitted to the red curve; (c) same as (b) but the polynomial has been subtracted from the red curve (d) PDFs obtained by Fourier sine transform of the structure factors in b.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2328 ACOM-TEM analysis of mineral particles ultrastructural organization in bone tissue

Verezhak M.1, Rauch E. R.2, Gourrier A.1 3
1Laboratory of Interdisciplinary Physics, Université Grenoble Alpes / CNRS, Saint Martin d’Hères, France, 2SIMaP laboratory, Université Grenoble Alpes / CNRS, Saint Martin d’Hères, France , 3European Synchrotron Radiation Facility, Grenoble, France
mariana.verezhak@ujf-grenoble.fr

Bone tissue has a complex hierarchical architecture that is self-assembled in order to perform diverse mechanical, biological and chemical functions. At the nanoscale it can be viewed as a composite material made up of two principal components: collagen fibrils of ~ 100 nm in diameter and platelet-shaped calcium phosphate mineral crystals of the 5 x 50 x 100 nm dimensions. The size, shape, organization, orientation and internal structure of mineral crystals has been a matter of disputes since bone sections were first studied by electron microscopy in the 1950’s [1].
Transmission electron microscopy (TEM) shed new light on this problem by allowing the direct visualization of bone structure. However, a lot of difficulties were faced related to image interpretation and to the choice of samples preparation technique. In collaboration with a medical team, we are now able to produce bone sections as thin as 70 nm. We are also currently exploring new bone sample preparation methods than ultramicrotomy as, e.g. tripod polishing and ion milling.
The novel use of the Automated Crystal Orientation Mapping with a TEM method (ACOM-TEM, also known as ASTARTM tool from NanoMEGAS) [2] to study the mineral particles ultrastructural organization in bone tissue with the spatial resolution of 20 nm is reported. The ACOM-TEM method operated in scanning mode and relied on the comparison between the high quality electron diffraction patterns collected at every scan position and the simulated patterns calculated for a given crystal in all possible orientations. This method, therefore, allows crystallographic indexing, high-resolution nanocrystal orientation (~ 1°) and crystal phase mapping.
The mineral particles in bone orientation 2-D mapping was, for the first time, analyzed and the presence of disorder, discontinuity and crystallinity degree variations is discussed. Current results are part of larger project aiming to understand the nanostructural characteristics of bone tissue and to identify key structural markers of pathological human bone [3], providing possible development of new diagnostic and pharmaceutical tools.

References:
1. Robinson R. A., Watson M. L. (1952). Collagen-crystal relationships in bone as seen in the electron microscope. Anatom Rec 114: 383–409.
2. Portillo J., Rauch E.F., Nicolopoulos S., Gemmi M., Bultreys D. (2010). Precession Electron Diffraction assisted Orientation Mapping in the Transmission Electron Microscope. Mater Sci Forum Vol. 644 pp 1-7.
3. Gourrier A., Li C., Siegel S., Paris O., Roschger P., Klaushofer K. and Fratzl P. (2010). Scanning small-angle X-ray scattering analysis of the size and organization of the mineral nanoparticles in fluorotic bone using a stack of cards model. J Appl Crystallogr 43, 1385-1392.

This project is supported by the NanoSciences Fondation (Grenoble, France), through the PhD Excellence Grant Programme for M. Verezhak.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2385 Investigation of dislocation structures by cross-correlation based EBSD mapping and TEM imaging

Kalácska S.1, Groma I.1, Ispánovity P. D.1
1Eötvös Loránd University
kalacska@metal.elte.hu

During unaxial compression of copper single crystals an inhomogeneous dislocation structure develops. With the use of cross-correlation based analysis of electron backscatter diffraction (EBSD) patterns it is possible to map plastic strain variations in deformed polycrystalline samples [1]. In this work this method is applied to visualize the dislocation structures and corresponding distortion fields in Cu single crystals compressed to different levels. The maps created by this method show inhomogeneous cell structure. Furthermore transmission electronmicroscopy is widely used to create micrographs that directly show dislocation arrangement within the sample.

Sample surface preparation plays a key role in creating ideal conditions for both TEM and EBSD measurements. Firstly, we applied various preparation techniques and investigated the efficiency of those methods. We used focused ion beam to create TEM foils of approximately 100 nm thickness. From samples with high dislocation content it's difficult to carve out such lamellas because during the thinning process the foil can spontaneously bend due to the inner stress field. We also made TEM samples with traitional electopolishing and ion polishing processes and compared the resultant TEM micrographs.

Then the distortion maps of the specimen are computed with the cross-correlation technique. This method is capable of detecting changes of the crystal orientation to higher accuracy than the commercial software provided for standard EBSD devices that analyse each EBSD pattern individually. The good qualitative agreement found between the two methods indicate that the cross-correlation method is capable of giving distribution characterization of the cellular dislocation structure. The results measured on the same surface area by cross-correlation based EBSD and TEM methods were compared and evaluated.

Reference:

[1] T.B. Britton and A.J. Wilkinson, High resolution electron backscatter diffraction measurements of elastic strain variations in the presence of larger lattice rotations. Ultramicroscopy 114 (2012) 82-95.

Special thanks to Károly Havancsák, Zoltán Dankházi and Gábor Varga for consultation and valuable suggestions. The help of Alajos Ö. Kovács and János Lábár is also appreciated.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2459 EBSD sample preparation: high energy Ar ion milling

Kalácska S.1, Baris A.1, Varga G.1, Radi Z.2, Lendvai A.2, Dankházi Z.1, Havancsák K.1
1Eötvös Loránd University, 2Technoorg Linda Ltd.
kalacska@metal.elte.hu

EBSD is a versatile tool providing grain size determination, orientation mapping, phase identification and 3D mapping. Since the EBSD information comes from a few tens of nanometers of the specimen surface regions the most critical issue of the EBSD measurement is the surface quality. The surface should be perfectly clean, free of amorphous or deformed surface layer and moreover it should be flat because of the shadowing effect. Lack of these factors can result either no or faded diffraction pattern.
As it is known, the usual mechanical grinding and polishing create an amorphous layer of (1-100) nm thickness on the surface. The commonly suggested colloidal silica polishment continues for hours and can embed residual polishing material in the surface grains. Electropolishing of the surface can also be tried, but this is a difficult and complex procedure, nevertheless in some cases it cannot lead to the desired result.
In the last decades a new surface milling method is spreading. This is based on energetic ion beam milling; the underlying physical process is the sputtering. One direction of this method is the focused ion beam technique (FIB) with ion energies up to 30 keV. The other direction uses near parallel inert gas (usually Ar) ion beams with energy up to 10 keV.
In this poster we present a newly developed Ar ion sample milling apparatus and show how advantageously it can be utilized to produce high quality sample surface. Surface quality development on series of metal samples was investigated using Technoorg Linda's SC-1000 SEMPrep Ar ion milling apparatus. The surface quality of samples was characterized by the image quality (IQ) parameter of the electron backscatter diffraction (EBSD) measurement. Ar ion polishing recipes have provided to prepare a surface appropriate for high quality EBSD mapping. The initial surfaces of samples were roughly grinded and polished. High quality surface smoothness could be achieved during the subsequent Ar ion polishing treatment. The optimal angles of Ar ion incidence and the polishing times were determined for several materials using a FEI Quanta 3D FEG SEM.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2531 Determination of dislocation density by electron backscattering diffraction and X-ray line profile analysis in ferrous lath martensite

Berecz T.1, Csóré A.1, Jenei P.2, Gubicza J.2, Szabó P. J.1
1Department of Material Science and Engineering, Budapest University of Technology and Economics, Budapest, Hungary, 2Department of Materials Physics, Eötvös Lóránd University, Budapest, Hungary
berecz@eik.bme.hu

Ferrous martensite can appear in several forms, such as lath, lenticular and plate, depending mainly on the composition. Among these martensite structures the lath martensite has high industrial significance because of its high strength and toughness. Lath martensite can appear usually in the technologically more important low (and medium) carbon, low cost and low alloyed steels.

The lath martensite morphology exhibits a characteristic multilevel microstructure. A parent austenite grain consists of several packets (the group of laths with the same habit plane). Each packet is divided into parallel blocks and a block is further subdivided into laths. The size of individual martensite laths is very small, therefore they cannot be seen by optical microscopes. There are high angle boundaries between the blocks and packets, while low angle (about 5-10°) boundaries between the single laths.

The strength and toughness of the lath martensitic steels strongly depend on the microstructure through packet and block sizes, as well as the size, shape and arrangement of the laths. The reason of their high strength and toughness is mainly the high angle boundaries between the blocks and packets which hinder the dislocation movements.

Dislocation density in the lath martensitic structure can be determined by both automated electron backscattering diffraction (EBSD) and X-ray line profile analysis (XLPA) method. Dislocations can cause local lattice distortion, which leads to misorientation between individual points in the lattice. Using automated EBSD, the local orientations are determined at individual points in a regular grid on a planar surface of a polycrystalline specimen.

From the difference between the neighboring orientations on planar surfaces the dislocation density can be calculated. XLPA is sensitive to microstrains around the individual dislocations, even if the dislocations are arranged into such configurations which do not yield any misorientation between the different volumes of the crystal. Thus, the dislocation density calculated by XLPA may be different from that measured by automated EBSD.

In our study dislocation densities are determined in individual laths and blocks by EBSD and these results are compared with the dislocation density measured by XRD in ferrous lath martensite.

This work was supplied by the Hungarian Scientific Research Fund (OTKA PD 101028).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2605 Quantitative local structure analysis of nanocrystalline FeAl by electron diffraction

Rentenberger C.1, Gammer C.2, Karnthaler H. P.1
1University of Vienna, Physics of Nanostructured Materials, Vienna, Austria, 2National Center for Electron Microscopy, LBNL, Berkeley, California, USA
christian.rentenberger@univie.ac.at

Profile analysis by X-ray diffraction has been proven to be able to obtain microstructural parameters averaged over a large sample volume (>10µm3). In nanocrystalline materials it is frequently the case that local information is required. This can be achieved by local quantitative analysis based on selected area electron diffraction (SAED). Using the method of PASAD [1] that provides profile analysis of SAED patterns we show that structural parameters can be deduced of volumes on a submicrometer scale (<0.01µm3).

Nanocrystalline B2 ordered FeAl with a mean grain size of about 35nm was made by high pressure torsion (HPT) followed by a heat treatment [2]. The achieved nanocrystalline material was exposed to a further HPT deformation (3 turns, 8 GPa). SEM studies indicate that the deformation occurs inhomogeneously in the form of shear bands. TEM studies were carried out using 200kV.

Fig. 1 shows a bright field image of a nanocrystalline FeAl sample after further deformation by HPT. The complex contrast variations are caused by orientation variations of individual grains and by lattice defects. The darker band in the middle of the image corresponds to a shear band (SB). The density of the dislocations is so high that it is not possible to determine it. Therefore, we use an alternative method. Fig. 2 shows an SAED pattern taken from the encircled area (cf. Fig. 1). The pattern consists of concentric rings. Using PASAD-tools [1] an intensity profile as a function of the diffraction vector g is obtained by integration along the rings (cf. inset Fig. 2). The broadening of the peaks (half-width at half maximum, HWHM) corrected for instrumental broadening was studied by fitting combined Voigt peak-functions. Since broadening by grain size and strain has different effects on the peak profiles both of them can be determined using the method of modified Williamson-Hall plots [3]. This is shown in Fig. 3(a) taking the contrast factors C of dislocations (slip system <111>{110}) into account. The slope of the curve is proportional to the square root of the dislocation density. The values of the slope were calculated from 35 SAED patterns arranged in a 5x7 array within the area indicated in Fig. 1. Fig. 3(b) shows a contour plot of the slope values as a function of the SAED positions. The values indicate that even in a nanocrystalline material the dislocation density within a shear band can be up to a factor 4 higher than in the neighbouring area.

[1] C. Gammer, C. Mangler, C. Rentenberger, H. P. Karnthaler. Scri. Mater 63 (2010) 312.

[2] C. Mangler, C. Gammer, H. P. Karnthaler, C. Rentenberger. Acta Mater 58 (2010) 5631.

[3] T. Ungar, A. Borbely. Appl. Phys. Lett. 69 (1996) 3173.

The authors acknowledge support by the Austrian Science Fund (FWF):[I1309, P22440, J3397] and C.G. by the National Center for Electron Microscopy, Lawrence Berkeley Lab, supported by the U.S. Dept. of Energy under Contract # DE-AC02-05CH11231.

Fig. 1: TEM bright-field image of nanocrystalline FeAl deformed by HPT. Structural parameters were measured by profile analysis of SAED patterns of 35 circular areas placed within the marked rectangle.

Fig. 2: TEM selected area electron diffraction pattern of the encircled area indicated in Fig. 1. The inset shows the corresponding intensity profile.

Fig. 3: (a) Modified Williamson Hall plot obtained from the intensity profile shown in Fig. 2. (b) Contour plot drawn from the slope values of the modified Williamson-Hall plots obtained from a 5x7 array of SAED patterns (of the area marked in Fig.1). The values are proportional to the square root of the dislocation density.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2650 Fluctuation electron microscopy of an amorphous-crystalline composite material

Ebner C.1, Gammer C.2, Karnthaler H. P.1, Rentenberger C.1
1University of Vienna, Physics of Nanostructured Materials, Vienna, Austria, 2National Center for Electron Microscopy, LBNL, Berkeley, California, USA
christian.ebner@univie.ac.at

Fluctuation electron microscopy (FEM) is a TEM technique that allows the characterization of the atomic structure in an amorphous material. It measures the spatial fluctuations in the scattering of electrons arising on a medium-range scale (1-3nm). Here, the FEM technique based on the acquisition of tilted dark-field images was applied to specimens containing nanometer sized crystalline regions embedded in an amorphous matrix.  
Intermetallic Co3Ti with the nominal composition of Co-23at.%Ti was made by mixing Co and Ti of high purity in an induction furnace under Ar atmosphere. The high oxidation tendency of Ti leads to the formation of some small titanium-oxide particles. After annealing at 950°C for ~100h under a static Ar overpressure to achieve the L12 long range ordered phase, the crystalline Co3Ti alloy was rendered amorphous by severe plastic deformation using high-pressure torsion (HPT with 20 turns at 8GPa).
Fig. 1a shows a TEM bright-field image of the Co3Ti sample subjected to HPT deformation. Dark dots (5-10nm in size) within a homogeneous speckle contrast characteristic for an amorphous sample indicate the presence of small crystalline particles. Some of these crystalline particles light up in the tilted TEM dark-field image (cf. Fig. 1b) when a certain scattering vector k is used. Fig. 2 shows the corresponding TEM diffraction pattern of a large area. The dominance of the diffuse rings is characteristic for the amorphous phase. The particles can be analysed by EELS but in this study we want to emphasize the capability of FEM. Therefore, FEM that is sensitive to spatial differences in diffraction was applied. Fig. 3 and 4 show the FEM results calculated from tilted TEM dark-field images taken from the entire reciprocal space by varying the direction and length of k. The images were analysed statistically by calculating the mean and the normalized variance V(k) of the image intensity I(k,r): V(k)=(<I(k,r)2>/<I(k,r)>2)-1, where <> means averaging over sample position r [1]. By averaging <I(k)> and V(k) of images taken with a given k, plots of <I(k)> and V(k) as a function of k are obtained (cf. Fig. 3).  In order to analyse the crystalline particles, V(k) values of two-phase areas V(k)tp are compared with the value of the amorphous area V(k)a. The plot V(k)tp - V(k)a shows peaks at positions corresponding to titanium-oxide lattice planes (cf. Fig. 4). The good agreement of the results by FEM and EELS reveals that FEM is able to identify crystalline particles and it underlines also the applicability of FEM for the characterisation of structural medium-range order in the amorphous phase.

[1] M.M.J. Treacy et al., J. Phys.: Condens. Matter 19 (2007) 455201.

The authors acknowledge support by the Austrian Science Fund (FWF):[I1309, P22440, J3397] and the National Center for Electron Microscopy, Lawrence Berkeley Lab, which is supported by the U.S. Department of Energy under Contract # DE-AC02-05CH11231.

Fig. 1: TEM bright-field (a) and tilted TEM dark-field image (b) of amorphous Co3Ti containing small crystalline particles.

Fig. 2: TEM diffraction pattern and the corresponding intensity profile of amorphous-crystalline Co3Ti.

Fig. 3: Plot of the mean intensity and the normalized variance Va of an amorphous area as a function of k. The position of the first peak in Va indicates the presence of Co3Ti-like structure on a medium-range scale.

Fig. 4: Plot of the normalized variance Vtp – Va. Depending on the orientation of the oxide particles in the taken area (area1-3) different peak positions corresponding to different lattice planes are observed.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2743 Illumination Wavefront Determination by Image and Diffraction Focal Series

McLeod R. A.1,2, Rouviere J.2, Zuo J.1,3
1Fondation Nanosciences, Grenoble, France, 2CEA, INAC/SP2M UJF-Grenoble Minatec campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France, 3University of Illinois at Urbana-Champaign, Champaign, USA
robbmcleod@gmail.com

In a transmission electron microscope (TEM), the geometric optics of the illumination system typically are unknown to the user, outside of some basic principles such as condenser lens underfocus or overfocus. When the intensity of a nanoprobe is measured, the phase shift across the probe is lost. The phase shift contains fine oscillations that affect how the probe propagates through the specimen. We present here a method to measure the optical parameters of the illumination system. With an optical model of the illumination mode, an estimate of the probe phase can be found for any lens conditions. The resulting complex entry-wavefunction can then be used for simulation and optimization of the instrument for nanobeam diffraction or coherent diffractive imaging (CDI).

The illumination system is modeled as a single compound lens using the paraxial approximation, with a demagnification of the source and limiting condenser aperture above the lens as shown in Fig. 1. The method calculates the three degrees of freedom: (1) the electron probe diameter b, (2) the convergence angle of the illumination α (or equivalently numerical aperture) and (3) the focal length of the illumination system f (shown in Fig. 2). The dependent parameters, (4) the condenser aperture optical diameter a, and (5) the defocus from specimen to the cross-over zf , are calculated in-addition. The demagnification can be estimated (1⁄M~60) for the given spot size from the nominal aperture diameter. By Fourier optics, the wavefront at the aperture can be numerically forward propagated by za to estimate the complex wavefunction at the specimen.

Our method relies on acquisition of focal series of the nanobeam probe in vacuum via the Python scripting interface. The objective lens excitation is fixed at the eucentric focus. The operating condenser lens, C3 in the case of a FEI Titan, is varied through a large range, forming a series of nanobeam probes at the specimen plane, as shown in Fig. 3. The range is from an image of the condenser aperture conjugate on the specimen plane (C3 = -0.25 in Fig. 3), to the illumination focused on the specimen plane (C3 = 0.02 in Fig. 3). The TEM is then placed in diffraction mode and a series of vacuum diffractograms over both diffraction lens (DL) excitations, and the same range of C3 excitations, is collected (not shown). The diffraction series allows the convergence angle α to be measured, and the combination of both series allows the focal length to be stated in nanometers rather than nominal units. The magnifications in image-mode and camera length in diffraction-mode at each C3 and DL were measured from a second series of images and diffractograms from a monocrystalline Silicon specimen.

RAM acknowledges the financial support of Fondation Nanosciences and CEA.

Fig. 1: The simplified TEM illumination model forms a probe on the specimen of diameter b and convergence angle α. The limiting aperture is demagnified by a factor 1/M. By varying the power of the lens for a series of focal lengths f, the unknowns of the model may be calculated and a conversion from nominal lens excitation to focal length made.

Fig. 2: Focal length of illumination f. The result for the two condenser apertures used, 10 μm and 30 μm, vary only slightly.

Fig. 3: A representative example set for the probe series in imaging mode for the condenser aperture in focus, at C3 = -0.25, to the focal point at C3 = 0.02, and just into overfocus, using the 10 m aperture.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2769 Automated crystal orientation mapping in TEM for the statistical analysis of microstructure evolution in nano-grained polycrystalline thin-films

Aebersold A. B.1, Hébert C.1, Alexander D. T.1
1Interdisciplinary Centre for Electron Microscopy (CIME), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, CH-1015, Switzerland
arthur.aebersold@epfl.ch

Non-epitaxial polycrystalline films are important in many technological applications. Characterization of their microstructure is crucial for understanding their growth mechanisms and improving their properties. Their microstructure formation typically begins by dense nucleation of randomly oriented grains. During film thickening these grains impinge on each other, resulting in the overgrowth of unfavorably oriented grains and the subsequent formation of a textured film with a columnar or V-shaped grain structure. In this work our aim is to develop a methodology for quantitatively determining the grain size and orientation distributions throughout the thickness of such films, in order to help create accurate simulation models and to correlate film microstructures to their macroscopic properties.
The methodology is based on the principle of orientation mapping (OM); given the nm scale of the film microstructures, in this contribution we apply OM by nano-beam diffraction in TEM using a 2–3 nm electron probe and the NanoMEGAS ASTAR [1]. The high spatial resolution of this technique comes at the cost of needing suitably electron-transparent samples. A quantitative analysis of microstructure evolution requires the sample to have large thin areas from different heights within the film. These requirements can be met by a special double-wedge sample geometry previously proposed by Spiecker et al. [2], which provides continuous plan-view sections throughout the film thickness (see Fig. 1). The heights in the film of the plan-view sections are determined by cross-correlating the position of the thin area to the thickness of the film after dimpling, which was measured from visible light interferences after the dimpling step. Furthermore, the plan-view sections are perpendicular to the direction of the grain elongation thereby minimizing the regions of grain overlap within the projection of the specimen. This improves the reliability of the ASTAR measurements.
Here we report the application of this methodology to nano-grained polycrystalline low-pressure chemical vapor deposited ZnO films used as transparent conductive oxide layers in thin-film solar cells [3]. The applied methodology allows us to extract quantitative in-plane data on the evolution of grain size, orientation, and boundary misorientation as a function of height in the film (see Fig. 2), which can be compared to existing theory and simulations and help to provide new insights into the growth mechanisms that create these films.

References:
[1] EF Rauch et al., Microscopy and Analysis 22 (6) (2008) p. S5.
[2] E Spiecker et al., Acta Mater. 55 (2007) p. 3521.
[3] S Faÿ et al., Thin Solid Films 518 (2010) p. 2961.

The authors acknowledge funding from the SNSF, Grant Number 137833. L. Fanni, Dr S. Nicolay and Dr A. Hessler-Wyser of the IMT PV-lab, EPFL are thanked for the samples and discussions.

Fig. 1: Illustration of double wedge sample geometry. a) ZnO film on glass, b) dimpling of ZnO film (first wedge), c) wedge polish, d) bright-field TEM imaging at different heights along the electron transparent edge (red solid line)

Fig. 2: Inverse pole figure maps overlaid with reliability index, extracted grain size and orientation distributions from two different heights in a ZnO thin film. The data demonstrate the ability of the methodology for obtaining quantitative data on nanocrystalline grain distributions along the height axis.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2894 Analysis of the ordering state of pyroxenes using precession electron diffraction.

Jacob D.1, Wouossaju S.1, Palatinus L.2
1UMET, UMR 8207 CNRS-Université Lille 1, Villeneuve d’Ascq, France, 2Institute of Physics of the Academy of Sciences of the Czech Republic, 182 21 Prague, Czech Republic
damien.jacob@univ-lille1.fr

The precession electron diffraction (PED) technique [1] has been originally developed for structure determination at a submicrometer scale in a transmission electron microscope (TEM). Since, many structures have been solved using PED, recently combined with the tomographic acquisition of 3D electron diffraction data [2]. Using PED, integrated intensities of the diffracted beams as a function of the rocking beam orientation are collected. The resulting intensities keep dynamical in nature, due to residual multiple scattering, but are more closely related to the strength of the scattering events and ranking of reflections as a function of their intensities is generally correlated to the structure factor values, which is crucial for structure solution.

Recently, it has been shown that PED could also be used for structure refinement [3]. In this case, experimental intensities have to be compared with dynamical simulations of diffracted intensities, taking into account the multiple scattering occurring when the electron beam is passing through the crystal. Applied to structures with mixed occupancies, the analysis can be used to refine atomic occupancies of specific sites of the structure, giving access to the ordering parameter. In the field of mineralogy, the PED refinement has thus been used to analyze the ordering state of orthopyroxene (OPX) samples. Results have enabled the distinction between an equilibrated sample (natural OPX (Mg0.60Fe1.40)Si206) and a non equilibrated one (heat-treated (1000°C, 48h) and quenched sample from the same origin), giving ordering parameter values in good agreement with those obtained at the grain scale using XRD [4].

To go further and use PED data to decipher the thermal history of the sample with sufficient precision, the sensitivity of the PED refinement method still appeal for a detailed quantitative evaluation. In this work we discuss the influence of experimental parameters such as the irradiation dose and/or heating of the sample under the electron beam. Analyses are performed on the previously studied equilibrated OPX sample. Our results show a noticeable evolution of the ordering parameter with the electron beam irradiation duration (Fig. 1), which assesses for the high sensitivity of the technique. Possible evolution of the ordering state associated with the in-situ heating of the sample will also be explored, opening the road to the study of intra-crystalline diffusion kinetics at a very local scale in a TEM using PED. [1] R. Vincent and P.A. Midgley, Ultramicroscopy 53 (1994) p. 271. [2] U. Kolb et al., Crystal Research and Technology 46 (2011), p. 542. [3] L. Palatinus et al. Acta Crystallographica A (2013), 69(2), P. 171. [4] D. Jacob et al., American Mineralogist 98 (2013) p.152.

We gratefully acknowledge C. Domeneghetti (Univ. Pavia) and F. Camara (Univ. Torino) for supplying the OPX samples together with their XRD structural analysis

Fig. 1: Plot of XFe(M2) vs. XFe(M1) in a natural OPX sample as obtained from PED dynamical refinement as a function of the duration of the electron beam illumination (200kV, LaB6 Tecnai 20 microscope). Dashed line corresponds to the constant composition line. Green triangle corresponds to XRD results obtained at the grain scale
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2929 Application of Large-Angle Convergent-Beam Electron Diffraction to APBs Recognition

Jezierska E.1
1Warsaw University of Technology, Faculty of Materials Science and Engineering, Woloska 141, 02-507 Warsaw, Poland
jezierska@op.pl

The Large-Angle Convergent-Beam Electron Diffraction (LACBED) technique was proposed by Tanaka in 1980 to improve the quality of the CBED patterns obtained with a large angle convergent incident beam (Kossel patterns) [1]. In this method a specimen is raised (or lowered) from its usual eucentric position in the object plane. The LACBED technique which uses a defocus incident beam has a unique property: the image of the illuminated area of the specimen is superimposed on the diffraction pattern composed of Bragg lines [2]. Therefore, the pattern is a mapping between the direct and the reciprocal spaces and “shadow image” of a defect is visible on the pattern.
TEM investigations were performed on JEM 3010 Jeol equipped with Gatan CCD camera. Conventional TEM studies and LACBED were used to elucidate the structure of ordered intermetallics. The antiphase domains structure in various ordered intermetallics with perfect L12 superstructure has been examined. For advanced studies of the nature of antiphase boundaries (APBs) LACBED method was employed.
Ordering of atoms occurs in a large number of alloys. Whereas in the disordered form the lattice sites are occupied at random, they will be occupied by atoms of a given chemical species in the ordered form. Ordering is accompanied by domains formation. The arrangement of domains is characteristic for ordered alloy and applied technology. Due to phase contrast the visibility of APBs is significant on TEM images. Using centered superlattice dark-field image the mapping of ordering can be achieved (Fig. 1). Perfect symmetry was confirmed from LACBED images and ordering is manifested in superlattice Bragg lines (Fig. 2). Any defects breaking the translational symmetry and perfect order can be visible on LACBED lines. For antiphase domain boundaries (APBs) in ordered compound the splitting of superlattice Bragg lines on LACBED images can be observed (Fig. 3). The superlattice excess line is split into two lines with equal intensity on bright-field LACBED pattern as well as on dark-field LACBED pattern if the domains are enough large to see the effect (Figs. 3-4). This splitting can be considered as typical and used to identify APBs. For very fine domains only subtle effect of affected Bragg lines can be noticed [3].
References:
[1] M. Tanaka, R. Saito, K. Ueno, Y. Harada, LACBED, Journal of Electron Microscopy, 29 (1980) 408-412.
[2] J.P. Morniroli, Large-Angle Convergent-Beam Electron Diffraction (LACBED). Applications to crystal defects, Sfμ , Paris (2002).
[3] E. Jezierska, J.P. Morniroli, Antiphase boundaries in Ni3Al ordered intermetallic – application of CBED method, Material Chemistry and Physics 81 (2003) 443-447

The financial support from the Polish Ministry of Science and Higher Education, Faculty of Materials Science & Engineering Warsaw University of Technology is gratefully acknowledged.

Fig. 1: TEM image of antiphase domains boundaries in (Al,Mn)3Ti ordered intermetallic phase with L12 superstructure (centered superlattice dark-field with 011 operating spot)

Fig. 2: LACBED image from perfect (Al,Mn)3Ti superstructure with [233] zone axis

Fig. 3: LACBED on antiphase domains boundary. The superlattice excess line is split into two lines with equal intensity on bright-field LACBED pattern

Fig. 4: DF LACBED of superlattice (01-1) Bragg line with splitting due to APB
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-2930 Quantitative CBED in a Nano-structured Material

Nakashima P. H.1, Bourgeois L.1,2, Etheridge J.1,2
1Department of Materials Engineering, Monash University, 3800 Victoria Australia, 2Monash Centre for Electron Microscopy, Monash University, 3800 Victoria, Australia
philip.nakashima@monash.edu

When rapidly quenched to room temperature from just below its melting point, aluminium can form octahedral voids of a few tens of nanometres in size, truncated with {001} facets. For convergent beam electron diffraction (CBED), this presents an interesting scenario that can be thought of as a “CBED sandwich”. For a focussed electron probe incident on a {001} void facet, the resultant CBED pattern is the product of diffraction from two totally coherent slabs of crystal, oriented along <001>, each slab sandwiching the free space in the void. Such a CBED pattern is not only sensitive to the thicknesses of the two slabs but also their separation across the void because the electron waves modified by the first slab of crystal then Fresnel propagate as they traverse the void.

In addition to highly constrained measurements of the thickness of the specimen, the dimension of the void in the beam direction and its position with respect to the entrance and exit faces of the specimen, quantitative CBED is used here to measure bonding-sensitive structure factors on either side of the void.

To investigate the sensitivity of the bonding electron density to the nanoscale geometry and size of these structures, we compare these results with recent work [1] where the same structure factors were measured with sufficient accuracy and precision as to be able to unequivocally determine the bonding electron density in aluminium. Our work takes advantage of the multislice formalism for electron scattering [2], which is conducive to the geometry of the “CBED sandwich” that a void in a metallic foil presents.

References:

[1] P.N.H. Nakashima, A.E. Smith, J. Etheridge, B.C. Muddle, Science 331 (2011), 1583.

[2] J.M. Cowley, A.F. Moodie, Acta Cryst. 10 (1957), 609.

The data collected for this work was obtained using the JEOL 2011 TEM in the Monash Centre for Electron Microscopy, funded by the ARC (RIEFP 99). PN thanks the ARC for grant FT110100427.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-3029 Strain Analysis by Nano-Beam Electron Diffraction (SANBED) in semiconductor nanostructures

Mahr C.1, Müller K.1, Erben D.1, Schowalter M.1, Zweck J.2, Volz K.3, Rosenauer A.1
1Universität Bremen, Otto-Hahn-Allee 1, 28359 Bremen (Germany), 2Universität Regensburg, Universitätsstraße 31, 93040 Regensburg (Germany), 3Philipps Universität Marburg, Hans-Meerwein-Straße, 35032 Marburg (Germany)
mahr@ifp.uni-bremen.de

The measurement of lattice strain is an important aspect in the characterisation of semiconductor nanostructures. As strain has large influence e.g. on the mobility of charge carriers, methods for accurate strain measurement with high precision are mandatory. In the present work [1] we measure strain from the positions of diffraction discs in convergent-beam electron diffraction (CBED) patterns using dedicated algorithms. Large one- and two-dimensional series of CBED-patterns (~3000) in semiconductor nanostructures have been recorded at an FEI Titan facility in STEM mode. Contrary to parallel illumination in conventional Nano-beam electron diffraction (NBED), we show that focusing the beam with a semi-convergence of 2.6 mrad increases the spatial resolution drastically by a factor of 5 to be 0.5 – 0.7nm. We determined the precision of this method to be 0.07%.

The rich inner structure of CBED-discs causes a big challenge to recognize their positions accurately. In particular, three different algorithms have been developed: As shown in figure 1, the first algorithm detects edges around each disc and iteratively deselects erroneous edges by circle-fitting. In this way the fit converges to the disc boundary. A disadvantage of this parameter-free method is a long computation time. An improvement of speed by a factor of 15 is achieved with the Radial Gradient Maximisation method. This method positions two sets of rings around the initially estimated disc position, one set of rings with smaller radii than estimated and one with larger radii, as illustrated in figure 2. Disc position and radius are determined by maximising the difference between the integrated intensity on inner and outer rings. The third method is a cross-correlation with different masks. As it is nearly a factor of 100 faster than the edge detection algorithm it is capable for in-situ strain measurement during CBED pattern acquisition. The left part of figure 3 shows two different masks. Mask A assumes fully illuminated CBED-discs, whereas with mask B the inner structure of the diffraction discs has less influence on the result. The right part of figure 3 shows the change in the correlation function if the disc is shifted. The disc position can be determined from the shift between mask and experiment. We compare the precision of the three different algorithms among each other and with respect to former approaches [e.g. 2, 3]. Finally we show by application, that specimen cooling, zero-loss energy filtering and aberrations of the projection system only weakly affect the measured strain.

[1] K. Müller and A. Rosenauer et al., Microsc. Microanal. 18 (2012), p. 995.

[2] F. Uesugi et al., Ultramicroscopy 111 (2011), p.995.

[3] A. Béché et al., Appl. Phys. Lett. 95 (2009), p. 123114.

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) under contracts numer RO2057/8-1, SCHO1196/3, V0805/4 and V0805/5.

Fig. 1: Disc position and radius recognition with selective edge detection. (a) Process from experimental raw data to fitted disc position. (b) Edge point deselection: Beginning with the first circle-fit to all edge points, the point with the largest distance to the fitted circle is deleted until only edge points on the disc boundary remain.

Fig. 2: Disc position and radius recognition with Radial Gradient Maximisation. Two sets of rings are positioned inside and outside an initial radius estimation and the intensity is integrated. Radius and position are fitted via maximisation of the difference between both sums.

Fig. 3: Disc-position recognition via cross-correlation with masks. Left: Masks for correlation. Right: Changes in correlation-function (down) when the experimental disc shifts. Disc-position recognition from shift between experiment and mask.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-3080 Aberration-compensated large-angle rocking convergent-beam electron diffraction (LARCBED)

Koch C. T.1, Ozsoy Keskinbora C.2, Mu X.2, van Aken P. A.2, Ishizuka K.3
1Institute for Experimental Physics, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany, 2Stuttgart Center for Electron Microscopy, MPI for Intelligent Systems, Heisenbergstrasse 3, 70569 Stuttgart, Germany, 3HREM Research Inc., 14-48 Matsukazedai, 355-0055 Higashimatsuyama, Japan
christoph.koch@uni-ulm.de

Convergent beam electron diffraction (CBED) is a very efficient technique for acquiring two-dimensional rocking curve information in a single exposure. This is possible, because, for crystal structures having very small unit cells, the space between the Bragg spots is large enough to provide space for many non-overlapping diffraction patterns. If the sample is thick enough (typically > 100 nm), the dynamical diffraction conditions between these diffraction patterns differ enough to produce strong variations in the diffraction intensities. For materials with larger unit cells, such rocking curves must be acquired sequentially, because the distance between reflections is much smaller. Also thin crystals (e.g. nanocrystals) require a much larger tilt range than thick crystals, in order for intensity variations to be significant [1]. For thin crystals with small unit cells one may therefore acquire many CBED patterns, each with a different beam tilt, and combine them to large angle CBED (LACBED) patterns [2]. However, at large beam tilts, care has to be taken to compensate for movement of the probe on the sample due to aberrations of the objective pre-field lens. Also, imperfections in the separation between beam tilt and shift will become significant at large beam tilts. Aberrations of the imaging system add to the complexity of precisely localizing the probe on the specimen. However, this problem has been solved by the commercially available QED plug-in for DigitalMicrograph (Gatan Inc.) [1,3] which allows for automated calibration and compensation of all aberrations up to 7th order.

Here we present results of acquiring large-angle rocking-beam electron diffraction (LARBED) patterns using the QED plug-in with a convergent probe. Fig. 1 shows two example CBED patterns from the data stack (Fig. 1a and c), as well as the sum of all CBED patterns in the stack (Fig. 1b). Note that the beam tilt range (radius of ‘beam tilt disc’) was 60 mrad in each direction. At such large beam tilts the central spot of the pattern would be outside the detector area if no de-scan (compensation of beam tilt by diffraction shift) would be have been applied.

Fig. 2 shows bright-field and dark-field LACBED discs with a diameter of 120 mrad (6.9°) that have been extracted from the data stack shown in Fig. 1 by simply placing each CBED disc at the position of the pattern where it would have been recorded without applying any diffraction shift.

[1] C.T. Koch, Ultramicroscopy 111 (2011) 828 – 840

[2] R. Beanland, et al., Acta Cryst. A69 (2013) 427–434

[3] http://www.hremresearch.com

The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under grant agreement n°312483 (ESTEEM2) and the Carl Zeiss Foundation.

Fig. 1: CBED patterns of SrTiO3 acquired using QED. a) [-110] zone axis pattern, b) sum of all patterns acquired in this experiment (data stack), and c) one of the CBED patterns acquired at high beam tilt. Aberrations of the illumination system have been compensated up to 5th order.

Fig. 2: Bright-field (BF) and dark-field (DF) LACBED discs extracted from the data stack shown in Fig. 1. Similar data can be extracted from any of the more than 70 reflections shown in Fig. 1b.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-3362 EBSD and EDS Characterisation of Vanadium rich β Phase Lamella in Advanced Titanium Alloys

Stephens C. J.1
1Thermo Fisher Scientific
chris.stephens1@thermofisher.com

EBSD has been the focus of significant interest in recent years, driven by advances in detector technology, computational power and indexing routines. The high spatial resolution of EBSD enables structural characterization of materials on the nanoscale, giving instant quantitative information such as orientation, phase and texture. This work discusses developments in EBSD and EDS for the characterisation of submicron grains within advanced titanium alloys, which have important applications in the aerospace sector. High resolution EBSD patterns are used to characterise the propagation of BCC lamella through HCP subgrains, within a larger HCP matrix. Such a system becomes an ideal tool to characterise the structural properties of advanced materials, where the size and orientation of the lamella are related to the formation processes.

EBSD structural information is cross-correlated with chemical information obtained simultaneously through energy dispersive x-ray spectroscopy (EDS), tracking the migration of trace transition elements towards grain boundaries. The detection of these additives presents a challenge for EDS analysis, due to severe overlaps and low concentrations. These are overcome through intelligent peak deconvolution routines and image filtering, with proprietary principal component phase analysis algorithms used to determine chemically unique phases at overall concentrations below 1%.

Ti alloys can exist in three phases, α, α + β and β. At lower temperatures, pure Ti is stable as a close-packed hexagonal crystal structure and at high temperature it undergoes allotropic transformation to the BCC phase. Al and V are respective stabilisers of the α and β phases, therefore accurate quantification is essential. The alloy described here is stable in the α +β phase at room temperatures and pressures, whereby slow to intermediate cooling rates allow the formation of α colonies within the β phase.

Figure 1 (left) shows a high resolution forescatter image of the α phase alongside α + β phase, revealing the different topographical natures of these regions. Figure 1 (right) reveals the BCC region overlaid onto the image quality (IQ) map, showing a distinct lamella structure. EDS analysis of V segregation within the BCC phase is shown in figure 2 (left), with the V rich beta phase again highlighted. The quantification of V requires peak deconvolution due to the low weight percentage and the overlap with the Ti spectrum. The contoured quantitative weight percentage map of the same grain is shown in figure 2 (right), in 0.8% step sizes up to a maximum concentration of 8.07% showing a migration of V towards the phase boundary. This example demonstrates the benefits that intelligent EDS can bring to EBSD analysis of complex materials.

Fig. 1: left: Forescatter SEM image showing a high degree oftopography in the upper left side associated with the α +β phase and a smoother region in the lower rightside of the image assosciated with the a phase. RightBCC Euler map overlaid onto the pattern quality map highlighting theposition of the BCC lamella within the α + β phase

Fig. 2: Left: Quantitative elemental EDS map of vanadium, associated with the β-phase, overlaid onto the forescatter SEM image. Right: Quantitative EDS map in gradients of 0.8 %, revealing a higher concentration of V at the phase boundary

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-3490 Composite crystal structures of MxCuO2 cuprates; (Mx = Li2, Ca.83, Sr.73, Ba.67, [(Sr/Ca)2Cu2O3]1/√2, Na)

Milat O.1, Salamon K.1, Demoli N.1
1Institute of Physics, Bijenička 46, HR 1000 Zagreb, Croatia
milat@ifs.hr

The MxCuO2 cuprates belong to class of composite crystals consisting of two subsystems[1]: „CuO2-chains“ and „Mx-cations”. An electron microscopy and diffraction study of a number of rare earth cuprates, is presented (Fig.1.) in relation with the corresponding charge ordering that is induced by variable cation valency and nonstoichiometric composition. Level of cation defficiency and the accompanying self doping affects structure modulation, as well as the average Cu-valency; it can range from Cu+2.30 for Ca.83CuO2, to Cu+2.66 for Ba.67CuO2, and even up to Cu+3.0 for NaCuO2, or down to Cu+2.0 for Li2CuO2. In the case of Mx = Ca.83, Sr.73, Ba.67, [(Sr/Ca)2Cu2O3]1/√2, the two subsystems are mutually incommensurate and modulated along the “chain” direction, while for the end cases of: Mx = Na, Li2, the structural unit cells are commensurate (Fig. 2.)

The underlying lattices of these subsystems have common a and b parameters while the ratio cCh/cM of their c-parameters along the chain-direction varies with x. For a particular case of Mx=[(Ca/Sr)2Cu2O3]1/√2, the so-called “chain-ladder” (Sr/Ca)14Cu24O41 compound is well known for its optical and magnetic properties[2],[3]. In this case, the cation subsystem consists of an extended structure: (Sr/Ca)2Cu2O3 -“ladders”. The building unit of the ladders is a pair of cations plus a pair of zigzag edge-sharing CuO4-squares, Fig. 2, that are connected along “rungs”, so that the cLd period is defined by the CuO4-square diagonal. For the chains, the CuO4 building units share opposite edges and the cCh period is equal to the CuO4-square edge. In the case of “chain-ladder” composite structure, the cLd/cCh ratio is found to vary slightly with cation composition, but is always close to √2, so that the formula [(Ca/Sr)2Cu2O3]xCuO2 (x≈1/√2) correctly represents compound's composite structure.

With increasing Ca-substitution the cLd/cCh ratio varies from 1.44 for pure Sr14Cu24O41, to 1.416 for highly substituted Sr0.6Ca13.4Cu24O41. This is accompanied by charge (hole) redistribution between the CuO2-chains and the Cu2O3-ladders[3].

[1] Milat O., Van Tendeloo G., Amelinckx S., Mebhod M., Deltour R. (1992), Acta. Cryst., A48, 618-625.

[2] Vuletić T., Korin-Hamzić B., Ivek T., Tomić S., Gorshunov B., Dressel M., Akimitzu J. (2006), Phys. Rep. 428, 169-258.

[3] Ilakovac V., Gougoussis C., Calandra M., Brookes N. B., Bisogni V., Chiuzbaian S. G., Akimitsu J., Milat O., Tomic S., Hague C. F. (2012), Phy. Rev. B 85, 075108.

S. Tomić from Zagreb (Croatia), A. Migliori from Bologna (Italy), and G. Van Tendeloo from Antwerp (Belgium) are acknowledged for collacoration and support. Financial support has been received from Croatian Ministry of Sciences, Education and Sport.

Fig. 1: EDP of MxCuO2 composite crystals for Mx= Ca.83 (a), Sr.73 (b), [Sr/Ca2Cu2O3]1/√2 (c), along the [1000] zones perpendicular to the “CuO2-chains”. Indexing in 4-D crystallography notation; the third index is for the “chain-”, the fourth one for the “cation-sublattice”. Two subsystem unit cells are indicated; mismatch corresponds to: (1-x)c*Ch.

Fig. 2: Schematic representtion of the composite crystal structures of Ca.83CuO2, (Sr/Ca/La)14Cu24O41, (with incommensurate “CuO2-chain” modulation), and NaCuO2, Li2CuO2 (with commensurate superlattices); in top view (up), and front view (down).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-5870 Error Analysis of the Crystal Orientations and Misorientations obtained by the Classical Electron Backscatter Diffraction Method

Ram F.1, Zaefferer S.1
1Max-Planck-Institut für Eisenforschung GmbH, Düsseldorf, Germany
ram@mpie.de

The orientations obtained by the classical two-dimensional Hough transform-based EBSD method are accompanied by error and imprecision. These measures gain importance when the retrieved orientations and misorientations are used as an input for further analysis --- e.g., in grain boundary analysis, dislocation density analysis, and microstructure-based crystal plasticity modeling. In this contribution, the extent of the error and precision of the retrieved orientations and misorientations will be presented. They are examined using descriptive statistical analysis applied to patterns simulated based on the dynamical theory of electron diffraction. For a ~1 Mpixel pattern reduced to 240×204 pixels, subjected to the two-dimensional weighted Hough transform with 0.25° resolution, and convoluted by a butterfly mask of 13×13 pixels dimensionality, the error in the retrieved orientation is 1°, and the precision of the retrieved orientation is 0.7°. The error in the retrieved misorientation is 1.6° and its precision is 0.2°. The accuracies of the retrieved orientations and misorientations are obtained analytically through the model-based inferential statistics. The error in the detected lattice planes is assumed to have a Fisher-von Mises distribution. To estimate the accuracy, this error is propagated to the retrieved orientation and misorientation. The result is a confidence region for the true orientation or misorientation. The accuracies are defined based on their corresponding confidence regions. It is shown that the obtained accuracies are reliable upper bounds for the corresponding error. The maximum level of over/underestimation is 0.3° for orientation; it is 0.9° for misorientation.

1. Krieger Lassen, N. C., D. Juul Jensen, and K. Conradsen. 1994. “On the Statistical Analysis of Orientation Data.” Acta Crystallographica Section A Foundations of Crystallography 50 (6) (November 1): 741–748.


2. Krieger Lassen, N. C. 1996. “The Relative Precision of Crystal Orientations Measured from Electron Backscattering Patterns.” J. Microsc. (Oxford, U. K.) 181 (1): 72–81.

Fig. 1: Schematic drawing of the error, precision, confidence region, and accuracy of a quantity.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-5883 Indexing electron diffraction patterns from randomly-oriented crystals

Wang Y. C.1, Wan W.1, Zou X. D.1
1Stockholm University, Stockholm, Sweden
yunchen.wang@mmk.su.se

Electron diffraction (ED) can be used to study nanometre-sized crystals and provides information about the unit cell, space group and even intensities for a complete structure solution. With a known unit cell, ED patterns can be indexed. The reflection indices are found after indexing and the reflection conditions can be used to derive the space group. Indexing is usually done with in-zone ED patterns from aligned crystals. Alignment of the crystal can be time consuming and for many electron beam sensitive materials it is very difficult, even impossible. Indexing ED patterns from randomly orientated crystals is thus necessary and it gives valuable information about the phase of the material, the space group and possibly quantitative intensities of the reflections. Similar problem has been studied in femtosecond X-ray crystallography using X-ray free electron laser [1]. However the algorithms cannot be adopted for electron data due to the differences in wavelengths. Here we propose a fast and robust approach for indexing ED patterns from randomly orientated crystals, given the unit cell parameters. Rather than measuring the d* values and mutual angles of the reflection vectors and matching them to the calculated values, we use instead the “difference vectors”, which are obtained by subtracting the 2D coordinates of pairs of diffraction spots. Difference vectors typically have shorter d* values than the original reflection vectors and are thus easier to index (less ambiguity). Angles between difference vectors are also included in the indexing to identify a unique solution. Zone axes given by the difference vectors are close (usually within 1-2°) to the actual zone axes along which the ED patterns are taken. They can be used to quickly narrow down the solution for indexing the original reflection vectors, resulting in a quick and reliable solution.

[1] H. N. Chapman, et al., Nature 470, 73(2011)

This project is supported by the Knut & Alice Wallenberg Foundation through the project grant 3DEM-NATUR and a grant for purchasing the TEM.

Fig. 1: Illustration of the “difference vector” approach for indexing ED patterns from randomly orientated crystals. The ED pattern was taken from a silicalite-1 crystal in a random orientation. Three reflections, (-7,1,-3), (-5,0,-1) and (-3,2,-4), generate difference vectors (4,1,-1) and (2,-1,2) which are of lower resolution and easier to index
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-9-P-5973 In situ observation of reverse transformation in steels using EBSD measurement at elevated temperature

Masaaki Sugiyama and Akira Taniyama
Innovative Structural Materials Association (ISMA), Futtsu branch located in Nippon Steel & Sumitomo Metal Corporation
sugiyama.88p.masaaki@jp.nssmc.com

In the advanced high strength steels, the phase transformation from a high temperature phase of austenite with fcc structure to the low temperature one of ferrite with bcc structure, including of bainite or martensite phases must be controlled to get a suitable microstructure with good mechanical properties during the manufacturing process. Since there are several kinds of heating and cooling treatments in the steel product line, the nucleation sites and growth behavior in the forward and reverse phase transformation are important controlling factors, which are strongly affected by a local inhomogeneity such as precipitation, segregation, and crystal orientations.

Using a field emission typed SEM equipped with the Electron Backscatter Diffraction (EBSD) detector, the resolution for the local crystal orientation area is improved up to about 20nm at room temperature. As a next step, the development of the EBSD measurement at elevated temperature is expected in order to carry out the in situ observation for the phase transformation, recrystallization and grain growth [1]. Since the observation temperature at a high temperature is depended by the capacity of the heating stage, we have applied a new heating stage developed by TSL solutions specified for the EBSD measurement. Figure 1 shows photographs of a part of SEM (JSM-7800), in which the heating stage has been installed. The sample size is 5.0 x 7.0 x 0.6 mm in dimensions. The thermocouples are mounted on both of a sample surface and the sample holder. The heating stage can be operated at tilting angles up to 70°and a radiation protected cover is fixed.

The reverse phase transformation from the ferrite to austenite phase in the bainitic steel has been investigated to make clear the austenite grain orientation memory effect [2]. Since the memory effect shows strong heating rate dependence and the heating temperature range, it is considered that the growth rate of retained austenite between the lath boundaries compete with the decomposition of cementite in the microstructure of the bainite phase. Figure 2 is one of examples showing the orientation imaging maps during the reverse transformation measured up to 1100℃. The in situ observation technique developed at elevated temperature with a stable drift control, it becomes possible to discuss the nucleation sites of the austenite and its orientation characteristics. The artifact arising from the vacuum experiment in SEM must be also discussed.

References

[1] S.Wright and M.M.Nowell ; Electron Backscatter Diffraction in Materials Science, A.J.Schwartz et al. (eds) , (2009) 329.

[2] T.Hara, N.Maruyama, Y.Shinohara, H.Asahi, G.Shigesato,M.Sugiyama, T.Koseki, ISIJ Int., 49(2009) 1792.

This work was supported by the New Energy and Industrial Technology Development Organization (NEDO) under the " Innovative Structural Materials Project (Future Pioneering Projects)".

Fig. 1: Photographs showing a SEM column and the heating stage fixed inside the chamber.

Fig. 2: Orientation imaging maps measured at high temperature using the heating stage. (a) Ferrite(BCC) map at 1000C, (b) Austenite(FCC) map at 1000C, (c) Ferrite(BCC) map at 1100C, (d) Austenite(FCC) map at 1100C.
↑ Go to the Table of content ↑

IT-10. Electron tomography

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-10-IN-1695 Colouring atoms in 3 dimensions

Bals S.1, Goris B.1, Altantzis T.1, Van Tendeloo G.1
1EMAT, University of Antwerp, Belgium
sara.bals@ua.ac.be

New developments in the field of nanoscience drive the need for 3 dimensional (3D) quantitative characterization techniques yielding information down to the atomic scale. The 3D resolution of electron tomography was recently pushed to the atomic level [1-3]. One approach is based on compressive sensing (CS), a technique specialized in finding a solution with a sparse representation to a set of linear equations. At the atomic scale, the approach exploits the sparsity of the object since only a limited number of voxels is occupied by atoms. The CS technique was applied to the 3D reconstruction of Au nanorods based on only 4 HAADF-STEM images. The crystal lattice of the nanorods was reproduced without prior knowledge on the atomic structure [3]. As shown in Figure 1, also the 3D visualization of crystal defects at the atomic scale is currently possible using the same technique.

Going further than determining the 3D positions of atoms, a crucial aim is identifying the type of individual atoms in hetero-nanoparticles. We recently investigated core-shell Au@Ag nanorods using the CS methodology [4]. A detailed analysis of the position and the atom type was performed using orthogonal slices through the 3D reconstruction (Figure 2). Individual Ag and Au atoms can be distinguished, even at the metal-metal interface, by comparing their relative intensities. These results demonstrate the feasibility of chemically sensitive 3D reconstructions with a resolution at the atomic scale. However, such experiments are experimentally and computationally still far from straightforward and very time consuming.

An alternative approach to resolve the chemical composition of complex nanostructures in 3D is by using energy dispersive X-ray (EDX) mapping. This is a suitable technique for electron tomography since the number of generated X-rays increases with sample thickness. Early 3D EDX experiments were complicated by the specimen-detector geometry [5], but recent efforts enable 3D EDX in an optimized manner [6]. A 3D EDX reconstruction of a Au@Ag nanocube is presented in Figure 3 and clearly illustrates the potential of 3D EDX mapping, but further challenges include extracting quantitative information from such reconstructions.

[1] S. Van Aert, K. J. Batenburg, M. D. Rossell, R. Erni, G. Van Tendeloo, Nature 470 (2011) 374

[2] M.C. Scott et al., Nature 483 (2012) 444

[3] B. Goris, S. Bals, W. Van den Broek, E. Carbo-Argibay, S. Gomez-Grana, L. M. Liz-Marzan, G. Van Tendeloo, Nature Materials 11 (2012) 930

[4] B. Goris, A. De Backer, S. Van Aert, S. Gómez-Graña, L. M. Liz-Marzán, G. Van Tendeloo, S. Bals, Nano Lett. 13 (2013) 4236

[5] G Möbus, RC Doole and BJ Inkson, Ultramicroscopy 96 (2003) 433

[6] P Schlossmacher et al, Microscopy Today 18 (2010) 14

We acknowledge support from the ERC ( “Countatoms-#24691”, and “Colouratoms-#335078”) and the FWO. We thank Prof. Liz-Marzán for providing the samples and useful discussions.

Fig. 1: Fig. 1 (a) 3D reconstruction of a Au nanodumbbell. At the tip, twin boundaries are present. (b) Orthogonal slices through the reconstruction of the tip of the nanodumbbell, presented in more detail in (c) enable one to determine the stacking across the twin boundary.

Fig. 2: Fig. 2 (a) Orthogonal slices through the reconstruction show the core-shell structure of the nanorod (b) Detailed view of a slice through the reconstruction perpendicular to the major axis of the nanorod (c) Intensity profile acquired along the direction indicated in (b), showing the capability to assign each atom to be either Ag or Au.

Fig. 3: Fig. 3 (a) 2D EDX map of a Au@Ag nanocube. A tilt series of these 2D EDX maps was acquired and a 3D reconstruction shown in (b) could be obtained.

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-10-IN-3013 Functional soft matter

Friedrich H.1
1Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Den Dolech 2, 5612AZ Eindhoven, The Netherlands
h.friedrich@tue.nl

The design and synthesis of materials with novel functional properties is a major focus of chemical research. This includes soft matter which are formed by the interactions that arise from (self)-organizing molecules, polymers, and clusters over length scales beyond typical small molecule dimensions. To understand and apply the processes that underlie the formation of nanoparticles and their self-organization into larger functional structures requires 3D nanoscale imaging [1,2]. We focus on the (liquid phase) (self)-organization of soft (in)organic materials and composites thereof using cryogenic (scanning) TEM electron tomography (ET). In this presentation I will take you through the complex and beautiful 3D nano and meso landscape of functional soft matter. Examples will include quantitative ET of a Ruthenium loaded carbon nanotube based heterogeneous catalyst (Figure 1a) [3], quantitative ET of the assembly process of organic solar cell bulk heterojunctions composed of P3HT and PCBM polymers (Figure 1b) [4], and cryogenic ET of liquid infiltration and drying processes in ordered mesoprorous silica (SBA-15) crystallites [5]. Since to-date, more frequently a detailed quantification understanding of particle sizes, size distributions, or particle location and distances is required, I will focus on this information contributes to determine the self-organization pathways [6,7]. Furthermore, I will discuss the effects of limited electron dose, applied angular sampling scheme, and reconstruction algorithm on the achievable 3D resolution (Figure 1d-h) [8,9]. Our findings suggest that for cryo conditions fewer images in the tilt-series are advantageous, contradictory to Crowther’s sampling-based resolution estimate [8,9]. Finally, I will conclude with an outlook on trends for 3D imaging.

[1] H. Friedrich et al, Angewandte Chemie International Edition 49 (2010) 7850.

[2] H. Friedrich et al, Chemical Reviews 109 (2009) 1613.

[3] H. Friedrich et al, ChemSusChem 4 (2011) 957.

[4] M. Wirix et al. Nanoletters (2014) accepted.

[5] T. M. Eggenhuisen et al, Chemistry of Materials 25 (2013) 890.

[6] G. Prieto et al, Nature Materials 12 (2013) 34.

[7] J. Zečević et al. ACS Nano 7 (2013) 3698.

[8] D. Chen et al. Journal of Physical Chemistry C 118 (2014) 1248.

[9] D. Chen et al. manuscript in preparation.

The author gratefully acknowledge the contributions of all (co)authors of the referenced manuscripts and especially, J. Zečević, D. Chen, M. Wirix, G. Prieto, T. M. Eggenhuisen, and funding from the NRSCC, NWO and the European Union.

Fig. 1: Quantitative 3D imaging examples: (a) Ru/CNT catalyst; (b) P3HT/PCBM bulk heterojunction; (c) infiltrated and dried SBA-15 crystallite; (d) simulation model to determine ET resolution and reconstructions using (e) SIRT; (f) TVM; , DART (d), and WBP (e) at a total electron does of 104 e/Å2 and tilt increments of 1°.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-1637 Combined tilt- and focal series scanning transmission electron microscopy: TFS 3D STEM

Dahmen T.1, Baudoin J. P.2, Lupini A. R.3, Kuebel C.4, Slusallek P.1, de Jonge N.5
1German Research Center for Artificial Intelligence GmbH, Saarbrücken, Germany, 2Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA, 3Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 4KIT –Karlsruhe Institute for Technology, Eggenstein-Leopoldshafen, Germany, 5INM – Leibniz Institute for New Materials, Saarbrücken, Germany
niels.dejonge@inm-gmbh.de

Tilt-series transmission electron microscopy (TEM) tomography is the method of choice to obtain nanoscale three-dimensional (3D) information from samples in biology and materials science. 3D data is acquired by mechanically tilting the sample stage and recording images at each tilt angle. However, the tilt range is limited to ±70° for most samples, and the tomographic reconstruction suffers from missing information and a limited resolution on account of the so-called “missing wedge”. Furthermore, the quality of the reconstruction critically depends on the precision of the alignment of the individual images. Alternatively, scanning TEM (STEM) focal series can be recorded avoiding tilting altogether but this method lacks axial resolution [1]. A new recording scheme to obtain 3D data is presented by combined tilt- and focal series (TFS) STEM. This method significantly reduces the two aforementioned limitations of tilt series tomography. The specimen is rotated in relatively large tilt increments, and for every tilt direction, a through-focal series is recorded (Fig. 1). Both the tilt-series and focal-series data are reconstructed into a 3D tomogram in the same software algorithm. The conical shape of the STEM probe is taken into account via forward- and backward projection operators. The TFS method exhibits reduced “missing wedge” artifacts and a higher axial resolution than obtainable using STEM tilt series [2]. Fig. 2 shows that the missing wedge is still present in the TFS but low spatial frequency signal components are now present (arrow) in the central vertical region. Streaks corresponding to the tilt directions are also less pronounced. The TFS reconstruction results in a superior shape representation and tolerates a much smaller number of tilt angles than tomography, which is beneficial for image stack alignment. A further advantageous application of TFS STEM is the imaging of micrometers-thick samples. With TFS it is possible to limit the overall tilt range while obtaining a higher axial resolution than for a tilt series alone.

We thank L. Marsallek and S.J. Pennycook for discussions, and E. Arzt for support through the INM. Electron microscopy performed at the SHaRE user facility at Oak Ridge National Laboratory, and at the Karlsruhe Nano Micro Facility, Helmholtz infrastructure, Karlsruhe Institute of Technology. Research supported by the U.S. Department of Energy, Basic Energy Sciences, and by NIH grant R01-GM081801.

Fig. 1: Schematic views of the TFS recording scheme with STEM. (left) A thin specimen is imaged pixel-by-pixel in dark field mode STEM using the annular dark field detector (ADF). (right) In a combined tilt- and focal series, images are acquired in a through-focal series at each tilt angle. The specimen stage is titled after each focus series.

Fig. 2: Comparison of tilt series STEM tomography with TFS STEM. (left) Spatial frequency spectrum (Fourier Transform) of a vertical (xz) slice of the conventional tilt series STEM tomography data. (right) Frequency spectrum of an xz slice of the TFS STEM dataset. The red lines mark the border of the missing wedge. With permission from [2].
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-1863 Towards mass contrast in 3D atomic resolution tomographic reconstructions from HRTEM images with the inverse dynamical electron scattering method

Van den Broek W.1, Koch C. T.1
1Institute for Experimental Physics, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
wouter.vandenbroek@uni-ulm.de

We have recently reported a novel reconstruction algorithm, inverse dynamical electron scattering (IDES), that retrieves the object’s electrostatic potential from a high resolution transmission electron microscopy (HRTEM) tilt series [1]. By reformulating the multislice algorithm as an artificial neural network IDES is able to very efficiently recover the scattering object using a gradient based optimization. Prior knowledge about the atomic potential shape and the sparseness of the object is accounted for naturally. In [2] we demonstrated the generality of this method with applications to simulated ptychography and scanning confocal electron microscopy data sets and the optimization of the images’ defocus values along with the object.

Reconstructions so far had been performed on simulations of a small Au nanoparticle that was 1.6nm wide. In this work a larger and more complicated system is treated: A PbSe-CdSe core-shell particle with 1963 atoms. The particle is derived from a CdSe rock-salt structure and has a cubic shape with sides of 3.7nm. The unit-cell parameter a equals 0.61nm. The Cd atoms in the cube’s interior octahedron are replaced by Pb-atoms and shifted by a vector [−a/4, a/4, a/4]; see [3].

The potential is reconstructed from a simulated HRTEM tilt series with the α-tilt varying from -10° to +10° in 2°-steps with zero β-tilt, and the β-tilt varying from -10° to +10° in 2°-steps with zero α-tilt. The images were given a signal-to-noise ratio of 10. See Fig. 1 for 6 typical simulations out of the total of 21.

The forward simulations have been carried out with the new FDES (forward dynamical electron diffraction) software that runs on the graphics processing unit. A solution was achieved with conjugate gradients optimization. The solution’s sparseness was increased through L1-norm regularization and through the use of a generalized potential, taken as the weighted average of the potentials of Se, Cd and Pb.

Although FDES was set to use more accurate approximations than IDES—specimen rotation is treated exactly in FDES, but is approximated by a shifted Fresnel propagator in IDES and FDES used a multislice slice thickness 5 times smaller than IDES—an atomic resolution reconstruction was still possible. The Pb-atoms can be isolated by thresholding the reconstructed gray values, thus demonstrating mass contrast. Fig. 2 shows a side-by-side comparison of the original and the reconstructed particle potential, visualized with IMOD [4].

[1] W. Van den Broek, C.T. Koch. Phys. Rev. Lett. 109 (2012) 245502.
[2] W. Van den Broek, C.T. Koch. Phys. Rev. B 87 (2013) 184108.
[3] S. Bals et al. Nano Lett. 11 (2011) 3420–3424.
[4] http://bio3d.colorado.edu/imod/index.html

The authors acknowledge the Carl Zeiss Foundation as well as the German Research Foundation (DFG, Grant No. KO 2911/7-1).

Fig. 1: Six typical simulations. The acceleration voltage is 80kV, the defocus is -20nm, the spherical aberration is 64μm, the pixel size is 0.025nm and the multislice slice thickness is 0.02nm. The point resolution is 0.17nm. The images are degraded by partial temporal and spatial coherence and the CCD’s modulation transfer function.

Fig. 2: Left: Pb-atoms in the original particle’s core. Right: The Pb-atoms in the reconstructed potential, identified by simple thresholding, thus demonstrating mass contrast. Due to the high level of noise in the measurements there are some false positives and false negatives. Lowering the threshold would also reveal the Cd and Se atoms.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-2126 Four-dimensional simultaneous EELS & EDX tomography of an Al-Si based alloy

Haberfehlner G.1, Albu M.1, Orthacker A.1, Kothleitner G.1
1Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology & Centre for Electron Microscopy and Nanoanalysis, Graz, Austria
georg.haberfehlner@felmi-zfe.at

Electron tomography is a powerful technique for 3D characterization at the nanoscale. Recent developments in electron tomography include the use of TEM imaging techniques based on inelastic scattering, such as EFTEM, EELS and EDX [1,2] as well as reconstruction algorithms, including prior information in the reconstruction process, such as total-variation (TV) minimization [3].

Modification of eutectic Si in Al-Si alloys by modifying elements such as Sr, Na or Yb is a frequently used method to improve mechanical properties of alloys [4]. Nevertheless the modification mechanisms are still a matter of debate and require understanding the elemental distribution down to the atomic level.

In this work we investigate an Al-5 wt.% Si alloy with 15 ppm Na and 6500 ppm Yb. On this alloy we demonstrate simultaneous EELS and EDX tomography. We apply a TV minimization reconstruction algorithm to elemental maps extracted from analytical TEM data and we reconstruct spectral EELS and EDX data, to get local spectra in three dimensions.

Tomography experiments were performed on a probe-corrected FEI Titan3 microscope operated at 300 kV, equipped with a Gatan GIF Quantum energy filter and a Bruker Super-X detector. Low-loss (-80 to 920 eV), core-loss EELS (1120 to 2120 eV) and EDX spectrum images of a FIB-prepared needle-shaped sample were acquired every 5° over a range of +/-75°. All analytical tomography data was aligned based on HAADF STEM images, acquired at the same time as the spectrum images. 2D elemental maps were extracted for each tilt angle from both EDX and EELS spectrum image data sets.

Reconstruction of the elemental maps was done with the simultaneous iterative reconstruction technique (SIRT) as well as using TV minimization. TV minimization is an efficient method for reconstruction from few and noisy projections, as is the case for elemental maps. It assumes locally constant regions in the reconstruction, a valid assumption as we expect sharp interfaces between Yb-rich precipitates, Si-particles and the Al-rich matrix. Fig. 1 shows reconstructions of elemental maps extracted from EDX data and Fig. 2 shows the same reconstructions of elemental maps for EELS core-loss data.

Additionally we reconstructed spectral EDX and EELS data. Using SIRT each spectral channel is reconstructed, which provides four-dimensional datasets containing EELS and EDX spectra for each voxel (see Fig. 3).

This work founds a basis for quantitative elemental mapping in three dimensions and can be extended towards 3D chemical fingerprinting or extraction of local electrical and optical properties.

[1] Jarausch et al, Ultramicroscopy 109:326, 2009

[2] Lepinay et al, Micron 47:43, 2013

[3] Leary et al, Ultramicroscopy 131:70, 2013

[4] Li et al, Philos. Mag. 92:3789, 2012

The authors would like to thank Jiehua Li and Peter Schumacher from Chair of Casting Research, University of Leoben for providing the samples, This work has been supported by the FFG OPTIMATSTRUCT project and within the European Union's 7th Framework Programme in the project ESTEEM2.

Fig. 1: Reconstructions of elemental maps extracted from EDX data. (a) Orthogonal slices through reconstructions of the Yb L-line, Si K-line and Al K-line using SIRT and TV minimization. (b) 3D surfaces extracted from the TV minimization reconstructions.

Fig. 2: Reconstructions of elemental maps extracted from EELS core-loss data. (a) Orthogonal slices through reconstructions of the Yb M45-edge, Si K-edge and Al K-edge using SIRT and TV minimization. (b) 3D surfaces extracted from the TV minimization reconstructions.

Fig. 3: Local reconstructed spectra from (a) Yb-rich precipitates, (b) Si-rich region, (c) Al-rich matrix. (1) Masks of the investigated regions, (2) core-loss EELS spectra, (3) EDX spectra. (2)-(3) show single voxel spectra (top) and spectra summed over all voxels of the masks shown in (1) (bottom).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-2253 Possibilities and limitations for atom counting using quantitative ADF STEM

De Backer A.1, Martinez G. T.1, MacArthur K. E.2, Jones L.2, Béché A.1, Nellist P. D.2, Van Aert S.1
1Electron Microscopy for Materials Science (EMAT), University of Antwerp, Antwerp, Belgium, 2Department of Materials, University of Oxford, Oxford, United Kingdom
Annick.DeBacker@uantwerpen.be

Advanced statistical methods can be used to count the number of atoms in each atom column of high-resolution ADF STEM images [1-3]. Here we discuss the possibilities and limitations of achieving single atom sensitivity.
Four images of the same Ir/Pt nanoparticle were recorded at different magnifications and electron doses. In order to allow comparison with simulations, the images were normalised with respect to the incoming electron beam intensity [4]. Next, using statistical parameter estimation theory, the total scattered intensities are quantified atom column–by–atom column. An example analysis for the image recorded at the highest magnification and electron dose is illustrated in Fig. 1; the total scattered intensities are visualised in the histogram. The number of significant components and their intensities were retrieved by evaluating the so-called integrated classification likelihood (ICL) criterion in combination with Gaussian mixture model estimation. These results allow us to quantify the number of atoms in each atom column. As shown in [3], the reliability of atom counts depends on the number of atom columns present in an image, the width of the components, and the performance of the ICL criterion. These parameters can be linked with the quality of the recorded images.
In Fig. 2, the intensities of the components resulting from the counting analyses are compared with the total scattered intensities resulting from simulated images using STEMsim. For image 3 an excellent match was found. However, analysing images of lower magnification and/or electron dose worsens the match with simulation. The same effect is observed when analysing an image composed of every second pixel of image 3. In this way, the lower magnification of images 1 and 2 is mimicked. This leads to less precise measurements of the total scattered intensities resulting in insufficient statistics for the determination of the number of components. However, when enhancing the statistics by combining the values of the scattered intensities of the four images collectively, the experimental intensities again match with simulated values. In addition, the statistical approach for atom counting provides us high precision leading to near single atom sensitivity for this combined set of images.
In conclusion, an advanced quantitative method to count the number of atoms is presented together with its possibilities and limitations. Single atom sensitivity may be achieved when the experimental images are of sufficient quality to yield sufficient statistics.

References

[1] S Van Aert et al., Nature 470, p 374 (2011)
[2] S Van Aert et al., PRB 87, 064107 (2013)
[3] A De Backer et al., Ultramicroscopy 134, p 23 (2013)
[4] A Rosenauer et al., Ultramicroscopy 109, p 1171 (2009)

Funding from the FWO Flanders, the EU FP7 (312483 - ESTEEM2), and the UK Engineering and Physical Sciences Research Council (EP/K032518/1) is acknowledged.

Fig. 1: Illustration of the atom counting procedure; a) experimental ADF STEM image, b) refined parameterised imaging model, c) evaluation of ICL as a function of the number of components, d) histogram of estimated scattered intensities together with the estimated Gaussian mixture model, e) quantification of the number of atoms in each atom column.

Fig. 2: Comparison of experimental and simulated total scattered intensities. The inset shows the specific pixel size and dwell time for the individual images of the Ir/Pt particle.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-2435 Towards 4-D EEL spectroscopic scanning confocal electron microscopy (SCEM-EELS) optical sectioning on a Cc and Cs double-corrected transmission electron microscope

Wang P.1, Boothroyd C. B.2, Dunin-Borkowski R. E.2, Kirkland A. I.3, Nellist P. D.3
1National Laboratory of Solid State Microstructures and College of Engineering and Applied Sciences, Nanjing University, Nanjing 210093, People’s Republic of China, 2ER-C and PGI5, Forschungszentrum Jülich, D-52425 Jülich, Germany, 3Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH, UK
wangpeng@nju.edu.cn

The spectrum imaging method of combining scanning transmission electron microscopy with electron energy-loss spectroscopy (STEM-EELS) has been widely used for materials characterization at the atomic-scale. Three dimensional (3-D) optical sectioning using scanning confocal electron microscopy (SCEM) [1], as shown in Fig. 1 a), has been developed as an alternative to tilt-series electron tomography [2]. The confocal imaging mode in STEM has been implemented with spherical aberration (Cs) correctors, which allow the use of substantially increased objective apertures and hence provide a considerably decreased depth of focus, typically below 10 nm. Both the theoretical basis of the image contrast [3] and the experimental implementation of the technique [4-6] have been studied. However, due to Cc-aberrations in the post-specimen optics, inelastically scattered electrons with different energy losses △E are focused at different focal length (Fig. 1a)), which causes an EEL spectrum to be out-of-focus away from the confocal energy loss [5], as shown in Fig. 2a). In order to avoid this problem, a Cc-corrector is need in addition to a double-aberration corrected TEM, as shown in Fig. 1b).
In this work, we propose a novel spectrum imaging mode by combining the SCEM and EELS techniques, which can potentially let one perform 4D EEL spectroscopic SCEM (or so called SCEM-EELS for short) optical sectioning, allowing quantitative chemical characterization over a full 3D specimen volume. Preliminary experiments have been carried out both on a non Cc-corrected Oxford-JEOL 2200MCO instrument with 3rd order double Cs correctors and on a Cc-corrected FEI Titan 60-300 PICO, which has an illumination-side Cs corrector, a Cs-Cc achro-aplanat image corrector and a post-specimen EEL spectrometer. Fig. 2b) shows 2D spectrum images recorded from an amorphous carbon film on the EELS CCD camera in a confocal configuration aligned for energy losses of 0 eV on a Cc-corrected TEM. It demonstrates that the inelastically scattered electrons are simultaneously in-focus on the EELS CCD camera over the entire energy loss range.

References:
[1] P.D. Nellist, P. Wang, Annual Review of Materials Research, 42, (2012), 125-143.
[2] P.A. Midgley, M. Weyland, Ultramicroscopy, 96 (2003) 413-431.
[3] A.J. D'Alfonso et al, Ultramicroscopy, 108 (2008) 1567-1578.
[4] P. Wang et al, Ultramicroscopy, 111 (2011) 877-886.
[5] P. Wang et al, Physical Review Letters, 104 (2010) 200801.
[6] P. Wang et al , Applied Physics Letters, 100 (2012) 213117.

P.W., A.I.K. and P.D.N. acknowledge financial support from the Leverhulme Trust (F/08 749/B), the EPSRC (EP/F048009/1); P.W. acknowledges financial support from the Thousand Talents Program.

Fig. 1: Schematic diagrams of confocal trajectories for SCEM with an EEL spectrometer behind a circular pinhole on a non Cc-corrected TEM (a) and a Cc-corrected TEM (b), respectively. Due to Cc aberration correction in the post-specimen lenses in (b), electron rays with an energy loss difference of △E can still be focused on the pinhole plane.

Fig. 2: 2D spectrum images recorded on the EELS CCD camera in a confocal configuration established for energy losses of 0 eV on a non Cc-corrected TEM from a Si slab (a) and on a Cc-corrected TEM from an amorphous carbon film (b), respectively.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-2543 On-axis electron tomography of needle-shaped biological samples

Saghi Z.1, Divitini G.1, Winter B.2, Spiecker E.2, Ducati C.1, Midgley P. A.1
1(1) Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge, CB3 0FS, UK, 2(2) Center for Nanoanalysis and Electron Microscopy (CENEM), Department Werkstoffwissenschaften / VII, Universität Erlangen-Nürnberg, Cauerstraße 6, 91058 Erlangen, Germany
saghizineb@gmail.com

A key challenge in structural biology is to image large volumes while maintaining sufficient resolution to identify small features in their original cellular context [1]. Electron tomography (ET) has contributed greatly to this field, but imaging sections thicker than a few hundred nanometers is difficult because of the sample geometry and microscope configuration: as the specimen is tilted to high angles, the thickness increases and the quality of the images deteriorates. Moreover, for geometric constraints, the tilt range rarely exceeds ±70°, leading to elongation and blurring of features, and an overall challenging volume to segment. Here, we show that preparing a needle-shaped sample rather than a flat section can alleviate many of the limitations encountered in biological ET. The technique is illustrated on a 500nm diameter needle extracted from an epoxy-embedded portion of the nucleus accumbens shell from a rat brain. The sample was prepared in a Helios NanoLab focused ion beam (FIB) machine and transferred to an on-axis tomography holder. An HAADF-STEM tilt series was then acquired from -90° to +90° with 1° increment, using an FEI TITAN microscope operating at 300kV, and Inspect3D was used for the alignment and reconstruction by weighted backprojection. Figure 1(a) shows a low magnification view of the needle and the region selected for tomography. A voxel projection and slice through the reconstructed needle (b,c) provide highly detailed structural information. In Figure 2, we compare the quality of ET results from -90°:2°:+90° and -60°:2°:60° acquisitions. Cross-sections through a portion of exitatory synapse and mitochondrion (positions 1 and 2 in Figure 1(c)) illustrate the advantages of a full tilt range on-axis ET experiment with enhanced signal-to-background ratio and isotropic sharpness of features. Note that the ±60° cylindrical volume shown here is still of better quality than a reconstructed section from similar tilt range, since the thickness remains constant throughout the tilt series.
Combining this novel sample preparation technique with advanced imaging modes (BF-STEM for example [2]) and sophisticated reconstruction algorithms such as compressed sensing [3], we anticipate that ET will provide a complementary method to serial sectioning and FIB-SEM slice-and-view techniques [4].

[1] W. Baumeister, Current Opinion in Structural Biology 2002, 12(5):679.
[2] A.A. Sousa et al., Journal of Structural Biology 2011, 174(1): 107.
[3] Z. Saghi et al., Nano Letters 2011, 11(11):4666.
[4] Samples provided by Andrea Falqui and Roberto Marotta, IIT, Genova, Italy.

The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483-ESTEEM2 (Integrated Infrastructure Initiative–I3), as well as from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC grant agreement 291522-3DIMAGE.

Fig. 1: (a) Needle-shaped biological sample, prepared by FIB. The rectangle indicates the area selected for electron tomography. (b) Voxel projection of the reconstruction from a full range acquistion. (c) A slice through the volume showing a detailed view of an exitatory synapse (1) and a mitochondrion (2).

Fig. 2: Cross-sectional slices through the synapse (a,b) and mitochondrion (c,d) with different tilt ranges. Isotropic resolution is observed for full tilt range on-axis tomography (left), as illustrated in the insets.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-2765 A method for quantitative analysis and improvement of 3D electrostatic nanopotentials reconstructed by electron holographic tomography

Wolf D.1, Lubk A.1, Lichte H.1
1Triebenberg Laboratory, Institute of Structure Physics, Technische Universität Dresden, Dresden, Germany
daniel.wolf@triebenberg.de

Electron holographic tomography (EHT), i.e. using off-axis electron holography (EH) as imaging mode for electron tomography (ET) in the transmission electron microscope (TEM), facilitates the 3D mapping of materials on the nanometer scale [1,2]. The phase shift of the electron wave that can be reconstructed by EH contains the projected electrostatic scalar potential and, for magnetic samples, the projected magnetic vector potential of the specimen [3]. Therefore, tomographic reconstruction of phase tilt series results in 3D maps of the electric potential (magnetic case is not considered here).

At nanometer resolution (1-10nm), the major contribution to tomograms reconstructed by EHT is the mean inner potential (MIP). Its value depends on the atomic species, the atomic packing in the unit cell, but also on the distribution of the valence electrons. Thus, the MIP represents a finger print of chemical composition and can be used to detect for example core-shell structures (e.g. AlGaAs-GaAs [2]) or gradients of composition in nanowires (NWs). Recently, the three-dimensional nanosponge structure of Si embedded in SiO2 has been revealed with EHT [4]. Furthermore, functional potentials, such as the built-in potentials across p-n junctions in semiconductors can be measured [1,2]. In this context, also surface and sub-surface effects, e.g. Fermi-level pinning [5], have been studied, quantitatively.

In order to extract quantitative information from the 3D reconstructions, it is indispensable to know their fidelity. Here, we show a procedure to proof the reliability of the tomograms by comparing their re-projections with the original ones (Fig. 1a)). By applying this procedure on an Ag, ZnO and Si NW and evaluating the potential averaged over the entire specimen, we determine the MIP values from the projection data (Fig. 1b)).

Moreover, the 3D reconstruction can be remarkably improved by normalizing it with the tomogram reconstructed from the projected thickness. The latter is obtained after step 3 in the procedure shown in Fig. 1a). Because its reconstruction is done from the same tilt range, the resulting tomogram contains very similar missing wedge artifacts as the original one. Therefore, such artifacts can be corrected to a great amount using this approach (compare in Fig. 2: a,b with c,d).

[1] P.A. Midgley and R.E. Dunin-Borkowski, Nature Materials 8, (2009), p. 271.
[2] D. Wolf, A. Lubk, F. Röder, and H. Lichte, Current Opinion in Solid State and Materials Science 17, (2013), p. 126.
[3] H. Lichte and M. Lehmann, Reports on Progress in Physics 71 (2008), p. 016102.
[4] R. Hübner, D. Wolf, D. Friedrich, B. Liedke, B. Schmidt, K.H. Heinig, at this conference.
[5] D. Wolf, A. Lubk, A. Lenk, S. Sturm, and H. Lichte, Appl. Phys. Lett. 103 (2013), p. 264104.

We thank M. Graf, TU Dresden for providing the Ag nanowire, and Z. L. Wang, Georgia Institute, Atlanta for providing the ZnO nanowire. The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative - I3).

Fig. 1: a) Procedure to determine from the projected potential tilt series the averaged potential tilt series on the example of an Ag nanowire. b) Potential averaged over the entire specimen vs. projection angle a. The mean of these values corresponds to the mean inner potential V0.

Fig. 2: 3D reconstruction with reduced missing wedge artifacts on the example of an Ag NW and a ZnO NW. a,b) Slice through 3D potential. c,d) Same slice as in (a,b) but normalized with the reconstruction of the projected thickness. e,f) Line profiles corresponding to the gray arrows in a-d).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-2787 EELS and tomography: from EELS Spectrum Images to Spectrum Volumes.

Yedra L.1,2, Eljarrat A.1, Rebled J. M.1,3, López-Conesa L.1, Dix N.3, Sanchez F.3, Estradé S.1,2, Peiró F.1
1Laboratory of Electron Nanoscopies (LENS)- MIND/IN2UB, Dept. d'Electrònica, Universitat de Barcelona, Barcelona, Spain, 2CCiT, Scientific and Technical Centers, Universitat de Barcelona, Barcelona, Spain, 3Institut de ciencia dels materials de Barcelona (ICMAB), Bellaterra, Spain
llyedra@el.ub.edu

In transmission electron microscopy (TEM), 3D tomographic reconstruction can be achieved by acquiring a series of images at different tilt angles. A different approach is obtaining 3D chemical reconstructions from energy filtered images in the TEM (EFTEM)[1-3], and more recently, by acquiring EELS spectrum images (EELS-SI), each pixel containing a complete EELS spectrum [4,5]. However, in both techniques only a limited amount of information is effectively reconstructed. In this paper we aim to derive a full EELS dataset in 4D, where every voxel of a whole volume contains a complete spectrum of energy losses, as schematized in Fig. 1. By analogy to the spectrum image notation, we will name this 4D dataset as EELS spectrum volume (EELS-SV).

Our approach to EELS-SV reconstruction is based upon SI, thus taking a single SI for every tilt angle. It takes advantage of Multivariate Analysis (MVA), and more precisely of blind source separation (BSS)[6], to find a new spectral basis (Fig. 2a) which can describe all the spectra in the dataset as a weighted sum of its components. Therefore only the 3D reconstructions of the weighting components (Fig. 2b) will be necessary to recover the spectra in each voxel (Fig. 2c-e). We will apply this approach to analyze a BFO/CFO nanocomposites, enabling the characterization of a CFO nanocolumn embedded in BFO matrix.

References

[1] G. Möbus, et al, Ultramic, 96 (2003) 433.

[2] M. Weyland, P.A. Midgley, Microscopy and Microanalysis, 9 (2003) 542.

[3] R.D. Leapman, et al, Ultramic, 100 (2004) 115.

[4] K. Jarausch, et al, Ultramic,. 109 (2009) 326.

[5] L. Yedra et al., Ultramic., 122 (2012) 12

[6] N. Dobigeon et al., Ieee Transactions on Signal Processing, 57 (2009), 4355

Fig. 1: Schematic of the 4D dataset, the EELS spectrum volume, consisting of 3 spatial dimensions plus an additional energy loss dimension. Here it is presented along with an extracted xy spectrum image, a spectrum line along z direction and a single spectrum from an inner voxel.

Fig. 2: EELS-SV reconstruction procedure. a) Components of the spectrum. b) 3D reconstructions, c) Schematic representation of two orthoslices and reconstructed SI for transversal and longitudinal orthoslices. d) Single spectrum and e) spectrum line extracted from the slices.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-2806 Electron cryo-tomography with a new type of phase plate

Danev R.1, Buijsse B.2, Fukuda Y.1, Khoshouei M.1, Plitzko J.1, Baumeister W.1
1Max Planck Institute of Biochemistry, Martinsried, Germany, 2FEI, Eindhoven, The Netherlands
danev@biochem.mpg.de

Recent years have shown an increased interest in the development and use of phase plates in cryo-EM. The oldest and the most productive type of phase plate is the carbon film Zernike phase plate. Despite its good performance the Zernike phase plate has a few pitfalls. One major practical hindrance is its short lifetime, typically about 10 days. Another disadvantage is the generation of fringes around high-contrast features in the image. Despite its shortcomings the Zernike phase plate has been the main motivation and experience generator in the last years.

We are currently working in collaboration with FEI on the development and testing of a new type of phase plate. It addresses both of the above mentioned shortcomings of the Zernike phase plate. Our tests indicate that the new phase plate lasts for more than six months inside the microscope. This is a big advantage in terms of servicing and up time of the microscope. Another big advantage of the new phase plate is that it produces fringe-free images which resemble in appearance light microscopy phase contrast images. The new phase plate is being developed as a part of a product package which will include new hardware – phase plate holder & phase plate, and new software for alignment, calibration and ease of use.

We tested the new phase plate with two automated acquisition software packages – FEI Tomography and SerialEM. Both packages work well and are able to automatically acquire tilt series with the phase plate. There are only a couple of additional steps that are required for setting up the phase plate before starting the tilt series acquisition. The reconstruction process for the phase plate tomograms is almost identical to that for conventional defocus phase contrast tomograms and can be performed with any existing reconstruction software package, such as IMOD. Because of the strong low frequency components in the phase plate images a simple weighted back-projection reconstruction was in most cases sufficient to produce sharp, high-contrast tomograms. No further processing, such as de-noising, was necessary. In a few thick specimen cases a SIRT reconstruction produced better looking tomograms and again no de-noising or other post-processing was necessary. Overall the new phase plate works very well for cryo-tomography and users with cryo-tomography experience require only a short training on how to use it.

The new phase plate was tested in two microscope models – a 200 kV FEI Tecnai F20 and a 300 kV FEI Titan Krios. The lifetime and image quality performance was equally good with both microscopes.

We thank Matthijn Vos from FEI for providing test samples.

Fig. 1: A slice through a phase plate cryo-tomogram of a vitrified primary culture neuron cell.
Fig. 2:
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-2812 Fast tomography acquisition for in situ 3D analysis of nanomaterials under variable gas and temperature conditions in Environmental-TEM

EPICIER T.1, 2, ROIBAN L.1, LI S.2, AOUINE M.2, SANTOS AIRES F. C.2, TUEL A.2, FARRUSSENG D.2
1MATEIS, INSA de Lyon, Université Lyon I, 69621 Villeurbanne Cedex, France, 2IRCELYON,Université Lyon I, 2, Av. A. Einstein, 69626 Villeurbanne Cedex, France
lucian.roiban@insa-lyon.fr

In the last two decades, tilted tomography in a transmission electron microscope (TEM) has become a widely used approach in order to quantify the three dimensional (3D) distribution of features in materials and nanomaterials[1, 2]. During the tilt series acquisition, a projection of the area of interest is recorded at each angle over a large angular amplitude, the final resolution along Z axis being directly related to the maximal tilting angle. The tilt series acquisition is usually performed automatically; depending on the employed acquisition method (automatic focusing, and cross-correlation based tracking), the total acquisition time typically ranges between 30 minutes to several hours. Such conditions are totally incompatible with in-situ experiments, where the materials are subject to changes under external mechanical or electrical solicitations as well as variable temperature and gas flow. Following the 3D evolution in such a context can be attempted by a ‘before/after’ strategy, where a first tomography analysis is performed on the object prior to any solicitation, then a second one after the solicitation as performed to track fuel cell nanocatalysts during electrochemical aging [3]. The recent development of commercial Environmental TEM (ETEM) [4] offers a wide range of in situ environmental studies of nanomaterials, such as oxidation / reduction at high temperature: this opens new opportunities to (try to) investigate in situ the 3D structure of nanomaterials. In this context, we are currently optimizing a fast acquisition method for tomography studies, based on video acquisition of tilted series in less than 1-4 minutes. We have applied this approach to the study of metallic Ag nanoparticles (NPs) encaged in silicalite hollow shells (silica-cages) for application in selective catalysis [5]. Single-tilt tomography and ETEM experiments were performed on a Cs-corrected TITAN ETEM, 80-300 kV, recently installed at CLYM in Lyon. Results are illustrated by figures 1 (fast acquisition performed over an angular amplitude of 116° in 3 minutes and 40 seconds) and figure 2 (ETEM experiments up to 700°C and oxygen partial pressure of 2 mbar). References [1] P.A. Midgley, R.E. Dunin-Borkowski, Nature Mat., 8 (2009) 271-280. <span>[2] T. Epicier, chap. 3 ‘Imagerie 3D en mécanique des matériaux’, ed. J.Y. Buffière, E. Maire, Hermès - Lavoisier, Paris, (2014). <span>[3] J. Jinschek, Microscopy and Analysis, Nanotechn. Issue November (2012) 5-10. <span>[4] Y. Yu, H.L. Xin, R. Hovden, D. Wang, E.D. Rus, J.A. Mundy, D.A. Muller, H.D. Abruña, Nano Lett., 12 9 (2012) 4417-4423. <span>[5] S. Li, L. Burel, C. Aquino, A. Tuel, F. Morfin, J.L. Rousset, D. Farrusseng, Chem. Comm. 49 (2013) 8507-8509.

 

Thanks are due to CLYM (www.clym.fr) for guidance of the ETEM project financed by CNRS, Région Rhône-Alpes, ‘GrandLyon’ and French Ministry of Research and Higher Education. The authors thank N. Blanchard and C. Langlois for fruitful discussions and L. Burel for her assistance.

Fig. 1: Fast single-tilt tomography; a-b): video frames extracted at 78° and - 38.5° from a continuous tilting series acquired in bright field mode in less than 4 minutes; c): surface rendering of the silica-cages (green) and size histogram of Ag NPs (red); only 3% are outside of the silica cages. Acquisition conditions: high vacuum, 20°C, 300 kV.

Fig. 2: a): Assembly of silica cages containing Ag NPs at 20°C under high vacuum; b): same area at 700°C under high vacuum: the Ag NPs have grown but are mostly still inside the silica-cages; c): other area at 450°C under 2 10-2 mbar of O2 flow: note that all Ag NPs are out of the cages on the carbon supporting film, contrarily to b).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-2946 Advanced 3-D Reconstruction Algorithms for Electron Tomography

Arslan I.1, Sanders T.2, Binev P.2, Roehling J. D.3, Batenburg K. J.4, Gates B. C.3, Katz A.5
1Pacific Northwest National Laboratory, Richland, WA, USA, 2University of South Carolina, Columbia, SC, USA, 3University of California-Davis, Davis, CA, USA, 4University of Antwerp, Antwerp, Belgium, and Centrum Wiskunde & Informatica, Amsterdam, The Netherlands., 5University of California–Berkeley, Berkeley, CA, USA
ilke.arslan@pnnl.gov

Electron tomography in the physical sciences has become a powerful tool for nanomaterial analysis. Recently, electron tomography is finding applications in more beam sensitive materials such as catalysts. For beam sensitive materials, the goal is to acquire the smallest number of images as possible but still maintain an accurate and high resolution 3-D reconstruction. Standard methods of 3D reconstruction, such as weighted back projection (WBP) and simultaneous iterative reconstruction technique (SIRT), are not equipped to handle this lack of information, and create significant blurring.  This gives rise to a search for new methods of reconstruction.  Two of the recent successful algorithms are the discrete algebraic reconstruction technique (DART) and total variation (TV) minimization within compressed sensing (CS).

 

DART uses an algebraic reconstruction method (e.g. ART, SIRT) and pairs it with the prior knowledge that there are only a small number (two or three) of different materials in the sample, each corresponding to a different gray value in the reconstruction.  An initial reconstruction is computed and rounded to the chosen fixed gray values based on some threshold, and iteratively refined using ART.  The method of TV minimization stems from the mathematical theory of compressed sensing and only recently became available due to new algorithms for solving the TV minimization problem.   The method considers the characterization of real images and encourages the reconstruction to minimize the number of jumps in gray values, creating clearer material boundaries than conventional methods (i.e. WBP or SIRT), hence creating a similar effect to that of DART.

 

The advantage of DART is that an accurate selection of the gray values and the rounding procedure for the reconstruction gives very accurate material boundaries, not available through any other reconstruction technique. However, the TV minimization procedure has fewer parameter selections, making initial reconstruction simpler and providing a more stable method for reconstruction. Moreover, the introduction of the TV norm has the potential for creating boundaries alternate to what a DART reconstruction would find.  Both methods are extremely valuable. In this presentation we discuss the pros and cons of each method, and show examples to illustrate when to use one method over the other.  

This research was funded in part by the DOE BES DE-SC0005822 and the LDRD and Chemical Imaging Initiative programs at PNNL. The Pacific Northwest National Laboratory is operated by Battelle under contract DE-AC05-76RL01830.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-10-O-2962 Incoherent and coherent imaging for tomography of nano particle

Chen F. R.1, Kisielowski C.2, Tsai C. Y.1, Van Dyck D.3
11. National Tsing Hua University, Hsin-Chu, Taiwan, , 22. NCEM & JCAP Berkeley, CA, USA, , 33. University of Antwerp, Belgium
fchen1@me.com

Transmission electron microscopy (TEM) has been well recognized for its power in spatial resolution to the sub-Å level, especially, with aberration-corrected optics. However, the ultimate goal of electron microscopy is not only to obtain nice images but also to advance materials science. This means that EM has to evolve from describing to understanding materials properties. It is well-known that all the structure-property relations are encoded in the positions of the atoms and the shape of particle, specially, in the case of catalysts and biological species. The drawbacks of high resolution TEM are two folds. First, it gives only 2D projected structural information. And second, the passband of the lens transfer at the low spatial frequencies is very poor and such that the information about shape is lost.
In my talk, I will show that how we develop a novel theory and method for incoherent and coherent TEM imaging technique to determine 3D shape of nano-object with atomic resolution. For coherent imaging, our approach is to retrieve the three-dimensional atomic structure of nanocrystals from the electron exit wave function of a single projection image. The method employs wave propagation to determine the local exit surface of a sample together with the mass of each atomic column. Intensities are scaled by the mean inner potential of the sample and single atom sensitivity is expected since aberration-corrected electron microscopes are now available with such extraordinary capabilities. The validity of the approach is tested with a simulated exit wave function of a gold wedge, as shown in the fig. 1(a) and 1(b). The fig. 1(c) shows the reconstructed tomogram for the wedge Au crystal. For incoherent imaging, we present a new route to enhance the contrast by hollow cone imaging technique for biological objects using thermal diffuse scattered (TDS) electrons. Hollow cone imaging is incoherent and thus does not interfere with the central beam therefore it generates the amplitude contrast. Furthermore, the TDS signal is linear to the mass-thickness and easy to interpret and so it is suitable for soft material tomography. Here Fig. 2. we report the first results on the application of TDS to single particle analysis of proteins. The proof of the concept of the method has been demonstrated experimentally for Chaperonin GroEL as a standard protein since it is stable, easy to obtain and the structure is well-known.

CK is supported by the Office of Science, Office of Basic Energy Sciences of the U.S. Department of Energy under Contract No. DE-AC02—05CH11231. D. Van Dyck acknowledges the financial support from the Fund for Scientific Research - Flanders (FWO) under Project nos. VF04812N and G.0188.08 . F.-R. Chen would like to thank the support from NSC 96-2628-E-007-017-MY3 and NSC 101-2120-M-007-012-CC1.

Fig. 1: Fig. 1(a) modulus of he simulated exit wave for Au wedge crystal (b) phase of simulated exit wave of Au wedge crystal (c) reconstructed tomogram from 1(a) and 1(b).

Fig. 2: Fig. 2 (a) Bright field image of GroEL (b) Hollow cone image (HCI) of GroEl (c) Intensity profile across the image in 2(a) and 2(b). (d) Reconstructed tomogram
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-1482 Accuracy and applications of electron-beam deposited nano-dot fiducial markers in electron tomography of rod-shaped specimens

Hayashida M.1, Kumagai K.1, Malac M.2 3, Bergen M.2
1National Institute of Advanced Industrial Science and Technology (AIST), 2National Institute of Nanotechnology, 3University of Alberta
misa-hayashida@aist.go.jp

Electron tomography is a method employed in a transmission electron microscope (TEM) to reconstruct a three-dimensional (3D) volume from a series of images acquired at suitable tilt increments. An easy, accurate alignment of the series is critical to obtain good quality 3D reconstruction of the sample. For tomography of biological samples, Au nanoparticles are usually used as fiducial markers, which are randomly placed on the sample from a suspension. For precise alignment, markers must be uniformly dispersed over the observed region of the specimen. However, it is difficult to obtain even dispersion because the colloidal Au nanoparticles are usually dense materials. On the other hand, for high resolution imaging of nanomaterials, rod-shaped specimen is usually used, because it allows us to obtain data without missing wedge.. It is, however, difficult to disperse colloidal Au nano-particles near the observing area while not interfering with the objects of interest in such samples. Electron-beam fabricated tungsten nano-dot fiducial markers, deposited in a standard scanning electron microscope with a gas delivery system, allows placing the fiducial markers at nearby locations that do not interfere with the area of interest. In particular we discuss the accuracy of alignment using the nano-dot fiducial markers and demonstrate the application of the method to some rod-shaped specimens with nanoparticles.

<Sample> An example of a typical rod-shaped specimen with Ag nanoparticles is shown in Fig. 1a. The rod-shaped specimens were fabricated by focused ion beam. Nano-dot markers were fabricated onto the specimen using electron beam induced-deposition of tungsten from W(CO)6 precursor.

<Accuracy> As seen in Fig. 2a, the nano-dot markers have nearly parabolic cross section. However, for automatic detection of positions of markers, cross correlation of a radially symmetric template is typically used. To improve alignment accuracy, the shape of the template was changed to better reflect the shape of the markers and their projected shape for various tilts of the specimen, as shown in Fig. 1c. The total error in marker position over the entire tilt range is taken as the minimum root-mean-square (RMS) error between the expected and measured marker position in the projected images. Using the improved shape of template, the RMS error was reduced from 2 to 1.6 pixels compared to radially symmetric template.

<Reconstructed image> Figure 2b shows the cross-sectional image of Ag nanoparticles. Not only the rod shaped sample boundary appears sharp, but the individual Ag nanoparticles with less than 5 nm diameter are clearly visible in the reconstructed images.

We are grateful for financial support of AIST in Tsukuba, Japan and NINT / NRC in Canada. The ongoing support of Hitachi High Technologies Canada contributed to success of this work. We are indebted to Dr. Takashi Nakamura (AIST) for Ag NP sample preparation and Martin Kupsta for support and for assistance on the Hitachi NB 5000.

Fig. 1: (a)Image of the entire specimen. The regions are (starting from the top): amorphous carbon with nano-dot markers, ordered array of Ag nanoparticles, Si wafer substrate.(b) Radially symmetric template image.(c) Selected template image was modified to fit the shape to actual markers' shape at every tilt for every marker.

Fig. 2: Fig.2 (a),(b) a cross section of reconstructed volume of Ag nanoparticles in a plane perpendicular to the tilt axis. The plane of the cross section is marked in Fig1a.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-1681 Quantitative 360° electron tomography analysis of mesoporous hematite nanoparticles

Winter B.1, Butz B.1, Distaso M.2, Dudák M.3, Kočí P.3, Klupp Taylor R. N.2, Peukert W.2, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy, FAU Erlangen-Nürnberg, Cauerstraße 6, 91058 Erlangen (Germany), 2Institute of Particle Technology, FAU Erlangen-Nürnberg, Cauerstraße 4, 91058 Erlangen (Germany), 3Institute of Chemical Technology, Technická 5, CZ 16628 Prague (Czech Republic)
benjamin.winter@ww.uni-erlangen.de

Metal oxide nanomaterials find application in diverse fields of research and industry such as catalysis, drug delivery, sensing, photo-electrochemistry, optical detection or as pigments [1,2].
The materials properties and hence application performance are affected by the particle size, shape, surface characteristics as well as by possible pore or defect structures. Tuning the parameters of the chosen synthesis technique can affect some or all of these properties and thus enables the tailoring of the particle function.
Here, wet-chemically synthesised mesoporous hematite nanoparticles (NPs) are investigated regarding their internal re-organization during calcination. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies showed that the calcination temperature has a pronounced effect on the pore morphology although a quantitative evaluation of the size distribution and connectivity of the pores was not possible (Fig. 1). Therefore, we carried out a detailed study using electron tomography (ET) in scanning TEM (STEM) imaging mode (enhanced mass-thickness contrast) in order to reveal the three-dimensional morphology, size distribution and interconnectivity of the pores inside the hematite NPs (Fig. 2 b,c,d). NPs in aqueous solution were directly drop-casted onto a pillar with an electron-transparent thickness that has been preliminarily thinned using focused ion beam (FIB) milling. Transferring this tip (Fig. 2 a) onto a 360° ET sample holder enables the acquisition of a tilt series with full tilt-angle range (Fig. 2 b). This permits the reconstruction of the particle morphology without a missing wedge of information and therefore improves the quality of the 3D reconstruction.
The tomograms were used to perform a quantitative analysis of the pore space. Virtual capillary condensation (VCC) [3] and maximum sphere inscription (MSI) [4] were applied to determine the pore size distribution of NPs as illustrated for one particle in Fig. 3. The results are compared with independent N2 sorption measurements. NPs calcined at lower temperatures tend to have a more open-porous structure with a higher degree of porosity, whereas with increasing temperature fewer and rather enclosed pores were found to occur more frequently.
The quantitative analysis of the pore morphology using 360° ET will help to get a better understanding of this promising class of material.

1. Wang et al., New J. Chem. 38 (2014), pp. 46.
2. Echigo et al., Jr., Am. Mineral. 98 (2013), pp. 154.
3. Štěpánek et al., Colloids Surf., A 300 (2006), pp. 11.
4. Novák et al., Chem. Eng. Sci. 65 (2010), pp. 2352.

DFG Research Training Group 1161, Cluster of Excellence “Engineering of Advanced Materials” (EXC 315), DFG SPP 1570, Czech Science Foundation (GACR P106/10/1568).

Fig. 1: a) SEM and TEM images of hematite nanoparticles calcined at 400°C and 500°C.

Fig. 2: a) Photo of a 360° ET tip with a droplet of hematite particles in solution; b) STEM image of Fe2O3 nanoparticles attached to this tip - green frames tag particles calcined at 400°C, yellow frames at 500°C; c), d) comparison of STEM images and the 3D reconstruction (volume rendering) of c) 400°C and d) 500°C particles.

Fig. 3: Reconstructed hematite nanoparticle calcined at 400°C showing a) a snapshot of the virtual capillary condensation (VCC) [3] (pores gradually filled with liquid (blue)), b) a slice displaying colour-coded results from the MSI analysis [4]; c) diagram showing the pore size distribution resulting from the MSI analysis and the VCC simulation.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-1698 The physical properties of plastic support films for 3-D transmission electron microscopy.

Daraspe J.1, Longo G.2, Kizilyaprak C.1, Humbel B. M.1
1University of Lausanne, Electron Microscopy Facility, Lausanne Switzerland, 2EPFL, Laboratory of Physics of Living Matter, Lausanne, Switzerland
jean.daraspe@unil.ch

In the last 10 years, the acquisition of large volumes of biological specimens at high resolution has regained importance, especially in the field of neurobiology. The analysis of large volumes has become essential to better understand cell-cell relationship and the interaction of subcellular organelles.
Though more modern and automated processes like serial block face scanning electron microscopy [1] and focused ion beam scanning electron microscopy [2] ease the process of gaining large volumes, traditional serial section has come to a revival [3].
Serial sectioning maintains its ability to image almost an unlimited 3D volume, up to mm2 at high resolution (X=Y=~1nm; Z=50-70nm). Further serial thick section TEM tomography can improve the Z resolution to about 2nm. The only limit is the skill and persistence of the operator.
For serial section image acquisition and for serial TEM tomography the stability of the plastic support film of the TEM grids, especially large slot grids, is crucial. The following quality criteria are required:
- Flatness, the films should not bend during pick-up of the sections.
- Resilient and strong, the films have to support mechanical tensions.
- Beam resistance, the films should not drift and disrupt in the electron beam.
- Temporal stability, these qualities should be maintained for a long time to allow storage of prepared grids.
To improve the stability of the section on the support film it is important to review the properties of the different polymers commonly used and to find the formulation, which best matches the quality criteria required.
We prepared support films from 6 different polymers and analysed their behaviour during pick-up and TEM imaging. Plastic films were casted on microscope slides and two films of equal thickness were separated. One was used as a support film on a TEM grid and the other was mounted on a slide for AFM analysis. In the AFM the thickness, stiffness and adhesion force was measured. In the TEM, drift measurements were done by following gold particles at high magnification using time lapse series acquisition. Thickness was also measured by TEM tomography.
In conclusion, two polymers emerged that fulfil the requested criteria for 3D investigations by serial sectioning.

1 - Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. Denk W et al.; PLoS Biol. 2004 Nov;2(11):e329
2 - Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. Knott G et al.; J Neurosci. 2008 Mar 19;28(12):2959-64
3 - Array Tomography: A New Tool for Imaging the Molecular Architecture and Ultrastructure of Neural Circuits. Micheva KD et al; Neuron. 2007 Jul 5;55(1):25-36

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-1846 A Remote Control/Observation System and an Operation Support System for the Ultrahigh Voltage Electron Tomography

Yoshida K.1, Nishi R.1, Yasuda H.1
1Research Center for Ultra High Voltage Electron Microscopy, Osaka University
yoshida@uhvem.osaka-u.ac.jp

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-1866 A memory efficient method for 3D object reconstruction with HAADF STEM depth sectioning

Van den Broek W.1, Rosenauer A.2, Van Aert S.3, Sijbers J.4, Van Dyck D.3
1Institute for Experimental Physics, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany, 2Institut für Festkörperphysik (IFP), Universität Bremen, Otto-Hahn-Allee 1, 28359 Bremen, Germany, 3Electron Microscopy for Materials Science (EMAT), University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 4iMinds - Vision Lab, Department of Physics, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium
wouter.vandenbroek@uni-ulm.de

In high angle annular dark field scanning transmission electron microscopy (HAADF STEM) depth sectioning, the object is illuminated with a beam with a small depth of field, caused by a large beam convergence angle, see Fig. 1.

In [Ultram. 110 (2010) 548--554], it is shown that the simultaneous iterative reconstruction technique (SIRT) applies to depth sectioning. The SIRT algorithm is given as

fk+1 = fk + AT [ ( q - A fk ) / A If ] / [ AT Ip ],

where k indicates the iteration number, A is the projection matrix, f is the object vector and q the experimental projection, arithmetic operators between vectors are elementwise and Ip and If each denote a vector of which each element equals 1 with a length equal to that of the projection and the object, resp.

The matrix A scales badly with object size and readily grows too large for the computer memory. Here, we propose to perform the matrix-vector multiplication A fk implicitly through a 2D convolution of each horizontal layer of the object with its corresponding single atom image, followed by a sum in the vertical direction over all horizontal layers. Implementation of the matrix-vector multiplication A If is analogous. The multiplication of AT with the vector ( q - A fk ) / A If can be carried out implicitly by stacking the image in an 3D array and convolving each of the layers with the corresponding single atom image. The matrix-vector multiplication AT Ip can be implemented analogously. The memory load is now reduced to storing the object.

A technique analogous to charge flipping [Acta Cryst. A64 (2008) 123--134] is added: After each iteration values below a certain positive threshold have their sign reversed.

The validity of these implicit matrix-vector multiplications is tested with a simulation of a 1.6nm Au particle. The microscope parameters are: Acceleration voltage: 200kV; C1: -2.67nm; C3: 3.54μm; C5: -1.13mm; C7: 10cm; convergence semi-angle: 86.8mrad. The object measures 1000 x 1000 x 125 voxels of 8 x 8 x 210 pm3. See Fig. 2. The particle is tilted away from the zone-axis. The single atom images encoded in the matrix A are a convolution of the Au atom potential and the probe intensity.

A total of 125 images with a defocus step of 0.21nm is simulated and used as input for SIRT with 64 iterations. In Fig. 3 it is shown that the severe elongation in the vertical direction, so typical for depth sectioning, is overcome. The authors have reported these results in [A memory efficient method for fully three-dimensional object reconstruction with HAADF STEM, Ultram., accepted].

W. Van den Broek: The Carl Zeiss Foundation and DFG, KO 2911/7-1; A. Rosenauer: DFG, AR 2057/8-1; S. Van Aert, J. Sijbers, D. Van Dyck: FWO, G.0393.11, G.0064.10, G.0374.13.

Fig. 1: Features of the object close to the beam crossover are well localized in the image while features further away are smeared out. Depth information is unlocked by varying the defocus, thus bringing other regions of the object in focus. The underlying assumption is that the image formation is incoherent.

Fig. 2: Upper left: Horizontal slice through the middle of the data set. Lower left: Average of all horizontal slices. Right: Average of the vertical slices. The white oval marks the particle's position.

Fig. 3: Depth-profiles of the simulated measurements, the reconstruction and the original object averaged over a disc of radius 1.6nm centered on the particle. The FWHM of the profile of the reconstruction (2.07nm) now approximates that of the original object (1.85nm).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-1875 Improving Depth of Focus in STEM Tomography using Focal Series

TREPOUT S.1, MESSAOUDI C.1, BASTIN P.2, MARCO S.1
1Institut Curie / INSERM U759, Campus Universitaire d'Orsay, Bât. 112, 91405 ORSAY cedex FRANCE, 2Institut Pasteur, CNRS URA 2581, Parasitology & Mycology Department, Institut Pasteur, 25, rue du Docteur Roux, 75015 PARIS, FRANCE
sylvain.trepout@curie.fr

Scanning Transmission Electron Microscopy (STEM) is a point to point imaging method which uses a focused electron beam to build a projection image of the sample. One of the advantages of STEM is that the focused electron beam can pass through thicker samples (up to 1 µm-thick on a 200 kV FEG) providing higher signal-to-noise ratio than standard TEM (wide spread beam). This makes STEM more suitable for electron tomography of thick samples. Nevertheless, the smaller is the size of the probe, the more reduced is the depth of focus (DOF). A way of solving this difficulty is to take advantage of raster scanning to get focused images line by line by dynamic focus [1]. However, in our experience, the recovery of full-focused images by dynamic focus is not helpful when the DOF cannot encompass a very thick sample (>0.5 µm). To circumvent this limitation we have developed an acquisition scheme and an image processing method in which we reconstruct full-focused images from STEM images recorded at different defocus.
This process of DOF correction can be used for both 2D studies and tomographic experiments. Briefly, it consists in two steps. For a 500 nm thick sample, i) five images are acquired at different focus values ranging from -300 nm up to 300 nm defocus (using 150 nm steps); ii) the images are processed using an ImageJ macro based on Turboreg [2] and Extended Depth of Field [3]. This macro consists of two steps: i) the images acquired at different focus are aligned; ii) regions of these registered images which are at focus are combined to compute a single image.
The performance, in terms of resolution, contrast and SNR, of the afore mentioned approach has been evaluated in silico by comparing 3D reconstructions computed from the projections of a phantom volume leading to two different datasets. In the first dataset the whole images are at focus whereas in the second one, parts of the images have been modified to mimic the focus changes observed in experimental data. The method was applied to compute the 3D reconstruction of a resin-embedded T. brucei 500 nm-thick section using five focal series. Sections of the reconstructed volumes without (1 focal series), with intermediate (3 focal series) and full (5 focal series) DOF correction on high-tilt images (±40°) are displayed in figure 1 showing the improvement in the observed details.

References:
1. Feng et al. “Automated electron tomography with scanning transmission electron microscopy”. J. Microsc., 2007, 228:406-12.
2. Thévenaz et al. "A Pyramid Approach to Subpixel Registration Based on Intensity". IEEE Tr. Im. Pro., 1998, 7: 27-41.
3. Forster et al. « Complex Wavelets for Extended Depth-of-Field: A New Method for the Fusion of Multichannel Microscopy Images”. Mic. Res. Tech., 2004, 65:33-42.

This work has been funded by the ANR grant 11-BSV8-0016.

Fig. 1: Comparison of 3D information recovered from depth of focus correction of high-tilt images. a, b, c) 1 nm-thick XY planes from reconstructions computed with only high-tilt images (±40°) without, with intermediate and full depth of focus correction respectively. d, e, f) 1 nm-thick YZ planes from the same reconstructions. Scale bar, 100 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-1904 Electron Holographic Tomography of Electric and Magnetic Stray Fields around Nanowires

Lubk A.1, Wolf D.1, Lichte H.1
1Triebenberg Laboratory, TU Dresden, Dresden, Germany
Axel.Lubk@triebenberg.de

One important consequence of the support theorem of the Radon transformation is that the so-called outer Radon problem (ORP), stating that the tomographic reconstruction of a scalar function in the exterior of a convex domain from projections along submanifold passing outside that domain, has a unique solution. Cast into the specific circumstances of electron holographic tomography that means that a reconstructed phase tilt series from outside a charged nanostructure, such as a nanowire (NW), is sufficient to reconstruct the corresponding potential in that outer region (Fig. 1). The topic of this contribution is the solution of the ORP, including its technical implementation and benefits for the determination of physical quantities within the framework of electron holographic tomography. Until now, only the interior Radon problem, i.e. the reconstruction of potentials within a convex (circular) domain containing e.g. a pn-junction has been treated. There, a number of issues which can be potentially tackled with the help of the ORP have been encountered: (A) Tilt angles within the TEM are often limited to within approx. -70° to 70°, rendering spatial frequencies in the corresponding missing wedge in Fourier space inaccessible. (B) Phase unwrapping algorithms cannot distinguish between unresolved phase gradients larger then π and phase jumps, producing artefacts at object boundaries (e.g. at FIB prepared specimen). (C) During tilting the proximity of a low-index zone axis can lead to dynamical scattering. (D) Magnetic field reconstruction suffers from the difficult alignment of 2 by 180° flipped tilt series required to seperate electric and magnetic phase shifts. The lowered influence of these issues in the ORP is of course purchased by disregarding any information from the blocked specimen region. However, the laws of electro-(magneto)statics relate the outside potentials to object charges and dipoles, thus rendering the fringing fields a valuable property. We will demonstrate a solution to the ORP including its potential to mitigate the above issues by reconstructing a small beam induced charging field around a core-shell GaAs-AlGaAs NW (Fig. 2) and a magnetic field emerging from the tip of a Co2FeGa Heusler alloy NW (Fig. 2) into vacuum. Both fields are very weak and could only partially recovered by means of the interior Radon problem due to the above issues.

The authors acknowledge financial support from the European Union under the Seventh Framework Program under a contract for an Integrated Infrastructure Initiative. Reference 312483 - ESTEEM2.

Fig. 1: Geometry and coordinate system employed in the ORP. D indicates the reconstruction domain containing the function f. The projection is denoted by F and in both functions f and F the dark gray region indicates the part excluded within the ORP.

Fig. 2: Electrostatic potential and axial component of the magnetic field reconstructed in the exterior of two NWs by solving the ORP.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-1980 Energy dispersive X-ray (EDX) tomography of bimetallic nanoparticles

Slater T. J.1, Macedo A. M.2, Burke M. G.1, O'Brien P.1, Camargo P. H.2, Haigh S. J.1
1The University of Manchester, Manchester, UK, 2Universidade de Sao Paulo, Sao Paulo, Brazil
thomas.slater-5@postgrad.manchester.ac.uk

Electron tomography can be used to provide spectroscopic analysis in three dimensions at nanometer resolution through a variety of imaging techniques. EDX tomography promises accurate simultaneous projections of all elements that fully meet the projection requirement. We present the latest results of three-dimensional elemental analysis using EDX tomography to investigate bimetallic nanoparticles.
The design of the Super-X detector [1] as included on the FEI Titan G2 80-200 has a large solid angle of detection (≈0.8sr) and therefore a count rate high enough to allow EDX tomography of many types of sample. EDX tomography of focused ion beam (FIB) prepared samples is now entirely feasible and results in minimal detector shadowing at any angle [2]. However, FIB prepared rods of free standing nanoparticles are not straightforward to prepare. Dispersing nanoparticles on to a standard TEM grid is a simple preparation method but the grid bars and sample holder shadow the EDX detectors at a range of angles. This prevented acquisition of EDX data at a large range of angles when using traditional single detectors but can be overcome when using the Super-X detector. To combat effects of detector shadowing we have used a simple acquisition-time varying sampling scheme that is guided by prior characterisation of the detector. We acquired EDX spectral images of a single AgAu nanoparticle at a range of tilt angles for a fixed acquisition time. Acquisition time at each tilt angle was then adjusted to provide similar Au counts at each angle (Fig. 1).
We have used our novel acquisition scheme to investigate elemental distributions in bimetallic nanoparticles synthesized via the galvanic replacement reaction, primarily AgAu nanoparticles. The elemental distribution in these nanoparticles is particularly important for their catalytic and optical properties. Two dimensional EDX mapping suggested that the extent of surface segregation in the AgAu nanoparticles varied with composition. Particles with low Au content (below 20 at% Au) appeared to display Au surface segregation and particles with high Au content (above 40 at% Au) displayed Ag surface segregation. However, two dimensional maps cannot categorically reveal surface compositions. For example, it is unclear whether intense lines of Au counts in Fig. 2a are situated on the surface of or within the nanoparticle. For this reason, we performed EDX tomography on nanoparticles that showed, separately, Au and Ag surface segregation (Fig. 2c,d). Through EDX tomography we were able to conclusively show a reversal in surface segregation in AgAu nanoparticles prepared via the galvanic replacement reaction.

References
1 von Harrach, H. S. et al. Microsc. Microanal. 15 (2009), 208-209
2 Lepinay, K. et al. Micron 47 (2013), 43-49

SJH thanks the USA Defense Threat Reduction Agency (grant number HDTRA1-12-1-0013) and Gates Foundation for funding support. PHCC and AM thank FAPESP and CNPq for funding support (grant numbers 2011/06847-0, 2013/19861-6 and 471245/2012-7, respectively).

Fig. 1: Figure 1. a) Counts of Au and Ag peaks as a function of tilt angle when using a fixed time acquisition scheme. (b) Varied acquisition-time scheme used in order to correct for detector shadowing by the sample holder. (c) Counts of Au and Ag peaks as a function of tilt angle when the varied acquisition-time scheme is used.

Fig. 2: Figure 2. a,b) Two dimensional EDX spectral images showing Au and Ag distributions within nanoparticles with Au and Ag surface segregation. c,d) Surface visualisations of reconstructed tomograms of Au and Ag in nanoparticles displaying Au and Ag surface segregation.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2061 Whole-Cell Imaging of the Budding Yeast Saccharomyces cerevisiae by High-Voltage Scanning Transmission Electron Tomography

Murata K.1, Esaki M.2, Ogura T.2, Arai S.3, Yamamoto Y.3, Tanaka N.3
1National Institute for Physiological Sciences, Okazaki, Aichi, 444-8585, Japan, 2Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, 860-0811, Japan, 3Ecotopia Science Institute, Nagoya University, Nagoya, Aichi, 464-8603, Japan
n-tanaka@esi.nagoya-u.ac.jp

High-voltage electron tomography provides three-dimensional (3D) information about cellular components in thicker sections beyond 1 μm, but image degradation caused by multiple inelastic scattering of transmitted electrons limits the attainable resolution. Scanning transmission electron microscopy (STEM) is believed to give enhanced contrast compared to conventional transmission electron microscopy (CTEM), and thicker samples up to around 1 μm can be analyzed with an intermediate-voltage electron microscope, because the depth of focus and the inelastic scattering are not the critical limitations.
      We have applied STEM to 1 MV high-voltage electron tomography to extend the limitation of the specimen thickness, and seamlessly investigated the whole-cell structure of the budding yeast, Saccharomyces cerevisiae, size of which was ~3 μm width. The high-voltage STEM tomography, especially with a bright-field mode, demonstrated sufficient enhanced contrast and more-intense signals compared to regular TEM tomography (Fig. 1), permitting segmentation of major organelles in the entire cell (Fig. 2). The technique also showed less specimen shrinkage. The current spatial resolution is limited with the specimen preparation and the relatively large convergence angle of the scanning probe, but the present new technique has a potential to solve longstanding problems of image blurring in thick biological specimens beyond 1 μm, and to open a new research field in cell structural biology.
[1]K. Murata et al., to be submitted (2014).

This study was supported by the program of Joint Usage/Research Center for Develpment Medicine at IMEG, Kumamoto University. H-1250M at NIPS and JEM-1000K RS at Nagoya University were used for observation.

Fig. 1: Tomogram slices of an whole budding yeast cell at xy (a) and xz (b) planes were calculated from the tilt series collected by TEM-BF(Blight field), STEM-BF, and STEM-ADF(Annular dark field), respectively. Scale: 1 μm.

Fig. 2: The major organelles of a budding yeast cell were successfully segmented in a 3D tomogram calculated from the STEM-BF tilt series. Scale: 1 μm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2098 Cryo-STEM Tomography for 3D Analysis of Cell Structure

Aoyama K.1,2
1Application Laboratory FEI Japan, Tokyo, Japan, 2Osaka University, Osaka, Japan
kazuhiro.aoyama@fei.com

Recently, several studies for observation of biological specimens as plastic section have been performed by using STEM, and the potential has been indicated. STEM tomography offers several important advantages including: (1) it is effective even for thick specimens, (2) ‘dynamic focusing’, (3) ease of using an annular dark field (ADF) mode and (4) linear contrasts. It has become evident that STEM tomography offers significant advantages for the observation of thick plastic specimens. In this study, the technique applied for Cryo-specimens. Of course, even in Cryo-Tomography, the advantages of STEM above mentioned are valid. Because STEM has advantage to resist specimen thickness, it is expected to be powerful method for observing whole cell structure in Cryo-microscopy without thin sectioning.

The insufficient contrast is one of the serious problems in Cryo-electron microscopy. Therefore, the image contrasts by TEM and STEM have to be compared carefully and quantitatively. Figure 1 shows the comparison of the image qualities. The specimen was vitreous ice on Quantifoil made by standard procedure of Vitrobot. Titan Krios equipped with 2 cameras and STEM system was used for the experiment. TEM images were taken by CCD camera (Gatan US4000) and direct detected CMOS camera (FEI Falcon). STEM image was taken in bright field mode. The imaging conditions, image pixel size and the number of irradiation electrons, were normalized. The average counts of the pixels and the standard deviation (SD) were measured for each image, and then SD/mean was calculated. The result was clear that the STEM image had very low back ground noise. This character can be explained theoretically, there are several seasons; (1) Short operation time for pixel (dual time vs. exposure time), (2) Large physical size of the detector, (3) Very small collection angle (same as very small objective aperture in TEM imaging).

By applying Cryo-STEM tomography, clear membrane structure of organelle appeared without staining and without sectioning.

Fig. 1: A comparison of the image quality in Cryo-EM
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2123 3D electron tomography analysis of silicon nanoparticles in SiC matrices by quantitative determination of EELS plasmon intensities

Xie L.1, Jarolimek K.2, Van Swaaij R.2, Thersleff T.1, Zeman M.2, Leifer K.1
1Uppsala University, Department of Engineering Sciences, Applied Materials Sciences, Box 534, SE-751 21 Uppsala, Sweden., 2Photovoltaic Materials and Devices, Delft University of Technology, P.O. Box 5031, 2600 GA Delft, The Netherlands.
ling.xie@angstrom.uu.se

Silicon nanoparticles (NPs) embedded in insulating or semiconducting matrices has attracted much interest for the third generation of photovoltaics, “all-Si” tandem solar cells. This study is to show how silicon NPs are distributed in 3D on a silicon carbide thin film using the electron tomography technique in the transmission electron microscopy (TEM). [2]

We first have assessed Si NPs distributions in such SiCx sample with a low degree of crystalline using bright field (BF) TEM tomography (figure 1) and found an average nearest neighbour spacing of two NPs of about 12nm. For more crystalline NPs, the projection requirement is no more fulfilled and only those Si NPs that are both crystalline and oriented to a Bragg reflection are detectable. [3] Therefore, in this case, conventional BF TEM signal is unsuitable for electron tomography and we applied spectrum imaging (SI) techniques: EELS SI imaging and EFTEM SI imaging. Since Si and SiCx have different plasmon energies, [4] we can extract Si plasmon and SiCx plasmon images from the spectrum images. We observed that only a proper fit of the plasmon spectrum with subsequent extraction of Si and SiCx plasmon images results in the correct Si ad SiCx distribution (figures 2 and 3), whereas just EFTEM images taken from windows around the Si and the SiC plasmon energy resulted in overlaps in the image.

For both, STEM and EFTEM SI signals, in figure 2 and 3, we are able to detect the entire population of NPs. In figure 3, the stripes like contrast inside of crystalline NPs shown in the BF TEM image persist in plasmon images. This is due to parallel beam illumination in EFTEM SI mode thus making the STEM SI imaging more suitable for tomography of these NPs. In Figure 2, for STEM SI, the contrast evolution during the tilting is thickness dependent, thicker part of the sample gives stronger contrast in the extracted plasmon images, and this nonlinear thickness effect can be corrected by introducing attenuation coefficient. [5]

In summary, to study the 3D distribution of Si NPs in SiCx matrix, we compared three signals from BF TEM, STEM and EFTEM SI signals. In order to overcome the non-linearity of contrast change during the tilting process, STEM-SI signal in combination with quantitative treatment of the plasmon spectra shows clear Si NP contrasts and overcomes limits set by the projection requirement.

[1] S. Perraud et al., Phys. Status Solidi A, 1–9 (2012).

[2] J. Frank, Electron Tomography: Three Dimensional Imaging with the Transmission Electron Microscope, Plenum, New York, London, 1992.

[3] P. A. Midgley et al., Ultramicroscopy 96 (2003) 413.

[4] R.F. Egerton, Electron Energy-Loss Spectroscopy in the Electron Microscope, 420, 2011.

[5] W. Van den Broek et al. Ultramicroscopy 116 (2012) 8–12

The authors acknowledge the support from the EU founded FP8 project “SNAPSUN”.

Fig. 1: Figure 1. (Left) 2D BF TEM image of Si nanoparticles embedded in amorphous Si riched SiC:H matrix, (Right) 3D model view of Si nanoparticles distributed in matrix, green spheres indicate Si nanoparticles, and reconstructed X, Y and Z images are also shown in the volume. Scale bar is 10 nm.

Fig. 2: Figure 2. (Left) 2D STEM-ADF image of Si nanoparticles embedded in SiCx matrix, (Middle) Si plasmon image, (Right) SiC plasmon image. Both plasmon images are extracted from STEM SI data set.

Fig. 3: Figure 3. (Left) 2D BF TEM image of Si nanoparticles embedded in SiCx matrix, (Middle) Si plasmon image, (Right) Reconstructed tomogram and the formation of Si networks were shown in the volume by using the isosurface. Plasmon images are extracted from EF TEM SI data set which is acquired with a 2 eV energy slit at 17 eV (Si).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2238 EELS CS tomography of FeO-Fe3O4 core-shell nanoparticles. An approach to recover 3D oxidation state distribution.

Torruella P.1, Arenal R.2,5, Saghi Z.3, Yedra L.1,4, Eljarrat A.1, de la Peña F.3, Midgley P. A.3, Estradé S.1,4, Peiró F.1, Estrader M.6, López-Ortega A.7, Salazar-Alvarez G.8, Nogués J.9,10,11
1LENS–MIND-IN2UB, Dept.d’Electrònica, Universitat de Barcelona, Barcelona, Spain , 2LMA, Instituto de Nanociencia de Aragon, Universidad de Zaragoza, Zaragoza, Spain, 3Department of Materials Science and Metallurgy, University of Cambridge, Cambridge, United Kingdom, 4CCiT, Universitat de Barcelona, Barcelona, Spain, 5Fundacion ARAID, Zaragoza, Spain, 6Departament de Química Inorgànica, Universitat de Barcelona, Barcelona, Spain, 7INSTM and Dipartimento di Chimica “U. Schiff”, Università degli Studi di Firenze, Firenze, Italy, 8Department of Materials and Environmental Chemistry, Arrhenius Laboratory, Stockholm University, Stockholm, Sweden, 9Departament de Física, Universitat Autònoma de Barcelona, Bellaterra (Barcelona), Spain, 10ICN2 – Institut Catala de Nanociencia i Nanotecnologia, Campus UAB, Bellaterra (Barcelona), Spain, 11Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
pautorruellabesa@gmail.com

The aim of this work was to characterize a sample consisting on FeO-Fe3O4 core-shell cubic-shaped nanoparticles. Because of the similarities in the composition and effective atomic number of the core and the shell, high angle annular dark field (HAADF) imaging could not be used to resolve the structure.

As an alternative, EELS fine structure can be used to obtain information on the oxygen and iron oxidation state, thus making it possible to distinguish between FeO and Fe3O4. However, there is the limitation that EELS projects the information of the 3D nanoparticle into a 2D map.

To overcome this limitation there is the possibility to consider EELS spectrum image data-sets as suitable for 3D tomographic reconstruction, not only containing information on the chemical composition of the sample (as in [1]) but also on the oxidation state of Fe at each voxel.

A tilt series of spectrum images (SI) was acquired on a probe corrected FEI Titan. Then the images were treated with Hyperspy to obtain independent spectral components from the iron edge, with could be correlated with the different iron oxides. In order to improve the quality of the reconstruction, a new reconstruction algorithm based on the mathematical theory of compressed sensing (CS) was used. To our knowledge this is the first time that the CS algorithm has been used to reconstruct an EELS core-loss spectrum image data-set.

The CS reconstructions show a shell thickness of 9nm around the core. The 3D reconstruction proves a total shell coverage of the core and that there has been no appreciable phase mixing.

[1] Ll. Yedra et al., Ulramicroscopy 122 (2012), pages 12-18.

The measurements were performed in the Laboratorio de Microscopias Avanzadas (LMA) at the Instituto de Nanociencia de Aragon (INA) - Universidad de Zaragoza (Spain). We acknowledge the support received from the European Union Seventh Framework Program under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative – I3) and under Grant Agreement 291522-3DIMAGE.

Fig. 1: Obtained spectral components from the iron edge after performing PCA and ICA with Hyperspy.

Fig. 2: Central orthoslice from CS reconstruction corresponding to ‘core’ component in figure 1 showing core thickness measurement.

Fig. 3: Central orthoslice from CS reconstruction corresponding to ‘shell’ component in figure 1 showing shell thickness measurement.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2280 Tomography in Analytical Transmission Electron Microscopy of Nanomaterials

Orthacker A.1, Haberfehlner G.1, Tändl J.3, Poletti M. C.3, Kothleitner G.1,2
1Center for Electron Microscopy, Graz, Austria, 2Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Graz, Austria, 3Institute for Materials Science and Welding, Graz University of Technology, Graz, Austria
angelina.orthacker@felmi-zfe.at

The engineering of specific material properties requires both a structural and chemical understanding, often obtained with electron microscopic techniques. While analytical scanning transmission electron microscopy (STEM), including energy dispersive x-ray (EDX) spectroscopy and electron energy loss spectroscopy (EELS), can offer the necessary chemical information, the integrative character of the signal acquired through transmission might hide important structural details of the material. Those details can be revealed by using electron tomography, where the data is acquired at different tilt angles and, after alignment, reconstructed to form a full 3D model of the investigated material. This technique on its own, however, lacks the important chemical information. The content of this work is the combination of both techniques, analytical STEM and tomography, which offers a more complete understanding of the material structure and composition.
Combining analytical signals in the form of spectrum images with a tomographic acquisition however, represents a major challenge. First of all there is no possibility to acquire a tilt series including EELS and EDX spectra for each data point automatically. Secondly, there is no simple way of reconstructing a four dimensional object, containing two spatial, one energy and one tilt angle coordinate. Having tackled this problem, the next issue arises due to the often very limited statistical quality of the analytical signals. In order to minimize acquisition times, dose and sample drift, dwell times per pixel are often in the order of a few milliseconds, necessitating special reconstruction algorithms that can handle noisy data.
The material studied in this work is an alloy containing scandium and zirconium rich nanoparticles embedded in an aluminum-magnesium matrix (fig.1 and fig.2). These nanoparticles increase the mechanical resistance of the material. Their sizes and chemical compositions can vary, depending on the aging process. Previous work reported that these particles can exhibit a core-shell structure.
As the acquisition of EDX and EELS spectra takes more time than non-spectroscopic imaging techniques special care needs to be taken concerning the stability of the sample. This can be achieved by reducing the number of tilt angles, which is possible if special reconstruction algorithms are used. Total variation minimization mathematically assumes minimized gradients which can reduce artefacts and noise. While this can be problematic if the sample itself exhibits gradients of concentrations, it can lead to smooth reconstructions if the sample consists of clearly separable phases.

We thank the Austrian Cooperative Research Facility, the European Union (7th Framework Programme: ESTEEM2), and the Austrian Research Promotion Agency FFG (TAKE OFF project 839002) for funding.

Fig. 1: aluminum alloy; left: EFTEM overview image at 40eV; right: HAADF image of Sc-rich nanoparticle

Fig. 2: extracted spectra of an EELS (left) and EDX (right) spectrum image of a nanoparticle in its matrix, supporting that the particle is Sc-rich
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2420 Tilt-less Electron Tomography

Oveisi E.1, Letouzey A.2, Lucas G.1, Cantoni M.1, Schäublin R.3, Fua P.2, Hebert C.1
1Interdisciplinary Centre for Electron Microscopy, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland, 2Computer Vision Laboratory, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland, 3Centre de Recherches en Physique des Plasmas, Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland
emad.oveisi@epfl.ch

Accurate three-dimensional (3D) knowledge of dislocation structures is more and more vital for the understanding of complex deformation mechanisms at the nanometer scale. In this respect, tomography in transmission electron microscopy developed in the area as a fruitful technique, but is demanding and sometimes inapplicable, as it requires the acquisition of multiple images within a large tilt range [1-4]. The major challenge to electron microscopists now is the development of efficient techniques to overcome these limitations and thus facilitate 3D reconstructions.
We have developed an efficient method that provides a highly reliable insight into the 3D reconstruction of dislocations, in which both image acquisition and reconstruction are addressed. This technique makes use of the convergent electron beam in scanning transmission electron microscopy (STEM) and provides from a single viewing direction a stereoscopic pair of micrographs. Our newly reconstruction algorithm allows us to derive the true structure of dislocations in three dimensions on the sole basis of the acquired stereoscopic micrographs. The reconstruction algorithm firstly extracts, from the STEM stereo images, the dislocation lines using state of the art curvilinear structures detection algorithm. These 2D representations of dislocations are then automatically matched between images. Finally, the algorithm, given the projection parameter and the corresponding virtual tilt angle of each image, can then reconstruct the 3D structure of the dislocations. The method is successfully demonstrated on the 3D visualization of dislocation arrangements in a Fe-10Cr model alloy.
With a proper input of the crystallographic axes of the specimen in the algorithm, the visualization tool allows determining the habit planes of the curves in the dislocation lines. The missing wedge effects are reduced in the 3D structure reconstructed via this algorithm, and “replaced” by a small uncertainty along the Z-axis, that thanks to purposely-designed smoothing techniques applied in the algorithm, can be kept in the range of few pixels.
In summary, this efficient and straightforward technique will markedly facilitate the 3D reconstruction and, if prior knowledge about the object can be ascertained, can be extended to a wide range of fields in which tilting associated problems, specimen thickness, and sensitivity to electron radiation are the limiting factors.

[1] P.A. Midgley, R.E. Dunin-Borkowski, Nature Materials 8 (2009) 271.
[2] J.S. Barnard, J. Sharp, J.R. Tong, P.A. Midgley, Science 313 (2006) 319.
[3] M. Tanaka, M. Honda, S. Hata, K. Higashida, Materials Transactions 49 (2008) 1953.
[4] M. Weyland, P.A. Midgley, Materials Today 7 (2004) 32.

Prof. P. Stadelmann, Dr. D. Alexander, and Q. Jeangros are gratefully acknowledged for stimulating discussions. This work was financially supported by Swiss National Science Foundation for scientific projects.

Fig. 1: Bright field image of dislocations in a Fe-10Cr model alloy.

Fig. 2: Volume-rendered image of dislocations viewed along various directions. The green, blue, and red arrows respectively correspond to the principal crystalogrphic [100], [010], and [001] axes.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2426 Nonlinear intensity attenuation in bright-field TEM images and its influence on tomographic reconstruction of micron-sized materials

Yamasaki J.1, Mutoh M.1, Ohta S.2, Yuasa S.2, Arai S.1, Sasaki K.1, Tanaka N.1
1Nagoya University, Nagoya, Japan, 2JEOL Ltd., Akishima, Japan
yamasaki@esi.nagoya-u.ac.jp

    Currently, tomography in transmission electron microscopes (TEM) is widely applied to three-dimensional (3D) analyses of nanometer-sized and sub-micron-sized materials. One of the next methodological targets should be quantitative 3D reconstructions in which not only the shape but also the internal density are correctly reproduced. This is hindered generally by the nonlinearity between projection thickness and image intensity. In the case of mass-thickness contrast in bright-field TEM (BF-TEM) images, the ideal exponential attenuation of the image intensity with increasing thickness is disturbed by multiple scatterings.
    In the present study, the nonlinear attenuation in BF-TEM images was analyzed using amorphous carbon microcoils (CMCs) [1] shown in Fig. 1(a). Their well-defined shapes and compositional homogeneity are quite useful for estimating the mass-thickness [2]. The intensity attenuation was measured along the line in Fig. 1(b), which was taken by the high-voltage electron microscope in Nagoya University [3]. The results measured at the acceleration voltages of 400, 600, 800 and 1000 kV were converted to the plots of the electron transmittance T in Fig. 2. At a glance, T at any voltages seems to undergo the linear attenuation. However, the least squares fitted line for the data at 400 kV exhibits a considerably negative intercept value at zero thickness. Such nonlinear attenuation should induce failures in conversion from intensity to thickness and thus inhibits correct 3D reconstructions of the internal density.
    The influence of the nonlinearity on tomographic reconstructions was examined using a 360°-tilt sample holder, which was specially developed for eliminating the missing-wedge effect [2]. Figure 3 shows the results of the reconstructions from the tilt series taken at 400 kV and 1000 kV. Although the 3D shape of the CMC has been reconstructed well in both cases, the internal density is not uniform but has a gradient from the center at 400 kV. Moreover, there is a slight increase in the vacuum level in the interior of the coil. The inaccurate density reconstruction should result from the nonlinearity shown in Fig. 2. Judging from the plot for 600 kV electrons in Fig. 2, the linearity is valid at least down to lnT = −0.4, which corresponds to the electron transmittance of about 2/3. This information should be beneficial in practical tomography experiments because one can foresee quality of the reconstruction from the minimum transmittance in a single BF-TEM image prior to the tilt series acquisition.

References
[1] S. Motojima et al., Appl. Phys. Lett. 56 (1990) 321.
[2] J. Yamasaki et al., submitted to Microscopy.
[3] N. Tanaka et al., Microscopy 62 (2013) 205.

The authors are grateful to Mr. M. Ohsaki in JEOL Ltd. for discussions about designing the sample holder and System In Frontier Inc. for discussions on precise 3D reconstruction procedures. We also thank Mr. Y. Yamamoto and Dr. C. Morita of HVEM laboratory in Nagoya University for their assistance with the experiments. One of the authors (N.T.) thanks Dr. S. Motojima for useful discussions.

Fig. 1: Carbon microcoils. (a) SEM image (Microphase Co., Ltd.) and (b) BF-TEM image taken by the HVEM.

Fig. 2: Attenuation of electron transmittance T in BF-TEM images with increasing thickness.

Fig. 3: 3D reconstructions of the CMC from the tilt series of BF-TEM images taken at (a) 400 kV and (b) 1000 kV.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2565 Using transmission electron tomography to unravel the structure of hybrid active layers in non volatile memory elements

Girleanu M.1,2, Nau S.3, Sax S.3, List-Kratochvil E.3,4, Soliwoda K.5, Celichowski G.5, Grobelny J.5, Brinkmann M.1, Ersen O.2
1Institut Charles Sadron, UPR-22 CNRS, 23 rue du Loess, BP 84047, 67034 Strasbourg cedex 2, France, 2Institut de Physique et Chimie des Matériaux de Strasbourg, UMR 7504 CNRS-UdS, 23 rue du Loess BP43, 67034 Strasbourg cedex 2, France, 3NanoTecCenter Weiz Forschungsgesellschaft mbH, Franz-Pichler-Straße 32, A-8160 Weiz, Austria, 4Institute of Solid State Physics, Graz University of Technology, Petersgasse 16, 8010 Graz, Austria, 5Department of Materials Technology and Chemistry, Faculty of Chemistry, University of Lodz, Pomorska 163, 90-236 Lodz, Poland
girleanu@ipcms.unistra.fr

The interest in plastic electronics has grown significantly over the last twenty years with the development of new electronic devices, particularly solar cells and field effect transistors. More recently, non-volatile memories (NVMEM) based on hybrid materials gained particular interest [1,2]. Typically such devices consist of an insulating or semi-conducting layer sandwiched between a bottom ITO electrode and an upper metallic electrode (silver or aluminum). Bias application on these devices switches them from a low current OFF state to an ON state at high current. The switching mechanism involves the formation of "conducting filaments" [3] in the active layer but no direct observation of such filaments in a hybrid device was demonstrated so far. In hybrid layers, the formation of filaments is eased by the presence of metallic nanoparticles. It is therefore important to understand how such nanoparticles are dispersed in a polymeric matrix. In this study, we have analyzed the morphology of hybrid layers of polystyrene loaded with functionalized Au nanoparticles (NPs). TEM tomography allows to determine the 3D distribution of the nanoparticles in the devices. Evidence is found for the formation of large clusters of Au NPs that are rejected to the top surface of the active layer during the film spin-coating. Moreover, a fraction of Au NPs is dispersed in the PS film but only within an interfacial layer close to the bottom ITO substrate whereas the largest part of the PS matrix does not contain any Au NPs. It is shown that TEM tomography is a valuable tool to unravel the 3D structure of the active layers in NVMEM.

[1] J.C. Scott et al., Adv. Mater. 2007, 19, 1452.

[2] B. Cho et al., Adv. Funct. Mater. 2011, 21, 2806.

[3] S. Nau et al., Adv. Mater. 2014, DOI: 10.1002/adma.201305369.

Fig. 1: Cross-sectional view of the active layer of a hybrid non volatile memory element obtained by tomography. The active layer is made of a polystyrene matrix loaded with Au nanoparticles.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2603 Analysis of bainitic transformation process in Cu-Al-Mn Alloy by using an orthogonally arranged FIB-SEM for precise 3D microstructure analysis

Motomura S.1, Hara T.2, Nishida M.3, Omori T.4, Kainuma R.5, Asahata T.6, Fujii T.7
1Interdisciplinary Graduate School of Engineering Science, Kyushu University, Kasuga, Fukuoka 8168580, Japan, 2Advanced Key Technology Division, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 3050044, Japan, 3Faculty of Engineering Science, Department of Engineering Science for Elec-tronics and Materials, Kyushu University, Kasuga, Fukuoka 8168580, Japan, 4Department of Metallugy, Materiale Science, and Materials Processing, Grad-uate School of Engineering, Tohoku University, Aoba-yama 02, Aoba-ku, Sen-dai 9808579, Japan, 5Department of Metallugy, Materiale Science, and Materials Processing, Graduate School of Engineering, Tohoku University, Aoba-yama 02, Aoba-ku, Sen-dai 9808579, Japan, 6Hitachi High-Tech Science, Corp. 36-1 Takenoshita, Oyama-cho, Sunto-gun, Shizuoka 4101393, Japan, 7Hitachi High-Tech Science, Corp. 36-1 Takenoshita, Oyama-cho, Sunto-gun, Shizuoka 4101393, Japan
MOTOMURA.Shunichi@nims.go.jp

In order to investigate a 3D microstructure of complex materials precisely, we have developed an orthogonally-arranged FIB-SEM instrument which is specially designed to obtain a high-quality serial sectioning SEM image-set. The most characteristic point of this instrument is that the SEM and the FIB are arranged orthogonally. Fig. 1 shows the concept of this instrument. The advantages of this arrangement are that high-resolution and high-contrast SEM images can be obtained with low accelerating voltage such as less than 1kV because of the uniform background intensity and the short working distance (2mm). Furthermore, since the analytical instruments (EDS, EBSD and STEM, etc. ) can be located ideally , multiscale analyses can be performed in the single instrument. Fig. 2 shows the arrangement of apparatuses around a specimen viewed from the top along the SEM axis. We applied this technique on the analysis of the microstructure of bainite phase in non-ferrous noble metal based alloys. Bainitic transformation has both characteristics of a diffusionless and a diffusional transformation. Many studies on bainitic transformation have been conducted in various alloy systems such as steel, a noble metal (Cu, Ag, and Au) based alloy systems. However, the mechanism of the bainitic transformation has still been unclear. In this study, in order to reinvestigate the bainite in Cu-Al-Mn alloy, several samples with varying aging condition are prepared and observed by the orthogonally-arranged FIB-SEM and other recent SEM and TEM techniques. Fig. 3 show the SEM secondary electron (SE) image of the sample aged at 503K for 10 min. We can see some capillary binite precipitations. As a result of serial sectioning, however, it was revealed that the shape of bainite is plate-like crystal. The results of the analysis of the transformation process with these new techniques will be discussed.

Fig. 1: Schematic illustration showing the configuration of the SEM and FIB in the orthogonally arranged system.

Fig. 2: Schematic illustration showing the arrangement of apparatuses around a specimen.

Fig. 3: SEM SE-image of Cu-Al-Mn alloy aged at 503K for 10 min sample.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2620 Tune Stem-ADF/HAADF conditions improving dislocations tomography

Ibarra A.1, Cuestas C.1, San Juan J.2, Nó M. L.2, Arnaudas J. I.1,3
1Laboratorio de Microscopías Avanzadas, Instituto de Nanociencia de Aragón, Universidad de Zaragoza, 50018 Zaragoza, Spain , 2Dptos. Física Aplicada II y Física Materia Condensada, Fac. Ciencia y Tecnología, Universidad del Pais Vasco Apdo. 644, 48080 Bilbao, Spain, 3Dpto. Física de la Materia Condensada, Universidad de Zaragoza, 50009 Zaragoza, Spain
aibarra@unizar.es

The 3D geometry of dislocations and their interactions govern the properties of many materials. Mechanical and electrical properties are controlled by the density, distribution and dynamics of defects at the micrometer level: dominant slip mechanisms, Lomer-Cottrell locks of dislocations… Transmission electron microscopy (TEM) enables image individual dislocations and understands the relationship between the defect structure of materials and their macroscopic properties. Conventional electron micrographs are, however, two-dimensional (2D) projections of three-dimensional (3D) structures. A solution to this problem is electron tomography, which has mainly been used for reconstructing the shapes of samples using mass-thickness contrast [1]. However diffraction contrast has been considered too complex to be used in tomography due to the change of the contrast when tilt the sample.
Recently, dislocation tomography was attempted using weak-beam dark-field (WBDF) contrast [2]. A successful reconstruction was produced but the process was needlessly difficult. An alternative approach was tried by using annular dark field (ADF) STEM image [3], which is less sensitive to small specimen misalignments, making it more suited to materials research. In addition, ADF STEM images are less dynamical than WBDF images and consequently other strong features such as bend contours, thickness fringes and striped dislocation contrast affect to ADF-STEM image in a minor way than in WBDF condition.
The aim of this work is to tune the microscope conditions in order to enhance the contrast and resolution of the image avoiding some artifacts. The first results have been carried out in CuAlNi Shape Memory Alloys (SMA) because of the interest of dislocations as nucleation points for the martensitic transformation and consequently on the thermo-mechanical properties of the alloys. We can observe in Fig. 1 dislocations loops parallel to the sample surface (001), carried out by STEM-HAADF technique adjusting the conditions in the microscope to improve the contrast when tilts the sample. A tomogram of the observed dislocations has been taken each degree between +60 and -60 and reconstructed by Simultaneous Iterative Reconstruction Technique (SIRT). A picture of the final reconstruction is showed in Fig. 2. Large edge dislocations with [001] vector line and mixed ones with [101] direction have been analyzed.

References:
[1] Weyland M and Midgley P 2003 Ultramicroscopy 96 413
[2] Barnard J S, Sharp J, Tong J R and Midgley P A SCIENCE VOL 313 21 JULY 2006
[3] Sharp J, Barnard J S, Kaneko K, Higashida K and Midgley P A Journal of Physics: Conference Series 126 (2008) 012013

Acknowledgments
This work has been supported by Gobierno de Aragón, Grants E81 and Fondo Social Europeo. Authors thank Spanish Ministry of Economy and Competitivity, MICINN projects MAT2012-36421 and Consolider-Ingenio CSD2009-00013.

Fig. 1: Fig. 1: STEM-HAADF image, the conditions in the microscope have been adjusted to enhance the contrast and avoid some artifacts. We can observe a prismatic loop dislocation, lines [100] and [010] with b = (1/2)[001] in plane (001) and dislocations out of plane.

Fig. 2: Fig. 2: Snapshot of the 3-D reconstruction. We can observe different steps in the prismatic loop and check the out-of-plane dislocations. Edge dislocations with vector line [001] and mixed dislocations with [101] direction.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2637 Combined 3D characterization of porous zeolites by STEM and FIB tomography

Beltrán A. M.1, Przybilla T.1, Winter B.1, Knoke I.1, Machoke A.2, Schwieger W.2, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Department of Materials Science and Engineering, Friedrich-Alexander-Universität Erlangen-Nürnberg, Cauerstrasse 6, 91058 Erlangen, Germany, 2Lehrstuhl für Chemische Reaktionstechnik, Friedrich-Alexander-Universität Erlangen-Nürnberg, Egerlandstrasse 3, 91058 Erlangen, Germany
Thomas.Przybilla@ww.uni-erlangen.de

Conventional transmission electron microscopy (TEM) techniques are usually limited to acquire information of specimen in two dimensions. In many cases, a three-dimensional (3D) characterization is required, as is the case for porous materials used in catalysis, for which a detailed knowledge of the 3D-morphology, size distribution and interconnectivity of the pores is crucial.
In this work we compare the 3D characterization of micro- and macroporous zeolite particles used as catalyst support (Fig. 1) with the aim of obtaining a detailed analysis of its porous structure by two different and complementary techniques, namely electron tomography (ET) based on annular dark-field (ADF) scanning TEM (STEM) and focused ion beam (FIB) tomography. The size of the particles is in the range of several micrometres, so both techniques are applicable with their specific advantages and disadvantages. ADF-STEM ET has been performed in a FEI Titan3 80-300 microscope at 200 kV, with a spatial resolution of 2.1 nm and a convergence semi-angle of 5 mrad for an increased depth of focus. Tilt series have been acquired in a tilt range from -72° to 72° (1.5° tilt increments). For the 3D reconstruction, the simultaneous iterative reconstruction technique (SIRT) algorithm has been applied with 50 iterations. ADF-STEM ET has revealed a porous structure (Fig. 2a) with mostly interconnected pores. However, due to the missing wedge, artefacts in the reconstruction can be observed due to the lack of information for the highest tilt angles (Fig. 2b).
In order to reconstruct larger volumes FIB tomography is a well suited alternative. In this case, a FEI Helios NanoLab 660 Dual Beam FIB is used to perform the sequential milling and imaging with a depth resolution of 20 nm by using a milling voltage of 5 kV and beam currents in the range of 10 - 40 pA. Fig. 3 exemplarily shows the SEM image of one slice recorded during a FIB tomography series. The pores are nicely resolved. However, in this case the well-known curtaining effect leads to artificial striations in the shadow of pores.
The combination of both tomography techniques is well suited for a more complete 3D characterization of such medium-sized structures. Further work is focusing on the combined application of 360° ET (full tilt-angle range) and (subsequent) FIB tomography to one and the same particle and a detailed comparison of the reconstructed volumes. The application of 360° ET prevents missing wedge artefacts and, therefore, improves the quality of the reconstruction.

Financial support from the German Research Foundation through the Priority Program 1570 and the Cluster of Excellence EXC 315 “Engineering of Advanced Materials”.

Fig. 1: a) SEM and b) STEM images of the studied zeolites.

Fig. 2: a) Reconstruction (surface rendering) and b) ortho slice view of the reconstruction showing the artefacts due to missing wedge and interconnections between pores of a zeolite particle by ADF-STEM ET.

Fig. 3: SEM image (tilted view) showing one single slice of the zeolite particle shown in Figure 1a) during FIB tomography series (milling was performed from top to bottom). Please note that milling artefacts occur due to curtaining below the pores (indicated by arrows).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2720 Prospects of Electron Holographic Tomography at Atomic Resolution : Linear Reconstruction of Dynamic Scattering using Simulated Tilt Series

Krehl J.1, Lubk A.1, Lichte H.1
1TU Dresden, Dresden, Germany
Jonas.Krehl@triebenberg.de

Electron Holographic Tomography (EHT) has been shown to be a powerful tool for directly measuring electric potentials at medium resolution in three dimensions (3D). Although Electron Holography enables the retrieval of waves also at atomic resolution, it has not yet been possible to reconstruct the 3D information from a specimen with atomic resolution. That is because tomographic reconstruction schemes generally assume a linear transfer of specimen information (e.g. the potential) into the recorded signal. At medium resolution and orientation out of zone axis this is valid for the phase of electron waves (in the Phase Grating Approximation). However, as dynamic scattering becomes dominant at atomic resolution this linear approximation becomes invalid. The aim of this work was to examine and clarify the artefacts and errors that are created by this disparity of applying standard tomographic reconstruction methods. The obtained results are important for the development of tomographic schemes suited for atomic resolution.

To that end tilt series of a single gold nanocrystal were simulated and subsequently reconstructed; the thusly acquired electric potential is analysed and compared to the original specimen potential. Care has been taken to avoid low-index zone axes during tilt. Furthermore, special attention was paid to the influence of regularisation on the reconstruction. In order to characterise the reconstruction quality several generic defects have been simulated apart from a pure crystal: a lattice vacancy, a substitute atom and a shifted atom.

The results show that the neglection of Fresnel diffraction of the wave, while transmitting through the specimen, in standard tomography is the dominating artefact in the reconstructed potential. It leads to broadening of the reconstructed atomic potentials and a characteristic dip at their centre (see Fig. 1); both artifacts depend on the distance to the focal planes of the individual waves of the tilt series. Apart from that, the reconstructed atomic potential information is well located around the original atomic position, as shown by the well-localized effect of the atom removal / substitution in the reconstruction (see Fig. 2).

Consequently, linear reconstruction schemes are not disqualified per se at the atomic level: If they could be augmented to include the Fresnel Propagation they may become a viable method for the reconstruction of experimental data. However, the numerous problems of the experimental acquisition of atomic resolution tilt series will prove to be additional hurdles on the path to 3D atomic resolution.

This work is funded by the European Union (ERDF) and the Free State of Saxony via the ESF project 100087859 ENano.

Fig. 1: A stripe of a cross section through the reconstructed potential of a gold monocrystal (diameter about 4 nm). The simulated projections, used in the reconstruction, were at 1° intervals from -90° to 89° and for each orientation the focus was set to the object exit plane.

Fig. 2: Equivalent areas of a cross section through the reconstructed potentials of gold monocrystals. (A) exclusively made up of gold atoms, whereas (B) one atom replaced with a silver. Their difference (C) = (A-B) shows the influence of the different Z of Gold and Silver.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-2985 Autofocus method with high-definition TV camera for ultrahigh voltage electron microscope tomography

Nishi R.1, Kanaji A.1, Yoshida K.1, Kajimura N.1, Nishida T.1, Isakozawa S.2
1Osaka University, Osaka Japan, 2Hitachi High-Technologies Corporation, Ibaraki, Japan
rnishi@uhvem.osaka-u.ac.jp

The 3 MV ultra-high voltage electron microscope (UHVEM) H-3000 at Osaka University has capability of observation for micrometers' thick-sliced biological samples. This fea¬ture is suitable for tomographic three-dimensional imaging [1]. While taking obtain a tilt series of electron tomography, acquiring a hundred images, their image position and focus must be accurately aligned automatically. We proposed the Auto-Focus system using image Sharpness (AFS) [2] is suitable for acquisition of UHVEM tomography series [3]. The method is that values of image sharpness corresponding to defocus values become to be maximized as shown in Fig.1. To find the maximized image sharpness, we use fitting five points with a different defocus value to quasi-Gaussian function [3]. Acquisition of images by the slow scan CCD (SS-CCD) camera is good image quality but the acquisition time is taken more than one minute for one autofocus operation getting five defocused images.
In this study, we use a high-definition TV camera (HDTV camera; effective image area is 1.2k × 1k size) instead of the SS-CCD camera (4k × 4k) for fast acquisition of images. The HDTV camera captures one image for only 1/30 second. However, S/N of the image and the resolution are lower than the SS-CCD camera. To improve poor S/N, we integrated the images for 22 frames so that each image sharpness is enough to fitting. For lower resolution than SS-CCD image, we selected the defocus step made to be larger to discriminate difference of sharpness with each defocused image. By using HDTV camera for autofocus process, it took 6 seconds during one autofocus procedure, which became shorter by one order. It took 30 seconds to record one image by SS-CCD after autofocus and position alignment. We can obtain the series of 61 images during 30 minute. So, we successfully decreased total acquisition time of tomography series in half.

[1] A. Takaoka, et al, Ultramicroscopy 108 (2008) 230-238.
[2] H. Inada, et al, Proc. of 8APEM (2004) Kanazawa, Japan, 60-61.
[3] R. Nishi, et al, Microscopy 62(5)(2013) 515–519.

This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, under a Grant-in-Aid for Scientific Research (Grant No. 23560024, 23560786).

Fig. 1: Image sharpness S(x) to relative object lens current. The unit of the abscissa axis is manually adjusted minimum focusing step. Red solid circles are measured image sharpness and solid curve is fitted curve with the inset equation.

Fig. 2: Relative objective lens current change with a tilt angle during acquisition of tomography series. Starting angle is -60 degree and end angle is +48 degree. Two series were acquired in the same area. The deviation was smaller than the minimum step by manual.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-3102 Compressed-sensing EDX Tomography of Composite Nanowires

Yeoh C. S.1, Saghi Z.1, Midgley P. A.1
1Department of Materials Science and Metallurgy, University of Cambridge, UK
csmy2@cam.ac.uk

The introduction of high solid angle EDX detectors has seen a renaissance in interest in EDX-based electron tomography [1]. The confined geometry of the TEM however makes the EDX spectrum prone to artefacts especially for samples tilted to high angles or mounted on copper grids [2]. Here we examine these issues further by analysing an EDX spectrum-image tilt series of a composite nanowire structure.

The sample, a Cobalt phthalocyanine (Co Pc) zinc oxide core-shell nanowire, has been studied using an FEI Tecnai Osiris equipped with a large solid angle (>0.9 srad) silicon drift detector (SDD). The ZnO nanowire shell is polycrystalline and the level of detail reconstructed in the tomogram should allow comparative assessments of spatial resolution and uniformity of grey levels in the reconstruction.

A series of EDX spectrum-images together with STEM HAADF images was recorded at 5° tilt increments with equal acquisition time (Fig 1a). The total counts in each spectrum-image for each element of interest are plotted as a function of tilt in Fig 1b. As has been seen previously the counts at high angles increase dramatically. At low angles there is a small drop in counts up to around ±20°; this is likely to be attributable to geometric shadowing of the EDX detector by the specimen holder.

The trend in the count increase is typified by the Cu signal which we believe arises primarily through the excitation (via scattered electrons) of x-rays originating from the copper support grid. As the grid is tilted the area of copper in line of sight of the scattered electrons increases by a simple geometric factor equal to approximately 1/cos(tilt angle), resulting in an increase in Cu signal. This function is plotted in Fig 1b and the fit to the Cu signal is good.

We decided to use the Zn signal in the nanowire as a test case; the increase in the signal mirrors to a large extent that seen in the Cu (and Co). As a first approximation, in order to use this tilt data for a reconstruction, we normalised the Zn signal at each tilt increment, given each map had the same acquisition time. We used compressed sensing (CS) methods [3] to reconstruct the Zn tomogram and compared the result with a more conventional SIRT reconstruction (see Fig 2). Two advantages of CS reconstruction are apparent: i) the morphology, seen in the cross-sectional slice, is more faithfully reproduced (c.f. STEM HAADF reconstruction) and ii) greyscales within the ZnO phase are more uniform.

Further work is underway to confirm the origins of the x-ray signal variation with tilt in order to move towards a true quantitative compositional tomogram.

[1] Möbus et al. Ultramicroscopy 2003,96(3-4),433-451
[2] Slater et al. Proceedings of EMAG Conference 2013
[3] Leary et al. Ultramicroscopy 2013,131,70-91

We thank Ana Borras, ICMS, Seville, Spain, for providing samples, Rowan Leary and Pierre Burdet for their help including CSET and EDX spectrum-image processing. We acknowledge support received from David Brown and Sasol Technology UK, and also the European Union Seventh Framework Program under Grant Agreements: 312483–ESTEEM2 (Integrated Infrastructure Initiative - I3) and 291522-3DIMAGE.

Fig. 1: (a) 0° tilt STEM HAADF image of Co Pc-ZnO core-shell nanowire mounted on 5 nm C-film, the tilt axis is vertical, (b) Total counts summed over EDX maps for Co, Cu and Zn Kα peaks for varying tilt angle with function 1/cos(tilt angle) fitted to Cu distribution. Acquired with a probe current of 0.7 nA, 45x45 pixels and dwell time of 40 ms.

Fig. 2: (a) and (b): Slices through tomographic reconstructions of the Zn Kα peak (integrated over 8.49 – 8.79 keV): (a) 30 iterations SIRT reconstruction performed in Inspect3D, (b) CSET reconstruction. (c) Slice through tomographic reconstruction of STEM HAADF tilt series using CSET.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-3208 Electron tomography in the scanning electron microscope

Ferroni M.1, Migliori A.2, Morandi V.2, Ortolani L.2, Pezza A.2, Sberveglieri G.1
1Department of Information Engineering, University of Brescia and CNR-INO, Via Valotti 9, 25123 Brescia - Italy, 2CNR-IMM Section of Bologna, via Gobetti 101, 40129 Bologna, Italy
matteo.ferroni@unibs.it

The achievements in the implementation of electron tomography in the scanning electron microscope (SEM) and the potential of this 3-D imaging technique are summarized and discussed.
In SEM, the 3D imaging strategies consist in slice-and view assisted by FIB or microtomy, for the investigation of large specimen volumes. Differently the best resolution is pursued for relatively small volumes through TEM at high beam voltage.
The proposed implementation of electron tomography in the SEM is appropriate to the investigation at nanometric resolution of specimen volumes in the intermediate range, namely 2000 (w) x 2000 (l) x 200 (thickness) nm, as it combines the reconstruction algorithm with the signal corresponding to incoherently scattered electrons in the Scanning-Transmission (STEM) imaging mode. STEM imaging takes advantage from some peculiar characteristics of the experimental set-up [3]. This approach attains nanometric resolution and is free from aberrations caused by post-specimen imaging lenses; it also allows to collect transmitted electrons over a wide angular range [4][5]. The optimization of detector design and performance makes the contrast comply with local variations of composition or projected thickness. The bright-field component of the transmitted electrons can be effectively separated from the dark-field one, by varing the detection strategy [4].
The STEM mode preserves the monotonic variation of the signal with specimen thickness and meets the basic projection requirement for the 3-D analysis of nanowires, carbon based nanostructures or ultrastructures of biological specimens. In addition, the large value for the maximum detection angle ensures a complete detection of the scattered electrons, even in case of relatively large specimen thickness. In the case of tomography, these features are essential to maintain the proper image contrast when the specimen is rotated.
Fig. 1 shows the reconstruction of a ZnO crystalline nanostructure from a 110° tilt series at 1° step. ImageJ [6] with the TomoJ plug-in was used [7]. The disposition of the wires, their uniform section and the tapered termination are properly retrieved. Similarly, carbon-based tubes, filled with cobalt nanoclusters, were reconstructed as shown in Fig. 2. The tomogram from the STEM tilt series featuring compositional contrast, clearly shows the cobalt clusters inside the tubes.
These results demonstrate the potential of the method and optimization of the experimental set-up is under development to consolidate this technique in the set of 3-D methods of electron microscopy.

REFERENCES

1 Merli et al. Ultramic. 88 (2001) 139.
2 Morandi et al , JAP 101 (2007) 114917.
3 Morandi et al. APL 90 (2007) 163113.
4 http://rsbweb.nih.gov/ij/
5 Messaoudii et al, BMC Bioinf. 8 (2007) 288.

The authors acknowledge the finacial support from TomoSEM (F97I12000120007)

Fig. 1: Up-Left) SEM image of ZnO nanowires. The ROI is boxed. Up-Right) TEM shows the regular hexagonal shape and the pyramidal termination of the ZnO single-crystal nanowires. Bottom) Visualization of the reconstructed volume (4.5 micron large) corresponding to a primary magnification of 50.000.

Fig. 2: (Left and Center) STEM Bright- and Dark- field compositional images of carbon tubes filled with Co nanoclusters - Beam energy 30 keV. – (Right) Tomogram of the 
cobalt clusters inside the carbon tubes. The inset shows one Co particle, demonstrating the chemical sensitivity and the monotonically variation of the contrast upon tilting.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-3212 Phase Contrast Cryo-Electron Tomography with a New Phase Plate

Khoshouei M.1, Danev R.1, Gerisch G.2, Ecke M.2, Plitzko J.1, Baumeister W.1
1Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Martinsried, 82152, Germany. , 2Max Planck Institute of Biochemistry, Department of Cell Dynamics, Martinsried, 82152, Germany.
maryamkh@biochem.mpg.de

There has been more than 60 years working on the concept of combining phase contrast method with electron microscopy. Many laboratories are involved in development of the phase contrast electron microscopy to improve the performance of transmission electron microscopes and to reduce the beam damage to frozen-hydrated biological specimens [1].

There are different types of phase plates e.g. thin film, electrostatic, Photonic, magnetic and anamorphotic phase plates. Each method has its own pros and cons but overall the most successful phase plate has been thin carbon film Zenrike phase plate. Thin carbon film Zenrike phase plate has its own drawback such as charging effect and having a short lifetime [2]. Recently, we developed a new type of the phase plate, which has much longer lifetime and in general better performance.

The aim of the current work is development and applications of the phase contrast method. The work is being carried out at Max-Planck Institute of Biochemistry in Germany in collaboration with FEI in the Netherlands.

The structure of the whole vitrified worm sperm from Lumbricus terrestris species has been studied using new generation of phase plate. Sperm motility is a critical factor for fertilization and highly depends on the ultrastructure of different parts of the mature sperm [3].

This large and filiform mature cell comprises of acrosome (Fig.1a), nucleus (Fig.1b), mitochondria (Fig.1c) and flagellum (Fig.1d). In the pertinent literature, some research has taken place based on ultrastructure of worm sperm using tissue fixation or plastic sectioning but not in cryo. This research is carried out in cryo in combination with phase contrast method.


References:

[1] R. Danev, S. Kanamaru, M. Marko and K. Nagayama, Journal of Structural Biology 171 (2010), p. 174-181.
[2] R. Danev and K. Nagayama, Journal of Structural Biology 161 (2010), p. 211-218.
[3] A. Rolando et al, International Journal of Morphology (2007), p. 277-284.

We would like to thank Julia Mahamid for her assistance in Cryo-electron tomography.

Fig. 1: Slices of tomograms from Earth worm sperm with the new generation of phase plate. Acrosome (a), Nucleus (b), Mitochondria (c) and Flagellum (d) [Titan Krios 300kV, energy filter, direct detector, def: -500nm, mag:26000]                                                                                                                              

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-3225 Dedicated and innovative system for tomography in the Scanning Electron Microscope

Morandi V.1, Migliori A.1, Ortolani L.1, Pezza A.1, Maccagnani P.1, Masini L.1, Ferroni M.2, Sberveglieri G.2, Rossi M.3, Vittori-Antisari M.4, Vinciguerra P.5, Pallocca G.5, Del Marro M.5
1CNR-IMM Bologna Section, Bologna, Italy, 2Dept. of Information Engineering, Brescia University, Brescia, Italy, 3Dept. of Basic and Applied Sciences for Engineering, Sapienza University, Roma, Italy, 4Unità Tecnica Tecnologie dei Materiali, ENEA Casaccia, Roma, Italy, 5Assing S.P.A., Roma, Italy
morandi@bo.imm.cnr.it

For 3D non-destructive materials characterization, two are the leading tomography techniques: X-Ray Computed Tomography and Electron Tomography implemented in TEM operated in STEM mode [1]. The first one is undoubtedly the most important for industrial applications, providing resolution of few tens of μm, for cm- to mm-scale objects, while, the second one is of great interest in many research fields, and is capable of 3D reconstruction of sub-μm-scale objects with a resolution up to 0.24 nm [2].
In this paper we will highlight the implementation and the capabilities of an alternative electron tomography system in a SEM operated in STEM mode, composed by dedicated sample holder, STEM detector and analogue/digital signal processing system.
This system aims to cover the range between the previously mentioned techniques, taking advantages of the flexibility of the SEM platform, and of the resolution and image quality capabilities of the STEM mode implemented [3]. We will show that the STEM-in-SEM tomography approach opens up the perspective for the 3D analysis of volumes up to 100 μm3, such as nanowires, carbon based nanostructures or biological specimens, with resolution up to 10 nm.
Fig. 1 shows the main building blocks of the of the innovative tomographic acquisition system. The principal constraint for the success of tomography using STEM imaging is to maintain the monotonic variation of the mass-thickness contrast over the whole tilt range. Therefore, the use of STEM for tomography requires an acquisition system capable of collecting the transmitted electron over a large and adaptable collection angles. The detector geometry with five independent circular active sectors permits to optimize the efficiencies of signal collection, as a function of the tuneable specimen-detector distance, energy and beam current. The dedicated specimen holder with rotation capability ensures the eucentricity of the observed detail and a reduced missing wedge, which are fundamental for 3D reconstruction purposes (Fig. 1a). Moreover, the dedicated signal processing system (Fig. 1b) performs faster than conventional STEM systems in the acquisition of the tilt series, preserving the amplification, conditioning and managing of the signals as required by tomographic reconstruction.
Finally, we will demonstrate the potential of the method with examples of 3D reconstruction of micro and nano-structures. In Fig. 2 is reported the tomographic reconstruction and the 3-D rendering of a bundle of human skin collagen fibers, as obtained with the first release of our dedicated system [4].

References

1. P.A. Midgley et al. J. of Ultram. 223 (2006) 185
2. M.C. Scott et al. Nature 483 (2012) 444
3. V. Morandi et al. App. Phys. Lett. 90 (2007) 163113
4. TomoSEM Project: F97I12000120007

Fig. 1: (a) Setup inside the SEM-chamber: specimen-holder on motorized stage and STEM detector on a specific holder with variable working distance capability. (b) SEM platform and external mixed signal boards for amplification and managing of the signals.

Fig. 2: Acquisition steps for a complete tomogram of a collagen fibers bundle. (a) STEM image of a human skin thin section. (b) Tilt series acquisition. (c) Reconstructed tomogram with highlighted the ROI shown in the 3-D rendering in (d).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-3301 Improved Electron Tomography Image Reconstruction usingCompressed Sensing based Adaptive Dictionaries.

AlAfeef A.1, Cockshott W. P.1, MacLaren I.2, McVitie S.2
1School of Computing Science, University of Glasgow, Glasgow G12 8QQ, UK, 2SUPA School of Physics and Astronomy, University of Glasgow, Glasgow G12 8QQ, UK
a.al-afeef.1@research.gla.ac.uk

Electron tomography (ET) is an important technique for studying the 3D morphologies of nanostructures using the electron microscope. ET involves the collection of a series of 2D projections over a wide tilt range, which are subsequently aligned and processed to obtain a 3D volume reconstruction. It is well known that the quality of the reconstruction obtained using established algorithms is significantly affected by artifacts when the maximum angular range (the “missing wedge” artefact) or the number of acquired projections is limited. The reconstruction quality can be enhanced by including additional prior knowledge about the specimen in the reconstruction process and this is the key point of the compressive sensing (CS) family of techniques[1]. Such approaches have recently been applied to ET[2] and showed excellent results with higher fidelity and reduced artefacts even with subsampled datasets. Such features give CS major advantages for ET such as reducing total irradiation dose. The key prior knowledge employed in CS is that the signal (i.e. images), needs to be sparse in a transform domain. If a suitable transform enables a sparse representation of the dataset, then the original signal can be accurately reconstructed from a significantly smaller set of measurements than that required by the classical sampling theorem. As sparsity is a key requirement for an accurate reconstruction, researchers have investigated a range of sparsifying transforms, including for ET. In spite of their success in some cases, such transforms may not apply for all cases (nanostructured objects), and real signals are not always compressible (sparse) in such transforms. Also, some sparsifying transforms have a limited ability to remove artifacts. One common sparsifying transform is Total Variation (TV). It is only effective for those samples that are well described in terms of sharp, discrete boundaries. Other drawbacks of using TV include over-smoothing of fine structures and the inability to separate true structures from noise. Consequently, it is essential to seek superior transforms. In this work, we propose an alternative image reconstruction algorithm for ET that learns the sparsifying transform adaptively (in a similar manner to how our visual cortex processes natural images[3]). This new technique ET data with higher fidelity than analytically based CS reconstruction algorithms. The proposed technique is tested using a simulated phantom, which is known to be difficult to reconstruct using the popular CS-TV techniques, together with an experimental tilt series from a polymer solar cell.

References
[1] Donoho, D.2006. IEEE T Inform Theory, 52 1289–1306.
[2] Saghi, Z. et al.2011 Nano Lett. 11 4666–4673
[3] Olshausen, A.et al. 1996 Nature. 381 607–609

This research was supported by a Lord Kelvin Adam Smith Scholarship of the University of Glasgow.

Fig. 1: Reconstructed images from simulated under-sampled tilt series. A) CS-Phantom- The reference image of the numerical simulation. B) Reconstruction using TV based approach (CSTV) and C) proposed dictionary learning based approach (DLET) from noisy 28 projections.

Fig. 2: Quality curves of different reconstruction experiments using WBP, CSTV and DLET with higher degree increment steps between projections. Used metrics are: Peak Signal-to-Noise Ratio (PSNR), visual signal-to-noise ratio (VSNR) and Entropy Correlation Coefficient (ECC).

Fig. 3: Adaptively learned dictionary consisting of 100 atoms of 55 patches used in the DLET.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-3455 Use your smartphone to calibrate your TEM's goniometer

Wollgarten M.1, Stapel H.1, Garcia-Moreno F.1
1Helmholtz Zentrum Berlin für Materialien und Energie GmbH, Hahn-Meitner-Platz 1, 14109 Berlin, Germany
wollgarten@helmholtz-berlin.de

For reconstruction of tomographic data sets the precise knowledge of the experimental parameters is mandatory. Besides the incident intensity [1], the tilt angles have to be known precisely. In a recent paper, Hayashida and co-workers [2] measured the accuracy of a TEM goniometer and found a total deviation of 4° over an angular range of about 180°. This shows that the goniometer might be a source of flawed input data with serious consequences for the reconstruction work. Thus, calibrating the TEM's goniometer can be essential for high quality tomography work.

For calibration, a digital protractor can be used[2]. However, smartphones provide a number of sensors, among them accelerometers.

To determine the tilting accuracy of our LIBRA 200 FE TEM the acceleration sensors were used. An android application was written which reads out the acceleration raw data along the SP´s x-, y-, z-axis and stores it to a file.

While orienting the SP such that two axes show zero acceleration, the third axis is expected to be parallel to the earth´s gravity field vector. In these positions accelerations different from 9.81 m/s² where found. As a first approach, we slowly rotated the device to find for each axis the maximum and minimum acceleration value and used this pair to linearly scale the reading to the interval [-1,1]. In a second step, the three component acceleration vector was normalized to length 1.0 g (= 9.81 m/s²).

To measure the tilting angles of the TEM, the SP was mounted on a Fischione (model 2040) tomography holder. A self written script within Gatan´s Digital Micrograph was used to tilt the holder from -76° to 76° with 1° increments. Each tilting step was followed by a 10 seconds rest.

The acquired data set was processed as outlined above resulting in a stepped curve (tilt versus time).

The difference between the measured and the set value is plotted in Fig. 1.

An error with a periodicity of about 15° of the goniometer's worm gear is evident. However, whereas the reproducibility within each group is very good, an offset is observed between the groups which we attribute to a insufficient calibration of the accelerometers.

Nevertheless the sensitivity of the sensors turns out to be enough to detect tilting deviations significantly smaller than 0.1°. Therefore, we aim at improving the sensor calibration. This will allow for precise calibration of the goniometer.

References
[1] Wollgarten, M., Habeck, M., Micron, in print, doi: 10.1016/j.micron.2014.02.005 (2014).
[2] Hayashida, M., Terauchi, S., Fujimoto, T., Rev. Sci. Instrum. 82, 103706, doi: 10.1063/1.3650457 (2011).

Fig. 1: Measured deviation from the nominal goniometer tilt angle. The squared markers represent the configuration for which the smartphone was vertically mounted at a nominal tilt of zero degree. For the other set it was fixed in horizontal orientation.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-5857 3D imaging of Si FinFETs by combined HAADF-STEM and EDS tomography

Qiu Y.1, 2, Van Marcke P.1, Richard O.1, Bender H.1, Wilfried V.1, 2
1Imec, Kapeldreef 75, B-3001 Leuven, Belgium , 2Instituut Kern-en Stralings Fysika, K.U.Leuven, B-3001, Leuven, Belgium
Yang.Qiu@imec.be

In last few decades electron tomography is intensively studied to recover the 3D shape of a wide range of nano-scale materials. High angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) tomography attracts also increasing attention to study the morphology of semiconductor nano-devices with dimensions below 10 nm scale and a truly 3D morphology as instead of 2D projection images it provides 3D reconstruction with sensitivity along the beam direction. Recent advances of TEM systems with high brightness field emission gun (FEG) coupled with high count rate X-ray energy dispersive spectrometers (XEDS) opens the possibility to analyze the 3D volume both in imaging and chemical analysis modes.
Samples with dense Si fins (45 nm pitch) after Si etch, oxide fill and recess and thin epi-Si growth are explored. Pillar shaped specimens with various diameters are deposited on top of standard TEM grids and analyzed with Fischione conventional tomography holder. The work is done without gold markers, cf. fig 1, in a Titan cube 60-300 double aberration corrected system with SuperX EDS detector. Compared to lamella samples, the pillar configuration allows to increase the maximum tilt angle range from ~±65º to ±80º so that missing wedge effects are minimized. HAADF-STEM images are acquired in 1º steps and EDS maps each 5º. The STEM images are aligned and reconstructed by Inspect3D. Fig 2 presents the volume rendered 3D visualizations of the HAADF-STEM reconstruction and the orthoslices indicated in the volume. Si, SiO2 and FIB-damaged Si can be easily differentiated by the intensity. Moreover, Si and O X-ray maps are reconstructed separately with the same alignment. Fig 3 shows the superposition of both Si and O reconstructed volumes and the same corresponding slices as shown in fig 2. The orthoslices from the EDS reconstruction agree well to the HAADF-STEM reconstruction and also reveals the oxide grown on the Si fins. Compared to the 2D projection images, the orthoslices from the reconstruction indicate that the Si etch depth varies as indicated by blue arrows in fig 2c, 2d and fig 3c, 3d.
We show that the alignment can be done without marker tracking. The reconstructed volume based on HAADF images and EDS maps can bring useful fully interpretable information in composition and morphology unlike the 2D images that suffer from projection effects. Aberration corrected TEM improves the spatial resolution. Further analysis will involve using 360º tilt holder to fully eliminate the missing wedge artifacts and quantification of the elemental reconstructed volume, as well as application to next FinFET device processing steps involving gate and metallization steps.

Fig. 1: Fig. 1 a) HAADF-STEM images of the pillar shaped specimen (diameter around Si fins area ≈ 240nm) on standard copper grid zoomed in b) and c)

Fig. 2: Fig. 2 a) Volume rendered 3D visualization of HAADF-STEM reconstruction and its corresponding orthoslices in xy plane (b), yz plane (c) and xz plane (d) respectively

Fig. 3: Fig. 3 a) 3D chemical rendering of the Si fins and its corresponding orthoslices in xy plane (b), yz plane (c) and xz plane (d) respectively (gray : Si, yellow/red : SiO2)
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-10-P-6047 Multi-Axis Electron-Holographic Tomography

Sturm S.1, Wolf D.1, Lubk A.1, Lichte H.1
1Triebenberg Laboratory, Institute of Structure Physics, Technische Universität Dresden, Germany
Sebastian.Sturm@Triebenberg.de

Electron holography has proven to be a suitable method for measuring electrostatic and magnetic fields of nanostructures in the TEM. In electron holographic tomography (EHT) [1], the intrinsic fields can be mapped in all three dimensions, thus providing quantitative access to the true inner potentials and not only their projections. However, single-axis (SA) electron tomography (ET) often suffers from loss of information in one dimension, due to the limited tilt range of common tomographic specimen holders. In Fourier space, this limitation leads to an unsampled area, the so-called “missing wedge”. Performing multi-axis ET by combining several SA tilt series of the same object, one can minimize this missing volume. In case of Dual-Axis Tomography (DA) for example (two series acquired with tilt-axes perpendicular to each other) the volume can be reduced to a “missing pyramid” [2]. Fig 1 shows the corresponding missing volumes in Fourier space, depending on the available tilt range. For usual tilt ranges of about +-70°, DA is expected to provide a much better resolution in the third dimension than SA.
Another potential benefit of Multi-Axis EHT is the possibillity to reconstruct more then only one component of the B-vector field of magnetic samples.
Here we report on the development and implementation of Multi-Axis EHT.
For the necessary alignment of the residual displacements within the tilt series, the centre of mass for the projected potential at each tilt has been determined [3]. This method has two major advantages: all tilt series are inherently aligned with respect to each other, and the (projected) tilt axis is automaticaly alligned.
For tomographic reconstruction, a self-written software program has been developed. It is based on the concept of a weighted simultaneous iterative reconstruction technique (WSIRT [4]) but is especially adapted to the peculiarities of multi-axis geometries (Fig 2). Combining all tilt series within the process of a 3D back-projection, before application of a weighting filter in 3D-Fourier space and applying the iteration loop, takes into account the projections of all tilt series at once, instead of applying the weighting and iteration loop for each 2D-slice independently with separate SA tilt series.
As an example in Fig 3 the 3D potential of barium titanate nanoparticles [5], reconstructed by DA EHT, is compared with the two corresponding 3D potentials, reconstructed from only one of the two SA tilt series of the complete dataset. In z-direction the DA tomogram exhibits sharper edges then its SA counterparts.

[1] D Wolf et al. Ultramicroscopy, 110(5) (2010), 390-399

[2] P Penczek et al. Ultramicroscopy, 60(3) (1995), 393-410

[3] S Sturm. Diploma thesis, TU Dresden (2011)

[4] D Wolf. Dissertation, TU Dresden (2010)

[5] BTO nanoparticles provided by D. Szwarcman and G. Markovich (Tel-Aviv University).

[6] Funded by the EU (ERDF) and the Free State of Saxony via the ESF project 100087859 ENano.

Fig. 1: Fig. 1: Missing wedge (a) and missing pyramid (b) in Fourier space and information loss according to the corresponding volumes.

Fig. 2: Fig. 2: 3D-WSIRT algorithm reconstructing several tilt series synchronously.

Fig. 3: Fig. 3: Dual Axis EHT reconstruction of BaTiO3 nanoparticles. The measured object edges in the tomogram are compared with single axis reconstructions of the two data subsets.

↑ Go to the Table of content ↑

IT-11. Electron holography and lens-less imaging

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-11-IN-1935 Atomic Resolution Electron Diffractive Imaging and 3D

Zuo J. M.1, Lyu X. W.1, Gao W. P.1, Meng Y. F.1
1Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA
jianzuo@illinois.edu

Electron diffractive imaging promises sub-angstrom resolution imaging in 3D. Key to electron diffraction imaging is coherent electron diffraction using a parallel beam for selected area diffraction. Lateral coherent length as large as ~500 nm in FEG TEM has been reported [1].
The principle of phase retrieval is based on finding solutions based on a set of constraints. One of the constraints is the object support. The phase retrieval is carried out iteratively. For electron diffractive imaging, use of phases recorded in electron images at the beginning of iteration helps with a number of experimental issues[2]. Sub-Å resolution imaging has been demonstrated for a number of materials, including carbon nanotubes[3], CdS[2], CeO2 [4], Si [5] and TiO2 [6]. Accurate phase retrieval at nm resolution was recently demonstrated by Yamasaki et al [9]. Electron diffractive imaging can also be easily extended to medium and low energy electrons [7, 8]. Dronyak and his co-workers experimentally determined the morphology of a single MgO nanocrystal using the measure 3D Bragg diffraction peak [10]. 3D reconstruction resolving atoms was reported by Chen et al. [12] using experimental STEM image data [13].
Here we report a new method of 3D reconstruction using Fienup’s hybrid input-output (HIO) algorithm. Electron diffraction patterns are centered in 3D reciprocal space in a single axis tilt series. For the 3D sample data, the computational cost increase dramatically in order to achieve higher resolution result. We overcome this challenge by GPU-acceleration. Simulation.3D structure of Au icosahedron is reconstructed from calculated diffraction patterns including missing angles and noise in order to test the algorithm performance. Experimental implementation and its challenge will be discussed.

Reference
[1] S. Morishita, J. Yamasaki, N. Tanaka, Ultramicroscopy 129, 10-17 (2013)
[2] W. J. Huang, J. M. Zuo et al., Nature Physics 5, 129-133 (2009).
[3] J.M. Zuo, J. Zhang, W.J. Huang, K. Ran, and B. Jiang, Ultramicroscopy 111, 817-823 (2011).
[4] A. J. Morgan et al., Phys. Rev. B 87, 094115 (2013)
[5] S. Morishita et al. Applied Physics Letters 93(18), 183103 (2008)
[6] L. De Caro et al., Nature Nanotechnology 5, 360-365 (2010)
[7] O. Kamimura et al., Ultramicroscopy 110(2), 130 (2010)
[8] T. Latychevskaia et al., (2103), arxiv.org/pdf/1305.1897
[9] Yamasaki, J et al, Appl. Phys. Lett, 101, 234105 (2012)
[10] Dronyak R et al., Appl. Phys. Lett., 96 , 221907 (2010)
[11] Chen, C.-C. et al., Nature 496, 74 (2013)
[12] Rez, P. & Treacy, M. M. J. Nature 503, http://dx.doi.org/10.1038/nature12660 (2013)
[13] J. Miao et al., Nature 503, E1–E2 doi:10.1038/nature12661, (2013)
[14] This work is supported by DOE BES DE-FG02-01ER45923

This work is supported by DOE BES DE-FG02-01ER45923.

Fig. 1: Figure 1, left, a schematic illustration of sampling in reciprocal space as used in a single axis tilt series of electron diffraction patterns. Right, reconstructed 3D image using GPU accelerated HIO algorithm from simulated diffraction patterns of an icosahedron nanoparticle with 1.2 Å information limit and 25º missing wedge and simulated noise.
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-11-IN-5757 Towards atomic resolution and three-dimensional mapping of electrostatic potentials and magnetic fields using off-axis electron holography

Dunin-Borkowski R. E.1
1Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Forschungszentrum Jülich, D-52425 Jülich, Germany
rafaldb@gmail.com

The ability to achieve high phase sensitivity with close-to-atomic spatial resolution in off-axis electron holographic measurements is offered by the latest generation of ultra-stable transmission electron microscopes, which are equipped with high brightness electron sources and aberration correctors. In this talk, I will discuss recent developments in the quantitative and three-dimensional characterization of electrostatic potentials and magnetic fields with close-to-atomic spatial resolution using electron holography. I will begin by describing two complementary approaches that can be used to measure the electrostatic potentials and electric fields of electrically-biased metal needles as a function of applied voltage in the electron microscope. The phase shift can be analyzed either by fitting the recorded phase distribution to a simulation based on lines of uniform charge density or by using a model-independent approach involving contour integration of the phase gradient to determine the charge enclosed within the integration contour. Both approaches typically require evaluation of the difference between phase images recorded at two applied voltages, in order to subtract mean inner potential (and magnetic) contributions to the phase. I will then describe recent progress in the development of a model-based approach that can be used to reconstruct the three-dimensional magnetization distribution in a specimen from a series of phase images recorded using electron holography. The approach involves the projection of three-dimensional magnetization distributions onto two-dimensional grids to simulate phase images of three-dimensional objects from any projection direction. This forward simulation approach is then used in an iterative model-based algorithm to solve the inverse problem of reconstructing the three-dimensional magnetization distribution in the specimen from a tilt series of phase images. Such a model-based approach avoids many of the artifacts that result from the use of classical tomographic techniques. Finally, I will consider challenges associated with the use of chromatic aberration correction of the Lorentz lens in the TEM to achieve higher spatial resolution in magnetic characterization. When considering experiments aimed at the retrieval of weak phase shifts, it is important to remember that the sample must remain clean, that electron-beam-induced charging can contribute to the measured phase shift and that the quantitative interpretation of phase shifts measured from crystalline specimens can require comparisons with dynamical simulations. If time permits, I will conclude with recent progress in the application of off-axis electron holography to obtain results during ultrafast switching processes in situ in the electron microscope.

M. Beleggia, T. Kasama, V. Migunov, J. Caron, J. Ungermann, A. Kovacs, A.H. Tavabi, P. Diehle, A. London, T.F. Kelly, D.J. Larson and M. Farle are thanked for their valuable contributions to this work.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-1464 Split-illumination electron holography

Tanigaki T.1, Aizawa S.1, Park H. S.1, Matsuda T.2, Harada K.3, Murakami Y.1,4, Shindo D.1,4
1Center for Emergent Matter Science (CEMS), RIKEN, Saitama, Japan, 2Japan Science and Technology Agency, Saitama, Japan , 3Central Research Laboratory, Hitachi Ltd., Saitama, Japan, 4Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan
tanigaki-toshiaki@riken.jp

  Off-axis electron holography [1] has been used for observing microscopic distributions of magnetic fields, electrostatic potentials and strains at nanoscale level and for aberration-corrected electron microscopy by detecting phase shifts of electron waves. An off-axis electron hologram is formed by overlapping an object wave transmitted through a sample with a reference wave passed through the reference area. The inherent problem with this method is that the distance D between the object and reference waves, or the hologram width W, is limited by the lateral coherence length R or by the brightness of the illuminating electron waves.

 

  We solved this long-standing problem by developing split-illumination electron holography (SIEH). Experiments were performed using a 300-kV cold field emission transmission electron microscope (TEM) (HF-3300X, Hitachi High-Technologies Co.).

 

  In our SIEH (Fig. 1), we can illuminate a sample by using two highly separated and yet coherent electron waves without reducing the density of electron and form high-contrast holograms at regions far from the sample edge. The separation distance D can be controlled by a condenser biprism in the illuminating system. The fringe spacing s and the width W of the hologram can be independently controlled as in double-biprism electron interferometry [2]. Using SIEH, a fringe contrast of 50% can be attained even if the object wave is as far as 17 μm from the reference wave in the sample plane [3].

 

  Recently, in order to improve precision of phase measurement in SIEH, double condenser biprism type SIEH without Fresnel fringes was developed (Fig. 2) [4]. Since demanded phase shifts to be measured in nanoscale are becoming smaller and smaller, it is important to improve precision of phase measurements to broaden the applications of the off-axis electron holography. The developed methods are used for varieties of applications and will be used for revealing electromagnetic phenomena in atomic scale.

 

References:

[1] A. Tonomura, “Electron holography”, (Springer-Verlag, 1999).

[2] K. Harada et al., Appl. Phys. Lett. 84 (2004) 3229.

[3] T. Tanigaki et al., Appl. Phys. Lett. 101 (2012) 043101.

[4] T. Tanigaki et al., Ultramicroscopy 137 (2014) 7.

The authors are grateful to the late A. Tonomura for his valuable discussions. This research was supported by a grant from JSPS through the “FIRST Program”, initiated by CSTP.

Fig. 1: Schematic diagrams of electron-optical method and fringe contrasts of holograms. (a) Conventional electron holography. (b) Split-illumination electron holography in which a coherent electron wave is split into two coherent partial waves. (c) Measured fringe contrasts C of holograms as function of distance D between object and reference waves.

Fig. 2: Schematic diagram of double condenser biprism (CB) type split-illumination electron holography without Fresnel fringes (a) and holograms (b, c) and phase images (d, e) of charged latex particles. (b, d): double CB, (c, e): single CB.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-1669 Electrostatic potential of single-layer graphene measured using electron holography and ab-initio calculations

Chang S.1, Dwyer C.1, Nicholls R.2, Boothroyd C. B.1, Bangert U.3, Dunin-Borkowski R. E.1
1Ernst Ruska-Centrum, Forschungszentrum Juelich, Germany 1, 2Department of Materials, University of Oxford, UK 2, 3Department of Physics and Energy, University of Limerick, Ireland 3
shery.chang@fz-juelich.de

Graphene, a single-layer, hexagonally-coordinated carbon material has attracted huge attention in a wide range of fields due to its unique structural properties [1]. For example, it has found applications in electronic devices, energy storage, and electrocatalysis [2]. Characterisation of graphene imposes a requirement for high sensitivity to image a thickness of one atom. High-resolution TEM and ADF-STEM have been used to study the atomic arrangements and defects in graphene (and its related materials). The electrostatic potential of a single layer of graphene, a fundamental quantity of a materials structure property, is however less explored.

Here we use electron holography and density functional theory calculations to accurately measure the electrostatic potential of a single-layer of graphene. A Cs and Cc aberration-corrected TEM (Pico), operated at 80kV, was used to take holograms of graphene. The biprism voltage was set to be 175V, giving interference fringes of spacing 0.04nm. The graphene was grown using chemical vapour deposition on a SiO2 substrate and then transferred onto a TEM grid.

Figure 1 shows the phase of a typical area of the graphene sheet. It can be seen that there is a band near the edge of the graphene and some patches across the graphene sheet with larger phase shifts, which are typical features of silicon oxide and other hydrocarbon contamination left on graphene from TEM specimen preparation. The edges of the graphene sheet are more than a one-layer thick although patches of single-layer graphene can be found.

Figure 2 shows a region of single-layer graphene near the edge, after 2-hours of electron beam illumination to form a hole for the reference wave. The modulus of the Fourier transform of the complex wave-function (shown in the inset of figure 2) shows that high spatial resolution information is present in the phase (with the 1-210 reflection visible). The phase shift from the single layer graphene was measured to be 58 mrad (with respect to the vacuum) and the phase profile is shown in the inset of figure 2.

In order to compare the experimental measurement of the electrostatic potential with theory, both all-electron (Wien2K) and density functional theory calculations (VASP) were used. The theoretical calculation gives good agreement with the experimental measurement. Further implications from the theoretical calculations will be discussed in the presentation.

References:
[1] K. S. Novoselov et. al., Science, 2004, 306, 666 [2] Y. Sun et. al., Energy Environ. Sci., 2011, 4, 1113

Fig. 1: Phase of the holographam of a typical region of a graphene sheet.

Fig. 2: Phase of the hologram of the single-layer graphene. Inset (right) shows the modulus of the fft of the complex wave, and the inset (left) shows the phase profile from the vaccum to the graphene region. 
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-1798 Low-voltage electron diffraction microscopy of multi-layer graphene

Kamimura O.1, Dobashi T.1, Maehara Y.2, Kitaura R.3, Shinohara H.3, Gohara K.2
1Central Research Laboratory, Hitachi, Ltd., 2Division of Applied Physics, Faculty of Engineering, Hokkaido University, 3Department of Chemistry & Institute for Advanced Research, Nagoya University
osamu.kamimura.ae@hitachi.com

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-1905 Electron Holographic Tomography of Mean Free Path Lengths with nm-Resolution

Lubk A.1, Wolf D.1, Röder F.1, Lichte H.1
1Triebenberg Laboratory, TU Dresden, Dresden, Germany
Axel.Lubk@triebenberg.de

In off-axis Electron Holography a Möllenstedt biprism is introduced slightly above an intermediate image plane (image coordinates R) in the TEM to generate an interference pattern (“hologram”) with the following sinusoidal intensity distribution Ihol(R) = I0+I(R)+2μA0A(R)cos(QR+φ(R)). Here, I0, I, μ, A0, A, Q and φ are the reference intensity, conventional image intensity, contrast damping factor due to camera MTF and partial coherence, reference amplitude, reconstructed amplitude, carrier frequency and reconstructed phase. Reconstructed phases have been successfully analyzed in terms of various electrostatic and magnetostatic potential characteristics. In this contribution we will show how to tomographically reconstruct elastic and inelastic mean free path lengths (MFPL) from the concomitantly reconstructed conventional image intensity I and amplitude A. Starting points are the following exponential attenuation laws ln(A(R)/A0) = 0.5∫1/λA(r)dz and ln(I(R)/I0) = ∫1/λI(r)dz with corresponding attenuation coefficients λ-1 holding under out-of-zone axis conditions employed in medium resolution Electron Holography. Based on fundamental electron scattering principles we relate the λs to elastic and inelastic MFPLs correcting a serious misinterpretation preventing quantitative analysis in the past. Noting that the attenuation laws represent a Radon transformation when performed over a π-range of tilt angles, we then develop adapted tomographic reconstruction schemes. That involves dedicated normalization and regularization in order to reduce the influence of the generally low SNR. We demonstrate the MFPL reconstruction at a GaAs-Al1/3Ga2/3As core shell nanowire grown by low pressure metal-organic vapor phase epitaxy (MOVPE) method using colloidal Au nanoparticles (NPs) as metal catalysts. The tilt series was recorded at a Cs-corrected FEI TITAN TEM at 300 kV. Amongst other features its reconstructed potential reveals the core-shell structure as well a potential slope of yet unknown origin towards the Au tip (Fig. 1). The reconstructed elastic MFPL data (Fig. 1) also reveals the core-shell structure as well as a chemical composition variation from AlAs to GaAs in the tapered region thereby facilitating an unambiguous interpretation of the above noted potential slope in terms of chemical composition change. The inelastic MFPL (Fig. 1) on the other hand only vaguely hinds the existence of the core-shell which is a result of the strong delocalization of the dominating bulk plasmon excitation in the inelastic MFPL. Both elastic and inelastic MFPL agree very well with theoretic predictions.

We thank N. Lovergine (University of Salento, Lecce) for providing the GaAs/AlGaAs nanowire and T. Niermann for helping with recording the tilt series. The authors acknowledge financial support from the European Union under the Seventh Framework Program under a contract for an Integrated Infrastructure Initiative. Reference 312483 - ESTEEM2.

Fig. 1: Short compendium of 3D data of GaAs-AlGaAs core-shell nanowire including reconstructed potential and both, elastic and inelastic, mean-free-path-length (MFPL) cross-sections with corresponding linescans.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-2018 Mapping the number of graphenes for whole micron-size flakes by mean of low voltage electron holography

Castro C.1, Ortolani L.2, Arenal R.3,4, Monthioux M.1, Masseboeuf A.1
1CEMES, University of Toulouse, Toulouse, France, 2CNR IMM-Bologna, Bologna, Italy, 3Laboratorio de Microscopias Avanzadas, Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, Calle Mariano Esquillor, Zaragoza, Spain, 4Fundacion ARAID, Zaragoza, Spain
celia.castro@univ-rouen.fr

Synthesis of graphene with controlled properties is a utopian goal to reach if a multiscale analyzing method is not developed. One of the challenges is to allow accurate counting of monoatomic layers at the nanoscale over a flake area. Therefore, the number of layers as well as their stacking configuration have been related to optical and electrical properties of few-layer graphene.1
The recent developments of aberration-corrected transmission electron microscopes (AC-TEM) working at low-voltage (LV) conditions, which limit the knock-on damage, make possible to obtain atomic-resolution information on carbon-based materials.2-3
Counting edges of graphene stacks or peeling them under the electron beam provide very local information and cannot be applied to thick stacks. Quantitative thickness mapping can be obtained by combining high angle annular dark field imaging (HAADF) and electron diffraction. HAADF intensity is thickness-dependent and electron diffraction provides a calibration tool by determining the signal of a monolayer related to the TEM settings.4
Another way for mapping the number of graphene layers is LV transmission electron holography. The phase shift of electrons induced by the surface electrostatic potential is proportional to the thickness. This phase shift is intrinsic to the mean inner potential of the individual graphene layer and directly represents the local number of layers.5
In the present study, this method is emphasised in the I2TEM machine, a new AC-TEM dedicated to electron holography developed by Hitachi with CEMES Lab. We take advantage of much larger holograms, free of Fresnel fringes keeping irradiation damages limited and still achieving nanometer scale resolution thanks to the unique combination of a double biprism configuration, a second stage unit located upper in the column (Lorentz mode), and the LV with cold field emission. First maps of quantized graphene layers over micronic field of view will be presented. A variety of graphene flakes obtained from CVD or exfoliated graphitewill be analyzed through parameters including stacking type, sample preparation and artifacts of carbon based materials on TEM. Two examples taken from graphite flakes are provided as figure 1 and 2, in which the number of graphenes is large. The method is however sensitive enough for mapping thickness variations related to single graphene.
1 Koshino M. New J. Phys. 15 2013 15010
2 Sasaki T., et al. J. Electr. Microsc. 59 2010 S7
3 Suenaga K. et al. Nature, 468 2010 1088
4 Meyer J.C. et al. Solid State Comm., 1–2 2007 10
5 Ortolani L. et al. Carbon, 49 2011 1423

The authors acknowledge for financial support the EU-7Framework Program 312483-ESTEEM2, the "Conseil Regional Midi-Pyrénées" and the European FEDER within the CPER program, the Transpyrenean Associated Laboratory for Electron Microscopy (TALEM) and the French National Research Agency for the ANR-10-EQPX-38-01 and the ANR GRAAL. 

Fig. 1: a. Electron hologram of a multi-graphene and folded flake b. Phase contour map every 10 graphene layers. Reported values represent local measurements.

Fig. 2: a. Phase contour map every 10 graphene layers. Reported values represent local measurements. b. Profile of number of layers extracted from figure 2a.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-2326 Quantitative comparison of experimental and calculated image waves at atomic resolution

Niermann T.1, Lehmann M.1
1Institut für Optik und Atomare Physik, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany
niermann@physik.tu-berlin.de

Nowadays, the image wave function within the transmission electron microscope can be experimentally obtained by off-axis electron holography with high quality. This becomes possible by recent progresses made in instrumentation as-well-as in the reconstruction of holograms [1].

By matching experimentally obtained wave functions with specialized empirical models, several structural information, like atomic column positions, can be obtained with high precision, e.g. [2]. Instead of using such empirical models, we report here on quantitative comparisons of the full reconstructed wave function with the full image wave functions as calculated by the Bloch-wave method and the normally applied formulation of wave propagation by the objective lens. The matching between experiment and simulation was done by least-square fitting, i.e. using the complex ℓ2-norm pixelwise.

As experimental (nuisance) parameters the origin of the lattice, the absolute amplitude, the global phase as well as a linear phase change over the field of view were optimized. Beside the global phase, these parameters should, in principle, already be experimentally determined by empty holograms that were taken as reference. However, a linear phase change easily happens due to slight charging of the specimen. Furthermore, the global amplitude might change due to drifts of the illumination.

Optimized specimen parameters are thickness (including a linear change of thickness over the field of view), specimen tilt, and strain (linear change of column distance over the field of view). Considered imaging parameters were focal spread, two-fold astigmatism, axial coma, and defocus as-well-as a linear change of defocus over the field of view (corresponding to an inclined exit surface of the specimen).

Fig. 1 shows an experimentally obtained wave function from a GaAs-wedge recorded in [110] zone axis and the corresponding matched calculation. The left hand sides of Fig. 1 shows the comparison with aberrations applied to calculations. The right hand side shows the same comparison with the experimental wave function corrected by these aberrations, which exhibits the familiar dumbbell contrasts of GaAs. Furthermore, we report on the properties of the minimum and investigate the variances and correlations of the parameters (Fig. 2 and 3), which manifest in the shape of the minimum, and investigate specimen regions of different thickness.

[1] Niermann & Lehmann, Micron (2014), DOI: 10.1016/j.micron.2014.01.008
[2] S. Bals et al., PRL 96 (2006) 096101

Support by the DFG within SFB787 is kindly acknowledged.

Fig. 1: Comparison between experimental wave function and calculation. The upper and lower rows shows the wave functions in amplitude and phase, respectively. In the left four panels, the residual lens aberrations are applied on the model, in the four panels on the right hand side, the experimental wave is corrected for the lens aberrations.

Fig. 2: Evaluation of mismatch in dependence of specimen tilt. The tilt is expressed by the coordinates of the center of the Laue circle, zone axis is [110]. The orientation of the reciprocal space is indicated in Fig. 1. The minimum is close to (0.5,-0.5,1.5). (Here only a constant thickness/defocus over the field was assumed).

Fig. 3: The shape of the minimum of error function in dependence of defocus and thickness shows the slight correlation between both parameters. The fit is more sensitive to thickness than defocus, since the minimum is more steeper in the former case. (Here only a constant thickness/defocus over the field were assumed).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-2383 Coherent imaging beyond detector area and Abbe limit, towards atomic resolution

Latychevskaia T.1, Fink H-W1
1Physics Institute, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
tatiana@physik.uzh.ch

In a typical imaging experiment, data analysis relies on the recorded data during the experiment. In coherent imaging, this could be a hologram or a diffraction pattern obtained with light, electrons, X-rays, or any other type of radiation with wave nature. The achievable resolution is determined by the numerical aperture of the experimental setup limited by the size of the detector area.
We present a method that allows extrapolation of an experimental record beyond the area detected during the experiment by using intrinsic wave properties, namely their continuity in space. The amplitudes of the scattered waves are mapped onto the detector area and allow retrieval of the phase distribution. Once the complex-valued distribution of the scattered waves are retrieved, we extrapolate them to the full space extend, far beyond the detector area. As a result, the object reconstructed from such extrapolated interference pattern exhibit a higher resolution than provided by the initial experimental record [1-2]. The most attractive feature of our technique is that it does not require a new experiment as it can be applied to an already existing experimental record. The interference pattern can numerically be post-extrapolated to the full 2π hemi-sphere leaving the wavelength as the only resolution limiting factor.
An example is shown in Fig. 1, where a small section of a noisy interference pattern created by two point sources displaying less than three interference fringes is extrapolated to a much larger interference pattern. As a result, the two point sources can be resolved in the reconstruction. Fig. 2-3 show application of the technique to experimental optical in-line holograms and diffraction patterns.
The post-extrapolation technique is especially interesting when applied to electron or X-ray interference patterns as it can reveal atomic resolution from low-resolution images. Moreover, the extrapolation can also be applied to crystalline structures where diffraction patterns exhibit distinct Bragg peaks, such as graphene, see Fig.4. Diffraction patterns of graphene [3], could be extrapolated to reveal higher-order Bragg peaks and achieve enhanced resolution in the reconstruction, as shown in Fig.4.
We will present the application of this extrapolation method towards holograms and diffraction patterns of both, non-crystalline and crystalline structures, demonstrating its application for different types of waves: electrons, X-rays and THz waves, and we will also address the possibility of three-dimensional extrapolation.

1. Latychevskaia, T. and H.-W. Fink, Applied Physics Letters, 2013. 103(20): p. 204105.
2. Latychevskaia, T. and H.-W. Fink, Optics Express, 2013. 21(6): p. 7726-7733
3. Longchamp, J.-N., et al., Phys. Rev. Lett., 2013. 110(25): p. 255501.

The work presented here is financially supported by the Swiss National Science Foundation (SNF).

Fig. 1: Fig.1. Extrapolation of an interference pattern. (a) Fraction of an interference pattern created by two point-scatterers. (b) Extrapolated interference pattern. (c) and (d): Image of two point sources reconstructed from (c) the fraction of the diffraction pattern and (d) from the extrapolated interference pattern [1].

Fig. 2: Fig.2. Extrapolation of an in-line hologram. (a) Scanning electron micrograph of the sample and (b) its experimental optical hologram. (c) Extrapolated hologram. (d) and (e): Object reconstruction from hologram (b) and the extrapolated hologram (c), respectively. The insets shows the intensity profiles [2].

Fig. 3: Fig.3. Extrapolation of a diffraction pattern. (a) Scanning electron micrograph of the sample. (b) Piece of its optical diffraction pattern. (c) Extrapolated diffraction pattern. Reconstructions of the sample obtained from (d) the piece of diffraction pattern and (e), the extrapolated diffraction pattern [1].

Fig. 4: Fig.4. Extrapolation of a simulated diffraction pattern of a crystalline structure. (a) Graphene patch containing 1003 carbon atoms with two divacancies and (b) its diffraction pattern. (c) Extrapolated diffraction pattern. (d) and (e): Reconstructions of the sample obtained from (b) and (c), respectively.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-2391 Electron exit wave reconstruction in Gaussian basis: from high resolution image to diffraction pattern

Borisenko K. B.1, Allen C. S.1, Warner J. H.1, Kirkland A. I.1
1Department of Materials, University of Oxford, Parks Road, Oxford, OX1 3PH, UK
konstantin.borisenko@materials.ox.ac.uk

Accurate electron exit wave reconstruction can offer not only increased resolution and improved signal to noise ratio but it can also provide some 3D information about the sample in high resolution transmission electron microscopy (HRTEM). Restoration of the exit wave from experimental data usually involves collecting a series of images with varying focus (a focal series). The exit wave can be also recovered by recording overlapping diffraction patterns (DP) and applying specially designed iterative numeric algorithms in a ptychographic approach. Both these approaches require a number of images or diffraction patterns to be collected, which increases the total electron dose that the sample needs to withstand. Such an approach can be difficult to implement for radiation sensitive materials, due to possible sample damage during acquisition of focal or diffraction series.

In the present work we suggest and test an approach that in principle allows reconstructing exit wave from a single image or diffraction pattern for a weak-phase object. One of the obstacles to using only one image or diffraction pattern for the reconstruction is that in this case the number of variables in the exit wave to be determined is comparable to the number of data recorded. Presence of the aberrations of the objective lens and experimental noise further complicates the analysis and can lead to multiple or unstable solutions. By representing the electron exit wave in Gaussian basis we greatly reduce the number of variables needed to find the solution. This approach also has an advantage of analytic representation of the DP and also derivatives needed for the reconstruction process. We test the suggested method on experimental HRTEM image of graphene and simulated diffraction pattern of a carbon nanotube. The reconstruction algorithm involves solving an overdetermined system of non-linear equations with either numeric or analytic derivatives and appears robust to noise. We compare the reconstructed experimental exit wave phase from graphene with the multislice simulations.

We thank the financial support from the European Union under the Seventh Framework Program under a contract for an Integrated Infrastructure Initiative (Ref 312483-ESTEEM2).

Fig. 1: Experimental image of graphene a) and the reconstructed exit wave phase b).

Fig. 2: Simulated input exit wave and corresponding target diffraction pattern (represented as a logarithm of the square root of the diffracted intensity) and the reconstructed exit wave.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-2513 Observation of electric field using electron diffractive imaging

Yamasaki J.1, Ohta K.1, Sasaki H.2, Tanaka N.1
1Nagoya University, Nagoya, Japan, 2Furukawa Electric Co., Ltd., Yokohama, Japan
p47304a@nucc.cc.nagoya-u.ac.jp

    Information on electromagnetic fields in and around nanometer-sized semiconducting or magnetic devices is obtained from phase shifts of illumination electron waves. Although the most established method for phase imaging is presently off-axis electron holography, another choice could be electron diffractive imaging (EDI). In the method, a complex wave field is reconstructed from a diffraction pattern through numerical iterations under some constraints in real space. So far we have succeeded in reconstructions of atomic structures of crystals [1, 2] and thickness maps of wedge-shaped Si [3]. Figure 1 shows an example of the results, in which phase image of the transmission electron wave undergoing dynamical diffractions is reconstructed from the primary spot in a selected-area diffraction (SAD) pattern. In the present study, we performed reconstructing electric fields around MgO nano particles and a p-n junction in GaAs.
    A 200kV thermal field-emission TEM (JEOL: JEM-2100F) was used for taking SAD patterns with spatially coherent illumination. A post-column energy filter (Gatan: GIF tridium) was utilized for removing inelastic background intensity from samples and also for achieving a camera length large enough for precise sampling of low-angle scattering intensity. Energy-filtered bright field TEM images were also recorded to use as the real-space constraints. Figure 2 shows the phase reconstruction around MgO particles isolating in the vacuum. In Fig. 2(c), the electric field, which radiates from the particles positively charged by electron beam irradiation, is clearly observed. The reconstruction of the p-n junction in GaAs is shown in Fig. 3. Although the junction is invisible in the TEM image (Fig. 3(a)), the potential change deforms the primary spot (Fig. 3(b)), which results in visualization of the junction in the reconstructed phase image (Fig. 3(c)). The width of the depletion layer and the offset across the junction agree well with the doping concentration and measurements by off-axis electron holography.
    Unlike off-axis electron holography, the present method needs neither electron biprisms nor a vacuum area adjoining to the field of view of interest. The present study exhibits the future possibility that EDI will become an alternative to electron holography in some cases for observing electromagnetic fields relating to nanometer-sized materials.

References
[1] S. Morishita, et al., Appl. Phys. Lett. 93 (2008) 183103.
[2] S. Morishita, et al., AMTC Lett. 2 (2010) 116.
[3] J. Yamasaki, et al., Appl. Phys. Lett. 101 (2012) 234105.

We thank Dr. S. Morishita in JEOL Ltd. for valuable discussions.　The present study was partly supported by JSPS KAKENHI (Grant No. 21760026), The Public Foundation of Chubu Science and Technology Center, and Toyoaki Scholarship Foundation.

Fig. 1: Reconstruction of the phase image of the wedge-shaped Si crystal by electron diffractive imaging. (a) Bright-field TEM image, (b) the primary spot in the SAD pattern, and (c) the reconstructed phase image.

Fig. 2: Visualization of electric field (arrows) around the charged MgO particles. (a) Bright-field TEM image, (b) the primary spot, and (c) the phase image.

Fig. 3: Visualization of the p-n junction in GaAs. (a) Bright-field TEM image, (b) the primary spot, and (c) the phase image.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-2667 Caustics and diffraction from two oppositely biased metallic tips imaged in the coherent transmission electron microscope

Tavabi A. H.1, Migunov V.1, Dunin-Borkowski R. E.1, Pozzi G.2
1Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Forschungzentrum Jülich, Jülich, Germany , 2Department of Physics and Astronomy, University of Bologna, Viale B. Pichat 6/2, 40127 Bologna, Italy
a.tavabi@fz-juelich.de

The coherence of a modern field emission transmission electron microscope (TEM) allows fascinating electron-optical phenomena to be observed, such as the fine structure of umbilic foci outlining the caustic of an astigmatic probe, the hyperbolic umbilic catastrophe produced by a coma aberration function [1] and cusped fan-like structures in defocused images of electrically biased nanotube bundles [2].
Here, we study bright-field TEM images of two oppositely-biased metallic tips, which show a rich structure that depends sensitively on applied bias and defocus as a result of a combination of electrostatic field topography and electron-optical phase shift and is strongly reminiscent of the elliptic umbilic diffraction catastrophe that occurs when visible light is refracted by a water droplet with a triangular perimeter [3].
An FEI Titan 60-300 field emission gun TEM was used to study two metallic tips that had been thinned electrochemically and mounted in a specimen holder equipped with piezo-electric drivers and electrical contacts. The tips were placed in front of each other at a separation of ~1 micron and a potential difference of up to 130 V was applied between them. The positively charged wire was found to act like a terminating convergent electron biprism, producing an overlapping region of intensity containing two-beam fringes, whereas the negatively charged wire acted like a terminating divergent biprism. The combined effect of the fields resulted in a highly complex interference pattern, which is shown in Fig. 1 for a nominal defocus of 9.5 mm and a potential difference of 130 V. The overlapping region has a triangular structure that is similar to the elliptic umbilic diffraction catastrophe. Figure 2 shows this region at a higher magnification, with the hexagonal structure of the spots and their modulation by the two-beam biprism fringes visible.
We have interpreted the key features in these images by using a simple model of two uniformly and oppositely charged lines placed in front of each other [4]. The shapes of the tips can then be approximated by suitably choosing two of the equipotential ellipsoidal surfaces. The resulting simulations shown in Figs 3 and 4 are in good qualitative agreement with the experimental results.

References
[1] T. C. Petersen et. al., Phys. Rev. Lett. 110 (2013) 033901.
[2] M. Beleggia et. al., Appl. Phys. Lett. 98 (2011) 243101.
[3] M. V. Berry, J. F. Nye, and F. J. Wright, Phil. Trans. Roy. Soc. London, Series A, 291 (1979) 453.
[4] M. Muccini et .al., Ultramicroscopy 45 (1992) 77.

We are grateful to C. Dwyer for valuable discussions.

Fig. 1: Figure 1. Bright-field TEM image recorded at a defocus of 9.5 mm from two metallic needles that have a potential difference of 130 V between them.

Fig. 2: Figure 2. Central region of Fig. 1 displayed at a higher magnification, revealing spots that have a hexagonal-like structure.

Fig. 3: Figure 3. Simulated image corresponding to the experimental conditions used to acquire Fig. 1.

Fig. 4: Figure 4. Central region of Fig. 3 displayed at a higher magnification.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-2781 Towards electron holography of 3D magnetization distributions in nanoscale materials using a model-based iterative reconstruction technique

Caron J.1, Ungermann J.2, Dunin-Borkowski R. E.1, Riese M.2
1Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Research Centre Jülich GmbH, Jülich, Germany, 2Institute of Energy and Climate Research – Stratosphere (IEK-7), Research Centre Jülich GmbH, Jülich, Germany
j.caron@fz-juelich.de

Electron holography is a powerful technique for recording the phase shift of a high-energy electron wave that has passed through a thin specimen in the transmission electron microscope. The phase shift is, in turn, sensitive to the magnetic field and electrostatic potential in the specimen. Here, we introduce an approach that can be used to reconstruct the three-dimensional magnetization distribution in a magnetic specimen from a series of phase images recorded using electron holography. We generate simulated magnetic induction maps by projecting the three-dimensional magnetization distribution onto a two-dimensional Cartesian grid. We use known analytical solutions for the phase shifts of simple geometrical objects to pre-compute contributions to the phase from individual parts of the grid, in order to simulate phase images of arbitrary three-dimensional objects from any projection direction, with numerical discretization performed in real space to avoid artifacts generated by discretization in Fourier space without a significant increase in computation time. This forward simulation approach is then used in an iterative model-based algorithm to solve the inverse problem of reconstructing the three-dimensional magnetization distribution in the specimen from a tomographic tilt series of two-dimensional phase images. The model-based approach avoids many of the artifacts that result from using classical tomographic techniques such as filtered back-projection, as well as allowing additional constraints and known physical laws to be incorporated.

The authors are grateful to the European Research Council for an Advanced Grant.

Fig. 1: Illustration of the simulation process: The projected two-dimensional magnetization distribution is sub-divided into pixels which are represented by simple geometries (e.g., a disc). The contribution to the phase shift from every pixel is calculated in the form of two pre-computed components, which are oriented along the axes of the grid.

Fig. 2: a) Simulated magnetic phase shift of a uniformly magnetized sphere with a radius of 64 nm in a 128nmx128nmx128nm volume. The magnetization direction is indicated by the arrow. b) Corresponding magnetic induction map (20x phase amplified). The colors represent the direction and magnitude of the phase gradient, according to the color wheel shown.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-11-O-3089 Electrical charge quantification by electron holography

Gatel C.1, Lubk A.2, Pozzi G.3, De Knoop L.1, Snoeck E.1, Hytch M. J.1
1CEMES-CNRS and Université de Toulouse, 29 rue Jeanne Marvig, 31055 Toulouse, France, 2Institute of Structure Physics, Technische Universität Dresden, Mommsenstr. 9, 01069 Dresden, Germany, 3Department of Physics and Astronomy, University of Bologna, Viale Berti Pichat 6/2, 40127, Bologna, Italy
gatel@cemes.fr

The distribution and movement of electrical charge are fundamental to many physical phenomena, particularly for applications involving nanoparticles, nanostructures and electronic devices. However, there are very few ways of quantifying charge at the necessary length scale. Beyond providing structural and chemical information at the atomic scale, TEM can also determine the electrostatic field at the nanometer scale with a dedicated technique known as electron holography (EH).
We recently developed a new quantitative method to count the elementary charges with a precision of one elementary unit of charge using aberration-corrected EH [1]. We achieve this by applying at the nanoscale the elegance and power of Gauss’s Law to phase images extracted from holograms. This method provides direct access to the total charge enclosed by a given contour without assuming further details about neither the position of the charges within or outside the field of view nor the material investigated, contrary to a model-based approach where the whole electrostatic potential has to be computed. The extra sensitivity is achieved by the high signal-to-noise of aberration-corrected instruments and our new methodology. We performed a statistical analysis to reveal the relationship between the size of the contours and the precision of the charge measurement. A dedicated software has been developed for performing the charge evaluation based on line integration.
We will present different examples to illustrate the principle and the precision of this method. Among them, we will show the charge measurements on different MgO nanocubes where we determined a surface distribution of these charges with the corresponding value due to the surface states or adsorbates acting as charge traps (Figures 1 and 2). Another example will concern the in-situ field emission of a biased carbon cone nanotip (CCnT) [2]. The CCnT was placed to a defined distance from an Au-anode plate. We then ramped up the voltage between the nanotip and the anode from 0 to 95 V until the electric field around the tip was strong enough to allow the electrons to tunnel through the barrier and a field emission current could be acquired. During the voltage ramping and the field emission, holograms were recorded at each voltage step (Figure 3). After extracting the phase images, we applied this method to determine the numbers of accumulated charges and the charge density on different place of the tip as a function of the applied voltage (Figure 4). We will then discuss of these values, particularly the charge density at the beginning and during the field emission process.

[1] C. Gatel et al. Phys. Rev. Lett. 111, 025501 (2013)
[2] L. de Knoop et al. Micron (2014) - Accepted

The authors acknowledge the European Union under the Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative Reference 312483-ESTEEM2

Fig. 1: Reconstructed phase image of a MgO nanoparticle.

Fig. 2: By contour enclosed charge as a function of the short side a as indicated on the Figure 1; the linear fit of contours within the particle and the constant fit outside of the particle are indicated by red and black lines respectively.

Fig. 3: Hologram of a biased CCnT for field emission. In white is represented the contour used to count the number of charges in the enclosed area.

Fig. 4: Enclosed charge as a function of the length of the enclosed area and the applied voltage between the CCnT and the Au-anode plate.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1472 Restoration of Singularities in Reconstructed Phase of Crystal Image in Electron Holography

Li W.1,3, Tanji T.2,3
1Graduate School of Engineering, Nagoya University, Nagoya, Japan, 2EcoTopia Science Institute, Nagoya University, Nagoya, Japan, 3Global Research Center for Environment and Energy Based on Nanomaterials Science, Nagoya, Japan
liwei00jp@yahoo.co.jp

Off-axis electron holography, which can be used to measure the inner potential of a specimen from its reconstructed phase image,　has been widely used recently for characterizing materials. Under severe conditions such as in-situ observation in gas atmospheres, steep or large phase changes such as crystal lattice images or bulk specimen edges, and low signal-to-noise ratio conditions, abrupt reversals of contrast from white to black may sometimes occur in a digitally reconstructed phase image, resulting in inaccurate information. This phase distortion is due mainly to the digital reconstruction process and weak electron wave amplitude in some areas of the specimen. Hence, a posterior image processing that correct imperfections are indispensable for obtaining accurate phase information. In this study, we apply digital image processing to the phase image of a crystal for the restoration of such abrupt phase contrast changes, and obtain relatively accurate phase information for the crystal structure from the same electron hologram. Figure1 show the restoration of W8Nb18O69 structure phase images obtained by electron holography. The phase image (Fig.1) which is simply reconstructed and corrected the aberration of the microscope includes many singularity points as shown indicated by arrowheads. Restoring such singularity points improves the quality of reconstructed image as shown in Fig.2. Figures 3 and 4 show them in wire frame mode.Further work is required to be accomplished in the practice. The present method of phase image restoration for simulation with Poisson and Gaussian noises contributes to the correctly phase reconstruction of the hologram with quit weak electron-wave amplitude and noisy circumstance.

This study was partially supported by the Global
Research Center for Environment and Energy Based on Nanomaterials Science

Fig. 1: Directly reconstructed phase image.

Fig. 2: Phase image after restoration.

Fig. 3: Wire-frame image of Fig.1.

Fig. 4: Wire-frame image of Fig.2.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1514 Observation of the magnetic flux and three-dimensional structure of skyrmion lattices by electron holography

PARK H. S.1, Yu X.1, Aizawa S.1, Tanigaki T.1, Akashi T.2, Takahashi Y.2, Matsuda T.3, Kanazawa N.4, Onose Y.5, Shindo D.1, 6, Tokura Y.1, 4
1RIKEN Center for Emergent Matter Science (CEMS), Wako, Saitama, Japan, 2Central Research Laboratory, Hitachi, Ltd., Hatoyama, Saitama, Japan, 3Japan Science and Technology Agency, Saitama, Japan , 4Department of Applied Physics and Quantum-Phase Electronics Center (QPEC), University of Tokyo, Tokyo, Japan, 5Department of Basic Science, University of Tokyo, Tokyo, Japan, 6Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan
hspark@riken.jp

Topological spin textures have been attracting increasing interest for use in studying quantum magneto-transport and for possible application to spintronics. Skyrmions are particularly attractive for use as information carriers in memory and logic devices because of the emergence of spin transfer torque at extremely low current densities (~106 A/m2) [1]. Several challenges must be addressed before the skyrmion can be applied to actual devices. They include realization of skyrmions at room temperature, clarification of their three-dimensional (3D) structures, and fabrication of thin films containing skyrmions. Despite recent theoretical studies, the 3D structures of skyrmions remain elusive. Observing the 3D structures of skyrmions at the microscopic level is a prerequisite for applications of skyrmions to spin-electronic devices.

Electron holography, using the wave nature of electrons, provides opportunities for directly detecting and visualizing, in real space, the phase shifts of the electron waves due to the electromagnetic fields [2]. However, precise phase measurement of weak phase objects such as skyrmions is very challenging because procedures are needed for averaging the phase images and separating the electric and magnetic vector potentials. Nevertheless, the advantage of electron holography compared to Lorentz electron microscopy and magnetic force microscopy, under just-focused condition, makes it possible to visualize a quantized magnetic flux with nanometer resolution, in addition to determining its density in the vicinity of skyrmions. Here we investigated the 2D magnetic flux distributions (Fig. 2) of skyrmion lattices in helimagnet Fe0.5Co0.5Si thin samples with a stepped thickness as shown in Fig. 1 and estimated the 3D structures of the helical and skyrmion phases by using high-voltage holography electron microscopes [3].

References:

[1] N. Nagaosa, Y. Tokura, Nat. Nanotech. 8, 899-911 (2013).

[2] A. Tonomura, Electron holography, 2nd ed., (Springer-Verlag, Tokyo, 1999).

[3] H. S. Park et al., Nat. Nanotech., in press (2014).

The authors thank the late Dr. A. Tonomura for his valuable discussions. This research was supported by the grant from the JSPS through the “FIRST Program” initiated by the CSTP.

Fig. 1: Fig. 1. Lorentz micrographs. (a) A thin sample produced by FIB technique and its illustration. Thickness differences are represented by different levels of contrast.  (b) Thickness dependence of skyrmion lattices along sample with field cooling at 25 mT and 12 K. The scale bar is 300 nm.

Fig. 2: Fig. 2. Handedness reversal of magnetic flux flow with change in direction of applied field. (a,b) Surface plots of phase image. Sign reversal of phase shift with change in applied field direction is clearly visible. (c) Enlarged surface plot in vicinity of skyrmion. Red and white arrows represent direction of lines of magnetic flux.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1535 Strain fields in the vicinity of SiGe nanopyramids evidenced by focus series phase retrieval imaging

Donnadieu P.1, Neisius T.2, Liu K.3,4, Aqua J. N.4, Ronda A.3, Berbezier I.3
1SIMAP CNRS, Université de Grenoble Alpes, Domaine Universitaire BP 75 38402 Saint Martin d'Hères France, 2Université Paul Cézanne - Campus St Jérôme - 13397 Marseille France, 3IM2NP – CNRS, Université Paul Cézanne 13397 Marseille France, 4INSP - UPMC - 4 place Jussieu, 75252 Paris France
patricia.donnadieu@simap.grenoble-inp.fr

Nanoscale characterization is a major issue for the development of nanostructures. Local composition and morphology are at stake as well as strain within the nanoobjects and in the near substrate since strains might be monitoring self organization. While local chemistry can be known using EDS or EELS methods, efforts are still required to determine morphology and strains by TEM. Off axis holography is able to provide such information but remains a quite dedicated technique. Hopefully among the numerous forms of holography, focus series can be used to derive phase image. The advantage of this in line holography is to be easy to carry out in the course of a classical TEM study and to provide phase image on a large scale (up to several microns). The method consists in acquiring images at different defocus (-Δ, 0, Δ) and deriving the phase image using a filtering type processing that realizes the inversion of the phase transfer function.

In the present work, the focus series method has been applied to SiGe nanostructures deposited on a Si substrate. These SiGe nanostructures are expected to have a pyramidal shape and an average composition Si20Ge80. Figure 1 displays the focus series obtained on a plane view sample.Using the image analysis described in (1), phase image with very unrealistic values were obtained while the method had been validated by the measurement of inner potential on gold nanoparticles. Actually it appears that depending on the nanoobject scale, the image analysis may require further optimization. In particular strain fields are long range effect that prevents from applying filters appropriate for a nanoparticle assembly. The filtering process has to be modified: instead of a Gaussian edge filter, a Tikhonov regularisation was applied (i.e. a q2/(q2+a2)2 filter). The phase image (Fig.2) results from this modified filtering. The nanopyramids are characterized by a central positive phase with four lobes of negative phaseshift located at the corners of the pyramids. This negative phase cannot be explained by chemical segregation since the inner potential VGe is higher than VSi but by a strain effect changing the local inner potential. However the relation between strain and phase requires more investigation.

At this point, we wish to emphasize on the phase retrieval method optimization and on the qualitative information like location of strains and the first stages of self organization, namely the striking observation of non-random islands assemblies. This behaviour is representative of preferential nucleation of islands that is now investigated with computations of both total energy and kinetic nucleation barrier as a function of the distance between islands.

(1) P. Donnadieu et al, Applied Physics Letters 94, 263116, 2009

The French CNRS-CEA network METSA is acknowledged for providing the access to TEM facilities

Fig. 1: Focus series TEM images of SiGe nanostructures deposited on Si (plane view sample prepared by chemical polishing on the Si side). Images were recorded at defocus -200 nm, 0, 200 nm. This defocus value gives the best compromise between image contrast and spatial resolution. For Δ=200 nm, the spatial resolution on the phase image is better than 5 nm.

Fig. 2: Phase image retrieved from the focus series using the modified image processing. The SiGe nanopyramids show a positive phase central part with lobes of negative phase at the corner. Remarkably the negative lobes of neighbouring pyramids are close to contact along <200> directions or overlapping along <110> directions.

Fig. 3: Sketch of a cross section of a SiGe nanopyramid deposited on Si substrate summarizing the interpretation of the phase images. The positive phase central part is consistent with a Si20Ge80 pyramid while the negative phase lobes at the pyramid corners can be interpreted by local strains in the Si substrate.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1545 A thin film Zernike phase plate micro fabricated using MEMS technology.

Konyuba Y.1, Iijima H.1, Abe Y.2, Suga M.1, Ohkura Y.1
1JEOL Ltd., 2Yamagata Research Institute of Technology
ykonyuub@jeol.co.jp

An image of biological and polymer samples with conventional transmission electron microscopy (TEM) shows relatively low contrast without staining because they are composed of light elements such as carbon, nitrogen and oxygen. Zernike phase contrast TEM (ZPC-TEM) is an answer to this problem [1]. So far, many types of phase plates (i.e., thin film, electrostatic, magnetic, etc.) for TEM have been proposed [2]. Above all, a thin film phase plate has produced promising results [3]. Accordingly, ZPC-TEM attracts increasing attention to Cryo-TEM applications such as cryo-electron tomography and single-particle analysis, since a specimen in the field of interest is composed of light elements and preferred to be unstained to avoid artifacts.

The thin film phase plate has however encountered a few problems. The most crucial one is charging that makes its reliability poor and its lifetime short. The second problem is that the fabrication of the phase plate has been made by human hands. For these problems we have tried to produce a phase plate through a mass production process of micro fabrication using micro electro mechanical system (MEMS) technology. At the first onset, we made a sandwich plate composed of silicon nitride (SiN) coated with metallic titanium (Ti) on both sides to reduce the charging. The entire process is shown in Fig.1. In the process the center hole, which is the most essential part for phase plate performance, was also produced by electron beam lithography and dry etching process. By virtue of these processes, the center hole is close to a perfect circle as shown in Fig. 2. And constant hole diameters in the mass-produced phase plates were assured. The left part of Fig. 3 shows a ZPC-TEM image of carbon thin film (Quantifoil) obtained with a Ti/SiN/Ti thin film phase plate using a field emission TEM (JEM-2200FS) and the right part shows its Fourier transform. According to the Fourier transform, we confirmed that the contrast increases in a low spatial frequency region.

Reference
[1] Danev, R. and Nagayama, K., Ultramicroscopy 88, 4, 243 (2001).
[2] Glaeser, M, Rev. Sci. Instrum. 84, 111101 (2013).
[3] Dai, W. et al, Nature, 502, 707 (2013).

Fig. 1: The micro fabrication process of a Zernike phase plate made of a Ti/SiN/Ti thin film. Micro electro mechanical system (MEMS) technology was utilized for the process.

Fig. 2: An SEM image (secondary electron) of the center hole in the thin film Zernike phase plate of Ti/SiN/Ti. The hole diameter is approximately 200nm.

Fig. 3: A ZPC TEM image of a carbon film using the phase plate made of a Ti/SiN/Ti thin film and its Fourier transform.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1659 Factors affecting phase noise in off-axis electron holography

Boothroyd C. B.1, Dwyer C.1, Chang S.1, Dunin-Borkowski R. E.1
1Ernst Ruska-Centrum und Peter Grünberg Institut, Forschungszentrum Jülich, D-52425 Jülich, Germany
ChrisBoothroyd@cantab.net

The amount of noise in the reconstructed wave of an electron hologram depends on the visibility of the interference fringes, which in turn depends on the coherence and intensity of the incident beam and the stability of the microscope[1]. Here we investigate the dependence of phase noise on condenser lens strength for holograms taken on a 300kV FEI Titan with two biprisms and no specimen present. We use a lower biprism voltage of 150V with no extra lens giving a fringe spacing of 0.08nm. The magnification was 450k and round illumination was used for reproducibility. A 4s exposure time, giving negligible biprism drift, was used. We did not change the gun extraction voltage[2]. The gun lens, spot size (C1 lens) and intensity (C2 & C3 lenses) were each varied starting from gun 3, spot 3 and intensity set so the beam filled the screen at 160k magnification.

For each illumination condition a hologram was taken with no specimen present, reconstructed in the standard way and the mean intensity within the hologram overlap region, the fringe contrast and the standard deviation of the reconstructed phase measured. Here the mean intensity is used as a simple measure of the coherence of the beam, a lower mean intensity is associated with a higher coherence. Fig. 1a shows a plot of the fringe contrast vs mean intensity. As the beam is made more coherent (lower mean intensity) using any of the gun lens, spot size or intensity the fringe contrast increases, as expected. The phase noise derived from the same holograms is shown in fig. 1b, from which it can be seen that the lowest phase noise is for a mean intensity of about 700 counts. When the coherence is increased so as to reduce the mean intensity below 700 counts, the increased fringe contrast is offset by increased noise due to fewer counts [3].

It can be seen from both figures that it does not matter whether the coherence is increased by increasing the spot size or the intensity, the resulting phase noise is the same. Increasing the gun lens has almost the same effect except that the fringe contrast and the phase noise are slightly worse for the highest gun lens than for the same coherence set with either the spot size or the intensity. While this observation is to be expected for a perfect microscope with no instabilities[4], it is an important result to demonstrate experimentally.

We thus conclude that for adjusting the beam coherence it makes no difference whether the intensity or spot size are used and that using the gun lens produces only slightly higher phase noise.

[1] H Lichte, KH Herrmann and F Lenz, Optik 77 (1987) 135
[2] A Lenk and H Lichte, Proc EMC 2012 ed DJ Stokes and JL Hutchison (RMS, 2012) 515
[3] WJ de Ruijter and JK Weiss, Ultramic 50 (1993) 269
[4] H Lichte, Ultramic 108 (2008) 256

Fig. 1: (a) Hologram fringe contrast (Imax-Imin)/(Imax+Imin) and (b) standard deviation phase noise plotted against mean intensity within the overlap region for different settings of the gun lens, spot size and intensity.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1673 Observation of Fraunhofer Diffraction Pattern with Electron Vortex Beam using Fork-Shaped Grating with Various Opening Shapes

Harada K.1, Kohashi T.1, Iwane T.1
1Central Research Laboratory, Hitachi Ltd., Hatoyama, Saitama 350-0395, Japan
ken.harada.fz@hitachi.com

      Electron vortex beams are considered as probes for next-generation electron beam machines, especially for transmission electron microscopes (TEM), because the vortex beams carry intrinsic orbital angular momentum. We expect that it will bring with it an unprecedented measurement capability.

      In order to generate a vortex beam, we fabricated a fork-shaped grating [1] made from a Si3N4-membrane with a 200-nm-thickness by using a focused ion beam machine (FB-2100, Hitachi High-Technologies Corp.). The maximum grating size in one direction was 10 μm. Electron diffractions from the gratings were observed with a 300-kV field emission electron microscope [2]. The optical system was constructed for small angle diffraction with a camera length of 150 m and was similar to the twin-Foucault imaging system [3].

      During the experiment, we noticed the shape of the grating openings was superimposed on the ring of diffraction spot, which is a typical shape of the vortex beam. We also noticed the opening size is inversely proportional to the diameter of the diffraction ring. This phenomenon is considered to be due to a combination of Fraunhofer diffractions from the grating and the opening. Figure 1 shows electron micrographs of circular-fork-shaped gratings (left panels) and their electron diffractions (right panels). The smaller the grating opening size, the larger the diameter of the diffraction rings.

      The left panels of Fig. 2 show TEM images of fork-shaped gratings with triangular, square, and pentagonal openings. The right panels show electron diffractions whose spot-shapes reflect those of the openings.

      The combination of the fork-shaped grating and its opening allowed us to observe the rotational phenomena of the diffraction rings in the through-focus condition. Figure 3 shows diffraction patterns from a fork-grating with a diamond-shaped opening for three different focuses. The diffraction spots on the right are rotated in the opposite azimuth direction to those on the left. Figure 4 plots the rotation angles of the first, second, and third diffraction rings (spots) versus the defocusing distance, Δf. They show the lower order rings rotated more. The phenomenological picture of this rotation is consistent with the phase distribution of the vortex beam. The rotation itself can be explained by the Gouy phase [4, 5].

 

References:
  [1] B. J. McMorran et al., Science, 331, 192 (2011).
  [2] T. Kawasaki et al., Jpn. J. Appl. Phys., 29, L508 (1990).
  [3] K. Harada, Appl. Phys. Lett., 100, 061901 (2012).
  [4] G. Guzzinati et al., Phys. Rev. Lett. 110, 093601 (2013).
  [5] B. J. McMorran et al., Laser Science (Frontiers in Optics, San Jose, CA, USA, 2011), LWL 1.

Fig. 1: Ring diameter of diffraction is related to opening size. Contrast of the diffraction pattern of the botom panel is enhanced.

Fig. 2: Opening shape reflects the shape of each diffraction ring.

Fig. 3: Diffractions from grating with diamond-shaped opening is rotated by defocusing.

Fig. 4: Rotation angles of the first, second and third diffraction rings on both sides versus defocusing, Δf.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1676 Noise estimation for off-axis electron holography

Röder F.1, Lubk A.1, Wolf D.1, Niermann T.2
1Triebenberg-Labor, Technische Universität Dresden, 01062 Dresden, Germany, 2Institut für Optik und Atomare Physik, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany
Falk.Roeder@Triebenberg.de

Off-axis electron holography provides access to the phase of the elastically scattered wave in a transmission electron microscope by formation of an interference pattern at the image plane (hologram) encoding amplitude and phase therein [1]. Quantitative interpretations of experimental phase shifts retrieved from these holograms additionally require the knowledge of the noise transferred through the detection and holographic reconstruction process. Only for the special case that assumes homogeneous samples, uncorrelated Poissonian distributed noise and a special reconstruction aperture corresponding to the real-space reconstruction scheme [2], noise transfer formulas were derived by F. Lenz [3]. Here, we present a general noise transfer formalism for off-axis electron holography providing access to the final covariance matrix of amplitude and phase for arbitrary objects and reconstruction apertures. As an initial condition, we need the covariance matrix of the detected hologram, which is determined by the noise transfer properties of the detector. This covariance is estimated by the recently developed noise spread function (NSF) [4, 5] using suitable approximations. To the general reconstruction formulas, we apply error propagation describing the transfer of the estimated covariance matrix of the acquired hologram into the reconstructed amplitude and phase images. We show that our derived formulas agree with the Lenz model [3], if the corresponding conditions are assumed. For the general case, we experimentally verify the presented noise transfer formulas for two different cameras (Gatan 1024x1024 CCD cameras of model MSC 794 equipped with different scintillators) with and without object. We compare the theoretically determined noise with experimentally measured noise, which is obtained by statistical evaluation of various hologram series. In Figure 1 the variances of amplitude (a) and phase (b) of empty holograms in dependence on the size of the reconstruction aperture q0 are depicted for two different cameras and show good agreement between experiment (dashed red) and theory (solid blue) within the errors. The off-diagonals of the covariance matrices of amplitude (c) and phase (d) are represented for q0 = 1/2 qc (carrier frequency), which are mainly determined by the size of the reconstruction aperture. Also these results exhibit good agreement within the errors of the measurements.

[1] H. Lichte, M. Lehmann, Rep. Prog. Phys. 71 016102 (2008)

[2] M. Lehmann, E. Völkl, F. Lenz, Ultramicroscopy 54 (1994) 335-344

[3] F. Lenz, Optik 79 (1988) 13-14

[4] T. Niermann, A. Lubk, F. Röder, Ultramicroscopy 115 (2012) 68-77

[5] A. Lubk, F. Röder, T. Niermann, C. Gatel, S. Joulie, F. Houdellier, C. Magénd, M. J. Hÿtch, Ultramicroscopy 115 (2012) 78-87

The authors gratefully acknowledge critical and inspiring discussions with Prof. Dr. Hannes Lichte (TU Dresden, Germany). The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative - I3).

Fig. 1: Sample variances for amplitude (a) and phase (b) depending on reconstruction aperture size q0 (squares and circles for different cameras). The corresponding off-diagonals for q0 = 1/2 qc (carrier frequency) are shown in (c) and (d)  (Δm as distance between two detector pixels). Experimental values are in red and calculated in blue. 

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1720 Operating principles and practical applications of hole-free phase plate imaging

Malac M.1,2, Kawasaki M.3, Beleggia M.4, Pollard S.5, Zhu Y.5, Egerton R.2,1, Okura Y.6
1National Institute of Nanotechnology, Edmonton, Canada., 2Department of Physics, University of Alberta, Edmonton, Canada., 3JEOL USA Inc., MA 01960, USA., 4Center for Electron Nanoscopy, Technical University of Denmark, Lyngby, Denmark., 5Brookhaven National Laboratory, Upton, New York, USA., 6JEOL Ltd., Akishima, Tokyo 198-8558, Japan.
marek.malac@gmail.com

Ideal Zernike phase plate (PP) imaging in a TEM could, in principle, provide a quantitative measure of the phase shift induced by the sample directly from the measured image intensity for weak phase objects. In practice, the contrast transfer in PP imaging is far too complicated to allow for reliable quantification of image intensity. Furthermore, most samples are not weak phase objects. On the other hand, PP imaging, even at its current stage of development, allows to decrease the irradiation dose needed for a desired signal to noise ratio (SNR) [1], and to obtain qualitative information about samples that would otherwise require more complicated methods, such as electron holography, or complicated sample preparation. Here we present novel examples and discuss operating principles of the hole-free phase plate (HFPP) implementation [2] of PP imaging.

The HFPP implementation of PP imaging uses a uniform thin film placed in the back focal plane of the objective lens that charges due to primary beam-induced secondary electron emission. The steady-state electrostatic potential resulting from the charges phase shifts the diffracted beams relative to the direct beam resulting in strong phase contrast.

Figure 1 a) shows an example of a mouse kidney sample about 70 nm thick. Generally, biological sample of this kind are stained to obtain sufficient contrast. Instead, HFPP imaging allows to obtain sufficient contrast to study and measure lateral dimensions of the object without the need for staining. Compared to the standard bright field TEM (BFTEM) in Fig 1. b) the HFPP contrast is significantly higher. The good transfer of low spatial frequencies by the HFPP is seen in the power spectra (insets) where the HFPP in a) shows a bright region at low frequencies that is not present in BFTEM shown in b). Figure 2 a) shows an example of hard magnetic material (PrFeB) imaged using a HFPP. When compared to Fresnel imaging in Fig 2 b), new information can be obtained: the edge of the sample is clearly visible in a) while Fresnel fringes make it difficult to detect the sample edge in b). The HFPP image in a) also allow the fringing magnetic field extending into vacuum to be observed [3]. We have shown that phase plate imaging using the hole-free phase plate set up allows to establish low-dose phase contrast from samples that, when observed in Fresnel mode, would require staining. We have also shown that HFPP imaging on magnetic samples provides information that is not possible to obtain in Fresnel mode.

[1] M. Malac et. al. Ultramicroscopy 108 (2008), p. 126.

[2] M. Malac et. al., Ultramicroscopy 118 (2012), p. 77.

[3] S. Pollard et. al. Appl. Phys. Lett. 102 (2013), p.192401.

JEOL Ltd. NSERC and NRC supported this work. The samples used for Fig.1 were provided by N. Hosogi and H. Nishioka, both JEOL Ltd.

Fig. 1: .
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1723 Direct Observation of Magnetic Domain Walls by Lens-Less Foucault Imaging (LLFI)

Taniguchi Y.1, Matsumoto H.2, Harada K.3
1Advanced Microscope Systems Design Dept., Hitachi High-Technologies Corp., Hitachinaka, JAPAN, 2Application Development Dept., Hitachi High-Technologies Corp., Hitachinaka, JAPAN, 3Central Research Laboratory, Hitachi Ltd., Hatoyama, JAPAN
taniguchi-yoshifumi@naka.hitachi-hitec.com

       Lorentz microscopy, categorized as having Fresnel and Foucault modes, is as a practical technique for observing magnetic properties by using a transmission electron microscope (TEM). The Fresnel mode is more popular because it does not require any special equipment in its electron optical system. The Foucault mode, however, requires a magnetic-field shielding lens and an off-center objective aperture. Although this mode can visualize magnetic domains under in-focus conditions, it has been considered impractical. Recently, however, a novel Foucault imaging method, named lens-less Foucault imaging (LLFI) [1], was developed for conventional TEM without any special equipment.

       Figures 1(a), (b) and (c) show the optical systems of different modes of LLFI in a 300-kV field emission TEM (HF-3300; Hitachi High-Technologies Corp.). Figures 1(a) and (b) are for observing Foucault images and (c) is for small angle electron diffraction (SAED) pattern. The objective lens was switched off and the electron beam was focused with a condenser lens to the crossover on the selected area (SA) aperture plane. The SA aperture was used as an angular aperture selecting for appropriate Foucault images, and the focal length of the magnifying lens was changed in order to observe the Foucault images and diffractions. The irradiated area on the specimen was set by selecting an appropriate diameter for the condenser aperture. Figure 1(d) is an example of SAED of a 90° ferromagnetic domain structure of La0.75Sr0.25MnO3 (LSMO). The observation was done with a camera length of 150 m. White circles in the lower panel of Fig. 1(d) indicate the diameters and positions of the SA aperture for the LLFI observations and lowercase letters of individual circles correspond to Foucault images in Figs. 1(e) – (l).

       Figures 1(e) – (h) show Foucault images of each dispersed deflection spot (see in Fig. 1(d)). The magnetization structure among the 90° domains is directly visualized. Figures 1(i) – (l) show Foucault images of the magnetic domain walls in the same area as Figures 1(e) – (h). Figure 1(i) shows 90°/180° domain walls imaged with four streaks including the optical axis by using an SA aperture 5 μm in diameter. Figure 1(j) is an image of 180° domain walls made using an SA aperture 3 μm in diameter. Figure 1(k) and (l) are 90° domain walls imaged with individual streaks from the upper and lower positions on the optical axis.

       The LLFI method is advantageous for observing not only magnetic domains and domain walls but also SAEDs less than 2×10-5 rad. In combination with high-angular-resolution imaging, it will open the way to developing new applications in Lorentz microscopy.

 

References:
 [1] Y. Taniguchi et al., Appl. Phys. Lett., 101, 093101 (2012).

 

The authors would like to thank Prof. S. Mori of Osaka Prefecture University for supplying the LSMO specimens and valuable discussion about the materials.

Fig. 1: (a) Optical system for domain observation, (b) for domain-wall, (c) for small-angle electron diffraction, (d) SAED from 90°/180° domain structure of LSMO, (e)–(h) Foucault images of 90°/180° domains from each single deflection spot, (i) –(l) Foucault images of domain walls from streaks, (j) 180° domain walls, (k) and (l) 90° domain walls.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1725 Twin-Foucault Imaging for Observing 180° Domains in Magnetic Materials

Harada K.1
1Central Research Laboratory, Hitachi Ltd., Hatoyama, Saitama 350-0395, Japan
ken.harada.fz@hitachi.com

     The conventional Foucault method can visualize magnetic domains under in-focus conditions, but it cannot observe the whole region irradiated by the incident electron beam at once. This is because an off-center objective aperture filters out one of the two deflected beams from materials that have 180° magnetic domains whose magnetizations are in opposite directions to one another. To solve this problem, the twin-Foucault imaging (TFI) method uses an electron biprism instead of an objective aperture to obtain two Foucault images simultaneously [1].

     Figure 1(a) shows the optical system for the TFI method using a 300-kV field-emission TEM. The electron biprism was installed between the objective and the first magnifying lenses. When a negative potential is applied to the filament electrode of the biprism, the two electron beams are deflected in dispersive directions away from the optical axis and form two individual Foucault images on the image plane simultaneously. Figure 1(b) shows small angle electron diffraction (SAED) spots from 180° magnetic domains and the shadow of the biprism.

     Figure 1(c) shows micrographs of La0.825Sr0.175MnO3 (LSMO) film taken by a CCD camera. The ordinary electron micrograph in the middle panel was divided into two series (upper/lower) of Foucault images with reversed contrast by applying negative/positive potentials to the biprism of ± 50 V and ± 100 V. The winding fringes in the central parts of these images are bend contours, and the vertical black and white stripes in Figs. 1(c) correspond to individual 180° magnetic domains. The Foucault images are switched by the polarity of the applied potential to the biprism.

     The TFI method can visualize magnetic domain structures. Figure 1(d) shows examples of processed images of the domain structures. The left panel is an image made by subtracting the right image from the left, i.e., l−r, and the right panel is an image made by subtracting the left from the right, i.e., r−l, in the uppermost panel of Fig. 1(c). In Fig. 1(d), the magnetic domain structures are clearly visible in the reversed contrast in images, whereas the bend contours and other contrasts have been eliminated.

     The TFI method can be used to extend the conventional Fresnel method when the applied potential to the biprism is switched off and the specimen is observed in defocused conditions. Figure 1(e) shows over- (left panel) and under-focused (right panel) Fresnel images and in-focus micrograph (center panel).

     The TFI method not only has the advantages described here; it can also be used, for example, to observe the dynamics of imaging domain switching. It will lead to new applications in Lorentz microscopy.

 

References:
[1] Harada, K., Appl. Phys. Lett., 100, 061901 (2012).

The authors would like to thank Prof. S. Mori of Osaka Prefecture University for supplying the LSMO specimens and valuable discussion about the materials.

Fig. 1: (a) Optical system for TFI method, (b) SAED pattern with shadow of a filament electrode of a biprism, (c) twin-Foucault images with different potentials from −100 V to 100 V, at intervals of 50 V, (d) 180° domain structures made by subtraction processing of twin-Foucault images, (e) Fresnel images and in-focus micrograph (center panel).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1727 Observation of Magnetic Field by Combination of Electron Tomography and Holography

Tsuneta R.1, Ikeda M.1, Ono S.1, Yamane M.1, Sugawara A.1, Harada K.1, Koguchi M.1
1Central Research Laboratory, Hitachi Ltd., Hatoyama, Saitama 350-0395, Japan
ken.harada.fz@hitachi.com

     Electron tomography has developed in the last decade with the progress of electron microscopy and the development of algorithms for the Radon transformation and image processing with interpolation [1]. On the other hand, electron holography is one of the standard techniques for observing phase maps of electron waves. The combination of tomography and holography has also led to elucidation of the distributions of the mean inner potential of materials [2]. In the case of the magnetic field, the component By parallel to the rotation axis (see Fig. 1(a)) can be calculated from the phase shift Δφy by using the conventional tomography algorithm [3], but the other two components (Bx, Bz) perpendicular to the rotation axis cannot be calculated, because they are mixed together with a rotation angle θ. In the case of a magnetic field in free space, however, the phase shift Δφθ projected to the optical axis can be described simply [4], for which Bx and Bz can be separated.

     Figure 1(a) is a schematic diagram of the tomography/holography experiment. A thin magnetic pillar made of C/CoFeB/SiO2 was prepared with a focused ion beam machine (see Fig. 1(b)) and mounted on the 360° rotation axis of the specimen holder. Two holograms projected from the front surface and back surface of the material were processed in order to divide the magnetic and electric fields around the specimen. Figures 1(c) and (d) are examples of reconstructed interferograms of the magnetic and electric fields. Holograms made with a double-biprism interferometer [5] for tomography were recorded every 10°. Eighteen phase maps corresponding to the projected magnetic field were reconstructed from thirty-six holograms. The phase maps were processed into a three-dimensional (3-D) magnetic field distribution in free space by using a modified tomography algorithm.

     Figure 2 shows the reconstructed 3-D magnetic field distribution on the plane perpendicular to the pillar-shaped magnet. Figure 3 shows the reconstructed magnetic field in three planes parallel to the pillar.

     To improve the precision and resolution of 3-D reconstructions of the magnetic field, especially inside the material, the two components of the magnetic field should be measured independently. A dual-axis 360° rotation specimen holder has already been developed for this purpose [6]. The results of an experiment using this dual-axis holder will be reported soon.

 

References:
[1] S. Ono et al., Appl. Phys. Express, 4, (2011) 066601.
[2] G. Lai et al., Appl. Opt., 33, (1994) 829.
[3] G. Lai et al., J. Appl. Phys., 75, (1994) 4593.
[4] H. Shinada et al., IEEE Transaction on Magnetics, 28, (1992) 1017.
[5] K. Harada et al., Appl. Phys. Lett., 84, (2004) 3229.
[6] R. Tsuneta et al., Kenbikyou, 48, (2013) 205 (in Japanese).

The authors would like to thank Mr. H. Hasegawa of the Central Research Lab., Hitachi Ltd. for supplying the C/CoFeB/SiO2 specimens and valuable discussion.

Fig. 1: (a) Experimental setup for tomography/holography, (b) scanning ion micrograph of C/CoFeB/SiO2 pillar, (c) reconstructed interferogram of magnetic field, (d) interferogram of electric field.

Fig. 2: Reconstructed 3-D magnetic field distribution in one horizontal plane.

Fig. 3: Reconstructed 3-D magnetic field distributions in three vertical planes (a, b and c).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1784 Accumulation Processing in Reconstruction for Electron Holography

Kasai H.1, Harada K.1
1Central Research Laboratory, Hitachi Ltd., Hatoyama, Saitama 350-0395, JAPAN
hiroto.kasai.qm@hitachi.com

 

     One of the long standing problems affecting electron holography, the lateral coherence limitation, has been solved by using the split illumination method with a specially customized TEM. The customized TEM has one or two biprisms in a condenser optical system [1, 2]. Conventional holography TEM, however, does not have any biprisms in the condenser system. The problem, therefore, remains unsolved in practice. In order to solve the problem by using a conventional TEM, an "accumulation processing" method was developed.

     Figure 1(a) explains the concept. The target sample is in the object region (n) far from the specimen edge, and the distance from the region (n) to the vacuum region (ref) exceeds the coherent length R. The first hologram is recorded with two waves in region (n) and region (n-1); the second hologram is also recorded with these waves but in regions (n-1) and (n-2). The specimen is shifted perpendicular to the hologram through a distance W that is equal to the hologram's width. The last hologram is recorded with the waves in region (1) and the reference region (ref).

     The reconstructed phase distribution Δηn is described as the difference between the phase distributions ηn of the waves: Δηn = ηn-ηn-1. After reconstruction of all of the holograms, the reconstructed phase distributions are summed one by one, i.e., ΣΔηn =Σ(ηn-ηn-1) =ηn-ηref= ηn. The summed result is the same as the phase distribution reconstructed from the hologram recorded in region (n) and the reference region (ref). This means that the problem is solved in principle.

     Figure 1(c) shows a conventional phase map corresponding to the magnetic lines of force from the apex of the magnetic force microscopy (MFM) tip (see Fig. 1(b)). When the hologram width was changed, the density of the magnetic lines of force changed. This means that the magnetic field from the tip leaked into the reference region. Thus, it is important to keep the reference wave far from the magnetic material.

     Figure 1(d) shows the phase distribution reconstructed by the accumulation method. All of the regions were reconstructed by using just one reference wave and put in order from (n) to (1). The reconstructed area was seven times as wide as the possible area reconstructed by conventional holography. Accordingly, magnetic lines of force not only from the apex of the tip, but also from other areas can be visualized.

     Figure 1(e) shows the subtracted phase distribution processed from (d). A comparison of the results in Figs. 1(c), (d) and (e) clarifies that conventional holography visualizes differentiation of the magnetic field around the tip.

 

References:
[1] T. Tanigaki et al., Appl. Phys. Lett., 101, 043101 (2012).
[2] T. Tanigaki et al., Ultramicroscopy, 137, 7 (2014).

Fig. 1: Fig. 1 (a) Principle of "accumulation processing", (b) SEM image of MFM tip, (c) magnetic lines of force (X 4) from the apex of the tip reconstructed by the conventional method, (d) magnetic lines of force (X 1) around the tip reconstructed by the accumulation method, (e) differential distribution from (d).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1790 Phase Reconstruction in Annular Bright Field STEM

Ishida T.1, Kawasaki T.1, Kodama T.2, Ogai K.3, Ikuta T.4, Tanji T.1
1Nagoya University, Nagoya, Japan, 2Meijo University, Nagoya, Japan, 3APCO Ltd., Hachioji, Japan, 4Osaka Electro-Communication University, Neyagawa, Japan
takafu_i@echo.nuee.nagoya-u.ac.jp

In scanning transmission electron microscopy (STEM), an annular bright field (ABF) imaging [1] enabled simultaneous imaging of light and heavy elements. The ABF image contrast does not change in a thick specimen. On the other hand, in a thin specimen, the ABF image contrast oscillates by thickness and defocus [2,3]. This effect seems to same as the conventional bright field phase contrast imaging. Exact interpretation of the ABF image needs to be reconstructed to phase and amplitude information on specimens. In STEM, a new phase reconstruction technique revealed phase and amplitude images using a multi-channel detector [4]. We apply this technique to the ABF. This is called ABF phase imaging. The ABF phase imaging requires an annularly arrayed 24 detectors. We will show that this new method works effectively for reconstructing the phase of electron wave.

In the present study, the STEM (Hitachi HD-2300S) equipped with a spherical aberration corrector and an annular condenser aperture. The STEM was operated at the acceleration voltage of 200 kV with a convergence semi-angle of 20 - 25 mrad. The annular array detector is placed under a post-specimen projection lens. The inner- and outer-side angles of the annular array detector were set to 20 and 25 mrad by changing the excitation of the post-specimen projection lens. The STEM system takes 24 images simultaneously with a scanning time of 8.3 second.

Figs. 1(a)-(c) show some parts of 24 images of the graphite and the number k in these images corresponds to the position of the detector used for forming these images, as shown in Fig. 1(d). Images obtained with different detectors show different direction lattice fringes of 0.34 nm. Fig. 2 shows the reconstructed amplitude and phase images. The phase image is clearly visible in 0.34 nm lattice fringes. In contrast, the amplitude image mainly shows the thickness of the specimen.

We also carry out Image simulation for atomic resolution ABF phase images using multislice method. The image simulation will be presented an image contrast is proportional to atomic number Z. These results show the capability of this new method for a high resolution electron phase retrieval technique.

 References
[1] S.D. Findlay, N. Shibata, H. Sawada, E. Okunishi, Y. Kondo, T. Yamamoto and Y. Ikuhara, Appl. Phys. Lett. 95 (2009) 191913
[2] R. Ishikawa, E. Okunishi, H. Sawada, Y. Kondo, F. Hosokawa and E. Abe, Nat. Mater. 10 (2011) 278–281
[3] S. Lee, Y. Oshima, E. Hosono, H. Zhou, K. Takayanagi, Ultramicroscopy 125 (2013) 43–48.
[4] M. Taya, T. Matsutani, T. Ikuta, H. Saito, K. Ogai, Y. Harada, T. Tanaka, Y. Takai, Rev. Sci. Instrum. 78 (2007) 083705.

This work was supported by JSPS KAKENHI Grant Numbers18GS0211, 24360020.

Fig. 1: (a)-(c) Observed images obtained by the annular array detector. (d) Schematic of the detector layout.

Fig. 2: Reconstructed (a) amplitude and (b) phase images, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1940 Experimental evaluation of magnetic phase reconstruction in Lorentz TEM using the ‘transport-of-intensity’ equation

Kohn A.1, Habibi A.1
1Department of Materials Engineering and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev
akohn@bgu.ac.il

Imaging the micromagnetic structure of materials at the nanometer scale is motivated by the scientific study of new magnetic phenomena, and the technological drive for new information storage devices, which increase the storage density.
The micromagnetic structure can be imaged using transmission electron microscopy (TEM) in a variety of contrast modes, termed ‘Lorentz TEM’; for example, Fresnel-contrast (defocused images). Magnetic imaging in the TEM is possible because in the presence of a magnetic (and electric) potential, the electron wave-function undergoes a phase shift. Therefore, for quantitative mapping of the magnetic induction, the phase shift of the electron-wave needs to be reconstructed.

The ‘transport-of-intensity’ equation (TIE) is a general phase reconstruction methodology that can be applied to Lorentz TEM through the use of Fresnel-contrast images. We present an experimental study of sub-micrometer sized Permalloy elements in order to test the application of the TIE for quantitative magnetic mapping. We find that quantitative phase reconstructions (e.g. Fig. 1) are possible for defoci distances ranging approximately between 200 and 800 μm. The lower defocus limit is attributed to competing sources of image intensity variations in the Fresnel-contrast images such as structural defects and diffraction contrast. The upper defocus limit is shown to originate from a numerical error in the estimation of the intensity derivative.

Three sources of magnetic phase information are compared: domain walls, element edges and vortex cores. The vortex cores are shown to enable quantitative phase reconstructions while the domain walls and element edges enable only qualitative phase reconstructions. Considering the above limitations, we show quantitative reconstructions of elements sized down to approximately 100 nm and 5 nm thick. Thus, the minimal detection of the product of the magnetic induction and thickness is 5 Tesla·nanometer and magnetic structures are spatially resolved down to a size of 12 nanometers.

Fig. 1: Calculated (a, b, c, d) and experimental (e, f, g, h) eqi-phase contour maps (spaced at 1 radian) for Permalloy elements: triangular, 1µm diagonal, 10nm thick (a, e), circular, 250nm in diameter, 20nm thick (b, f), square, 130nm edge, 10nm thick (c, g) and circular, 1µm in diameter, 5nm thick (d, h).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1945 Dopant mapping in thin FIB prepared silicon samples by off-axis electron holography

Kohn A.1, Pantzer A.1,2, Vakahy A.2, Eliyahou Z.1, Levi G.2, Horvitz D.2
1Department of Materials Engineering and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , 2Micron Semiconductors Israel Ltd.
akohn@bgu.ac.il

Modern semiconductor devices function due to accurate dopant distribution. Off-axis electron holography (OAEH) in the transmission electron microscope (TEM) can map quantitatively the electrostatic potential in semiconductors with high spatial resolution. For the microelectronics industry, ongoing reduction of device dimensions, 3D device geometry, and failure analysis of specific devices require preparation of thin TEM samples, under 70 nm thick, by focused ion beam (FIB). Such thicknesses, which are considerably thinner than the values reported to date in the literature, are challenging due to FIB induced damage and surface depletion effects.
We report1 on preparation of TEM samples of silicon PN junctions in the FIB completed by low-energy (5 keV) ion milling, which reduced amorphization of the silicon to 10 nm thick. Additional perpendicular FIB sectioning (e.g. Fig. 1) enabled a direct measurement of the TEM sample thickness in order to determine accurately the crystalline thickness of the sample. Consequently, we find that the low-energy milling also resulted in a negligible thickness of electrically inactive regions, approximately 4 nm thick. The influence of TEM sample thickness, FIB induced damage and doping concentrations on the accuracy of the OAEH measurements were examined by comparison to secondary ion mass spectrometry measurements as well as to 1D and 3D simulations of the electrostatic potentials. We conclude that for TEM samples down to 100nm thick, OAEH measurements of Si-based PN junctions, for the doping levels examined here, resulted in quantitative mapping of potential variations, within ~0.1V. For thinner TEM samples, down to 20nm thick, mapping of potential variations is qualitative, due to a reduced accuracy of ~0.3V (Fig. 2).

1. A. Pantzer et al., Ultramicroscopy 138 (2014) 36–45

We thank A. Ripp for advising and implanting PN junction samples; M. Sokolovsky for SIMS analysis; I. Amit and Y. Rosenwaks for assistance with ‘Sentaurus’ 3D simulator.

Fig. 1: Example (50 nm thick sample) of additional perpendicular FIB sectioning to measure layer thicknesses directly: (a) bright-field TEM overview image of the sample; (b) High-resolution TEM image of the region denoted schematically by the black rectangle in (a). A 6 nm thick amorphous Si surface layer is measured and 154 nm thick crystalline Si layer.

Fig. 2: Comparison of potential profiles for sample thicknesses varying between 20 and 100 nm as derived from OAEH to the band potential as calculated by a 1D Poisson simulation using data from SIMS measurements.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-1954 Specimen Charging Measured by Off-axis Electron Holography

McLeod R. A.1, Beleggia M.2,3, Malac M.4
1Fondation Nanosciences, Grenoble, France, 2Denmark Technical University, Lyngby, Denmark, 3Hemholtz-Zentrum-Berlin, Berlin, Germany, 4National Institute for Nanotechnology, Edmonton, Canada
robbmcleod@gmail.com

In off-axis electron holography, the acquisition of hologram series for the purpose of averaging is becoming popular to improve the signal-to-noise ratio. Image series can also be considered as a form of tomography, with the 3rd axis being time. In this case, a hologram series can measure dynamic electric and magnetic characteristics of a specimen.


Hologram series have a total exposure time of minutes. During this period, interaction with the electron beam will cause many secondary electrons to be emitted from the specimen. The generated holes are screened by the mobile charges and the dielectric response of the material, generating a screening Coloumb potential. If a metal is nearby, the potential is further screened by an image charge, resulting in a phase shift of dipole character. Thus the holes left-behind by SE emission constitute a form of radiation damage for phase retrieval techniques. The holes will be refilled at a rate determined by the conductance and morphology of the specimen. The hole half-life relative to the total exposure time governs the ultimate accuracy of the measured phase. The technique is expected to improve understand of charging behavior, especially in insulating specimens.


Here we have conducted experiments with a series of short exposure (0.25 – 0.5 s) off-axis holograms at 300 keV of latex nanoparticles on a lacey carbon support. The beam was initially blanked for ~15 minutes. Based on the Berriman effect, we expect the latex particle to only partially discharge over this time period. The beam was unblanked at the start of the hologram series acquisition. The experiment was repeated at three current density levels: high (1240 A m-2), medium (260 A m-2, shown in Fig. 1), and low (70 A m-2). The phase shift difference inside the particle boundaries between the start and end of the series measured for the high (0.8 rad) and moderate (0.2 rad, shown in Fig. 2) current densities. For the low current density series the phase error (~0.12 rad) was too high to directly estimate the phase shift. To estimate the charge on the latex particle as a function of dose, the vacuum projected potential was found from the rotational average of the vacuum phase around the particle. A least-squares best-fit to a model of a surface-charged sphere plus image charge yielded an estimate of the relative charge, as shown in Fig. 3. The measured phase is relative rather than absolute because the average phase of the entire series was used as a reference to remove fringing fields. The high and medium current densities charged the particle, whereas for the low current density there was actually discharge during illumination. The time-resolved behavior is visualized by videos, which will be shown at the conference.

RAM acknowledges the financial support of Fondation Nanosciences and CEA and MM the financial support of NINT. RF Egerton provided valuable discussion.

Fig. 1: Phase contours (0.1 rad each) for the averaged phase shift over the medium dose-rate series.

Fig. 2: Phase difference between first seven and last seven holograms for medium dose-rate series.

Fig. 3: Accumulated electron charge as a function of cumulative dose for the three current density levels. The total series exposure time was 119 s for the low and med cases and 141 s for the high case.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-2006 Solving the transport of intensity equation using flux-preserving boundary conditions

Parvizi A.1, Koch C. T.1
1Institute for Experimental Physics, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
amin.parvizi@uni-ulm.de

The Transport of Intensity Equation (TIE) is a non-interferometric phase reconstruction method which overcomes disadvantages of interferometric methods such as coherent illumination and interferometer stability [1]. The TIE is a Poisson type equation which relates a modified Laplacian of the phase of the wave to the intensity variation along the optical axis (see eqn.(a.1) in Fig 1). In the absence of singularities in the principle (central) plane of focus, many approaches of solving the TIE exist. The problem common to all these approaches is that the necessary boundary conditions are not known. The most popular approach is based on the Fast Fourier Transform (FFT) and solves the TIE non-iteratively in the frequency domain. The implicitly assumed periodic boundary conditions are the main drawback of this method [1]. Another method is based on the multigrid approach for solving partial differential equations, yielding an exact solution of the TIE in the spatial domain. This approach allows us to define zero-flux boundary conditions (Fig1,eq. (a.2), where n ⃑ is the normal to the boundary) and the a vanishing phase shift within regions of vacuum. Since the phase in vacuum is constant, a circle (the number or the shape is arbitrary) with Dirichlet boundary condition can be placed in an area containing vacuum. Figure 1b shows the graphical representation of above-mentioned boundary conditions. Fig.2a and b show under and over focused images which are used to compute the intensity variation along the optical axis (Fig.2c).
Fig. 3 compares phase maps reconstructed from the simulated data by different methods. Fig. 3c shows the phase recovered by the Fourier method and our finite element multigrid solution obtained by using the software package COMSOL is shown in Fig. 3b. This figure shows clearly that especially the low frequency details of the phase reconstructed by the flux-preserving approach agree better with the phase used for simulating the images, than the reconstruction obtained by the Fourier transform method. In our presentation we will show applications of this method to experimental data acquired in the TEM and also the optical microscope. 
[1] D. Paganin and K.A. Nugent, Non-interferometric phase imaging with partially-coherent light Phys. Rev. Lett., 80, 2586-2589 (1998)

This work was supported financially by the Carl Zeiss Foundation as well as the German Research Foundation (DFG, Grant No. KO 2911/7-1).

Fig. 1: a) (a.1)TIE equation and (a.2) Equation defining the zero-flux boundary condition b) Graphical representation of the combination of boundary conditions used for the reconstruction scheme presented here.

Fig. 2: Under-focused image b) Over-focused image c) dI/dz approximated by the difference of the images shown in a and b.

Fig. 3: a) Original phase, b) Phase reconstructed by the method presented here c) Phase reconstructed by the FFT method for solving the TIE.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-2111 Optimisation of spatial and phase resolution of off-axis electron holography for detection of single dopant atoms.

Mayall B.1, McLeod R. A.2,3, Cooper D.1
1CEA-LETI, Minatec, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France, 2Fondation Nanosciences, Grenoble, France, 3CEA-INAC, Minatec, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France
benjamin.mayall@cea.fr

The reduction of the size of semiconductor devices leads to use of higher dopant concentrations. The scale of these devices implies that what was known as a very high dopant concentration will be only a few tens of atoms. The small numbers of these dopants mean that the just one of these atoms out of place will become statistically significant with respect to the electrical properties of the device. Thus, it is becoming increasingly important to be able to characterise the position and activity of these individual dopants.

Assuming a perfect specimen and vacuum, simulations show a phase sensitivity of better than 2π/2000 will be required in order to detect a single ionised dopant atom in silicon. The current state of the art for atomic resolution is around 2π/200, thus an improvement of an order of magnitude is required. The phase sensitivity of a reconstructed phase image is given by the relation,

Δφ≈(2/NV2)0.5

where N is the number of electron counts and V is hologram contrast. In order to achieve the best spatial resolution at atomic resolution it will not be possible to acquire holograms for very long periods due to specimen drift. Thus different approaches must be employed to improve the sensitivity.

Off-axis electron holography has been performed using a double aberration corrected FEI Titan Ultimate TEM equipped with an X-FEG. To improve the number of counts, a large series of holograms can be added together [1]. Fig. 1 shows a phase image of a silicon calibration specimen delta-doped with boron atoms. In Fig. 1(a) a single phase image acquired for 8 seconds is shown whereas (b) shows improvements from summing 25 holograms. In order to improve the contrast of the holograms, a monochromator can be used. Fig. 2 shows the effects of using the monochromator on the fringe contrast measured on reference holograms. An increase from 25 to 35 % will make a significant step towards the target of 2π/2000. Other approaches have been used to improve the reconstruction procedure. The spatial resolution of holograms containing strong phase objects is usually one third of the carrier frequency to avoid cross-talk from the centreband. The suppression of centreband in Fourier space by using phase shifting holography [2] improves the spatial resolution for a given carrier frequency and relaxes the electron biprism bias, resulting in higher V.

In this presentation we will show how combinations of these different methods of optimising phase noise and spatial resolution have been combined and then applied to wedge polished silicon specimens that contain different types of dopant atoms.

[1] R. McLeod et al. In press Ultramicroscopy (2013)

[2] V. Volkov et al. Ultramicroscopy 134 p175 (2013)

This work has been performed on the nanocharacterisation platform (PFNC) at Minatec. DC thanks the European Research Council for the Starting Grant “Holoview”.

Fig. 1: (a) Secondary Ion Mass Spectrometry (SIMS) profile of boron doped delta layers. (b) Reconstructed phase image from single hologram acquired for 8 seconds. (c) Reconstructed phase image from 25 holograms summed together.

Fig. 2: Electron holograms acquired without (a) and with (b) monochromator active. (c) Fringe intensity profiles acquired from hologram and (d) measured contrast as a function of C2 with and without monochromator.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-2202 Toward a 3D strain mapping at nanometer scale with Dark-Field Electron Holography

Javon E.1, Gatel C.2, Hÿtch M. J.2, Bals S.1, Van Tendeloo G.1
1EMAT, University of Antwerp, Antwerp, Belgium, 2CEMES-CNRS and Université de Toulouse, Toulouse, France
elsa.javon@uantwerpen.be

Dark-Field Electron Holography (DFEH) is a technique developed to map strain at the nanometre scale with a large field of view. The principle is based on measuring the variation phase of diffracted beam due to small strain which is the so called geometric phase1. Due to the diffraction conditions used for obtaining dark-field holograms, it is not possible to combine classical electron tomography with DFEH as it was done with electron holography for 3D magnetic and electric field mapping. Until now and similarly to GPA, two diffracted beams were selected to map 2D strain field projected on the electron direction. Here, we propose to combine 3 or 4 non collinear diffracted beams to reach the 3D strain map of a multilayer sample constituted by the repeated stacking of SiGe/Si layers grown along the [001] direction.
In order to keep the same dynamical conditions, all the diffracted beams belong to the {220} family. Two of them (0-22) and (02-2) are included in the [100] zone axis and two others (20-2) and (-20-2) in the 90° tilted zone axis namely [010] as shown in the Fig.1. Since DFEH imposes very strict constraints relating to the sample such as flatness and uniformity of the thickness on both reference and strain areas, specific FIB techniques have been developed for 2D strain mapping with DFEH. Here, we created a needle sample with a squared cross section in order to obtain a symmetric configuration and exactly the same thickness in both zone axis (Fig.2). The value of the thickness was chosen such that the crystalline thickness corresponds to a half integer of the extinction distance2 ξ220 and that the field of view was large enough.
The strain maps corresponding respectively to [100] and [010] are presented in the Fig.3. Since sample edges yield relaxation effects which are even stronger in strain regions, the measured profiles does not exactly correspond to the expected bulk values. However, the similarity between the strain profiles from both zone axis proves of the symmetry of the boundary conditions. All the projections that are required to reconstruct the 3D strain map are now known and the information common of both zone axis serves to validate the method.

In this study we present a method yielding for the first time a 3D reconstruction of the strain at nanometer scale. These results are also compared with the new 2-beams theory considering dynamical effects and resulting simple projection rule for the measurement of the geometric phase within the thickness. To carefully develop this method we chose a well characterized strained sample, however, the scope of this new technique is of high importance for entirely controlling the electron mobility of new nano devices.

1.M.Hÿtch et. al Nature453(2008)
2.A.Lubk, E.Javon et. al Ultramic.136(2014)

The authors acknowledge financial support from the European Union under the Seventh Framework Program under a contract for an Integrated Infrastructure Initiative (ESTEEM2) and the French National Agency (ANR) in the frame of its program in Nanosciences and Nanotechnologies.

Fig. 1: Schematic illustration of the needle preparation: a squared cross section yielding the same thickness in both [100] and [010] zone axis.

Fig. 2: (a) SEM image of a FIB of the squared needle. (b) Conventional dark field image of the multilayer SiGe.

Fig. 3: Strain map εzz corresponding respectively to the (a) [100] and (b) [010] zone axis. (c) Comparison of both strain profiles from the [100] and [010] zone axis along the [001] direction with the bulk value.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-2409 Low-keV electron microscope based on a single-atom electron source

Chang W. T.1, Lin C. Y.1, Hsu W. H.1, Chen Y. S.1, Hwang I. S.1
1Institute of Physics, Academia Sinica, Taipei, Taiwan, R.O.C.
wtchang@phys.sinica.edu.tw

Imaging of biological and organic molecules is a challenge in current electron microscopes due to insufficient contrast. Low-energy electrons emit from a single-atom tip1 (SAT) provide better contrast in light element materials and low radiation damage, and wave phases scattered from an object can be used to reconstruct images of the object due to its full spatial coherence2. Therefore, development of low-energy electron microscopes based on a reliable single atom emitter is highly desirable in study of light element materials.
We have built a low-energy electron point projection microscope (PPM) to evaluate emission characteristics of single atom emitter, as shown in Fig. 1. The beam divergence angle, measured at the half maximum intensity [Fig. 2(a)], becomes larger and the beam size at extractor becomes smaller with decreasing emitter-extractor separation [Fig. 2(b)]. This measurement may provide useful information for constructing an electron gun based on a single-atom source.
The beam energy used in PPM is always less than 500 eV and is not suitable for imaging molecules of 1 nm or thicker because the inelastic mean free path is less than 1 nm. Here we propose a low-keV (500 eV~ 5 keV) electron microscope based on a single-atom electron gun and a focusing lens, as shown in Fig. 3(a). In this electron energy range, samples with thickness of 3 nm or thinner can be imaged. It allows different imaging modes, including SEM imaging, coherent electron diffractive imaging, and holographic imaging.
A new challenge for the design is alignment. A narrow beam emitted from a single-atom source facilitates focusing, but also makes the alignment of the electron beam become critical, because misalignment by merely 1˚ causes a significant decrease in the beam intensity. Design for fine alignment of the source is essential for the successful extraction of the electron beam. Therefore, a piezo-stage with precision linear and angular adjustments is used to fine position the emitter. The custom-made stage enables building a compact and rigid system and provides the freedom to align and optimize the performance the entire system, as shown in Fig 3(b). Fig 3(c) presents a diffraction pattern of graphene with an imaging area around ~20 μm. We are improving the lens system to get better focusing of the system. This new instrument may allow determination of the atomic structures of individual thin nano-objects, such as graphene, carbon nanotubes, DNA molecules, or protein molecules.

Ref:

1.  Nano Lett. 4 (2004), 2379.

2. Nanotechnology 20 (2009), 115401.

This work was sponsored by the Academia Sinica. We pay our great thanks to their financial support

Fig. 1: Schematic diagram of a point projection microscope based on a single-atom emitter.

Fig. 2: (a) Intensity profile of an extracted beam from a single-atom electron source; (b) Opening angle & beam size v. s. the emitter-extractor separation.

Fig. 3: (a) Schematic diagram of the low-keV microscope. (b) Photograph of the microscope. (c) Diffraction pattern of graphene.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-2437 Reconstruction of the projected crystal potential using HRTEM – prospects for materials science investigations

Lentzen M.1, Barthel J.2
1Ernst Ruska Centre, Research Centre Jülich, Jülich, Germany, 2Central Facility for Electron Microscopy, RWTH Aachen University, Aachen, Germany
m.lentzen@fz-juelich.de

Potential reconstruction is the logical continuation of wave function reconstruction [1–3] in high-resolution electron microscopy. It aims at eliminating the problems in the structural interpretation of reconstructed wave functions, chiefly imposed by the effects of dynamical electron diffraction. These effects cause a non-linear relation of atomic scattering power and modulation of the wave function [4], and the local modulation near atomic columns can be further obscured through delocalisation and asymmetries induced by crystal tilt.

A series of investigations using the channelling model of dynamical electron diffraction [4] and a rapid and stable potential reconstruction algorithm revealed that the projected crystal potential can be determined for thick objects [5]. Object thickness, residual defocus aberration of the wave function, and phenomenological absorption, parameters often unknown in experiment, can be fitted self-consistently together with the projected potential [6], as well as crystal tilt [7].

In a materials science investigation of a thin Ba0.5Sr0.5Co0.8Fe0.2O3 (BSCF) crystal a through-focus series of 20 images was recorded with an aberration-corrected TITAN 80-300 microscope operated at 300 kV. After wave function reconstruction and numerical aberration correction up to the information limit of 0.08 nm (Fig. 1, right) the projected potential (Fig. 2, left) was reconstructed with a best fit of 8.4 nm object thickness, 1.6 nm residual defocus, 7.0 nm–1 crystal tilt, and a small residual of S = 4.5%. The potential map is free from non-linear contrast modulation, and the effects of tilt are strongly reduced. Column-by-column measurement of the potential maxima at the oxygen sites reveals through a histogram single oxygen atom precision of 2.6 volt per atom (Fig. 2, right). The three maxima of the distribution indicate a high concentration of oxygen vacancies.

[1] H Lichte, Ultramicroscopy 20 (1986), p. 293.

[2] W Coene et al, Phys. Rev. Lett. 69 (1992), p. 3743.

[3] A Thust et al, Ultramicroscopy 64 (1996), p. 211.

[4] K Kambe, G Lehmpfuhl and F Fujimoto, Z. Naturforsch. A29 (1974), p. 1034.

[5] M Lentzen and K Urban, Acta Cryst. A56 (2000), p. 235.

[6] M Lentzen, Ultramicroscopy 110 (2010), p. 517.

[7] M Lentzen, Proceedings MC2011 Kiel (2011), IM2 P133.

JB gratefully acknowledges funding from the German Federal Ministry of Economics and Technology within the COORETEC initiative.

Fig. 1: (left) High-resolution image of BSCF at bright-atom pass-band conditions, red: Ba/Sr, green: Co/Fe, blue: O. (right) Phase of reconstructed exit wave function of BSCF. Frames are 3.4 nm × 3.4 nm.

Fig. 2: (left) Reconstructed projected potential of BSCF, frame 3.4 nm by 3.4 nm, red: projected unit cell, blue: oxygen columns used for histogram analysis, dashed: Σ3 twin boundaries. (right) black: frequency of oxygen potential maxima versus maximum of oxygen potential (volt), grey: fit of the distribution with three gaussians.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-2687 Direct Imaging of Two-Dimensional Electron Gas at Oxide Interfaces using Inline Electron Holography

Song K.1, Ryu S.2, Lee H.2, Choi S.3, Paudel T. R.6, Koch C. T.4, Rzchowski M. S.5, Tsymbal E. Y.6, Eom C.2, Oh S.1
1POSTECH, Pohang, Republic of Korea, 2University of Wisconsin-Madison, Madison, USA, 3Korea Institute of Materials Science, Changwon, Republic of Korea, 4Ulm University, Ulm, Germany, 5University of Wisconsin-Madison, Madison, USA, 6University of Nebraska, Lincoln, USA
ksong@postech.ac.kr

Recently, a variety of new physical properties and phenomena have been discovered to emerge at atomically engineered interfaces of complex oxide systems. One example is the two-dimensional electron gas (2-DEG) forming at the interface between two insulating perovskite oxides, LaAlO3 (LAO) and SrTiO3 (STO). Theoretically, the electron concentration at this atomically-controlled interface can be manipulated by means of the polarity-induced electric field, which is facilitated by simply changing the film thickness of LAO on a STO substrate. The resulting conducting “interface material” is known to be localized within a few nm from the interface. Although the existence of 2-DEG has been proved and utilized in many prototype devices, there are still compelling debates related to the origin, spatial distribution, and electrostatic compensation of this “interface material”. Here, we directly visualize and quantify the 2-DEG forming at the interface of LAO/STO by using inline electron holography. By combining electron energy loss spectroscopy (EELS) and quantitative measurements of the atomic displacement of cations the possible origin of 2-DEG will be discussed.
A wide area 2-D charge density map with sub-nanometer resolution (~0.8 nm) was obtained by applying a Laplacian image filter to the electrostatic potential map as shown in Fig. 1. The electrostatic potential maps were retrieved by carefully calibrating the mean inner potentials and local thicknesses of LAO and STO. Whilst the charge density map obtained from the 3 unit cell (u. c.) sample, which is below the known critical thickness of 4 u. c., seems not to host any significant charge density near the interface (Fig. 1a), the 10 u. c. sample exhibits the negative charges beneath the interface (Fig. 1b). The width of 2-DEG, measured at full width at half maximum, is 0.82 ± 0.34 nm. In order to extract a correct density value of 2-DEG from the total charge density map, one has to take account of a change of the dielectric constant of STO near the interface due to a large intrinsic electric field. The calibration of the dielectric constant using Landau theory yields a 2-DEG density close to the theoretical expected value of ~3.3×1014 e cm-2 corresponding to the transfer of 0.5 e per unit cell. The scanning transmission electron microscopy (STEM) analysis combined with EELS indicates that the origin of this interfacial 2-DEG is most likely related with the oxygen vacancies formed at the LAO surface (Fig. 3), which agrees well with the recent first principles calculations.

This works has been supported by the AFOSR under Grant numbers FA2386-13-1-4136 and FA9550-12-1-0342.

Fig. 1: Charge density maps across the LaAlO3/SrTiO3 interfaces obtained by using inline electron holography for: a. 3 u. c. and b. 10 u. c. LaAlO3 samples.

Fig. 2: Charge density profile extracted from the charge density map of 10 u. c. LaAlO3/SrTiO3 shown in Fig. 1b.

Fig. 3: High-angle annular dark field (HAADF) image of a 10 u. c. LaAlO3/SrTiO3 and EELS spectra of Ti-L2,3 and O-K edges obtained from 2D line scans.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-2718 80 kV double biprism electron holography

Genz F.1, Niermann T.1, Lehmann M.1
1Technische Universität Berlin, Institut für Optik und Atomare Physik, Straße des 17. Juni 135, 10623 Berlin, Germany
florian.genz@physik.tu-berlin.de

Off-axis electron holography is a powerful method to retrieve the image phase in high quality[1]. Recently, off-axis electron holography performed with an acceleration voltage of 80 kV has moved into the focus of interest because electrons accelerated with 80 kV produce less knock-on damage and hence can be used to investigate, e.g., carbon-based materials [2]. The performance of a double biprism setup at 80 kV acceleration voltage and possible advantages in comparison to 300 kV were investigated.
A FEI Titan transmission electron microscope equipped with a high-brightness Schottky field-emission gun, an image Cs-corrector and a 2k by 2k camera (Gatan US1000) was used for this work. The camera's modulation transfer function (MTF) was determined with the edge method (fig. 1). The MTF is significantly improved at an acceleration voltage of 80 kV compared to 300 kV, probably due to a smaller electron scattering volume in the scintillator. The improvement is best for a sampling rate of about 10 pixels per fringe, giving an improvement factor of 1.9. In experimental electron hologram series with different fringe spacings, but keeping a sampling rate of 10 pixels per fringe realized by accordingly adjusting the magnification, about the same improvement of the standard deviation of the reconstructed phase was measured (fig. 2). Thereby, other effects like, e.g., electron energy dependent detection quantum efficiency of the camera, seem to be negligible.
To further decrease the standard deviation of the reconstructed phase and hence improve the phase sensitivity, a double biprism setup [3] was used. It allows the minimization of the applied biprism voltages. With the resulting increased stability of the holographic system, longer exposure times are possible, leading to an observed standard deviation of the reconstructed phase of about 2π/740 in a single empty hologram with an exposure time of 20 s.
The 80 kV off-axis electron holography is demonstrated using a thin GaN-foil oriented along [11-20] zone axis. Here, to avoid image resolution due to specimen drift, ten holograms with an exposure time of two seconds were made and averaged. The residual lens aberrations in the reconstructed image wave were corrected to retrieve the object exit wave. For comparison with the experiment, a GaN foil with thickness 1.9 nm, a chromatic aberration of 1.3 mm and an energy spread of 0.8 eV was simulated (fig. 3) [4]. Amplitude and phase of the object exit wave show a good match to the simulation.

  1. M. Lehmann et al., Microscopy and Microanalysis 8 (2002), p. 447-466.
  2. M. Linck et al., Microscopy and Microanalysis 18 (Suppl 2) (2012), p. 478.
  3. K. Harada et al., Applied Physics Letters 84 (2004), p. 3229.
  4. P. A. Stadelmann, Ultramicroscopy 21 (1987), 131.

Fig. 1: Damping by the modulation transfer function (MTF) of the camera as a function of the sampling frequency g at acceleration voltages of 80 kV and 300 kV.

Fig. 2: Standard deviation in the reconstructed phase of electron holograms with a sampling rate of 10 pixels per hologram fringe as a function of the inverse fringe spacing s related to the object plane. The exposure time was 2 s. A smaller standard deviation is equivalent to a higher phase sensitivity.

Fig. 3: Comparison of experimental and simulated object exit-wave of a thin GaN foil in [11-20]-orientation with a thickness of approximately 1.9 nm. Both amplitude and both phase images have the same grey levels.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-2738 Nanoscale strain analysis by dark-field electron holography of shallow trench isolation structures for MOSFET technology

Ravaux F.1, Cherkashin N.2, Lolivier J.3, Alexander D. T.1
1Interdisciplinary Centre for Electron Microscopy (CIME), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland, 2CEMES-CNRS, Toulouse, France, 3EM Microelectronic SA, Marin, Switzerland
duncan.alexander@epfl.ch

As microelectronic devices shrink, mechanical deformations induced by the various fabrication process steps play a growing role in their technological properties. These parameters must be understood and controlled because strained silicon can significantly boost transistor performance [1], but can also lead to product failures. Of interest in this study are transistors based on shallow trench isolation (STI) structures (Fig 1.) used in metal-oxide-semiconductor field effect transistor (MOSFET) technology. The main goal is to understand the mechanisms responsible for strain formation inside the active silicon area.
Off-axis dark field electron holography (DFEH) in TEM is used to perform the strain analysis on dedicated test structures that simulate the mechanical deformation on the active silicon areas that are supposed to receive the MOSFET. The choice of removing the MOSFET was made in order to analyze/evaluate purely the influence of the STI structure, within a systematic study of the influence of STI fabrication steps. The DFEH technique [2] is based on the interference of diffracted beams coming from two different regions of the sample with the aid of an electrostatic biprism (Fig. 2). If the two regions present a lattice constant difference, a phase difference will be measured from the holographic fringes and the strain information can be retrieved.
For the experiments, classical focused ion beam (FIB) TEM lamellae were prepared but thickness variations due to the STI geometry and lamella bending due to strain relaxation prevented the extraction of the strain tensor from the entire structure. For this reason, a dedicated sample preparation method has been developed to match the requirements imposed by the DFEH method (i.e. uniform 120 nm sample thickness with no specimen bending). Backside FIB milling [3] combined with an innovative “double-bar” rigid-thin TEM lamella geometry succeeds in avoiding these artefacts (Fig. 3). Measurements made on the HITACHI I2TEM at CEMES-CNRS (Toulouse, France) now permit the extraction of the complete sample-plane strain tensor in the active silicon area sandwiched by two STI structures (Fig. 4). The results are in accordance with values obtained by CBED and TCAD simulations in the literature [4].
[1] M. Cai et al., IEEE Trans. Electron Devices, vol. 57 (2010) 1706–1709
[2] M. Hÿtch et al., Nature, vol. 453 (2008) 1086–1089.
[3] J. Gazda et al., Microsc. Microanal., vol. 16 Supplement S2 (2010) 230–231.
[4] A. Steegen and K. Maex, Mater. Sci. Eng. R Rep., vol. 38 (2002) 1–53.

The authors acknowledge funding by the Swiss Commission for Technology and Innovation (CTI), Franc Fort project number 13496.1 PFFLE-NM, and ESTEEM2 for Transnational Access to the I2TEM at CEMES. Marco Cantoni at CIME is thanked for many useful discussions.

Fig. 1: Schematic figures of CMOS transistor (a) and STI structures (b).

Fig. 2: Principle of the dark-field electron holography (DFEH) technique.

Fig. 3: The double-bar “rigid-thin” TEM lamella geometry (a). SEM images of the thin part (b) and the rigid frame (c). Bright-field TEM image of the region of interest (d).

Fig. 4: Strain measurement of the active silicon area sandwiched by STIs.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-2745 Electron Holography and Electron Energy Loss Spectroscopy for Studying Electrochemical Reactions at Electrode/Electrolyte Interfaces in a Lithium-Ion Battery

Hirayama T.1, Yamamoto K.1, Shimoyamada A.1, Yoshida R.1, Iriyama Y.2
1Nanostructures Research Laboratory, Japan Fine Ceramics Center, 2Department of Materials, Physics and Energy Engineering, Nagoya University
t-hirayama@jfcc.or.jp

It is essential that detailed knowledge about the electrochemical reactions near the interfaces between electrodes and electrolyte be obtained to find clues for the development of more efficient batteries [1]. For this purpose, measurement of electric potential distributions and direct observation of ion distributions are of fundamental importance. Here we report our results of observations of such phenomena in all-solid-state lithium-ion batteries.

Electron holography is a powerful technique to map electric potential distributions in working batteries [2,3], while electron energy loss spectroscopy (EELS) is an effective technique to directly detect lithium distributions. The two methods provide a powerful means of revealing the electrochemical reactions that occur at electrode/electrolyte interfaces.

Figure 1(a) shows a schematic of the model battery used in our experiments. Fig. 1(b) shows the electric potential distribution measured at the negative side by electron holography after the first charge-discharge cycle. The negative potential indicates that a negative electrode region was formed in situ during this cycle [2]. Fig. 2(a) and 2(b) respectively show a transmission electron micrograph of a region near the negative electrode and corresponding spectrum obtained from Spatially Resolved Electron Energy Loss Spectroscopy (SR-EELS) measurements for a specimen that had undergone 50 charge-discharge cycles. This spectrum indicates that the in situ negative electrode contains excess lithium.

In summary, we have successfully observed distributions of electric potentials and lithium ions at the negative electrode side of an all-solid-state lithium-ion battery. These microscopy techniques provide new insights into the electrochemical reactions that take place in all-solid-state batteries during cycling, and should aid the design of high-performance devices.

References

[1] M. Armand and J.-M. Tarascon, Nature 451, 652-657 (2008).

[2] K. Yamamoto, et al., Angew. Chem. Int. Ed. 49, 4414-4417 (2010).

[3] K. Yamamoto, et al., Electrochem. Commun. 20, 113-116 (2012).

The authors would like to thank Dr. Y. Sugita and Mr. K. Miyahara of Chubu Electric Power Co., Inc., and Dr C. Fisher of Japan Fine Ceramics Center for valuable discussions. SR-EELS measurements were performed as part of the RISING project of NEDO, Japan. We are grateful to Profs. T. Abe, Y. Uchimoto and Z. Ogumi of Kyoto University for their encouragement and useful suggestions.

Fig. 1: FIG. 1. (a) Schematic of the model battery sample used in the experiments. An area corresponding to that enclosed by the red rectangle was observed by electron holography. (b) Electric potential distribution obtained by electron holography.

Fig. 2: FIG. 2. Spatially Resolved Electron Energy Loss Spectroscopy (SR-EELS) measurements. (a) Transmission electron micrograph of the region at the negative electrode and electrolyte Li1+x+3zAlx(Ti,Ge)2-xSi3zP3-zO12 (LICGC) interface; (b) the corresponding spectrum obtained by SR-EELS.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-2987 Distribution of electric field and mechanical vibrations of CdS nanocombs during field emission

Migunov V.1, Duchamp M.1, Liu R.3, Kamran M. A.3, Zuo B.3, Li Z.2, Farle M.2, Dunin-Borkowski R. E.1
1Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Gruenberg Institute, Forschungszentrum Juelich, Juelich, Germany, 2Faculty of Physics and Center of Nanointegration (CeNIDE), University of Duisburg-Essen, Duisburg, Germany, 3Beijing Key Lab of Nanophotonics and Ultrafine Optical Systems, School of Physics, Beijing Institute of Technology, Beijing, China
v.migunov@fz-juelich.de

CdS comb-like nanostructures (Fig. 1a) were recently shown to have remarkable optical properties and possible applications as waveguides [1]. On the other hand, nano-cone-based field-emission guns (FEGs) have shown a strong improvement of the coherence compared to classical W tips [2] that is of particular interest for electron holography for example. As CdS-based nanostructures are also used in optically-induced field emission [3], it combination with wave guiding properties may allow their use in FEGs, in which the light that triggers the emission of electrons is focused onto a different part of the nanostructure to that from which the electrons are emitted. With this an increase of the local temperature at the electron emission point that results in increase of electron energy spread can be avoided. Here, we study the field emission properties of such nanocombs in situ in the transmission electron microscope (TEM) using off-axis electron holography combined with electrical biasing.

The experiments involved mounting a CdS nanocomb onto a W needle and bringing a movable W probe towards CdS comb in situ in the TEM (Fig. 1b). Bias voltages of between 0 and -140V were applied to the nanocomb. Off-axis electron holograms were acquired before and during field emission from the apex of one of the branches of the comb. The phase shift measured using electron holography is proportional to the projected electrostatic potential. The density of the measured equiphase lines shown in Fig. 2 facilitates identification of the region from which electrons are emitted. This information can be correlated directly with the morphology of the apex. We measured threshold bias voltages for field emission for different electrode configurations. Unexpectedly, the nanocomb was observed to vibrate when field emission was initiated, as shown in Fig. 3.

[1] Liu R. et al., Nano Letters, 2013, 13, 2997−3001

[2] Houdellier F. et al., 15th European Microscopy Congress Manchester Central, 2012

[3] Zhang J. et al., Sci. China Phys. Mech. Astron., 2011, 54 (11), 1963–1966

We thank Giulio Pozzi for fruitful discussions.

Fig. 1: a) Scanning electron micrograph of a CdS nanocomb deposited on a holy carbon grid. b) Bright-field TEM image showing the geometry of the field emission experiment.

Fig. 2: Equiphase contours recorded using electron holography for a bias voltage of -23 V between the apex of one tooth of the CdS nanocomb (bottom) and the W probe (top).

Fig. 3: Frames from a video recorded in a TEM, showing the end of a tooth of the CdS comb (bottom) before the onset of field emission at a bias voltage of -32 V (left) and during field emission at a bias voltage of -33 V with a current of 4 nA (right). The contrast of the tooth is blurred due to the onset of mechanical vibrations with MHz range frequency.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-3018 Experimental electron holographic tomography of magnetic vector fields in nanoscale materials

Diehle P.1, Caron J.1, Kovacs A.1, Ungermann J.2, Kardynal B.3, Dunin-Borkowski R. E.1
1Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute 5, Forschungszentrum Jülich, Jülich, Germany, 2Institute of Energy and Climate Research, Forschungszentrum Jülich, Jülich, Germany, 3Institute of Semiconductor Nanoelectronics and Peter Grünberg Institute 9, Forschungszentrum Jülich, Jülich, Germany
p.diehle@fz-juelich.de

It is important to develop a characterization technique that can be used to measure three-dimensional magnetization distributions in nanoscale magnetic materials, which are of interest for a variety of applications that include future information storage technologies [1].

Off-axis electron holography allows the phase shift of the electron wave that has travelled through a thin sample to be measured in the transmission electron microscope (TEM) [2]. The phase shift is, in turn, sensitive to the in-plane component of the magnetic flux density within and around the specimen integrated in the electron beam direction. Although conventional tomographic reconstruction algorithms can in principle be used to reconstruct the magnetic flux density within and around a TEM specimen from two tilt series of magnetic phase images, significant artefacts can result from the difficulty of acquiring two ideal tilt series of images about independent axes over a complete range of specimen tilt angles.

We have therefore chosen to develop a different approach, which is based on the use of a model-based reconstruction algorithm to reconstruct the three-dimensional magnetization distribution in a TEM specimen, rather than the magnetic flux density, from two tilt series of phase images recorded using off-axis electron holography. Our approach involves repeated forward calculation of magnetic phase images until a best-fitting simulated magnetization distribution to the experimental images is obtained.

Practical challenges include the need to subtract the mean inner potential contribution to the phase shift recorded at each specimen tilt angle, the need to take into account the perturbed reference wave in the simulations, the minimization of contributions to the recorded phase from diffraction contrast and the preparation of a TEM specimen that is neither too thick nor obscured by another part of the specimen or the specimen holder when it is tilted to high angles about two axes.

Figure 1 shows simulated magnetic induction maps and magnetic phase images of an elliptical magnetic element for two different specimen tilt angles. Figure 2 illustrates the use of an electron-transparent silicon nitride membrane to acquire two independent tilt series of electron holograms of a lithographically patterned magnetic element. We will compare simulated and experimental results from both lithographically patterned elements and more three-dimensional nanoscale magnetic specimens and discuss the influence of noise and experimental artefacts on the final reconstructed magnetization distribution.

[1] S. S. P. Parkin et al., J. Appl. Phys. 85, 5828 (1999)
[2] D. Gabor, Proc. Roy. Soc. A 197, 454 (1949)

RDB acknowledges the European Commission for an Advanced Grant.

Fig. 1: Simulated magnetic phase shift and magnetic induction map of a uniformly magnetized elliptical element (saturation magnetisation = 3T) with semi-axis lengths of 600nm and 200nm and a thickness of 50nm for two different specimen tilt angles. The colors represent the direction and magnitude of the phase gradient, according to the color wheel shown.

Fig. 2: Illustration of the design of lithographically patterned magnetic elements that can be tilted to high angles about two independent axes.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-3140 Electron Beam induced Currents in GaN p-n Junctions

Park J. B.1, Niermann T.1, Lehmann M.1
1Institut für Optik und Atomare Physik, Technische Universität Berlin, Straße des 17. Juni 135, D-10623 Berlin, Germany
park@physik.tu-berlin.de

Off-axis electron holography (EH) is a unique approach for analyzing potential variation down to nanometer scale. In absence of magnetic fields and in kinematical diffraction condition, the phase modulation is proportional to the potential (sum of mean inner potential and built-in potential Vpn) and the specimen thickness. However, quantitative measurement of Vpn in GaN shows a huge deviation with the theoretical value. Additionally, it seems that the hypothetical “dead layer” on both surfaces affected by focused ion beam (FIB) preparation is not the only reason for the discrepancy since the measured Vpn from the bulk (n-GaN)/shell (p-GaN) geometry of the GaN p-n junction is not affected by the FIB preparation [1].
In this study, we assess the influence of the electron beam on the measured Vpn at GaN p-n junctions. Two GaN p-n junctions of comparable dopant concentration were prepared by FIB. Thereby, a needle-like shape geometry is deliberately chosen to ensure that the whole specimen is illuminated during the experiment [2]. Moreover, a defined path of the induced current arising from electron-hole pair generation and secondary electron emission is provided. Low voltage (5 kV) is applied for the final polishing step to mitigate the surface damaging by FIB.
Fig. 1 shows the FIB milled needle with a sketch of the layer structure. In order to enhance the signal to noise ratio of the phase, series of holograms separately for each illumination intensity were recorded and subsequently averaged. The reconstructed phase depicts a clear phase jump at the p-n junction (Fig. 1 (c)).
Two electron beam induced effects are observed. Firstly, in Fig. 2 the measured Vpn increases with reducing the electron dose rate. This illumination dependency of Vpn can be quantitatively explained by a solar-cell model. Hence, Vpn = Vpn, expected – Vbias is fitted on the data set, where Vpn, expected is determined by the doping concentration (sample A: 3.41 V, sample B: 3.43 V), Vbias is the voltage drop across the p-n junction, which is affected by illumination induced currents owing to secondary electron emission and e-h pair generation [3]. Additionally, fluctuation of Vpn is depicted by two needles, which are prepared from a same wafer and with the same FIB preparation steps. However, both needles (I and II) show the same behaviour of Vpn over the electron dose rate. Secondly, beam damage of the specimen is observed in Fig. 3. The Vpn diminishes over a period of time and might converge to a constant value, which is about the half of the initially measured Vpn.

1. S. Yazdi et al., journal of physics: Conf. Ser. 471 (2013), p.012041.

2. A. Lenk, Dissertation, Dresden (2008).

3. S.M. Sze in “Physics of Semiconductor Devices”, 2. Edition, John Wiley & Sons (1981).

This work is carried out within the framework of the DFG collaborative research center SFB787 semiconductor nanophotonics.

Fig. 1: (a) SEM image of the needle-like shape specimen after FIB preparation. (b) The layer structure of the specimen. (c) The reconstructed phase of (a). The phase jump at the p-n junction due to different doping type is clearly visible. The large-area undulation of the phase change along the specimen is caused by thickness variation.

Fig. 2: The measured built-in potential Vpn over the electron dose rate for two needle specimens of sample A (FIB needle I and II). The Vpn is enhanced over roughly 30% by reducing the electron dose rate by a magnitude of 2.

Fig. 3: The Vpn is measured directly after FIB preparation over a period of time (about 1 hour) at sample B. We observe a decrease of Vpn by a factor 2 over time.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-3191 Hybridization of Off-Axis and Inline High-Resolution Electron Holography

Ozsoy Keskinbora C.1, Boothroyd C. B.2, Dunin-Borkowski R. E.2, van Aken P. A.1, Koch C. T.3
1Max Planck Institute for Intelligent Systems, Stuttgart, Germany, 2Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Forschungszentrum Jülich, Jülich, Germany, 3Institute for Experimental Physics, Ulm University, Ulm, Germany
ozsoy@is.mpg.de

In conventional TEM experiments, only the intensity (i.e., the square of the amplitude) of the wave function can be measured. The phase information gets lost when the electron is detected by the CCD camera. Denis Gabor introduced an approach that could be used to solve this problem 66 years ago [1]. In Gabor’s original setup, which is the pioneering scheme for inline holography, the wave that has been scattered by the specimen (the object wave) interferes with a reference wave propagated along the same axis. Using laser light, Leith and Upatnieks [2] showed that separation of the axes of propagation of the reference and object waves could be used to solve the twin-image problem. Möllenstedt later translated this idea back to electron microscopy, creating the field of off-axis electron holography [3,4]. Inline electron holography, or focal series reconstruction, is now a common method in high-resolution TEM. Although it is very efficient for recovering high spatial frequency variations in phase, it is inefficient for recovering phase information at low spatial frequencies. In contrast, high-resolution studies are very challenging for off-axis holography, because the interference fringes must be at least twice as fine as the finest feature of interest in the object to be resolved.
In this study, we present a new approach that combines off-axis and inline holography and allows reliable phase information to be recovered for all spatial frequencies. For a desired signal-to-noise ratio, the required total exposure time is lower than that for traditional high-resolution off-axis electron holography.
All holographic data were acquired using round illumination with a FEI Titan TEM operated at 300 kV and using a bi-prism voltage of 97.4 V for off-axis electron holography. Figure 1 shows phase and amplitude images of a gold particle obtained using inline and off-axis electron holography and the hybrid method, respectively, for a total exposure time of 7s. Although the noise level in the vacuum region is slightly higher than for inline electron holography (0.055π vs. 0.046π), the recovery of low spatial frequencies is far better than for inline holography alone. Such noise levels are difficult to achieve using off-axis holography for the exposure time utilized here.
[1] D. Gabor, Nature vol. 161 (1948), p. 777–778.
[2] E.N. Leith, J. Upatnieks, J. Opt. Soc. Am. vol 52 (1962), p. 1123–1130.
[3] G. Möllenstedt and H. Düker, Naturwissenschaften vol. 42 (1955), p. 41–41
[4] G. Möllenstedt and H. Wahl, Naturwissenschaften vol. 55 (1968), p. 340–341

The authors thank to; John Bonevich for offering free public use of HolograFREE reconstruction software. Wilfried Sigle, Luis M. Liz-Marzan and Cristina Fernandez-Lopez for samples. The research leading to these results received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under grant agreement no312483 (ESTEEM2) and the Carl Zeiss Foundation.

Fig. 1: a)-c) Reconstructed phase, d-f) reconstructed amplitude images from inline, off-axis and hybrid methods, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-11-P-3505 Electron holography for magnetic and electric in situ imaging

Ponce A.1, Cantu-Valle J.1, Diaz Barriga E.2, Luna C.2, Mendoza-Santoyo F.1, Jose Yacaman M.1, Eder J.1
1University of Texas at San Antonio, 2Universidad Autónoma de Nuevo León
arturo.ponce@utsa.edu

In this work we report the local magnetic behavior of multi-segmented Cox-Ni1-x nanowires by off-axis electron holography as well as electric contribution in ZnO nanostructures. The nanowires were grown by electrode deposition, by alternating cycles that produce the multi-segmented structure. The crystalline phase of each segment and magneto-crystalline anisotropy will be resolved by the phase maps obtained by electron holography.
Samples were studied using JEOL JEM ARM-200F. Holograms were obtained under two different conditions; first following the dual-lens imaging system, using a voltage in the objective lens of 1V, and secondly under Lorentz mode, with the objective lens turned off.
The ZnO nanorods were studied under external bias applied to the sample in situ TEM. The experimental setup consists in a connection from the holder (Nanofactory electrical holder) to the external source. The holograms have been live recorded and the electric variation is observed in the reconstructed phase.
In order to select the optimum parameters for the holograms reconstruction, the fringe spacing, interference width and fringe contrast were measured for different biprism voltages. The holograms were recorded in-focus with a biprism voltage of about 20-23V and interference fringe spacing of 5nm and a fringe contrast of 22%. The quality of the holograms will depend in a high fringe contrast and number of electrons on the holograms, which imply better signal to noise ratio, this will be reflected in the reconstructed phase and amplitude images. The holograms acquisition was obtained using specialized software from Gatan, Digital Micrograph (DM) and processed by the latest version of HoloWorks, which includes a new feature to extract the magnetic induction and magnetic contours from a phase image.

This project was supported by grants from the National Center for Research Resources (5 G12RR013646-12) and the National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health. The authors would also like to acknowledge the NSF PREM # DMR 0934218. A special recognition to Holowerks LLC., and Dr. Edgar Voelkl for their guidance and support.

↑ Go to the Table of content ↑

IT-12. Surface microscopy and spectroscopy

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-12-IN-2866 Addressing Fundamental Problems in Information technology: Opportunities for X-Ray Photoelectron Spectromicroscopy

Schneider C. M.1,2
1Peter Gruenberg Institute PGI-6, Research Centre Juelich, D-52425 Juelich, Germany, 2Faculty of Physics and CENIDE, University Duisburg-Essen, D-47057 Duisburg, Germany
c.m.schneider@fz-juelich.de

Modern information technology must exploit the full potential of complex material systems for the meticulous control of state variables. These state variables are used to encode an information bit and may be electron charges in semiconductor nanoelectronics, electron spins in the case spintronics, or local redox configurations in resistive switching elements. Consequently, the materials encompass intermetallic compounds, oxides or chalcogenides, elementary and compound semiconductors or even molecular components. In addition, the functional elements, for example, individual memory cells or transistor structures often involve nanometric dimensions and operate on nanosecond timescales or even below. This imposes considerable challenges on the characterization of electronic, chemical and magnetic states in the steady state or during operation.

Immersion lens microscopy with synchrotron radiation has matured into a versatile and powerful tool to investigate a broad range of issues in condensed matter physics and materials science. It combines high-resolution imaging with spectroscopic capabilities in a unique fashion. The excitation with photons from the soft to the hard x-ray regime ensures element selectivity and variable information depth. The polarization state of the synchrotron radiation enables a distinction of different magnetic orderings (Fig. 1), whereas the intrinsic time structure of the synchrotron radiation permits the study of processes with picosecond time-resolution.

In this contribution we will review the present status of x-ray photoemission spectromicroscopy with emphasis on applications in information technology. In particular, we will cover model systems in spintronics and in resistive switching (Fig. 2). The results will cover both static properties and dynamic processes. We will also discuss new developments, such as photoemission microscopy with hard x-rays and imaging spin polarimetry.

I would like to thank N. Barrett, S. Cramm, R. Dittmann, W. Drube, M. Escher, V. Feyer, A. Gloskovskii, A. Kaiser, J. Kirschner, A. Koehl, I. Krug, Ch. Lenser, M. Merkel, M. Patt, L. Plucinski, J. Rault, O. Renault, Ch. Tusche, N. Weber, R. Waser, and C. Wiemann for their cooperation. Financial support through the Deutsche Forschungsgemeinschaft (SFB 917) is gratefully acknowledged.

Fig. 1: Ferro- (left) and antiferromagnetic domain pat- terns in the system NiO/Fe3O4(110) exploiting XMCD (Fe) and XMLD contrast (Ni) at the indicated photon energies. Wide arrows indicate the local spin alignment axis.

Fig. 2: Spatially resolved hard x-ray photoemission from a Fe:SrTiO3. The dark squares result from 7nm thick Au electrode pads deposited for the resistive switching experiments.
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-12-IN-3176 Towards 1.5λ resolution with low energy electrons

Tromp R. M.1,2
1IBM T.J. Watson Research Center, Yorktown Heights, NY, 2Kamerlingh Onnes Laboratory, Leiden University, The Netherlands
rtromp@us.ibm.com

The highest resolution aberration-corrected electron microscopes today, operating at 300 keV, achieve a spatial resolution of 50 pm, or about 25 times the wavelength of the electron, l. With the third-order spherical aberration of the objective lens compensated, this resolution is limited by chromatic aberration, fifth-order spherical aberration, parasitic aberrations, and various microscope instabilities. On the other end of the spectrum, Low Energy Electron Microscopy (LEEM) without aberration correction has achieved a spatial resolution of 4 nm at 3.5 eV, or about 6l. The resolution in such instruments is primarily limited by the spherical and chromatic aberrations of the uniform electrostatic field between sample and cathode objective lens. This uniform field is the first (virtual) image-forming element of the microscope. When used as a Photo Electron Emission Microscope (PEEM) resolution is usually limited to the range of 10-20 nm, depending on the details of the imaging conditions. In LEEM/PEEM aberration coefficients are strongly energy-dependent, and must be readily adjustable even in a single experiment, so as to track the aberrations as they change with electron energy.

Over the last several years we have developed an aberration-corrected LEEM/PEEM instrument[1], using a relatively simple catadioptric (i.e. electrostatic lens + mirror) correction system which provides independent control over the lowest order spherical and chromatic aberration coefficients, and the focal length of the correction optics. We have demonstrated the practical feasibility of aberration correction in LEEM/PEEM, achieving spatial resolution below 2 nm for the first time [2]. Detailed studies of the wave-optical image formation process show that resolution well below 1 nm is possible in principle [3].
In this talk I will review challenges and recent progress towards reaching the goal of a spatial resolution of just 1.5 times the wavelength of the electron in LEEM.

1. A new aberration-corrected, energy-filtered LEEM/PEEM instrument. I. Principles and design, R.M. Tromp, J.B. Hannon, A.W. Ellis, W. Wan, A. Berghaus, O. Schaff; Ultramicroscopy 110 (2010) 852

2. A new aberration-corrected, energy-filtered LEEM/PEEM instrument. II. Operation and results; R.M. Tromp, J.B. Hannon, W. Wan, A. Berghaus, O. Schaff; Ultramicroscopy 127 (2013) 25-39

3. A Contrast Transfer Function approach for image calculations in standard and aberration-corrected LEEM and PEEM, S.M. Schramm, A.B. Pang, M.S. Altman, R.M. Tromp; Ultramicroscopy 115 (2012) 88-108

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-12-O-1893 Secondary electron quasi-simultaneous observation of energy selective imaging and diffraction in DualEEM

Grzelakowski K. P.1
1OPTICON Nanotechnology, Wroclaw, Poland
k.grzelakowski@opticon-nanotechnology.com

We present the first results of the AES application in the novel technique based on PEEM [1] and LEEM [2] concepts: DualEEM [3].It utilizes the idea of the imaging α- Spherical Deflector Analyzer (α-SDA) [4] with the total deflection 2π.The image returns exactly to its origin on the optical axis independently of the starting angle and energy.As a consequence,the object of filtration and its image are invariant in the 2π deflection process. Additionally,the final angles of incidence at this plane change the sign after the full angle deflection,which indicates mirroring-like effect.This mathematical analogy to the classical mirror operator is further enriched by the unique property of the α-SDA analyzer: the direction of electron propagation before “reflection” at the symmetry plane is preserved after the 2π deflection process is completed.Therefore, contrary to the classical electrostatic mirror,the propagation direction on both sides of the mirror plane is preserved.This could be referred to as a unique “through the looking-glass” electron optical effect.Thus, the α-SDA imaging analyzer exhibits all the advantages of the electrostatic mirror without the loss of beneficial linear geometry.The α-SDA assures not only the selection of characteristic energy for imaging, but also a beam separation into two imaging channels: energy-selective real image and reciprocal (diffraction) image and their quasi-simultaneous acquisition.The microscope is equipped with an Auger electron gun located inside the immersion objective lens that allows for an unique electron beam sample illumination and thus,opens a new application field for electron spectromicroscopy under laboratory conditions.For the first time that unique kind of the sample illumination is used for the energy selective Auger electron imaging and diffraction. Both are visualized at two independent imaging channels:one for the real and the other for the reciprocal image.These images are acquired quasi-simultaneousely through software based switching of on and off potentials of the one of hemispheres of the α- spherical deflector analyzer.The first results are reported and discussed.                                                                       

1 E. Brueche, Z.Phys. 86  (1933) 448,

2 E. Bauer, in Proc.of the 5th Int. Congr.for El.Micr.,(Academic, N.Y., 1962, p.D-11)

3 K.P. Grzelakowski, Ultramicroscopy, 130 (2013) 29; 4 Ultramicroscopy 116 (2012) 95

The author acknowledges the financial support by the NCBR in Warsaw. My thanks are also due to Krzysztof Wojcik and his team at “Metob” for the excellent machining and advice. I am very grateful to Prof.Ernst Bauer for his valuable suggestions and discussions. I would like also to express my gratitude to Janusz Krajniak for his dedication to this project and Dariusz Mirecki for his support.

Fig. 1: Black and blue areas indicate α-rays and γ-rays, respectively, p1 and p2 denote the symmetry and diffraction planes of the α-SDA, respectively: (a) energy selective k-projection, upper hemisphere switched off, (b) energy selective real image mode, lower and upper hemisphere switched on. PEEM mode: α-SDA switched off (right hand part of Fig.b).

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-12-O-2156 Novel development of very high brightness and highly spin-polarized electron gun with a compact 3D spin manipulator for SPLEEM

Koshikawa T.1, Yasue T.1, Suzuki M.1, Tsuno K.1, Goto S.2, Jin X.3, Takeda Y.4
1Osaka Electro-Communication Univ. , Osaka, Japan, 2San-yu Electronic Corp. , Tokyo, Japan, 3School of Engineering, Nagoya Univ., Nagoya, Japan , 4Aichi Synchrotron Radiation Center, Seto, Japan
kosikawa@isc.osakac.ac.jp

We have already developed a novel very high brightness and high spin-polarized low energy electron microscope (SPLEEM) and applied it to clarify the magnetic property of [CoNix]y/W(110) and Au/CoNi2/W(110) during growth of ultra thin films[1-5]. Such thin multi layered films are important for current-driven domain-wall-motion devices. Our developed SPLEEM can make us the dynamic observation of the magnetic domain images possible. However the size of the spin-polarized electron gun is large and we have started to develop a new compact spin-polarized electron gun with a novel idea. In principle two devices are necessary to operate 3-dimensional spin direction; one is a spin manipulator which changes the out-of-plain spin direction and another one is a spin rotator which can change the in-plain spin direction. We have proposed a multi-pole Wien filter which enables 3-dimensional spin operation with one device. The developed 3D multi-pole spin manipulator which has 8 poles in the present development and the magnetic and electric field in the multi-poles Wien filter as shown in Fig.1. Uniform field can be obtained at the center part of the Wien filter with 8 poles and 12 poles, however 4 poles filter gives non-uniform field even at the center. In the present development 8 poles Wien filter has been adopted. The results of magnetic images and asymmetries of Co(4ML)/W(110) vs. polar and azimuthal angles are shown in Fig.2. The results clearly show that spin direction can be operated three dimensionally with one device.
1)  X.G. Jin et al., Appl. Phys. Express 1, 045002 (2008).
2)  N. Yamamoto et al., J. Appl. Phys.103, 064905 (2008).
3)  M.Suzuki et al., Appl. Phys.Express 3, 026601 (2010).
4)  M.Suzuki et. al., J.Phys. Cond. Matt. 25, 406001-1-8 (2013) (Short News on IOP web and IOP select).
5)  K. Kudo et al., J.Phys. Cond.Matte. 25, 395005-1-6 (2013).

This work was supported by a Grand-in-Aid for Scientific Research (A) (Grand No. 23246015) from the Japan Scoiety for the Promotion of Science (JSPS) and the System Development Program for Advanced Measurement ans Analysis from Japan Science and Technology (JST).

Fig. 1: 3D multi-pole spin manipulator and uniformity of magnetic and electricField.

Fig. 2: The magnetic images and the asymmetries vs. the polar and azimuthal angles for Co(4ML)/W(110).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-12-O-2690 Time-of-Flight Momentum Microscopy with Imaging Spinfilter: Dirac-Type States on Clean and Oxygen-Covered W(110) and Mo(110)

Medjanik K.1, Schönhense G.1, Chernov S.1, Schertz F.1, Nepijko S. A.1, Elmers H. J.1, Oelsner A.2, Tusche C.3, Kirschner J.3
1Institute of Physics, Johannes Gutenberg-University Mainz, Germany , 2Surface Concept GmbH Mainz, Germany, 3Max Planck Institute for Microstructure Physics, Halle, Germany
medyanyk@uni-mainz.de

We present a novel method for k-space mapping of electronic bands with utmost efficiency. The instrument combines the k-imaging properties of a cathode-lens microscope with the superior resolution of ToF spectroscopy and the parallel acquisition capability of ToF-PEEM [1]. For the first experiments a frequency-doubled Ti-sapphire laser was used for excitation by two-photon photoemission (2hv = 5.8 – 6.6 eV). A delay-line detector serves for rapid single-event counting with 150 ps time resolution and 10 Mcps maximum count rate. The dispersion behavior of the bands is observed in a 3D (kx,ky,E) matrix as schematically depicted in Fig.1, which is confined by the photoemission horizon (condition k_I_=0) in the shape of a E-kII paraboloid. The kII- and energy-range are presently limited by the low excitation energy. Mapping a complete data set with good statistics requires only few minutes of acquisition time. An integral Ir-based imaging spinfilter [2] yields spin resolved 3D-maps.

The low excitation energy is well suited to study surface states close to the centre of the SBZ. As first system we chose the highly anisotropic Dirac-type surface state recently discovered on W(110) [3] and the analogous state on Mo(110). These states arise in a pocket-shaped partial bandgap region, nevertheless the existence of Dirac states on metals was very surprising [3,4]. Fig. 2 shows kx-ky sections at EF (a,b) and E-kII sections displaying the crossover points for clean W(110) at about EB=1.25eV (c) and at 0.6eV for oxygen-covered Mo(110) (d). Hole doping by oxygen shifts the Dirac state to lower binding energy; in addition we found a pronounced pattern of very similar surface states on W(110)-O(1x1) and Mo(110)-O. No bulk bands are visible in this region of k-space. In all cases the Dirac state is highly anisotropic (2mm symmetry), revealing massless behavior (i.e. linear band dispersion) along one mirror plane and a high effective mass (flat band region) along the second mirror plane (g,h), similar as measured and calculated for clean W(110) [3,4]. For the oxidic surfaces we observe a complex 3D k-space behavior that is hard to elucidate in conventional ARPES. Using s-, p- and circular polarization we probe orbital symmetries and hybridization effects of the band states. The dichroism pattern in Fig. 2(e) is antisymmetric with respect to all mirror planes and can be understood in terms of a simple dz2-orbital model.

[1] G. Schönhense et al., Surf. Science 480 (2001) 180;

[2] C. Tusche et al., Appl. Phys. Lett. 99 (2011) 032505; D. Kutnyakhov et al., Ultramicroscopy 130 (2013) 63

[3] K. Miyamoto et al., Phys. Rev. Lett. 108 (2012) 066808;

[4] H. Mirhosseini et al., New J. of Phys. 15 (2013) 033019.

Project funded by BMBF (05K12UM2 and 05K12EF1).

Fig. 1: Scheme of the experiment; the 3D (kx,ky,E) time-resolving single-electron counting detector registers each electron within the E-kII paraboloid with a maximum count rate of 107 counts per second.

Fig. 2: Sections through the (kx,ky,E) matrix (at 2hv=6.6 eV). a,b: Fermi surfaces of W(110) and Mo(110)-O, respectively; c,d corresponding E-kII sections. e: circular dichroism in the region above the Dirac point of clean W(110), g: anisotropic shape of the Dirac point; f,h: calculated patterns for EB=0.9 and 1.2 eV [4].

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-12-O-2910 Shadow Dark-Field LEEM and Scanning Micro-LEED of Epitaxial Graphene on Ru(0001) and Ir(111) Surfaces

Yu K. M.1, Man K. L.1, Altman M. S.1
1Hong Kong University of Science and Technology, Hong Kong, China
phaltman@ust.hk

Spatially resolved measurements using cathode lens microscopies have made notable contributions to the understanding of graphene layers that are customarily spatially inhomogeneous [1]. We have applied low energy electron microscopy (LEEM) and complementary micro-low energy electron diffraction (μLEED) to study the structure and morphology of single layer graphene (g) on Ru(0001) and Ir(111) surfaces, examples of strongly and weakly interacting substrates, respectively. Our investigations of g/Ru(0001) reveal rich structural non-uniformity that depends strongly on preparation conditions. When the g/Ru(0001) layer is prepared using chemical vapor deposition (CVD) by exposure to ethylene at high temperature, we observe strong streaking of superstructure diffraction spots (Fig. 1(a),(c)) for large area (3μm) illumination. This indicates the proliferation of small angle (<0.25°) lattice rotations in the graphene layer. Corresponding small-angle lattice rotational domains are visualized in “shadow” dark field LEEM images (Fig. 1(b),(d)) that are formed by selecting the rotated edge of the streaked superstructure spots using the contrast aperture. The presence of rotation domains also causes the sharp sets of μLEED superstructure diffraction spots around each integer order spot to rotate to-and-fro as a group about their respective stationary foci when the small μLEED illumination beam (250nm) is scanned across the surface. These scanning μLEED measurements provide detailed information about the rotation angle distribution and even indicate a net deviation from perfect alignment between graphene and substrate.


Although the length scale of the rotational domains in g/Ru(0001) can be pushed up to the sub-micron length scale by increasing the growth temperature (Fig. 1(d)), further improvements are limited by the diminishing growth rate at increasingly higher temperature for accessible ethylene pressure. On the other hand, massive single rotation domains can be fabricated by CVD if the crystal is first pre-loaded with carbon by dissolution of a single graphene layer. Although near perfect rotational order was observed (Fig. 1(e),(f)), scanning μLEED revealed substantial spatial variation of lateral periodicity that was not seen when small-angle rotational domains were present. Hence, fabrication of optimal uniform g/Ru(0001) is still elusive. Experiments on g/Ir(111) reveal that small angle rotational microstructure is similarly prevalent when the graphene lattice is nominally aligned with the substrate, but it is substantially suppressed in macro-domains with larger misalignments.

Reference
[1] K.L. Man and M.S. Altman, J. Phys.: Condens. Matter 24, 314209 (2012).

Financial support from the Hong Kong Research Grants Council under Grant No. HKUST600113 is gratefully acknowledged.

Fig. 1: (a),(c),(e) LEED patterns obtained from the areas indicated in (b),(d),(f) shadow dark-field LEEM images of g/Ru(0001). Graphene was prepared by CVD at (a),(b) 1100K; (c),(d) 1270K; (e),(f) 1300K on a preloaded substrate. Contrast fine structure in (b),(d) is due to small angle rotation domains. A uniform rotation domain is seen in (f).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-12-P-3198 Detecting the topographic, chemical and magnetic contrasts with nanometer spatial resolution

Zanin D. A.1, Erbudak M.1, De Pietro L. G.1, Cabrera H.1, Kostanyan A.1, Vindigni A.1, Pescia D.1, Ramsperger U.1
1Laboratory for Solid State Physics, ETH Zürich, Switzerland
dzanin@phys.ethz.ch

During the last decades, magneto-imaging techniques based on the analysis of secondary electrons helped the discovery of many interesting phenomena related to magnetic-domain patterns, such as re-entrant topological transitions. For those studies, a typical spatial resolution of some tens of nm, achieved e.g. in Scanning-Electron-Microscopy with Polarization Analysis (SEMPA), was more than enough. Nowadays, the quest to resolve magnetic textures in direct space at atomic scale is triggered by novel fundamental and applicative issues. Domain walls, in relation to their potential use in spintronic devices, represent one example. Inspired by the Russel Young topografiner we redesigned the SEMPA setup by replacing the primary electron beam source and the probing method. We dubbed this new technique Near Field-Emission Scanning Electron Microscopy (NFESEM). In NFESEM the sample surface is typically investigated by scanning at constant height with a primary electron beam energy in the range between 20eV and 100eV. A suitable detector analyzes secondary electrons scattered by the surface. We present the resolution improvement on topographic mapping of Fe-patches evaporated on W(110) substrate (Figure 1) and advances in energy analysis of secondary electrons (Figure 2). Moreover, we report on recent efforts to endow NFESEM with the polarization analysis of the detected secondary electrons that emphasize the true potential of this new technique. In particular, the characteristic spatial resolution and the sizeable secondary electrons yield (see Figures 1 and 2) support the technical feasibility of electron spectroscopy and magnetic-domain mapping at nanometer scale with NFESEM.

We thank Andreas Fognini, Thomas Michlmayr and Yves Acreman for the scientific support, Thomas Bähler for technical assistance and the Swiss National Science Foundation and ETH Zurich for financial support.

Fig. 1: (Left) STM Map of 0.4 atomic layers of FE on stepped W(110), showing atomic Fe-patches (bright) residing on the terraces and decorating the steps. (Right) The same surface spot recorded in NFESEM mode. Although the Fe-patches are on top of the W-substrate they appear darker - both the patches on the terraces and along the steps.

Fig. 2: (Left) Energy spectrum of a GaAs(110) surface for a tip-sample distance of 100 nm, both the secondary electron cascade an the elastic peak are clearly distinguishable. (Top right) Map of a GaAs(110) decorated surface produced by secondary electrons with 13 eV energy for a tip-sample distance of 12 nm. (Bottom right) STM reference image.
↑ Go to the Table of content ↑

IT-13. Focused ion beam microscopy and techniques

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-13-IN-2711 Advances in FIB Nanotomography

Cantoni M.1, Knott G. W.1, Burdet P.2
1EPFL-CIME,Lausanne, Switzerland, 2Department of Materials Science and Metallurgy, EM Group University of Cambridge, United Kingdom
marco.cantoni@epfl.ch

FIB-tomography is used in materials science for 3D-analysis of nanostructured materials [1]and in life science for the analysis of complex structures like brain tissue [2]. This presentation summarizes recent technological improvements, which include advancements in detector technology for electron imaging and elemental analysis, scan generator technology for high throughput imaging, and automated drift correction for reliable 3D reconstruction. New in-column detectors have a higher sensitivity for low energy electrons, which is the basis for a very high resolution down to a few nm voxel size. The low kV imaging can be combined with energy filtering in order to detect a pure signal of backscattered electrons (BSE), which improves the reliability of phase segmentation and quantitative analysis. The quality of the 3D reconstructions can also be improved with refined procedures for drift correction based on reference marks. In addition, with the new scan generators image acquisition and ion milling can be performed synchronously. In this way the acquisition speed increases further. Finally, spectral and elemental mapping (XEDS) based on Silicon Drift Detectors (SDD) provides higher X-ray count rates. Increased acquisition rates open new possibilities in chemical analysis that provide larger data cubes with higher representativeness. The new possibilities of FIB-tomography are illustrated with the following examples: a) Reliable phase segmentation is discussed for a superconducting material with trapped pores that cannot be filled with resin. b) Combined analysis of SE- and BSE stacks reveals the complex microstructure of a Sn-solder with different nano-sized precipitates [3] and c) High throughput elemental analysis is performed of a NiTi stainless steel with a complicated multi-phase microstructure [4]. The examples document the recent advancements in resolution, contrast, stability and throughput, which are necessary for reliable and representative 3D-analysis.

References
1. L. Holzer, M. Cantoni, in Nanofabrication Using Focused Ion and Electron Beams—Principles and Applications, I. Utke, S. Moshkalev, P. Russell, Eds. (Oxford University Press, New York, 2012), pp. 410–435.
2. M. Cantoni, C. Genoud, C. Hébert and Graham Knott, Microsc. & Anal. 24(4): 13-16 (2010)2010.
3. M. Maleki, J. Cugnoni, J. Botsis, Acta Mater. 61 (1), (2013).
4. P. Burdet, J. Vannod, A. Hessler-Wyser, M. Rappaz, M. Cantoni, Acta Mater. 61 (8), 3090 (2013).

Fig. 1: Fig 1. Three-dimensional representation of the two different intermetallic phases in the SnCuAg-type solder, segmented based on simultaneously acquired secondary electron and backscattered electron image stacks.

Fig. 2: Fig 2. Complex chemical microstructure of a NiTi—stainless-steel weld with different phases. The yellow and red phases are chemically very close and required segmentation based on the secondary electron image contrast.
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-13-IN-5748 Development of cryogenic FIB-SEM based processes for organic and inorganic samples

Antoniou N.1
1ReVera Inc., Santa Clara, USA
nicholas@cns.fas.harvard.edu

Development of cryogenic FIB-SEM based processes Cryogenic EM is one of the best techniques we have to fix in place samples for EM that would otherwise be destroyed in the vacuum system [1]. Even though FIB-SEM has enabled a rapid, site specific and relatively easy way for TEM sample preparation it has not been easily adopted for cryogenically prepared samples. The impetus to develop a cryogenic sample preparation process with all the advantages of the room temperature one was high. However, the liftout and attach steps of the processes do not work in a cryogenic environment so either a more limited process had to be developed [2] or new equipment and new processes developed. Both have been achieved and we present here on the latter. This technique replicates the room temperature process but in cryo so that all of the developments surrounding FIB-SEM sample prep are available at cryogenic temperature. This not only requires a cold stage in the instrument but also sample transfer capability within and in-between each instrument involved. We describe this process, the equipment and modifications to it as well as applications. It was also discovered that certain inorganic material could benefit from cryogenic processing in FIB-SEM but for completely different reasons. Most FIB systems use gallium liquid metal as the ion sources (LMIS) and this species (Ga) can react chemically with certain compounds such as semiconductors to form undesirable side effects such as spheres and dots [3]. Milling under cryogenic conditions reduces these undesired reactions and in some cases this was the only way we could prepare the sample for TEM imaging (InN for example). We further investigated the effect of warm up after cryo milling for inorganic material and found that in most cases the undesirable side effects were minimized enough so that they did not interfere with our ability to image the sample. References [1] Adrian M., et al., “Cryo-Electron Microscopy of Viruses,” Nature 308, (01 March 1984), pp 32 - 36 [2] Rigort, A., et al., “Focused Ion Beam Micromachining of Eukaryotic Cells for Cryoelectron Tomography,” PNAS, (March 20, 2012), Vol. 109, No. 12, pp. 4449-4454. [3] Grossklaus, K. A., “Mechanisms of Nanodot Formation Under Focused Ion Beam Irradiation in Compound Semiconductors,” Journal of Applied Physics, Vol. 109 , Issue: 1, 2011, pp. 014319 - 014319-11.

The author acknowledges funding from the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN), which is supported by the NSF (under award no. ECS-0335765). CNS is part of Harvard University. I would also like to thank Dr. Ilan Shalish and Cheryl Hartfileld for their numerous contributions to this work.

Fig. 1: InN nanoparticles grown in a forrest and felled for easier pickup.

Fig. 2: A tungsten tip was sharpened to about 10 nm and used to pick up a single InN nanoarticle using only natural forces (Van der Waals). After pickup, the particle was placed on a TEM grid for milling in cryogenic conditions.

Fig. 3: GaN sample milled at room temperature using 1.5 nA FIB probe. The formation of dropplets is observed at room temperature and impedes clean milling of GaN.

Fig. 4: GaN sample milled at -145 C using 1.5 nA FIB probe as in Fig. 3. The dropplets did not form as in the room temperature milling.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-13-O-2039 FIB-SEM Tomography of biological samples: Strategy of preparation to resolve high-resolution 3-D volumes

Kizilyaprak C.1, Daraspe J.1, Longo G.2, Humbel B. M.1
1University of Lausanne, Electron Microscopy Facility, Lausanne, Switzerland. , 2EPFL, Laboratory of Physics of Living Matter, Lausanne, Switzerland.
caroline.kizilyaprak@unil.ch

Elucidating the three-dimensional (3-D) spatial distribution of organelles within cells is essential for investigating numerous cellular processes. Tomography in the transmission electron microscope (TEM) is the method of choice for 3-D imaging of cellular structures down to 3nm resolution [1]. However, TEM tomography is typically limited to 500 nm thick sections making the reconstruction of an entire eukaryotic cell very challenging [2]. There is a need for a technology that can be used for rapid 3-D imaging of large mammalian cells to provide information at nanometre resolution. The most promising technology at the moment is the FIB-SEM tomography of fixed biological samples embedded in resin [3-7]. A FIB-SEM microscope is a scanning electron microscope combined with a focused ion beam (FIB) such that both beams coincide at their focal points. This combination enables bulk resin samples to be locally sectioned by ion milling, producing new block face imaged with the electron beam. This process can be repeated allowing 3-D analysis of relatively large volumes with a field of view of several micrometres.

Any fixed and embedded resin samples prepared for TEM examination can be used for FIB-SEM tomography. However, considerations have to be given to artefacts and surface damages induced by FIB milling and imaging [8]. In this study, different protocols of sample fixation and staining were explored in order to improve the signal/noise ratio, preserve the ultra-structure and reduce charging effects of biological samples. In addition, the behaviour of specific resin formulations [9] was investigated in the FIB-SEM microscope. The milling rate was measured and the damages caused by the ion impact on the resin were analysed. The most stable resin was used to improve the milling conditions. Finally, the geometry of the sample was optimized to improve the imaging conditions using detection of the backscattered electrons with the through-the-lens detector (BSE-TLD).

In conclusion, we propose a sample preparation and imaging strategy for high-resolution FIB-SEM tomography (Figure 1).

References:

1. Baumeister,et al.,Trends in cell biology, 1999.9(2): p.81-5.

2. Noske, A.B., et al.,Journal of structural biology, 2008.61(3): p.298-313.

3. Heymann, J.A., et al.,Journal of structural biology, 2009.166(1): p.1-7.

4. Knott, G., et al.,Journal of visualized experiments : JoVE, 2011(53): p.e2588.

5. Bushby, A.J., et al.,Nature Protocols, 2011. 6(6): p.845-58.

6. Villinger, C., et al.,Histochemistry and cell biology, 2012.138(4): p.549-56.

7. Wei, D., et al.,BioTechniques, 2012. 53(1): p.41-8.

8. Drobne, D., et al.,Microscopy research and technique, 2007.70(10): p.895-903.

9. Luft, J.H.,The Journal of biophysical and biochemical cytology, 1961.9: p.409-14.

Fig. 1: FIB-SEM cross section of liver cell imaged at 2kV in backscatter electron mode. This image (4096 x 3536 pixels) comes from a series of 430 images with a voxel resolution of 3 x 3 x 10 nm3.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-13-O-2051 Modeling of Ion Generated Secondary Electrons

Huh U.1, Ramachandra R.2, Joy D. C.3
1University of Tennessee, Knoxville, TN USA, 2University of California,San Diego, CA, , 3Oak Ridge National Laboratory, Oak Ridge, TN, USA
djoy@utk.edu

The arrival of high performance ion beam scanning microscopes has made it essential to have a quantitative model of the ion beam interactions with specimens and their contribution to the generation of the ion induced secondary electron signal (iSE). We have developed an enhanced Monte Carlo simulation, based on our earlier IONiSE program (1) , which is designed to better understand the physics of ion-solid interactions and to perform quantitative simulations. Two key pieces of data are required for this model. The first is the stopping power of the incident ion in the chosen target. Here we use recent data from Berger et al, (2) whose ASTAR program provides stopping power and other data for the He+ ion . ASTAR stopping power profiles were computed for He+ energies from 10keV to 105keV and for elements with atomic number of 90 as seen in figure 1. The second step is to be able to compute the generation rate, the range, and the subsequent transport of the iSE deposited in the sample which has been done by a Monte Carlo method. The Bethe (3) model of secondary electron production requires two parameters, e which represents the generation rate of iSE in the target material, and l which determines the probability of the generated iSE signals reaching the sample surface and ultimately escaping from the specimen surface. So far there is only limited experimental data for iSE yields as a function of their landing energy but good agreement has been found with what little data is available.

References

1, Ramachandra R, Griffin B, Joy DC, (2009), ‘A model of secondary electron imaging in the

Helium Ion scanning microscope’, Ultramicroscopy 109, 748-757

2. Berger M J, Coursey, J S, Zucker M.A,and J. Chang, J, (2011). ‘Stopping-Power and Range

Tables for Electrons, Protons, and Helium Ion’. This is freely available from:

http://www.nist.gov/pml/data/star/index.cfm

3. Bethe H, (1941), The generation of Secondary Electrons, Phys Rev. 59, 940-942

This work was partially supported by the Center for Materials Processing, University of Tennessee

Fig. 1: Composite plot of the variation in stopping power (eV/cm2/1015) predicted by ASTAR for a helium ion source as a function of its beam energy (keV) and of the target material. The black line shows the averaged stopping power for all materials tested as a function of ion energy
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-13-O-2227 Temperature Evolution during FIB Processing of Soft Matter: From Fundamentals towards TEM Lamella Preparation

Schmied R.1, Froech J. E.1, Orthacker A.1, Kraxner J.1, Hobisch J.2, Trimmel G.2, Plank H.1,3
1Center for Electron Microscopy, Graz, Austria, 2Institute for Chemistry and Technology of Materials, University of Technology, Graz, Austria, 3Institute for Electron Microscopy and Fine Structure Research, University of Technology, Graz, Austria
roland.schmied@felmi-zfe.at

During the last decade focused ion beam (FIB) processing became a well-established technique for the site-specific preparation of ultrathin lamellas for transmission electron microscopy (TEM) but also for sub-surface 3D metrology and 3D prototyping on the nanoscale. Beside the undoubted advantages of straightforward implementation, FIB processing entails unwanted side effects, such as ion implantation, amorphization and partial high thermal stress [1]. While the former two are intrinsic properties and therefore invariable, local heating effects have been shown to depend strongly on the patterning strategy. The minimization of this technically induced heating is essential for low melting materials but requires deeper understanding of thermal effects during scanning.
Therefore, accessing local temperatures, its spatial and temporal evolution together with their consequences is essential for FIB processing of sensitive materials with respect to chemical damage and morphological instabilities. In the first part we present an approach, which uses ion trajectory simulations as input data for a thermal spike model which allows the prediction of local temperatures and its lateral distribution during FIB processing (see Figure 1a). Taking into account the thermal behavior of polymers, combined simulations and calculations reveal very good agreement with FIB experiments on polymers (see Figure 1b) confirming the suitability of this combined approach to predict local temperatures and its spatial and timely evolution.
In second step we apply the gained knowledge together with an alternative patterning strategy for the preparation of TEM lamellas, which minimizes technically induced temperature effects [2]. By this careful adaption of the patterning strategy we will show morphological stabilization, characterized via scanning electron microscopy (SEM) and atomic force microscopy (AFM), and demonstrate the reduced chemical damage via IR-Raman spectroscopy. Based on these results, TEM investigations of polymeric layer systems and organic transistors will be shown which confirms stabilized morphologies and minimized chemical damage.
The study demonstrates the massive thermal stress a polymer is exposed during FIB processing and the capabilities of adapted FIB processing for low melting materials which can be easily implemented in most FIB systems. By that, new possibility for FIB processing capabilities for low melting materials open up which have been considered as very complicated or even impossible in the past.
1. J. Mayer, et al., MRS Bulletin 2007, 32, 5, 400 – 407
2. R.Schmied et al. RSC Adv., 2012, 2 (17), 6932 – 6938

The authors gratefully acknowledge the valuable support provided by Prof. Ferdinand Hofer, Prof. Gerald Kothleitner, Dr. Boril Chernev, and Martina Dienstleder. The authors also thank FFG Austria and the Federal Ministry of Economy, Family and Youth of Austria for their financial support.

Fig. 1: (a) simulated 2D temperature distribution in PMMA showing different degrees of material modification (pristine (green), modified (yellow) and volatized (red)); (b) experimental data of minimum line widths for varying pixel dwell times (squares) during standard FIB processing on PMMA compared to the simulated minimum line width(grey band).
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-13-O-2698 A focused Xe+-ion column for fast materials sputtering at high spatial resolution to carry out time-of-flight mass spectrometry with nanoscale precision within a scanning electron microscope

Sedláček L.1, Hrnčíř T.1, Latzel M.2,3, Hoffmann B.2, Jiruše J.1, Christiansen S.2,4
1TESCAN Brno, s.r.o., Brno, Czech Republic, 2TDSU Photonic Nanostructures, Max Planck Institute for the Science of Light, Erlangen, Germany, 3Institute of Optics, Information and Photonics, University of Erlangen-Nuremberg, Erlangen, Germany, 4Helmholtz Center for Materials and Energy, Berlin, Germany
libor.sedlacek@tescan.cz

A unique combination of a high resolution scanning electron microscope (SEM) and a high current focused ion beam (FIB) using a plasma Xe ion source (FERA from TESCAN company) permits extremely high milling and material removal rates [1,2] while simultaneously being able to watch the process so that a precise end-point detection is at hand. Additional analytical add-ons such as energy dispersive x-ray detection (EDX), electron back-scatter diffraction (EBSD) and orthogonal Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) (TOFWERK company) [3] permit a novel quality of correlated microscopies/spectroscopies.

For TOF-SIMS the high performance focused Xe-ion beam is used to remove the analyte material with the spatial resolution of a FIB. TOF-SIMS provides secondary ion imaging as well as depth profiling, so that a full three-dimensional isotopic images with better than 100 nm lateral resolution are possible.

Compared to a FIB based on Gallium primary ions, the Xenon ion source provides a better detection limit for most of the elements. A quantitative analysis has been demonstrated using a Xe plasma source for material sputtering and alkali elements, such as Li, Na, K constituting the analyte [4]. Detection limits below 2 ppm have been achieved for these species. Moreover, there is no interference when using Xe-FIB instead of Ga-FIB between the analyte and source Gallium ions for material that contain elements such as e.g. Ce, Ge, Ga. Therefore the Xe-FIB is more suitable e.g. for the analysis of important and widely used semiconductor materials and compounds such as SiGe, (In)GaAs and (In, Al)GaN.

Performance of the TOF-SIMS instrument relying on Xe-FIB materials removal has been demonstrated on samples with light-emitting-diode (LED) structures composed of GaN with InGaN Quantum Wells (QWs). A stack of five QWs, each with a thickness of 2.4 nm has successfully been detected (Fig. 1). The focused e-beam of the SEM has been used during TOF-SIMS measurements to account for charge compensation. An analysis of a different LED layer stack showed that a comparably rough interlayer structure is present inside the multi-QWs as demonstrated by the monitoring of TOF-SIMS 27Al+ intensity which is related to a covering AlGaN layer. A 3D reconstruction of an Al rich layer that covers the QWs is shown in Fig. 2.

References:

[1] T Hrnčíř et al, 38th ISTFA Proceedings (2012), p. 26.

[2] J Jiruše et al, Microscopy and Microanalysis 18 (Suppl. 2) (2012), pp. 652-653.

[3] J A Whitby et al, Adv. Mat. Sci. Eng. (2012), 180437.

[4] F A Stevie et al, Surf. Int. Anal. (in press).

The research leading to these results has received funding from the European Union Seventh Framework Program [FP7/2007-2013] under grant agreement No. 280566, project UnivSEM.

Fig. 1: TOF-SIMS depth profile of a GaN/InGaN multi-QW LED sample. Xe primary ion beam current of 550 pA at 30 kV was used for materials sputtering and SEM e-beam current of 1,2 nA at 10 kV was applied to account for charge compensation. Normalized depth profiles of 115In+ and 69Ga+ show well resolved 2.4 nm thick In rich quantum wells.

Fig. 2: 3D reconstruction of TOF-SIMS 27Al+ signal that shows an Al rich layer covering InGaN/GaN QWs in LED structures. View from top (left) and bottom (right) are shown. The z-axis has been expanded 25 times to highlight the interfacial roughness. Field of view is 60 µm x 36 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-1477 Development of Cryo-FIB technique for the structural characterization of liquid samples

Tsuchiya M.1, Iwahori T.2, Morikawa A.3, Nagakubo Y.4
1Hitachi High-Technologies Corporation
tsuchiya-miki@naka.hitachi-hitec.com

It is important to observe and characterize the three-dimensional distribution of materials in liquid samples such as; cosmetics, functional paint, catalysts and similar products. Furthermore the requirement for investigating the structure inside of liquids at a microscopic level such as the interface of a dispersoid and dispersant has increased. In order to meet these requirements, we have developed a fully compatible cryo transfer holder for FIB and (S)TEM systems. Another area of development for this holder centers on controlling the temperature of the specimen during either the fabrication or observation and which makes it possible to transport a specimen in the frozen condition. This holder can be cooled to 100K by liquid N2 for observation or fabrication of the sample in a controlled thermal state. To demonstrate the capabilities of this holder a liquid foundation sample was investigated. The liquid foundation was first plunge frozen on a specimen stub and transferred to the holder. The holder containing the frozen foundation sample was then placed into a Hitachi NB5000 FIB-SEM for FIB fabrication.

As a result 100nm thin lamella was produced in the FIB and we were able to observe the structure of the microscopic dispersoid, and its distribution as first viewed in the NB5000 and then a HD2700 shown in Figure1.

Figure 2 shows the EDX maps of the thin foil specimen (Thickness: approximately 100nm) of the frozen foundation. In this result it is possible to clearly see each dispersoid, such as the Fe needle crystals and the granular Ti containing crystals. The results confirm the low temperature and high stability performance of this cryo-transfer holder.

In conjunction to this cryo holder a modified method for the micro-sampling technique which allows a micrometer sized sample to be taken from a millimetre sized specimen is required. For this a new mechanical cryo-probe was developed for sample extraction. This mechanical cryo probe is able to be cooled to 120K.

Figure 3a show the cross-section SEM images of a frozen facial foundation liquid which was lifted out by using a room temperature probe needle. Some hollows appeared inside the micro sample due to the water sublimating from the sample when contacted by the room temperature needle probe.

Figure 3b shows the cross-section SEM image of the same sample as figure 3a but lifted out by the mechanical cryo probe at a temperature of 120K. By using the mechanical cryo probe it is possible to pick up the frozen micro sample while maintaining its shape and we can observe clearly the pigments and dispersant in the frozen liquid foundation.

Fig. 1: BF-STEM images of liquid foundation at an accelerating voltage of 30kV(a) and 200kV(b). (a) Instrument: NB5000 FIB-SEM, Cooling temperature: 100K, Magnification: x3,500. (b)Instrument: HD-2700 STEM, Cooling temperature: 100K, Magnification: x50,000.

Fig. 2: EDX maps of liquid foundation using the 200kV STEM with the cryo transfer specimen holder. dispersoid dispersantInstrument: HD-2700, Acquisition Pixel: 256×200, Acquisition time: 30 min, Cooling temperature: 100K.

Fig. 3: Cross-section SEM images of liquid foundation. With the mechanical probe at room temperature(a), and with the cryo mechanical probe at 118K(b). Instrument: NB5000 FIB-SEM, Acc. Volt.: 1.5 kV, Magnification: x10,000
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-1484 Finding the needle in the haystack: FIB-SEM combined with array tomography to achieve higher Z-resolution in selected areas

Wacker I. U.1, Bartels C.1, Grabher C.2, Schertel A.3, Schröder R. R.4
1Center for Advanced Materials, Universität Heidelberg, Heidelberg, Germany, 2Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany, 3Carl Zeiss Microscopy, Oberkochen, Germany, 4Cryo-EM, CellNetworks, BioQuant, Universitätsklinikum Heidelberg, Heidelberg, Germany
irene.wacker@bioquant.uni-heidelberg.de

Problems in cell or developmental biology often ask for ultrastructural characterisation of a small volume such as a rare event or a specialized substructure inside a large bulk specimen. We propose an intelligent workflow consisting of hierarchical imaging cascades, potentially also relying on different imaging modalities for different resolution ranges. Based on array tomography (AT) [1,2] this allows a stepwise zooming in to a structure of interest from light microscopy via conventional SEM to FIB-SEM.
As a first example we studied a mixed population of cells, a coculture of human tumor cells with immune cells isolated from Zebrafish. Ribbons of serial sections from chemically fixed, epon-embedded cell pellets were placed on silicon wafers and inspected in a reflected light microscope (Fig. 1a). Cell pairs consisting of a large tumor cell and a small fish cell (circle in Fig. 1a) were then imaged in a FEG-SEM (Fig. 1b) revealing immunological synapses between fish immune cells and human target cells. To further characterize their contact region we applied FIB-milling to selected sections to analyze at higher z-resolution only those regions of interest that enclosed centrosomes, Golgi complex, and other membrane-bound organelles (Fig. 1c).
Next we used our approach to identify a rare structure – the neuromuscular junction (NMJ) – within a large tissue block. Tibialis muscle from mouse was chemically fixed, embedded, and serially sectioned. In a single cross section containing hundreds of muscle cells usually only a few cells exhibit part of an NMJ (circle in Fig. 2a). Once an NMJ was found it was imaged in xy on the surface of the section, which in this case was nominally 1µm thick (Fig. 2b). Then FIB-stacks were produced from a 10µm x 10µm area with 10nm step size. Figure 2c shows several images of such a stack with one postsynaptic fold on the left and actomyosin filaments on the right. After alignment the 3D volume can be resliced in xy (Fig. 3a) or volume rendered (Fig. 3b).
Currently we are recording more stacks from corresponding regions of interest in consecutive sections. Fusion of individual stacks into a larger 3D volume allows observing the convoluted network of the postsynaptic folds at a resolution that allows unambiguous tracking of the membranes.
A combination of AT with FIB-SEM is a good approach whenever it is not necessary for a given problem to create a quasi-native molecular atlas of a cell or a total wiring diagram as needed in brain connectomics approaches. In many cases the region of interest is small enough to be amenable to analysis by FIB-SEM.

[1] Micheva and Smith (2007), Neuron 55, 25
[2] Wacker and Schröder (2013), J Microscopy 252, 93

We thank the German Federal Ministry for Education and Research, project NanoCombine, grants FKZ: 13N11401 and FKZ: 13N11403 for financial support.

Fig. 1: (a) Immunological synapse between Zebrafish immune cell and human cancer cell preselected in reflected light microscope; (b) imaged in SEM (Zeiss Ultra); (c) volume rendering of Golgi complex and centrosome in Amira, scale bars: 10µm (a), 1µm (b)

Fig. 2: Imaging of NMJ (Zeiss Auriga): (a) overview of a cross section from mouse leg muscle, circle shows muscle cell containing part of an identified NMJ; (b) postsynaptic folds (orange overlay) imaged on surface of 1µm thick section; (c) postsynaptic folds (orange) in FIB-stack; scale bars: 100µm in (a), 1µm in (b), (c)

Fig. 3: 3D reconstruction from FIB-stack: (a) stack resliced in xy; (b) volume rendering in Chimera
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-1517 Cross sectional sample preparation of nanowires for TEM analysis using FIB

Lenrick F.1, Ek M.1, Jacobsson D.2, Wallenberg L. R.1
1nCHREM / Center for Analysis and Synthesis, Lund University, Box 124, SE-221 00 Lund, Sweden, 2Division of Solid State Physics, Lund University, Box 118, SE-22100 Lund, Sweden
filip.lenrick@polymat.lth.se

Structuring of materials on the nanoscale is a common way to increase performance in a wide variety of devises. Nanostructures are a challenge for transmission electron microscopy (TEM) sample preparation, as they typically extend from the substrate without surrounding material. One solution has been to remove the nanostructures from their substrate and place them on a TEM grid. Although being simple and time efficient preparation method, it is not always adequate. If the nanostructure-substrate interface is of interest, the structures have a thickness of more than a few hundreds nm, or a TEM projection direction along a long axis is required, an alternative preparation method is necessary.
Here we report on methods for FIB sample preparation for TEM analysis of GaAs-GaInP core shell nanowires. By using polymer resin as support and protection we are able to produce cross-sections both perpendicular to and parallel with the substrate surface with minimal damage. Consequently nanowires grown perpendicular to the substrates could be imaged both in plan and side view, including the nanowire-substrate interface in the latter case. The nanowires, which are roughly 1.5 µm high and 350 nm in diameter, were grown on a GaAs substrate using metalorganic vapour phase epitaxy.
For plan view cross-section, the nanowires were first casted in a tablet-shaped mold using Spurr’s epoxy. The tablet was mechanically polished on one side until a section of the GaAs substrate was exposed. A TEM lamella was extracted using standard in-situ lift out method.
For side view cross-section, the nanowires were first covered in polymer resin by spin coating. Lithography resist proved to be a suitable resin as recipes for precise thickness are available from the resist manufacturers. The viscosity proved low enough not the bend the nanowires during spin coating. After spin coating the resin was soft baked on a hot plate, which increased the viscosity without affecting the nanowires. Finally, a TEM lamella was extracted using standard in-situ lift out method.

Fig. 1: a) TEM overview of plan view cross-section lamella. b) TEM image of upper nanowire marked with a black arrow in a). Shell and core have {011} facets. c) STEM HAADF image of lower nanowire cross-sections marked with a black arrow in a). The shell have both {011} and {112} facets. d) STEM XEDS maps from the red rectangle in a) (scale bar is 100 nm).

Fig. 2: a) TEM overview of side view cross-section lamella. b) HRTEM image of interface marked in a). c) diffractogram of image b). d) DFTEM images, colors correspond to diffractions spots marked in c). e) TEM image from tip of nanowire marked in a). f) DFTEM image from a wurtzite diffraction spot. g) DFTEM image from a zincblende diffraction spot.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-1667 Method For Improving FIB Prepared TEM Samples By Very Low Energy Ar+ / Xe+ Ion Mill Polishing

Kauffmann Y.1, Cohen-Hyams T.1, Kalina M.1, Kaplan W. D.1
1Department of Materials Science & Engineering, Technion IIT, Haifa, Israel
mtyaron@tx.technion.ac.il

The great progress in development of new transmission electron microscopes (TEM) during the last two decades has reached a point where the main limiting factor for obtaining fully quantitative and reliable information at the atomic scale is not the optics or the stability of the microscopes but rather the quality of the investigated specimen. The quality of a TEM specimen is determined by how thin and transparent it is to electrons, the surface roughness (variation in local thickness), and the amount of amorphization of the free surfaces (top, bottom and edges) of the specimen.

Well established methods for preparing TEM samples, such as mechanical polishing and electro-chemical polishing, are available. These methods provide very good quality samples when large structures or interfaces are present. When nano-scale site-specific investigation is needed, the best method available is the dual focused ion beam (FIB). This method uses a focused Ga+ ion beam to thin the area of interest down to few tens of nanometers. The ion bombardment of the specimen surface can introduce various artifacts, such as surface amorphization, Ga+ ion implantation, cratering and material re-deposition. These artifacts can be partially reduced by lowering the Ga+ ion energy down to 2 KeV.

For fully quantitative high resolution TEM studies, one needs to get the thinnest possible sample and remove completely all the artifacts introduced by the FIB. This can be achieved by further milling of the FIB sample using well controlled low energy (0.2-0.5 KeV) Ar+ / Xe+ ion milling.

Here we present a method to improve various FIB prepared TEM samples using low voltage ion polishing. This method provides a quick and fairly easy way to prepare high quality TEM samples for fully quantitative and reliable studies.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-1686 Focused ion beam sample preparation for atom probe tomography

Cherezova V.1, Chelpanov V.1, Kurushin V.1, Filatov A.1
1Systems for Microscopy and Analysis LLC, Moscow, Russia
v.a.kurushin@gmail.com

In recent times nanostructured multicomponent materials for which spatial distribution of chemical elements is crucial have been widely adopted. The most acceptable method for these materials is atom probe tomography, which allows identifying atom nature and its position in the volume of interest providing 3D imaging with sub-nanometer resolution. During material researching by tomographic atom probe the evaporation of atoms from the sample surface takes place in the process of high electric field. For the achieving of true information the main requirements are preferred for sample geometry: needle-shaped, tip radios, uniform circular cross-section, etc. The focused ion beam sample preparation permits to meet the before-mentioned needs in the optimum way. Now we describe existing FIB-based needle preparation technique (“lift-out”) from particle-reinforced materials for atom probe tomography. This procedure includes following stages: protective layer deposition onto the region of interest (ROI); milling of two regular cross section patterns on both sides of the ROI; the tilt of the stage and cut the lamella almost free from the bulk sample. Then the preparation is proceeded with using in-situ micromanipulator: it is inserted to the ROI; the lamella is attached to the probe using ion beam assisted Pt deposition and cut completely; the lamella is then transferred to a pre-prepared micropost. The next stage includes the lamella attaching (in preferred orientation) to the micropost and milling to detach from the micromanipulator. The final step is thinning of the made sample in order to sharpen its tip to aimed parameters and processing of the needle with low-energy ion beam to remove amorphous layer.
The advantage of chosen method of samples preparation for atom probe tomography is an opportunity to both orient them in parallel with initial surface and cut the sample from the need thickness.

Fig. 1: SEM micrographs (Quanta 3D FEG) of lamella, attached to micromanipulator and maneuvered to the micropost

Fig. 2: SEM micrographs of work piece attached to the micropost

Fig. 3: SEM micrographs of the needle during initial thinning

Fig. 4: SEM micrographs of the needle after final thinning
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-1712 Sample preparation using Xenon IC-Plasma FIB: benefits and problems.

Audoit G1, Estivill R2, Mariolle D1, Blot X1, Grenier A1, Barnes J P1, Cooper D1
1CEA, LETI, MINATEC Campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France., 2STMicroelectronics, 850 rue Jean Monnet, 38926 Crolles, France.
guillaume.audoit@cea.fr

For many types of specimens it is necessary to remove a large amount of material in order to provide an electron transparent region or a needle structure for atom probe tomography (APT). This is particularly true in the case of 3D Integrated Circuit development and manufacturing. This requirement has become sufficient to bring to the market a new generation of commercially available focused ion (FIB) tools that are equipped with inductively coupled Xenon plasma ion sources. This technology allows the generation of beam currents that are twenty times higher than those available using conventional FIBs that use a liquid metal (gallium) ion source. The use of xenon ions to prepare samples such as scanning electron microscope (SEM) cross-sections, transmission electron microscope (TEM) lamellae or even APT needles is attractive because of the theoretical reduced damage of xenon when compared to gallium. Figure 1 shows SRIM simulations of Xe, Ga and Ar ions with different energies in silicon at an angle of incidence of 5° normal to the specimen surface [1]. The simulations suggest that the range of the ions is significantly less for Xe ions. Although the simulations do not account for effects such as channeling in a crystalline specimen, the link between specimen damage and the SRIM simulations has been verified [2]. In this presentation we will introduce the Xe plasma milling system and present measurements of the implantation of Xe ions in Silicon compared to Ga and Ar. For example Figure 2 shows amorphisation and ion implantation profiles have been measured using TEM and APT measurements as a function of the accelerating voltage on silicon and compare to TRIM calculations. We will show the results of specimen preparation using Xe ions, for example of materials that are sensitive to gallium like GaAs and InP which tend to form eutectic compounds that precipitate under Gallium implantation and local heating. Scanning spreading resistance microscopy (SSRM) measurements on InP/GaAs samples cross sectioned with Xenon ions have been compared to Ga-FIB prepared samples in order to compare the sample surface in terms of roughness and dead layer for electrical measurements. Xenon Plasma-FIB specimen preparation also has drawbacks due to the large beam size diameter that has been quantified in this study. We believe that using Xe plasma FIB could open up applications for site specific time of flight (ToF)-SIMS, Auger and XPS analysis, for which the use of a Ga-FIB is impracticable for the production of large enough surface areas, i.e. few hundreds of micrometers.

We thank the Recherche Technologique de Base (RTB) program (national network of large facilities for Basic Technological Research) and the nanocharacterization platform (PFNC).

Fig. 1: Figure 1(a): SRIM simulation of Ar, Xe, and Ga range in silicon as a function of the energy for an angle of incidence of 5°. (b) Magnified plot indicated in (a) by the red shaded region.

Fig. 2: Figure 2: SEM view of an APT needle of silicon milled with 30kV xenon ions and a reconstructed volume showing the Xenon penetration.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-1838 FOCUSED ION BEAM LITHOGRAPHY OF SUBWAVELENGTH PHOTONIC 3D-CHIRAL STRUCTURES

Artemov V. V.1, Rogov O. Y.1, Gorkunov M. V.1
1Institute of Crystallography RAS
artemov@ns.crys.ras.ru

The focused ion beam (FIB) milling is a powerful tool for fabricating nanoscale photonic structures [1]. As the next step after successful fabrication of lamellar optical gratings with subwavelength periods [2], we employ the FIB technique to produce truly 3D patterned nano-scale structures. The report describes fabrication and analysis of periodic subwavelength arrays of 3D-chiral holes in a freely suspended silver film.
In order to generate digital templates for FIB lithography (‘stream files’) a special numerical routine has been developed. The templates contain the ion beam waypoints’ coordinates and their ‘dwell time’. Accordingly, all the desired characteristics such as the form, dimensions and the etching depth of a single element can be set. Employing this method allowed us to fabricate periodic arrays of 3D-chiral holes in the freely suspended 200 nm thick silver film with a total processed area of 30x30 μm2 and one element size of 300 nm. Fig. 1a features a 3D lithography model of a single chiral element. The X, Y, and Z coordinates correspond to those of waypoints in the template and the dwell time, respectively. Fig. 1b shows a micrograph of the fabricated structure tilted by 52°, and Fig. 1c shows a normal view of the structure. The fabricated structures have proven to exhibit significant optical activity and circular dichroism.

[1] C. Enkrich, F. Perez-Williard, D. Gerthsen, J. Zhou, T. Koschny, C.M. Soukoulis, M. Wegener, S. Linden, Focused-ion-beam nanofabrication of near-infrared magnetic metamaterials, Adv. Mater. 17 (2005) 2547.
[2] M.V. Gorkunov, V.V. Artemov, S.G. Yudin, S.P. Palto, Tarnishing of silver subwavelength slit gratings and its effect on extraordinary optical transmission, Phot. Nanostr. Fund. Appl. (2013) http://dx.doi.org/10.1016/j.photonics.2013.10.001.

This research was financially supported by the RFBR No. 13- 02-12151 ofi_m and the RAS Presidium program 24. We are grateful to A. L. Vasiliev for the access to the FEI Helios microscope.

Fig. 1: 3D model of the chiral structure unit cell as implemented into the FIB milling digital template (a), SEM micrograph of the fabricated structure tiled by 52° (b), normal view of the fabricated structure (c). 
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-1861 Measurement of TEM lamella thickness and Ga implantation in the FIB

Lang C.1, Hiscock M.1, Dawson M.2, Hartfield C.2, Statham P. J.1
1Oxford Instruments NanoAnalysis, High Wycombe, UK, 2Oxford Instruments NanoAnalysis, Dallas, USA
matthew.hiscock@oxinst.com

Accurate control over sample thickness and quality is paramount in order to take full advantage of the ever increasing resolution in aberration corrected TEMs. For instruments combining a focused ion beam with an electron beam methods based on either back scattered electron contrast [1] or transmissivity of electrons [2] have been demonstrated for measuring the sample thickness. However, these methods only work on homogenous samples without compositional variations. They also don’t provide any information on the degree of ion implantation.

Here we show a method that uses X-rays generated by the electron beam - lamella interaction to accurately and rapidly measure the lamella composition and thickness. In order to measure the thickness and composition of the lamella, we used Oxford Instruments’ AZtec LayerProbe software [3] and X-Max 150 EDS detectors to acquire and process EDS spectra. LayerProbe refines a starting model of the sample structure against the EDS spectra to calculate the film thickness and composition of the layers. The first layer is defined as the material comprising the lamella. The top layer can be defined to contain the element used as the ion source (e.g. Gallium) to obtain a measure of the degree of ion implantation in the specimen.

Fig. 1a shows an electron image of a TEM lamella prepared from a Ni based superalloy . Fig. 1b shows a surface plot of the lamella thickness and Fig. 1c the Ga thickness calculated from a grid of EDS spectra. The thickness of the lamella is clearly decreasing from the area close to the weld towards the free end of the lamella with the lowest thickness of the lamella measured at around 75nm. The Ga thickness profile shows a different trend with an increase Ga thickness close to the left lower corner and also close to the weld. Fig. 2 shows an X-ray map of a TEM lamella prepared from a silicon semiconductor device. The device structures containing Cu and W are clearly visible in the X-ray maps. One of the Cu lines fades and disappears from the right side to the left of the lamella indicating that the line runs at an angle to the direction of the FIB cut. With LayerProbe it is possible to measure the projected Cu thickness and Si thickness from X-ray spectra reconstructed from the X-ray map. By comparing measurements taken from the right side of the lamella with measurements towards the left side we can see how the thickness increase of the lamella affects the ratio of device vs surrounding Si matrix for both the W and Cu rich device areas.

[1] A. R. Hall, Microscopy and Microanalysis 19 (2013), p. 740.
[2] U. Golla-Schindler, Conference Proceedings EMC (2008), p 667.
[3] C. Lang et al., Microscopy and Microanalysis 19 (2013), p. 1872.

Fig. 1: (a) shows an electron image of a TEM lamella of Ni superalloy 600 and the area for which the lamella thickness in (b) and the equivalent Ga thickness (c) have been calculated.

Fig. 2: The local lamella thickness as well as the contribution of different device layers to this thickness was calculated from spectra reconstructed from an X-ray map.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-1915 Advances in ex situ lift out

Giannuzzi L. A.1
1EXpressLO LLC
lucille.giannuzzi@expresslo.com

The focused ion beam (FIB) ex situ lift out (EXLO) technique for scanning/transmission electron microscopy (S/TEM) specimen preparation was historically the first lift out technique developed [1]. EXLO is well known for its ease, speed, and reproducibility, and is perfectly suited for manipulation of electron transparent specimens to carrier devices developed for in situ S/TEM testing as shown in figure 1a. Using EXLO for manipulation to a conventional carbon coated grid limits the specimen from being further FIB milled and inhibits certain S/TEM techniques. The development of a patent pending grid design and technique called EXpressLO™ allows EXLO and manipulation without needing a carbon film support [2-4]. The specimen is lifted out and manipulated directly to a slotted S/TEM grid surface such that the specimen may be directly analyzed and/or further FIB milled, broad beam ion milled or plasma cleaned. Using this new grid design, a specimen can also be manipulated easily into a backside orientation which avoids curtaining artifacts after further FIB milling [3]. The Xe+ ion plasma FIB (PFIB) is capable of producing electron transparent specimens for S/TEM [5]. The EXpressLO™ method can also be used for manipulating large PFIB prepared specimens as shown in figure 1b where a 50 micrometer long specimen is manipulated to a grid [6]. The 1 micrometer thick PFIB specimen manipulated to the EXpressLO™ grid can be further milled using conventional Ga+ ion FIB or a PFIB. EXLO is now flexible and continues to be fast and reproducible which saves labor and FIB instrumentation time, ultimately reducing the cost per specimen.

References:

[1] L.A. Giannuzzi, J.L. Drown, S.R. Brown, R.B. Irwin, F.A. Stevie, Mat. Res. Soc. Symp. Proc. Vol. 480, Workshop on Specimen Preparation for TEM of Materials IV, (1997), Materials Research Society, p. 19-27.

[2] L.A. Giannuzzi, Microscopy and Microanalysis 18, Supp 2, (2012) 632-633.

[3] Lucille A. Giannuzzi, Proceedings of ISTFA, ASM International. (2012), 388-390.

[4] L.A. Giannuzzi, Microscopy and Microanalysis 19, Supp 2, (2013) 906-907.

[5] L.A. Giannuzzi and N.S. Smith, in press, Microscopy and Microanalysis 20, Supp 2, (2014)

Qiang Xu from DENSsolutions provided samples and the in situ MEMS carrier device shown in figure 1a. Noel Smith from Oregon Physics provided the PFIB prepared specimen shown in figure 1b.

Fig. 1: (a) electron transparent specimen manipulated to a DENSsolution in situ carrier device via EXLO. (b) PFIB specimen manipulated via EXLO using the EXpressLO™ method and grid.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-1929 Influence of FIB milling on the determination of sp2/sp3 ratio of carbon materials

Zhang X.1, Schneider R.1, Müller E.1, Mee M.2, Meier S.2, Gumbsch P.1, 2, Gerthsen D.1
1Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany, 2Fraunhofer Institute for Mechanics of Materials IWM, Freiburg, Germany
xinyi.zhang2@kit.edu

In focused-ion-beam (FIB) preparation of TEM samples, the energetic Ga+ beam may have damaged the original structure of both sides of the cross-section specimen. Damaged cover layers of amorphous structure are expected for FIB lamellae of carbon materials with modified bonding configurations. Quantitative ELNES technique is well-established for bonding-configuration analysis of carbon. However the sensitivity of this technique is limited by the inevitable FIB-induced damage. Here we propose a simple mathematical model to correct this damage influence on the determination of sp2/sp3 ratios of carbon materials. And the model is tested for HOPG and DLC films with different fractions of sp2 bonds.
The bonding configuration throughout the sampled material column can be considered as a linear combination of those of the damaged layers on both sides and the bulk. Assuming that the damaged cover layers are of uniform thickness and ignoring the local difference in the bonding configurations in the damaged layers and the bulk material, a linear relationship can be derived between the Iπ*/Iσ* ratio for HOPG (or sp2 % for DLCs) obtained from the C-K edge spectra and 1/t, where t indicates the relative thickness obtained from corresponding low-loss spectra. Consequently, the intercept is the real Iπ*/Iσ* ratio (or sp2 %) for the bulk.
FIB preparation for HOPG and DLC samples was followed by a standard lift-out technique. 30 keV Ga+-ions were used for thinning and during the final stage a high tension of only 5 kV was applied to minimize the damage. C-K edge EELS spectra were taken at magic angle (MA) conditions. The cleaved HOPG specimen was largely kept perfect in graphite crystallinity and thus provides as a standard for the FIB-prepared HOPG.
The difference between the FIB-prepared HOPG and the standard is reduced from as high as 20 % to 4 % after the correction. Fig. 1 demonstrates the original quantitative EELS results of two DLCs as a function of 1/t. The DLC (a-C:H) with high sp2 % (69 %) shows little discrepancy with the thickness variation (see red symbols in Fig. 1) and is in accordance with the Raman study (70 %). Therefore, it could imply that the damaged a-C layer contains the same fraction (~ 70 %) of sp2-hybridized C-atoms. Seen from the black symbols in Fig. 1, the ta-C film with lower fraction of sp2 bonding shows a larger dispersion of sp2 % from 39 % to 60 % with respect to t ranging from 0.4 to 1.4, however a linear relationship is indeed found and the sp2 % is corrected to 33 ± 1.3 % by the model. Further assuming that the sp2 % of the FIB-damaged layer is ~ 70 % for all carbon specimens, the damaging depth on each side are estimated to be ~ 15 nm for the HOPG lamella and ~ 10 nm for the ta-C one.

XZ acknowledges funding from China Scholarship Council (CSC) (No. 2010606030). PG acknowledges support from Deursche Forschungsgemeinschaft DFG (project grant Gu 367/30).

Fig. 1: MA-EELS quantification of sp2 % for the a-C:H and ta-C DLCs as a function of the reciprocal of the relative thickness (1/t). Dashed lines are linear fitting results for each sample.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2055 Optimized Detection Limits in FIB-SIMS by Using Reactive Gas Flooding and High Performance Mass Spectrometers

Wirtz T.1, Dowsett D.1, Philipp P.1, Eswara Moorthy S.1
1Department “Science and Analysis of Materials” (SAM), Centre de Recherche Public – Gabriel Lippmann, 41 rue du Brill, L-4422 Belvaux, Luxembourg
wirtz@lippmann.lu

FIB-based instruments play a crucial role in materials science and also in life science. While such FIB instrumentation is an ideal tool for high resolution imaging and nanofabrication, its analysis capability is currently limited. By contrast, Secondary Ion Mass Spectrometry (SIMS) is an extremely powerful technique for analyzing surfaces given its excellent sensitivity, high dynamic range, high mass resolution and ability to differentiate between isotopes. Adding SIMS capability to FIB instruments offers not just the prospect of obtaining SIMS information limited only by the size of the probe-sample interaction (~10nm) but also enables a direct correlation of such SIMS images with high resolution secondary electron images of the same zone taken at the same time (correlative microscopy).

Past attempts of performing SIMS on FIB instruments were rather unsuccessful due to unattractive detection limits, which were due to (i) low ionization yields of sputtered particles, (ii) extraction optics with limited extraction and collection efficiency of secondary ions and (iii) mass spectrometers having low duty cycles and/or low transmission. In order to overcome these limitations, we have investigated the use of reactive gas flooding during FIB-SIMS and we have developed compact high-performance magnetic sector mass spectrometers with dedicated high-efficiency extraction optics.

Our results show that the yields obtained with Ga+, He+ and Ne+ bombardment, which are intrinsically low compared to the ones found in conventional SIMS, may be drastically increased (up to 4 orders of magnitude) by using reactive gas flooding during analysis, namely O2 flooding for positive secondary ions and Cs flooding for negative secondary ions (Figure 1) [1-3]. The resulting detection limits vary from 10-3 to 10-6 for a lateral resolution between 10 nm and 100 nm (Figure 2).

The emitted secondary ions are extracted by dedicated optics which we have designed for several FIB-based instruments and injected into a specially designed compact high-performance magnetic sector double focusing mass spectrometer. The obtained extraction efficiency ranges from 40% to 100% while successfully avoiding any artefacts (broadening or distortion) regarding the primary ion beam. The specifications of the mass spectrometer include highest transmission (100%), high mass resolution (M/DM > 2000), full mass range (H-U) and parallel detection of several masses.

Here we will present the FIB-SIMS systems we have developed, give an overview of the obtained performances and present typical examples of applications.

References

[1] P. Philipp et al., Int. J. Mass. Spectrom. 253 (2006) 71

[2] T. Wirtz et al., Appl. Phys. Lett. 101 (2012) 041601

[3] L. Pillatsch et al., Appl. Surf. Sci. 282 (2013) 908

Fig. 1: Enhancement of secondary ion yields using reactive gas flooding under He+ and Ne+ bombardment.

Fig. 2: Detection limit using a Ga+ FIB with and without Cs0 flooding vs. minimum feature size: example for the detection of Si-.

Fig. 3: Ga+ FIB-SIMS image of the Ca distribution in skin cells (field of view: 50x50 µm2)
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2068 Characterization of carbonaceous contamination and the cleaning capability of atomic hydrogen during focused ion beam processing

Steiger-Thirsfeld A.1, Basnar B.2, Tomastik C.3, Pongratz P.4, Lugstein A.2
1University Service Center for Transmission Electron Microscopy, Vienna University of Technology, Vienna, Austria, 2Institute of Solid State Electronics, Vienna University of Technology, Vienna, Austria, 3AC2T Research GmbH Austria, Wr. Neustadt, Austria, 4Institute of Solid State Physics, Vienna University of Technology, Vienna, Austria
steiger@ustem.tuwien.ac.at

During focused ion beam (FIB) milling undesired peculiar depositions (Fig. 1a), from mainly carbon compounds of the residual gas at the edge of ion beam exposed regions, occur. Surface diffusion of residual gas molecules and the reduced ion dose are causing enhanced residual gas deposition rates at the boundary area of ion beam exposed regions [1]. Especially for sputtered structures with dimensions in the nanometer range, these unwanted ion beam induced depositions (IBID) can be of the same order of magnitude as the intended structures, therefore they must be eliminated.

Preliminary, we have characterized the dynamics of this contamination growth. Crystalline (c-Si) and amorphous Si (a-Si) were irradiated by a 50 kV Ga+ ion beam, with various scanning parameters, under high vacuum conditions (2.7 × 10-7 mbar). A refresh time variation in several steps from 23 ms to 15 s was performed with a dwell time of 1 μs (Fig. 1b). Atomic force microscopy (AFM) measurements reveal that for refresh times longer than 1 s, IBID entirely inhibits the net volume loss by sputtering. The residual gas deposition rate saturates for a refresh time of about 5 s (Fig. 2a). Dwell time variations from 0.5 μs to 10 μs at a refresh time of 5 s show the expected decreasing in deposition rate. The pronounced indented shape of the deposits exhibits the influence of surface diffusion. A surface diffusion constant in the order of 10-9 cm2/s was roughly estimated. Auger electron spectroscopy (AES) depth profiles of the chemical composition of low dose ion irradiated c-Si show a mixture of Si, C, and O with decreasing C and O concentrations from top to the bottom of the deposits (Fig. 2b). The investigated depositions consist roughly of of 22 at% C and 2 at% O in addition to Si.

When an atomic hydrogen (H*) gas beam, generated by a thermal cracker source [2], has been delivered to a c-Si surface during FIB processing, reduced swelling heights in the range estimated from the difference in mass density of amorphous and crystalline Si were determined (Fig. 3a). Moreover, the H* delivery suppresses the residual gas deposition, even for refresh times in the range of seconds (Fig. 3b). We conclude that simultaneous Ga+ and H* bombardment removes adsorbed carbon compounds and the additional H* delivery impedes the surface diffusion of adsorbed residual gas molecules. Thus a significant build up of contamination can be avoided.

[1] J.B. Wang, A. Datta, Y.L. Wang; Applied Surface Science 135 (1998) 129-136
[2] K. G. Tschersich, J.P. Fleischhauer, H. Schuler; Journal of Applied Physics 104, 034908 (2008)

This work has been supported by the EC (FP6, CHARPAN, Contract no.: IP 15803-2).

Fig. 1: AFM images of ion beam induced residual gas depositions. 1 (a) Undesired IBID at the edge of a dot pattern. 1 (b) Indented IBIDs on c-Si for fluence values in the range of 6.9 × 1013 ions/cm2 to 2.1 × 1016 ions/cm2 with a refresh time of 5 s.

Fig. 2: Characterization of ion beam induced residual gas depositions. 2 (a) Deposition height dependence on ion fluence with the refresh time as parameter measured by AFM. 2 (b) AES depth profile of the chemical composition of low fluence (6.9 × 1014 ions/cm2) ion irradiated c-Si.

Fig. 3: Cleaning effect of atomic hydrogen during FIB processing. 3 (a) Comparison of height (depth) characteristics of FIB processed areas in c-Si and a-Si with and without additional H* delivery. 3 (b) Comparison of height (depth) characteristics of FIB processed areas at a refresh time of 5 s with and without additional gas delivery.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2075 Nanopatterning Plasmonic Structures Using Focused Ion Beam and E-Beam Lithography

Cohen Hyams T.1, Spektor G.2, Gal L.2, Orenstein M.2
1Department of Materials Science & Engineering, Technion, Haifa, Israel, 2Department of Electrical Engineering, Technion, Haifa, Israel
tzipic@technion.ac.il

The Focused Ion Beam (FIB) system uses a Ga+ ion beam to raster over the surface of a sample in a similar way as the electron beam in a scanning electron microscope (SEM). One of the capabilities of FIB is its ability to mill complex nanopatterns (including bitmapped images), making it the ideal tool for precise 3D maskless nanopatterning of a wide variety of materials. The FIB is the ideal tool for prototyping a wide range of devices in the R&D stage of product development, since it offers high reproducibility and scalable throughput. However, the ion bombardment of the specimen surface can introduce various artifacts, such as surface amorphization, Ga+ ion implantation, cratering and material re-deposition.

Electron-beam lithography is an alternative tool for 3D nanopatterning. E-Beam lithography uses a focused beam of electrons to “write” patterns on a surface covered with an electron sensitive film (resist). The main advantage of electron-beam lithography is that it can direct-write with nm resolution. This form of maskless lithography has high resolution and low throughput.

These two techniques are considered to be the best methods for fabricating structures for surface plasmon coupling and manipulation. The structures can be used to obtain confined longitudinally polarized plasmonic focal spots and other higher order effects.
In this study, we present a comparison between FIB nanopatterning and E-beam lithography to fabricate various nano engraved plasmonic structures in gold comprising different spiral types and their engagements.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2432 Optimization of the sample preparation method for semiconductor dopant contrast observation with SEM

Druckmüllerová Z.1,2, Kolíbal M.1,2, Vystavěl T.3, Šikola T.1,2
1Institute of Physical Engineering, Brno University of Technology, Brno, Czech Republic, 2CEITEC, Brno University of Technology, Brno, Czech Republic, 3FEI Company, Brno, Czech Republic
zdenadr@seznam.cz

Since semiconductor devices are being scaled down to dimensions of several nanometers, there is a growing need for techniques capable of quantitative analysis of dopant concentrations at nanometer scale in all three dimensions. Therefore we optimized the sample preparation methodology for imaging dopant contrast by scanning electron microscopy (SEM) at incident electron energies about 1keV [1], which enables to visualize and analyze dopant concentration changes. SEM analysis at such conditions became widely used providing promising results, but many unresolved issues hinder its routine application for device analysis, especially in case of buried layers where the site-specific sample preparation is challenging. We report on optimization of a site-specific sample preparation by the focused Ga ion beam (FIB) providing an improved dopant contrast in SEM. As a testing sample we used differently doped multilayer structure deposited on Si (see Fig. 1). Similarly to the lamella preparation for transmission electron microscopy by FIB, a polishing sequence with decreasing ion energy is necessary to minimize the thickness of the electronically dead layer [2]. We have achieved the contrast values comparable to the cleaved sample, being able to detect dopant concentrations down to 1x1016 cm-3.(see Fig. 2). A theoretical model shows that the electronically dead layer corresponds to an amorphized Si layer [3] formed during ion beam polishing. Our results also demonstrate that the contamination caused by electron beam scanning is significantly suppressed for focused-ion-beam treated samples compared to the cleaved ones.

References:
[1] Chakk Y. & Horvitz D. (2006). Contribution of dynamic charging effects into dopant contrast mechanisms in silicon. J. Mat. Sci. 41, 4554-4560.
[2] Giannuzzi L. A., Geurts R. nad Ringnalda J. (2005). 2kV Ga+ FIB milling for reducing amorphous damage in silicon. Microsc. Microanal. 11 suppl.2, 828-829.
[3] Kazemian P., Twitchett A. C., Humphreys J. C. & Rodenburg. C. (2006b). Site-specific dopant profiling in a scanning electron microscope using focused ion beam prepared specimens. Appl. Phys. Lett. 88, 212110.

Sample was provided by Cornelia Rodenburg, PhD., University of Sheffield, Great Britain. This work was supported by the Grant Agency of the Czech Republic (P108/12/P699) and by European Regional Development Fund – (CEITEC - CZ.1.05/1.1.00/02.0068). M. K. and Z.D. acknowledge the support of FEI Company.

Fig. 1: Description of the sample used: SEM image of the cleaved multilayer structure deposited on n-doped silicon substrate is in the middle, a schematic description of alternating layers of intrinsic silicon (150 nm) with p-doped layers in depicted on the left. The contrast profile normalized to n-doped substrate is shown on the right.

Fig. 2: SEM contrast improvement of sample cut by Ga FIB using decreasing final polishing energy.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2440 Channeling contrast: a cost effective alternative to EBSD orientation mapping in scanning focused probe instruments (SEM/FIB) ?

Langlois C. T.1, Yuan H.1, Douillard T.1, Van de Moortele B.2, Descamps-Mandine A.3, Blanchard N.4, Epicier T.1
1MATEIS Laboratory, INSA Lyon, France, 2LGL laboratory, Ecole Normale Supérieure Lyon, France, 3INL Laboratory, INSA Lyon, France, 4Light Matter Institute, Claude Bernard University of Lyon, France
cyril.langlois@insa-lyon.fr

Generally, for grains at the micron scale, orientation maps are obtained by Electron Back Scattered Diffraction (EBSD) in a Scanning Electron Microscope (SEM). For various reasons, orientation maps with the EBSD technique can be challenging: material properties (conductive or not, preparation problems), geometrical setup (impossibility to collect the signal), pseudo-symmetry Kikuchi diffraction patterns, nano-sized structures, large area mapping, etc. In this context, it is worth investigating other means to map the crystallographic orientations of grain.
Channeling contrast is a well-known phenomenon allowing grains of a polycrystalline sample to be distinguished, even if only one phase is present. Depending on the orientation of the crystallographic planes, the secondary and backscattered electron yields vary from one grain to the other, resulting in different intensities received by the detectors. For this reason, the contrast of each grain varies when the orientation of the sample is changed [3].
We show in this study how it is possible to use this channeling effect (with an electron or an ion beam) to obtain the three Euler angles characterizing the orientation at a given point of the sample surface. The main concept is to obtain an intensity profile at that point, to compare the intensity profile to a semi-empirical database of profiles and to find the best fit, i.e. the three Euler angles associated with this point. Comparing with EBSD measurements on the same area allowed us to determine the precision of the indexation, which is better than 5°. We discuss then the best way to obtain an intensity series, either by tilting or rotating the sample, with regards to acquisition stability and unicity of the indexation. The issue of acquisition time is also discussed, and an example of our indexation method based on a 30 sec movie over 360° is shown. We conclude by evaluating the pros and cons of using ions or electrons for such indexation purposes.
[1] Estimation of recrystallized volume fraction from EBSD data, J. Tarasiuk, Ph. Gerber and B. Bacroix, Acta Materialia (2002) 50 1467–1477
[2] Characterization of the Grain-Boundary Character and Energy Distributions of Yttria Using Automated Serial Sectioning and EBSD in the FIB, S.J. Dillon and G.S. Rohrer, J. Am. Ceram. Soc. (2009) 92 1580–1585
[3] Crystallographic orientation contrast associated with Ga+ ion channeling for Fe and Cu in focused ion beam method, Y. Yahiro, K. Kaneko, T. Fujita, W.-J. Moon and Z. Horita, J. Electron Microscopy (2004) 53 571–576

Fig. 1: Snapshot of the software written to extract intensity profiles from a tilt series and Euler angles from an EBSD map acquired on the same area

Fig. 2: Snapshot of the software written to compare the experimental profile (green), the semi-empirical profile corresponding to EBSD orientation (red), and the best fit semi-empirical profile found in our database (blue). In this case, the disorientation between the EBSD orientation and the ‘best fit’ orientation is around 3°only
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2481 Cryogenic FIB Lift-out as a preparation method for damage-free soft matter TEM imaging 

Parmenter C. D.1, Fay M. W.1, Hartfield C.2, Amdor G.2, Moldovan G.2
1University of Nottingham, 2Oxford Instruments Nanoanalysis
christopher.parmenter@nottingham.ac.uk

We have demonstrated that it is possible to prepare and remove a thinned lamella and transfer to the TEM, whilst maintaining cryogenic conditions. Once further refined, this method offers the possibility of compression and stain artefact free imaging of soft matter samples (cells, tissues, plant samples, polymers, gels etc) preserved and maintained at cryogenic temperatures. Biological samples contain a high degree of water, which dehydrate under vacuum. Solutions are: critical point drying, resin impregnation with heavy metal stains or cryogenic fixation. Once stabilised the samples can be prepared with an ultramicrotome to yield electron transparent sections, however, they commonly suffer from compression and/ or knife artefacts. In addition, there is a desire to move away from staining or methods which can induce structural re-arrangement. The removal of a thinned lamella from a bulk sample for Transmission Electron Microscopy (TEM) analysis has been possible in the Focused Ion Beam Scanning Electron Microscope (FIB-SEM) for over 20 years either via in-situ (by use of a micromanipulator) or ex situ lift-out approaches [1]. Both are currently only applied to samples at room temperature as there are a number of technological and sample handling issues for cryogenic samples. Recent efforts have demonstrated cryo lift-out is possible for materials samples[2]. This work further extends the development of cryo lift-out to allow label and damage-free imaging of soft and biological structures. To preserve the vitreous nature of the water in cryo-preserved samples the temperature should be maintained below -140°C and the probe tip held by the manipulator cooled to at least -130°C. To achieve this, an OmniProbe 100 was modified with a thermal break and cooling braid, which was attached to the cold finger of the cryo stage (Quorum PPT 2000).

Prior to lamella extraction, an alginate-collagen hydrogel, was sputter-coated with platinum and a tungsten layer from a gas injector. The gel was milled using a modified TEM lamella protocol to approximately 2μm thickness, before the lamella was attached to the cooled tip by cryo-condensation of water via a gas injector (figure 1). The lamella was subsequently secured to a TEM (lift-out) support grid (figure 2) and further thinned to electron transparency (figure 3). The sample was transferred under liquid nitrogen to a cryo-TEM holder and imaged at 200 kV in both bright and dark field imaging (figure 4). 

[1] L Giannuzzi et al. in “Introduction to Focused Ion Beams: Instrumentation, Theory, Techniques and Practice”, ed. LA Giannuzzi and FA Stevie, (Springer, 2005) Chapter 10, p.201-228.

[2] N Antoniou et al, Conf. Proc. 38th Int. Symp. Testing and Failure Analysis (2012) p. 399-405.

Many thanks to Dr James Dixon (University of Nottingham, CBS) for supplying the hydrogel samples.

Fig. 1: Cryo-FIB milling of a bulk sample to prepare a thin lamella, scale bar 5 µm

Fig. 2: Extraction of the lamella by the cooled manipulator after attachment and release of lamella, scale bar 10 µm.

Fig. 3: Micrograph of the attached and thinned lamella, scale bar 5 µm

Fig. 4: Dark field TEM image of collagen fibrils in an alginate hydrogel matrix, scale bar 100 nm
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2485 Characterization of Ga+ FIB Damage in Electron Beam Induced Deposited Platinum, Tungsten and Carbon Chemistries for In-situ S/TEM Sample Preparation

Van Leer B.1, Landin T.1, Wall D.1, Roussel L.1
1FEI
brandon.van.leer@fei.com

TEM/STEM sample preparation by focused ion beam (FIB) and SEM/FIB instrumentation has become routine in the last several years. Technology advances in automation and in-situ techniques have reduced preparation times for sub-50 nm lamellae to less than an hour and with state-of-the-art technology less than 30 minutes [1]. DualBeams (FIB-SEM) are also often used for micro- and nanoprototyping applications.  For S/TEM sample preparation electron or ion beam induced deposition (EBID, IBID) is required to planarize the region of interest to minimize artifacts generated by the FIB [2].

Many studies have investigated surface and sidewall lamella damage in Silicon by FIB [3]. In addition to sidewall damage by FIB for cross-sections or FIB processed S/TEM samples, surface damage must also be considered for FIB preparation especially when characterization of the sample surface is required. It has been shown that low energy electron beam induced deposition (EBID) imparts the smallest surface damage when compared to Ga+ ion beam induced deposition (IBID) [4]. However, the rate of deposition with EBID is ~ 20X slower than IBID, thus understanding the damage depth into EBID layer during the FIB deposition process will reduce process time for cross-section or S/TEM sample preparation.

Approximately 100 nm EBID C, W and Pt layers were deposited onto Si using 5 keV; 6.3 nA (C and W) and 2 keV; 6.3 nA (Pt). 30 keV Ga+ IBID C, W and Pt layers were deposited over the layers of interest and FIB prepared for STEM in SEM analysis. A 5 keV Ga+ FIB Pt IBID over 2 keV Pt EBID sample was also prepared. Each face of the lamellae was FIB milled using Ga+ ions at 30 keV and 88.5 degrees incident angle, followed by 5 keV at 85 degrees incident angle. Figures 1a and 1b are STEM in SEM images of the Pt and C experiments respectively. Measurements (Figure 2) reveal that IBID tungsten penetrated approximately 15 nm into the EBID tungsten while the IBID platinum penetrated more than 3X deeper into EBID platinum. This value decreased to approximately 16 nm when 5 keV IBID was employed for Pt.

[1] D. Wall, “Ultra-Fast In-Situ Sample Preparation.” FEI P/N 04AP-FR0111, FEI Company, 2007.
[2] Introduction to Focused Ion Beams, eds. L.A. Giannuzzi and F.A. Stevie, Springer (2005).
[3] L. A. Giannuzzi et al., Micros. Microanal., 11(Suppl 2) (2005), p. 828.
[4] B.W. Kempshall et al., J. Vac. Sci. Tech. B, 20(1) (2002) 286.

Fig. 1: 30 keV STEM in SEM images of surface 30 keV Ga+ FIB damage during Pt IBID into EBID Pt

Fig. 2: 30 keV STEM in SEM images of surface 30 keV Ga+ FIB damage during C IBID into EBID C

Fig. 3: Average FIB damage depth (nm) during IBID of Pt, W and C over EBID Pt, W and C
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2498 FIB/SEM tomography for 3D visualization of virus infected fibroblasts with TEM-like resolution.

Villinger C.1, 2, Neusser G.3, Kranz C.3, Walther P.2, Mertens T.1
1Institute of Virology, University Medical Center Ulm, Germany, 2Central Facility for Electron Microscopy, Ulm University, Germany, 3Institute of Analytical and Bioanalytical Chemistry, Ulm University, Germany
clarissa.villinger@uni-ulm.de

Keywords: FIB/SEM tomography, high pressure freezing, freeze substitution

Focussed ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel electron microscopy technique that is increasingly used within life sciences. Here we present its application for ultrastructural visualization of fibroblasts infected with human cytomegalovirus (HCMV). For that we employed optimized sample preparation protocols including high pressure freezing and freeze substitution. The result was an improved ultrastructural preservation and a high image contrast. Additionally, our well established embedding procedure of cell monolayers in Epon allows not only ultrathin sectioning but also sample preparation for FIB/SEM tomography. The detection of the secondary electron signal allows us to resolve cellular and viral ultrastructures down to the level of lipid bilayers (Fig. 1).

The main focus of our work is the egress of HCMV capsids from the nucleus (capsid formation and genome packaging) into the cytoplasm (further virion maturation). The diameter of virus capsids exceeds the diameter of nuclear pores. Hence, the virus has to find an alternative path to leave the nucleus. It is known that this process is made possible by an envelopment and de-envelopment process at the inner (INM) and outer nuclear membrane (ONM), respectively [1, 2]. We visualized a portion (z=5 µm) of an HCMV infected nucleus with FIB/SEM tomography. The resulting 3D reconstruction showed that the surface area of the INM was increased through large infoldings which can extend deep into the nucleoplasm (Fig. 2). DNA free and DNA filled capsids were both present within these infoldings (perinuclear capsids). This model has already been postulated based on 2D TEM images [3]. Our 3D data now confirm this model. Additionally, the slice thickness of 20 nm allowed imaging of every nuclear as well as perinuclear capsid within the analyzed volume. This gives the opportunity to analyze the capsid distribution in 3D, thus, making the results and interpretation of 2D TEM images more accurate. In conclusion, our FIB/SEM data provide a detailed image of the nuclear stages of HCMV morphogenesis, from capsid assembly and DNA packaging to capsid egress.

[1] Skepper JN et al. (2001).J. Virol. 75, 5697–5702.

[2] Mettenleiter TC et al. (2013). Cell. Microbiol. 15, 170–178.

[3] Buser C et al. (2007). J. Virol. 81, 3042–3048.

Fig. 1: Mature HCMV particle within a vesicle. The resolutions of the SEM and the TEM images are comparable. The two leaflets of the lipid bilayers are resolved in both images. The high resolution in the SEM image is gained by a primary acceleration voltage of 5kV and detection of the secondary electron signal. Diameter of virus particle approx. 200 nm.

Fig. 2: 3D reconstruction of HCMV infected nucleus. DNA free (orange) and DNA filled (green) capsids are enclosed by large infoldings of the INM (blue). Only the outline of the infolding is depicted in (A), (B+C) show complex internal membranes. (B) The infoldings can be spherical and/or tubular.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2590 Enabling future Nanotomography and Nanofabrication with Crossbeam technology

Schulmeyer I.1, Kienle M.1
1Carl Zeiss Microscopy GmbH, Carl-Zeiss-Str. 22, 73447 Oberkochen
ingo.schulmeyer@zeiss.com

A Focused Ion Beam (FIB) combined in one instrument with a Field Emission Scanning Electron Microscope (FE-SEM) has become a powerful instrument for numerous Standard and cutting edge applications in Research and Industry. The FIB is mainly used to open up the third dimension to a SEM. The ion beam is not only able to cut slices of a samples surface for tomographic imaging, but can also be used to create new materials or functional structures with superior properties. With increasing application maturity also the demand for faster systems, complete detection, more precise structuring and a wider application range rises.

3D-imaging and –analytics allow a complete characterization of a samples volume. It is widely used in Materials and Life Sciences and allows better understanding of compound materials, brain tissues, electronic devices and other samples. To achieve representative information of a sample, the analyzed volume needs to be sufficiently large and the resolution has to be high. The voxel resolution in tomography is mainly limited by the thickness of the slices cut with the ion beam [1]. The Crossbeam 540 allows the thinnest slices down to 5 nm and below. To keep the thickness homogenous over thousands of slices, a long-term stable FIB comes along with a sophisticated Software solution including adaptive slice thickness tracking (Figure 1). In interaction with a charged particle beam, each material behaves different. We introduce solutions to process, image and analyze all kinds of samples, including charging, outgassing or dirty and even magnetic samples. We will discuss our latest developments in detector technology to improve the signals especially at low acceleration voltages and the acquisition time.

Preparation of samples for TEM or other imaging techniques is one of the main applications for modern FIB-SEM instruments. Not only the quality, thickness and homogeneity of the prepared specimen matters, but also an fast, easy and integrated workflow. The quality of the results in TEM and STEM depend not only on the instrument, but to a high degree on the analyzed specimen. The unique X² method combined with a FIB that performs excellently at low voltages provides homogenous TEM lamellas with < 10nm thickness and minimum amorphous layer [2] (Figure 2).

Modern FIB-SEM systems cover not only the typical application range but are also used to host and integrate numerous instruments and components for advanced experiments. The Zeiss FIB-SEM allows integration of many components for In-Situ analysis like heating, cooling and tensile stages, SIMS, EBIC and CL, different manipulators, e.g. for probing and liftout, a Laser option for micropatterning or fast removal of large amounts of material and many more.

[1] L. Holzer and M. Cantoni, Review of FIB-tomography, Nanofabrication Using Focused Ion and Electron Beams: Principles and Applications (2011), p. 410ff

[2] L. Lechner, J. Biskupek and U. Kaiser, Improved Focused Ion Beam Target Preparation of (S)TEM Specimen - A Method for Obtaining Ultrathin Lamellae, Microscopy and Microanalysis 18 (2012), p. 379-384

Fig. 1: Innovative thermo-electrical generator transforming exhaust heat into electrical power (Mg2Sn-Mg2Si). 3D stack was acquired on Crossbeam running ATLAS3D. 8nm Voxel Size. The diffusion zone thickness varies between 10 to 25µm

Fig. 2: large 50µm x 20µm TEM lamella prepared automatically in 25 min (left). Lamella thinned using the X²-method (centre). HRTEM image of a lamella demonstrating atomic resolution at 20kV. The spotty contrast variations in the image are caused by strong dynamic diffraction contrast that cannot be avoided at 20 kV [2] (right).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2616 In situ FIB/SEM micro-compression tests of layered crystals

Schweizer P.1, Niekiel F.1, Butz B.1, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Department of Materials Science and Engineering, University of Erlangen-Nürnberg, Cauerstr. 6, 91058 Erlangen, Germany
peter.schweizer@ww.stud.uni-erlangen.de

Keywords: micro-compression, layered crystals, FIB, transition metal dichalcogenides

With the recent surge of interest in two-dimensional materials other than graphene, transition metal dichalcogenides (TMDCs) have gained a lot of attention because of their outstanding properties ranging from superconductivity to the formation of charge density waves [1, 2]. However not only monolayers of TMDCs are of interest, since the bulk form also shows strongly anisotropic behavior in most physical properties, which makes TMDCs promising candidates for many applications such as solid lubrication [3]. The anisotropic behavior in these layered crystals arises from the contrast between the weak Van der Waals bonding between the layers and the strong ionic/covalent bonds within the layers. Despite the amount of attention that this class of material has gained, the mechanical properties are largely unknown. This is due to the difficulty of preparing samples that are suitable for traditional mechanical testing. Consequently compression tests of micro-pillars are emerging as a novel way to measure the mechanical properties of materials on a micro scale [4].
In this contribution we show the preparation of micro-pillar samples from layered crystals, choosing vanadium diselenide (VSe2) as model material system. An FEI Helios Nanolab 660 DualBeam has been equipped for both sample preparation and in situ compression of the studied pillars. The pillars have been prepared using the focused ion beam (FIB), compression has been performed using a Kleindiek micromanipulator while imaging in situ using SEM. Force measurement is enabled using a Kleindiek SpringTable. Image correlation is used to determine the deflection of a cantilever, which corresponds to a force via a known spring constant. Deformation is measured tracking the difference in displacements between substrate and pillar. The complete indentation setup is shown in Figure 1.
Figure 2 shows the compression of an exemplary VSe2 pillar, cut at an angle of 20 degrees from the basal planes. The inset shows the resulting force displacement diagram. As expected a preferential slip along the basal planes is clearly visible. The sample preparation and micro-compression testing route established in this work on the example of VSe2 is anticipated to provide a deeper insight on the mechanical properties of TMDCs and other more complex layered crystals , like, e.g., misfit layer compounds.

References
[1] Q H Wang et. al., Nat. Nanotechnol., 2012, 7, 699–712
[2] X Huang et. al., Chem. Soc. Rev., 2013, 42, 1934
[3] L Rapoport et. al., J. Mater. Chem., 2005, 15, 1782–1788
[4] M D Uchic et. al., Science, 2004, 305, 986

The Authors gratefully acknowledge financial support by the German Research Foundation (DFG) via the research training group 1896 „In situ microscopy with electrons, X-rays and scanning probes”.

Fig. 1: The Kleindiek indenter setup inside the chamber of the focused ion beam.

Fig. 2: SEM-image of a vanadium diselenide pillar (prepared at an angle of 20° relative to the basal planes) being compressed by a diamond flat punch and the resulting force displacement curve (inset).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2701 High performance nanomachining using the new analytical FIB-SEM system

Jiruše J.1, Havelka M.1, Haničinec M.1, Polster J.1, Hrnčíř T.1
1TESCAN Brno, s.r.o., Brno, Czech Republic
jaroslav.jiruse@tescan.cz

A new analytical tool GAIA, combining high performance Focused Ion Beam (FIB) column with ultra-high resolution Scanning Electron Microscope (SEM), has been developed. The SEM resolution has been improved down to 1 nm at 15 kV and 1.4 nm at 1 kV, see Figure 1, thanks to a new objective lens of a single-pole immersion type [1, 2]. It creates a strong magnetic field surrounding the sample and decreasing optical aberrations. Intermediate lens enables to work in the magnetic-field-free mode suitable for analysis, magnetic sample imaging and observation during FIB machining. FIB milling process can be controlled by SEM imaging simultaneously, because of two independent scanning generators and sophisticated TESCAN detection system. Besides chamber detectors for detection of secondary (SE), backscattered (BE), transmitted (TE) electrons and secondary ions (SI), InBeam SE and InBeam BE detectors placed in the column give the free space around the sample.

The new non-magnetic Cobra FIB column with high resolution of 2.5 nm at 30 kV [3] and great performance at high currents has been designed to protect from the influence on the magnetic field of the immersion SEM objective lens. DrawBeam software allows drawing of patterns in CAD-like GUI for electron and ion beam lithography. The patterns are generated by ultra-fast scanning generator with pixel dwell time down to 20 ns. The novel milling strategy is included in DrawBeam software for 2.5 times faster FIB cross sectioning, see Figure 2. The technique is based on the correction of the intended shape to maximize the milling rate and to minimize the redeposition effects. The new AutoSlicer software for the automated cross sectioning and TEM lamella preparation increases FIB performance even further, see Figure 3. FIB column control is greatly simplified by using TESCAN In-Flight Beam TracingTM technology, which newly enables to compute and optimize FIB column settings.

GAIA instrument is prepared for fabrication and observation of non-conductive samples. Charge accumulation on the surface caused by FIB milling can be neutralized by integrated electron flood gun or SEM electron beam. SEM imaging without charging artifacts can be performed at critical energy, below 4 keV, with improved resolution. TESCAN beam deceleration technology, applying negative voltage on the sample, allows automatic control of the electron landing energy down to 50 eV (manually down to 0 eV) and it further improves SEM resolution at low beam energies, see Figure 1. The new control module provides sample discharge and touch-alarm protections.

References:

[1] Z Shao et al, Rev. Sci. Instrum. 60 (1989) p. 3434.

[2] J Jiruše et al, Microsc. Microanal. 19 (Suppl 2) (2013) p. 1302.

[3] A Delobbe et al, EFUG 2011, http://www.imec.be/efug

The research leading to these results has received funding from the European Union Seventh Framework Program [FP7/2007-2013] under grant agreement No. 280566, project UnivSEM.

Fig. 1: Ultra-high resolution at low beam energies: TiO2 nanotubes at 1 kV (left) and polymer nanofibers at 20 V (right).

Fig. 2: Different approaches for cross section preparation: (a) basic top-down “staircase” strategy, (b) a single-pass cross sectioning with high redeposition, (c) a fully optimized cross section object with 2.5 times higher milling speed, the highest depth and the best shape without redeposition. Milled using 12 nA FIB current at 30 keV.

Fig. 3: Automated TEM lamella preparation utilizing innovative fast cross section milling approach.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2704 Focused ion beam patterning of boron-doped diamond electrodes: Influence of patterning parameters on the heterogeneous electron transfer behavior

Eifert A.1, Langenwalter P.1, Higl J.2, Lindén M.2, Nebel C.3, Mizaikoff B.1, Kranz C.1
1Institute of Analytical and Bioanalytical Chemistry, University of Ulm, Ulm, Germany, 2Institute of Inorganic Chemistry II, University of Ulm, Ulm, Germany, 3Fraunhofer Institute for Applied Solid State Physics, Freiburg, Germany
alexander.eifert@uni-ulm.de

Diamond with its large band gap of 5.49 eV can be transformed into a metallic-like semiconductor by doping with very high boron concentrations. This new electrode material outmatches many common electrode materials concerning potential window, chemical inertness and signal-to-noise-ratio. The superior chemical and physical properties in combination with the possibility to fabricate microelectrodes or microelectrode arrays for example with FIB [1, 2], renders them highly suitable for bioanalytical applications. The electrochemical behavior of BDD electrodes depend on a variety of parameters, such as doping level, defects, carbon impurities, crystal orientation, surface termination/modification and grain boundaries [3]. Patterning of BDD with FIB technology leads to damages due to the ion bombardment such as amorphization and hence changes the overall electrochemical behavior.
In this contribution we present the influence of different FIB patterning parameters on the electrochemical properties such as heterogeneous electron transfer rate constant and peak separation of the obtained BDD microelectrode arrays. Post fabrication electrochemical treatments will restore to a certain extent the electrochemical properties. Next to electrochemistry also Raman spectroscopy was applied to characterize the ion irradiated sample, which will also be presented.

References
[1] J. Hees, R. Hoffmann, A. Kriele, W. Smirnov, H. Obloh, K. Glorer, B. Raynor, R. Driad, N. Yang, O. A. Williams, C. E. Nebel, ACS nano 5 (2011) 3339–3346.
[2] A. Eifert, W. Smirnov, S. Frittmann, C. E. Nebel, B. Mizaikoff, C. Kranz, Electrochem. Commun. 25 (2012) 30–34.
[3] K. B. Holt, A. J. Bard, Y. Show, G. M. Swain, J. Phys. Chem. B 108 (2004) 15117–15127.

The Focused Ion Beam Center UUlm, which is supported by FEI Company (Eindhoven, Netherlands), the German Science Foundation (INST40/385-F1UG), and the Struktur- und Innovationsfonds Baden-Württemberg are greatly acknowledged. This work was supported by the project "Methoden für die Lebenswissenschaften P- LSMeth/23" funded by the Baden-Württemberg Stiftung.

Fig. 1: Rate constants calculated from recorded CVs after electrochemical treatment. Electrochemical measurements were recorded in a solution containing 10 mmol/l K4[Fe(CN)6], 0.016 mol/l Tween 20 and 0.1 mol/l KCl. The influence of dwell time dependency was investigated at an accelerating voltage of 30 kV and a beam current of 15 nA.

Fig. 2: Raman spectra recorded after different fabrication steps of the microelectrode arrays. Raman spectra were acquired using an excitation laser with 532 nm and 10 mW power. For detection a 100x objective with a 0.9 numerical aperture was used.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2724 Novel strategies towards faster and smoother FIB cross-sectioning

Hrnčíř T.1, Dluhoš J.1, Haničinec M.1, Hrachovec V.2
1TESCAN Brno, s.r.o., Libušina třída 1, Brno, Czech Republic, 2ON Semiconductor Czech Republic, 1. máje 2230, Rožnov pod Radhoštěm, Czech Republic
tomas.hrncir@tescan.cz

The Focused Ion Beam (FIB) and the Scanning Electron Microscopy (SEM) are essential techniques for many applications. FIB modifies the sample by the milling or, when accompanied by the Gas Injection System (GIS), by the deposition; SEM is used for imaging of resulting shapes at the high resolution, for charge compensation, or as a source of electrons for other analytical techniques. Common FIB-SEM instruments allow creation and imaging of a broad range of shapes. The most important shape is the cross section, which is used both for sectioning the sample and TEM sample preparation, by milling two cross sections at both sides of TEM sample [1]. Two parameters of the cross section are crucial – the fast milling rate and the high quality of the surface, with no damage or artifacts.

The milling rate depends on the sample material and on the beam incidence angle (Fig. 1). An optimized scanning strategy for the cross section is introduced, to keep the slope of the sample surface under the ion beam closer to the maximum rate. Apart from increasing the milling rate, this method also reduces the redeposition artifacts to avoid obstacles limiting the effective depth. The resulting cross section shape is greatly improved and around 2.5 times deeper when compared to classical stair shape (Fig. 2). This shortens the cross-sectioning and TEM sample preparation time significantly.

The common width and depth of the cross section, milled by Ga FIB, are ~10 µm. For larger cross sections with dimensions up to ~100 µm, Xe plasma FIB is much more efficient [2]. As the surface milled by Xe plasma FIB is often not as smooth as the surface milled by Ga FIB, the quality has to be improved by tilting the sample and milling from several directions [3]. This makes cross-sectioning more difficult and less accurate. To overcome these drawbacks, a novel method was developed to greatly improve the surface quality, while keeping the milling process easy and accurate. Commonly used eucentric sample stages allow the tilting only around the axis perpendicular to both FIB and SEM columns. The novel multi-tilt sample stage allows an additional tilting also in the plane of the cross section. Unlike the solution described in [3], where the eucentric stage was used, the proposed method allows to control the whole milling/tilting process by SEM imaging, which is essential for the precise end-point detection (Fig. 3). Greatly improved results were obtained on polycrystalline material samples and on semiconductor samples, like solder bumps (Fig. 3), packaged ball-bond Au wires (Fig. 4) and TSV.

References
[1] MHF Overwijk et al, J. Vac. Sci. Technol. B 11 (1993), 2021.
[2] T Hrnčíř et al., ISTFA Conf. Proc. (2012), 26.
[3] F Altmann, et al., ISTFA Conf. Proc. (2012), 39.
[4] http://www.srim.org

The research has been supported by the Technological Agency of Czech Republic *TE 01020233 (AmiSpec).

Fig. 1: Ga FIB milling rate on Si when changing the incident angle, modeled by SRIM [4]. Beam energy is 30 keV.

Fig. 2: Increasing cross-sectioning rate by optimizing the scanning strategy. A) Classical stair strategy, which gives a nice shape but it is slow; B) One-pass polishing, where the shape is corrupted by redeposition artifacts; C) Novel optimized strategy, with the nicest shape and the highest depth of the cross section.

Fig. 3: Xe plasma FIB polishing of the solder bump by alternating the stage tilt in the cross section plane. Arrows point in FIB direction and the polishing process is controlled by SEM imaging, allowing to stop the process in the center of the bump accurately.

Fig. 4: Cross section through the packaged semiconductor sample (PWM controller from ON Semiconductor) by Xe plasma FIB, which was practically impossible to perform by Ga FIB previously.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2741 Ultra-Fast Three Dimensional Microanalysis Using the Scanning Electron Microscope Equipped with Xenon Plasma Focused Ion Beam

Dluhoš J.1, Petrenec M.1, Peřina P.1, Reinauer F.2, Kopeček J.3, Hrnčíř T.1, Jiruše J.1
1TESCAN Brno, s.r.o., Brno, Czech Republic, 2EDAX-AMETEK GmbH, Wiesbaden, Germany, 3Institute of Physics ASCR, v. v. i., Prague, Czech Republic
jiri.dluhos@tescan.cz

The three dimensional microanalysis became a widely accepted analytical method during the past few years, gaining from the ability to describe the materials structure and composition as it exists in real components.

A high resolution scanning electron microscope (SEM) combined with a focused ion beam (FIB) is used for a high precision tomographical method based on FIB slicing and SEM observation of the slice. The FIB-SEM can be further equipped by analytical methods like energy-dispersive X-ray spectrometer (EDS) for elemental composition or electron backscattered diffraction analyzer (EBSD) for crystal orientation mapping, resulting in 3D microanalysis, i.e. 3D EDS and 3D EBSD [1].

However, the main limitation of this tomographic method so far has been the speed of data acquisition. This influences also the volume which can be analyzed in reasonable time of several hours. A novel solution for rapid 3D microanalysis is introduced in this paper using a high performance Xe+ plasma focused ion beam. Such a system allows FIB-SEM tomography on objects with dimensions of hundreds of microns easily within few hours [2, 3], newly combined also with high speed EDS and EBSD. Utilizing the recently developed “static position” approach [4], the speed of 3D EDS and 3D EBSD acquisition can be maximized.

The conventional Ga+ FIB systems have a limitation of maximum beam current of about 60 nA. For practical FIB-SEM tomography, the volume is limited to units or maximum several tens of microns. Contrary to that, the Xe+ plasma source FIB incorporated in the TESCAN’s FERA3 FIB-SEM allows ion beam currents up to 2 µA [5]. Together with the higher sputtering yield of accelerated Xe ions it reaches about 50 times higher milling rates than Ga+ based FIB.

Examples of 3D EDS and 3D EBSD obtained using the Xe+ plasma FIB-SEM are shown. The 3D EBSD was acquired on a Cu wire commonly used in microelectronics, see Fig. 1. A total volume of 100×100×30 µm was analyzed in about 2.5 hours. Data acquisition time was about 1 min for FIB slicing at 30 keV beam energy with 1 µA beam current and 4 min for 200×200 points EBSD map acquisition for each of the 30 slices.

The 3D EDS example in Fig. 2 shows the volume of 100×70×45 µm of Sn60Pb40 solder processed in about 2.3 hours. Acquisition of 45 slices was done at SEM beam energy 20 kV with lateral resolution 0.5 µm. EDS maps were stored with full spectra at each point. Elemental ROI maps using Sn Lα, and Pb Mα peaks were used for 3D visualization.

References:

[1] S Zaefferer, Book of abstracts EMAS Workshop (2009) p 123.

[2] T Hrnčíř et al, Microsc.Microanal. 19 (Suppl 2) (2013) p. 860.

[3] T Hrnčíř et al, 39th ISTFA Proceedings (2013) p. 27.

[4] Patent CZ 301692 (2009).

[5] T Hrnčíř et al, 38th ISTFA Proceedings (2012) p. 26.

The research leading to these results has received funding from the European Union Seventh Framework Program [FP7/2007-2013] under grant agreement No. 280566, project UnivSEM.

Fig. 1: 3D EBSD reconstruction of a copper wire used in microelectronic. The volume 100x100x30μm was analyzed and all data were acquired within 2.5 hours using FERA3 Xe+ Plasma FIB-SEM equipped with EBSD by EDAX . Crystal orientation was mapped using a color coded inverse pole figure (IPF-Z).

Fig. 2: 3D EDS reconstruction of a Sn60Pb40 solder the FERA3 Xe+ Plasma FIB-SEM. Volume of 100x70x45µm was analyzed and all data were acquired in about 2.3 hours. a) Reconstruction of a composite 3D elemental map using Sn Lα, and Pb Mα peaks. b) Separation of Pb phase reveals formation of dendritic structure.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2874 Targeted 3D-CLEM workflow on cultured cells

Steyer A. M.1, Schieber N. L.1, Duishoev N.1, Pepperkok R.1, Kirmse R.2, Schertel A.2, Schwab Y.1
1EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg Germany, 2Carl Zeiss Microscopy GmbH, Oberkochen
anna.steyer@embl.de

Correlative light and electron microscopy (CLEM) experiments uniquely provide a highly accurate link between the imaging of living cells and their 3D ultrastructure (4). However, CLEM generally suffers from a low throughput. The major hurdles include tracking the object throughout the different imaging modalities, the tedious procedures for sample preparation and the lack of automation in the data acquisition by electron microscopy. We aim to overcome these issues by developing an automated correlative workflow that links live cell imaging to high resolution 3D electron microscopy using FIB-SEM. The approach we will develop enables collecting statistically significant data from a large number of cells in a heterogeneous population, clearing the way to statistical analysis of important mechanisms in cell biology. Aiming to develop a flexible and versatile approach, we foresee applications of our method in other biological areas such as pharmacology, developmental biology or virology.

Currently, our workflow is developed and tested on cultured cells and employed to in a high-throughput study of Golgi apparatus organization. In a tight collaboration with the team of R. Pepperkok (EMBL Heidelberg), we visualize at the ultrastructural level how specific mutations influence the morphology of the Golgi apparatus. Using an automated light microscopy platform, large genome wide siRNA knockdown screens have led to the identification of key genes for the morphogenesis and function of the Golgi apparatus (3). Light microscopy was utilized to screen for specific phenotypes, fluorescent microscopy to find cells of interest and electron microscopy to look at the ultrastructure.

References:

1) Briggman K. L. and D. D. Bock (2012). "Volume electron microscopy for neuronal circuit reconstruction" Curr Opin Neurobiol 22(1): 154-161.

2) Colombelli J., et al. (2008). “A correlative light and electron microscopy method based on laser micropatterning and etching.” Methods Mol Biol. 457:203-213

3) Simpson J. C., et al. (2012). "Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway" Nat Cell Biol 14(7): 764-774.

4) Spiegelhalter C., et al. (2010). “From dynamic cell imaging to 3D ultrastructure: novel integrated methods for high pressure freezing and correlative light-electron microscopy” PLOS One 5(2): 203-213

5) Villinger C., et al. (2012). "FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells" Histochem Cell Biol 138(4): 549-556.

Many thanks to Team Schwab, Team Pepperkok and our collaboration partner Zeiss.

Fig. 1: Correlative light and electron microscopy workflow using a Focused Ion Beam-Scanning Electron Microscope i) 10x transmitted light image, j) 40x transmitted light image with laser cuttings around the area of interest, k) 40x fluorescent microscopy image, m) SEM image, o) Golgi network model acquired with a FIB-SEM
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2936 Characterisation of the FIB Induced Damage in Diamond

Rubanov S.1, Suvorova A.2
1University of Melbourne, Melbourne, Australia, 2The University of Western Australia, Perth, Australia
sergey@unimelb.edu.au

Despite diamond’s extreme properties a TEM sample from diamond can be relatively easy prepared using FIB milling [1]. However, FIB milling results in formation of amorphous damage layers [2-3]. In addition, the rearrangement of broken diamond sp3 bonds into graphitic sp2 bonds is possible.
To study the initial stages of the damage formation (001) diamond sample was irradiated with 30 keV Ga ions with doses ranging from 2×1014 to 1016 ions/cm2. Continuous milling effect has been studied using rectangular trenches 4×4 µm2 and 2 µm deep formed using 100 pA beam current. The near surface regions of the trenches contained two types of damage: the bottom-wall, where the ion beam was normal to the surface and the side-wall, where it was at low angle to the trench walls.
For the dose 2×1014 ions/cm2 the point defect density was below amorphisation threshold and implanted region remains crystalline. For the dose 4×1014 ions/cm2 and above most of implanted region had defect density above amorphisation threshold and became amorphous (Fig. 1). The bottom part of the implanted layer remains crystalline but distorted due to still large number of point defects (Fig. 1b). EELS examination showed the presence of both sp2 and sp3 bonding in the damage corresponding to two different chemical states of carbon. The swelling of the amorphous damage layer shown in Fig. 1a is related to diamond’s sp3 bonds conversion to sp2 bonds with significant decrease in density. Using a mass balance calculation the density of the amorphous layers was determined. The density decreased with ion dose increased, and reached density of graphite (2.24 g/cm3, 80% sp2) for highest dose. For continues milling the thicknesses of the amorphous damage layers were measured to be 16 nm for side-walls and 44 nm for the bottom-walls (Fig. 2a). Concentration of Ga atoms was found to be 20 and 32 at.% for side-wall and bottom-wall damage layers. The thickness of the initial amorphous damage layers exponentially grows with ion dose (Fig. 2b) and has a tendency to saturate at the value which was measured for continuous milling.
The FIB induced damage in diamond comprises amorphous and crystalline components and is a result of complex process of ion penetration, swelling and sputtering. Amorphisation in diamond results in transition of sp3 bonds to sp2 corresponding to two different chemical states of carbon with accompanying density reduction. High concentration of Ga atoms is a result of accumulation of implanted atoms in damage layers due to short penetration depth and low sputtering yield in diamond.
References
[1]    S. Rubanov, AMTC Letters 2 (2010) p. 104.
[2]    J.F. Walker and R.F. Broom, Inst.Phys.Conf.Ser. 157 (1997)  p. 473.
[3]    S. Rubanov  and. P.R. Munroe, J. Microsc. 214 (2004) p. 213.

Fig. 1: TEM image showing damage in diamond after implantation of 4×1015 Ga/cm2 (a) and mag-nified TEM image of amorphous-diamond interface (b).

Fig. 2: (a) TEM image showing damage in diamond after continues milling; (b) the measured thick-ness of the amorphous damage layers as a function of the implantation dose.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-2986 High quality site specific TEM cross section preparation of structured materials

Graff A.1, Hübner S.1, Simon-Najasek M.1, Altmann F.1
1Fraunhofer Institute for Mechanics of Materials / Center for Applied Microstructure Diagnostics (CAM), Halle, Germany
andreas.graff@iwmh.fraunhofer.de

A requirement for every TEM investigation is the preparation of electron transparent samples. TEM preparation by focused ion beams (FIB) is nowadays widely used to produce site specific sections from the region of interest. Various approaches for TEM sample preparation using FIB have been developed. The most flexible are lift-out techniques where a lamella is directly made from the original sample, transferred to a support grid and finally thinned to electron transparency.
In this paper, we demonstrate an improved workflow for TEM sample preparation by FIB for extremely thin and distortion-free lamellas. By using a special TEM grid with clamps and an active griper for sample transfer the welding and cutting procedure necessary for the standard lift out technique (Fig. 1 left) can be avoided [1]. An additional advantage is the fixation of the TEM lamella on both sides, thus reducing the bending of the lamella in the final stages of the thinning. The special design of the holder allows preparing the sample from different directions without damaging the clamps. A TEM grid holder construction with an additional rotation axis perpendicular to the sidewalls of the TEM lamella is presented where the angle of incidence can be varied independently for the front and the backside (Fig. 1 right). The result of this kind of preparation is an electron transparent window inside a mechanically stable bar of the specimen (Fig. 2). The transparent window has a trapezium shape with adjustable angles between 0 and 90 degrees. A possible variation of the angles can be used to control and reduce curtaining effects often appearing in structured multi-material systems. To control the residual thickness of the lamella inside the window, thickness measurements are performed during thinning by electron backscatter imaging using a cross beam instrument (NVision, Zeiss). Thus, the plan parallel shape and the thickness of the sample can be controlled during the final milling to reach a well-defined homogeneous lamella thickness [2]. TEM investigations of the samples prove the reduction of curtaining and wrapping in the ultra-thin transparent window (Fig. 3).
The workflow was successfully applied to different material systems which are discussed in the present contribution. The efficiency of the process and the high quality of the TEM samples are shown.
References:
1. Altmann F., et al., Microscopy and Microanalysis, Volume 17, Supplement S2, 2011, pp 626-627
2. Salzer R., Lechner L., Microscopy and Microanalysis, Volume 18, Supplement S2, 2012, pp 654-655

Fig. 1: Left: SEM image of the lamella transfer into the special clamp holder by active gripper. Right: Sample holder mounted on a Kleindiek RotTip to realize a second tilt axis.

Fig. 2: Left: Color coded thickness map of a semiconductor sample. Right: TEM overview of the sample. Curtaining is hardly visible.

Fig. 3: Left: Bright field image of the grain structure of a Tungsten plug. Right: HRTEM image of the silicon SiO2 interface in ultrathin area of the lamella (Thickness less than 40nm).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-3033 About the accuracy of post-mortem alignment methods in FIB/SEM nano-tomography

Yuan H.1,2, Van de Moortèle B.2, Epicier T.1,3
1University of Lyon, MATEIS, umr CNRS 5510, Bât. Blaise Pascal, INSA Lyon, 69621 Villeurbanne Cedex, France, 2University of Lyon, LGLTPE, umr CNRS 5276, ENS Lyon, 69364 Lyon Cedex 07, France, 3University of Lyon, IRCELYON, umr CNRS 5256, 2, Av. A. Einstein, 69626 Villeurbanne Cedex, France
bertrand.van.de.moortele@ens-lyon.fr

Within the last decade, FIB/SEM tomography has become a commonly used tool for 3D microstructural investigation of materials at a sub-micrometer level [1, 2]. It consists in a true tomographic approach; in a Focused Ion Beam (FIB) microscope coupled with a Scanning Electron Microscope (SEM), the ion beam is used to mill the sample and prepare fresh surfaces which are successively imaged with SEM. The stack of acquired SEM images is then further aligned in order to restore the analysed volume of matter. By alignment, one intends both the drift correction during the acquisition itself [3] and a final post-mortem numerical alignment. The present contribution focuses on this final step: the post-mortem alignment before 3D reconstruction.

Basically, typical post-mortem alignment of the image stack is performed using cross-correlation based algorithms allowing the successive images to be aligned with respect to a reference image. Several methods have thus commonly used [1-5]: the most intuitive method is to define a Region of Interest (ROI) as a reduced frame in the field of view, with the option to locate this ROI near a lateral edge of the scanned area, or at the interface with the top surface of the sample in order to take benefit of an assumed fixed feature which will improve the accuracy of the alignment (fig.1). Further refinements introduced markers, i.e. holes or small trenches machined with the ion beam which will serve as markers facilitating the alignment. It will be demonstrated here that these alignment routines may easily fail, although the final reconstructions generally look correct. The principal issue of all methods mentioned above is due to the fact that they rely on the assumption that the markers, or the microstructure itself, are isotropic, intrinsically ‘fixed’ (with respect to the bulk sample) and not deformed, modified or erased during the 3D acquisition. This is generally not true and this will be demonstrated by dedicated test examples. As illustrated by fig.2 and 3, we will investigate alternative methods in order to improve the alignment and consequently the reliability of the reconstructed volumes. Among these methods, a promising one consists in a correlation with the top surface of the sample which can be reconstructed by stereoscopy prior to the 3D FIB-SEM acquisition and matter removal.

References

[1] L. Holzer,et al., J. of Microscopy 216 (2004), 84.

[2] M. Schaffer, et al., Ultramicroscopy 107 (2007), 587.

[3] H. Yuan in “EMC 2012: Proc.15th EMC, Vol. 2”, ed. D.J. Stokes and J.L. Hutchison, RMS: London, (2012), p. 135.

[4] H. Iwaia, et al., J. Power Sources 195 (2010), 995.

[5] M.D. Uchic et al., Ultramicroscopy 109 (2009), 1229.

[6] K. Lepinay et al., M and M, 19, (2013) 85.

We kindly acknowledge the financial support of Carl Zeiss SAS. Thanks are due to the CLYM (http://www.clym.fr) for the access to the Nvision 40 FIB instrument.

Fig. 1: Front view of the sample (serpentine) during the 3D-FIB analysis. Different ROIs (numbers, frames and arrows) are used for the post-mortem alignment (see fig. 2): ROI3 consists in FIB-milled lateral trenches filled with W (white contrast), whereas ROI4 corresponds to the top surface underlined by a W layer.

Fig. 2: Reconstruction of the top surface topography from a stereoscopic SEM analysis using 3 images taken at -10°, 10° (shown) and 0° with the help of the MEX program (Alicona SARL, Les Ulis, France).

Fig. 3: Superimposition of the top surface as reconstructed by the MEX© procedure (in green) and from the 3D-FIB analysis (in red) for the 4 ROI defined in figure 1. The best correspondence is by far obtained from the alignment based on the surface topography (case 4).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-3258 FIB as Fabrication Tool for Advanced Analytical Infrared Sensing Schemes

Sieger M.1, Neusser G.1, Schaedle T.1, Kranz C.1, Mizaikoff B.1
1Institute of Analytical and Bioanalytical Chemistry, University of Ulm, 89081 Ulm, Germany
gregor.neusser@uni-ulm.de

Within the last decade, focused ion beam (FIB) technology emerged as a universal tool, for maskless prototyping via highly localized milling and deposition processes. FIB prototyping is particularly interesting for analytical devices such as novel optical sensing structures for the mid infrared (MIR) regime and functionalized scanning probes [1].

The MIR (3-12µm) spectral range is particularly interesting for biosensing applications, since it provides inherent molecular selectivity. MIR photons interact with most organic and inorganic molecules by excitation of vibrational und rotational modes [2-5].

Quantum cascade lasers and semiconductor thin-film waveguides facilitate highly sensitive optical biosensors for the MIR regime. Among those biosensors, the Mach-Zehnder interferometer (MZI) is one of the most promising sensor as it can be fully integrated in a lab-on-a-chip microsystem and provides high sensitivity.

The developed MIR-MZIs are chip-integrated solid-state devices based on GaAs/Al0.2Ga0.8As technology wave guide fabricated via conventional optical lithography and reactive ion etching (RIE). Since optical lithography is limited to a resolution of about 2 µm, the microfabricated devices were further structured via FIB milling for minimizing scattering losses at the Y-junction, and to increase the optical throughput.The radius of the Y-junction edges was reduced from about 2 µm to less than 100nm, thereby leading to a throughput enhancement of more than 30% of incident light in comparison to structures without FIB milling (Fig. 1)[6].

Grating couplers are another way for minimizing coupling losses, especially for wave guide with micro- to nanoscale dimensions. FIB milling of grating couplers is a reproducable and reliable strategy for launching radiation into a wave guide structure and improving the overall performance of the optical device [7].

References:

[1] C. Charlton, B. Temelkuran, G. Dellemann, B. Mizaikoff, Appl. Phys. Lett., 86, 194102 (2005).

[2] C. Charlton, M. Giovannini, J. Faist, B. Mizaikoff, Anal. Chem., 78, 4224-4227 (2006).

[3] C. Young, S.-S. Kim, Y. Luzinova, M. Weida, D. Arnone, E. Takeuchi, T. Day, B. Mizaikoff, Sens. &Act. B, 140(1), 24-28 (2009).

[4] X. Wang, S.-S. Kim, R. Roßbach, M. Jetter, P. Michler, B. Mizaikoff, Analyst, 137, 2322-2327 (2012).

[5] A.Eifert, W. Smirnov, S. Frittmann, C.E. Nebel, B. Mizaikoff, C. Kranz, Electrochem. Commun. 25 (2012) 30-34

[6] M. Sieger, F. Balluff, X. Wang, S.-S. Kim, L. Leidner, G. Gauglitz, B. Mizaikoff, Anal. Chem., 85, 3050-3052 (2013).

[7] T. Schädle, A. Eifert, C. Kranz, Y. Raichlin, A. Katzir, B. Mizaikoff Applied Spectroscopy, 67, 1057-1063 (2013).

[8] X. Wang, M. Sieger, B. Mizaikoff, Proc. SPIE 8631, Quantum Sensing and Nanophotonic Devices X, 86312M (2013)

The authors gratefully acknowledge support for parts of this work by the Kompetenznetz Funktionelle Nanostrukturen Baden Wuerttemberg, Germany. Finally, the Focused Ion Beam Center UUlm is thanked for providing prototyping and characterization facilities during the present studies.

Fig. 1: Figure 1. Scanning electron microscopy (SEM) images of GaAs/AlGaAs MIR-MZI waveguides with different opening angles α and a constant distance between the arms d: (a) top view of a Y-junction before, and (b) after FIB milling process [8].
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-3393 USING THE FOCUSED ION BEAM MICROSCOPE TO DEVELOP DIFFRACTION GRATINGS FOR QUANTUM WELL INFRARED PHOTODETECTORS

Rodrigues W.1, Schmidt W.1
1Microscopy Center from Federal University Minas Gerais
wesller@hotmail.com

Quantum well infrared photo detectors (QWIP) has several advantages when compared to other kinds of IR detectors, but it requires an optical coupling in order to work due to the quantum selection rule. These detector must has a Electrical Component of radiation (TE) parallel with structures grown direction. Diffraction gratings are one of the several types of optical coupling suitable to be implemented on these detectors. In this work we opt by diffractions gratings because the best optical coupling if compared with other techniques (1).

Due to the wavelengths of interest in this work (4,1um) and the refraction index the detector's material (n = 3.2) the features of the such gratings are almost sub-micrometric (2). The processes of optical lithography currently available in Brazil do not allow the fabrication of these diffraction gratings, essential to the functioning of these IR detectors, with the theoretical dimensions recommended, especially for near and mid infrared (3). The Focused Ion Beam microscope (FIB) allows the fabrication of these structures at resolutions more than enough (4) (5) (6).

The study and fabrication of these gratings are the objects of this work. FIB was used in the manufacture of gratings with several dimensions, from the theoretical dimensions until the optical lithographic dimensions (Figure 1) on QWIP mesa devices fabricated by usual optical lithography, enabling the investigation of the possible loss of electrical response of the QWIP´s detectors if they were textured with the available optical lithographic processes currently in Brazil.

To characterize the dimensions of arrays was used Atomic Force Microscopy (Figure 2). After fabrication diffraction gratings was deposited In each individual mesas a thin Gold film (180nm) to make the electric contacts by gold wire bonding (Figure 2a,b ).

This chip was assembly in header where the electric response the individual mesas will be measured and compared (Figure 3). The features of dimensions these gratings and their effect on the electric response of the devices will be presented.

Fig. 1: Figure 1 – SEM image of the photodetector mesas after fabrication of diffraction gratings

Fig. 2: Figure 2a – AFM Image of a diffraction grating. Figure 2b – SEM image of one already textured mesa with the deposited ohmic contact

Fig. 3: Figure 3 – SEM image of the chip assembled on the header and SEM image of the wire bonded mesas on the header
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-5975 LIFE CELL IMMUNOGOLD LABELLING OF RNA POLYMERASE II visualized by Focused Ion Beam Scanning Electron Microscopy (SEM-FIB)

Orlov I.1, Schertel A.2, Zuber G.3, Drillien R.1, Weiss E.4, Schultz P.1, Spehner D.1
1Integrative Structural Biology, IGBMC, UMR 7104, 67404 Illkirch, France, 2Carl Zeiss Microscopy GmbH, ASC, D-73447, Oberkochen, Germany, 3LCAMB UMR 7199, Faculty of Pharmacy, 67401 Illkirch, France, 4Biotechnology and Cell Signaling, UMR 7242, IREPS, 67412, Illkirch, France
spehner@igbmc.fr

The intracellular localization and dynamics of proteins involved in cellular processes are often studied in living cells at light microscopy resolution by monitoring proteins fused to fluorescent tags. These methods have nevertheless intrinsic drawbacks. First, the level of expressed fusion proteins is difficult to match with endogenous levels and since the endogenous protein is generally not extinct, the fusion protein acts on top of its native counterpart. The second restriction comes from the limited spatial resolution of light microscopy which, despite the spectacular development of super-resolution light microscope modalities, does not attain molecular dimensions.
Electron microscopy is an invaluable method to improve spatial resolution and to describe the cellular context of proteins of interest but relies on electron dense probes or reagents to detect the labeled macromolecule. The most successful and widely used method consists in conjugating primary or secondary antibodies to gold particles (Faulk and Taylor, 1971) which have a high electron scattering power and create an easily recognizable highly contrasted round shape.
Here we exploited the ability of cells to internalize macromolecules with a method named “live cell immunogold labeling” which takes advantage of lipid-based protein delivery agents compatible with cell viability to internalize the probes (Futami et al., 2012, Freund et al., 2013).
We used this method to label RNA polymerase II with electron dense labels suitable for EM localization studies and demonstrate for the first time that antibodies coupled to 0.8 nm ultrasmall gold particles (Van de Plas P. and Leunissen J.L., 1993) can enter the nucleus and be detected after amplification. Cells grew normally for more than 8 hours after probe uptake. The label was detected, after silver enhancement, by transmission electron microscopy and by scanning electron microscopy coupled to Focussed Ion Beam slicing (SEM-FIB)(Schroeder-Reiter E.et al.,2009). These methods open the new possibility to label nuclear or cytoplasmic antigens in living cells, and to immunolocate them in the whole cell volume using the SEM-FIB technology.
Faulk, W. P. & Taylor, G. M. Immunochemistry 8, 1081-1083 (1971)
Futami, M. et al. Bioconjug Chem 23, 2025-2031, doi:10.1021/bc300030d (2012)
Freund, G. et al. MAbs 5, 518-522, doi:25084 [pii]
Van de Plas, P. & Leunissen, J. L. Methods Cell Biol 37, 241-257 (1993)
Schroeder-Reiter E. et al., J. Struct. Biol., 165, Issue 2, (2009)

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-6027 Structural characterization of hybrid-organic nanocomposites via focus ion beam preparation and electron microscopy

Ankah G. N.1, Büchele P.2, Tedde S. F.2, Adam J.1, Torrents O.1, Pfaff M.1, Poulsen K.3, Koch M.1, Gimmler C.3, Schmidt O.2, Kraus T.1
1INM - Leibniz Institute for New Materials, Campus D2.2, 66123 Saarbruecken, Germany, 2Siemens AG Corporate Technology, Günther-Scharowsky-Str. 1, 91058 Erlangen, Germany, 3Centrum für Angewandte Nanotechnologie (CAN) GmbH, Grindelallee 117, 20146 Hamburg, Germany
GenesisNgwa.Ankah@inm-gmbh.de

Nanocomposites of conductive polymers and functional nanoparticles have recently been employed in applications such as photodetectors [1-3]. The particles convert light or x-rays into charge-carrier combinations that travel through a polymer blend to the contacting electrodes. In order to correlate device performances with the distribution of the nanoparticles in the organic polymer matrix, it is necessary to perform structural investigation at particle-level resolution. Focused ion beam (FIB) sample preparation is a prerequisite when specific regions are to be analyzed at such resolutions. In this work, composites of P3HT, PCBM and PbS nanoparticles or other inorganic nanoparticles were prepared by spray coating. Trenches and lamellae were prepared via FIB at several positions of the sprayed area. Their microstructures were analyzed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM).
The distribution of nanoparticles inside the nanocomposite affects the properties of electronic materials. Conductive pathways, optical adsorption lengths and optical scattering depend on particle arrangement and affect sensor performance. Agglomerates and fully demixed particle phases make it harder for charge carriers to enter the polymer blend. Voids in the composite hinder transport and scatter light. We discuss the occurrence of such defects depending on processing.
FIB cuts through the soft polymer matrix that intersect hard nanoparticles can cause artefacts in the microstructure such as ridges, grooves, etc. The high energy ion bombardment may lead to local melting or the creation of amorphous layers. We discuss ion-beam related artefacts and their dependence on the preparation. We studied the extent of beam damage by comparing FIB cuts with samples prepared differently (e.g. using a microtome).


[1] Rauch T., Böberl M., Tedde S. F., Fürst J., Kovalenko M. V., Hesser G., Lemmer U., Heiss W., Hayden O., Nature Photonics 3, (2009) 332.
[2] Wagner B. K., Kang Z., Nadler J., Rosson R., Kahn, B., Proc. of SPIE 8373, (2012).
[3] Saunders B. R., Turner M. L., Advances in Colloid and Interface Science 138, (2008) 1.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-13-P-6033 Structural and mechanical characterization of human dental tissues across multiple scales at the Oxford Multi-Beam Laboratory for Engineering Microscopy (MBLEM)

Sui T.1, Ying S.1, Lunt A. J.1, Baimpas N.1, Landini G.2, Korsunsky A. M.1
1MBLEM, Department of Engineering Science, University of Oxford, UK, 2The School of Dentistry, College of Medical and Dental Sciences, University of Birmingham, St Chad's Queensway, Birmingham B4 6NN, UK
alexander.korsunsky@eng.ox.ac.uk

The combination of focused ion and electron beams within the vacuum chamber of the Tescan LYRA3 XM instrument at the Oxford Multi-Beam Laboratory for Engineering Microscopy (MBLEM) provides a set of versatile tools for structural and mechanical characterisation of natural and engineered materials. In the present study we report the use of SEM for imaging, and FIB for ion beam milling and TEM lamella preparation of samples of human dentine and enamel, the two principal tissues used by nature to build teeth. We also pay particular attention to the dentine-enamel junction, the DEJ, a functionally and structurally graded interface that accommodates significant change in the degree of mineralisation (nearly two-fold), stiffness (nearly five-fold) and hardness (two-fold). We report using the combination of synchrotron X-ray diffraction with FIB ring-core milling to determine the variation of the lattice parameter of the mineral content, the nanocrystalline hydroxyapatite (HAp) particles. The use of FIB-DIC allows the separation of lattice parameter variation into chemical changes and mechanical effects (residual elastic strains) in the vicinity of the DEJ. In addition, the analysis of TEM lamella extracted from the sample made it possible to visualise the fractures observed in the dental tissues, and to explain the toughening mechanisms that operate at the nano-scale in these materials. The combination of synchrotron X-ray diffraction with FIB ring-core milling was also used to determine the variation of the hydroxyapatite (HAp) lattice parameter and elastic strains in the vicinity of the DEJ.

We acknowledge the support of colleagues at Tescan in UK and CZ: Ray Codd, Zora Strelcova, Jiri Dluhos and many others.

↑ Go to the Table of content ↑

IT-14. Scanning probe microscopy and near-field microscopies

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-14-IN-2450 Advances in quantitative and three-dimensional mapping of soft matter by bimodal force microscopy

Garcia R.1
1Instituto de Ciencia de Materials de Madrid, CSIC
r.garcia@csic.es

Force microscopy is considered the second most relevant advance in Materials Science since 1960. Despite the success of AFM, the technique currently faces limitations in terms of three-dimensional imaging, spatial resolution, quantitative measurements and data acquisition times. Atomic and molecular resolution imaging in air, liquid or ultrahigh vacuum is arguably the most striking feature of the instrument. However, high resolution imaging is a property that depends on both the sensitivity and resolution of the microscope and on the mechanical properties of the material under study. Molecular resolution images of soft matter are hard to achieve. In fact, no comparable high resolution images have been reported for very soft materials such as those with an effective elastic modulus below 10 MPa (isolated proteins, cells, some polymers). Similarly, it is hard to combine the exquisite force sensitivity of force spectroscopy with molecular resolution imaging. Simultaneous high spatial resolution and material properties mapping is still challenging. This presentation reviews some of the above limitations and some recent developments based on the bimodal operation of the AFM to address and overcome them.

Recent References

E.T. Herruzo, A. P. Perrino and R. Garcia, Nat. Comm. 5, 3126 (2014)
R. Garcia and E. T. Herruzo, Nat. Nanotechnol. 7, 217-226 (2012).
E. T. Herruzo, H. Asakawa, T. Fukuma, R. Garcia, Nanoscale 5, 2678 (2013)
H. V. Guzman, A.P. Perrino, R. Garcia, ACS Nano 4, 3198 (2013)

We thank the financial support from the Ministerio de Economía y Competitividad, project CSD2010-00024 and the European Research Council, project 340177- 3DNanoMech.

Fig. 1: Bimodal AFM 3D images of solid-water volumes. a, 3D map of a mica-water interface. The stripes are associated to the presence of hydration layers. b, 3D map of a GroEL patch-water interface. The side view shows a slightly rough landscape with variations of the amplitude of about 1 nm.
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-14-IN-3211 Theory of Near-Field SEM: lateral resolution, scaling properties and compact equations

Xanthakis J. P.1
1National Technical University of Athens, Athens, 15700 Greece
jxanthak@central.ntua.gr

A new form of lenseless microscopy called near-field emission scanning electron microscopy (NFESEM) has been devised at ETHZ. In this form of microscopy the emitting tip is placed at a distance d of a few nm away from the sample (anode) with no focusing device in between. Besides the obvious advantages of price, NFESEM can perform DOS analysis (as STM) but also chemical identification by looking at the backscattered (or secondary reemitted) electrons.

The vertical resolution of this form of microscopy is as good as that of other forms of microscopy but the lateral resolution is about 3 nm at d=25nm and can be improved to 1 nm at smaller d. The latter constitutes a surprisingly good result considering the absence of a lens but it is not understood with conventional field emission theory. In this paper we present ab initio 3-dimensional WKB calculations applied to an ellipsoidal emitting tip that can explain the good lateral resolution capabilities of NFESEM. In particular, we show that the electron trajectories converge faster to the vertical direction compared to spherical emitting tips. This process begins in the classically forbidden (or tunneling) region where, contrary to accepted wisdom, electron paths may bend (see figure 1). As an end result of our calculations the beam spot size as a function of tip-anode distance d is obtained (figure 2)

The current-voltage (I-V) characteristics of this device are obviously d dependent. However for d>R=radius of curvature of the emitter, these characteristics show a remarkable property: all I-V curves fall onto each other when the applied voltage V is scaled (multiplied) by a single scaling function S(d), see figure 3. The explanation of this is rudimentary for image rounded linear potentials but for non-linear tunneling potentials U as those of nanoscopic emitters it demands a thorough investigation of the non-linear terms of U which we give.

Finally, in the course of our investigations, we have managed to produce- by strict mathematical proof- a generalized Fowler-Nordheim equation which is valid for any shape of surface. The accuracy of our equation – estimated by comparison to ab initio calculations- depends on R. For R>10nm it gives excellent results, for 5nm<R<10nm it gives satisfactory results and for R<5nm it does not work so well but still it gives much better results than the traditional FN equation, see figure 4.

This work would not have been possible without the substantial contributions of my former postdoc Dr Gerry Kokkorakis and my present PhD student Andreas Kyritsakis

Fig. 1: Comparison of beam-spot diameters by usuing straight line paths and curved paths calculated by a 3-Dimensional WKB method

Fig. 2: Lateral resolution of an ellipsoidal NFESEM emitter as a function of tip-anode distance d. The various curves are for different combinations of the large and small radii of the elliptical tip.

Fig. 3: Scaling properties of NFESEM voltage-current (I-V) characteristics.Main figure: scaled I-V curve, Inset: I-V curves for different d

Fig. 4: Comparison of Fowler-Nordheim plots calculated by our compact generalized FN equation and those obtained by ab initio WKB  calculations.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-14-O-1627 Using SPM nanomanipulation to discover new materials and properties

Barboza A. P.1, Guimaraes M. H.1, Oliveira C. K.1, Massote D. V.1, Fernandes T. F.1, Archanjo B. S.2, Lacerda R. G.1, Batista R. J.3, Oliveira A. B.3, Mazzoni M. S.1, Chacham H.1, Neves B. R.1
1Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 2Inmetro, Duque de Caxias, Brazil, 3Universidade Federal de Ouro Preto, Ouro Preto, Brazil
berneves@gmail.com

In this work, Scanning Probe Microscopy (SPM) was employed for matter manipulation at the nanoscale in ambient conditions. More specifically, the SPM nanomanipulation potential is illustrated by two recent works of our group: in the first one, a new material – diamondol – is proposed and its realization is evidenced via SPM experiments [1]. In the second one, the SPM nanomanipulation was used to both induce and discover a general property of solid lubricants: a negative dynamic compressibility [2].
According to our ab initio calculations, the diamondol, or hydroxylated diamond, would be a new 2D material, formed via compression-induced diamondization of two, or more, graphene layers stabilized by hydroxyl ions (see Fig. 1a). The experimental observation of diamondol was carried out in a series of SPM experiments on mono-, bi-, and multilayer graphene in a controlled environment (humidity and temperature). Using electric force microscopy (EFM) to both inject and monitor charges and to apply pressure on the sample [3] (see Fig. 1b), we observed a pronounced inhibition on the charging efficiency for bilayer and multilayer flakes as the tip pressure increased, while monolayer charging was pressure-independent (Fig. 1c). The influence of the water content on the sample surface was tested in a series of charge injection experiments carried out at different temperatures (25°C and 120°C). The ensemble of experimental results can be well accounted for by the diamondol hypothesis, thus giving strong evidence of its experimental realization.
In the second study, a novel mechanical response of solid lubricants (few-layer graphene, h-BN, talc and MoS2) to the simultaneous compression and shear by a SPM tip is observed. The response is characterized by the vertical expansion of these 2D layered materials upon compression (see Figs. 2a-d). Such effect is proportional to the applied load, leading to vertical strain values (opposite to the applied force) of up to 150% (Fig. 2d). The effect is null in the absence of shear, increases with tip velocity, and is anisotropic. It also has similar magnitudes in these solid lubricant materials, but it is absent in single-layer graphene and in few-layer mica and Bi2Se3 (non-lubricant layered materials). We propose a physical mechanism for the effect where the combined compressive and shear stresses from the SPM tip induce dynamical wrinkling on the upper material layers, leading to the observed flake thickening (Fig. 2e). The new effect (and, therefore, the proposed wrinkling) is reversible in all four solid lubricants where it is observed.

Financial support from Fapemig, Capes, CNPq, Rede Nacional de Pesquisas em Nanotubos de Carbono, and INCT/Nano-Carbono is acknowledged.

Fig. 1: (a) Upon compression and in the presence of hydroxyl ions, two graphene layers undergo a sp3 re-hybridization, creating a layer of diamondol (b) SPM setup used to make and identify the diamondol. (c) Graph of the amount of injected charges as a function of the tip compression force which indicates diamondol creation.

Fig. 2: Contact Mode AFM images of a graphene flake under increasing tip loads: (a) 10 nN, (b) 195 nN, and (c) 391 nN. (d) Height profiles from (a), (b), and (c) evidencing the negative strain of graphene. (e) Upon compression and shear by the AFM tip, the topmost layers of a solid lubricant slide and wrinkle, creating a net expansion of the material.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-14-O-1830 In-situ Dynamic SPM Studies of Organic Semiconductor Thin Film Growth on Silicon Oxide

Chiodini S.1, Straub A.1, Donati S.1, Borgatti F.1, Albonetti C.1, Biscarini F.2
1Consiglio Nazionale delle Ricerche, Istituto per lo Studio dei Materiali Nanostrutturati (CNR-ISMN), Bologna, Italy, 2Life Science Dept., Università di Modena e Reggio Emilia, Modena, Italy
schiodini@bo.ismn.cnr.it

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-14-O-1831 Exploring the Local Photovoltaic Mechanisms in Organic Bulk Heterojunction Nanostructures by means of Scanning Probe Microscopy

Sebaihi N.1, Moerman D.1, Letertre L.1, Douhéret O.2, Lazzaroni R.1,2, Leclère P.1
1Laboratory for Chemistry of Novel Materials, Center for Innovation and Research in MAterials and Polymers (CIRMAP), Research Insitute for Materials Science and Engineering, University of Mons (UMONS), Mons, Belgium, 2Materia Nova R&D Center, Mons, Belgium.
philippe.leclere@umons.ac.be

Recent research and progress in organic photovoltaic (OPV) repeatedly insist on the importance of the molecular organization of the compounds forming the active bulk-heterojunction (BHJ) blends. The morphology of the blend has been to tremendously affect both the charge transfer at the donor-acceptor interface and the carrier transport to the electrodes. And still, for each material combination, much remains to be understood to fully assess its specific and ultimate morphology. For this purpose, high resolution characterization methods are of primary interest to locally depict the different electrical mechanisms ruling the photovoltaic process. Conductive Atomic Force Microscopy (C-AFM) and Kelvin Probe Force Microscopy (KPFM) have already proven to be of significant help to yield nanoscale two-dimensional mapping of electrical properties.
C-AFM and related PeakForce TUNA emerged as powerful technique to electrically evidence phase separation in blends. An additional key feature lies in local I-V curve providing useful information about the charge transport mechanisms within the materials forming the blends. Quantitative measurements leading to local determination of hole mobility have already been reported. It appears that upon illumination the technique has shown to be sensitive to photocurrent. With photoconductive-AFM (pc-AFM), a dedicated external calibrated module  has been recently introduced allowing full quantitative mapping of photovoltaic mechanisms. In this study, we will present and discuss the obtained results on two well-known samples: (i) poly(3 hexyl thiophene)(P3HT):[6,6]-phenyl-C61-butyric acid methyl ester (PCBM) and (ii) poly[2-methoxy-5-(3′,7′-dimethyloctyloxy)-1,4-phenylenevinylene])(MDMO-PPV):[6,6]-phenyl-C61 -butyric acid methyl ester (PCBM).
KPFM is mainly used to delineate phase separation and potential variations at interfaces. Upon illumination, photovoltage can also be evidenced. Yet, in organic electronics, KPFM still suffers from harsh operating environment (ultra-high vacuum and low temperature) to reach satisfactory spatial resolution and lacks for modeling for quantitative measurements. Augmenting KPFM with the PeakForce TappingTM technology allows a drastic improvement in the spatial resolution for KPFM measurements in ambient conditions. With the additional external calibrated illumination module, mapping of photovoltage in BHJ blends can be obtained, opening the doors of local characterization of charge transfer at donor-acceptor interfaces, where crucial processes are occurring in photovoltaic devices.

Research on conjugated polymers in Mons is supported by the Science Policy Office of the Belgian Federal Government (BELSPO PAI 7/05), the OPTI2MAT Excellence Program of Région Wallonne, and FNRS-FRFC. Ph.L. is Research Associate of F.R.S.-FNRS (Belgium).

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-14-O-2891 Gaseous nanobubbles on immersed surfaces: Properties and imaging by AFM in situ and ex situ

Janda P.1, Tarábková H.1
1J. Heyrovský Institute of Physical Chemistry ASCR v.v.i., Prague, Czech Republic
pavel.janda@jh-inst.cas.cz

Hydrophobic surfaces immersed in water are often densely occupied by gaseous nanodomains – nanobubbles and nanopancakes, with the size ranging between 10 – 100 nm, but it can exceed 1000 nm. The first direct proof of their existence came in the year 2001 in a form of in-situ atomic force microscopic (AFM) image made by J.W.G. Tyrrell and P. Attard [1] . Nevertheless, nanobubbles were still considered as artifacts, mostly due to their rather peculiar behavior, seeming disobeyance of Young Laplace law and hence nonexistence of plausible physical model. As various fields became affected by nanobubble existence, including interfacial physical chemistry, biophysics, microbiology, material sciences, nanofluidics, heterogeneous (electro) catalysis, immersion lithography and others, etc, nanobubbles started to attract growing attention. Their full impact is however, still to be disclosed.
Recent work performed in our laboratory [2] [3] [4] revealed the nanobubble ability to significantly rearrange solid surfaces which they adhere to. Nanobubbles can act as surface nanopatterning elements, changing in a significant manner its nanomorphology even at very mild conditions - in deionized water, at room temperature and pressure variations not exceeding 10 kPa. Besides nanobubble imaging by AFM in situ and distinguishing nanobubbles from solid nano-objects, we are presenting our novel technique, which can be called „nanobubblegraphy“[4], due to its ability to allow ex situ recognition and ex-post imaging of nanobubbles on dried surface as imprints developed after relatively short (sub-second) exposition in polymeric matrix (Figs 1 - 4).
Nanobubble properties and their utilization for imaging in situ and ex situ are discussed in relation to current physical models.

References

[1] J. W. G. Tyrrell and P. Attard: Phys. Rev. Lett. 87, 176104 (2001)
[2] P. Janda, H. O. Frank, Z. Bastl, M. Klementová, H. Tarábková, L. Kavan, Nanotechnology 21 (2010) 095707 (7pp)
[3] Viliam Kolivoška, Miroslav Gál, Magdaléna Hromadová, Štěpánka Lachmanová, Hana Tarábková, Pavel Janda, Lubomír Pospíšil, Andrea Morovská Turoňová: Colloids and Surfaces B: Biointerfaces 94 (2012) 213– 219
[4] H. Tarábková, P. Janda: J. Phys.: Condens. Matter 25 (2013) 184001

Acknowledged project support GACR P208/12/2429

Fig. 1: AFM image of polystyrene film as received by spin-coating. 

Fig. 2: AFM image of polystyrene film after exposition to nanobubbles in deionized water. Net-like nanopattern corresponds to 2D nanofoam imprinted into polystyrene matrix.

Fig. 3: Profile analysis of polystyrene film as received by spin coating (data from Fig. 1)

Fig. 4: Profile analysis of polystyrene film after exposition to nanobubbles in deionized water (data from Fig. 2)
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-1654 Investigating electrical charged samples by scanning probe microscopy: the influence to atomic force microscopy and magnetic force microscopy image artifacts.

Costa C. A.1, Lanzoni E. M.1, Piazzetta M. H.1, Galembeck F.1, Deneke C. F.1
1Laboratório Nacional de Nanotecnologia (LNNano), Centro de Pesquisa em Energia e Materiais (CNPEM), Campinas, São Paulo, Brazil.
carlos.costa@lnnano.cnpem.br

The electric state of a surface is of great importance for its chemical and physical properties, e.g. bounding of molecules, charge transfer or dielectric properties. Various scanning probe microscopy techniques based on atomic force microscopy (AFM) map electric surface potentials, electrostatic forces as well as the topography to distinguish electrostatic from van der Waals forces. However, the signal acquired from van der Waals forces can also show artifacts arising from electrostatic forces. Even so such effects are of great importance to interpret image obtained from charged surfaces, the effect is not discussed in detail in literature.

In this work, AFM artifacts resulting from electrical charged surfaces are investigated. In a detailed and systematic study, the influence of an electric field gradient above sample to topography, phase and magnetic force microscopy (MFM) images is investigated. Images were acquired with a commercial AFM using a lithographical patterned Kelvin force microscopy (KFM) calibration sample (Fig. 1). Our results show that electrical charges give rise to a signature in topography (Fig. 2) and phase signal. In order to minimize these artifacts, they are studied in regard of various acquisition parameters. We find that either using a low relative set point or high free vibration amplitude during images acquisition reduces the influence to the AFM measurements. Both approaches can sufficiently negate the effect by increasing the tip/sample interaction (either by getting the tip closer to the sample surface or by larger tip vibration amplitudes). As a trade off to these approaches, the sensitivity to topography features is reduced.

Finally, commercial metalized MFM cantilevers are studied in regard their sensitivity to electrical charge present on the sample surface. We observe the appearance of a MFM contrast for the non-magnetic KFM test structure (Fig. 3) for such conditions. The electrical charges give rise to a MFM signature indistinguishable from a magnetic signature exhibiting a strong correlation to KFM images obtained from our sample. The results indicate that great care has to be taken in the interpretation of topographic, phase contrast or magnetic images, when electrical fields are present on the sample surface.

This research was financially supported by Ministério da Ciência, Tecnologia e Inovação (MCTI) - Brazil.

Fig. 1: Illustration of KFM test sample consisting of interdigitated Al stripes on a glass substrate.

Fig. 2: AFM topography images (a) 0V, (b) 5V and (c) 10V applied between the finger pairs.

Fig. 3: (a)Topographic image and “MFM” phase shift for our KFM test sample (b) 0 V and (c)5V applied between the finger pairs.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2107 High-Sensitivity Imaging in Liquid by Torsional Resonance Mode Atomic Force Microscopy utilizing Lorentz Force Actuation

Yang C. W.1, Ding R. F.1, 2, Lai S. H.1, Liao H. S.1, Lai W. C.1, Huang K. Y.2, Chang C. S.1, Hwang I. S.1
1Institute of Physics, Academia Sinica, Taipei, 11529, Taiwan, 2Department of Mechanical Engineering, National Taiwan University, Taipei, 10617, Taiwan
yangcw@phys.sinica.edu.tw

Atomic force microscopy (AFM) [1] is a widely used technique for characterizing the structures and mechanical properties of material surfaces. However, operation in aqueous solution is still very challenging because the force sensitivity of dynamic AFM modes in liquid is usually much reduced compared with that in air or in vacuum. It has been shown that torsional resonance (TR) mode of vibrating cantilever is only affected by the tip-sample lateral force gradient, and not sensitive to the long-range normal forces [1]. The resonance characteristics (amplitude, phase, or frequency) start to change only when the tip gets in contact with the sample, which allows clear detection of the contact point and maintaining a soft contact between the tip and the sample. However, up to now, only very few types of cantilevers can be excited with pure torsional resonance in water [1,2].

Here we present a design based on Lorentz force induction to excite pure torsional resonances of different types of micro-cantilevers in air and in water [3]. Figure 1 shows the schematic of a rectangular cantilever actuated by the Lorentz force. An oscillating current passes through a cantilever which is mounted near two permanent magnets. The induced Lorentz force is equal and opposite on the two cantilever beams, resulting in a net torque to excite the torsional resonance [4]. With this actuation, pure torsional resonance of different types of cantilevers can be excited in air as well as in water, as shown in figure 2. To demonstrate the imaging capability, phase-modulation torsional resonance (PM-TR) mode is employed to resolve fine features of purple membranes in a buffer solution, as shown in figure 3. Most importantly, as shown in figure 4, force-versus-distance curves using a relatively stiff cantilever (k~ 40 N/m) can clearly detect hydration layers at a water-mica interface, indicating high force sensitivity of the torsional mode. Thus, the high resonance frequencies and high quality-factors for the tosional mode may be of great potential for high-speed and high-sensitivity imaging in aqueous environment. Moreover, this mode has a good potential and application for in-plane material characterization.

[1] Yang C. W.; Hwang I. S.; Nanotechnology 2010, 21, 065710
[2] Mullin N.; Hobbs J., Appl. Phys. Lett. 2008, 92, 053103.
[3] Yang C. W et al, Nanotechnology 2013, 24, 305702
[4] Byeonghee Lee et al, Nanotechnology 23, 055709 (2012).

This research is supported by the National Science Council of ROC (NSC99-2112-M-001-029-MY3 and NSC101-2221-E-002-022) and Academia Sinica.

Fig. 1: Schematics of Lorentz force actuation

Fig. 2: Torsional resonance curves of different cantilevers in air and in water

Fig. 3: AFM images of purple membrane taken with PM-TR mode in a buffer solution.

Fig. 4: Force curves of the phase lag and the amplitude of a vibrating cantilever versus the tip–sample distancemeasured on a freshly cleaved mica surface in DI water
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-1878 Thermal conductivity reduction measurement on Si and P3HT nanowires : diameter size effect

Grauby S.1, Munoz Rojo M.2, Martin Gonzalez M.2, Dilahire S.1
1Univ. Bordeaux, LOMA, UMR 5798, F-33400 Talence, France, 2Instituto de Microelectrónica de Madrid, IMM-CSIC, 8 PTM 28760 Tres Cantos, Madrid, Spain
stephane.grauby@u-bordeaux.fr

Nanostructuration has induced a renewed interest for thermoelectric materials whose performance can be evaluated through their figure of merit ZT=(S2σT)/λ where S is the Seebeck coefficient, σ the electrical conductivity and λ the thermal conductivity. Indeed, in a nanostructured material, the dimension reduction could possibly induce a thermal conductivity reduction and consequently an increase of their figure of merit. This can be partially ascribed to phonon boundary scattering appearing when one dimension of the nanostructured material becomes smaller than the phonon mean free path. The nanomaterial behaves than as a phonon glass and an electron crystal.

The work presented here deals with the study of the variation of the thermal conductivity of nanowires when reducing their diameter size due to confinement effects. The thermoelectric device is actually made of nanowires embedded in a matrix. We have studied two different kinds of nanowires with varying diameters: on one hand inorganic semiconductor Si nanowires in a SiO2 silica matrix, on the other hand poly(3-hexylthiophene) (P3HT) nanowires, an organic semiconductor polymer, which have been proven to have good thermoelectric properties[1], in an alumina matrix.

Measuring the thermal conductivity of individual nanowires embedded in a matrix is still challenging and nowadays there are not many techniques able do it[2]. Nevertheless, we have developed a technique based on an AFM associated to a thermoresistive tip and called 3ω-Scanning Thermal Microscopy (3ω-SThM). The thermoresistive tip is used both as a heater and a sensor. A current passing through it heats the tip. Depending on the thermal conductivity of the scanned material, the heat flux passing from the tip to the scanned sample varies, inducing a tip temperature variation. Then, the tip resistance changes, which induces a tip voltage variation. As a consequence, measuring the tip voltage variation enables to deduce the material thermal conductivity[3]. This mode enables to simultaneously obtain a topographical image and a thermal conductivity contrast image.

We show that a thermal conductivity reduction is observed when reducing the diameter of the nanowires for both silicon and P3HT nanowires. The thermal conductivity is reduced by 4 for P3HT nanowires when their mean diameter is reduced from 350nm to 120nm and up to a factor 10 for silicon nanowires when the mean diameter is reduced from 300nm to 50nm.

References:

1. C. Bounioux et al, Energy & Environmental Science, 2013, 6, 918-925.

2. M. Muñoz Rojo et al, Nanoscale, 2013.

3. E. Puyoo et al, J. Appl.Phys., 2011, 109, 024302-024309.

Fig. 1: Si nanowires: Scanning Electron Microscopy images respectively before (a) and after (b) encapsulation in the SiO2 matrix; 5µmx5µm 3ω-SThM images (c) topographical image, and (d) thermal conductivity contrast image.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-1892 Conduction and Dissipation of Electrostatic Charges: Fundamental Study by Scanning Probe Microscopy

YIN J.1, NYSTEN B.1
1IMCN-Institute of Condensed Matter and Nanosciences (BSMA - Bio and Soft Matter Division), Université Catholique de Louvain, Belgium
jun.yin@uclouvain.be

Static electricity is a well-known and often observed physical phenomenon. It can cause dangerous problems in many applications, such as dust filters, chemistry, sophisticated electronics, cable insulation, charge based data storage, etc. Although many contributions have been done, the understanding of conduction and dissipation behaviors, charge transfer to, and retention on, surface or charges leakage over surfaces is far from being completed. Since most of studies are at the macroscopic scale, a microscopic and systematic study is of importance to understand these phenomena. Thanks to the development of scanning probe microscopy, a number of new electrical modes using a conductive probe have been developed and used to characterize the different microscopic electrical properties, such as Current-Sensing AFM (CS-AFM), Kelvin Probe Force Microscopy (KPFM). The objective of our project is to make a microscopic, detailed and systematic study of the phenomena of electrification, charge, discharge, conduction and dissipation mechanisms of electrostatic charges. The materials studied are two different kinds of fibers used in antistatic filters: polyester fiber and stainless steel conductive fiber commercially named Bekinox® fiber.

Surface properties of stainless steel conductive fiber are first studied (Fig.1). The surface topography and surface roughness are studied by standard AFM, the surface electrical resistance distribution is measured by CS-AFM, and the surface potential distribution is measured by KPFM. I-V spectroscopy is performed statistically to investigate the different charge transport mechanisms from different surface states. Second, the mechanisms responsible for charge conduction and dissipation between two fibers are studied. It can be noted that when a conductive fiber is put in non-galvanic contact with an other polarized conductive fiber, a dynamic behavior of surface potential variation can be measured by KPFM on the first conductive fiber (Fig.2). The quantified charging and discharging curves can be fitted to obtain relaxation times. Different contact systems, including different types of fibers, galvanic and non-galvanic contacts, are investigated systematically in order to deeply understand the mechanisms of conduction and dissipation.

This research is supported by FRIA (Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture) of FRS-FNRS (Fonds de la Recherche Scientifique).

Fig. 1: Topography, surface current distribution, surface resistance distribution images obtained at the same location on Bekinox® fiber (a) topography, (b) (c) (d) electrical current distribution at 2 V, -2 V and 2 V again, (e) (f) (g) electrical resistance distribution at 1 V, 3 V, 4 V and 5 V, respectively. Image size is 5x5 µm².

Fig. 2: Topography and successive KPFM images on  Bekinox® fibers in non-galvanic while changing the applied voltage from 0 to 8 V (a) topography, (b) (c) successive KPFM images, the white arrows present the scanning direction (d) (e) two profiles from KPFM images, the average value of profile (d) is higher than profile (e).
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2022 A ferrule-top optomechanical probe to collect topographic and near field information about a sample at the nanometer scale

van Hoorn C. H.1, Chavan D. C.1, Tiribilli B.2, Margheri G.2, Mank A. J.3, Ariese F.1, Iannuzzi D.1
1Faculty of Sciences, LaserlaB, Vrije Universiteit, Amsterdam, The Netherlands, 2Institute of Complex Systems, National Research Council, Sesto Fiorentino, Italy, 3Philips Innovation Services, Eindhoven, The Netherlands
c.h.van.hoorn@vu.nl

Atomic force microscopy (AFM) is an excellent technique for obtaining high-resolution images of the topography of a sample. The impact of AFMs in nanotechnology could be even more significant if the imaging capabilities were supported by an accurate mapping of the optical field in the close proximity of the surface. Scanning near field optical microscopy (SNOM), for example, has been shown to be able to combine AFM imaging with the possibility to collect optical information at the nanoscale. Yet, because of the complexity of its working principle, SNOM has been so far only used in specialized research laboratories and has been mostly limited to the analysis of surfaces in dry environments. To solve this limitation, in a previous work [1] some of us have proposed an all-optical device obtained by carving a tipped cantilever on top of an optical fiber. The opposite end of the fiber can be coupled to a readout system that was shown to be able to detect any tiny movement of the cantilever and to collect the SNOM signal coming from a prism illuminated under total internal reflection conditions. Here, we present another similar all-optical probe for AFM+SNOM imaging. The probe is based on ferrule-top technology [2-4], which relies on the possibility to fabricate a small cantilever on top of a ferruled fiber. This design keeps the overall advantages of the previous version (small dimensions, ease of use, easy integration in harsh environments) while significantly reducing the fabrication costs. Using this probe, we were able to obtain the SNOM profile and, simultaneously, the topographic image of a test grating (NT-MDT SNG01) kept in air and illuminated from below via an evanescent field. The AFM height resolution and the SNOM lateral resolution resulted to be comparable to conventional SNOM systems. Interestingly, measurements were repeated in water, with no major deterioration of the overall performance. This result paves the way for AFM+SNOM imaging on biological samples.

References:

1. B. Tiribilli, G. Margheri, P. Baschieri, C. Menozzi, D. C. Chavan, D. Iannuzzi, Journal of Microscopy 2011, 242, 10–14.

2. G. Gruca, K. Heeck, J. H. Rector, and D. Iannuzzi, Optics Letters 2013, 38, 1672.

3. G. Gruca, D. C. Chavan, J. H. Rector, K. Heeck, and D. Iannuzzi, Sensors and Actutators 2013, A190, 77.

4. D. C. Chavan, G. Gruca, S. de Man, M. Slaman, J. H. Rector, K. Heeck, D. Iannuzzi, Review of Scientific Instruments 2010, 81, 123702.

This work was funded by the European Research Council and by NanonextNL.

Fig. 1: Fabrication procedure of a ferrule-top probe. A glass ribbon is glued on top of a ferrule (a, b). The length of the cantilever is adjusted using a laser ablation machine (c). A tipped fiber is glued to the cantilever and the cantilever is released by focussed ion beam milling (FIB). Finally, a fiber is inserted into the borehole (d).

Fig. 2: A schematic view of the experimental setup. A blue (473 nm) laser beam impinges the top surface of a prism to create an evanescent field. The probe was scanned over the sample surface, in order to obtain a topographic and SNOM image simultaneously.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2058 High-sensitivity high-resolution elemental 3D analysis by in-situ combination of SIMS and SPM

Fleming Y.1, Eswara Moorthy S.1, Wirtz T.1, Gerard M.1, Gysin U.2, Glatzel T.2, Meyer E.2, Maier U.3
1Department “Science and Analysis of Materials” (SAM), Centre de Recherche Public – Gabriel Lippmann, 41 rue du Brill, L-4422 Belvaux, Luxembourg, 2Department of Physics, University of Basel, Klingelbergstr. 82, Basel, Switzerland, 3Ferrovac GmbH, Thurgauerstr. 72, CH-8050 Zürich, Switzerland
wirtz@lippmann.lu

Owing to its excellent sensitivity, its high dynamic range and its good depth resolution, Secondary Ion Mass Spectrometry (SIMS) constitutes an extremely powerful technique for analyzing surfaces and thin films. In recent years, considerable efforts have been spent to further improve the spatial resolution of SIMS instruments. As a consequence, new fields of application for SIMS, e.g. nanotechnologies, biology and medicine in particular, are emerging [1-2].

State-of-the-art SIMS instruments allow producing 3D chemical mappings with excellent sensitivity and spatial resolution. However, several important artifacts arise from the fact that the 3D mappings do not take into account the sample’s surface topography. The traditional 3D reconstruction assumes that the initial sample surface is flat and the analyzed volume is cuboid. The produced 3D images are thus affected by a more or less important uncertainty on the depth scale and can be distorted. Moreover, significant field inhomogeneities arise from the surface topography as a result of the distortion of the local electric field. These perturb both the primary beam and the trajectories of secondary ions, resulting in a number of possible artifacts, including shifts in apparent pixel position and changes in intensity.

In order to obtain high-resolution SIMS 3D analyses without being prone to the aforementioned artifacts and limitations, we developed an integrated SIMS-SPM instrument, which is based on the Cameca NanoSIMS 50 [2]. This instrument, an in-situ combination of sequential high resolution Scanning Probe Microscopy (SPM) and high sensitivity SIMS, allows topographical images of the sample surface to be recorded in-situ before, in between and after SIMS analysis. Hence, high-sensitivity high-resolution chemical 3D reconstructions of samples are possible with this extremely powerful analytical tool [3-4].

In addition, this integrated instrument allows a combination of SIMS images with valuable AFM (Atomic Force Microscopy) and KPFM (Kelvin Probe Force Microscopy) data recorded in-situ in order to provide an extended picture of the sample under study. The known information channels of SIMS and AFM/KPFM are thus combined in one analytical and structural tool, enabling new multi-channel nanoanalytical experiments. This opens the pathway to new types of information about the investigated nanomaterials.

This paper will present the prototype instrument with dedicated software, its performances and some typical examples of application.

References:

[1] Y. Fleming et al., Appl. Surf. Sci. 258 (2011) 1322-1327

[2] T. Wirtz et al., Surf Interface Anal. 45 (2013) 513-516

[3] T.Wirtz et al., Rev. Sci. Instrum. 83 (2012) 063702

[4] C. L. Nguyen et al., Appl. Surf. Sci. 265 (2013) 489-494

Fig. 1: Combined SIMS-SPM 3D reconstruction of an Al (100nm) / Si sample exposed to a plasma streamer (Field of view: 15x15 µm2): (a) Al- signal (b) Si- signal

Fig. 2: PS/PMMA blend (Field of view: 22.3x17.3 µm2): (a) Combined SIMS-SPM 3D reconstruction of the 12C- secondary ion signal. (b) Combined SIMS-SPM 3D reconstruction of the 16O- signal, which is characteristic of PMMA [3].
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2144 Imaging nanostructures of nitrogen/oxygen molecules at HOPG-water interfaces with different atomic force microscopy modes

Hwang I.1, Yang C.1, Lu Y.1, Fung C.1, Ko H.1
1Institute of Physics, Academia Sinica, Taipei, 11529, Taiwan
ishwang@phys.sinica.edu.tw

Nanobubbles, cap-shaped soft nanostructures, and micropancakes, quasi-2D layered structures, have been reported at the interfaces between hydrophobic solid surfaces and water based on atomic force microscopy (AFM) studies [1]. Previous studies have indicated that these interfacial structures contain gases because they are formed under water saturated or supersaturated with gases. They were considered as novel gaseous states by many researchers. However, there are several puzzles about them, such as the high stability, the nature, the rather flat morphology, etc.


We have investigated these interfacial structures on highly ordered pyrolytic graphite (HOPG) surfaces in pure water with different atomic force microscopy (AFM) modes, including the frequency-modulation (FM), the tapping, and PeakForce techniques. The FM mode provides more accurate measurement of the surface profiles of nanobubbles than the other two imaging modes (Fig.1). The height obtained with PeakForce mode is smaller than the true height of nanobubbles due to a snap-in when the tip touches a nanobubble, as shown in the force vs the tip-sample separation curve (Fig. 2a). This is because a positive peak force is required to achieve stable imaging. The resonance frequency shift vs the tip-sample separation curve (Fig. 2b) shows a sharp increase in the resonance frequency when the tip touches a nanobubble, thus the snap-in has little effect on the height measurement in the FM mode. Similar force curves are seen on micropancakes. Combining AFM images obtained with these modes, models for nanobubbles and micropancakes are proposed, which can provide a better explanation for the high stability of these interfacial structures.

[1] Seddon J. R. T. and Lohse D.; J. Phys: Condens. Matter 2011, 23, 133001.

This research is supported by the National Science Council of ROC (NSC96-2628-M-001-010-MY3 and NSC99-2112-M-001-029-MY3) and Academia Sinica.

Fig. 1: Topographic imaging of nanobubbles on HOPG in DI water taken with (a) the FM mode, (b) PeakForce mode, (c) the tapping mode (TM). (d) Horizontal height profiles across the center of nanobubble 3 taken with the FM PF, and TM modes.

Fig. 2: Approaching force curve measured on nanobubbles at HOPG-water interfaces. (a) Force vs the tip-sample separation curve. (b) Resonance frequency shift vs the tip-sample separation curve.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2422 Instrument induced artifacts in scanning probe microscopy.

Lanzoni E. M.1, Costa C. A.1, Barboza V. A.1, Galembeck F.1, Deneke C. F.1
1Laboratório Nacional de Nanotecnologia (LNNano), Centro de Pesquisa em Energia e Materiais (CNPEM), Campinas, São Paulo, Brazil.
evandro.lanzoni@lnnano.cnpem.br

In the last three decades, scanning probe microscopy (SPM) techniques have been established as the major way to directly probe the 3 dimensional structure of a sample surface. The images are acquired by accurate movements of a sharp tip (probe) above the sample surface, controlled by a scanning electronic. For topography images van der Waals interaction between tip and sample is generally used as feedback mechanism. The  two major classes of topography images artifacts are: the tip-sample convolution resulting in a broadening of the observed structures as well as digitalization artifacts arising from the analog-digital conversion carried out during image acquiring.

In this work, we carry out a detailed analysis of the tip-sample convolution artifacts to topographic image formation in regard of the finite resolution implied by the analog-digital conversation. We discuss possible ways to identify these artifacts and wrote a software module to identify them in obtained images. As shown in Fig. 1, the resolution in the X-Y is limited by the tip-sample surface convolution depending on the geometry of the probe-scan-plane and sample-surface-plane. Commonly, the real tip-sample contact occurs on the tip side and not at the tip apex. Furthermore, the tip scans over the surface and the microscopy converts the obtained analog signal to a digital image with a certain number of points. Hence, the lateral resolution depends on the number of points for a given scan size as illustrated in Fig. 2. As illustrated in Fig. 3, for a small enough pixel size, the tip-sample convolution dominates the maximal obtainable resolution as the real contact point is not the tip apex. Furthermore, the tip-sample convolution in conjunction with the finite pixel size results in an interpolation of the surface, which is shallower than the real surface feature and is determined by the tip geometry.

We implemented a software module in the free SPM software Gwyddion that analysis the sample surface inclination in regard of such sample-tip convolution gradients. By assuming a certain tip radius and tip slope, we mark areas in the obtained topographic image, which are most likely exhibiting the wrong topographic information. The artifact analysis allows a better understanding of the instrument or acquisition parameters, i.e. tip radius needed to obtain artifact free images (e.g. use of super sharp tips), inclination of scan/surface planes, number of points needed for a image, or dynamic scanner range.

This research was financially supported by the Ministério da Ciência, Tecnologia e Inovação (MCTI) - Brazil.

Fig. 1: Illustration of tip apex /sample surface geometry convolution. The black line shows the surface profile obtained due to the convolution.

Fig. 2: AFM topography images of InGaAs surface in the same area scanned with (a) 32 X 32 pixels and (b) 256 X 256 pixels.

Fig. 3: Illustration of a profile interpolation resulting from the pixel size.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2536 Surface potential investigation of AlGaAs/GaAs heterostructures by Kelvin Force Microscopy

Pouch S.1, Chevalier N.1, Mariolle D.1, Triozon F.1, Niquet Y. M.2, Melin T.3, Borowik Ł.1, Delaye V.1
1CEA, LETI, MINATEC Campus, 17 rue des Martyrs, 38054 GRENOBLE Cedex 9, France, 2CEA, INAC, MINATEC Campus, 17 rue des Martyrs, 38054 GRENOBLE Cedex 9, France., 3Institut d’Electronique de Microélectronique et de Nanotechnologie, CNRS-UMR 8520, Avenue Poincaré, BP 60069,59652 Villeneuve d’Ascq Cedex, France.
sylvain.pouch@cea.fr

The Kelvin force microscopy (KFM) provides a spatially resolved measurement of the surface potential, which is related to the energetic band structure of a material. The goal of this work is to investigate the surface potential measured by KFM on AlGaAs/GaAs heterostructures.

For this study, we selected a certified reference sample BAM-L200 [1] composed of epitaxial layers of AlGaAs and GaAs, with a decreasing thickness (600 to 2 nm) and an uniform Si (n) doping (5.1017 cm-3). The surface potential measurement is performed with an Omicron XA VT AFM, under ultra-high vacuum (of 10-11 mbar). Two scanning modes are used: the amplitude modulation (AM-KFM), sensitive to the electrostatic force and the frequency modulation (FM-KFM), sensitive to its gradient. [2] Three kinds of tips have been used for this study: Platinum coated silicon tips (BudgetSensors), Au nanoparticles coated silicon tips (Next Tip) and super sharp silicon tips (Nanosensors).

We will present the measurements obtained with these different tips on the sample area containing the narrowest layers. The relevant result is the fact that the contrast decreases with diminution of the layer thickness. With Pt-coated Si tips, a maximum contrast of about 270 meV was observed, whereas for super sharp Si tips the maximum contrast equals 290 meV [Fig. 1]. This contrast vanishes when layer thickness becomes thinner than 5 nm for Pt-coated SI tips and 3 nm for super sharp Si tips. This loss of contrast can be explained primarily by the resolution limit of our instrument but also the band bending length scale at the AlGaAs/GaAs interface, related to the dopant concentration. The contribution of band bending between the layers to the measured potential is evaluated by a self-consistent simulation of the electrostatic potential, accounting for the free carriers distribution inside the sample and for the surface and interface dipoles. As shown in Fig. 2, the electric fields of the narrow layers recover each other, resulting in the partial or total loss of the intrinsic sample structure. Simple comparison of simulation with KFM surface potential measurements provides information that KFM measurements represent real values and are not influenced by KFM resolution limit.

All measurements were made on the CEA Grenoble nanocharacterization platform (PFNC).

[1] M. Senoner, T. Wirth, W. Unger, W. Österle, I. Kaiander, R. L. Sellin and D. Bimberg, BAM-L002 - a new type of certified reference material for length calibration and testing of lateral resolution in the nanometre range, Surface and Interface Analysis 36, 1423-1426 (2004)
[2] S. Sadewasser and T. Glatzel, Kelvin Probe Force Microscopy (2012)

Fig. 1: KFM images obtained on BAM-L200 with super sharp silicon tips, with respect to sample schema: (1) Topographic image; (2) Surface potential images.

Fig. 2: Theoretical simulation of the electric potential at 5 nm above the surface of the sample, and averaged section of previous KFM image.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2566 DESIGN, IMPLEMENTATION AND CHARACTERIZATION OF A 3D-PRINTED AFM HEAD WITH PIEZOTUBE AND ELECTROMAGNETIC ACTUATORS FOR BIOMOLECULAR APPLICATIONS

Sevim S.1, Özer S.2, Feng L.2, Crawford K.2, Karaca O.2, Torun H.2
1Department of Mechanical Engineering, Boğaziçi University, Bebek/Istanbul, Turkey, 2Department of Electrical and Electronics Engineering, Boğaziçi University, Bebek/Istanbul, Turkey
semih.sevim89@gmail.com

An atomic force microscope (AFM) has been developed for biomolecular force spectroscopy. Design, implementation and characterization of the AFM head are described here. The head is portable and was manufactured at low cost using stereolithography. A flexible software-based controller was implemented that can be adapted easily for different applications. The AFM head, made of a rigid polymer material (Rigid Opaque, Stratasys, Ltd., MN, USA) is shown in Fig. 1(a). The head houses a piezotube actuator, a laser diode, a quadrature photodetector and an AFM cantilever. The cantilever is mounted to the piezotube using a customized holder. The incident laser beam (fiber pigtailed, Oz Optics, Ottowa, Canada) is directed to the cantilever using a kinematic mount. Reflected laser light is directed to the quadrature detector (Pacific Silicon Sensor, Westlake Village, CA, USA) via a mirror, which has a one degree-of-freedom of rotation. The photodetector is placed on a translational microstage with two degrees-of-freedom. The space below the cantilever plane is empty so that the head can be integrated with an inverted microscope. In addition, an electromagnet was employed with the head for direct cantilever actuation and for other magnetic applications. A one-dimensional actuation coil is integrated to the head as shown in Fig. 1(b). The system allows cantilever actuation using the piezotube actuator and the electromagnet. Fig. 2(a) shows a sample force curve obtained using the piezo actuator. The drive signal in various waveforms was generated by the customized software-based controller to actuate a commonly used AFM cantilever (SNL-10D, Bruker Probes, Santa Barbara, CA) on a silicon sample at various frequencies, from 10 mHz to 1 kHz. In addition, Fig. 2(b) shows a typical current signal input to the electromagnet the corresponding photodetector signal. A commercially available MFM cantilever (MESP, Bruker Probes, Santa Barbara, CA) was actuated by a square wave in air at 1 kHz. The head was designed and optimized for force spectroscopy experiments. The force noise of the system using typical AFM cantilevers has been characterized as 6.8 pN within a bandwidth of 1 kHz. Finally, a biomolecular force spectroscopy experiment to probe interactions between FGF-2 and heparin was performed using the piezotube actuation. Fig. 3(a) shows a typical force curve, exhibiting an unbinding event with a force strength of 500 pN, whereas in Fig. 3(b) there is another force curve indicating no adhesion/rupture events.

Authors would like to acknowledge funding from the EC (ICT FET-Open) under the MANAQA Project.

Fig. 1: Fig. 1 (a) The photograph of assembled AFM Head, manufactured using stereolitography. (b) The photograph of AFM head employed with an electromagnet to actuate the MFM cantilevers.

Fig. 2: Fig. 2 (a) Typical force curves, taken with 10 Hz piezo actuation over a silicon wafer. (b) Electromagnetic actuation of a MFM cantilever in air at 1 kHz.

Fig. 3: Fig. 3 (a) A typical force curve exhibiting an unbinding event between FGF-2 and Heparin molecules. (b) A force curve which is indicating no adhesion/rupture event.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2717 Integration of SPM module inside FIB-SEM instrument

Rudolf M.1, Sedláček L.1, Jiruše J.1
1TESCAN Brno, s.r.o., Brno, Czech Republic
libor.sedlacek@tescan.cz

In the nanotechnology field, SPM (Scanning Probe Microscope) integrated into a SEM (Scanning Electron Microscope) offers a completely new opportunities [1]. Recently, TESCAN integrated a SPM with the lateral scan range up to 50 µm and Z scan range 8 µm [2]. Its compact construction is optimized for operating in a confined space of a FIB-SEM chamber without affecting the performance of both the electron or ion beams. Such dedicated SPM design allows the investigation of the same place on the sample by SPM, FIB (Focused Ion Beam) and high resolution SEM with a spot size down to 1 nm simultaneously without the need to perform an additional sample re-positioning.

We developed an intuitive software module to simplify the SPM navigation on the sample. It is possible to save the region of interest to a memory and recall it later on either by SPM, the SEM or both. Saved positions are shown along with their title in a live SEM window, see Fig. 1. The past experience has proved that the SPM module inside the FIB-SEM microscope is useful for several applications, such as process optimization of electron and ion beam lithography [3, 4] or TOF-SIMS (time-of-flight secondary ion mass spectroscopy) where the SPM can provide complementary information about the depth profile.

Fig. 2 shows an example how such a combination of the SEM, FIB and SPM was utilized. Hydrogenated Diamond-like Carbon (H:DLC) layer prepared by PECVD (Plasma Enhanced Chemical Vapor Deposition) method on a Si wafer was locally milled by FIB in order to uncover the Si-DLC interface, and the thickness of the DLC layer was measured using SPM. The damage caused by FIB milling is only local and it has no disturbing effects on measurements performed later on the same sample.

In another example we utilized in-situ cooperation of SEM/FIB/TOF-SIMS/SPM techniques together. FIB and TOF analyzer were used to create a concentration depth profile of elements contained in H:DLC layer deposited on an Si wafer, and the SPM navigated by the SEM provided an additional information about the true depth profile, see Fig. 3.

References:

[1] W Heichler, Microsc. Microanal. 19 (suppl. 2) (2013) p. 350.

[2] M&M 2011 trade show. See also [online]. [cit. 2014-03-03]. <http://www.specs.de/cms/upload/PDFs/SPECS_Prospekte/new_design/20130312_Curlew_brochure_final_web.pdf>

[3] J Jiruše et al, MC Proceedings Part 1 (2013) p. 154.

[4] J Jiruše et al, Proceedings 57th EIPBN (2013) p. 01-07.

The authors acknowledge funding from the European Union Seventh Framework Program [FP7/2007-2013] under grant agreement No. 280566, project UnivSEM.

Fig. 1: Positions on the sample surface are saved to the memory and shown in a live SEM window. Position in a memory can be recalled either by SMP, SEM, or both.

Fig. 2: DLC layer deposited on the Si wafer sputtered by the FIB. The DLC layer thickness is determined by the SPM module. (a) SEM image, (b) AFM image, (c) AFM profile.

Fig. 3: Concentration depth profile of carbon and silicon in H:DLC layer measured by TOF-SIMS with the depth information provided by SPM. From the SPM depth measurements and the sharp increase in the silicon signal intensity the thickness of a DLC layer can be obtained.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2845 In situ characterization of the growth mechanism of PEDOT films with electrochemical atomic force microscopy

Reggente M.1, Passeri D.1, Angeloni L.1, Rossi M.1,3, Tamburri E.2, Orlanducci S.2, Terranova M. L.2
1Department of Basic and Applied Sciences for Engineering, Sapienza University of Rome, Rome, Italy, 2Department of Chemical Science and Technology - MINASlab, University of Rome 'Tor Vergata', Rome, Italy, 3Center for Nanotechnology Applied to Engineering of Sapienza (CNIS), Sapienza University of Rome, Rome, Italy
melania.reggente@uniroma1.it

Conductive polymers (CP) belong to an attractive class of materials with plastic-like mechanical properties and electric conductivity typical of metals, which have awakened an increasing interest for several applications, e.g. sensing, electronic and energy. One of the most studied CP is the Poly(3,4-ethylenedioxythiophene) (PEDOT) due to its well-known properties and the advantage of being synthesized as thin-film directly on the substrates of interest. PEDOT thin films can be realized by electrochemical synthesis performed in an aqueous media containing a small quantity of the monomer 3,4-ethylenedioxythiophene (EDOT) and a suitable supporting electrolyte [1-2]. It is known that the process parameters influence both the structural and the electrical properties but further studies on the nucleation and growth mechanisms of the film formation are still required. Thus, it can be useful to monitor in real-time the synthesis process of PEDOT films in order to tune the process parameters and produce films with reproducible specific properties. Up to now, atomic force microscopy (AFM) based techniques have been employed to underlying the morphological features of the films at different steps of the deposition process and their related conductive properties but results of an in situ AFM investigation are not yet reported.
In this work, the growth mechanism of electrodeposited PEDOT films are investigated by using the electrochemical atomic force microscopy (EC-AFM) in order to determine the correlation between their morphological features and the electrochemical parameters of the process. In EC-AFM, a standard AFM apparatus is equipped with a three-electrode electrochemical cell whose working electrode is the sample surface where the electrodeposition takes place. Thus, a real time study of the electrochemical reactions occurring at the surface of the sample is achieved and the in situ surface morphology evolution is monitored by using an unbiased AFM probe. In particular, the electropolymerization of EDOT is observed performing a cyclic voltammetry and controlling the evolution of current flowing through the electrode surface, together with a standard AFM image. By varying the supporting electrolyte concentration, the voltammetry scan rate and the working electrode surface, the nucleation and growth mechanisms of the film are investigated and the results are compared with the already hypothesized growth model.
Overall, this work demonstrates the capability of the EC-AFM to deepen the growth mechanism of electrodeposited polymeric films with tunable and reproducible properties.
[1] E. Tamburri et al., Synthetic Metals, 159 (2009) 406–414.
[2] V. Castagnola et al., Synthetic Metals, 189 (2014) 7–16.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2847 Atomic force microscopy techniques for the detection of nanomaterials incorporated in biological systems

Reggente M.1, Passeri D.1, Angeloni L.1, Scaramuzzo F.1, De Angelis F.2, Barteri M.2, Rossi M.3
1Department of Basic and Applied Sciences for Engineering, Sapienza University of Rome, Rome, Italy, 2Department of Chemistry, University of Rome Sapienza,Rome, Italy, 3Center for Nanotechnology Applied to Engineering of Sapienza (CNIS), Sapienza University of Rome, Rome, Italy
melania.reggente@uniroma1.it

The capability of detecting nanomaterials (NMs) in biological samples represents one of the main challenges in bionanoscience, as it would allow the monitoring of cell-NMs interactions at the nanoscale, which is of primary importance in several fields of application, from drug delivery to nanotoxicology. To this aim, Atomic Force Microscopy (AFM) has been proposed as a versatile platform for the detection of NMs in biological matrices. By detecting the inhomogeneity of the mechanical, electrical or magnetic properties of the host-guest systems, the presence of NMs can be revealed [1].
In this work, different AFM-based techniques are employed to investigate the interactions between Magnetic Nanoparticles (MNPs) and cells. We show the possibility to reveal the presence of MNPs in biological system by detecting both the magnetic and the mechanical properties of the samples. First of all Magnetic Force Microscopy (MFM), a two-pass AFM-based technique which requires a tip coated with a magnetic film to obtain images reflecting the local magnetic properties of the samples, is employed for the imaging of MNPs internalized in cells. In addition to this, buried MNPs in soft biological matrices are visualized using three different AFM-based techniques in which the contrast reflects the non homogeneous mechanical properties of the host and guest systems. In particular, images of the local sample stiffness are obtained using the AFM force-volume imaging mode allowing the detection of force-distance curves. Moreover, the Contact Resonance Frequencies (CRFs) of the cantilever in contact with the sample surface, which are related to the local elastic modulus of the sample, are recorded employing the Atomic Force Acoustic Microscopy (AFAM). Following an offline procedure, the semi-quantitative CRFs maps are then converted into quantitative indentation modulus maps by assuming a proper model for the cantilever-tip-sample system. Furthermore, online maps of the local indentation modulus of the samples are recorded using Torsional Harmonics AFM (TH-AFM) which allows the evaluation of the local sample stiffness by acquiring force-distance curves in tapping mode. Finally, the mechanical properties evaluated with these three different techniques are compared and the influence of the penetration depth of each technique on the results is discussed and rationalized.
A careful comparison between the images obtained using all these techniques based both on the magnetic and the mechanical contrast allows to detect NMs incorporated in biological matrices and represents a clear indication of the AFM powerfulness in the field of nanobiotechnology.
[1] Atomic Force Microscopy in Cell Biology, B.P. Jena, J.K.H. Hörber (Eds), Academic Press, San Diego California USA (2002).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2848 Bacterial adhesion force measurements by microbial cell probe Atomic Force Microscopy

Angeloni L.1, 2, Passeri D.1, Reggente M.1, Pantanella F.3, Schippa S.3, Mantovani D.2, Rossi M.1
1Department of Basic and Applied Sciences for Engineering, University of Rome Sapienza, Via A. Scarpa 16, 00161 Rome, Italy, 2Lab. for Biomaterials and Bioengineering (CRC-I), Dept. Min-Met-Materials Eng. & University Hospital Research Center, Laval University, Quebec City, Canada , 3Department of Public Health and Infectious Diseases, University of Rome Sapienza, Piazzale A. Moro 5, 00185 Rome, Italy
livia.angeloni@uniroma1.it

The ability of bacteria to adhere to solid surfaces, proliferate and make a biofilm is the primary cause of food contamination, hospital infections and failures of long-term biomedical implants. Consequently, there is an increasing interest in developing surfaces with antibacterial properties in many areas such as food processing and health-related fields like medicine and dentistry.
In order to design antibacterial surfaces it is necessary to understand the physical and molecular interactions governing the bacterial adhesion to a surface, which is the first crucial step of biofilm formation. Several methods have been developed to evaluate these mechanisms and to identify the main influencing parameters. Static adhesion assays can provide experimental samples suitable for the qualitative or semi-quantitative measurements of bacterial adhesion. Fluid shear systems have been used to simulate the in vivo dynamic mechanical stress and to obtain global probabilistic measurements of the bacterial adhesion strength [1].
Nevertheless, the physical interactions involved in bacterial adhesion have not been understood in detail and the development of experimental procedures for further investigations is essential.
For a more focused investigation on the adhesion mechanisms, techniques comprising the manipulation of single bacteria are more appropriate.
Atomic Force Microscopy (AFM) can be used to obtain local information about the first physicochemical interaction phase of bacterial attachment to a surface, by the measurement of force-distance curves. The process can be studied by two different experimental approaches: i) by measuring the interaction forces between bacterial cells and a standard AFM tip [1] or ii) by measuring the interaction forces between bacteria on AFM tip and different surfaces [2].
In this work we develop an experimental procedure to obtain quantitative measurements of bacterial adhesion to different surfaces, by recording force-distance curves using probes coated with different bacterial species. Force-distance curves measurements are carried out, in air and in liquid, on substrates with different properties (chemical composition, hydrophobicity, charge)
The influence of the experimental conditions on the results is analyzed with the aim of assessing the most appropriate procedure.
Also, the results are discussed focusing on the influence of different physicochemical properties of bacteria and surfaces in the adhesion mechanism.
Overall, this work represents a preliminary study on the capability of AFM force distance curves measurements to investigate the physicochemical mechanism involved in the bacterial adhesion to abiotic surfaces.
[1] M. Katsikogianni et al., Eur Cell Mater 8 (2004) 37-57
[2] Y.J. Oh et al., Ultramicroscopy 109 (2009) 874–880

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-2849 Quantitative characterization of magnetic nanoparticles properties by Magnetic Force Microscopy

Angeloni L.1, 2, Passeri D.1, Reggente M.1, Marianecci C.3, Mantovani D.2, Rossi M.1
1Department of Basic and Applied Sciences for Engineering, University of Rome Sapienza, Via A. Scarpa 16, 00161 Rome, Italy, 2Lab. for Biomaterials and Bioengineering (CRC-I), Dept. Min-Met-Materials Eng. & University Hospital Research Center, Laval University, Quebec City, Canada, 3Department of Drug Chemistry and Technologies, University of Rome Sapienza, Piazzale A. Moro 5, 00185 Rome, Italy
livia.angeloni@uniroma1.it

The development of techniques for the characterization of magnetic nanomaterials has great interest by reason of the specific properties that occur in magnetic materials when their dimensions are reduced to the nanoscale. These properties, coupled with the nanometric size, make magnetic nanomaterials suitable for several biomedical applications. Magnetic nanoparticles (MNPs) can be used as carriers for drug delivery systems, mediators for magnetic hyperthermia treatments, contrast agents for Magnetic Resonance Imaging (MRI), markers for cell labeling [1].
The design of these techniques requires a detailed knowledge on the magnetic and structural properties of the adopted nanomaterials. For example the magnetic hyperthermia heating effect, the translational force exerted on drug delivery carriers, the drag force in cells magnetic separation systems are strongly dependent on the size and the magnetic properties of the nanoparticles, like the magnetic susceptibility χ, the saturation magnetization Ms, and the magnetic dipole m.
Standard techniques, like Superconducting Quantum Interference Devices (SQUID) or Vibrating Sample Magnetometer (VSM), allow the detection of global magnetic properties of nanoparticles populations. But the detection of magnetic properties of single particles is not possible and the evaluation of these properties in dependence of the particles size is not explicit.
In this work we develop an experimental procedure to obtain quantitative measurements of nanoparticles magnetic properties (χ, Ms, m) and to directly relate these characteristics with the particles size, by using Magnetic Force Microscopy (MFM). MFM is a particular non-contact scanning probe technique, based on the detection of the magnetostatic interaction between a magnetic AFM probe and a magnetic sample [2]. Thanks to its nanometric lateral resolution and its capability to detect weak magnetic fields, MFM is a powerful tool for the characterization of single nanoparticles dimensions and magnetic properties. However, MFM measurements are also affected by non magnetic tip-sample interactions. Consequently the quantitative magnetic characterization of nanomaterials requires the accurate analysis and interpretation of MFM data. In this respect, the study is also focused on the evaluation of the influence, on the MFM measurements, of non-magnetic tip-sample interactions, like electrostatic forces, with the aim of assessing an experimental procedure to detect only magnetic tip-sample interactions.
Overall, this work represents a preliminary study on the applicability of MFM technique in the quantitative measurement of properties of magnetic nanomaterials.
[1] Q. A. Pankhurst et al., J. Phys. D: Appl. Phys. 36 (2003) R167–R181
[2] P. Grutter, Ultramicroscopy, 47 (1992) 393-399

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-3249 Water meniscus investigated at nanoscale contacts with a heated atomic force microscope (AFM) cantilever probe

Assy A.1, Lefèvre S.1, Chapuis P. O.1, Gomes S.1
1Université Lyon 1, CETHIL, UMR5008, F-69621 Villeurbanne cedex, France
ali.assy@insa-lyon.fr

While working under ambient conditions, Scanning Probe Microscopy (SPM) techniques face up to a liquid meniscus when the probe gets into contact with the sample. The meniscus is formed due to the capillary condensation of the ambient environment. This meniscus could be a barrier and prevents exploiting the performed measurements or an advantage for some applications like the “Dip-Pen Nanolithography”. In the case of some applications where BioMEMS or NEMS/MEMS are involved, the meniscus problem is referred as to the stiction. In our case, the stiction is the large lateral force required to initiate relative motion between the probe and the sample. In order to find out a solution to this problem, we present an investigation of the volume and the radii of the water meniscus at different temperatures of the probe. The probe is mounted on an atomic force microscope (AFM) for its 3D positioning and displacement and for controlling the force between the probe and the sample. A resistive element is located at the tip apex and serves to heat the probe depending on the electrical current. The probe temperature is verified through a Wheatstone bridge and is maintained constant during the approach of the probe to the sample. The variations of the capillary forces are measured at different probe temperatures on different hydrophilic and hydrophobic samples. The measurements as a function of the probe temperature show a progressive evaporation of the meniscus. Moreover, and simultaneously to these variations, the heat conductance to the sample is measured. A correspondence between the thermal signal and the capillary forces is evidenced as shown in Figure 1. Based on theoretical models found in the literature, the meniscus interaction radius is evaluated from the capillary forces. Afterwards, the heat conductance at different probe temperature levels is linked with the evolution of the capillary forces, e.g. the meniscus volume. The experimental results obtained with different probes are compared and in accordance with literature values. The effect of roughness on the capillary forces is shown for different samples. For each used probe, we introduce a model that takes into account all the heat transfer mechanisms that operate simultaneously between the probe and sample. The transposition of these results could be interesting for many related applications such as BioMEMS and NEMS/MEMS.

Fig. 1: An example of the correspondence between the pull-off forces and the heat flux ratio as a function of the probe mean temperature (Tm). The measurements shown here are between a Pt/Rd microprobe and a Ge sample. (IT) and (DT) stand up for increasing temperature and decreasing temperature respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-3424 Surface Potential Distribution on Quartz Crystal Surfaces by AFM Silica-Probing

Yelken G. O.1, Polat M.1
1Izmir Institute of Technology, Department of Chemical Engineering, Urla, Izmir, Turkey
gulnihalyelken@iyte.edu.tr

Interaction forces between colloidal particles play an important role in numerous physicochemical systems in mineral, ceramic, and environmental sciences since they determine stability, rheology, and forming characteristics. Control and manipulation of these properties depend on detailed analysis of the interactions among the particles. Interparticle interactions can be divided into two main categories; van der Waals(vdW) and Electrical Double Layer [1,2].
Proper use of these theories and their comparison with the experimentally measured force values require knowledge of such material properties as Hamaker constants and charge on the interacting surfaces. The surface potential at the point of measurement could then be determined from the electrostatic component. Multiple AFM force measurements on carefully selected locations on the surface could be used to generate a surface charge/potential map of the surface using appropriate theories [3,4,5].
In this study we used a powerful surface analysis tool, AFM, to determine the surface charge or surface potential on quartz single crystal surfaces in aqueous solutions. This use of AFM is new and novel and requires insightful use of theory and experiment. Using AFM to map the charge distribution on surfaces in solution is different than the EFM measurements in air since measuring surface potential in air or in vacuum is a straightforward process which has been used for years using different devices. The methodology, we used is basically depends on a point by point comparison of measured interaction force between a surface and the AFM tip of known characteristics with the theoretical force predicted for the same system. The results were confirmed with separate electrokinetic measurements of all surfaces.

References
1. [1] Derjaguin, B.V., L. Landau, Physicochim, URSS, No:14, 633,1941.
2. Verwey, E.J.W., J.T.G. Overbeek, Theory and Stability of Lyophobic Colloids, Elsevier, Amsterdam,1948..
3. Sader, J.E., Chon, J.W.M., Mulvaney, Calibration of rectangular atomic force microscope cantilevers, Rev. Sci. Instrum., No: 70, 3967-3969, 1999.
4. Polat, M., H. Polat, Analytical solution of Poisson–Boltzmann equation for interacting plates of arbitrary potentials and same sign, J.of Colloid and Interface Science, 341,1, 178-185, 2010.
5. Yelken,G. O., Polat, M., Determination of electrostatic potential distribution by atomic force microscopy (AFM) on model silica and alumina surfaces in aqueous electrolyte solutions, Applied Surface Science, 2014. http://dx.doi.org/10.1016/j.apsusc.2014.02.022

The support from The Scientific and Technological Research Council of Turkey (TUBITAK) under the project grant TUBITAK 109T695 is acknowledged.

Fig. 1: Surface potential distributions on a 5 μm × 5 μm portion of the quartz (0001) surface at  pH=2 values in 10−3 M KCl solution.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-14-P-5952 Photothermal Excitation for Reliable and Quantitative AFM

Johann F.1, Labuda A.1, Walters D.1, Bocek D.1, Rutgers M.1, Cleveland J.1, Proksch R.1
1Asylum Research, an Oxford Instruments Company
florian.johann@oxinst.com

Since the advent of atomic force microscopy, cantilevers have predominantly been driven by piezos for AC imaging and data acquisition. The ease of use of the piezo excitation method is responsible for its ubiquity. However, the well-known “forest of peaks”, which is clearly observed while tuning a cantilever in liquids, renders AC imaging in liquids problematically because the peaks move around with time (see Figure 1). Effectively, these shifting peaks result in a setpoint that changes with time causing stability problems while AFM imaging. Furthermore, the same “forest of peaks” prevents the quantitative interpretation of forces in liquids[1], air[2], and vacuum environments[3], even if the cantilever tune looks clean. Dissipation studies in all these environments have especially suffered due to piezo excitation of the cantilever.

Photothermal excitation is an alternative method for exciting a cantilever by heating/cooling the base of the cantilever to drive the cantilever. Photothermal excitation results in repeatable, accurate and time-stable cantilever tunes, as seen in the Figure. Therefore, the setpoint remains truly constant while imaging, preventing tip crashes, or unwanted tip retractions. True atomic resolution images of calcite in water, shown in Figure 2, were made for hours with no user intervention, testifying to the stability of photothermal excitation. Unlike other specialized drive methods, photothermal excitation is compatible with almost any cantilever and with all AFM techniques. The introduction of a blue laser into the AFM also enables several other functionalities, such as tuning the temperature of the cantilever. Furthermore, because the photothermal tune represents the true cantilever transfer function, existing AFM theories can be applied to accurately recover conservative and dissipative forces between the tip and the sample. This is especially important for force spectroscopy, dissipation studies, as well as the frequency modulation AFM techniques.

Our recent developments in perfecting photothermal excitation and its benefits to the AFM community will be shown.

[1] A. Labuda, K. Kobayashi, et al. AIP Advances 1, 022136 (2011)
[2] R. Proksch and S. V Kalinin, Nanotechnology 21, 455705 (2010)
[3] A. Labuda, Y. Miyahara, et al. Phys. Rev. B 84, 125433 (2011)

Fig. 1: Since the amplitude and phase do not drift with time, blueDrive delivers stable imaging. Piezo drive, on the other hand, has a time varying amplitude and phase which requires constant intervention by the user to maintain stable imaging conditions.

Fig. 2: A freshly cleaved crystal face of calcite was imaged in ultrapure water during three hours. No user intervention was necessary throughout the experiment, because the drive amplitude was remained stable.
↑ Go to the Table of content ↑

IT-15. X-ray, neutron and other microscopies

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-15-IN-1716 Chemically selective spectromicroscopy by soft x-ray scanning transmission x-ray microscopy

Hitchcock A. P.1
1Dept. of Chemistry & Chemical Biology McMaster University, Hamilton, Canada
aph@mcmaster.ca

Soft X-ray scanning transmission X-ray microscopy (STXM) uses natural near edge X-ray absorption spectral contrast for chemical speciation and quantitative chemical & orientation mapping (geometric & magnetic) in 2d & 3d with <20 nm spatial resolution. Recently STXM capabilities have been expanded to include electron detection for surface studies, X-ray fluorescence for enhanced sensitivity, and ptychography. STXM is ideal for wet samples since soft X-rays readily penetrate a few microns of water. I will outline instrumentation, data analysis, and capabilities of soft X-ray STXM. Examples will include:

Biomagnetism. STXM with circularly polarized light [CLS 10ID1 or ALS 11.0.2] measure magnetism by X-ray magnetic circular polarization (XMCD). We use this to study magnetotactic bacteria [1] which biomineralize intra-cellular chains of ~50 nm magnetite single crystals. In most cases all magnetic moments in a chain point in the same sense. Recently we found cases where there is internal reversal -the magnetic field of one part points opposite to other parts of the chain (Fig. 1). The gap region exhibits an Fe L3 spectrum similar to that of magnetite but without XMCD [2]. These are situations where either magnetite bio-mineralization has failed or the chain is in the act of growing. Our studies provide insights into biomineralization. Use of ptychography to measure XMCD with improved spatial resolution (<10 nm) will be described.

Automotive hydrogen fuel cells. Polymer electrolyte membrane fuel cells (PEM-FC) are being developed for near-future mass production automotive applications. The performance, efficiency and lifetime of PEM-FC depend on composition and nanostructure of electrodes. Optimization is critical for the cathode where the rate limiting oxygen reduction reaction takes place. STXM is a powerful tool to study a wide range of issues in PEM-FC optimization including mapping ionomer in cathodes [3,4]. Most studies to date have been carried out on dry, microtomed samples at ambient temperature (25 C, 0 % RH) which are very different from typical operating conditions of PEM-FC (70 C, 80 % RH). The nanostructure change with temperature and hydration. Instrumentation and methods to examine PEM-FC under more realistic conditions are needed. We have developed an environmental cell for in situ STXM measurements under controlled relative humidity (0-100%) and temperature (-30 - 80 C) (Fig. 2). We study water saturation in cathode and membrane [5] and changes on freezing.

1. K.P. Lam, et al. Chem. Geology 270 (2010) 1101; S. Kalirai et al. ibid 300 (2012) 14.
2. S. Kalirai et al. PLOS One 8 (2013) e53368.
3. V. Berejnov et al PCCP 14 (2012) 4835.
4. V. Berejnov, et al. ECS Trans., 50 (2012) 361.
5. V. Berejnov et al., ECS Trans. 41 (2011) 395.

Research supported by NSERC, CFI, OIT, Canada Research Chair funding and AFCC. CLS is supported by NSERC,CIHR, NRC and U. Saskatchewan. ALS (LBNL) is supported by BES, DoE.

Fig. 1: Internal magnetic reversal in a magnetotactic bacterium (MTB). (a) Fe L3 STXM-XMCD of an MV-1 MTB measured with circular polarization parallel (green), anti-parallel (red) to magnetic vector of chain. (b) TEM of cell with interrupted chain. (c) XMCD spectra of 3 sub-chains. (d) STXM image at 710 eV. (e) color coded XMCD signal.

Fig. 2: (a) cartoon of the in situ STXM environmental cell. (b) photo in ALS 5322 STXM. (c) O 1s spectra of 3 phases of water. (d) color coded composite (cathode, PFSA, liquid water) from O 1s stack of a PEM-FC membrane electrode assembly at 85% RH. (e) color coded composite (water vapor, PFSA, liquid water) from the same stack.
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-15-IN-3325 Imaging live cells by X-ray laser diffraction

Nishino Y.1, Kimura T.1, Joti Y.2, Bessho Y.3
1Research Institute for Electronic Science, Hokkaido University, Sapporo, Japan, 2Japan Synchrotron Radiation Research Institute/SPring-8, Hyogo, Japan, 3Academia Sinica, Taipei, Taiwan
yoshinori.nishino@es.hokudai.ac.jp

Coherent imaging is a growing field in optical science. It requires no lens for image formation, but instead numerically reconstructs object images from the coherent diffraction data. It is, therefore, advantageous for x-rays, for which it is difficult to fabricate lenses with a high numerical-aperture. Coherent imaging has been demonstrated to be a powerful tool to visualize cells and organelles using synchrotron radiation [1,2]. Recently emerging X-ray free-electron lasers (XFELs) further extends the ability of coherent imaging to achieve spatial resolution beyond the conventional radiation-damage limitation. Because the pulse duration of XFELs is in the femtosecond range, X-ray interaction with the sample occurs before radiation damage becomes obvious. XFELs also allow us to image samples in solution in close-to-natural conditions [3]. We are developing a method which we refer to as pulsed coherent x-ray solution scattering (PCXSS). We performed PCXSS experiments using a Japanese XFEL facility SACLA. We will present some early results of our PCXSS experiments performed for inorganic and biological samples [4].

References:
[1] “Imaging whole Escherichia coli bacteria by using single-particle x-ray diffraction”: J. Miao, K. O. Hodgson, T. Ishikawa, C. A. Larabell, M. A. Le Gros & Y. Nishino, Proc. Natl. Acad. Sci. U.S.A. 100, 110–112 (2003).
[2] “Three-Dimensional Visualization of a Human Chromosome Using Coherent X-Ray Diffraction”: Y. Nishino, Y. Takahashi, N. Imamoto, T. Ishikawa & K. Maeshima, Phys. Rev. Lett. 102, 018101 (2009).
[3] “Advances in X-ray scattering: from solution SAXS to achievements with coherent beams”: J. Pérez & Y. Nishino, Curr. Opin. Struct. Biol. 22, 670–678 (2012).
[4] “Imaging live cell in micro-liquid enclosure by X-ray laser diffraction”: T. Kimura, Y. Joti, A. Shibuya, C. Song, S. Kim, K. Tono, M. Yabashi, M. Tamakoshi, T. Moriya, T. Oshima, T. Ishikawa, Y. Bessho, &Y. Nishino, Nature Commum. 5, 3052 (2014).

This research was partially supported by the X-ray Free Electron Laser Priority Strategy Program from MEXT; CREST from JST; KAKENHI Grant Numbers 23651126, 22310075, 22540424, and 23860001 from JSPS; and the Cooperative Research Program of ‘Network Joint Research Center for Materials and Devices’. We thank the operation and engineering staff of SACLA for helping perform the PCXSS experiments.

Fig. 1: Schematic of pulsed coherent X-ray solution scattering (PCXSS)

Fig. 2: Coherent x-ray diffraction pattern from a living Microbacterium lacticum cell exposed to a single XFEL pulse

Fig. 3: Reconstructed image of a living Microbacterium lacticum cell 
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-15-O-2379 Lessons learnt from four years of experience with diffractive imaging at X-ray Free Electron Laser sources

Strueder L.1, Hartmann R.1, Holl P.1, Huth M.1, Schmidt J.1, Soltau H.2
1PNSensor, Munich, Germany, 2PNDetector, Munich, Germany
lothar.strueder@pnsensor.de

Fourth generation accelerator-based light sources, such as VUV and X-ray Free Electron Lasers (FEL), deliver ultra-brilliant (~1012 -1013 photons per bunch) coherent radiation in femtosecond (~10 fs to 100 fs) pulses and, thus, require novel focal plane instrumentation in order to fully exploit their unique capabilities. As an additional challenge for detection devices, existing FELs (FLASH, Hamburg, LCLS, Menlo Park; SACLA, Hyogo) cover a broad range of photon energies from the EUV to the X-ray regime with significantly different bandwidths, intensities and pulse structures.

In order to meet these challenges, a novel, large area, broadband (50 eV to 25 keV), high-dynamic-range, intensity and spectroscopic imaging X-ray detector based on the pnCCDs has been established [1]. The sensor covers an area of 60 cm2 with 1024 x 1024 pixels and 10.000 x 10.000 spatial resolution points, including a hole in the center for the non-scattered X-rays. They have been operated up to 120 Hz in a full frame high resolution mode. The pnCCD detectors have been used in experiments from 30 eV (FLASH) up to 9.5 keV (LCLS, SACLA). The sensitive thickness of the fully depleted, fully sensitive CCDs is 450 µm. As the detectors are back-illuminated, an ultra-thin radiation entrance window has been developed to achieve clean energy spectra and high quantum efficiency for the lowest to the highest energies. Some of the detectors are equipped with integrated light blocking filters to avoid signal deterioration through visible light (see Fig. 1).

Different classes of experiments have been performed, each going towards the physical limits of measurement precision of the detectors: highest energy resolution (see Fig. 2) (atomic physics), the highest dynamic range (nano-crystallography), imaging of biological samples and X-ray scattering experiments (Bond orientational order of liquid and supercooled water) requiring a position resolution well below 10 µm. For all of the above experiments optimizations have been realized to fulfill the experimental requirements. The deep subpixel resolution and the controlled extraction mode of the detectors have already been demonstrated at the light sources [2]. Fig.3 shows the improvement of the charge handling capacity from 3x105 electrons per pixel to more than 1.5x106 measured at LCLS. The better understanding of the detector physics and data analysis leads to an optimization of operation modes for specific experiments, enabling for the development of new and more precise measurement methods. Detectors of this type will be used in X-ray microscopy this summer. Measurements from this application will be shown equally.

[1] L. Strüder et al., Nucl. Instr. and Meth.A 614 (2010),483-496
[2] S. Send et al., Nucl. Instr. and Meth.A711(2013)132-142

Fig. 1: Image of the pnCCD detectors on a 150 mm Si-wafer. The central chip has an area of 60 cm2, a pixel size of 75x75 µm2 and a format of 1024x1024. The sensitive thickness is 450 µm. It has a center hole for the passage of the non-scattered X-rays.

Fig. 2: X-ray emission of highly excited Xe –atoms (35+) at an LCLS experiment. The excitation energy was 1.5 keV, the energy resolution is approx. 100 eV (FWHM) at 1.5 keV, integrated over an area of 60 cm2 with a frame rate of 120 Hz of a 1024 x 1024 format of the pnCCD spectroscopic X-ray imaging array.

Fig. 3: Left: charges over flooding neighboring pixels. The max. charge handling capacity (CHC) in approx. 3x105electrons per pixel for this standard setting. Right: The same scattering process with a CHC of approx. 1.5x106 electrons per pixel due to different operating conditions of the same detector.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-15-O-2550 Diffraction Contrast Tomography as an Additional Characterization Modality on a 3-D Laboratory X-ray Microscope

Feser M.1, Merkle A.1, Holzner C.1, Fahey K.1, Lauridsen E.2, Reischig P.2, Poulsen H. F.2
1Carl Zeiss X-ray Microscopy Inc., Pleasanton CA 94566, USA, 2Xnovo Technology ApS, 4600 Køge, Denmark
michael@feser.org

We introduce a novel method to add grain position, orientation and size information to absorption 3-D x-ray microscope imaging for poly-crystalline samples. This imaging modality will be available on a commercial x-ray microscope and will open the way for routine, non-destructive studies of time-evolution of grain structure to complement destructive EBSD end-point characterization. Grain sizes down to 40 micrometers can be studied using this non-destructive image modality.

Crystallographic imaging (i.e. imaging of crystallites/grains in polycrystalline materials) are primarily known from electron microscopy, and particularly the introduction of the electron back-scattering diffraction (EBSD) technique in the early 1990’s, has made it a routine tool for research and/or development related to metallurgy, functional ceramics, semi-conductors, geology etc. The ability to image the grain structure in such materials is instrumental for understanding and optimization of material properties and processing. However, the destructive nature of 3D EBSD prevents the technique from directly evaluating the microstructure (and grain-orientation) evolution when subject to either mechanical, thermal or other environmental conditions. Non-destructive x-ray diffraction imaging methods allow for such ‘4D’ time dependent studies, and to date have been primarily the domain of a limited number of synchrotron facilities.

Here, we present a novel method to acquire, reconstruct and analyze grain orientation and related information from polycrystalline samples on a commercial laboratory x-ray microscope (ZEISS Xradia 520 Versa) that utilizes a synchrotron-style detection system. Known as laboratory diffraction contrast tomography (DCT), this technique may be efficiently coupled to in situ environments within the microscope or subject to an extended time evolution experiment (across days, weeks, months), which remains a unique strength of laboratory (non-synchrotron) experiments. Following an evolution experiment, the sample may be sent to the electron microscope or focused ion beam (FIB-SEM) for destructive but complementary investigation of the same volume of interest.

We will show a selection of results of laboratory DCT, discuss the boundary conditions of such a method, and point to the future to discuss ways in which this can be correlatively coupled to related techniques for a better understanding of a materials structure evolution in 3D at multiple length scales.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-15-O-2821 Three-Dimensional architecture of Hepatitis C virus replication factory studied by soft X-ray cryo-tomography

Perez-Berna A. J.1, Rodriguez M. J.2, Friesland M.2, Sorrentino A.1, Chichon F. J.2, Carrascosa J. L.2, Gastaminza P.2, Pereiro E.1
1ALBA Synchrotron Light Source, MISTRAL Beamline – Experiments Division, 08290 Cerdanyola del Vallès, Barcelona, Spain, 2Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Campus Cantoblanco, 28049 Madrid, Spain
ajperezberna@gmail.com

Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability and inefficient infection systems have severely limited the analysis of the HCV life cycle and the development of effective antivirals and vaccines. HCV is a positive strand RNA that replicates its genome in intracellular membranes forming a complex membranous web. Nevertheless, the three-dimensional structure of this membranous web in whole infected cells is still unknown.

In this study we have performed full-field cryo soft X-ray tomography (cryo-SXT) in the water window photon energy range (Schneider et al. 2010; Chichon et al. 2012) to investigate in whole, unstained cells, the morphology of the membranous rearrangements induced by the HCV replicon in conditions close to the living physiological state. We have obtained the first complete cartography of the dramatic cellular modification caused by the stable subgenomic HCV replicon transfected in cell culture (Kato et al, 2003). Moreover, in order to identify the viral proteins allocation in the different subcellular compartments, we have correlated the three-dimensional structure obtained with X-rays with electron microscopy immunelabeling and confocal immunofluorescence. The morphology of the membranous HCV factory web is a cytoplasmic accumulation of large and small heterogeneous vesicles, mitochondria and lipid droplets.

The understanding of the membranous replication factory of HCV provides a powerful tool for the analysis of host-virus interactions that should facilitate the discovery of antiviral drugs and vaccines for this important human pathogen.

Schneider G, Guttmann P, Heim S, Rehbein S, Mueller F, Nagashima K, Heymann JB, Müller WG, McNally JG. Three-dimensional cellular ultrastructure resolved by X-ray microscopy. Nature Methods 2012, 7: 985-987

Chichón FJ, Rodriguez MJ, Pereiro E, Chiappi M, Perdiguero B, Guttmann P, Werner S, Rehbein S, Schneider G, Esteban M, Carrascosa JL. Cryo X-ray nano-tomography of vaccinia virus infected cells. J. Struct. Biol. 177, 202-211 (2012).

Kato T, Date T, Miyamoto M, Furusaka A, Tokushige K, Mizokami M, Wakita T. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology. 2003 Dec;125(6):1808-17.

These experiments were performed at MISTRAL beamline at ALBA Synchrotron Light Facility with the collaboration of ALBA staff.

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-15-O-3352 Laboratory Full-Field Transmission X-ray Microscopy and Applications

Dehlinger A.1,2,3, Seim C.1,2, Legall H.1,2,3, Stiel H.1,3, Rancan F.4, Meinke M.4, Rehbein S.5, Wiesemann U.6, Kanngießer B.1,2
1Berlin Laboratory for innovative X-ray technologies (BLiX), Berlin, Germany, 2Technische Universität Berlin, Institut für Optik und Atomare Physik, Berlin, Germany, 3Max-Born-Institut, Berlin, Germany, 4Charité Berlin, Clinical Research Center for Hair and Skin Science, Berlin, Germany, 5Helmholtz-Zentrum für Materialien und Energie, Berlin, Germany, 6Bruker ASC GmbH, Cologne, Germany
aurelie.dehlinger@mbi-berlin.de

X-Ray microscopy in the water window allows imaging with resolutions in the nanometer regime as well as a high natural contrast between carbon and oxygen. Hence, it is possible to examine aqueous biological samples with up to 10 µm thickness in their natural state. Apart from cryo fixation of the specimen, which is usually required in order to avoid radiation damage, extensive sample preparation is not necessary. The use of highly brilliant laboratory X-ray sources has allowed the transfer of this technology, previously limited to synchrotron facilities, into the laboratory. This transfer inures to the benefit of a broader scientific community for applications in various fields such as medicine, biology and environmental sciences.

We introduce the plasma driven laboratory full-field transmission X-ray microscope (LTXM) located at the Berlin Laboratory for innovative X-ray technologies [1]. The half-pitch resolution of ∆x = (31 ± 3) nm is comparable to resolutions achieved at synchrotron facilities. The used wavelength at 2.478 nm is close to the absorption edge of oxygen and thus offers the best contrast within the water window. The large penetration depth and the short exposure times of less than one minute reached by the microscope, make soft X-ray cryo tomography feasible. An overview of first applications, like measurements on cryo-frozen yeast cells and human skin slices, will be given.

References

[1] H. Legall, G. Blobel, H. Stiel, C.Seim et al., “Compact x-ray microscope for the water window based on a high brightness laser plasma source,” Opt.Express, 20(16), 18369-18369 (2012).

This project was funded by the BMBF (#13N8913) and the BMVBS (WTW #03WWBE106).

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-1449 Single flash imaging of live hydrated biological cells by a contact soft x-ray microscope coupled with an intense laser-plasma soft x-ray source

Kado M.1, Kishimoto M.1, Tamotsu S.2, Yasuda K.2, Aoyama M.2, Shinohara K.1
1Quantum Beam Science Directorate, Japan Atomic Energy Agency, 2Division of Natural Science, Nara Women's University
kado.masataka@jaea.go.jp

We have developed a contact soft x-ray microscope combined with an intense laser-plasma soft x-ray source to achieve flash imaging of live hydrated biological cells. Laser-plasma soft x-ray source produced by a high power pulsed laser is extremely bright and very suitable for biological x-ray microscopy to capture an image of living specimens for which require a single flash exposure to avoid imaging any damages on the specimens. We also have invented to use a fluorescent microscope to identify the cellular organelles in the images obtained with the soft x-ray microscope. The biological cells were cultivated directly onto the PMMA photo resists and observed with the soft x-ray microscope and the fluorescent microscope at the same time. The obtained soft x-ray images and fluorescence images of the cells were directly compared and each cellular organelle such as mitochondria, actin filaments, and chromosomes in the soft x-ray images were clearly identified. Since the soft x-ray microscope has higher spatial resolution than that of the fluorescent microscope, fine structures of the cellular organelles in the hydrated biological cells were observed.
Shown in figure 1 are the soft x-ray image (a) and the fluorescence image (b) of the live biological cells. Appearing blue in the fluorescence image were chromatin, red were mitochondria, and green were actin filaments. The both images were clearly identical and each cellular organelle in the soft x-ray image could be identified directly comparing with the fluorescence image.
Shown in figure 2 are the soft x-ray images of one of the cells (a) shown in Fig.1 and enlarged images of surrounding area of the nucleus (b) and mitochondria (c) in the same cell. The cellular organelles such as chromatin and mitochondria in the images were identified comparing directly with the fluorescence image. All of the bright spots surrounding the nucleus in Fig. 2(a) were recognized to be mitochondria. Shown in Fig. 2(c) is the soft x-ray image of a single mitochondrion picked up from the Fig. 2(b) and detailed structure of the mitochondrion was obtained.

This research was partially supported by the Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research (C), 25390134, 2014.

Fig. 1: Soft x-ray image (a) and fluorescence image (b) of live hydrated biological cells. Appearing blue in the fluorescence image were nuclei, red were mitochondria and green were cytoskeletons.

Fig. 2: Soft x-ray images of one of the cells (a) shown in Fig. 1 and enlarged images of surrounding area of the nucleus (b) and mitochondria (c) in the same cell. The cellular organelles such as chromatin and mitochondria in the images were identified comparing directly with the fluorescence image.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-1509 At-wavelength observation of phase defect embedded in extreme ultraviolet lithography mask

Amano T.1, Terasawa T.1, Watanabe H.1, Toyoda M.2, Harada T.3, Watanabe T.3, Kinoshita H.3
1EUVL Infrastructure Development Center, Inc., Ibaraki, Japan, 2Tohoku University, Miyagi, Japan, 3University of Hyogo, Hyogo, Japan
tsuyoshi.amano@eidec.co.jp

Extreme ultraviolet (EUV) lithography is considered to be the most promising next-generation lithography after the point where 193-nm immersion lithography would cease to deliver smaller features. However, the path to establish the EUV lithography is not without technical difficulties. Issues with insufficient light-source power, defect-free mask fabrication, and resist material development are to be resolved. Regarding the types of mask defects, the nature of the pattern defects in the EUV mask is mostly same as in the case of optical masks except for those defects that are classified as reflective multilayer defects, such as bump or pit phase defects that propagate through the multilayer during its deposition on the substrate surface and it is hard to repair. Therefore, to reduce the effect of the phase defect on wafer printing image, two methods are suggested. One method is to cover the phase defects beneath the absorber pattern by shifting the location of device pattern during mask patterning. The other is to eliminate the influence of the phase error by removing the absorber away from the close proximity of the phase defects after fabricating the device pattern. To make these methods success, it would be necessary to be able to pinpoint the location of the phase defects and the affected areas.
In this presentation, influence of the phase defect structures on EUV microscope images were examined to predict the inclination angle dependency of the phase defect impact on wafers since the phase defect does not always propagate in a vertical direction from the substrate surface through the multilayer. Figures 1(a) and 1(b) show photograph of the EUV microscope and illustration of the imaging optics, developed by Tohoku Univ. that was utilized in this study. The EUV light was sourced from a beam line BL3 of the New SUBARU synchrotron facility at the Univ. of Hyogo. A programmed phase defect EUV mask was prepared. Figure 2(a) shows the cross-sectional transmission electron microscope (TEM) images of the vertical and inclined grown phase defects. The calculated inclination angles of the phase defects were 0 and 4 degrees. Figure 2(b) represents the scanning probe microscope (SPM) images of the phase defects with half-pitch 88 nm lines-and-spaces (L/S). The L/S with the phase defects were observed using the EUV microscope. Figure 2(c) show the EUV microscope images and their intensity profiles. The impacts of the inclination angles on EUV microscope images were significant even though the positions of the phase defect relative to the absorber line, as measured by scanning prove microscope, were same. As a result, the EUV microscope could identify the positional shift of the effective defect position caused by the inclined propagation through the multilayer.

This work was supported by New Energy and Industrial Technology Development Organization (NEDO).

Fig. 1: (a) Photograph of the EUV microscope. (b) Schematic model of the EUV microscope optics.

Fig. 2: (a) Cross-sectional TEM images of the vertical- and inclined-grown phase defects. (b) SPM images of the phase defects in half-pitch 88 nm L/S. (c) EUV microscope images and intensity profiles.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-1561 Online Tools for Microscopy and Microanalysis Facilities.

Apperley M. H.1, Whiting J.1, Cribb B.2, Frost C.2, Ceguerra A.3, Liddicoat P.3
1Australian Microscopy and Microanalysis Research Facility, The University of Sydney, NSW, 2006, Australia, 2Center for Microscopy and Microanalysis, The University of Queensland, QLD, 4072, Australia, 3Australian Center for Microscopy and Microanalysis and the School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, NSW, 2006, Australia
miles.apperley@sydney.edu.au

Australian Microscopy and Microanalysis Research Facility (AMMRF) is a national grid of equipment, instrumentation and expertise in microscopy and microanalysis that provides nanostructural characterisation capability and services, from widely used optical, electron, X-ray and ion-beam techniques to world-leading flagship platforms. One of the benefits of the network of core facilities is the ability to collaborate to develop online tools that are then accessible by all the laboratories in the network. These tools assist researchers to identify the techniques they need to use, facilitate training and enable data analysis & management.
The Technique Finder (TF) is a web application that enables prospective facility users to identify the techniques most suited to their research, based on a researcher-centric approach and terminology as opposed to instrument-oriented jargon.
MyScope: Training for Advanced Research, is an online suite of education tools for teaching and learning in the area of microscopy and microanalysis. The modules in MyScope contain a number of components including: an interactive questionnaire to allow the user to assess their knowledge, guide choices and tailor the learning environment for flexible learning; self guided tutorials with videos, animations and glossary to prepare students with knowledge and specialist language; virtual instrument platforms to practice use of instrumentation; and online competency testing to demonstrate readiness for hands-on experience.
A Data Management System (DMS) addresses the needs of an increasing number of AMMRF users who are using high-end instruments to produce large datasets. Those users are facing the demands of a new wave of data-intensive instruments and software that enable: higher spatial resolution; higher chemical resolution; 3D and 4D+ approaches; more rapid dynamic processes; and multi-dimensional analyses.
A specific analysis platform being developed is the Atom Probe Workbench. This tool is a component of a larger national eResearch project in Australia, that is aiming to integrate existing tools and techniques with a network of specialised cloud-based computing systems and data-storage facilities. This integration will enable the atom probe research community to access and create valuable tools, accelerating the research process.

The authors acknowledge support from the National Collaborative Research Infrastructure Strategy; National eResearch Architecture Taskforce; Office of Learning and Teaching; and National eResearch Collaboration Tools and Resources (NeCTAR) project.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-1564 MyScope: On-Line Microscopy and Microanalysis Training and Education in Core Facilities

Apperley M. H.1, Munroe P. R.1, White T.2, Shapter J.1, Muhling J.1, Soon L.1, Ringer S. P.1, Grinan E.1, Frost C.1, Cribb B.1
1Australian Microscopy and Microanalysis Research Facility, Sydney, Australia, 2School of Materials Science and Engineering, Nanyang Technological University, Singapore
miles.apperley@sydney.edu.au

The Australian Microscopy and Microanalysis Research Facility (AMMRF) is a national grid of equipment, instrumentation and expertise in microscopy and microanalysis that provides nanostructural characterisation capability and services, from widely used optical, electron, X-ray and ion-beam techniques to world-leading flagship platforms. One of the principal activities of the AMMRF is to provide research training in microscopy and microanalysis. Until recently, much of this training was provided either in the classroom or through one-to-one training at the instrument itself, however, these approaches faced limitations.

Firstly, the large number of researchers requiring training places pressures on the core facilities to balance the need to maximise the beam-time of expensive and complex instrumentation for research purposes with that for training new users, who will ultimately perform the research. Prioritising instrument time for research reduces time available for training and vice versa.

Another common challenge in such facilities is diversity of the student body. In the case of our project the cohort requiring training had a variety of backgrounds and goals: undergraduate students with different educational backgrounds seeking an overview of topic; final year students requiring specific techniques for project work; future career or postgraduate students; and also professional researchers, educators and managers. A more flexible approach to training and education was needed.

To address these challenges and improve the training outcomes of researchers, the AMMRF developed MyScope: Training for Advanced Research. MyScope is an online suite of education tools for teaching and learning in the area of microscopy and microanalysis. The modules in MyScope provide a novel advancement in online training. They contain a number of components including: an interactive questionnaire to allow the user to assess their knowledge, guide choices and tailor the learning environment for flexible learning; also, tailoring capability for academics and trainers; self guided tutorials with videos, animations and glossary to prepare students with knowledge and specialist language; virtual instrument platforms to practice use of instrumentation; and online competency testing to demonstrate readiness for hands-on experience.

The authors acknowledge funding from the Office for Learning and Teaching, Australian Government Department of Education, CG10-1490.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-2171 Spectroscopic imaging with a new pnCCD camera: improved dynamic range, position resolution, anti-blooming and compactness

Ihle S.1, Eckhardt R.2, Hartmann R.1, Holl P.1, Huth M.1, Kalok D.1, Ryll H.1, Schmidt J.1, Simson M.2, Soltau H.2, Soltau J.2, Steigenhöfer D.1, Thamm C.2, Strüder L.1
1PNSensor GmbH, Munich, Germany, 2PNDetector GmbH, Munich, Germany
sebastian.ihle@pnsensor.de

pnCCDs are well known as radiation detectors for spectroscopic imaging for X-rays in many fields of science: X-Ray Fluorescence analysis (XRF), X-ray astronomy, X-ray Free Electron Laser science and at synchrotrons. pnCCDs are radiation detectors on high resistivity 450 µm fully sensitive silicon. They are back-illuminated, with a thin, homogeneous radiation entrance window, enabling the detection of X-rays from 30 eV up to 30 keV with high quantum efficiency. As all pnCCDs are equipped with a fully column parallel readout, frame rates of more than 1.000 frames per second are achieved, keeping the read noise level at 3 electrons. Some of the key performance figures are e.g. a quantum efficiency above 90% from 1 keV up to 10 keV, extreme radiation hardness, operation at temperatures around -20 °C or warmer, energy resolution of less than 130 eV (FWHM) at 6 keV and 37 eV (FWHM) at 90 eV. These properties have enabled a variety of spectacular measurements. The following improvements were made recently:

(a) New Colour X-ray camera module: The new CXC module (see Fig. 1) was designed to fit in small and tight surroundings. The new camera can be operated in vacuum without any entrance filter or with a Be filter in normal environments. Capillary optics can be coupled to the entrance window.

(b) High dynamic range mode: The previously applied standard operating modes were able to handle about 300.000 electrons in a single pixel. The new settings allow confining and transferring more than 2.5 million electrons in the CCD.

(c) Controlled charge extraction: If the amount of signal charge overcomes the charge handling limit the surplus charges can be taken out in a controlled way to avoid overflowing electrons to spoil the information content of the neighbouring pixels. A direct electrical access to the pixels allows to define a saturation level of the pixels (anti-blooming). We have tested this mechanism experimentally with a charge load of 2 billion electrons per pixel. An example is shown in Fig 2.

(d) Subpixel resolution: The low noise of the pnCCD system enables to centroid the signal charge cloud with a position precision of 2.5 µm (rms) (see Fig 3). This is achieved by increasing the charge cloud diameter by reducing the electric field during the charge collection process and therefore increasing the charge diffusion process.

All the above improvements are delivering new qualities to the compact X-ray camera stimulating new methods for spectroscopic imaging measurements.

The authors would like to thank the technical staff of PNSensor and PNDetector for their outstanding support.

Fig. 1: Photo of the new Colour X-ray Camera (CXC) module.

Fig. 2: Demonstration of anti-blooming operation. The pnCCD is illuminated with a bright spot. In normal operation (left) the signals spills into the neighboring pixels. In anti-blooming mode the excess charge is removed in a controlled way.

Fig. 3: Measured position precision as a function of operating parameters.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-2231 Multilayer Laue Lenses as High Resolution hard X-ray Optics

Kubec A.1, 2, Niese S.1, Melzer K.1, 2, Braun S.2, Patommel J.1, Leson A.2, Zschech E.3
1Technische Universität Dresden, Dresden, Germany, 2Fraunhofer IWS Dresden, Dresden, Germany, 3Fraunhofer IKTS-MD, Dresden, Germany
adam.kubec@tu-dresden.de

X-ray Microscopy provides a high resolution sample analysis method while demands on sample preparation are typically less restrictive than for transmission electron microscopy. Resolutions are still limited by the available optics but improvements in this field can easily be implemented in existing setups and will thus directly influence and improve measurements. Multilayer Laue lenses (MLL) are promising x-ray optics to achieve high efficiency focusing with small spot sizes down to sub-10 nm with current and down to sub-1 nm with improved geometries.
We have deposited multilayer stacks with layer thicknesses according to the zone plate law and a total deposition thickness of more than 50 micrometer. Deposition processes using magnetron sputtering took up to 83 hours and the stacks containing up to 6500 individual layers with thicknesses down to 5 nm have been obtained.
These parameters require long term system process stability on the one hand and a high precision in zone deposition on the other hand. The actual one dimensionally focusing lens is subsequently fabricated with mechanical preparation and focused ion beam milling. Two of these lenses have to be placed perpendicularly in a distance of about 30 micrometers from each other to obtain point focusing [Fig. 1]. Two of these lenses were crossed perpendicularly at a distance of 30 µm to obtain a point focus [Fig.1].
We have successfully demonstrated several focusing and imaging experiments using crossed MLLs. In synchrotron beam times at ID13, ESRF in Grenoble, France and P06, PETRA III in Hamburg, Germany spot sizes down to 39x49 nm2 at 20 keV x-ray energy have been shown using the coherent diffraction imaging method of Ptychography [Fig. 2 and 3]. Furthermore we have demonstrated measurements with a wedged MLL realizing the improved geometry. Global diffraction efficiency was enhanced by more than 50% on average over the entire aperture of the lens. In addition, full field imaging was shown for the first time using multilayer Laue lenses at a laboratory microscope with a rotating copper anode.

References:
[1] A. Kubec, S. Braun, S. Niese, P. Krüger, J. Patommel, M. Hecker, A. Leson and C. Schroer: Ptychography with Multilayer Laue Lenses and their Initial Characterization with a Laboratory Based X-ray Microscope, to be published

The work has been supported by the BMBF within the cool silicon project and is partly funded by the European Regional Development Fund and the Free State of Saxony via the ESF project 100087859.
Portions of this research were carried out at the light source PETRA III at DESY and on the ID13 beamline at the European Synchrotron Radiation Facility (ESRF), Grenoble, France.

Fig. 1: A pair of crossed multilayer Laue lenses with the designated beam direction.

Fig. 2: A phase reconstruction of the test sample used for the ptychography measurements. [1]

Fig. 3: Amplitude reconstruction of the focal plane. [1]
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-2249 Multilayer Laue Lenses as High Resolution High Efficiency X-ray Focusing Optics

Kubec A.1, 2, Niese S.1, Melzer K.1, 2, Braun S.2, Patommel J.1, Leson A.2
1Technische Universität Dresden, Dresden, Germany, 2Fraunhofer IWS, Dresden, Germany
adam.kubec@tu-dresden.de

Multilayer Laue Lenses (MLL) are a promising approach based on diffraction to focusing hard x-rays and promise to open the path to nanometer spot sizes [1]. Limitations implied by the fabrication process of zone plates regarding possible zone widths and aspect ratios are circumvented. Using thin film deposition techniques alternating zones of two different materials are deposited onto a flat substrate with thicknesses according to the zone plate law. A lamella then cut out of the coating using Focused Ion Beam milling. This segment is the actual lens and produces a focal line. Combined with a second perpendicularly aligned lens a point focal is achieved. The structures accommodating the lenses are glue-bonded directly onto each other. The distance between the lenses is approximately 30 µm (fig. 1). The pair of lenses is then fixed onto a single mount. Only two precise tilting stages are necessary as well as two stages for coarse position alignment. At the ESRF beamline ID13 and the PETRA III beamline P06 we have shown such setups of pairs of crossed MLLs. The lenses were characterized using Ptychography [2]. According to the reconstructions of the complex wave field focal spots with a FWHM of about 50 x 50 nm2 and less have been achieved. In addition the local diffraction efficiency of a wedged MLL was compared to a regular tilted geometry lens. The results show an increase of intensity in the first focusing order of more than 30%. Particularly the local diffraction efficiency of the zones with less than 10 nm zone width increased noticeably.

References
[1] J. Maser et al., Optical Science and Technology, the SPIE 49 Annual Meeting (2004)
[2] S. Hönig et al., Optics Express, Vol. 19, Issue 17, pp. 16324-16329 (2011)

 

The work has been supported by the BMBF within the cool silicon project and is partly funded by the European Regional Development Fund and the Free State of Saxony via the ESF project 100087859.
Portions of this research were carried out at the light source PETRA III at DESY and on the ID13 beamline at the European Synchrotron Radiation Facility (ESRF), Grenoble, France.

Fig. 1: Process photograph of the Magnetron Sputter Deposition.

Fig. 2: SEM image of a pair of crossed MLL with a distance of approximately 30 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-2471 THERMODYNAMİCS OF ZİRCON AND SOLİD SOLUTİON REACTIONS

Kılıç A. D.1, Ateş C.2
1Fırat University Geology Department, Elazığ, Turkey
didem7399@hotmail.com


Zircon (ZrSiO4) from granitic gneisse and ortho gneisse in the Pütürge metamorphites were mineralogically characterized by inductively couples plasma mass spectrometry (ICP-MS), X-ray powder diffraction and Cathodolüminescence (CL) analyses show that the zircon grains have developed isostructural solid solutions with coffinite (USiO4), throite (ThSiO4). These zircons have different thermodynamic and processing. This processes are chemical reequilibration of crystalline zircon solid solutions. Zircons have textural and chemical variety which presents characteristic of metamict and partly metamict. Metamict zircons are high REE, U, Th content. Whereas partly zircons are more low REE, U, Th. The chemical characteristic of zircons can produce both aqueous fluids and melts. İn the zircons are observed by porous structure and inclusion rich spaces. The inclusion rich and porous zircon grains are featured by lower concentrations of trace elements. This were interpretationed dissoution-reprecipitation process intrastructure of zircons. This show that its reacts with an aqueous fluid, dissolution-reprecipitation process will produce more less trace elements than the other zircons.
İn order to constrain the timing of metamorphism, 39Ar/40Ar dating were performed on four biotite. The four samples is 83.21±0.069 Ma. Accordingly, greenschist and amphibolite facies metamorphism occured at Santonien. U-Pb cristallization age from zircon probably correspond to the timing of fluid influx or anatexis rather than to the age of peak metamorphic conditions.
Keywords: Solid solution inclusions, zircon, high metamorphism, CL analyses, 39Ar/40Ar biotite dating, U-Pb isotope

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-2661 Evaluating the detection of cracks in underwater wet welds by x-ray microtomography

Paciornik S.1, Silva L. F.1, Santos V. R.1, Bernthaler T.2
1DEMa PUC-Rio - Brazil, 2Institut für Materialforschung - Hochschule Aalen - Germany
sidnei@puc-rio.br

Despite efforts to improve the mechanical properties of wet welds, welding in direct contact with water still presents critical problems. High cooling rates and the presence of hydrogen derived from water dissociation leads to the formation of defects, such as pores and cracks in the weld metal (WM) and in the heat affected zone (HAZ) which adversely affect mechanical properties. The occurrence of hydrogen assisted cold cracking is considered as one of the most important factors for the usual low ductility in the WM [1, 2].
During cooling, weld beads contract both in transverse and longitudinal directions. It is well established that longitudinal contractions are responsible for higher residual stress after welding. As a consequence, the low WM toughness associated with hydrogen embrittlement can lead to nucleation of cracks with a predominant orientation transverse to the weld axis.
In previous works, both Optical Microscopy (OM) [3] and x-ray microCT [4,5], linked to Image Analysis (IA) have been used to characterize crack size, density, shape and orientation. Cracks are challenging to image and measure from microCT due to their strong anisotropy and the small dimension of the tip, which can go beyond the resolution of microCT.
In this work, a weld sample with varying cross-section (Fig. 1) was imaged by microCT before and after being submitted to a tensile test up to failure. The variation in strain due to varying thickness led to changes in crack tip opening and crack length. Fig. 2 shows a typical reconstructed layer of the weld with cracks. The presence of noise and limited contrast hinder the detection of cracks. After noise filtering and segmentation the cracks were rendered, as shown in Fig. 3, and measured, allowing the estimation of detection limits for this kind of defect by microCT.

REFERENCES

1. A. Q. BRACARENSE et al., “Comparative study of commercial electrodes for underwater wet welding”, (In International Congress of the International Institute of Welding, São Paulo, 2008).
2. V. R. SANTOS et al., “Recent Evaluation and Development of Electrodes for Wet Welding of Structural Ship Steels” (In 29th International Conference on Ocean, Offshore and Arctic Engineering, Shangai, 2010).
3. M. H. P. MAURICIO et al., “Quantitative Hydrogen Cracking Evaluation by Digital Optical Microscopy” (In IMC17, Rio de Janeiro, 2010).
4 . S. PACIORNIK ET al. “Characterization of Pores and Cracks in Underwater Welds by ct and Digital Optical Microscopy”. Proc. 1st International Conference on 3D Materials Science, 2012. p. 177-182.
5. PADILLA, E. et al . Image analysis of cracks in the weld metal of a wet welded steel joint by three dimensional (3D) X-ray microtomography. Mat Charac, 83, 139-144, 2013.

The financial support of CNPq, CAPES and FINEP, Brazilian agencies is gratefully acknowledged. 

Fig. 1: Tensile test specimen with varying cross section (dimensions in mm).

Fig. 2: Reconstructed microCT image of part of the sample in Fig. 1, revealing cracks.

Fig. 3: 3D rendering of cracks, after noise filtering and segmentation.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-5855 Nanoscale characterization of hierarchical biological materials using synchrotron quantitative scanning-SAXS imaging

Gourrier A.1,2,3, Burghammer M.3, Reiche I.4, Boivin G.5
1Univ. Grenoble Alpes, LIPHY, F-38000 Grenoble, France, 2CNRS, LIPHY, F-38000 Grenoble, France, 3European Synchrotron Radiation Facility (ESRF), Grenoble, France, 4Laboratoire d’Archéologie Moléculaire et Structurale, UMR 8220 CNRS Université Pierre et Marie Curie, Sorbonne Universités, Ivry-sur-Seine, France., 5INSERM U1033, Université de Lyon, France
aurelien.gourrier@ujf-grenoble.fr

The characterization of biological materials often proves challenging due to their high degree of structural hierarchy and their composite nature at the nanoscale. Bone is a typical example which presents an additional level of complexity because of the variety of morphologies encountered from the nanometer to the centimeter scale. This stems from the physiological processes associated with the synthesis, mechanical adaptation to external loads and self-healing. As a result, there is a growing consensus in the biomedical field over the necessity of multiscale approaches for the evaluation of the effects of bone pathologies. The molecular and supra-molecular levels, in particular, are currently receiving a lot of attention. At these scales, bone consists of complex arrangements of collagen microfibrils mineralized with calcium phosphate nanoparticles. Precisely how this nanoscale organization affects the mechanical properties of the higher hierarchical levels is still poorly understood.

In this paper, we will highlight the potential of quantitative scanning-SAXS imaging1,2,5 for such studies. This technique relies on scanning a sample with a monochromatic X-ray beam much smaller than the sample dimensions (typically 100 nm- 10 μm), and recording the scattered intensity in forward geometry. The images acquired at small scattering angles (SAXS) provide atomic to nanoscale resolution. They are reduced to scalar values by various algorithms based on the theory of SAXS and mapped as a function of scan coordinates to produce the final images. Using state-of-the-art X-ray optics and detectors with synchrotron sources, nanoscale fluctuations in density within a size range of ~1-100 nm can be mapped with very high spatial resolution over sample regions comparable to histology (cm2). This new method is therefore highly competitive and bridges the gap between TEM or AFM and high resolution microscopies.

Various results will be presented from fundamental, biomedical and archaeological studies to demonstrate the potential of this method. In particular, the size, organization and orientation of the mineral nanoparticules in bone will be described in various healthy and pathological/altered conditions.

Various results will be presented from fundamental, biomedical2,4 and archaeological3 studies to demonstrate the potential of this method. In particular, the size, organization and orientation of the mineral nanoparticules in bone will be described in various healthy and pathological/altered conditions.

Fig. 1: qsSAXS Image of the particle size (nm) of a thin section (6 (H) x 10 (V) mm2 x 50 μm) illiac crest biopsy of a sheep model.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-5878 A Hard X-ray Nanoprobe at Diamond Light Source

Cacho-Nerin F.1, Parker J. E.1, Peach A.1, Wilkin G.1, Quinn P.1
1Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxon. OX11 0DE, UK
fernando.cacho-nerin@diamond.ac.uk

Beamline I14 is the hard X-ray nanoprobe beamline currently under construction at Diamond Light Source in Oxfordshire, UK. It is scheduled to come into operation in 2017. The beamline will be a dedicated facility for nanoscale microscopy and micro-nano SAXS, serving two endstations housed in a new external building approximately 175m from the main synchrotron ring. The nanoprobe endstation aims to achieve the smallest possible focus (initial aim 50nm) with the capability to exploit future optics developments. The optical design is optimised for scanning X-ray fluorescence, X-ray spectroscopy and diffraction. The mesoprobe endstation will be optimised to carry out simultaneous small and wide angle X-ray scattering studies as well as scanning fluorescence mapping, with a variable focus beam in the range 5µm – 100 nm. The beamline will complement electron and optical microscopy and enable new science in a number of areas spanning materials science, biology, engineering and earth science.

The I14 beamline will be housed in the same building as the new UK national electron microscopy facility, which provides 4 state-of-the-art electron microscopy suites covering the physical and life sciences. This facility combines staff and expertise from a number of different areas which we believe will allow us to make exciting progress in sample preparation techniques and correlative x-ray and electron microscopy studies. Here we present the design and key specifications of Beamline I14, and highlight potential applications.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-15-P-6022 Microscopy in the extreme ultraviolet and soft X-ray spectral region and its applications

Herbert S.1, Danylyuk S.1, Loosen P.1, Maryasov A.2, Wilson D.2, Bußmann J.2, Rudolf D.2, Juschkin L.2, Küpper L.3, von Wezyk A.3, Bergmann K.3, Lebert R.4
1Chair for the Technology of Optical Systems, RWTH Aachen University and JARA - Fundamentals of Future Information Technology, 2Chair for the Experimental Physics of EUV, RWTH Aachen University and JARA – Fundamentals of Future Information Technology, 3Fraunhofer Institute for Laser Technology, 4Bruker ASC
stefan.herbert@ilt.fraunhofer.de

Since the invention of first optical microscopes, applications pushed the further development of microscopes. Constant push for higher resolution leads to continuous decrease of the working wavelength of microscopes from visible light to UV, VUV and finally to EUV and X-ray spectral regions. Numerous applications of such short-wavelength microscopes are being investigated in Aachen. In the past decade we have built several industrially relevant microscopes, based on laboratory plasma sources, for different applications and demonstrated their potentials. In this contribution a transmission microscope for 13.5nm wavelength (Fig.1), a reflection dark field microscope for 13.5nm (Fig.2), a water window microscope for 1-5nm (Fig.3) and a lensless Microscope for 17.3nm will be presented and discussed in detail.

Hereby the microscopes for 13.5nm have been developed for tasks, connected to deployment of the upcoming extreme ultraviolet (EUV) lithography and therefore are designed for investigations of defective multilayer mirrors or thin films. The use of multilayer mirrors in EUV lithography as imaging optics requires an actinic (at wavelength) inspection, as defects inside the multilayer stack could not be detected with surface techniques. Moreover, the independence of such an inspection tool from a synchrotron and a flexible table top design is crucial for industrial realization of EUV lithography.

Another application in the short wavelength region, which becomes more and more applicable as the power of computers increases is lensless imaging. The benefit of not having manufacturing limited optics in the beam path and with that having a diffraction limited microscope strikes the malus of required high computational power. The application of lensless microscopy with incoherent plasma sources has to our knowledge not been realized elsewhere.

For investigations of organic samples a microscope in the water window has been developed. In this spectral region of 1-5nm carbon and phosphor are absorbing and oxygen is transmitting the light, which gives excellent contrast for water-based samples. Extension of the microscope from 2D to tomographic measurements will be discussed.

Furthermore we present concepts and first experiments of a time resolved microscope for 17.3nm and a microscope, which can measure the magneto-optical contrast of materials at absorption edges, e.g. cobalt 3p at 20.3nm (Fig.4). The time resolved microscope targets sub-100nm spatial resolution and a time resolution of around 4ns, achieved by a triggered micro-channel plate. The microscope for magneto-optical investigations is for the first time realized using a laboratory plasma source, which can enable broad application of the technique, not limited to high brilliance synchrotrons.

Fig. 1: Extreme ultraviolet Schwarzschild-objective based transmission microscope for operation in brightfield or darkfield mode with an optional second magnification step, achieving a spatial resolution of around 100 nm.

Fig. 2: Extreme ultraviolet Schwarzschild-objective based darkfield reflection microscope for defect inspection of mask blanks with high troughput and moderate spatial resolution

Fig. 3: Laboratory zone plate based water window soft x-ray microscope, achieving a spatial resolution of around 40 nm

Fig. 4: Extreme ultraviolet microscopic setup for experiments on magneto-optical contrast of elements
↑ Go to the Table of content ↑

IT-16. Electron microscopy theory and simulations

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-16-IN-1783 Calculation and Simulation in Determining Site-specific Magnetic Structure by Dynamical Electron Diffraction-EMCD

Zhu J.1, Wang Z. Q.1, Song D. S.1, Zhong X. Y.1, Yu R.1, Cheng Z. Y.1
1National Center for Electron Microscopy in Beijing, School of Material Science & Engineering, Tsinghua University, Beijing 100084, China
jzhu@mail.tsinghua.edu.cn

Quantitatively determining the magnetic structure of material on a nanometer scale is a potential task for future transmission electron microscope (TEM). Site-specific electron energy-loss magnetic chiral dichroism (site-specific EMCD) method is come up with to get the crystallographic site-specific magnetic information of nanostructures.[1-2]
This presentation will briefly introduce how we process calculations, simulations and experiments for determining the site-specific magnetic structure of a nanostructure of NiFe2O4. By constructively combining using the dynamical electron diffraction and EMCD methods, to calculate the coefficients of Bloch waves and draw out the relative EMCD intensity mappings in momentum space, in which the effect of asymmetry of the dynamical electron diffraction needs to be considered;[3] then to select the optimum experimental parameters in EMCD experiments, by adjusting dynamical diffraction conditions to enhance site-specific EMCD signals; with sample’s site-specific magnetic circular dichroism spectra, and the site-specific spin/orbital magnetic moments extracted.
Compared with X-ray magnetic circular dichroism, the site-specific EMCD method shows its unique capability for solving the crystallographic site-specific magnetic structure on nano-scale.
Reference:
[1] Schattschneider P, Rubino S, Hebert C, et al. Detection of magnetic circular dichroism using a transmission electron microscope. Nature 441, 486–488 (2006).
[2] Wang ZQ, Zhong XY, Yu R, Cheng ZY, Zhu J. Quantitative experimental determination of site-specific magnetic structures by transmitted electrons. Nature Communications, 4, 1395 (2013).
[3] Dongsheng Song, Ziqiang Wang and Jing Zhu, Effect of the asymmetry of dynamical electron diffraction on intensity of acquired EMCD signals, unpublished.

This work is financially supported by National 973 Project of China and Chinese National Nature Science Foundation. This work made use of the resources of the Beijing National Center for Electron Microscopy.

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-16-IN-1882 Thermal magnetic field noise and electron optics - more experiments and calculations

Uhlemann S.1, Müller H.1, Zach J.1, Berger C.1, Haider M.1
1CEOS Corrected Electron Optical Systems, Heidelberg, Germany
uhlemann@ceos-gmbh.de

The simultaneous correction of the spherical (Cs) and the chromatic aberration (Cc) in transmission electron microscopy (TEM) has been implemented for a broad range of beam voltages: 20-300kV [1-3]. In such an instrument the effects of the lateral and temporal incoherence of the illuminating electron beam are largely suppressed. The measured remaining focus spread for instance is by far small enough to allow information transfer beyond g=20/nm - even at 300kV where Cc-correction is most challenging [1]. However, during the development of the corrector hardware we recognized, that an additional incoherence mechanism deteriorates the contrast of the recorded images. By careful measurements of the contrast transfer we found that an envelope function of the form exp[-2(πσ|g|)2] perfectly matches the observations. It turned out, that an isotropic image spread σ reduces the image contrast. Image spread can be understood as a stochastic, high-frequency image displacement during acquisition. Recently, it could be proven experimentally that the origin of this image spread is magnetic field noise (Johnson-Nyquist noise) emitted from the conducting parts around the electron beam. The thermodynamic nature of this noise was clearly demonstrated by cooling beam tubes (made from stainless steel or permalloy) from room temperature down to liquid nitrogen temperature [4].

In the experiments we measure the standard deviation σ of this image shift. Its variance σ2 is proportional to the product of the field correlation length ξ along the path and the variance <B2> of the transversal magnetic field [4]. Here, we report on the progress we made to understand the experimental results theoretically.

Surprisingly, magnetic materials (μr>1) introduce more integral noise than non-magnetic materials like stainless steel. Hence, we were very much interested in the common situation were magnetic material is placed outside a liner tube made from stainless steel, see Figure 1. The question arose, if the thin stainless steel tube is transparent for the stronger noise emitted from the magnetic components. Here we also report on the experiment “tube-in-tube”: A thin-walled (0.15mm) stainless steel liner tube with 3mm outer diameter is placed in a stack of permalloy tubes, see Figure 1. Beside numerical strategies, a semi-analytical approach to understand the compound system is presented, see Figures 2+3.

After all, electron optical design - especially the design of extended corrector optics - has to take into account the existence of magnetic field noise emitted from the conducting parts. We discuss scaling rules and why hexapole-type aberration correctors are collecting less image spread from thermal magnetic field noise than quadrupole-octupole-type Cc-correctors.

References:

[1] M. Haider et al, Microsc.Microanal. 16 (2010) p. 393.
[2] H. Sawada et al, AIEP 168 (2011) 297.
[3] http://www.salve-project.de
[4] S. Uhlemann et al, Phys.Rev.Lett. 111 (2013) 046101.

Fig. 1: Experiment to compare the thermal magnetic field noise of a thin stainless steel liner tube with the compound system. Permalloy tube fitted over a 3mm stainles steel liner tube (a), tube stack and outer holder tube (b), end view of the compound sample (c), dimensions (d), copper cooler and two samples: bare liner tube and the compound sample (e).

Fig. 2: Theoretical treatment by means of the fluctuation-dissipation theorem. The power-loss induced within a conducting structure by a fluctuating magnetic dipole is calculated. In rare cases with high symmetry Maxwell's equations can be solved directly by separating variables: A sheet of non-magnetic material in contact with a magnetic half-space.

Fig. 3: For thin conducting sheets (thickness t, resistivity ρ) a boundary-element method for a triangular mesh covered with t/ρ is the preferable way to calculate the frequency spectrum of the magnetic field noise.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-16-O-1476 Numerical Treatment of the Full, Non-Approximated Schrödinger Equation at Low Energies

Wacker C.1, Schröder R. R.1
1CryoEM, CellNetworks, Universitätsklinikum Heidelberg, Germany
christian.wacker@bioquant.uni-heidelberg.de

The imaging of samples at energies as low as 20 kV has attracted a lot of attention, recently [1]. With decreasing accelerating voltage even chemical elements with low atomic numbers (e.g. carbon, oxygen, nitrogen) must be treated as strong scatterers. Hence, simulations of the image formation process of organic molecules must include a correct description of high-angle scattering and inelastic scattering processes. In this work we present a new approach to deal with the first problem.

The conventional multislice (CMS) algorithm is derived from the Schrödinger equation using the high-energy or paraxial approximation. This approximation neglects the second derivative of the wave function along the optical axis and thereby replaces the Ewald sphere by a parabola. However, a careful mathematical analysis of the full Schrödinger equation allows developing a rigorous multislice (RMS) scheme without resorting to the high-energy approximation [2]. Recent implementations and numerical analyses of the RMS scheme demonstrated, that the CMS method is not accurate for accelerating voltages below 100 kV [3,4].

Therefore, it is interesting to note, that a numerical solution of the full Schrödinger equation without sophisticated mathematical treatment is also possible. Essentially, the Schrödinger equation can be regarded as a second-order ordinary differential equation. This enables us to use well-known numerical algorithms like the classical Runge-Kutta method. Similar to the multislice algorithms the wave is incrementally propagated through the sample. But instead of using integrated atomic potentials, the Runge-Kutta method integrates the potentials on-the-fly. Fig. 1 shows the amplitude-diffraction patterns of SmBa2Cu3O7-x calculated with this method at three different accelerating voltages. For reference, fig. 2 depicts the same sample but calculated with the CMS method implemented in real space. Besides, the Runge-Kutta method can also be applied to the high-energy approximation of the Schrödinger equation yielding comparable results to the CMS method (fig. 3).

<span>The computational effort increases with decreasing accelerating voltage, as the step size must be reduced to compensate for the higher scattering angles. Thus, we are currently investigating different implementations and parallelization approaches in order to reduce the required wall time and to allow for more complex samples.

[1] U. Kaiser, J. Biskupek, J.C. Meyer, J. Leschner, L. Lechner, H. Rose, M. Stöger-Pollach, A.N. Khlobystov, P. Hartel, H. Müller, M. Haider, S. Eyhusen, G. Benner, Ultramicroscopy, 111 (2011) 1239-1246

[2] J.H. Chen, D. van Dyck, Ultramicroscopy 70 (1997) 29-44

[3] C.Y. Cai, J.H. Chen, Micron 43 (2012) 374-379

[4] W.Q. Ming, J.H. Chen, Ultramicroscopy 134 (2013) 135-143

CW gratefully acknowledges the Studienstiftung des Deutschen Volkes for a PhD scholarship.

Fig. 1: Amplitude-diffraction patterns for SmBa2Cu3O7-x calculated by applying the Runge-Kutta method to the full Schrödinger equation.

Fig. 2: Amplitude-diffraction patterns for SmBa2Cu3O7-x simulated using the conventional multislice algorithm (CMS) in real space.

Fig. 3: Amplitude-diffraction patterns for SmBa2Cu3O7-x calculated by applying the Runge-Kutta method to the Schrödinger equation in the high-energy approximation.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-16-O-1624 Possible tuned laser boosting of spatial and energy resolution in EELS

Howie A.1
1University of Cambridge, Cambridge CB3 0HE , UK
ah30@cam.ac.uk

The potential advantage of combining the spatial resolution of electron energy loss spectroscopy (EELS) with the spectral resolution of photons has long been apparent [1] but, apart from cathodoluminescence studies [2,3], has made little progress.  More striking has been photon-induced electron microscopy (PINEM) pioneered primarily as a pump-probe technique for time-resolved imaging capability [4].  Fast electrons passing close to a carbon nanotube in coincidence with an intense pulse of laser illumination at frequency ω, experienced energy losses and gains nhω (-5 < n < 5).  These results were explained through the e-beam interaction in the near field region of the wave emitted by the nanotube in response to the laser pulse [5].

Sacrificing the time resolution of pulsed operation, more systematic exploration of the dielectric resonances of nanostructures could be provided by combining continuous tuned laser illumination with EELS (fig. 1). The z-dipole as well as the x-dipole shown here could be used. Initial computations [6] suggest, at not too high laser power, a substantial boosting of EELS signals with its own dipolar angular dependence (fig.2). Non-linearity could limit laser pumping of object boson oscillator modes (eg plasmons and phonons) but could result in stimulated emission more than proportional to laser intensity.  Studies of the loss and gain intensities as a function of laser power and frequency could usefully clarify the basic physics involved here and show how the near field mechanism combines with the usual excitation theory of EELS [7].  Significant boosting of EELS losses could obviously be useful for very weak losses and also for probing otherwise inaccessible Raman or catalytic hot spots (fig. 3).  More generally it could counter the severe loss of intensity experienced when the minimum momentum transfer in low loss EELS is increased in order to improve spatial resolution [8].  A minimum in lateral momentum tranfer hqmin can be set by off-axis spectroscopy or better by use of STEM hollow cone illumination with pre-spectrometer lens tuning to match aperture gaps precisely (fig. 4).

[1] Howie A, (1999) Inst. of Physics Conf. Series 161, 311.

[2] Yamamoto Y, Araya K and Garcia de Abajo FJ, (2007) Phys. Rev. B 64, 205219.

[3] Tizei LHG and Kociak M, (2013) Phys. Rev. Lett. 110, 153604.

[4] Barwick B, Flannigan DJ and Zewail AH, (2009) Nature 409, 902.

[5] Garcia de Abajo FJ and Kociak M, (2008) New J. Phys. 10, 073035.

[6] Adenjo-Garcia A and Garcia de Abajo FJ, (2013) New J. Phys. 15, 103021.

[7] Talebi N, Sigle W, Vogelgesang R and van Aken P, (2013) New J. Phys. 15, 053013.

[8] Muller DA and Silcox J, (1995) Ultramicroscopy 59, 195. 

I thank Professor Javier Garcia de Abajo for several illuminating discussions.

Fig. 1: Schematic representation of laser excitation of the x-polarised dipole mode of a nano particle.  The emitted dipole radiation interacts with an electron travelling in the z direction. 

Fig. 2: Contour plot (one quadrant only) of the laser stimulated x-dipole EELS loss intensity in the x-y plane.  The angular dependence determined by the laser E-field is not observed for the usual e-beam stimulated losses.

Fig. 3: With appropriate choice of laser beam direction and polarisation it may be possible to generate sufficient excitation for e-beam energy losses and gains to be detected at hot spots where normal EELS excitation is impossible or very weak.

Fig. 4: The use of an annular aperture for STEM hollow cone illumination and a non-overlapping EELS collection aperture defines a minimum lateral momentum transfer hqmin.  The usual loss excitation profile for the z-polarised dipole (blue) is thereby more localised (red) for qmin = 3ω/v. 
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-16-O-1997 Validities of three multislice algorithms for quantitative low-energy transmission electron microscopy

Ming W. Q.1, Chen J. H.1
1Hunan University, Changsha, Hunan, China
suokesi_ming@163.com

Three different types of multislice algorithms, namely the conventional multislice (CMS) algorithm, the propagator-corrected multislice (PCMS) algorithm and the fully-corrected multislice (FCMS) algorithm, have been evaluated in comparison with respect to the accelerating voltages in transmission electron microscopy. Detailed numerical calculations have been performed to test their validities. The results show that the three algorithms are equivalent for the accelerating voltage above 100 kV. However, below 100 kV, the CMS algorithm will introduce significant errors, not only for higher-order Laue zone (HOLZ) reflections but also for zero-order Laue zone (ZOLZ) reflections. The differences between the PCMS and FCMS algorithms are negligible and mainly appear in HOLZ reflections. Nonetheless, when the accelerating voltage is further lowered to 20 kV or below, the PCMS algorithm will also yield results deviating from the FCMS results. The calculation efficiency of the PCMS is illustrated to be much higher than the FCMS, while the accuracy of numerical calculation can be compared to the CMS. Therefore, the PCMS can be an alternative for fast simulation of HRTEM images. The present study demonstrates that the propagation of the electron wave from one slice to the next slice is actually cross-correlated with the crystal potential in a complex manner, such that when the accelerating voltage is lowered to 10 kV, the accuracy of the algorithms is dependent of the scattering power of the specimen.

This work is supported by the National Basic Research (973) Program of China (No. 2009CB623704); the National Natural Science Foundation of China (No. 51171063, 51071064); Instrumental Innovation Foundation of Hunan Province (No. 2011TT1003); PhD Programs Foundation (20120161110036).

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-16-O-2356 Measuring structure parameters from electron microscopy images : what are the limits?

Van Aert S.1, Gonnissen J.1, De Backer A.1, Sijbers J.2, den Dekker A.2,3
1Electron Microscopy for Materials Science (EMAT), University of Antwerp, Antwerp, Belgium, 2iMinds-Vision Lab, University of Antwerp, Antwerp, Belgium, 3Delft Center for Systems and Control, Delft University of Technology, Delft, The Netherlands
sandra.vanaert@uantwerpen.be

State-of-the-art electron microscopy combined with advanced model-based methods can provide reliable numbers for unknown structure parameters. Aberration correction greatly improves the quality of experimental images, new STEM data collection geometries allow one to visualise light atoms, and detectors start to behave as ideal quantum detectors. In combination with statistical parameter estimation theory to analyse experimental data, electron microscopy then performs at its ultimate limits. For example, atomic column positions can be measured down to picometer scale precision [1], differences in averaged atomic number of only 3 can be detected from HAADF STEM images [2], and the number of atoms in an atomic column can be counted with single atom sensitivity [3,4] (see fig. 1). The question then arises: how far can we go?

Ultimately, the attainable precision with which unknown structure parameters can be estimated is set by the unavoidable presence of electron counting noise. For continuous parameters such as atom positions, this limit is expressed by means of the Cramér–Rao lower bound (CRLB). For discrete parameters such as the number of atoms, the probability of error can be derived (defining e.g. the probability of reporting an atom when there is none or reporting no atom when there is one) [5].

Using these expressions, we show that the precision of the 3D atom positions estimated from depth sectioning data is poor under realistic exposure times. However, when simplifying the problem to the estimation of the vertical position of each atomic column with known number of atoms, picometer precision can be reached. The performance of depth sectioning can now be compared with HAADF STEM tomography. Furthermore, evaluating the probability of error helps us to determine STEM detector settings resulting into the highest detectability of light atoms (see fig. 2). Even so, we can define the minimally required electron dose in order to attain a maximum allowable error for miscounting atoms. This is of great importance when studying beam sensitive structures.

In conclusion, statistical parameter estimation theory is used to explore fundamental limits with which structure parameters can be estimated. The CRLB and the probability of error not only outperform classical performance criteria (including resolution, contrast or SNR), they also allow us to predict attainable limits under given experimental conditions and to explore the optimal experimental settings.

[1] S. Van Aert et al., Advanced Materials 24 (2012), p.523

[2] S. Van Aert et al., Ultramicroscopy 109 (2009), p.1236

[3] S. Van Aert et al., Nature 470 (2011), p.374

[4] S. Van Aert et al., Physical Review B 87 (2013), 064107

[5] A.J. den Dekker et al., Ultramicroscopy 134 (2013), p.34

The authors kindly acknowledge funding from the Fund for Scientific Research, Flanders (FWO).

Fig. 1: Examples of quantitative analysis using statistical parameter estimation theory. (a)Measurement of Ti displacements of ca. 5 pm inside a twin boundary in CaTiO3 [1]. (b)Quantitative characterisation of a La0.7Sr0.3MnO3-SrTiO3 interface from an HAADF STEM image [2]. Counting results of Ag (c) and Au atoms (d) with single atom sensitivity [3,4].

Fig. 2: Probability of error as a function of the inner radius of an annular detector for an aberration corrected microscope (300kV acceleration voltage, 21.7mrad probe forming angle, 100mrad outer detector radius, 12000 incident electrons/Å2). Green, red, and blue correspond to the problem of detecting Li, H, and Al/Ti in LiV2O4, YH2, and SrTiO3/LaAlO3.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-1553 Rutherford scattering of electron vortices

Van Boxem R.1, Partoens B.2, Verbeeck J.1
1EMAT, University of Antwerp, Belgium, 2CMT, University of Antwerp, Belgium
ruben.vanboxem@uantwerpen.be

Vortex beams have been met with great interest in various fields like optics, telecommunication, acoustics, and more recently in electron microscopy. Recently, techniques for the manipulation of the electron wave’s phase have received a boost1,2. Electron vortex beams show promise as a new tool to detect material properties at the nanoscale in a novel way. One feature which might contribute is the additional magnetic moment induced by a vortex electron’s orbital angular momentum (OAM).

Theoretically, various studies have been done describing free space electron vortices3 and how they behave in electromagnetic fields4,5. Relativistic aspects and the electron spin coupling to the OAM have also been considered6,7. On the other hand, basic scattering theory with electron vortex beams has not yet been fully understood, and that is why elastic scattering of an electron vortex beam on a screened Coulomb potential is considered here. This work8 introduces the incoming beam’s OAM (and associated transverse momentum) into the first Born approximation of quantum scattering theory. The influence of a beam’s OAM and corresponding spatial shape on the elastic scattering amplitude has been analyzed using the derived analytical formula.

Using the results here, we can propose scattering experiments in which high values of transverse momentum of the initial beam expose these novel features, proving the treatment here lives up to its intent: generalize plane wave scattering to cylindrically symmetric beams, including those with OAM. With this result established, more complicated scattering amplitudes can be calculated, leading to a complete electron vortex scattering theory.

1 A. Béché, R. Van Boxem, G. Van Tendeloo, and J. Verbeeck, Nature Physics, vol. 10, pp. 26–29, Jan 2014.
2 L. Clark, A. Béché, G. Guzzinati, A. Lubk, M. Mazilu, R. Van Boxem, and J. Verbeeck, Phys. Rev. Lett., vol. 111, p. 064801, Aug 2013.
3 P. Schattschneider and J. Verbeeck, Ultramicroscopy, vol. 111, no. 9-10, pp. 1461–1468, 2011.
4 K. Y. Bliokh, P. Schattschneider, J. Verbeeck, and F. Nori, Phys. Rev. X, vol. 2, p. 041011, Nov 2012.
5 K. Y. Bliokh, Y. P. Bliokh, S. Savel’ev, and F. Nori, Phys. Rev. Lett., vol. 99, p. 190404, Nov 2007.
6 K. Y. Bliokh, M. R. Dennis, and F. Nori, Phys. Rev. Lett., vol. 107, p. 174802, Oct 2011.
7 R. V. Boxem, J. Verbeeck, and B. Partoens, EPL (Europhysics Letters), vol. 102, no. 4, p. 40010, 2013.
8 R. V. Boxem, J. Verbeeck, and B. Partoens, to be published in PRA.

RVB acknowledges support from an FWO PhD fellowship grant (Aspirant Fonds Wetenschappelijk Onderzoek Vlaanderen).

JV acknowledges support from the ERC Starting Grant 278510 VORTEX.

Fig. 1: Schematic of the convergent beam scattering experiment. The relation of the transverse momenta and the aperture dimensions is shown, and an on-axis pinhole detector is shown.

Fig. 2: Transverse wave functions for the Bessel, aperture far field, and Laguerre-Gaussian beams. They all contain a first order vortex. The Laguerre-Gaussian has two intensity lobes (n=2), clearly showing the strongest localization of the three in the transverse plane.

Fig. 3: Elastic Coulomb scattering amplitude for fixed transverse momentum, and several values of OAM.

Fig. 4: Zeroeth order elastic scattering amplitude for several values of the transverse momentum. The limit to the plane wave result is clearly visible.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-1689 A new approach to determine excess free volume at high-angle grain boundaries – a proof of concept

Buranova Y. S.1, Rösner H.1, Divinski S. V.1, Wilde G.1
1Institute of Materials Physics, University of Münster, Wilhelm-Klemm-Str. 10, D-48149 Münster, Germany
buranova@uni-muenster.de

Grain boundaries (GBs) have a significant impact on the physical, especially mechanical properties of polycrystals. The GBs are typically characterized by structure units which are different compared to crystalline unit cells. It is assumed that grain boundary excess free volume plays an important role since it can be related to the grain boundary energy and has a significant influence on the transport and thermodynamic properties (diffusion/segregation). It has been reported that different GBs exhibit rather different excess free volumes, i.e. different specific mass densities from the crystalline bulk [1,2].
In this study we describe a new approach to determine the excess free volume from high-resolution transmission electron microscopy (HRTEM) images. For this purpose, an image analysis tool has been designed that allows determination of the local mass density from HRTEM images. Thereto the intensities of the GB regions have been compared with that of the grain interiors and the difference identified to be proportional to the density change, Δρ =(I-Igb)/I, where I is the intensity of the grain interior and Igb is the intensity of the GB.
In order to prove this concept, symmetrical tilt GBs with zone axes along the [100], [110] and [111] directions have been generated using molecular dynamics simulation (applying the LAMMPS software [3]) and subsequently taken as input for the simulation of HRTEM images using the Kirkland code [4,5]. The maximal density change of the GB has been estimated to be around -6% for the analyzed GBs. Calculations show that this approach works for pure samples with thicknesses up to 15 nm including aluminum oxide layers. The reliability of this approach is evaluated for different artificially chosen configurations including chains of vacancies and solute atoms.
Experimentally, well-defined aluminum bi-crystals have been investigated using aberration-corrected HRTEM. The results are discussed with respect to the relation between local structure, excess free volume and specific excess energy density.
[1] HB Aaron, GF Bolling, Surf. Sci. 31 (1972) p. 27.
[2] D Wolf, Scripta Metall. 23 (1989) p. 1913.
[3] S Plimpton, J Comp Phys. 117 (1995) p.1
[4] DL Olmsted, SM Foiles, EA Holm Acta Mater. 57 (2009) p. 3694.
[5] EJ Kirkland: Advanced computing in electron microscopy. 2nd ed. New York, Springer (2010).

Fig. 1: Example of a symmetrical [100] tilt GB (left) and the work flow of the approach (right).

Fig. 2: Characteristic plot of the intensity across a symmetrical [100] tilt GB shown in Fig.1.

Fig. 3: Intensity change versus misorientation angle of simulated symmetrical [100] tilt GBs. Solid lines represent least squares fits to the data points.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-1767 A Multislice Theory of Electron Scattering in Crystals including Backscattering and Inelastic Effects

Spiegelberg J.1, Rusz J.1
1Department of Physics and Astronomy, Uppsala University, Box 516, S-751 20 Uppsala, Sweden
jakob-spiegelberg@gmx.de

In order to interpret diffraction patterns obtained in transimission electron microscopy, scattering processes in the crystal have to be understood theoretically. Among all existent approaches to describe electron scattering, the multislice method has proven to be among the most versatile ones. To the best of our knowledge, existing multislice formalisms can not treat backscattering and inelastic processes simultaneously, what is of particular importance in electron microscopes working at reduced acceleration voltages. Furthermore, if the aim is to describe experiments using inclined illumination, e.g. reflection high-energy electron diffraction (RHEED), the conventional multislice method is inapplicable.

By combining Yoshioka's theory of inelastic scattering [H. Yoshioka, J. Phys. Soc. Jpn. 12, 6 (1957)] and van Dyck's approach for backscattering [J. H. Chen and D. Van Dyck, Ultramicroscopy 70, 29-44 (1997)], a general multislice formalism incorporating these phenomena can be derived. The new method is based on the slice transition operator technique defining an operator S(j-1) linking the wavefunctions of two adjacent slices, the jth and (j-1)th slice. In this case, S(j-1) is a 2n dimensional matrix where n is the number of inelastic excitations considered. Due to the complexity of the elements of S(j-1), however, a self-consistent solution of the matrix equation

          Φ(j)= S(j-1) · Φ(j-1)

is computationally very costly. Making the single inelastic scattering approximation, we propose a computational scheme allowing control over the number of backscattering events considered. In a nutshell, one identifies the contributions to forward and backward scattering in S(j-1) and propagates the wavefunction forward using the corresponding operators from S(j-1). During the propagation, contributions to the backward scattered beam arise - the backward propagation can be started using the corresponding parts of S(j-1). Since the backward propagation creates contributions to the twice backscattered, forward traveling beam, this cycle can be repeated until convergence is reached.

We present a more detailed description of the computational scheme and the derivation of the slice transition operator matrix. Moreover, we compare predicted elastic diffraction patterns of our new approach with those predicted by conventional multislice [J. M. Cowley and A. F. Moodie, Acta Cryst. 10, 609 (1957)] and the recent development [C. Cai and J. Cheng, Micron 43, 374 (2012)]. Special interest is taken into the single backscattering approximation and higher order backscattering.

Fig. 1: Left Column: Wavefunction of the electron beam after passing a 10 nm thick sample of bcc iron as predicted by the conventional multislice method and the differences between the CMS and RSMS wavefunction or the wavefunction predicted by the new formalism presented here. Right Column: Corresponding Fourier transforms.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-1962 Slice-by-slice simulations of absorption potential for high-angular resolution electron channeled X-ray spectroscopy

Ohtsuka M.1, Muto S.2
1Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan, 2EcoTopia Science Institute, Nagoya University, Nagoya 464-8603, Japan
m-ohtsuka@nucl.nagoya-u.ac.jp

Characteristic X-ray spectra vary with the change in the symmetries of the Bloch waves propagating preferentially along particular atomic sites, depending on the incident beam direction. The technique, high-angular resolution electron channeled X-ray spectroscopy, is accessible to atomic site-selective elemental analysis by beam rocking (i.e. scanning reciprocal space), as a good counterpart of the atomic column-by-column analysis using the state-of-the-art scanning transmission electron microscopy (i.e. scanning real space).

The incoherent channeling pattern (ICP), a 2D intensity distribution obtained by a beam-rocking EDX technique, can be quantitatively analyzed by comparing with the inelastic scattering cross-section using e.g., the Bloch-wave method [1], which is, however, time-consuming for calculating 2D ICPs, particularly in calculating the cases where the sample contains an extended defects such as a surface, interface or planar defect etc. We have thus developed a computationally more efficient algorithm where two kinds of absorption potentials are introduced [2]: the absorption potential Uall incorporating the inelastic contributions such as phonons, plasmons and core excitations, and Upartial = Uall − UEDX, where UEDX is the core excitation of a particular atom to calculate its subsequent X-ray emission. The wavefunctions Ψall and Ψpartial are calculated by solving the Schrodinger equations with Uall and Upartial, respectively. The difference in the total intensities between these two wavefunctions should be related to the characteristic X-ray intensity. However, Ψpartial tends to be overestimated for larger thickness and the attenuation of the electron densities propagating through the particular atoms is underestimated. In order to avoid this problem, we introduce a slice-by-slice method which divides the specimen into many thin slices. The X-ray intensity is evaluated by taking the slice-by-slice difference between the corresponding wave functions. The wavefunctions are connected at the entrance surface of each slice.

Figure 1 shows the calculated thickness dependence of the relative Sr-L line intensities of SrTiO3 at the exact [001] incident. Figure 2 shows calculated ICPs, tilted around the [001] axis. These results show the calculated ICP with the sufficient number of slices is nearly identical to that of the conventional method. The present scheme is easily extended to the multislice method which is particularly suitable for the cases where the system of interest contains lattice defects.

References
[1] M. P. Oxley, and L. J. Allen, J. Appl. Cryst., 36, 940 (2003).
[2] K. Watanabe et al., Phys. Rev. B, 63, 085315 (2001).

A part of this work was supported by a Grant-in-Aid on Innovative Areas "Nano Informatics" (Grant number 25106004) from the Japan Society of the Promotion of Science.

Fig. 1: Thickness dependence of Sr-L line intensities of SrTiO3 at [001] incident, calculated by the present method with single slice (black line), and many thin slices by dividing 150 nm thick into 769 slices (red line), and conventional inelastic cross-section method (open circles).

Fig. 2: Two-dimensional Sr-L ICPs around [001] of 150 nm thick SrTiO3, calculated by the conventional inelastic cross-section method (a), and present method with single slice (b), and 769 slices (c), respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2001 Modified Random Walk Algorithm for Monte Carlo Modeling of EBIC and Cathodoluminescence

Priesol J.1, Šatka A.1
1Institute of Electronics and Photonics, Slovak University of Technology, Ilkovičova 3, 812 19 Bratislava, Slovakia
juraj.priesol@stuba.sk

The aim of this contribution is to discuss the results obtained by Monte Carlo (MC) modeling and simulation of the interaction of accelerated primary electrons with semiconductors and semiconductor structures. In electron microscopy practice, MC method is routinely used for quantitative assessment of spatial distribution of energy deposited into the semiconducting material by primary electrons [1], [2], but there is a lack of papers dealing with the MC simulations of consequent diffusion and recombination processes of generated charge carriers [3], [4]. Diffusion of minority carriers has a radical impact on diffusion sensitive methods like electron beam induced current (EBIC) and cathodoluminescence (CL) and therefore it is important to pay a proper attention to it. Due to the complexity of MC simulations, diffusion is usually considered as a random motion of particles according to the random walk algorithm [5], i.e. each generated carrier passes constant distance Δs in random direction constant number of times k. Based on this simple model, three dimensional MC simulations of random diffusion from point source with initial number of N0 generated carriers were executed. MC simulations reveal non-exponential decrease of the carriers from the point source, which is not in agreement with analytical approximation and it has its origin in erroneous assumption of equal lifetime for each simulated carrier. It has been found out that this discrepancy has only a little effect on CL accuracy whereas it is significant for simulation of EBIC line profiles. To overcome this, a modification of random walk algorithm was proposed, where the value of k was determined using probability density function according to normal statistical distribution. The application of adapted model and its influence on the results of MC simulations of EBIC (Fig. 1) and CL, as well as the comparison of simulation and experiment (Fig. 2) performed on III-N semiconductor structures will be presented and discussed.

 

References

[1] Joy, D. C.: Monte Carlo modeling for electron microscopy and microanalysis. Oxford University Press, Inc., 1995, 224 pp., ISBN: 0-19-508874-3.
[2] Demers, H. et al, Scanning 33 (2011), p.135–146.
[3] Ledra, M. - Tabet, N., Superlattices and Microstructures 45 (2009), p.444–450.
[4] Doan, Q. T. - El Hdiy, A. - Troyon, M., J. Appl. Phys. 110 (2011), p. 124515.
[5] Pearson, K., Nature, no. 1865, vol. 72, (1905) p. 294.

This work has been supported by the Slovak Research and Development Agency (contract No. APVV-0367-11) and by Slovak Grant Agency (project VEGA No. 1/0921/13).

Fig. 1: Spatial distributions of 105 carriers recombining in GaN sample around the circular Shottky contact with radius rc = 2500 nm (left) and 150 nm (right) and  simulated for electron beam energy Epe = 5 keV, diffusion length of minority charge carriers L = 196 nm and surface recombination velocity vs = 0.

Fig. 2: Simulated EBIC line profiles at the circular Schottky contact with radius rc for different diffusion lengths L of minority carriers at Epe = 5 keV (left), and EBIC line profiles extracted from EBIC maps measured at reverse polarized Schottky contact formed by tungsten needle set onto the undoped GaN layer (right); fit shows diffusion length ~260nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2085 Calculations of elastic and inelastic scattering processes of relativistic electrons in oriented crystals

Hinderks D.1, Kohl H.1
1Physikalisches Institut der Universität Münster, Münster, Germany
dieter.hinderks@uni-muenster.de

Many modern electron microscopes operate at acceleration voltages of several hundred kV. The accelerated electrons thus reach velocities approaching the speed of light. Therefore the scattering processes have to be treated relativistically. We focus on inelastic scattering in crystals.

In a non-relativistic treatment the movement of the electrons inside the crystal is described using Bloch waves. Before the electrons enter into the crystal they are described by simple plane waves. This view is used in non-relativistic calculations in many cases. The periodic potential of a crystal provides Bloch waves as solutions of the Schrödinger equation. To ensure the boundary conditions at the interface of crystal and vacuum, the transmitted electrons are described using a superposition of plane waves. To obtain a reliable result for the scattering process, many excited Bloch waves have to be considered. The scattering process is mathematically described using matrix elements [1]. The computational complexity depends strongly on the number of Bloch waves considered. The general solution for the wave function of the incident electrons in the crystal is a superposition of many Bloch waves, which are excited at the same time. The excitation of every single Bloch wave is weighted with a excitation coefficient. The number of excited Bloch waves which have to be taken into account depends on the geometry of the crystal.

In this work we focus on an extension of this treatment for relativistic electrons. In contrast to the non-relativistic case the wave functions of the fast incident electrons and the atomic electrons have to be calculated using the Dirac equation. Therefore the incident electrons are described by relativistic four-component Bloch waves (Fig. 1). In our approach we use the relativistic propagator theory where the atomic electrons are seen under influence of a scalar and a vector potential generated by the fast incident electrons via their charge and current (Fig. 2). Furthermore retardation is considered in this relativistic treatment. This approach has previously been used for relativistic plane waves [2]. To consider crystalline materials the incident electrons are described by the relativistic Bloch waves. Consequently the matrix elements contain different sums over reciprocal space and the different single relativistic Bloch waves. The fourier coefficients of these Bloch waves depend on the crystal structure and can be calculated analoguously to the non-relatvistic treatment.

[1] A. Weickenmeier and H. Kohl, Phil. Mag. B60 (1989) 467.

[2] R. Knippelmeyer et al.,Ultramicroscopy 68 (1997) 25-41.

Fig. 1: Relativtistic Bloch wave

Fig. 2: Scattering Matrix
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2124 The dependence of SNR, contrast and resolution on electron dose and sampling

Lee Z.1, Rose H.1, Lehtinen O.1, Biskupek J.1, Kaiser U.1
1Universität Ulm, Materialwissenschaftliche Elektronenmikroskopie
zhongbo.lee@uni-ulm.de

Due to the practical application of hardware aberration correctors, the instrumental resolution of transmission electron microscopes has been remarkably improved. In order to achieve the highest resolution in aberration-corrected (AC) high-resolution transmission electron microscopy (HRTEM) images, high electron doses are required. In the case of high accelerating voltages, materials can be damaged predominantly via the knock-on damage mechanism, where atoms are displaced by direct impacts of the energetic incident electrons. However, when reducing the accelerating voltage, ionization can become the dominating damage mechanism, as the inelastic scattering cross section increases [1]. Effective ways of reducing ionization damage may be cooling of the specimen [2], or conductive coating [3]. However, such approaches are not always feasible. In both, high and low accelerating voltages, images need to be acquired with limited electron doses.

In this work we have performed dose-dependent AC-HRTEM image calculations (Fig. 1), and the dose related noise is treated as stochastic fluctuations around the ideal electron counts on each image pixel, instead of the additive noise. We have studied the dependence of the signal-to-noise ratio (SNR), atom contrast and resolution on electron dose and sampling. Graphene is used as the example material due to the simplicity of its structure, as it is the thinnest and lowest Z-number crystalline material, which allows most straight forward interpretation of the results. We have introduced a dose-dependent contrast definition, which can be used to evaluate the visibility of objects under different dose conditions. Based on our calculations, we have determined optimum samplings for high and for low electron dose imaging conditions.

Our calculation shows: SNR, atom contrast and resolution, all improve with increasing electron dose, converging towards their values obtained at infinite dose. As the sampling increases, the SNR increases and the resolution decreases; the atom contrast improves as long as the damping of MTF is negligible. We have determined optimum sampling under high-dose and low-dose conditions. Under high-dose conditions, the optimum sampling depends mainly on the required specimen resolution; under low-dose conditions, the best sampling is determined by our criteria that the required specimen resolution should be achieved with the minimal electron dose.

[1] R. Egerton, Microsc. Res Tech. 75(2012)1550-1556.

[2] Y. Chen and W. Sibley, Physical Review, 154(1967) 842.

[3] L. Reimer and H. Kohl, Transmission Electron Microscopy: Physics of Imaging Formation. Imperial College Pr. 2008.

[4] Z. Lee et al., Ultramicroscopy (2014), http://dx.doi.org/10.1016/j.ultramic.2014.01.010.

This work was supported by the DFG (German Research Foundation) and the Ministry of Science, Research and the Arts (MWK) of Baden-Württemberg in the frame of the (Sub-Angstrom Low-Voltage Electron microscopy) (SALVE) project.

Fig. 1: Calculated HRTEM images of graphene for different doses and samplings with a usable aperture of 50 mrad under 80 kV. The last row shows the CTF (purple) for different samplings. The PCTF function (blue), focus spread envelope (red) and image spread envelope (yellow) are the same for each column. Reproduced from the reference [4].

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2225 Inelastic scattering of electron vortex beams: mechanism and optimal conditions for EMCD measurements

Rusz J.1, Bhowmick S.2
1Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden, 2Department of Materials Science and Engineering, Indian Institute of Technology, Kanpur, India
jan.rusz@physics.uu.se

Electron vortex beams (EVBs) have attracted a lot of attention since their introduction to the transmission electron microscopy [1,2,3]. In [2] it was reported that EVBs should allow measurement of electron magnetic circular dichroism (EMCD; [4]). Our recent simulations of scanning transmission electron microscopy (STEM) seem to rule out utility of EVBs for measurement of EMCD, unless performed in atomic resolution [5].

The distribution of the EMCD signal in an intrinsic EMCD experiment [4] is anti-symmetric with respect to the mirror symmetry axes. As a consequence, a detector centred on a transmitted beam will not detect any net EMCD signal due to cancellation of positive and negative contributions. In contrast, vortex-induced EMCD can be measured at the transmitted beam. At the level of scalar-relativistic theory and assuming dipole-allowed transitions, we show that this only happens, when the discs in convergent beam electron diffraction (CBED) pattern overlap. Simulations in Fig. 1 illustrate the principle. The top row shows real-space probe wavefunction after passing through 10nm slab of bcc iron oriented in (001) zone axis. The beam diameter (FWHM is indicated in the top) is gradually reduced from left to right. Corresponding elastic CBED pattern shows discs, which eventually start overlapping, as the beam diameter is reduced. In the third row there is energy-filtered Fe-L3 diffraction pattern, which acquires chirality once the CBED discs start overlapping. Finally, bottom row is the distribution of EMCD signal in the diffraction plane. Note the symmetric component of EMCD developing in the middle, once CBED discs start overlapping.

There are several parameters, which influence the inelastic scattering of EVBs: acceleration voltage, beam diameter, EVB angular momentum and a distance of the beam from an atomic column. Optimum may vary as a function of crystal thickness, structure and orientation. We fixed the latter two to (001) zone axis of bcc iron. All the other parameters were independently varied. The results of the optimization are summarized in a condensed form in Fig. 2. The best conditions are predicted for 10nm slab of bcc iron using an EVB of FWHM diameter 1.6Å with an angular momentum 1ħ at acceleration voltage 200keV, using an annular detector of inner (outer) diameters of 1G (5G), respectively, G=(100). These values appear to be reachable with state-of-the-art STEM instruments available today [6].

[1] M. Uchida and A. Tonomura, Nature 464 (2010), 737.
[2] J. Verbeeck, H. Tian, and P. Schattschneider, Nature 467 (2010), 301.
[3] B. J. McMorran et al., Science 331 (2011), 192.
[4] P. Schattschneider et al., Nature 441 (2006), 486.
[5] J. Rusz and S. Bhowmick, Phys. Rev. Lett. 111 (2013), 105504.
[6] O. Krivanek et al., submitted.

We acknowledge Swedish Research Council and Swedish National Infrastructure for Computing.

Fig. 1: Scattering of EVB with angular momentum 1ħ on a 10nm thick bcc Fe crystal oriented along (001) zone axis. Beam diameter is shown by a violet label. Top rows shows scattered EVB wavefunction, elastic diffraction pattern (2nd row), energy filtered diffraction pattern at Fe-L3 edge (3rd row) and the EMCD distribution in the diffraction plane (bottom).

Fig. 2: Optimization of vortex-beam EMCD as a function of acceleration voltage Vacc and angular momentum <Lz>. Optimal beam diameter and inner aperture diameter Rin are shown in 4th and 3rd row. Resulting absolute and relative strength of EMCD are shown in the 1st and 2nd row, respectively. The overall optimum is the white square in the top left panel.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2289 An accurate parameterization for the scattering factors for neutral atoms that obey all physical constraints

Lobato I.1, Van Dyck D.1
1Emat, University of Antwerp , Groenenborgerlaan 171, B-2020, Department of Physics, Antwerp, Belgium
Ivan.Lobato@uantwerpen.be

In electron microscopy and electron diffraction the high energy electrons interact with the atoms of the sample trough their electrostatic Coulomb potential. This interaction is described by the high-energy Schrödinger equation. The simplest approach to describe the electrostatic specimen potential is by linear superposition of the spherically symmetric electrostatic potentials of each atom in the specimen. The atomic potential is related to the atomic charge distribution via Poisson's equation. The electron charge distribution can be computed from the knowledge of the atomic wave function, which can be obtained by numerically solving the Dirac equation. From the electron charge distribution, one can then calculate the X-ray scattering factor. By accounting for the contribution of the nucleus to X-ray scattering factor using the Mott-Bethe formula, one can then calculate the electron scattering factor.

In the simulation programs, one can use the numerical values for the scattering factors and interpolate them to obtain the atomic potential or scattering factor at the required points. But in order to reduce the data, one parameterizes the scattering factors using basic functions such as Gaussians and Lorentzians [1-3]. The drawback of all these parameterizations is that they are obtained by fitting with a discrete set of numerical values from which the asymptotic behavior is then extrapolated.

However, it becomes increasingly clear that electron scattering and simulation programs should also include scattering at very large angles. Indeed, high-angle annular dark-field (HAADF) STEM calculations are done using a frozen lattice model in which the atoms remain sharp and the scattering factors are not dampened by a Debye-Waller factor. The same argument holds for accurate computations of scattering into Higher Order Laue Zones (HOLZ). This means that reliable quantitative simulations for TEM or STEM can only be done using an accurate parameterization for the electron scattering factors that are valid up to very large angles.

We developed a new parameterization of the electron scattering factor using the analytic non-relativistic hydrogen electron scattering factors as a basis functions. The inclusion of the correct physical constraint in the electron scattering factor and its derived quantities allow using the new parameterization in different fields. A comparison between the new parameterization [4] and the previous analytical fittings are shown through figures 1-3.
References
1. A. Weickenmeier, H. Kohl, Acta Cryst. A24(1991), 390.
2. L.M. Peng, G. Ren, S.L. Duvared, J. Whelan, Acta Cryst. A52(1996), 257.
3. E.J. Kirkland, Advanced Computing in Electron Microscopy, Plenum Press, New York, 1998.
4. I. Lobato, D. Van Dyck to be published.

Fig. 1: The root mean square values of the deviation e between the numerical and fitted electron scattering factors vs. atomic number Z using four different parameterizations.

Fig. 2: Comparison among different fittings of the electron and X-ray scattering factors vs. scattering angle for copper. The green markers are the tabulated values of the scattering factors of Kirkland.

Fig. 3: Comparison among different calculated atomic potentials and electron densities vs. the three dimensional radius for copper.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2511 Recent improvements in the STEM-CELL software

Grillo V.1,2, Rotunno E.2, Campanini M.2, Spadaro M. C.2,3, D'addato S.2,3
1CNR-Istituto Nanoscienze, Centro S3, Via G Campi 213/a, I-41125 Modena, Italy, 2CNR-IMEM, Parco delle Scienze 37a, I-43100 Parma, Italy, 3Università di Modena e Reggio Emilia, via G Campi 213/a, I-41125 Modena, Italy
vincenzo.grillo@cnr.it

TEM and STEM analytical studies are going in toward a stronger involvement of computing for the image interpretation. Geometric Phase Analysis [1], for example, has proved to be a useful tool to evaluate the strain in different structures. TEM Image simulations are another very important tool to be compared with complicated structures to obtain quantitative interpretation of the contrast. Moreover the simulation of STEM images is a fundamental step to perform quantitative HAADF measurements [2]. Finally phase retrieval methods in TEM and probe deconvolution in STEM are useful tools to improve the image information [3]. Many simulation software are already available, however it is difficult to find a free and graphical tool that permits to perform both simulation and analysis on the same platform. For this reason we created the STEM CELL project [3][4]. The proposal of STEM CELL is

1) to facilitate multislice simulations by creating, manipulating complicated cells, facilitating the selection of the simulation parameters and interface with simulation routines (the work was based on Kirkland routines [5])

2) to implement analysis methods on both simulated and experimental images so that simulation can be more directly used as a benchmark for experiments.

3) To implement new simulation/analysis methods.

Among the most recent new features it is worth mentioning the probe deconvolution in STEM HAADF images, the phase reconstruction by means of the transport of intensity equation, the simulation of diffraction patterns for any unit cells and the column by column quantitative analysis of the HAADF contrast.

We show in fig. 1 a simulation of two GaAs tetrapods and a Ni particle. A layer of amorphous carbon has been also added to provide a more realistic effect. The sample has been constructed within STEM_CELL and simulated using the embedded Kirkland’s software. Fig. 2 is an example of experimental analysis of the contrast of a CeO2 nanoparticle. The reported experimental image has been obtained by deconvolution of the original image (not shown). The contrast of each column is interpreted approximately in terms of thickness (i.e. number of Ce atoms per column) using a “quick” calibration based on the analysis of the intensity histogram and of the minimal intensity step. Fig. 3 is an example of the transport of intensity equation application on magnetic particles [6].

[1] F M Hytch, et al Ultramicroscopy 74, (1998) 131.

[2] E. Carlino et al. Physical Review B 71 (2005) 235303.

[3] V. Grillo et al. Ultramicroscopy 125 (2013) 112

[4] V. Grillo et al. Ultramicroscopy 125 (2013)97

[5] E.J. Kirkland, Advanced Computing in Electron Microscopy, Plenum Press, NY 1998.
[6] V.V. Volkov et al. Ultramicroscopy 98 (2004) 271

Fig. 1: Model and HREM Simulation for 2 GaAs tetrapods and a Ni particle

Fig. 2: a) Deconvoluted STEM image of a CeO2 nanoparticles and b) quantitative column by column analysis of intensity

Fig. 3: Example of TIE (transport of intensity equation) analysis and graphical representation of magnetic field of two different groups of magnetic nanoparticles. The hue and brightness refer to different B direction/modulus. The line of the field are also shown.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2612 A Perturbation Theory Study of Electron Vortices in Electromagnetic Fields: the Case of Infinitely Long Line Charge and Magnetic Dipole

Xie L.1, Wang P.1, Pan Q. X.1
1National Laboratory of Solid State Microstructures and College of Engineering and Applied sciences, Nanjing University, Nanjing 210093, People’s Republic of China
wangpeng@nju.edu.cn

Electron vortex beam with a quantized orbital angular momentum l and half-integer spin angular momentum has been intensively studied since its introduction intro transmission electron microscopy (TEM) and scanning-TEM because it leads to appreciable number of potential applications in electron microscopy and nanomanipulation [1-3]. Until now, the fundamental physics of electron vortices is still in its infancy and only a few work concerning the basic interactions between electron vortex beams and matter were reported [4][5]. In this work, we study three fundamental interactions: the electron-electric potential interaction, the electron-magnetic potential interaction and the spin-orbit-coupling (SOC) of electron vortex beams in electromagnetic fields based on the relativistically corrected Pauli-Schrӧdinger equation and the perturbation theory.
We first study the interactions between the vortex beam and an infinitely long line charge with a line charge density δE. According to our calculation with an accelerating voltage of 300 kV and a convergence angle of 20 mrad electron vortex beam, both the electron-electric potential and SOC interactions are proportional to δE and decrease monotonically as a function of the topological charge l, as shown in Fig 1. Compared with the electron-electric potential interaction, which is several eV, the SOC interactions are much weaker and are in the range of 10-5~10-3 eV. Next we investigate the interactions between the vortex beam with an infinitely long magnetic dipole with a magnetic moment density δM polarized along z-direction. Fig. 1 shows the electron-magnetic interaction is also in the range of 10-5~10-3 eV. Our calculations indicate that the SOC and electron-magnetic potential interactions are too weak to be observed practically. Nevertheless, it is theoretically predicted that these weak interactions can be raised if a large convergence angle is used. In Fig. 2, we show the calculated results with an accelerating voltage of 300 kV and a convergence angle of 100 mrad electron vortex beam and it is evidently that both the SOC and electron-magnetic potential interactions are dramatically increased by one order, which is favorable for the observation of their effect in future aberration-corrected electron microscopy.

References:
[1] Bliokh, K. Y., et al, Phys. Rev. Lett. 99, (2007), 190404.
[2] Verbeeck, J., et al, Nature (London) 467, (2010), 301-304.
[3] McMorran, B. J., et al, Science 331, (2011), 192-195.
[4] Bliokh, K. Y., et al, Phys. Rev. Lett. 107, (2011),174802.
[5] Lloyd, S. M., et al, Phys. Rev. Lett. 109, (2011), 254801.
[6] Xie, L., et al, Micron, Accepted, (2014).

The authors would like to thank 1000 young talent plan program of China..

Fig. 1: Eel, Esoc and Emag as a function of topological charge l for 300 kV, 20 mrad electron vortex beam. Note that Esoc and Emag are ×10000 scaled.

Fig. 2: Eel, Esoc and Emag as a function of topological charge l for 300 kV, 100 mrad electron vortex beam. Note that Esoc and Emag are ×10000 scaled.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2621 Realistic amorphous carbon model for high resolution microscopy and electron diffraction simulations

RICOLLEAU C.1, LE BOUAR Y.2, AMARA H.2, LANDON-CARDINAL O.2, ALLOYEAY D.1
1Laboratoire Matériaux et Phénomènes Quantiques, CNRS-UMR 7162, Université Paris Diderot-Paris 7, Case 7021, 75205 Paris Cedex 13, France, 2Laboratoire d’Etude des Microstructures, UMR CNRS/ONERA 29, avenue de la Division Leclerc, 92322 Châtillon, France
christian.ricolleau@univ-paris-diderot.fr

Amorphous carbon and amorphous materials in general are of particular importance for high resolution electron microscopy, either for bulk materials, generally covered with an amorphous layer when prepared by ion milling techniques, or for nanoscale objects deposited on amorphous substrates. In order to quantify the information of the high resolution images at the atomic scale, a structural modeling of the sample is necessary prior to the calculation of the electron wave function propagation. It is thus essential to be able to reproduce the carbon structure as close as possible to the real one. The approach we propose here is to simulate a realistic carbon from an energetic model based on the tight-binding approximation in order to reproduce the important structural properties of amorphous carbon.
In this work, we propose a new method to model in a more realistic way amorphous carbon (a-C) that accurately accounts for its 3D structure. It is based on an energetic approach with a tight-binding (TB) potential in which the electronic band structure of the material is calculated with the recursion technique. The main advantage of this model is that it gives a very good description of the sp, sp2, and sp3 hybrid bonds and their competition [1].
At first, the model and the main structural properties of the generated carbon will be presented and compared with a simple model of carbon, where the atom positions are generated randomly. We have shown that the limit thickness for the wave phase approximation if 30% overestimated if we consider the random carbon model (Fig. 1). In a second step, we have studied the influence of the carbon model on the contrast of single Cu, Ag and Au atoms deposited on amorphous carbon substrate. Our work does not indicate any significant influence of the carbon structure on single-atom contrast when statistically relevant measurements are performed. Finally, we have compared both model structures for the determination of the long-range order parameter in a small CoPt nanoparticles deposited on a-C layer. We have clearly shown the importance to use realistic amorphous carbon model to obtain quantitative values for the diffracted intensities. As it can be observed on Figure 2, diffuse scattering intensity due to the 3D atomic arrangements of the realistic carbon has a significant contribution to the scattered information especially at low spatial frequencies [1].
This work emphasizes the necessity to use realistic carbon model for TEM image and diffraction simulation in order to extract very sensitive quantitative information, particularly in diffraction experiments.

[1] Ricolleau C., Le Bouar Y., Amara H., Landon-Cardinal O. and Alloyeau D., J. Appl. Phys., 114, 213504 (2013).

*Currently address: Département de Physique, Université de Sherbrooke, Sherbrooke, Québec J1K 2R1, Canada.

Fig. 1: Comparison of the (a) phase and (b) intensity of the transmitted beam (i.e., the unscattered part of the electron wave) calculated using Random and tight-binding carbon structures as a function of the thickness layer. The slice thickness is 0.25 nm. The simulation box size in the layer plane is 5nmx5 nm with a 1024x1024 sampling.

Fig. 2: Electron diffraction calculation of a CoPt NP with a LRO of 0.4 deposited on a 10 nm thick amorphous carbon layer simulated by (a) the random model and (b) the tight-binding model. (c) Radially integrated intensity profile as a function of the scattering vector for both diffraction patterns (a) and (b) in black and red, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2686 Remove the CCD influence from high-resolution electron microscopy images

Lin F.1, Jin C.2, Yang Y.1
1College of Science, South China Agricultural University, Guangzhou, Guangdong 510642, PR China, 2State Key Laboratory of Silicon Materials, Department of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, PR China
flin_163@163.com

Slow-scan charge-coupled device (CCD) camera is now an important recording medium for high-resolution transmission electron microscopy (HRTEM). However, the pixel of CCD chips has a certain size, and then the point spread effect cannot be neglected in image. In frequency domain, signal transfer is described by a modulation transfer function (MTF). In addition, Possion noise is inherent in electron images. The noise transfer is described by noise transfer function (NTF), which gives the attenuation of noise power relying on its frequencies [1].
To remove the MTF from HRTEM image, the MTF should be calculated firstly from experiments. All experimental images were recorded on a JEM-2010F TEM equipped with a Gatan-894 CCD camera. The beam-stopper image is shown in Fig. 1(a). Through averaging the image intensities of positions equally distancing from the edge, we could get the edge profile shown in Fig. 2(b). Deconvoluted from the filtered profile of edge, the PSF of CCD was resolved. We show it in Fig. 1(c) and use the Fourier transform to get the MTF of CCD.
To measure the NTF, 16 uniform-illumination images without beam-stoppers and 8 dark-current images were recorded. The ‘NTF 1’ and ‘NTF 2’ in Fig. 2 are estimated from the 16 uniform illumination images of totally ~2190 and ~4760 counts, respectively. Although the electron doses are different, the noise characteristics of NTF are almost the same. The NTF is mainly affected by the accelerated voltage and exposure time.
Based on an improved Wiener deconvolution filter, the restored image I’(u,v) in frequency domain of (u,v) is calculated as [2],
I'(u,v)=I(u,v)MTF*(u,v)/{|MTF(u,v)|2+Pn(u,v)/[PD(u,v)-Pn(u,v)]},
in which, I(u,v) is a HRTEM image, MTF(u,v) is the MTF of CCD in 2-dimensional space, and PD(u,v) and Pn(u,v) are the mean power spectral densities of detected image I(u, v) and additive noise, respectively. PD(u,v) is actually the image diffraction pattern multiplied by its conjugate, and Pn(u,v) is estimated from NTF. The NTF provides the power distributions of noise at various frequencies and is measured from uniform-illumination images. For weak scattering objects, such as few-layer graphene or boron nitride, the image is considerably “uniform” because of the weak scattering of atoms. Fig 3(a) gives a raw HRTEM image of graphene. After deconvolution, the lattices in center region are resolved from noise.

Authors acknowledged supports from the National Science Foundation of China (61172011 and 51222202), National Basic Research Program of China (2014CB932500), the Program for Innovative Research Team in University of Ministry of Education of China (IRT13037), the Fundamental Research Funds for the Central Universities (2014XZZX003-07) and Guangdong Natural Science Foundation (10151064201000006).

Fig. 1: (a) The beam-stopper image. The white line indicates one of the beam-stopper edges. (b) The blue line is the edge profiles averaged from one edge in (a). The red line gives a filtered edge profile. (c) The CCD’s PSF calculated from (b). Ideal step function convoluting with this PSF is the red line in (b).

Fig. 2: The NTF of CCD. ‘NTF 1’ and ‘NTF 2’ are calculated from the images uniformly illuminated under different electron doses after normalization.

Fig. 3: (a) A raw HRTEM image of graphene. (b) The image restored with the improved Wiener deconvolution filter.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2731 Methods for Scanning Transmission Electron Microscopy High Angle Annular Dark Field based for three dimensional analysis of the local composition in solid alloys

Rotunno E.1, Grillo V.1,2, Markurt T.3, Remmele T.3, Albrecht M.3
1CNR-IMEM, Parco delle Scienze 37a, I-43100 Parma, Italy, 2CNR-Istituto Nanoscienze, Centro S3, Via G Campi 213/a, I-41125 Modena, Italy , 3Leibniz Institute for Crystal Growth, Max-Born-Strasse 2, 12489 Berlin, Germany
vincenzo.grillo@cnr.it

We report on a novel approach to quantitatively reconstruct the number and the three-dimensional distribution of guest atoms inside a host matrix by STEM HAADF technique. Since the position of the guest atoms in the column strongly affects the chemical quantification for each column [1][2] this technique allows in addition for an improved quantification of the composition.

Our method is based on the joint analysis of a set of experimental data gained with variable beam convergence and/or defocus. It allows to invert the intensities into an atomic distribution along the columns for any dependence of the probe intensity on the thickness. It is therefore well suited to incorporate channeling effects that are usually neglected in other approaches [3]. We focus here on the systematic variation of the beam convergence that permits to set the maximum of the channeling oscillations at different depth [4].

From Fig 1, showing the dependence of the probe intensity with depth inside an InGaN sample for convergence angles of 9, 15 and 21 mrad, it is evident that with changing the convergence angle we are probing different parts of the sample.

To extract detailed information we represent the guest atom distribution in a given column mathematically as a continuous profile and develop it in terms of harmonic components. The components can be determined from the experimental contrast at variable probe conditions by an inversion of the functional dependencies between these parameters.

To test the application of the method we used simulations with the multislice Frozen Phonon algorithm. The results are shown in fig 2 a,b,c. Using the inversion algorithm we were able to retrieve the map of the number of atoms per column. In fig 2 d,e the real composition per column and that retrieved by the algorithm are compared, showing a substantial agreement.

While the use of a single image permitted to obtain the quantification for a single column with a confidence level less than 60%, the new paradigm permits to obtain a confidence level of 95%. .

We are also able to retrieve an estimation of the guest atoms distribution along z. Fig 2f shows the superposition of the estimated distribution and the actual position. We propose the method as a general framework to perform 3D quantitative analysis to be used in all multiple STEM-HAADF experiments including through-focal experiments.

[1] P.M. Voyles, et al. Ultramicroscopy 96 (2003) 251–273

[2] E. Carlino V. Grillo Physical Review B 71 (2005) 235303.
[3] K.v. Benthem, et al. Appl. Phys. Lett. 87, 034104 (2005)

[4] Y. Peng, et al. Journal of Electron Microscopy 53 (2004) 257

Fig. 1: Probe intensity vs depth inside an InGaN sample for convergences of 9 (a), 15(b) and 21 mrad (c). In fig d the profile in the center of the probe are plotted as a function of z.

Fig. 2: Simulation of a STEM-HAADF image of InGaN with convergence of of 9 (a), 15(b) and 21 mrad (c). Comparison of actual d) and retrieved e) column by column composition map. f) Retrieved “continuous” distribution of In atoms in a column compared with the actual distribution
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2748 The forward dynamical electron scattering (FDES) software; a graphics-processing-unit accelerated multislice algorithm

Van den Broek W.1, Koch C. T.1
1Institute for Experimental Physics, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
wouter.vandenbroek@uni-ulm.de

The forward dynamical electron scattering (FDES) software is a multislice algorithm taking advantage of the graphics processing unit (GPU) to speed up computation. Further acceleration is attained through efficient computation of the projected atom potential and an approximation of Poisson noise by the inverse Anscombe transform. Thermal diffuse scattering can be taken into account by the frozen phonon approximation. In the following, the three most time consuming processes are labelled A, B and C and their runtimes are compared.

FDES was written in the CUDA programming language [1] (with the CUFFT, CUBLAS and CURAND libraries) and run on a Tesla K20c GPU (NVIDIA).

Calculating the projected potential in real space is not optimal since the potential’s divergence in the origin requires special treatment; furthermore, one faces a trade-off between accuracy and computation speed in deciding on the projected potential‘s cut-off radius. Both problems are overcome in FDES. The four pixels nearest to the atoms are assigned the value of their intersection with a pixel-sized square centered on that atom (A), thus ensuring sub-pixel accuracy of the atom positions, see Fig. 1. Next, the result is convolved with the projected potential and deconvolved with the pixel-sized square (B). Since this (de)convolution is implemented as a multiplication in Fourier space where the scattering factors and the sinc-function are well-behaved, no numerical difficulties are encountered.

The multislice algorithm proper, i.e. the computation of the propagation of the electron wave through the sample (C), is sped-up by computing the necessary Fourier transforms on the GPU.

Since normally distributed values require no pre-processing on the central processing unit, FDES applies the inverse Anscombe transform p = round( 0.25 n2 – 3/8 ) [2] to variables n drawn from a normal distribution with mean 2 ( μ + 3/8 )1/2 − 0.25 μ−1/2 and variance 1 − exp( − μ / 0.78 ) to yield approximate Poisson distributed variables p with expectation value μ. This is dubbed Anscombe noise. See Fig. 2.

The runtime was measured on a simulation of a 25x25x15nm3 Al crystal with an FCC lattice (no advantage was taken of this symmetry) with a lattice constant of 0.405nm. The pixels were 0.025nm wide and the slice thickness was 0.1nm. The processes A, B and C took 0.022s, 0.191s and 0.488s respectively, between them accounting for 97% of the total runtime of 0.723s.

[1] NVIDIA CUDA C Programming Guide Version 5.0, 2012.
[2] F.J. Anscombe, Biometrika 35 (1948), 246–254.

The authors acknowledge the Carl Zeiss Foundation as well as the German Research Foundation (DFG, Grant No. KO 2911/7-1).

Fig. 1: Each entry in the list of atoms is assigned to a different processor on the GPU that calculates to which slice the atom belongs and what its four nearest neighboring pixels are in that slice. These pixels values are set to the intersection with a pixel-sized square centered on the atom.

Fig. 2: Left: Histograms of Anscombe distributed values with mean 5.0 and of Gaussian distributed values of mean 5.0 and variance 5.0, contrasted to a Poisson distribution of mean 5.0. (N = 107). Right: Absolute values of the difference of the histograms with the Poisson distribution; the error is nearly always lower for the Anscombe noise.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2768 Influence of the delocalization of inner-shell excitations on atomic-resolution elemental maps

Park M.1, Majert S.1, Kohl H.1
1Physikalisches Institut und Interdisziplinäres Centrum für Elektronenmikroskopie und Mikroanalyse (ICEM), Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Straße 10, 48149 Münster, Germany
parkmi@uni-muenster.de

Using elemental maps with atomic resolution obtained by energy dispersive x-ray spectroscopy (EDX) in a scanning transmission electron microscopy (STEM), it is possible to investigate the elemental distribution in a specimen. To analyse the experimental results it is necessary to compare them with calculations in order to distiguish specimen properties from imaging artifacts. We investigate the influence of channeling and of the delocalized excitation of inner-shell electrons on the elemental map.

For our calculations, we use the multislice method [1], which describes the behaviour of electrons passing through a thick specimen. In this theorythe specimen is divided into thin slices treated as a pure phase object.

In a first approximation, we assume that the number of x-ray quanta emitted by an atom is proportional to the electron intensity at its position. This means that the atoms are approximated as being point-shaped. We then replace the point-shaped description of atoms with the delocalized excitation function [2].

To estimate the influence of an atom exicitation probability on elemental maps we also compare the results of both approximations with each other and these with experimental results.

An interface between a strontium titanate crystal (SrTiO3) and a lead titanate crystal (PbTiO3) serves as an example of our calculation.

The results of the simulation with the localized approximation of Ti, Sr and Pb signals in an elemental map of this sample are shown in Figure 1. For this calculation we used an acceleration voltage of 200 kV, assumed an objective lens free of astigmatism, a spherical aberration coefficient Cs = 0,5 mm, and a 15 mrad aperture semiangle [3]. These results are compared with the experimental data from L. J. Allen et al. [4].

The major difference between both results is that the position of the boundary is clear in the simulation but not in the experimental results. This could be the result of the unevenness of interface in the experiment and the use of the localized approximation in the calculation. For a better interpretation of the results we currently investigate how the radius of the atomic column and the boundary changes when considering a delocalized excitation function in the simulation.

[1] Earl J. Kirkland “Advanced Computing in Electron Microscopy” (Plenum Press, New York, 1998) p. 157

[2] D. Von Hugo, H. Kohl, and H. Rose, Optik 79 (1988) p. 19

[3] S. Majert “Simulation atomar aufgelöster Elementverteilungsbilder mit der Multislice Methode”, BSc thesis (2012)

[4] L. J. Allen et al. “Chemical mapping at atomic resolution using energy-dispersive x-ray spectroscopy” (MRS BULLETIN, Volume 37, January 2012) p. 47

Fig. 1: Elemental map of 25 slices (≈10 nm) of an interface between a strontium titanate crystal (SrTiO3) and a lead titanate crystal (PbTiO3) in [001]-orientation simulated with the localized approximation
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2950 The partial spatial coherence function and the distribution of scattered electrons

Nguyen D. T.1, Findlay S. D.2, Etheridge J.1,3
1Department of Materials Engineering, Monash University, VIC 3800, Australia, 2School of Physics, Monash University, VIC 3800, Australia, 3Monash Centre for Electron Microscopy, Monash University, VIC 3800, Australia
dan.nguyen@monash.edu

The development of aberration correctors has made images with sub-Ångstrom spatial resolution in scanning transmission electron microscopy (STEM) routinely possible, greatly advancing our knowledge of the relationships between a material's atomic structure, composition, and its properties. It has been noted, however, that possessing atomically-resolved features does not guarantee an image can be quantitatively analysed column-by-column, especially for inhomogeneous materials [1,2]. The mechanism limiting our ability to attribute the spatial origin of the signal to specific atomic sites is the spreading of the electron probe as it travels through the specimen. The finite effective source profile, also referred to as the partial spatial coherence function, has been reported to have a large impact on the intensity distribution in STEM images [3,4]. However, the consequences of the effective source distribution for the spreading of the electron probe have not been much explored.

We investigate the implications of the partial spatial coherence function for quantitative analysis in STEM, especially for interpreting the spatial origin of imaging and spectroscopy signals. In particular, we examine how the shape of the source distribution, especially the length of its “tails”, influences the degree to which the electron probe scatters onto adjacent atomic columns. This was explored via the use of three different source distribution models applied to a GaAs crystal case study. The shape of the effective source distribution was found to have a large influence not only on the STEM image contrast, but also on the distribution of the scattered probe through the specimen and hence on the spatial origin of the detected electron intensities.

This has implications for our ability to extract column-by-column information via annular dark field, X-ray and electron energy loss STEM imaging, as precise knowledge of the spatial origin of the measured signal is a prerequisite for any high precision determination of structure, bonding and chemical composition at the atomic scale.

 

References
1. C. Dwyer and J. Etheridge, Ultramicroscopy, 96 (2003) 343-60.
2. P. M. Voyles, J. L. Grazul and D. A. Muller, Ultramicroscopy, 96 (2003) 251-73.
3. J.M. LeBeau, S.D. Findlay, L.J. Allen, S. Stemmer, Phys. Rev. Lett., 107 (2008) 206101
4. C. Dwyer, C. Maunders, C.L. Zheng, M. Weyland, P.C. Tiemeijer, J. Etheridge, Appl. Phys. Lett., 100 (2012) 191915

 

This research was supported under the Discovery Projects funding scheme of the Australian Research Council (Project Nos. DP120101573 and DP110101570).

Fig. 1: Comparison between a Gaussian (G), Gaussian + Lorentzian (G+L) and Lorentzian (L) source profile, with the FWHM chosen to give a 60% reduction in contrast with respect to a coherent probe. The resultant ADF-STEM images are shown below it (from left to right: Gaussian, Gaussian + Lorentzian, Lorentzian)

Fig. 2: The proportion of ADF signal generated from a single Ga atomic column in GaAs <001> recorded in a Voronoi cell around the column (top) and in an empty cell immediately adjacent to it (bottom). P refers to a diffraction-limited probe. G, G+L and L correspond to a Gaussian, Gaussian + Lorentzian and Lorentzian effective source, respectively.

Fig. 3: Cross-sectional intensity map of the scattered probe (using three different source profiles) through a GaAs crystal of <110> and <112> orientation, and the corresponding plot of the normalised probe intensity on the Ga atomic column (integrated within a radius of 0.5 Å). The arrows point to the centre Ga column and the closest As column.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-2955 Removing the effects of elastic and thermal scattering from spectrum images in scanning transmission electron microscopy

Lugg N. R.1,2, Neish M. J.1, Haruta M.3, Kothleitner G.4, Grogger W.4, Hofer F.4, Kimoto K.3, Mizoguchi T.5, Findlay S. D.6, Allen L. J.1
1School of Physics, The University of Melbourne, Melbourne, Australia, 2Institute of Engineering Innovation, The University of Tokyo, Tokyo, Japan, 3National Institute for Materials Science, Tsukuba, Japan, 4FELMI, Graz University of Technology, Graz, Austria, 5Institute of Industrial Science, The University of Tokyo, Tokyo, Japan, 6School of Physics, Monash University, Melbourne, Australia
nrlugg@sigma.t.u-tokyo.ac.jp

Scanning transmission electron microscopy (STEM) has proven to be a powerful tool for the acquisition of atomic-column-resolved elemental maps using electron energy-loss spectroscopy (EELS) and, more recently, energy-dispersive x-ray (EDX) spectroscopy. Furthermore, in EELS, given the ability to study how spectra change on the atomic scale, there has also been much interest in mapping not only the positions of atoms, but also their bonding states by studying changes in the fine structure of EELS data [1-3]. Specifically, atomic-resolution oxygen EELS data in transition metal oxides can potentially provide information about the entire electronic structure of a material since oxygen atoms bonded to different transition metals will display distinct spectra, reflecting the local bonding state. In atomic-resolution EDX, given the localized nature of the interaction potential involved and that such maps are not further complicated by subsequent elastic and thermal scattering after ionization, there is huge promise in quantitatively measuring elemental densities at atomic resolution.

However, due to the complex elastic and inelastic scattering of the electron probe, direct qualitative and quantitative interpretation of both elemental and bond maps is difficult. In bond mapping, the scattering of the electron probe causes the spectra from inequivalently bonded atoms to become mixed, and the features that distinguish them to become ambiguous. In elemental mapping, the highly non-linear response to atomic-column densities due to electron scattering denies any direct correspondence between signal and atomic-column densities [4].

Recently, a method has been developed to remove the effects of elastic and thermal scattering from spectrum images [5]. Using the quantum excitation of phonons model [6], the cross-section expression for inelastic scattering in STEM may be expressed as an inverse problem, and the elastic and thermal scattering deconvolved from experimental data. Here we show applications of this method in both EELS [7] and EDX [8] data of SrTiO3.

[1] DA Muller et al, Science 319 (2008) 1073
[2] H Tan et al, PRL 107 (2011) 107602
[3] M Haruta et al, APL 100 (2012) 163107
[4] BD Forbes et al, PRB 86 (2012) 024108
[5] NR Lugg et al, APL 101 (2012) 183112
[6] BD Forbes et al, PRB 82 (2010) 104103
[7] MJ Neish et al, PRB 88 (2013) 115120
[8] G Kothleitner et al, PRL 112 (2014) 085501

Fig. 1: (a) HAADF map of SrTiO3 [001] (projected structure inset). (b) EELS map using the O K edge (potential obtained from inversion inset). (c) SrTiO3 structure showing the two inequivalent O atoms in the [001] projection. Spectra from inequivalent O columns obtained from (d) background-subtracted data, (e) after inversion and (f) Wien2k calculation.

Fig. 2: SrTiO3 [001] experimental and simulated (inset) (a) HAADF (structure overlayed) and EDX (b) Sr K, (c) Ti K (d) O K edge maps. Numbers inset show atomic densities (atom/nm3) obtained from: the ideal structure (averaging the entire EDX map) [averaging specific columns] {inversion}. (e) EDX colour composite with masks used for column specific average.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-3028 Phase mapping at the interface retrieved by FFT based transport of intensity equation

Zhang X.1, 2, Oshima Y.2, 3
1Tokyo Institute of Technology, Tokyo, Japan, 2JST-CREST, Tokyo, Japan, 3Osaka University, Osaka, Japan
zhang.x.am@m.titech.ac.jp

   Phase retrieval using transport of intensity equation (TIE) is convenient and powerful to obtain electrostatic potential of materials, because it needs only three transmission electron microscope (TEM) images taken at different foci. In our previous results, potential map of gold nanoparticles adsorbed on a thin amorphous carbon (a-C) film was successfully obtained with high spatial resolution (1nm). However, it is difficult to obtain potential map of two-phase interface. In this study, we investigate how to obtain the phase map of the boundary between a-C and vacuum regions quantitatively.
  TEM observation was taken by 50pm-resolved R005 equipped with cold field emission gun and double aberration-correctors. Figure 1(a-c) shows three TEM images of under-focus 1000 nm, just-focus and over-focus 1000 nm. The TIE retrieved phase map using these three images is given in (d). Figure 1(e) and (f) are the line profiles along the blue lines indicted in (a) and (d). We notice that the mean intensity of vacuum, thin C-film and thick C-film regions are different in the original TEM image and the intensity profile does not reflect the expected phase shift among these regions. Moreover, a strong low-frequency contrast appeared in the phase map.
  We found the reasons of such discrepancy. Firstly, the periodic boundary condition required for FFT process of solving the TIE equation influences the retrieved phase map. Secondly, brightness difference exists in the focal-series TEM images, which is caused by the variation in objective lens current. Since the intensity difference applied to TIE is obtained by subtracting the over-focus TEM image from the under-focus one, the difference in image brightness causes deviation in the intensity differences. For example, all the values of ΔI are negative in the vacuum area. However, they should only oscillate around zero according to the wave theory.
  We consider that applying padding or mirror methods to the process is effective to satisfy the boundary condition, and aligning the current center rather than the voltage center of objective lens can eliminate the inhomogeneous illumination when taking a focal-series TEM images.

Fig. 1: (a-c) Three TEM images taken at under-focus 1000 nm, just-focus and over-focus 1000 nm. (d) The TIE retrieved phase map obtained using (a-c). (e,f) The line profiles along the blue lines indicted in (a) and (d), respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-3032 Computer vision in the service of Crystallography: Automated analysis of atomic-resolution images

Klinger M.1
1Institute of Physics ASCR, Prague, Czech Republic
miloslav.klinger@seznam.cz

An automated tool for a crystallographic analysis of HRTEM (High resolution Transmission electron microscopy), HRSTEM (High resolution Scanning Transmission electron microscopy) and diffraction images is proposed. Algorithms of artificial intelligence and computer vision are employed to detect features carrying the information and to process them in order to determine or estimate crystallographic quantities. This shall result in an expedited analysis, possibly higher precision and little to no human effort compared to manual analysis.

In the case of SAD (Selected area diffraction) images, diffraction spots or disks are detected in the widest possible area of the pattern and the zone axis is calculated. If the observed material is not known, the tool can choose the most probable candidate from a list candidates. HREM (High resolution electron microscopy) images can be segmented to separate individual grains depicted. If the image contrast features directly correspond to positions of atomic columns, the zone axis is determined and the crystallographic planes and direction in the image are identified. Dislocation detection and quantification can be performed as well as a grain misorientation estimation, reconstruction of positions of individual atoms and so on. If the image contrast features do not correspond directly to the atomic column positions, the atomic columns can be found using HREM simulations.

The proposed tool (implemented in MATLAB) has been successfully tested on number of real world images and diffraction patterns. It has proven its ability to autonomously provide correct results.

I would like to thank Professor Michael Mills and the National Center for Electron Microscopy for providing HRTEM images. Financial support offered by GACR GBP108/12/G043 and MEYS LM2011026 is appreciated.

Fig. 1: Segmented grains in HRTEM image of alluminium. Original image acquired in the National Center for Electron Microscopy.

Fig. 2: Three dimensional reconstruction of grains depicted on Fig. 1.

Fig. 3: Dislocation detected in alluminium. Burgers circuit can be seen on the left and visualization of inserted plane on the right. Original image acquired by Professor Michael Mills.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-3168 Simultaneous thickness and orientation mapping by dark-field transmission electron microscopy

Tyutyunnikov D.1, Koch C. T.1
11. Institute for Experimental Physics, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
dmitry.tyutyunnikov@uni-ulm.de

Transmission electron microscopy (TEM) is a powerful tool for investigating the atomic structure and morphology of nano- and micro-objects. To reveal the structure of a material the specimen prepared for TEM should be reasonably thin in order to be transparent for the electron beam. The thickness of the sample is an important parameter one should account for when analyzing images acquired in TEM. The probability for electrons to scatter multiple times increases with the specimen thickness. This effect has, for example, a strong influence on the contrast of high-resolution TEM images. There are several techniques available to estimate the thickness by TEM [1]. These methods can be divided into several categories, e.g. imaging methods, Convergent Beam Electron Diffraction (CBED) method, electron energy-loss spectroscopy (EELS) - based methods, and methods based on X-ray spectroscopy (EDXS). The most popular technique among them is based on EELS [2], which can be used for a variety of samples and is easy to implement computationally. However if one has thin crystalline samples one often has to deal with bending due to lattice relaxation. Bending can strongly influence the thickness values obtained by EELS. Here we report about a new technique which simultaneously delivers thickness and specimen surface orientation maps. Our approach is based on analysis of rocking curves extracted from experimental dark-field (DF) images acquired at different specimen tilts. We fit the parameters that affect dynamical electron diffraction rocking curves to experimental DF images. In its simplest version, our approach uses 2-beam theory, for which the intensity of the diffracted beam is given according to C.Humhreys 1979 review [3]. To determine the thickness we fit the power spectra to a sum of Gaussians. Since the rocking curves usually exhibit oscillatory behavior reflecting the thickness of the specimen one should observe peaks in power spectra of rocking curve. Fitting was done in MATLAB by using the unconstrained nonlinear optimization routine fminsearch [4] and the Matlab Curve Fitting Toolbox. As an example the mapping was done for a commercial semiconductor device. Figure 1 shows a single slice from DF image stack of this device acquired for the {220} reflection. Figure 2 illustrates how a dark-field tilt series samples reciprocal space to demonstrate the oscillatory behavior of the extracted rocking curve (inset in Fig. 1).

[1] D.B. Williams and Carter C.B. Transmission Electron Microscopy. Springer Science+Business Media, 2009

[2] T. Malis, et al. J. Electron Microsc.Tech., 1988

[3] C. J. Humphreys. Rep. Prog. Phys., 42, 1979.

[4] J.C. Lagarias, et al. SIAM Journal of Optimization, pages 112-147, 1998

The authors acknowledge financial support by the Carl Zeiss Foundation as wellas the German Research Foundation (DFG, Grant No. KO 2911/7-1). The authors also acknowledge Stuttgart Center for Electron Microscopy (StEM) for sample preparation and possibility to carry out the experiment.

Fig. 1: Single slice from a DF image stack acquired for the {220} reflection of Si in a MOSFET structure. The red square shows the ROI used for thickness and orientation mapping. The sample was prepared by automatic tripod polishing and consequent low-voltage Ar ion milling at T of liquid N2

Fig. 2: The Dark-field tilt series intersects the diffraction signal with the Ewald sphere indicated as green arks at different positions along kz axis. The diffraction signal was calculated by FFT for a 20 nm thick slab.

Fig. 3: Thickness map in Å. The data was binned before fitting. The thickness fitted outside the crystalline area is meaningless, of course.

Fig. 4: Map of the misorientation a0.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-3170 Structure factor refinement from electron diffraction for structures with arbitrarily large unit cells

Feng W.1, Kazzazi A.1, Koch C. T.1
1Institute for Experimental Physics, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
feng.wang@uni-ulm.de

Structure-factor refinement by quantitative convergent-beam electron diffraction (QCBED) [1] has been able to reveal the charge distribution responsible for the bonding between atoms [2]. However, in order to be able to fit a set of complex structure factors by comparing dynamical electron diffraction simulations to the contrast within CBED discs, the sample must typically be at least 100 nm thick, and the lattice parameters should not exceed 1 nm. Also, in order to extract the elastic scattering signal it must be assumed that the incoherent background (mainly thermal diffuse scattering (TDS)) varies only slowly, an assumption that is not generally correct. In contrast, large-angle rocking-beam electron diffraction (LARBED) [3] data (see, for example Fig. 1) can be acquired for arbitrarily large unit cell structures and reveals features that are clearly due to dynamical electron diffraction even at specimen thicknesses below 10 nm. This makes structure factor refinement from nanocrystals possible.

As is common in QCBED work we write the scattering matrix S in the form S=eiTA, by assuming previous knowledge of the excitation errors (diagonal of A), we reduce the problem to finding the factor T, which is acceleration voltage and specimen thickness related, and all the off-diagonal entries Ug-h in A, from a series of observed norms of entries in one column of S (the squared norms correspond to the diffraction intensities shown the example pattern in Fig. 1). To efficiently solve such a nonlinear programming problem up to several hundreds of variables, a gradient-based iterative method is critical.

In this work, we design a two-layer model to accelerate the calculation of the expensive Bloch-wave simulation and the cumbersome gradient approximation. We also compare and discuss the convergence and performance of different optimization algorithms applied to this problem.

[1] C. Deininger, G. Necker, J. Mayer, Ultramicroscopy 54 (1994) 15-30

[2] J.-M. Zuo, M. Kim, M. O’Keefe, J.C.H. Spence, Nature 401 (1999) 49

[3] C.T. Koch, Ultramicroscopy 111 (2011) 828 – 840

The authors acknowledge the Carl Zeiss Foundation as well as the German Research Foundation (DFG, Grant No. KO 2911/7-1)

Fig. 1: Bright-field and dark-field large-angle rocking convergent beam (LARBED) patterns of SrTiO3 in the [-110] zone axis, acquired on the Zeiss SESAM operated at 200 kV. This kind of data can be used to fit dynamical scattering equations to, even for very thin samples.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-3277 BlochSim: A new, free to use, open source Bloch Wave Simulation program

Evans K. L.1, Roemer R. A.1,2, Beanland R.1
1Department of Physics, University of Warwick, Coventry, West Midland, CV4 7AL, 2Centre for Scientific Computing, University of Warwick, Coventry, West Midlands, CV4 7AL
keith.evans@warwick.ac.uk

We present a new software package developed at the University of Warwick for simulation of dynamical diffraction in transmission electron microscopes using the Bloch wave method [1]. While the Bloch wave method is well established [2], freely available software to scientists is limited, less still is open source [3]. This prevents further development and adoption by the general microscopy community. The X-ray crystallography community has benefited hugely from open source and freely available packages such as SHELX [4], and availability of similar platforms in electron crystallography are very desirable. Our new software is intended to help bridge this gap by being open source and free of charge in order to facilitate greater usage throughout the community and ease of development.

The software package allows for simulation of Convergent Beam Electron Diffraction (CBED) patterns as well as the recently developed Digital Large Angle CBED (D-LACBED) [5]. More advanced features include crystal thickness determination, structural refinement and electron density mapping. All of these features take can advantage of the superior data range of D-LACBED [5].

Historically, structure solution and refinement has been dominated by X-ray methods. However, X-rays lack the nanometre size resolution required for the study of nanostructured materials such as interfaces, grains, nanobeams etc. Electrons, due to their much smaller wavelengths and stronger interaction with matter, have the required resolution and hence there is a niche for structural refinement using electron diffraction. We will describe the use of BlochSim with structure refinement strategies, in particular identifying and taking advantage of regions within large electron diffraction datasets which have enhanced sensitivity to structural parameters.

BlochSim uses a set of base tools that are compatible with X-ray crystallography and a key aim is to make the software multiplatform and user friendly. It will be configured to accept many forms of input structure (.cif, .pdb, .xyz) in order to reach a large user base. We encourage use and further code development by new users.

1. Bloch, F., Über die Quantenmechanik der Elektronen in Kristallgittern. Zeitschrift für Physik, 1929. 52(7-8): p. 555-600.

2. Stadelmann, P.A., A Software Package for Electron-Diffraction Analysis and HREM Image Simulation in Materials Science. Ultramicroscopy, 1987. 21(2): p. 131-145.

3. Tsuda, K. 2013; Available from: www.tagen.tohoku.ac.jp/labo/terauchi/personal/tsuda/mbfit_win.zip.

4. Sheldrick, G.M., A short history of SHELX. Acta Crystallographica Section A, 2008. 64: p. 112-122.

5. Beanland, R., et al., Digital electron diffraction - seeing the whole picture. Acta Crystallographica Section A, 2013. 69: p. 427-434.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-3287 Electron vortex beam diffraction via multislice solutions of the Pauli equation

Edström A.1, Rusz J.1
1Department of Physics and Astronomy, Uppsala University
alexander.edstrom@physics.uu.se

Electron magnetic circular dichroism (EMCD) has gained plenty of attention as a possible route to high resolution measurements of, for example, magnetic properties of matter via electron microscopy. However, certain issues, such as low signal-to-noise ratio, have been problematic to the applicability. In recent years, electron vortex beams\cite{Uchida2010,Verbeeck2010}, i.e. electron beams which carry orbital angular momentum and are described by wavefunctions with a phase winding, have attracted interest as potential alternative way of measuring EMCD signals. Recent work has shown that vortex beams can be produced with a large orbital moment in the order of l = 100 [6, 7]. Huge orbital moments might introduce new effects from magnetic interactions such as spin-orbit coupling.

The multislice method[2] provides a powerful computational tool for theoretical studies of electron microscopy. However, the method traditionally relies on the conventional Schrödinger equation which neglects relativistic effects such as spin-orbit coupling. Traditional multislice methods could therefore be inadequate in studying the diffraction of vortex beams with large orbital angular momentum. Relativistic multislice simulations have previously been done with a negligible difference to non-relativistic simulations[4], but vortex beams have not been considered in such work.

In this work, we derive a new multislice approach based on the Pauli equation, Eq. 1, where q = −e is the electron charge, m = γm0 is the relativistically corrected mass, p = −i ∇ is the momentum operator, B = ∇ × A is the magnetic flux density while A is the vector potential and σ = (σx , σy , σz ) contains the Pauli matrices. ψ±(r) represent wavefunctions for spin up (+) and down  (-) electrons. A two component fast electron equation of the form given in Eq. 2 is found. The solutions of this equation are studied computationally via a real-space multislice[1] approach. Results are presented for large orbital angular momentum vortex beams passing through model systems, such as bcc Fe, and are compared to results from the traditional Schrödinger equation  based calculations.

Fig. 1: Equations referred to in the text.

Fig. 2: References referred to in the text.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-3331 Atomically Resolved Low-Loss Imaging of Graphene in the Aberration-Corrected STEM

Oxley M. P.1, Kapatenakis M. D.1, Prange M. P.3, Zhou W.2, Idrobo J. C.2, Pennycook S. J.4, Pantelides S. T.1
1Vanderbilt University, Nashville, TN, USA, 2Oak Ridge National Laboratory, Oak Ridge, TN, USA., 3Pacific Northwest National Laboratory, Richland, WA, USA., 4University of Tennessee, Knoxville, TN, USA.
oxleymp@gmail.com

Aberration correction in the scanning transmission electron microscope (STEM) has led not only to improved resolution but also increased contrast and sensitivity at lower accelerating voltages. Imaging of beam sensitive two dimensional materials at atomic resolution has been enabled by operating at energies below the knock on threshold. In this way, single atom impurities have been imaged in BN using annular dark field (ADF) imaging [1] and electron energy-loss spectra (EELS) obtained in graphene with high spatial resolution [2]. Improved spectroscopic sensitivity has even allowed the measurement of energy-loss near-edge spectra (ELNES) providing information about local bonding of single impurity atoms in graphene [3]. We extend recent theoretical developments allowing the simulation of core-shell ELNES as a function of probe position to examine inelastic image formation based on low-loss spectroscopy [4].

In Fig. 1 we show an ADF image and simultaneously acquired low loss spectrum of graphene obtained using ORNL’s Nion UltraSTEM100 operating at 60 kV. Spectrum images derived from the energy ranges of 20-26, 34-40 and 50-56 eV are also shown. The 34-40 eV image shows resolved atomic columns while the other images show no apparent contrast. It should be noted that the intensity variation is of the order of 5% and the image would be considered delocalized by most commonly used definitions. Such low levels of contrast are only visible due to the increased sensitivity of modern instruments.

Preliminary image simulations based on first-principles dynamical electron scattering and density functional theory are shown in Fig. 2. Statistical noise at a level of 5 % has been added to show the effects of experimental noise on such low contrast images. While the simulated lower energy images both show graphene-like contrast, the addition of noise significantly reduces the visibility of the graphene structure at the lowest energy, while the graphene ring structure is still evident at the higher energy. Graphene like contrast is not observed at the higher energy loss. We will discuss the mechanisms underlying image contrast for inelastic STEM imaging based on low-loss spectroscopy.

References

[1] O.L. Krivanek et al., Nature 464, (2010), 571.

[2] W. Zhou et al., Microsc. Microanal. 18, (2012), 1342

[3] W. Zhou et al., Phys. Rev. Lett. 109, (2012), 206803.

[4] M. P. Prange et al., Phys. Rev. Lett. 109, (2012), 246101.

This work was supported by DOE Grant No. DE-FG02-09R46554, by the DOE Office of Basic Energy Sciences, Materials Sciences and Engineering Division, by NSF grant No. DMR-0938330.

Fig. 1: Experimental ADF image and low-loss spectrum of graphene. Images derived from the indicated energy-loss regions of the spectrum are also shown. Scale bars are 1 Å.

Fig. 2: Theoretical spectrum and images derived from the indicated portions of the spectrum. Noise has been added to allow visual comparison with experiment.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-3434 Super-Resolution applied to Magnetite boundaries images

Bárcena G.1, Guerrero M.1, Guerrero E.1, Kepaptsoglou2 D.2, Gilks D.3, Lari L.3, Lazarov V. K.3, Galindo P. L.1
1Department of Computer Science and Engineering, Universidad de Cádiz, 11510 Puerto Real, Spain, 2SuperSTEM Laboratory, STFC Daresbury Campus, Warrington, WA4 4AD, United Kingdom, 3Department of Physics, University of York, Heslington, York, United Kingdom
guillermo.barcena@uca.es

High-Angle Annular Dark-Field (HAADF) imaging is a useful tool to understand the nature of the interaction between different materials domain boundaries, but one must overcome the limitations imposed by the characteristics of the microscope that directly affects resolution. An approach to face this problem is to deal with super-resolution techniques. These techniques attempt to obtain high-resolution images from several observed low-resolution images captured from the same scene, thus the resolution of an image can be improved by bringing out details that might otherwise not be seen.
In this work we illustrate the application of super-resolution techniques to a series of 10 low resolution HAADF images of Magnetite (Fe3O4), oriented along the 001 direction.
Since classical super-resolution reconstruction programs running on a standard computer may take up to 6 hours to get the results, a specialized software suite running in GPUs [1] has been developed to speed up this process, and now results can be obtained just in 10 minutes.

Figure 1 show an experimental low resolution image of Magnetite where the presence of noise is noticeable. Three atoms of Fe have been marked in figure 1 and the corresponding intensity profile is plotted in figure 2, but just two intensity peaks can be appreciated.
In the super-resolution approach two steps are applied: alignment and reconstruction. In this work the alignment process is carried out by filtering the image with a Gaussian filter and then applying the Vandewalle’s modification [4]. Then, a variant of the Non Local Mean algorithm [2,3] is used in order to obtain a high-resolution image, where noise has been substantially reduced, so that the three atoms of Fe can be clearly identified, as shown in figure 3. This fact is made apparent in the corresponding intensity profile shown in figure 4.
These results indicate that super-resolution techniques can provide enhanced HAADF images in terms of resolution, quality and details definition.

[1] Bárcena-González, G. M. Sc. Thesis. Study and application of Superresolution's algorithms in electron microscopy images. October, 2013

[2] Binev, P., Blanco-Silva, et al (2012). High-quality image formation by nonlocal means applied to high-angle annular dark-field scanning transmission electron microscopy (HAADF–STEM). Modeling Nanoscale Imaging in Electron Microscopy, 127-145.

[3] Buades, A., Coll, B., & Morel, J. (2005). A non-local algorithm for image denoising. Computer Vision and Pattern Recognition, 2005. CVPR 2005. IEEE Computer Society Conference on, 2 60-65.

[4] Vandewalle, P. et al (2004). Double resolution from a set of aliased images. Proc. SPIE 5301, Sensors and Camera Systems for Scientific, Industrial, and Digital Photography Applications V, 374

Fig. 1: An experimental low resolution image of Magnetite, three atoms of Fe has been marked with a red circle. The presence of noise is noticeable, in fact, just two atoms can be clearly appreciated.

Fig. 2: Intensity profile corresponding to the marked area in figure 1, Two of the three intensity peaks can be observed.

Fig. 3: High-resolution image obtained by super-resolution techniques, noise has been substantially reduced, so that the three atoms of Fe marked with a red circle can be clearly observed.

Fig. 4: Intensity profile corresponding to the marked area in figure 3. The three atoms of Fe can be clearly identified.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-3481 Chiral-dependent electron vortex energy loss spectroscopy

Yuan J.1, Lloyds S.1, Babiker M.1
1Department of Physics, University of York, Heslington, York, YO10 5DD, United Kingdom.
jun.yuan@york.ac.uk

Chiral electron-vortex beams, carrying a well-defined orbital angular momentum (OAM) about the propagation axis, are potentially useful as probes of magnetic and other chiral materials. In particular, it has been proven that, unlike the optical vortex beams, electron vortex beams can directly induce dipole transitions between states normally assessable only with circular polarised light absorption. This has lead to the expectation that electron vortex beams can be used to acquire chiral dichrotism spectroscopy which will be particular useful for characterization of magnetic materials. Experimentally, the situation is confusing despite initial result [2,3]. To understand this, we present a theory [4] based on an effective operator, expressible in a multi-polar form, describing the inelastic processes in which electron-vortex beams interact with atoms, including those present in Bose-Einstein condensates, involving exchange of OAM. We show clearly that the key properties of the processes are dependent on the dynamical state and location of the atoms involved as well as the vortex-beam characteristics. The later is due to the extrinsic nature of the orbital angular momentum and distinguish chiral electron energy vortex beam energy loss spectroscopy from circular magnetic dichrotic spectroscopy. Our results can be used to identify optimal experimental schemes in which chiral-specific electron-vortex spectroscopy can probe magnetic sublevel transitions normally studied using circularly polarized photon beams with the advantage of atomic-scale spatial resolution. One of the schemes is shown in Fig. 1 where localization of the energy-loss signal is achieved through confocal arrangement and dipole transition selected through orbital-angular-momentum analyzer. [1] S. Lloyds, M. Babiker and J. Yuan, Phys. Rev. Lett. 108 (2012), 074802 [2] J Verbeeck et al, Nature 467 (2010), p. 301. [3] P. Schattschneider et al, Ultramicroscopy, 136 (2014) p81-5 [4] J Yuan, S. Lloyds and M. Babiker, Phys. Rev. A88, 031801

The authors gratefully acknowledge funding from the UK EPSRC (Grant No. EP/J022098).

Fig. 1: The experimental arrangement optimized for spatially resolved chiral-dependent electron vortex electron energy loss spectroscopy.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-16-P-5927 Simultaneous Imaging of O Atoms and relative heavier Sm atoms in Iron-based Superconductor SmFeAsO0.85F0.15 by HRTEM

Wang Y M Ge B H Che G C
Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of sciences, Beijing, China
wangym@iphy.ac.cn

High-resolution imaging of light elements using electron microscopy has always been a challenge. Image resolution is mainly limited by lens aberrations, especially the spherical aberration of the objective lens. Image deconvolution, as a special kind of image processing in High-resolution transmission electron microscopy could remove the image distortion by lens aberrations and transform an arbitrary image intuitively into the structure map, the resolution of which is limited by the information limit of electron microscope. Electron diffraction, which is not restricted by lens aberrations could overcome the resolution limitation. Here we show a combination of electron diffraction and image deconvolution to study the crystal structure of iron-based superconductor SmFeAsO1.85F0.15. Usually it is unexpected to distinguish O atoms in HRTEM images taken with a conventional microscope, especially those adjacent to relative heavier atoms at a very close distance. For the iron-based superconductor SmFeAsO1.85F0.15, every atom, not only heavy atoms Fe and As with atomic space 1.36 Å but also light O and relative heavier Sm atoms with 1.17 Å can be resolved individually in the final image (Fig.3) by using the image deconvolution combined with the corrected electron diffraction data. This is for the first time to image the oxygen atoms which is adjacent to the heavier atoms with the distance of 1.17 Å using the conventional electron microscope by this approach. It allows us not only to determine the crystal structure at atomic level but also to simultaneously reveal light and heavy atoms with a relatively big difference in atomic number and a much smaller atomic distance than the microscope resolution for related compounds.

[1] F.H. Li, Phys. Status Solidi A 207 (2010) 2639-2665.

[2]H.F. Fan, Z.Y. Zhong, C.D. Zheng, F.H. Li, Acta Cryst. A41 (1985)163-165.

 

This project is supported by the National Natural Science Foundation of China (Grant No. 51102275) and by the National Basic Research Program of China (973 Program, No. 2011CBA00101).

Fig. 1:  Fourier filtering image

Fig. 2: Deconvoluted image  at defocus value -600 Å. Rectangles indicate the projections of unit cells.

Fig. 3: Projected potential map obtained from Fig. 2 after phase extension in combination with the diffraction intensity correction.
↑ Go to the Table of content ↑

IT-17. Atom probe and non-traditional microanalytical tasks

↑ Go to the Table of content ↑
Type of presentation: Invited

IT-17-IN-3294 Correlative microscopy applied to atom probe specimen preparation – application to selected metallurgical problems

DANOIX F.1, GOUNE M.4, CUVILLY F.1, CAZOTTES S.2, ALLAIN S.3, ZAPOLSKY H.1
1Université de Rouen, GPM, Avenue de l’université, 76801 St Etienne du Rouvray - France, 2INSA de Lyon, MATEIS Bât. B. Pascal, 7 Avenue Jean Capelle 69621 Villeurbanne Cedex - France, 3Matériaux-Métallurgie-Nanosciences-Plasmas-Surfaces, IJL (Institut Jean Lamour) Parc de Saurupt 54000 NANCY - France, 4Université de Bordeaux, Institut de Chimie de la Matière Condensée de Bordeaux, UPR CNRS 9048, 87 Avenue du Docteur Schweitzer, 33608 PESSAC cedex - France
frederic.danoix@univ-rouen.fr

For more than 10 years, site specific specimen preparation for atom probe tomography using FIB and lift out techniques has drastically widened the possible applications of the technique. In particular, it made it possible the preparation of semiconductor or insulator specimen that laser assisted instrument can now analyze almost routinely. FIBs are systemically implanted in SEM chambers, where other imaging devices are also present. In particular, EBSD is now a common equipment in many labs. It provides complementary crystallographic information to SEM surface micrographs, which can be used in different ways. However, very little has been done using these crystallographic information provided by EBSD for APT specimen preparation. In this presentation, we show the various advantages of combining lift out and EBSD techniques, illustrated by different applications from localization of low volume fraction phases (metallographic aspect of EBSD) to ageing under external load and internal interfaces selection, where the orientation of each specific grain is an important parameter. In addition, the preparation of specimens with specific crystal orientation is a new approach to study and control specific experimental artefacts, such as chromatic aberrations, local magnification effect and surface diffusion.
Selected examples in various material science fields will illustrate theses experimental aspects, from carbon redistribution between phases in modern steels, the effect of the crystal orientation on the phase separation under uniaxial load in FeCr model alloys, and the chemical segregation at internal interfaces and defects.

Fig. 1: Influence of the crystal orientation on the internal nanostructure in a FeCr model alloy aged at 500°C
↑ Go to the Table of content ↑
Type of presentation: Invited

IT-17-IN-6090 Atomic scale studies of nanostructured and nanoscale materials with atom probe tomography

Cairney J. M.1, Felfer P. J.1, Scherrer B.1,2
1Australian Centre for Microscopy & Microanalysis, The University of Sydney, Sydney, NSW 2006 Australia, 2Nanometallurgy, ETH Zurich, 8093 Zurich, Switzerland
julie.cairney@sydney.edu.au

Atom probe tomography is a powerful microscopy technique that provides atomic-resolution 3D maps that show the precise location of the atoms within a volume of material [1]. It has seen widespread use for the characterisation of bulk metals and alloys, but new developments in specimen preparation and the use of lasers have now made it applicable to the study of a much wider range of material types. This presentation will provide an overview of a range of different studies involving ‘non-traditional’ functional and nanoscale materials. This includes bulk Pt/ZrO2-multilayers, which are model systems for applications like the newest generation of micro solid oxide fuel cells and sensors metallic glass nanowires. In these samples, which consist of nanocrystalline layers 10-40 nm thick, we are interested in the solubility and diffusion of oxygen in the noble metal layers. However, the study of such layers in atoms probe is subject to limitations due to the large difference in the field evaporation behaviour between the metal and the oxide. We will discuss how these issues have been addressed in our study, and the observed location of the oxygen atoms. We will also show recent results from the study of bimetallic Au@Ag core-shell nanoparticles. The catalytic performance of these particles greatly influenced by the distribution of the atoms of each element within the particle and on the particle’s surface. However, almost no quantitative, experimental data is currently available on the precise location of the individual atoms within particles less than 100 nm in size. We will demonstrate atom probe can be used to quantitatively determine the distribution of the individual chemical elements in 3D both inside and on the surface of nanoparticles extracted directly from a suspension. Other examples will include ion-irradiated bulk metallic glass nanowires, in which the distribution of embedded Ga ions is being investigated in order to understand their influence on the plasticity of the nanowires. In each case we will discuss how challenges around the specimen preparation have been overcome, any artifacts that are expected to arise in the data (and how these have been addressed), and the new analysis methods applied. References [1] B. Gault, S.P. Ringer, M.P. Moody, J.M. Cairney, Atom Probe Microscopy, Springer, 2012.

The authors acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Australian Centre of Microscopy & Microanalysis

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-17-O-2516 Glass analysis with Atom Probe Tomography utilizing a rapid nanotip preparation technique

Bell D. C.1, Magyar A. P.2, Graham A. C.2
1Harvard University, School of Enginnering and Applied Sciences, Cambridge MA USA, 2Harvard University, Center for Nanoscale Systems, Cambridge MA USA
dcb@seas.harvard.edu

The analysis of glass samples with high resolution electron microscopy is challenging, because these techniques are not ideal for imaging amorphous samples. Visualizing the nanoscale elemental distribution and aggregation within glasses can lead to better modeling and understanding of their thermal, electrical, and mechanical properties. Advances in glass technology have lead to “gorilla glass”, the practically indestructible material found in the screens of many mobile devices. Understanding the nanoscale properties of materials like “gorilla glass” can lead to the development of new even stronger materials.

In the local electrode atom probe (3D LEAP) traditionally amorphous glasses have been difficult to run, with a particularly high failure rate. Until now the predominant approach for the preparation of such samples has been FIB liftout from a bulk specimen, which is time consuming and costly. We have developed technique for drawing glass rods or capillaries into sub 100 nm atom probe tips (Fig.1). This rapid and low expense technique means that even though the possible failure rate in the atom probe may be high, a significant amount of time is not wasted with sample preparation.

We found that after coating the glass nanotips with metal we were able to use voltage mode on the atom probe to successfully characterize a glass specimen (Fig . 2) which shows a very well defined Boron concentration surface . Further development of this technique could lead to new understandings of the structure property relationships in glasses and provide new pathways for studying materials doped or encapsulated within glasses.

The authors greatfully Acknowledge the support of the National Science Foundation through  NSF MRI:1040243

D.C. Bell. gratefully acknowledges funding through the National Science Foundation award  (NSF DMR-1108382)

Fig. 1: SEM Image of the rapid preparation Glass APT nanotip, showing diameter less than 100nm  (Inset) Glass nanotip mounted  in the analysis chamber

Fig. 2: APT Reconstruction of Glass nanotip showing an Isosurface of Boron concentration in the glass sample
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-17-O-3072 An atom probe insight on corrosion of stainless steels

La Fontaine A. J.1, 2, Cairney J.1, 2
1School of Aerospace, Mechanical, Mechatronic Engineering, The University of Sydney, NSW 2006, Australia, 2Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006, Australia
alex.lafontaine@sydney.edu.au

For high temperature applications, such as in new-generation energy technologies, high-performance stainless steels offer an attractive combination of economy and mechanical / corrosion properties. For example, concentrated solar power (CSP) requires cost-effective and corrosion resistant materials that can operate for extended periods at high temperatures and withstand thermal cycling between 900°C and room temperature.

Stainless steels develop a passive layer protecting the steel from detrimental corrosion. When used in extreme conditions at high temperature in ultra-corrosive atmosphere, with thermal cycling or high pressure, this passive layer can be destroyed and leave the steel exposed to corrosion resulting in material failure.

The analysis of the chemical composition and microstructure of oxide layers using traditional analytical spectroscopy techniques has some limitation due to either limited lateral resolution or mass resolution. Atom probe tomography (APT), however, is a unique 3D technique allowing for atomic scale chemical characterization.

In the last decade, local electrode atom probe (LEAP) was applied to study oxide layers. For example, surface oxide layers in stainless steels or nickel-based alloys [1-3] were characterized by using laser-pulsed LEAP. The current work demonstrates our efforts in studying by laser-pulsed LEAP the complexity of oxides layers in intergranular corrosion cracks formed in a commercial austenitic stainless steel during thermal cycling.

References

[1] Lozano-Perez S, Saxey DW, Yamada T, Terachi T. Scripta Materialia 2010;62:855.
[2] Kruska K, Lozano-Perez S, Saxey DW, Terachi T, Yamada T, Smith GDW. Corrosion Science 2012;63:225.
[3] Baik S-I, Yin X, Seidman DN. Scripta Materialia 2013;68:909.

This work was made possible by an ARENA PhD scholarship. The authors acknowledge scientific and technical input and support from the Australian Microscopy & Microanalysis Research Facility (AMMRF) at The University of Sydney as well as CSIRO Energy Centre.

Fig. 1: (a) Photo of the concentrated solar power (CSP) plant used to heat treat the samples (b) Z-contrast back-scattered electron image of cross section of the sample (c) EBSD pattern quality map of IGC area with b.c.c. phase highlited in red, EDS O-K map - EDS Cr-K maps, atom probe volume of grain boundaries in chromite (Cr,O and Fe)

↑ Go to the Table of content ↑
Type of presentation: Oral

IT-17-O-3061 3D Field Ion Microscopy for Characterization of Atomic Scale Radiation Damage in Fusion Related Materials

Dagan M.1, Roberts S. G.1, Gault B.1, Bagot P.1, Moody M. P.1
1Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH, United Kingdom
michal.dagan@materials.ox.ac.uk

Prospective materials for plasma facing components of fusion reactors must withstand conditions of high temperature and radiation dose. Tungsten is a leading candidate for this role. Hence, an extensive atomic-scale investigation of tungsten behavior under extreme conditions is critical. However the detection of nanoscale damage to the material’s atomic lattice is particularly challenging even for advanced microscopy techniques. The formation of dislocations, nano-voids, self-interstitials and clustering effects requires the ability to directly view the arrangements of individual atoms on the crystal lattice.
Here we present a technique for the atomic scale study of tungsten utilizing field ion microscopy (FIM). Conventionally, FIM is a 2D imaging technique: only the surface of the needle-shaped specimen is imaged by evaporating image-gas atoms from atomic sites on the surface. However, by increasing the voltage applied on the tip during imaging, it is possible to field evaporate constituent surface atoms from the specimen, in a similar manner to atom probe tomography (APT). The result is a series of highly resolved 2D FIM images that can be tomographically stacked to retrieve a 3D reconstruction of the sample. FIM-based 3D reconstruction has the potential to significantly exceed the resolution of APT analysis as it is not subject to the same detection efficiency limitations.
In this work a new 3D atom-by-atom reconstruction procedure for FIM is described. A significant challenge is automating the identification of atoms within an image. Furthermore, every atom can potentially appear across hundreds of FIM images before it is evaporated. Hence, the progress of each atom must also be automatically tracked throughout the sequence. An automated identification approach has been developed based on clustering together high intensity pixels within each image and across the sequence. The results of this clustering algorithm, applied to identification of atoms within two consecutive (244) planes in a tungsten sample are presented in Figure 1. All atoms are successfully identified and represented by different colors. A smearing effect in the direction along the depth of the sample is the result of different evaporation rates of individual atoms.
In complementary work to study radiation damage, tungsten samples have been ion-irradiated. Figure 2 demonstrates the increased spatial resolution offered by FIM in comparison to APT. A radiation induced dislocation is clearly imaged using FIM while the APT reconstruction of tungsten atoms does not directly indicate its presence. Interestingly, carbon impurities segregate to this region and indirectly reveal the dislocation in the APT data. However, to image single vacancies only FIM has the necessary atomic resolution.

Fig. 1: a: 150 stacked FIM images of the evaporation of two (244) planes in a tungsten sample. The colors represent intensity ranges of the pixels. b: Atoms are identified automatically by clustering together high intensity pixels. Each atom appears in a different color and is smeared in proportion to its evaporation rate.

Fig. 2: a-c: FIM and APT of irradiated tungsten. a: FIM image of a dislocation. b(c): APT analysis of tungsten(carbon) atoms. Tungsten reconstruction shows no sign of dislocations however carbon segregation reveals their presence. d-f:lower dose irradiated tungsten. d:FIM of vacancies. e(f): tungsten(carbon) APT reconstructions, no indication of vacancies.
↑ Go to the Table of content ↑
Type of presentation: Oral

IT-17-O-3384 Nucleation and lateral growth of NiSi phase

El Kousseifi M.1, Hoummada K.1, Epicier T.2, Panciera F.1, Mangelinck D.1
1Aix-Marseille Université, CNRS, IM2NP, Case 142, 13397 Marseille Cedex 20, France, 2Université de Lyon, MATEIS, umr 5510, Bât. B. Pascal, INSA de Lyon, F-69621
mike.elkousseifi@im2np.fr

The Ni-based self-aligned silicide is widely used as contacts and interconnections in very large-scale integrated circuits [1]. They are obtained by solid state reaction between Ni thin film and Si substrate. Therefore, the fundamental mechanisms related to their formation, including the first stages of the nucleation, phase formation sequence, the growth kinetics, and the microstructures of the silicide, are of great interest for applications. NiSi is the desired phase in the Ni silicide sequence as the contact material in advanced integrated circuits [2]. However, a major disadvantage of NiSi is its degradation at high temperature. The addition of percent-wise Pt to Ni film increases significantly the stability of NiSi on Si substrates [3].
In this work, in situ-XRD annealing followed by atom probe tomography (APT) and in-situ transmission electron microscopy (TEM) analysis were used to study the reaction between 10 nm Ni (10% Pt) alloy film and Si(100) substrate. Isothermal annealing in in-situ XRD at different temperatures (200°C, 215°C and 230°C) have shown a nucleation-controlled behavior for NiSi growth at the epitaxial θ-Ni2Si/Si interface in contrast to the diffusion-controlled growth usually reported for NiSi [4]. TEM measurements have provided information about the NiSi nuclei shape and their distribution in the sample (Figure 1(a)) and additional information about the growth kinetics, while APT analyses were used to determine the 3D distribution of Ni, Si, and Pt atoms (Figure 1(b)). The Pt distribution was obtained in two cases: (1) in the θ-Ni2Si phase without the presence of NiSi nuclei and (2) inside the NiSi nuclei. A model for nucleation and lateral growth of NiSi at θ-Ni2Si/Si interface is proposed.

1. R. W. Mann, et al. IBM J. Res.Dev. 39, 403 (1995).
2. R. Mukai, et al., Thin Solid Films 270, 567 (1995).
3. D. Mangelinck, et al. Appl. Phys. Lett. 75 1736 (1999).
4. F. d’Heurle, et al. J. Appl. Phys. 55 4208(1984).

Thanks are due to the french METSA (www.metsa.fr) network for access to TEM at the CLYM platform (www.clym.fr), and to B. Van De Moortele (LGL, ENS-Lyon) for his assistance in sample preparation

Fig. 1: Figure 1: a) TEM image showing the NiSi nuclei inside the θ-Ni2Si phase. The cylinder represents approximately the region of APT analysis. b) APT concentration profile across θ-Ni2Si and NiSi phase.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-17-P-2954 Atom Probe Workbench for Materials Science & Engineering

Ceguerra A. V.1, Stephenson L. T.1, Apperley M.2, Goscinski W. J.3, Ringer S. P.1
1ACMM, and School of AMME, The University of Sydney, NSW Australia, 2AMMRF, The University of Sydney, NSW Australia, 3Monash eResearch Centre, Monash University, VIC Australia
anna.ceguerra@sydney.edu.au

The Atom Probe Workbench (APW)[1] is a tool for analysing Atom Probe Microscopy data[2] for materials science. Developed under the National eResearch Collaboration Tools and Resources (NeCTAR)[3] Characterisation Virtual Laboratory (CVL)[4] project, the APW collates existing atom probe tools[5] within an Australian NeCTAR cloud infrastructure. APW is deployed on top of the CVL fabric, a software infrastructure common to all CVL application drivers. Features of the APW include those that are inherent to open source programs utilised as part of the workbench, which are Galaxy workflow engine[6] and MyTardis[7]. Additional developed features of the APW include usage tracking & reports, citation reporting[8]. Initial reports from users during development show the potential of APW to be used by researchers around Australia and worldwide.

1 A. Ceguerra, P. Liddicoat, M. Apperley, W. Goscinski, and S. Ringer, Atom Probe Workbench version 1.0.0: An Australian cloud-based platform for the computational analysis of data from an Atom Probe Microscope (APM), used for chemical and 3D structural materials characterisation at the atomic scale. (2014) 10.4227/11/53014684A67AC.

2 B. Gault, M.P. Moody, J.M. Cairney, and S.P. Ringer, Atom probe microscopy (Springer, 2012) 9781461434351.

3 NeCTAR, National eResearch Collaboration Tools and Resources, http://www.nectar.org.au (18 March 2014).

4 CVL, NeCTAR Characterisation Virtual Laboratory, http://www.massive.org.au/cvl (18 March 2014).

5 A.V. Ceguerra, A.J. Breen, L.T. Stephenson, P.J. Felfer, V.J. Araullo-Peters, P.V. Liddicoat, X. Cui, L. Yao, D. Haley, M.P. Moody, B. Gault, J.M. Cairney, and S.P. Ringer, The rise of computational techniques in atom probe microscopy, Current Opinion in Solid State and Materials Science 17, 224 (2013) 10.1016/j.cossms.2013.09.006.

6 D. Blankenberg, G. Kuster, N. Coraor, G. Ananda, R. Lazarus, M. Mangan, A. Nekrutenko, and J. Taylor, Galaxy: A web-based genome analysis tool for experimentalists, Current Protocols in Molecular Biology Chapter 19 (2001) 10.1002/0471142727.mb1910s89.

7 S. Androulakis, J. Schmidberger, M.A. Bate, R. DeGori, A. Beitz, C. Keong, B. Cameron, S. McGowan, C.J. Porter, A. Harrison, J. Hunter, J.L. Martin, B. Kobe, R.C.J. Dobson, M.W. Parker, J.C. Whisstock, J. Gray, A. Treloar, D. Groenewegen, N. Dickson, and A.M. Buckle, Federated repositories of X-ray diffraction images., Acta crystallographica. Section D, Biological crystallography D64, 810 (2008) 10.1107/S0907444908015540.

8 A.V. Ceguerra, P.V. Liddicoat, W.J. Goscinski, S. Androulakis, and S.P. Ringer, A Tool for Scientific Provenance of Data and Software (2013) Computer and Information Technology Conference, Sydney, Australia 10.1109/CSE.2013.89.

We acknowledge the support of NeCTAR, AMMRF, The University of Sydney, Monash University, Intersect Australia.

For the full text of the acknowledgements, please see: http://dx.doi.org/10.4227/11/53014684A67AC

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-17-P-2966 Crystallographic calibration of Si-based atom probe reconstructions for enhanced short-range ordering information.

Breen A. J.1, 2, Ceguerra A. V.1, 2, Araullo-Peters V. J.1, 2, Moody M. P.3, Ringer S. P.1, 2
1Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006, Australia, 2School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, NSW 2006, Australia, 3Department of materials, The University of Oxford, Parks Road, OX13PH, Oxford, UK
andrew.breen@sydney.edu.au

Atom probe tomography (APT) enables the position and chemical identity of millions of atoms to be reconstructed in 3D with sub-nm precision. However, the spatial resolution is still generally not high enough to unequivocally determine the lattice positioning of atoms in crystalline materials. This presents a significant roadblock in understanding structure-property relationships at the atomic level. An important example is the need to more accurately characterise the dopant positioning within the ultra-shallow junctions of the latest generation of transistor devices that are now usually only several nm deep. Subtle differences in dopant positioning can have significant influence on device performance and this must be controlled more accurately if continual size reductions are to occur.  

In this study, we have developed methods to study the short-range ordering of dopants within silicon in unprecedented detail using APT. Latent crystallographic structure can be detected within the reconstructions with the help of newly developed crystallographic mapping tools. After doing this, it is apparent that the detected crystal structure is slightly different to the theoretical structure in size and shape, even after careful calibration using the method described by Gault et al. (Gault, et al., 2009). This is most likely due to assumptions used in the initial reconstruction algorithm. Further crystallographic calibration, using linear transformations of the reconstructed atom co-ordinates, is then used to perfectly restore the latent crystal structure. However, we have found that the spatial noise present is still high enough to be detrimental to the measured short-range ordering. Lattice rectification approaches (Moody, et al., 2011), where atoms are repositioned to the closest lattice site, are then used to restore this short-range order and the results are compared and discussed. This work provides a significant contribution to lattice based atom probe studies of doped silicon.

Gault, B., Moody, M.P., de Geuser, F., Tsafnat, G., La Fontaine, A., Stephenson, L.T., Haley, D. & Ringer, S.P. (2009). Advances in the calibration of atom probe tomographic reconstruction. Journal of Applied Physics 105(3).

Moody, M.P., Gault, B., Stephenson, L.T., Marceau, R.K.W., Powles, R.C., Ceguerra, A.V., Breen, A.J. & Ringer, S.P. (2011). Lattice Rectification in Atom Probe Tomography: Toward True Three-Dimensional Atomic Microscopy. Microscopy and Microanalysis 17(2), 226-239.

The authors acknowledge scientific and technical input from the AMMRF node at The University of Sydney, particularly Baptiste Gault, Leigh Stephensen, Takanori Sato and Julie Cairney. The authors are also grateful to the ARC for providing funding. We also thank the ANFF at the University of NSW, particularly Joanna Szymanska, for Bosch processing of Si wafers. 

Fig. 1: The latent crystallographic information within a Sb doped Si reconstruction. (a) The tomographic atom probe reconstruction. A thin red slice has been cropped out for crystallographic analysis. (b) 2D density map of Si. (C) 1D spatial distribution maps (SDM) of detected planes within the reconstruction (d) 2D SDM of {001} planes.

↑ Go to the Table of content ↑
Type of presentation: Poster

IT-17-P-3236 Improving Yield and Data Quality in Atom Probe Tomography

Larson D. J.1, Ulfig R. M.1, Prosa T. J.1, Lawrence D. F.1, Martin I. Y.1, Giddings A. D.1, Olson D. P.1, Kelly T. F.1
1CAMECA Instruments Inc., 5500 Nobel Drive, Madison, WI 53711 USA
rulfig@hotmail.com

The electric fields used in atom probe tomography (APT) generate stresses normal to the specimen axis proportional to the electric field squared [1]. As such, specimen fracture has long been a serious problem for APT [2-3]. There are many methods to improve yield, often with a trade-off required in experimental quality or convenience in some other area. Methods to improve yield include: 1) decrease ion detection rate, 2) increase specimen temperature, 3) use laser pulsing, 4) increase laser pulse energy, 5) increase background chamber pressure, 6) change feature orientation [4], 7) coat a sharpened specimen with a thin layer of material [3,6], and 8) slightly anneal the specimen. Figure 1 schematically illustrates several of these methods to improve APT specimen yield. The current work explores the use of thin coatings to modify the thermal and/or optical properties of 302 stainless steel and a Si/SiO2(10nm)/Si/Ni test structure in attempts to improve yield.

Although method number 3+4 above works well to improve yield, substantial increases in laser energy are often limited in materials with poor thermal diffusivity due to a degradation in data quality (e.g., long “thermal tails” in the mass spectrum, non-uniform detector hit-maps, etc.) [7,8]. An example of this effect is shown in the non-uniform detector hitmaps in a poor thermal diffusivity material (302 stainless steel). Figure 2 shows the effects of increasing laser pulse energy and suggests an increasing trend in azimuthal asymmetry (note laser incidence is lower left). Figure 3 presents the mass resolving power (MRP) at tenth maximum, uniformity in the form of XY hitmap signal correlation (lower values are desirable) [6], and spectral background as a function of laser energy.

In order to investigate the yield improvement of coatings, we have developed a test structure of uncoated Si/SiO2(10nm)/Si/Ni on which we can reach data collection conditions where this sample both does and does not yield. These data have been reproduced by successful data collection from the entire structure multiple times. Statistically, yield changes are difficult to prove unequivocally; however we have good evidence for an improvement for this oxide-based structure with thin metal coatings in Figure 4.

1. D. G. Brandon in, Field Ion Microscopy, eds. J. Hren, S. Ranganathan (Plenum, 1968) p.64.
2. T. J. Wilkes et al., J. Phys. D: Appl Phys. 5 (1972) p.2226.
3. S. Kölling and W. Vandervorst, Ultramicroscopy 109 (2009) p.486.
4. D. Lawrence et al., Microsc. Microanal. 14(S2) (2008) p.1004.
5. G. L. Kellogg, J. Appl. Phys. 53(9) (1982) p.6383.
6. D. J. Larson et al., Microsc. Microanal. 20(S2) (2014) in press.
7. J. H. Bunton et al., Microsc. Microanal. 13 (2007) p.418.
8. B. Gault et al., Ultramicroscopy 110 (2010) p.1215.

The authors would like to thank the team at CAMECA Instruments Inc. for continued software and hardware improvements to the LEAP®.

Fig. 1: 1)Methods to improve APT yield. 2)Effects of increasing laser pulse energy suggesting a trend in azimuthal asymmetry laser incidence is LL). 3)Mass resolving power at tenth maximum, uniformity in the form of XY hitmap signal correlation (lower=better). 4)Yield differences on a difficult to run specimen as a funciton of detection rate and coating.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-17-P-3469 The effects of laser wavelength and pulse energy on the measured oxide compositions by atom-probe tomography

Kruska K.1,2, Lozano-Perez S.1, Schreiber D. K.3
1Department of Materials, University of Oxford, Parks Road, Oxford, OX1 3PH, United Kingdom, 2Fundamental and Computational Science Directorate, Pacific Northwest National Laboratory, Richland, WA, United States, 3Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, United States
karen.kruska@pnnl.gov

Atom-probe tomography (APT) has been shown to be a useful tool to study environmental degradation, and in particular oxidation and corrosion, of steels and Ni-base alloys frequently used in the high temperature corrosive environments. In these corroded samples, APT is uniquely able to capture highly localized changes in composition with both high chemical sensitivity and near-atomic spatial resolution in 3D. In principle, it is possible to correlate the local O concentration with specific oxide phases. In practice, however, the measured O concentration is erroneously O deficient. This “loss” of oxygen atoms in relevant oxides is neither well documented nor understood.

In this study bulk Fe and Ni oxides – NiO, FeO and NiFe2O4 – have been systematically analyzed in two complementary APT systems with a green (λ=532 nm) and a UV (λ=355 nm) laser, respectively, to better understand the measured O deficiency. These oxides were selected primarily for their relevance to corroded microstructures. The laser pulse energies were varied and repeated in increasing and decreasing series to eliminate additional effects of increasing tip radius during data collection. The measured composition at a given pulse energy was found to be consistent, repeatable, and independent of tip radius. The consistency of measured compositions was also remarkable between multiple tips.

In all cases the measured O concentration increased with decreasing pulse energies, which is consistent with previous studies on other oxide and nitride materials. Figures 1a and b show this with both the green and the UV wavelength lasers. The ratio of O:O2 ions within the measured O was also studied (see Figure 1b and 2b),and was found to increase with increasing O concentration, suggesting that more molecular O are lost at higher laser energies, likely through sublimation of neutral molecular O2.

Figure 2 shows data from the spinel oxide, NiFe2O4, acquired with the UV laser. Although the O content increased and the overall metal content decreased with decreasing laser pulse energy, irregularities were observed in the Fe:Ni ratio (see Figure 2c). While it remained constant at high laser pulse energies it starts to drop at a laser pulse energy of 1 pJ. It appears that Fe cations are lost at low laser pulse energies in this case.

These results show that the evaporation behavior in the studied oxides is strongly cation dependant. It appears that the O:O2 ratio cannot be used as an indicator for the accuracy of the composition. A more promising indicator may be the cation ratio, especially in mixed spinel oxides.

A portion of the research was performed using EMSL, a national scientific user facility sponsored by the Department of Energy's (DOE) Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. DKS was funded by the US DOE Office of Basic Energy Science.

Fig. 1: a) O concentration evolution depending on the laser power in NiO and FeO for both green and UV laser systems. b) O concentration in dependence of O:O2 ratio in both green and blue laser systems.

Fig. 2: a) O concentration evolution depending on the laser power in NiFe2O4 for UV laser system. b) O concentration in dependence of O:O2 ratio UV laser system. c) Cation ratio normalised to the nominal Fe and Ni contents in NiFe2O4 in dependence of laser pulse energy.
↑ Go to the Table of content ↑
Type of presentation: Poster

IT-17-P-5928 Quantitative evaluation of C, Mn and Si in martensite steels by atom probe tomography, electron microscopy and X-ray diffraction

M. Kozuka 1 S. Otani 1 Y. Aruga 1
Materials Research Laboratory, Kobe Steel, Ltd., 1-5-5 Takatsukadai, Nishi-ku, Kobe, Japan 1
kozuka.masaya@kobelco.com

Martensite steels have been widely used due to high strength, which strongly depends on the microstructural factors such as solute carbon content in martensite phase. Due to the high spatial resolution and capability for analyses of light elements, atom probe tomography (APT) is one of the promising methods to quantitatively evaluate the solute carbon content of martensite phases. However, it is well known that the solute carbon content evaluated by APT apparently depends on the measurement parameters such as specimen temperature and peak identification of the mass spectrum [1]. Although Miyamoto et al. recently proposed that the dependence of apparent solute carbon content in Fe-C binary alloys on the specimen temperature is due to the detection loss of iron ions [2], the quantitative evaluation of the solute carbon content still has remained issues. For example, micro-alloying elements will affect the evaporation behavior and may change the APT analysis results. In this study, we conducted APT measurements of Fe-C, Fe-C-Mn and Fe-C-Si martensite steels with varied specimen temperature and pulse fraction. The effects of a micro-alloying element on evaluation of the solute carbon content were investigated in terms of measurement parameter and interpretation of mass spectrum. The steels were austenitized at 1203 K for 360 s followed by water quenching. Fig. 1 is a secondary electron image of the as-quenched Fe-1.0C-1.5Si steel etched by picral, showing that the alloy has martensitic structure and no mm-sized carbon segregation. Fig. 2 shows APT maps of carbon and silicon in the same steel. Although nm-sized segregation is confirmed in the carbon atom map (Fig. 2 (a)), the alloy seems to be suitable for the quantitativity check of the APT measurement because the carbon segregation looks uniform in the steel. In the presentation, quantitative evaluation results of electron probe microanalyser and X-ray diffraction are also shown to discuss their complementary use.

[1] J. Takahashi et al., Ultramicroscopy 111 (2011) 1233-1238.

[2] G. Miyamoto et al., Scripta 67 (2012) 999-1002.

We would like to thank Dr. T. Murakami (Kobe Steel, Ltd) for beneficial discussion.

Fig. 1: A secondary electron image of the Fe-1.0C-1.5Si steel.

Fig. 2: APT elemental maps of (a) C and (b) Si in the Fe-1.0C-1.5Si steel.
↑ Go to the Table of content ↑

MS-1. Nanoobjects and engineered nanostructures, catalytic materials

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-1-IN-1523 Atomic Ordering and Phase Separation in Magnetic Alloy Nanoparticles

Sato K.1, Konno T. J.1
1Institute for Materials Research, Tohoku University, Sendai, Japan
ksato@imr.tohoku.ac.jp

Recent demands for ultrahigh density magnetic storage technology require the development of novel recording media with higher magnetocrystalline anisotropy energy (MAE), with the aim of increasing storage density and reducing a recording noise. For such a purpose, L10-type CoPt ordered alloy nanoparticles are one of the candidate materials: the hard magnetic properties of this alloy can be attributed to the tetragonal ordered structure with a high MAE. Therefore, the atomic ordering and the stability of the ordered phase are the key issues for the magnetic properties. We hence intend to examine kinetics of ordering in CoPt nanoparticles. On the other hand, structure and properties of alloy nanoparticles can be tuned as well by taking advantage of phase separation. For this purpose, we focused on immiscible Co-Au system. Bimetallic nanoparticles were fabricated by sequential electron-beam depositions of Pt (or Au), Co and Al2O3 onto NaCl(001) substrates kept at 520-653 K. For CoPt nanoparticles, we carried out post-deposition annealing for promoting atomic ordering with different cooling rates. The structure and morphology of the nanoparticles were characterized using TITAN80-300, JEM-3011, and ARM200F (S)TEM. Figures 1 (a)-(c) and (a’)-(c’) show CS-corrected HRTEM images of the CoPt nanoparticles with sizes of ~5 nm and ~3 nm in diameter, respectively. The annealing conditions are as follows: (a, a’) 873 K-1h, slow cooling (1.5 K/min), (b, b’) 873 K-1h, rapid cooling (110 K/min), and (c, c’) 973 K-1h, rapid cooling. For 5 nm-sized particles (left panel), clear (110) atomic planes of the L10-ordered structure can be seen. In contrast, size dependence of the atomic ordering was found in the specimens followed by rapid cooling: the disordered phase was observed in nanoparticles smaller than 3 nm in diameter. For example, a nanoparticle shown in Fig. 1(b’) is the disordered phase, characterized by crossed {200} atomic planes. The population of the disordered particles was found to be 14% (Fig.1(e)). Thus, this study offers a kinetic explanation for size-dependent atomic ordering, in addition to the thermodynamic explanation of a reduced stability for the ordered phase in nanoparticles [1]. Figure 2 (a) shows an SAED pattern of Co-Au nanoparticles. The cube-on-cube orientation epitaxy is seen between Au and fcc-Co. An example of a HAADF-STEM image of a Co-Au particle is shown in Fig. 2(b). Formation of core-shell structure (Au-core) is clearly seen due to the large atomic number difference. As the particle size reduces (<8 nm), nanoparticles with Au-shell are also formed as shown in Fig. 2(c). It is expected that smaller sized Au-shell particles have a potential to nanocatalyst applications. [1] K. Sato et al. Philos. Mag. Lett. 92 (2012) 408.

This study was partially supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

Fig. 1: CS-corrected HRTEM images and the corresponding FFT patterns of the CoPt nanoparticles (average alloy composition: Co-61at%Pt): (a-c) ~5 nm in diameter, (a’-c’) ~3 nm in diameter. (d) a structure model (truncated octahedron) and a simulated image, (e) particle size range, where the disordered nanoparticles are observed, is marked in the histogram.

Fig. 2: (a) SAED pattern of Co-Au nanoparticles (average alloy composition: Co-46at%Au). (b, c) Z-contrast images by HAADF-STEM for Co-Au nanoparticles with core-shell contrasts: (b) a Au-core particle (D~13 nm), (c) a Au-shell particle (D~7 nm). It is presumed that the lower surface tension of Au than that of Co is responsible for the Au-shell formation.
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-1-IN-5776 Morphology and inner structure of anisotropic nanoparticles studied by advanced TEM and FIB techniques

Spiecker E.1, Butz B.1, Winter B.1, Niekiel F.1, Vieweg B.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Universität Erlangen-Nürnberg, Erlangen, German
Erdmann.Spiecker@ww.uni-erlangen.de

Extensive research in nanoscience is currently devoted to the synthesis and characterization of complex and anisotropic nanoparticles (NP), with particular focus on low-symmetry NP, patchy structures as well as hybrid particle assemblies. Anisotropy and increased complexity provide additional degrees of freedom for tailoring the physical and chemical properties, making such NP ideal candidates for enhanced chemical, catalytic, and optical applications. In this contribution, microscopic studies of anisotropic and complex NP are presented which illustrate the importance of combining advanced TEM and FIB techniques for comprehensive characterization.

Ge/Si patchy NP formed by gas phase synthesis in a two-stage hot wall reactor [1,2] have been studied by combining HAADF-STEM, STEM-EELS and HRTEM, as illustrated in Fig. 1. The microscopic investigations focused on two key questions, namely the accommodation of misfit (~ 4 %) by defect formation and possible interdiffusion, addressed by HRTEM and STEM/EELS, respectively. Another experimental aspect is the oxidation of the particle surface which could be suppressed by transfer via glove box and vacuum transfer holder.

PbSe quantum dots grown on single wall carbon nanotube (SWCNT) bundles for optoelectronic applications [3] have been studied by combining electron tomography and HRTEM at 80 kV. During wet-chemical synthesis the PbSe NP grow around the SWCNT bundles as revealed by the tomogram in Fig. 2a. In combination with HRTEM contributing crystallographic information (Fig. 2b) a 3D atomistic model is derived (Fig. 2c). By comparison of experimental and simulated HRTEM images the 3D model of the PbSe NP can be further refined.

Ag nanorods formed by wet chemical synthesis [4] are used to illustrate a new FIB method [5] which enables site and orientation specific cross-sectioning of anisotropic NP (Fig. 3). By employing a shadow geometry (Fig. 3b) in which a Si substrate protects the NP from the Ga+ ion beam no protection layer has to be deposited on the NP. Inspection of the NP during milling enables precise positioning of the final cross section. In the case of the Ag nanorod, HRTEM of the final cross section nicely reveals the characteristic five-fold twin structure with additional defects indicating partial strain relaxation (Fig. 3c).

[1] Körmer et al., Crystal Growth & Design 12 (2012), 1330.
[2] Mehringer et al., Journal of Aerosol Science 67 (2013), 119.
[3] Schornbaum et al., Chemistry of Materials, 25 (2013), 2663.
[4] Damm et al., Small 7 (2011), 147.
[5] Vieweg et al., Ultramicroscopy, 113 (2012), 165.
[6] http://www.gmp.ww.uni-erlangen.de/nanoSCULPT.php

The authors gratefully acknowledge collaboration with the Institute for Particle Technology (Prof. Peukert) and the Nanomaterials for Optoelectronics Group (Prof. Zaumseil). They thank Prof. Bitzek for providing the software nanoSCULPT [6] used for constructing the atomistic model of the PbSe NP. Financial support by the DFG through the projects EXC315 and GRK1161 is gratefully acknowledged.

Fig. 1: TEM study of Ge/Si patchy NP produced by gas phase synthesis [2]. a) HAADF-STEM image of aggregated NP with Si cores (dark) and Ge islands (bright). b) Concentration profiles across an interface (line in a)) from EELS line scan. c) Map of (111) lattice plane spacing derived from a HRTEM image (not shown) by geometric phase analysis.

Fig. 2: Morphology and atomic structure of PbSe NP grown on SWCNT bundle [3]. a) Tomogram of PbSe NP. b) HRTEM image of the same NP in [011] zone axis. c) Atomistic model obtained by filling the 3D volume with the PbSe structure in correct crystallographic orientation derived from HRTEM. d) HRTEM image simulation based on atomistic model.

Fig. 3: FIB technique for site and orientation specific sectioning of anisotropic NP [5]. a) TEM image of Ag nanorod with desired cross section indicated by black rectangle. b) Shadow-FIB geometry used for cross-sectioning without protection layer. c) HRTEM image of nanorod cross section showing the characteristic five-fold twin structure.
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-1-IN-5815 STEM Investigation of Nanostructured, Ceria-based, Surface Phases: Novel Catalysts with Outstanding Redox Properties

Calvino J. J.1, Sánchez-Gil J. J.1, Tinoco M.1, Arias D. C.1, Yeste M. P.1, Muñoz M. A.1, Hungría A. B.1, Hernández-Garrido J. C.1, Pérez-Omil J. A.1, Cauqui M. A.1, Blanco G.1, Trasobares S.1, Bayle-Guillemaud P.2, López-Haro M.2, Carlsson A.3
1Departamento de Ciencia de los Materiales e Ingeniería Metalúrgica y Química Inorgánica, Facultad de Ciencias, Universidad de Cádiz, Campus Río San Pedro, 11510-Puerto Real, Cadiz, Spain., 2Univ. Grenoble Alpes, F-38000 Grenoble, France, CEA-INAC/UJF-Grenoble 1 UMR-E, SP2M, LEMMA, Minatec Grenoble, F-38054, France, 3FEI Company, FEI Europe B.V., Achtseweg Noord 5, 5651 GG Eindhoven, Netherlands
jose.calvino@uca.es

Access to Rare Earth Elements (REE) and Platinum Group Metals (PGMs) is nowadays considered a major limiting factor for the development of Green Technologies. Aiming to contribute to the improvement in the efficient use of such strategic materials in the field of Heterogeneous Catalysis, novel nanocatalysts, based on ceria (CeO2) are being investigated in our lab, featuring the following characteristics: low lanthanide contents and a noble-metal free formulation.

The strategy to synthesize the new materials consists in structuring the ceria component as nanometer thick surface layers coherently grown onto the surface of a carrier oxide (ZrO2, YSZ, MgO). The analysis of the Redox properties of this new type of catalysts, which play in fact a key role in their catalytic performance in a variety of reactions, indicates a large improvement with respect to materials based on bulk ceria [1]. A better performance is observed both in H2-reducibility at low temperatures as well as in the stability of the redox response against aging treatments at very high temperatures.

STEM analysis of these new materials has been key both to check the success in the nanostructuration targets proposed for their synthesis and, what´s more important, to understand the behavior observed at macroscopic level in their redox properties. Thus, by combining experimental HAADF-STEM and HREM images recorded on these materials, Figure 1, with simulated ones, Figure 2, it has been possible to detect the presence of a variety of nanostructures ranging from isolated, atom-like, Ce-species, up to 3D, well-faceted, nanoparticles, going through patch or raft-like 2D nano-objects. Such exotic, highly dispersed, nanostructures pose challenges not only to their detection but also to their ultimate 3D-characterization by Electron-tomography and to the analysis of their chemical nature by STEM-EELS or STEM-XEDS. All these aspects will be covered during the lecture.

References

[1] D.C. Arias et al., J. Mat. Chem. A., 1 (2013) 4836

Financial support from MICINN/FEDER IMAGINE (CSD2009-00013) and FP7-I3 ESTEEM 2 (Grant Agreement 312483) Projects is gratefully acknowledged. Authors would like to thank the nanocharacterisation platform (PFNC) of CEA-Grenoble for access to their FEI-Titan3 Ultimate.

Fig. 1: (a) HAADF-STEM image of a 2-atomic planes thick CeOx surface structure recorded on a 4% mol. CeO2/MgO catalyst; (b) HREM view of the same catalyst showing at the same time both highly dispersed, atom-like, Ce-species and nanosized CeOx-rafts.

Fig. 2: Structural models (a,c) and HREM simulated images (b,d) of isolated Ce-species (top row) and 1-atom thick CeOx nano-rafts (lower row) supported on MgO.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-1571 Influence of Strain State on the Formation of Short-Period InGaN/GaN Nanowire Superlattice by Electron Energy-Loss Spectroscopy

Woo S. Y.1, Gauquelin N.1,2, Kociak M.3, Nguyen H. P.4, Mi Z.4, Botton G. A.1
1Department of Materials Science & Engineering, Brockhouse Institute for Materials Research, and Canadian Centre for Electron Microscopy, McMaster University, Hamilton, ON, Canada, 2Now at EMAT, University of Antwerp, Antwerpen, Belgium, 3Laboratoire de Physique des Solides, Université Paris-Sud XI, Orsay, France, 4Department of Electrical & Computer Engineering, McGill University, Montreal, QC, Canada
woosy@mcmaster.ca

Ternary InGaN alloys have been sought-after for various optoelectronic device applications, including their prospect as highly efficient phosphor-free white light-emitting diodes (LEDs). The growth of high quality InGaN epilayers over the entire compositional range, however, faces a few obstacles impeding the realization of their full visible wavelength range tunability. The large InN/GaN lattice mismatch can induce a high density of threading dislocations, and the InGaN miscibility gap leads to inhomogeneity and difficult indium incorporation. Therefore the determination of composition, in particular quantitative elemental mapping at high spatial resolution, is imperative to further understanding the formation of III-N heterostructures.
The growth of high quality III-N heteroepitaxy in a nanowire (NW) geometry is a promising alternative, as shown in the recently developed InGaN/GaN quantum dot (QD) superlattice towards controlled light emission across the entire visible spectrum [1]. In this work, multiple InGaN/GaN dot-in-a-wire nanostructures grown on Si(111) substrates by molecular beam epitaxy were characterized by aberration-corrected scanning transmission electron microscopy (STEM) to correlate their structural to optical and electrical properties. High-angle annular dark-field (HAADF) Z-contrast imaging showed that the 10 InGaN QDs are centrally confined within the active region, embedded between n- and p-doped GaN in the NW LED structure. Core-loss EELS spectrum imaging is used to evaluate the elemental distribution in the NW heterostructures. The In-content is quantified using a multiple linear least squares (MLLS) fitting routine, with combined internal and external reference spectra to fit the N K (399 eV) and In M4,5 (451 eV, in close proximity to the N K) edges in the spectrum image. A surface plot of the thickness-corrected In-map clearly illustrates the non-uniformity of InxGa1-xN composition between the 10 dots (Fig. 1), which has occurred systematically despite the constant growth conditions of the QDs. Geometric phase analysis (GPA) of corresponding atomic-resolution STEM images was used to extract the local strain components at the nanoscale. Along the growth direction (Fig. 2(c)), a direct correlation between a GaN barrier’s strain state and the amount of In incorporated into the subsequent QD can be deduced. Examining the strain distribution of the QDs aids to elucidate their formation as governed by the incorporation of In. In addition, effects of the varying composition on emission wavelength in single NWs using nm-resolved STEM-cathodoluminescence will also be shown.
[1] Nguyen et al., Nano Lett., 12(3), 1317-1323 (2012)

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).

Fig. 1: (a) HAADF-STEM image of a NW studied using STEM-EELS spectrum imaging, followed by subsequent MLLS fitting using combined internal and external reference spectra. (b) Surface plot of the thickness-corrected In-content map, generated from normalizing the MLLS-fitted In-map with the N-map, showing InxGa1-xN composition ranging between x=0.12–0.38.

Fig. 2: (a) MLLS-fitted relative In-content map from EELS with internal references. (b) Corresponding HAADF-STEM image with its strain maps along the growth (c) and in-plane (d) directions, and dilatation matrix (e). The maps show that there is a direct correlation between the In-content as highlighted in (a) and the strain along the growth direction (c).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-1786 Catalytically active structures in Au nanoparticulate catalysts studied by quantitative environmental TEM

Kuwauchi Y.1, Yoshida H.1, Takeda S.1
1The Institute of Scientific and Industrial Research, Osaka University
takeda@sanken.osaka-u.ac.jp

To validate the usefulness of in-situ environmental TEM (ETEM) in catalyst chemistry, there remain several issues to be addressed such as electron irradiation effects, heterogeneity of real catalysts, temperature and pressure gaps [1]. It is recently shown that these issues in ETEM observation can be settled in supported gold nanoparticulate catalysts (AuNP catalysts) and others by quantitative data analyses [2-4]. These quantitative analyses can confirm that an area of interest under atomic resolution ETEM observation acts as catalyst. Based on the analyses, we show some recent results that can only be derived by quantitative atomic resolution ETEM.
We used a prototype ETEM [1] that is equipped with a corrector for the spherical aberration of the objective lens and was operated at 80, 200 and 300 kV. The basic part of the prototype ETEM is commercially available as FEI Titan ETEM G2. The sample was Au/CeO2 powder that has exhibited high catalytic activity for the oxidation of CO even below room temperature. Details of the samples were already described before [2].
We have studied dynamic structures of AuNP catalysts. The observation proved that catalytically active AuNPs move reversibly and stepwise by approximately 0.09 nm on CeO2 support surface at room temperature and in a reaction environment (Fig. 1). The lateral displacements and rotations indicate that AuNPs are loosely bound to oxygen-terminated CeO2. The AuNPs are likely anchored to oxygen-deficient sites [5]. Observations indicate that the most probable activation sites in AuNP catalysts, which are the perimeter interfaces between an AuNP and a support, are not structurally rigid. It is also shown that the surfaces of AuNPs were structurally reconstructed under reaction conditions, via interactions with CO molecules [6]. CO molecules were observed on the surfaces of catalysts under reaction conditions (Fig. 2). We present more details on in-situ ETEM observation of the catalysts and others.

References
[1] S. Takeda and H. Yoshida, Microscopy. 62 (2013) 193-203.
[2] T. Uchiyama, H. Yoshida, Y. Kuwauchi, S. Ichikawa, S. Shimada, M. Haruta, and S. Takeda, Angew. Chem. Int. Ed. 50 (2011) 10157-10160.
[3] Y. Kuwauchi, H. Yoshida, T. Akita, M. Haruta, and S. Takeda, Angew. Chem. Int. Ed. 51 (2012) 7729-7733.
[4] H. Yoshida, K. Matsuura, Y. Kuwauchi, H. Kohno, S. Shimada, M. Haruta, and S. Takeda, Appl. Phys. Express 4 (2011) 065001/1-3.
[5] Y. Kuwauchi et al., Nano Lett. 13 (2013) 3073-3077.
[6] H. Yoshida, Y. Kuwauchi, J. R. Jinschek, K. Sun, S. Tanaka, M. Kohyama, S. Shimada, M. Haruta, S. Takeda, Science 335 (2012) 317-319.

This study was partially supported by a Grant-in-Aid for Scientific Research (A) , Grant No. 25246003 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This research was partly supported by the Management Expenses Grants for National Universities Corporations from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).

Fig. 1: Stepwise displacement and rotation of a AuNP supported on CeO2 [5]. (a) In-situ observation. Observation time is indicated. (b) Instantaneous structural models that are depicted in lateral (b) and top (c) views.

Fig. 2: Glimpse of gas molecules (CO) on the reconstructed surface of a AuNP [6]. In reaction gas in (b), the {100} facet, indicated by rectangle is structurally reconstructed, while in vacuum in (a) the facet is unreconstructed. Faint image contrast on the reconstructed facet in (b) can be accounted by adsorbates (CO molecules).

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-1802 3D elemental mapping of the atoms in bimetallic nanocrystals

Goris B.1, De Backer A.1, Van Aert S.1, Van Tendeloo G.1, Liz-Marzan L. M.2, Bals S.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 2BioNanoPlasmonics Laboratory, CIC biomaGUNE, Paseo de Miram´on 182, 20009 San Sebastian, Spain
bart.goris@uantwerpen.be

A three dimensional (3D) characterization of complex heterostructures is required for an optimized understanding of their properties. For example, it is known that the optical properties of bimetallic Au@Ag nanostructures are largely determined by the presence of certain surface facets and interfaces. Furthermore, effects such as alloying or intermixing of the atoms at the interfaces results in a shift of the plasmon resonances, urging the need for a thorough 3D study at the atomic scale. [1] Electron tomography is a technique to obtain 3D reconstructions based on a series of 2D projection images and recently, different approaches enable a 3D investigation at the atomic scale as well. [2-4]
Here, we apply a tomography approach which is based on compressive sensing to reconstruct the atomic lattice of Au@Ag bimetallic nanoparticles having important applications in the field plasmonics. In order to obtain a reliable reconstruction, 5 high resolution HAADF-STEM projection images are acquired along different orientations of the nanorod and used as an input for a tomographic reconstruction. More detailed information about the experimental set-up can be found in [5]. 3D visualizations of the results are illustrated in figure 1, where a visual distinction can be made between the Au core (yellow) and the surrounding Ag shell (blue). Figures 1a-c correspond to visualizations where the sample was tilted by 0º, 45º and 90º around the [010] axis, respectively. The resulting Fourier transforms correspond to the expected symmetry for a fcc crystal structure and the atomic lattice can clearly be recognized from the visualizations themselves.
Since the reconstruction is based on HAADF-STEM projection images, the intensity of the reconstructed atoms scales with their atomic weight. Therefore, Ag and Au atoms can be identified by analysing intensity profiles through the reconstruction. An example of such an intensity profile is presented in figure 2 which enables the labelling of each atom in the cross sections presented in figures 2b and 2c to be either Ag or Au. The result is shown in figures 2d and 2e yielding a correct indexing of the facets composing the interfaces. We conclude that the interface between the Au core and the Ag shell is sharp, without intermixing and mainly composed of {520} facets where bevels are observed at the <100> and the <110> directions. This type of detailed information is crucial to understand and optimize the physical properties of these materials. [6]
[1] M.B. Cortie and A.M. McDonagh, Chemical Reviews 11 (2011) 3713-3735
[2] S. Van Aert et al., Nature 470 (2011) 374-377
[3] M.C. Scott et al., Nature 483 (2012) 444-447
[4] B. Goris et al., Nature Materials 11 (2012) 930-935
[5] B. Goris et al., Nano Letters 13 (2013) 4236-4241

The authors acknowledge support from the European Research Council (ERC Grants # 24691-COUNTATOMS and #335078-COLOURATOMS) and from the Flemish Fund for Scientific Research.

Fig. 1: (a-c) 3D renderings of the reconstruction viewed along different directions where the sample was tilted around the [010] axis for 0º, 45º and 90º. The atoms in the Au core are rendered yellow whereas the surrounding Ag shell is shown in blue. The Fourier transforms of these projected views correspond to a fcc crystal lattice.

Fig. 2: (a) Three orthogonal slices through the reconstruction show the structure of the nanorod. (b,c) Detailed view of the slices through the reconstruction. An intensity profile is acquired along the direction indicated by the white rectangle in (b). (d,e) Slices corresponding to (b) and (c), in which each Au atom is indicated by a yellow circle.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2106 Using electron beam to investigate metal-carbon catalyst

Su D.1, Zhang B.1
1Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, China
dssu@imr.ac.cn

Carbon (e.g. carbon nanotube and graphene) supported metal nano-structured catalysts with superior performance has drawn considerable attentions in heterogeneous catalysis.[1] However, “metal-carbon support interaction” is much less understood. For instance, it is not clear that how to tune metal-carbon support interaction for preparing the supported metal nanoparticles with optimal morphologies and structures, stabilizing the chemical environment in interfacial area of metal and supports, and keeping the catalytic performance in severe conditions. Advanced techniques in transmission electron microscopy (TEM) are powerful research tools for probing the metal-carbon support interaction. It can provide the fine surface/interface structures at atomic and sub-electron-volt level, such as defects, location of doped atoms, coordination state, functional group species and electronic structures.[2-3] Here, selected several examples will be demonstrated in this presentation. Figure 1 shows typical high angle annular dark field- scanning TEM (HAADF-STEM) image of Pd rings on oxygen functionalized carbon nanotube (O-CNT) and the schematic representation of the formation of Pd rings on O-CNT. Metal–support interactions between Pd nanoparticles (NPs) and functionalized CNTs were established for controlling the metal-size distribution. Thermal detrapping permits nanosized Pd to possess similar dynamics as generated carbonaceous species under electron irradiation and results in a carbon metal hybrid structure with Pd rings on the edges of the nanobead.[4] Figure 2 shows the surface/interface changes of Pd supported on L-CNT (a) and H-CNT (b) after one hour of Suzuki–Miyaura reactions. Corresponding to the commonly reported high reactivity in homogeneous catalysis, carbon–carbon couplings with high efficiency can be achieved on supported Pd NPs by improving surface functionalization and the dispersibility of the catalyst. Such a system offers opportunities for characterizing surface catalysis with atomic precision, which is crucial for detecting dynamic changes on catalytically active species and understanding catalysis pathways.[5]

Reference
[1] D.S. Su, S. Perathoner, G. Centi, Chem. Rev. 2013, 113, 5782-5816.
[2] B. Zhang, D.S. Su, C.R. Physique 2014, in press (DOI: 10.1016/j.crhy.2013.11.001).
[3] L. Shao, B. Zhang, W. Zhang, D. Teschner, F. Girgsdies, R. Schlögl, D.S. Su, Chem. Eur. J. 2012, 18, 14962-14966.
[4] B. Zhang, L. Shao, W. Zhang, D.S. Su, ChemCatChem 2013, 5, 2581-2585.
[5] L. Shao, B. Zhang, W. Zhang, S.Y. Hong, R. Schlögl, D.S. Su, Angew. Chem. Int. Ed. 2013, 52, 2114-2117.

We gratefully acknowledge the financial support provided by NSFC of China (21133010, 21203215, 51221264, 21261160487), MOST (2011CBA00504), Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA09030103) and the China Postdoctoral Science Foundation (2012M520652).

Fig. 1: a) HAADF-STEM image of Pd rings/O-CNT. Schematic representation of the formation of Pd rings on O-CNT: b) Morphology of Pd/O-CNT in the initial stage. c) Coupled with heating (red ribbons, bottom), the electron-beam induces a significant rearrangement in the Pd NPs. The Pd rings are formed at the borders of the irradiated areas.[4]

Fig. 2: HRTEM images after 1 hour catalysis of: a) a Pd NP on L-CNTs, b) a Pd NP on H-CNTs. CNTs annealed at 700 oC have more defects than CNTs annealed at 1500 oC, HNO3 treatment introduced a high functionalization (H-CNTs) on defective CNTs and a low functionalization (L-CNTs) on graphitized CNTs.[5]
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2191 Visualising the Three-dimensional Morphology and Surface Structure of Metallic Nanoparticles at Atomic Resolution by Automated HAADF STEM Atom Counting

Jones L.1, Fauske V. T.2, MacArthur K. E.1, van Helvoort A. T.2, Nellist P. D.1
1Department of Materials, University of Oxford, Oxford, UK, 2Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway
lewys.jones@materials.ox.ac.uk

Because of their large proportion of surface atoms and favourable chemical activity, metallic nanoparticles are used to catalyse a wide range of technologically important reactions. However, many utilise expensive or rare metals, leading to the desire to account for their content and efficiency at the atomic scale. Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) proves a powerful tool here, with readily interpretable mass-thickness, or Z, contrast images facilitating analysis on an atomic column by column basis. In this work, pure platinum nanoparticles were imaged, such that the image intensity depends on the sample thickness. High-resolution images were recorded using a JEOL-ARM200F whose ADF detector was also calibrated to allow the data to be expressed in units of ‘fractional beam-current’ [1] (E = 200kV, convergence and detector angles of 27 and 69–279mrad).
After magnification calibration, the raw data (Fig 1, left) and the detector efficiency scan were passed to the in-house ‘Absolute Integrator’ software. This software automatically identifies the image peak-positions, normalises the data to units of fractional beam-current, performs a locally adaptive background subtraction (to account for the amorphous carbon-black support), divides the image into Voronoi cells and integrates the signal at each atomic column to yield a map of the absolute scattering cross-sections [2] (Fig 1, right). Comparing these with simulation (multi-slice, 30 phonon runs), the number of atoms per column was identified and a provisional three-dimensional (3D) model was built. Owing to the beam-sensitivity of the particles and the desire for high-throughput analysis, tomography was not possible; instead to obtain the likely z-positions an energy minimisation was performed.
Columns in the starting model were positioned symmetrically about the mid-plane (z = 0) with x-y positions taken from the peak-finding results. The model contained 238 atomic columns with 1656 atoms in total, was 11 atoms high at it thickest, and was assumed to contain no vacancies. This was then energetically relaxed using a modified Lennard-Jones potential; Fig 2 (left) represents the result after around 3½ hours.
From this 3D model the number of nearest-neighbours were calculated and used to colour-code the visualisation. A histogram of these coordination numbers (Fig 2, right) then directly indicates the ratios of the various crystal facets. This ability to observe the relative areas of surface facets opens new possibilities for surface science on an individual particle level and for exploring this in relation to catalytic performance.
[1] Lebeau & Stemmer, Ultramicroscopy 108 (2008) 1653–8
[2] E et al., Ultramicroscopy 133 (2013) 109–19

The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3).

Fig. 1: HAADF image of [110] oriented pure platinum nanoparticle (left) and the associated scattering cross-section analysis for each resolvable atomic column (right).

Fig. 2: Relaxed three-dimensional structure of the nanoparticle with colour indicating the nearest-neighbour coordination (left) and accompanying histogram analysis indicating ratios of faceting types.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2203 Plasmon energy from strained GaN quantum wells

Sigle W.1, Benaissa M.2, Korytov M.3, Brault J.4, Vennéguès P.4, van Aken P. A.1
1Max Planck Institute for Intelligent Systems, Stuttgart Center for Electron Microscopy, Stuttgart, Germany, 2CNRST, Rabat, Morocco, 3Leibnitz Institute for Crystal Growth, Berlin, Germany, 4CNRS-CRHEA, Valbonne, France
sigle@is.mpg.de

Monochromated valence electron energy-loss spectroscopy (VEELS) has been used to study the plasmon energy (Ep) from strained GaN quantum wells (QWs) embedded in AlN matrix [1]. The QWs were grown by molecular beam epitaxy. The width of the studied wells was 4, 3, and 2 nm, respectively, separated by a 30 nm thick AlN layer (Fig.1). EFTEM data were recorded in the Zeiss SESAM microscope [2] between 17 and 26 eV using a 0.3 eV energy slit. A Gaussian function was fitted to the volume plasmon peak at each image pixel. After integration parallel to the QW the plasmon energy profile is obtained (Fig.2). Plasmon energies are plotted in Fig.3 (red symbols) versus the inverse square of the well width. It shows a distinct blue-shift of the plasmon peak position with decreasing QW width. In order to take account of the influence of the AlN/GaN interfaces, we solved the relativistic expressions for the begrenzungs effect given by Bolton et al. [3] and Moreau et al. [4]. The interfaces induce an apparent blue shift of the plasmon with decreasing layer width. However, after correction of this shift the dependence of Ep on QW width is still marked (Fig.3, blue symbols). In a second step we considered the influence of the compressive strain of the GaN layers. Such strain is also known to cause a blue shift of the plasmon [5]. Because the critical thickness is about 3 nm the 2-nm- and 3-nm-QWs are completely strained whereas the strain relaxation in the 4-nm-QW is about 0.12 % [6]. Assuming a square-root dependence of the plasmon energy on unit-cell volume, we corrected the measured data (Fig.3, green symbols). The resulting data show reasonably well a linear trend which is consistent with the concept of quantum confinement. Thus, the use of high-resolution valence electron imaging offers the possibility to distinguish the interplay of different confined properties in strained GaN QWs, which is very promising for understanding and exploiting bandgap engineering of nowadays sophisticated devices.

[1] M. Benaissa et al.: Appl. Phys. Lett. 103 (2013) 021901.

[2] C. T. Koch et al.: Microsc. Microanal. 12 (2006) 506.

[3] J. P. R. Bolton and M. Chen: J. Phys.: Cond. Matter 7 (1995) 3389.

[4] P. Moreau et al.: Phys. Rev. B 56 (1997) 6774.

[5] J. Palisaitis et al.: Phys. Rev. B 84 (2011) 245301.

[6] B. Damilano et al.: Appl. Phys. Lett. 75 (1999) 962.

The research leading to these results has received funding from the European Union Seventh

Framework Programme [FP7/2007-2013] under grant agreement n°312483 (ESTEEM2).

Fig. 1: STEM bright-field image showing a set of 3 GaN quantum wells with nominal widths of 4 nm, 3 nm, and 2 nm, respectively, separated by 30 nm-thick AlN barrier layers.

Fig. 2: Volume plasmon energy profile across the three GaN quantum wells.

Fig. 3: As-measured plasmon energies as a function of the QW width (red). Blue symbols are data after correction for interface effects, green symbols after correction of strain effects. The dashed line is a linear fit to the final data.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2269 HR-STEM investigations of metallic nanoparticles grown with superfluidal He-droplets

Knez D.1, Volk A.2, Thaler P.2, Fisslthaler E.1, Grogger W.1, Ernst W. E.2, Hofer F.1
1Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Austria, 2Institute of Experimental Physics, Graz University of Technology, Austria
daniel.knez@felmi-zfe.at

Metallic nanoparticles have attracted more and more interest in recent years as they exhibit completely new physical and chemical properties compared to bulk materials. Over the years numerous synthesis methods, mostly based on wet chemical processes, pyrolysis or evaporation have been developed. In contrast, the nanoparticles used for our investigations were synthesized by using superfluid helium nanodroplets (composed of 103 to 106 helium atoms) at around 0.4 K under ultra-high vacuum (UHV) conditions.1 This approach provides exceptional advantages over conventional methods like sequential addition of a wide range of materials.
Thus, nanoparticles can be synthesized with any composition and different structures, with extremely high purity, which cannot be achieved by other known methods. Figure 1 shows a schematic of the synthesis facility.2 Knowledge of the morphology, dimension and composition of the produced particles are not only essential for understanding their physical and chemical properties, but also for optimizing the synthesis parameters. By using a probe corrected, monochromated FEI Titan3 60-300 equipped with a Super-X detector (EDX) and a Gatan Quantum energy filter we performed analytical high resolution STEM (HR-STEM) in order to characterize metal nanoparticles with respect to their morphology and chemistry.  The HR-STEM investigation of Ag nanoparticles on 3 nm carbon (prepared by the He-droplet method) reveals very small Ag clusters (3-6 nm in size) exhibiting a decahedral structure.

Furthermore, bimetallic clusters with a gold-silver core-shell structure were synthesized. STEM images (a and b in Fig. 3) of a AuAg particle reveal that it grew from single spherical particles inside the He droplet. Elemental analysis of the nanoparticles by EELS and EDX clearly showed that Ag and Au, which were added to the droplet sequentially, are not alloyed. The elemental distribution of this particle is shown in images f and g of Fig. 3.
Finally, the nano-optical properties of these metallic clusters will be studied via low-loss EELS measurements in the plasmon regime, which depend on their size, structure and morphology.3, 4 In order to quantify the influence of the underlying substrate, we will also compare conventional carbon films with mechanical exfoliated monolayer substrates (graphene and hexagonal boron nitride).

References:

1. P. Thaler et al., J. Chem. Phys. 140, 44326 (2014).

2. A. Volk et al., J. Chem. Phys. 138, 214312 (2013).

3. F.-P. Schmidt et al., Nano Lett. 12, 5780 (2012).

4. B. Schaffer et al., Micron 40, 269 (2009)

Our research is supported by the European Union within the 7th Framework Programme (FP7/2007-2013) under Grant Agreement no. 312483 (ESTEEM2) as well as by the Austrian Research Promotion Agency (FFG).

Fig. 1: The helium droplets (blue) are produced in the source (1) by evaporation. After passing the skimmer (2) they collide with atoms or molecules (red) evaporated by the thermal evaporator (3). The particles congregate in the center of the droplet and finally land on the target (4) (e.g. a TEM-grid)

Fig. 2: HR-STEM images (a: HAADF, b: BF) of a silver nanoparticle on a 3 nm amorphous carbon film with decahedral morphology

Fig. 3: a: HAADF image of the AuAg core-shell particle; b: STEM BF image showing that the particle grew from single spherical particles; c-d: EDX elemental maps for Au (c) and Ag (d); e: EELS Ag map (calculated via MLLS fitting); f-g: RGB maps illustrating the elemental core-shell distribution with data from (c) and (e) in (f) and from (c) and (d) in (g)
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2278 Fabrication of ordered nanopatterns in AlOx thin films by a single UV laser pulse

Szívós J.1,2, Serényi M.1, Gergely-Fülöp E.1, Sáfrán G.1, Lohner T.1
1HAS, RCNS, Institute for Technical Physics and Materials Science, 2University of Pannonia, Doctoral School of Molecular and Nanotechnologies
szivos.janos@ttk.mta.hu

Nano-scale modification of materials received wide research interest, recently. Most of the techniques for the fabrication of ordered nanostructures suffer from low throughput and high costs. Here we report a fast and cheap method to prepare ordered nanopatterns directly, or to prepare masks and imprint molds for nanolithography.

This applies a template of a monolayer of hexagonally self-assembled silica nanospheres (Langmuir-Blodgett (LB)). The sample surface is treated with a single UV laser (l=248 nm) pulse through the LB film. According to our simulations the nanospheres of the LB film focus the laser light as individual lenses [Fig. 1 (a)]. This provides an array of highly intense spots for the fabrication.

RF and DC magnetron sputtered amorphous AlOx layers, as potential masks, were subjected to laser patterning. Structure, morphology and optical properties of the films were characterized by Atomic Force- (AFM), Transmission Electron Microscopy (TEM) and Ellipsometry.

The intensity distribution of the laser spot was mapped by means of a GaP UV photodiode. The distribution is Gaussian-like, as it is shown in Fig. 1 (b). For a reasonably uniform exposure the inner part of the spot, marked with rectangle, was chosen for the treatment.

According to the Selected Area Electron Diffraction (SAED) and TEM results the RF and DC magnetron sputtered layers are fully amorphous [Fig. 2 (a)] and contain Al nanocrystals (nc-Al) embedded in an amorphous AlOx matrix [Fig. 2 (b)], respectively. Ellipsometry revealed that the absorption coefficient (α) of the nc-Al/AlOx layers is about 3 times higher than that of the fully amorphous layers.

The formation of the observed patterns was revealed by AFM and cross sectional (X) TEM. A wide, shallow pit of ~210 nm diameter obtained in the fully amorphous AlOx can be seen in the XTEM image [Fig. 3 (a)]. It is suggested to form by a volume decrease caused by the implosion of nanovoids due to the intense electromagnetic field and shock wave of the UV laser pulse. An AFM image of the pattern of these pits is shown in Fig 3 (b).

Fig. 4 (a) – (c) illustrates the formation of the patterns, at different laser energies, within the nc-Al/AlOx film: (a) shows a formed hillock that contains separate, small bubbles indicating moderate energy impact. An increase of the local energy is suggested to ignite plasma and gas release blowing up large bubbles (b). Further energy increase causes the burst of the bubble forming a crater in the layer (c). This refers to a series of holes observed by AFM in Fig. 4 (d) that is typical for the patterned nc-Al/AlOx film.

Our results show that by applying silica nanosphere LB films and carefully controlled UV laser pulses masks can be fabricated suitable for nanopatterning various thin films.

Z. Szabó’s help with the simulations and B. Fodor’s contribution with ellipsometry are acknowledged. This work was partially supported by National Development Agency grant TÁMOP-4.2.2/B-10/1-2010-0025.

Fig. 1: (a) The simulated lateral intensity distribution right beneath the LB film. White circles mark the nanospheres of the film. (b) The measured intensity distribution map of the UV laser spot. The black rectangle shows the quasi-homogenous area that had been chosen for patterning.

Fig. 2: Plan view dark field TEM images of the RF sputtered layers (a) and the DC sputtered layers (b). The insets are the Selected Area Electron Diffraction (SAED) of the samples. Inset in (a) represents an amorphous diffraction pattern, while additional rings of nanocrystalline Al can be realized in the SAED in (b).

Fig. 3: (a) The cross-sectional TEM image of a pit fabricated in the fully amorphous AlOx layer. (b) An AFM image (about 10 µm x 10 µm) of the ordered pattern of the obtained pits.

Fig. 4: (a) – (c): Pattern formation in the nc-Al/AlOx layer. Moderate local intensity forms a hillock (a). Bubble on a sample treated with higher fluence (b). Sample treated with even higher intensity: a burst bubble (crater) is created (c). Insets: EELS elemental maps of O (red), Si (green) and Al (blue). (d): Typical AFM image of the pattern obtained.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2359 Temperature-induced core-shell reconfiguration of FexO/CoFe2O4 nanocrystals in ordered 2D nanocrystal arrays*†

Yalcin A. O.1, Tichelaar F. D.1, van Huis M. A.2, Zandbergen H. W.1
1Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands, 2Soft Condensed Matter, Debye Institute for Nanomaterials Science, Utrecht University, Princetonplein 5, 3584 CC Utrecht, The Netherlands
a.o.yalcin@tudelft.nl

A large variety of single- and multi-component nanocrystals (NCs) can now be synthesized and integrated into nanocrystal superlattices.1,2 These superstructures and their components have a limited thermal and temporal stability, though, which often hampers their application as functional devices. On the other hand, temperature-induced reconstructions can also reveal opportunities to manipulate properties and access new types of nanostructures.3-5 In-situ atomically resolved monitoring of nanomaterials provides insight into the temperature induced evolution of the individual NC constituents within these superstructures at the atomic level.6 Here, we investigate the effect of temperature annealing on 2D square and hexagonal arrays of FexO/CoFe2O4 core/shell NCs (Figure 1) as a model for many complex oxides by in-situ heating in a transmission electron microscope (TEM). The FexO core has a rock salt structure with some cation deficiencies (x = 0.83-0.95)7 and the lattice constant varies between 0.4255 nm and 0.4294 nm, depending on the oxidation state.7 The CoFe2O4 shell has a spinel crystal structure (lattice constant 0.846 nm).8 Both structures have a face centered cubic (FCC) oxygen sublattice with a lattice mismatch of only 3 %.7 Both cubic and spherical NCs undergo a core-shell reconfiguration at a temperature of approximately 300 ⁰C, whereby the FexO core material segregates at the exterior of the CoFe2O4 shell, forming ‘snowman’-like particles (asymmetric dumbbells) with a small FexO domain attached to a larger CoFe2O4 domain (Figure 2). During reconfiguration, the core volume is filled by the CoFe2O4 shell material. Upon continued annealing, the segregated FexO domains form bridges between the CoFe2O4 domains, followed by coalescence of all domains resulting in loss of ordering in the 2D arrays. Annealed FexO domains contain Co traces as well (Figure 3).

[1] Redl, F.X. et al. Nature 2003, 423, 968–971.
[2] Talapin, D.V. et al. Nature 2009, 461, 964–967.
[3] van Huis, M.A. et al. Nano Letters 2011, 11, 4555–4561.
[4] Figuerola, A. et al. Nano Letters 2010, 10, 3028–3036.
[5] De Trizio, L. et al. ACS Nano 2013, 7, 3997–4005.
[6] van Huis, M.A. et al. Advanced Materials 2009, 21, 4992–4995.
[7] Pichon, B.P. et al. Chemistry of Materials 2011, 23, 2886–2900.
[8] Song, Q. & Zhang, Z.J. Journal of the American Chemical Society 2004, 126, 6164–6168.

* Yalcin, A.O. et al. Nanotechnology 2014, 25, 055601.

† This work has appeared on the journal cover (Nanotechnology Volume 25, Issue 5) as the featured article (http://ej.iop.org/pdf/nano/vol25/na2505-webcover.pdf).

This work is part of the research programme of the Foundation for Fundamental Research on Matter (FOM), which is part of the Netherlands Organization for Scientific Research (NWO).

Fig. 1: TEM images of different types of 2D arrays. a) cubic FexO/CoFe2O4 core/shell NCs forming a square 2D array. Inset figure was taken with an objective aperture inserted (diffraction contrast). Core-shell contrast is observed better in this way. b) Spherical FexO/CoFe2O4 core/shell NCs array forming a hexagonal 2D array.

Fig. 2: TEM images of FexO/CoFe2O4 ‘initially’ core/shell NC arrays; a) cubic NCs at 335 ⁰C, and b) spherical NCs at 360 ⁰C. The insets in the images were taken when the objective aperture was inserted (diffraction contrast). The (200) spacing of FexO is 0.21 nm, and (220) and (311) spacings of CoFe2O4 are 0.3 nm and 0.255 nm respectively.

Fig. 3: Energy filtered TEM Co-mapping of initially spherical NCs. Figure 3a is the zero-loss image and Figure 3b is the corresponding Co map. The dotted lines were used to clarify different domains. Arrows show the FexO domain with Co presence.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2547 Advanced Transmission Electron Microscopy Investigation of Epitaxy-Enabled Morphology Controling ITO Nanowires

Lebedev O. I.1, Shen Y.2, Turner S.3, Van Tendeloo G.3, Wu T.4
1Laboratoire CRISMAT,UMR 6508, CNRS ENSICAEN, F-14050 Caen, France, 2Division of Physics and Applied Physics, Nanyang Technological University, Singapore 637371 , 3EMAT, Department of Physics, University of Antwerp, B-2020, Antwerpen, Belgium, 4Materials Science and Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia
oleg.lebedev@ensicaen.fr

Controlling nanowire morphology in bottom-up synthesis and allowing the assembly of nanowires on planar substrates is of tremendous importance for device applications in electronics, photonics, sensing and energy conversion. To date, there has however been only limited success in reliably achieving these goals, hindering both the fundamental understanding of the growth mechanism and the integration of nanowires in real-world technologies. In this work, we will show the impact of transmission electron microscopy (TEM) on this domain, as an extremely versatile and powerful technique.

Novel dual-metal Au-Cu alloy nanoparticles were used as a catalyst for tin-doped indium oxide (ITO) nanowire growth. The enhanced mobility of the catalyst nanoparticles (NPs) enables in situ seeded growth of branched ITO nanowires (NWs). The dynamically tuned chemical potentials in the catalyst NPs selectively stabilize a rare cubic indium-tin-oxide phase (ISO), forming epitaxial heterojunctions within individual NW branches. This methodology of selecting phases and forming compositionally abrupt axial heterojunctions in NWs departs from the conventional synthesis routes, giving unprecedented freedom to navigate phase diagrams and promising novel nanomaterials and devices [1]

Here we report that growth of planar, vertical and randomly oriented ITO nanowires can be realized on yttria-stabilized zirconia (YSZ) substrates via the vapor-liquid-solid (VLS) mechanism, by simply regulating the growth conditions, in particular the growth temperature. [2]. TEM and reciprocal space mapping experiments reveal the indispensable role of substrate-nanowire epitaxy in the growth of oriented planar and vertical nanowires at high temperatures, whereas randomly oriented nanowires without epitaxy grow at lower temperature. Further control of the orientation, symmetry and shape of the nanowires was achieved through use of YSZ substrates with (110) and (111), in addition to (001) surfaces. Based on these insights, we succeeded in growing regular arrays of planar ITO nanowires from patterned catalyst nanoparticles. Overall, our discovery of unprecedented orientation control in ITO nanowires advances the general VLS synthesis, providing a robust epitaxy-based approach towards rational synthesis of nanowires.

[1] –J.Gao, O.I.Lebedev, S.Turner, Y.F. Li, Y.H.Lu, Y.P.Feng, P.Boullay, W.Prellier, G.Van Tendeloo, T.Wu Nano Letters 12 (2012) 275-280

[2] - Y. Shen, S. Turner, P. Yang, G. Van Tendeloo, O. I. Lebedev, T. Wu Nature Communications (Submitted) (2014)

This work was also supported in part by the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3).

Fig. 1: (a) Low-magnification STEM-HAADF image of the in-plane nanowires and (b) corresponding SAED pattern (c) GPA patterns along [100] and [010] directions. (d) HR STEM-HAADF cross-section image of ITO nanowire and (e) image of the tri-junctions of the ITO, YSZ and Au particle. (f) STEM-ABF image of ITO / YSZ interface with overlayed structural model

Fig. 2: ) Low magnification ADF STEM (top) and BF TEM(bottom) images of off-plane ITO NWs, (b) - high resolution HAADF-STEM image of the ITO-YSZ interface, (c) ABF-STEM image of the top part of an ITO NW. Notice the presence of the ISO phase.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2563 Quantitatively Following growth processes of CdSe@CdS core-shell particles on the atomic scale 

Mangel S.1, Aronovitch E.1, Enyashin A. N.2, Houben L.3, Bar Sadan M.1
1Chemistry Department, Ben Gurion University of the Negev, Beer Sheba, Israel, 2Institute of Solid State Chemistry UB RAS, Ekaterinburg, Russian Federation, 3Peter Grünberg Institut 5 and Ernst Ruska Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany
barsadan@bgu.ac.il

Colloidal core-shell crystals of II-IV semiconductors are one of the most extensively researched systems in nanoscience. CdSe@CdS nanoparticles are investigated due to their size dependent optical properties and although they have been known for the last two decades, even today new fabrication routes are still explored to improve their optical properties. While reports on the optical properties of single particles are available, the quantitative characterization of atomic order on a single particle level and the growth mechanism that resulted in that specific rearrangement, are still generally missing. The majority of characterization procedures are performed on ensembles that average properties and may hinder the understanding of fundamental aspects in the colloidal synthesis.
Moreover, atomic resolution analysis, which has emerged with aberration corrected instruments, have mainly provided analysis of few particles per sample. It is now, due to the Cc correction that offers superior resolutions in low voltages that the atomic ordering can be achieved on a routine basis to deliver new statistical data. The application of the low voltage reduces the rate of radiation damage so that both surface and bulk structure. We follow the growth process of the CdSe nanoparticles and the formation of the CdS shell covering them. For the first time, statistical atomic-scale information on dozens of individual nanoparticles is correlated with ensemble measurement data.
Previous research showed a proof of concept of determining the polarity and faceting of the nanoparticles by both TEM and HAADF STEM. The assignment of polarity to individual particles gives detailed understanding of when and where stacking faults form, and the new knowledge can be merged with the known kinetics of the reaction. The Cd- terminated edge, where growth is slower, produces more stacking faults or preserves more of the disorder of the original nucleus. The Se edge, which is the fast growing edge, produces almost a perfect W structure. Upon the deposition of the shell, the core is further annealed and stacking faults concentrate at both edges of the particle. However, the annealed sections may acquire larger fractions of the pressure induced ZB symmetry which is quite rare in the pure CdSe nanoparticles.
This analysis shows that high resolution electron microscopy can serve as a routine tool to understand growth kinetics and it may also be applied to the growth of other hybrid nanoparticle structures where kinetic procedures determine the interfaces nature and properties.

Fig. 1: Phase image of exit-plane wavefunctions reconstructed from through-focus series. (a) CdSe cores and (b) CdSe@CdS core shell particle. The wavefunctions were Fourier filtered to eliminate the background pattern of the periodic hexagonal graphene support film.

Fig. 2: Atomistic models of three possible stacking fault sequences, altering between Wurtzite (W) and Zinc Blende (ZB) crystal structures. Cd in violet, S/Se in green.(right) Overlay of the atomistic models on a phase image of a CdSe@CdS core-shell particle exhibiting the three possible atomic stacking fault configurations. (left)
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2583 Atomic Structure and Composition Analysis of Pt0.8Ni De-alloyed Nanocatalysts for Proton Exchange Membrane Fuel Cells – Aberration Corrected STEM Study

Rasouli S.1, Sharman J.2, Martinez A.2, Fongalland D.2, Hards G.2, Yamamoto T.3, Myers D.4, Higashida K.3, Ferreira P.1
1University of Texas at Austin, Austin TX , USA, 2Johnson Matthey Technology Centre, Sonning Common, Reading, UK, 3Kyushu University, Fukuoka, JAPAN, 4Argonne National Laboratories, Lemont, IL, USA
ss.rasouli@gmail.com

Proton exchange membrane fuel cells (PEMFCs) are promising energy conversion devices for transport and stationary applications. Pt nanoparticles are currently used as the catalyst in the anode and cathode of the fuel cell, respectively. However, alloys of Pt with base metals are being investigated to replace Pt on the cathode as a way to improve the efficiency of the fuel cell, and reduce cost [1].
In this work Pt-Ni nanoparticles were de-alloyed by acid leaching to produce 5.8 nm size Pt.8Ni catalyst nanoparticles. In order to better understand the relationship between the elemental distribution and the nanoparticle shape and structure, the nanoparticles were characterized by aberration-corrected scanning transmission electron microscopy (STEM), using high-angle annular dark-field (HAADF) imaging [2]. The Pt and Ni compositional distribution of the de-alloyed nanoparticles was investigated using EDS mapping in STEM mode. In order to better understand the three dimensional shape of the nanoparticles and the carbon support, 3-D electron tomography of the nanoparticles was performed in a JEOL JEM ARM 200F. A total of 61 STEM images were collected over a tilt range of -60 to +60 degrees, with a 2° tilting step. The final tilt series was aligned, reconstructed and visualized using Inspect 3D and Amira 4.1, respectively.
Figure 1 shows two aberration-corrected HAADF STEM images of the de-alloyed Pt0.8Ni nanoparticles projected along the [110] beam direction. Although most of the particles exhibit a truncated octahedron shape (Fig. 1a), there are also some particles with long {111} facets (Fig. 1b). The absence of superlattice reflections in the FFTs (insets of Figs 1a and 1b) shows that Pt and Ni are in solid solution, forming a face-centered cubic structure. However, as shown in Figures 1c and 1d, the nanoparticles exhibit regions of bright and dark contrast, which indicate that the composition of Pt and Ni is not uniform throughout the particle. This heterogeneous distribution among the various particles is a result of the de-alloying process. Moreover, although most of the particles are a Pt-Ni solid solution, there are also some particles exhibiting {100} supperlattice reflections in the FFTs, indicating a partially ordered structure (Figs 1e and 1f). In addition, Pt seems to segregate to the surface of the nanoparticles, as confirmed by EDS Mapping (Fig. 2). Finally, 3-D electron tomography confirms that most of the particles exhibit a truncated octahedron shape (Fig. 3).

References
[1] S. C. Ball et al. ECS Transactions, 11(1) (2007), p.1267.
[2] S. Chen, et al. J. Phy. Chem. C. 113(3) (2009), p.1109.

The authors gratefully acknowledge funding from the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Fuel Cell Technologies Office (Nancy Garland, Technology Development Manager).

Fig. 1: (a) and (b) Aberration corrected HAADF images of Pt0.8Ni nanoparticles and corresponding FFTs (insets). (c) and (d) normalized intensities across the nanoparticle (along the red line). (e) and (f) Aberration corrected HAADF images of the nanoparticles with supperlattice reflections in FFTs (insets).

Fig. 2: HAADF STEM image and EDS mapping of Pt0.8Ni nanoparticles

Fig. 3: 3D reconstructed image of the Pt0.8Ni nanoparticles
Fig. 4:
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2588 High Spatial/Energy Resolution Cathodoluminescene Spectroscopy: A Powerful Tool for Profound Characterization of the Physical Properties of the Advanced Nanostructures

FU X.1, FU Q.1, HAN X.1, LIU C.1, ZHANG J.1, LIAO Z.1, YU D.1
1Department of Physics, State Key Laboratory for Mesoscopic Physics, Peking University, and Collaborative Innovation Center of Quantum Matter, Beijing 100871, P. R. China
yudp@pku.edu.cn

    High special/energy resolution cathodoluminescence (CL) spectroscopy is now becoming more and more important in investigations of the optical properties of the low-dimensional structures. Two representative examples of the application of the CL in study were summarized in this presentation as follows:

    Elastic engineering strain has been regarded as a low-cost and continuous variable manner for altering the physical and chemical properties of materials, and it becomes even more important at low-dimensionality because at micro/nanoscale, materials/structures can usually bear exceptionally high elastic strains before failure. The elastic strain effects are therefore greatly “magnified” in micro/nanoscale structures and should be of great potential in the design of functional devices. The purpose of this presentation is to present a summary of our recently progresses in the energy band engineering of elastically strained ZnO micro/nanowires. First, we present the electronic and mechanical coupling effect in bent ZnO nanowires. Second, we summary the bending strain gradient effect on the near-band-edge (NBE) emission photon energy of bent ZnO micro/nanowires. Third, we show that the strain can induce exciton fine-structure splitting and shift in ZnO microwire. Related publications are presented in Figure 1.

    Surface plasmon polaritons (SPPs) show great potential for application in future nanoscale photonic systems due to the strong field confinement at the nanoscale, intensive local field enhancement, and interplay between strongly localized and propagating SPPs. A template stripping method combined with PMMA as a template was successfully developed to create extraordinarily smooth metal nanostructures with a desirable feature size and morphology for plasmonics and metamaterials. The advantages of this method, including the high resolution, precipitous top-to bottom profile with a high aspect ratio, and three-dimensional characteristics, make it very suitable for the fabrication of plasmonic structures. The confined modes of surface plasmon polaritons in these nanocavities have been investigated and imaged by using cathodoluminescence spectroscopy, which has been turned out to be a powerful means to characterize the resonant SPPs modes confined in metal nanocavities. The mode of the out-of-plane field components of surface plasmon polaritons dominates the experimental mode patterns, indicating that the electron beam locally excites the out-of-plane field component of surface plasmon polaritons.

This work was supported by MOST (Nos. 2013CB934600, 2013CB932602), NSFC (Nos. 11274014, 11234001), and the Program for New Century Excellent Talents in University of China (No. NCET-12-0002).

Fig. 1: Recent publications of CL spectroscopy summarized in the first part of the presentations demonstrating the advantages compared to the conventional optical methods.

Fig. 2: Publications related to the second part of the presentations via CL for characterization of the SPP modes confined in metal nanocavities, which is impossible to do it via other optical approaches.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2629 Insight into the structural, electrical and photoresponse properties of individual Fe:SrTiO3 nanotubes

Žagar K.1, Fabrega C.2, Hernandez-Ramirez F.2,3, Prades J. D.3, Morante J. R.2,3, Rečnik A.1, Čeh M.1
1Jožef Stefan Institute, Ljubljana, Slovenia, 2Catalonia Institute for Energy Research, Barceloba Spain, 3University of Barcelona, Barcelona, Spain
kristina.zagar@ijs.si

Titanates are suitable for many applications such as oxygen sensing and tunable high temperature superconducting microwave filters. The potential advantages of the nanostructured forms have been scarcely explored compared to other oxides. We report on the structural and electrical properties of individual iron-doped strontium titanate nanotubes (Fe:SrTiO3). The Fe:SrTiO3 nanotubes were assessed for the first time, showing high stability and reproducibility [1].

Fe:SrTiO3 nanotubes were synthesized using sol-gel electrophoretic deposition (EPD) technique [2]. The Fe:SrTiO3 sol, where 2 mol% of Ti was replaced by Fe, was deposited into the anodic alumina template while a potential was applied between the AAO/Al working electrode and Pt counter electrode. After the deposition samples were annealed at 700 °C for 1 h with subsequent template removal. Resulting Fe:SrTiO3 nanotubes were characterized by electron microscopy techniques. To study electrical properties, Fe:SrTiO3 nanotube devices were fabricated by focused ion beam nanolithography techniques [3].

Obtained Fe:SrTiO3 nanotubes with lengths between 5 and 10 µm and diameters of approximately 200 nm were polycrystalline, dense and made up of cubic grains ranging between 10 and 20 nm in size (Figure 1). Their chemical composition explored by Energy-dispersive X-ray (EDX) analysis showed the presence of Sr, Ti and Fe; and confirmed that Fe was effectively incorporated into the perovskite structure.

For the electrical characterization the prototype device was formed by integration of individual Fe:SrTiO3 nanotubes into simple circuit architecture and the electrical resistivity of approx. 35 ohm∙cm was calculated (Figure 2). This value was significantly lower than the values for intrinsic bulk SrTiO3 samples due to the presence of Fe. This result opens the door to the future synthesis of Fe:SrTiO3 nanotubes suitable for monitoring small trace level of oxygen. Furthermore, some devices were tested as UV-detectors with the final aim to explore the optoelectronics characteristics and validate their suitability for device integration. The dynamic behavior of the photoresponse obtained with a single Fe:SrTiO3 nanotube as a function of different UV photon fluxes is shown in Figure 3. Repeatable and reversible responses were found in all cases, demonstrating that our devices are nice proof-of concept systems showing that an Fe:SrTiO3 nanotube can be used as a UV photodetector.

References:

1. K. Zagar et al., J. Mat. Chem. Phys. 141 (2013), p. 9.

2. K. Zagar et al., Nanotechnology 21 (2010) p. 375605.

3. F. Hernandez-Ramirez et al., Chem. Phys. 11 (2009), p. 7105.

The research was also supported by the Framework 7 program under the project S3 (FP7-NMP-2009-247768) and European Union Seventh Framework Programme [FP7/2007-2013] under grant agreement n°312483 (ESTEEM2).

Fig. 1: (a) Bright-field image of a uniformly shaped and polycrystalline Fe:SrTiO3 nanotube. (b) Higher-magnification bright-field TEM image of polycrystalline Fe:SrTiO3 nanotube with grain sizes in the range from 10 to 20 nm.

Fig. 2: (a) Fe:SrTiO3 nanotube electrically contacted in 2-probe configuration using FIB lithography. (b) I-V curve of the contacted Fe:SrTiO3 nanotube. An ohmic response is found at room temperature and open air atmosphere.

Fig. 3: (a) Photoresponse Iph of a Fe:SrTiO3 nanotube as function of different UV photon intensities (b) Dynamic response of Iph as function of different UV photon fluxes.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2638 Core-shell GaAs/AlGaAs nanowires grown on Si (111)

Kehagias T.1, Florini N.1, Walther T.2, Moratis K.3, Hatzopoulos Z.3, Pelekanos N. T.3
1Physics Department, Aristotle University of Thessaloniki, GR-54124, Thessaloniki, Greece, 2Department of Electronic and Electrical Engineering, University of Sheffield, Mappin St, Sheffield S1 3JD, UK, 3Materials Science & Technology and Physics Departments, University of Crete and IESL/FORTH, GR-71003 Heraklion, Greece
kehagias@auth.gr

Precise control over III-V compound semiconductor nanowires (NWs) growth is crucial for the fabrication of advanced nanoscale electronic and optoelectronic devices. Core-shell GaAs/AlGaAs NWs were grown on Si (111) by plasma assisted molecular beam epitaxy (PAMBE). Initially, the NWs were grown via the vapor-liquid-solid mechanism, using Ga droplets as catalyst, for 20 min. Subsequently, the Ga droplets were removed by exposing the NWs to As flux and growth continued for another 40 min, varying the fluxes of the Al, Ga, and As, in order to form an AlGaAs shell around the GaAs initial core.

The structural features of the NWs were characterized by transmission electron microscopy (TEM) methods. TEM observations and selected area diffraction analysis showed that NWs are zinc-blende (ZB) single crystals grown epitaxially along the [111] direction normal to the Si substrate, despite the presence of a 1-3 nm thick amorphous SiO2 layer on the Si surface. Simultaneously, an interfacial GaAs layer is formed between the NWs, comprising large epitaxial and {111} twin related crystals [Fig. 1(a)]. The emanation point of the NWs is located on small heavily twinned GaAs crystals [Fig 1(b)], which evolve into NWs and finally, through the growth process usually merge with the GaAs crystals of the interfacial layer. In addition, “parasitic” NWs emerging from either the interface, or the original NWs were observed along the inclined <111> and/or the <100> directions. Mirror twins normal to the [111] growth direction can be observed throughout the length of the NWs [Fig. 1(c)]. In fact, NWs grow for several micrometers under a continuous succession of mirror twins. No wurtzite structure was observed.

The weak absorption contrast of high-resolution TEM (HRTEM) in conjunction with the minimal difference of the AlGaAs and GaAs lattice parameters turn HRTEM images unsuitable for visualizing the core-shell structure. Hence, the chemically sensitive 200 reflection for mass contrast TEM imaging [Fig. 1(d)], in addition to annular dark-field (ADF) scanning TEM (STEM) imaging [Fig. 1(e)], were used. These revealed the core-shell configuration of the NWs, where the AlGaAs shell spans from one half to 2/3 of the projected diameter of the NWs ranging from 80 nm to 180 nm. Furthermore, energy dispersive X-ray (EDX) analysis confirmed the core-shell morphology of the NWs and was used to estimate the NW shell composition.

Research co-financed by the European Union (European Social Fund-ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Research Frame (NSRF)-Research Funding Program: ARISTEIA II, project “NILES”.

Fig. 1: (a) TEM image, taken off the [1-10] zone axis, showing the NWs morphology. (b) A NW emerging from a small defected GaAs crystal. (c) HRTEM image, along the [1-10] direction, where mirror twins are depicted by arrows. The amorphous shell is attributed to oxidation. (d)&(e) TEM and ADF STEM images revealing the GaAs/AlGaAs core-shell structure.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2680 Understanding Symmetry Breaking in Anisotropic Nanoparticle Growth

Walsh M. J.1, Barrow S. J.2, Tong W.2, Funston A. M.2, Etheridge J.3
1Department of Materials Engineering, Monash University, Australia, 2School of Chemistry, Monash University, Australia, 3Monash Centre for Electron Microscopy, Monash University, Australia
michael.j.walsh@monash.edu

The highly promising optical, catalytic and electronic properties of gold nanoparticles, particularly nanorods, have made them a major area of research in recent years. In plasmonics, the light absorption and scattering properties of biocompatible Au nanorods make them effective biosensors, whilst their tuneable aspect ratio allows the localised surface plasmon resonant (LSPR) frequency to be shifted into the biologically transparent near infra-red range, opening up potential applications within drug delivery and photothermal hyperthermia treatments [1].

Gold nanorods are typically synthesised via a seed-mediated approach, in which silver ions and halides are used as surfactants [2]. There is currently little agreement on a mechanism for anisotropic growth, and few insights into the fundamental symmetry breaking event that is a prerequisite for shape anisotropy. The question remains as to what causes an essentially spherical seed particle, with a cubic lattice, to develop a preferential growth direction? Here we present an aberration corrected electron microscopy study of nanoparticles at the embryonic stages of growth to provide direct atomic scale insights into the onset of anisotropy.

Seed particles were found to be predominantly single crystal. Overgrowth of these seeds with and without the presence of silver ions is shown in figure 1; clearly revealing that it is the crucial addition of Ag+ that induces symmetry breaking. This event occurs at particle diameters of between 4-6 nm, and only for single crystal structures. We suggest a mechanism for symmetry breaking in which Ag stabilises small {110} truncations in the seed particle structure. The various stages of anisotropic growth are described in figure 2, in which particle size and morphology are characterised at regular intervals during nanorod synthesis. After the initial symmetry breaking event, rapid growth along the rod axis results in a variety of particle sizes. Nano-dumbbells are formed after several minutes, corresponding to a maximum redshift in the longitudinal LSPR frequency. Reduced growth in the length direction cause the dumbbell to transition to a nanorod morphology, with a subsequent blueshift in LSPR.

These observations are consistent with a silver under-potential deposition mechanism, and we discuss this and possible synergistic effects of AgBr complexing as driving forces for anisotropic growth. Precise understanding of the growth mechanism and resulting structure-property relationships of nanoparticles should allow for high yields of particles with size, shape and crystal surfaces tailored to a variety of potential applications.

References
[1] S. Abalde-Cela et al. J. R. Soc. Interface. 2010, 7, 435
[2] S. E. Lohse and C. J. Murphy. Chem. Mater. 2013. 25, 1250

This work was supported by the Australian Research Council (ARC) grant DP120101573 and used microscopes at the Monash Centre for Electron Microscopy funded by ARC Grant LE0454166

Fig. 1: Standard seed particles (left), overgrown seeds (centre) and seed particles overgrown in the presence of Ag ions (right).

Fig. 2: The stages of seed mediated Au nanorod growth after 0, 2, 20 and 60 minutes, with corresponding UV-Vis spectra.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2712 Spectral unmixing of electron energy-loss spectra of ~5 nm InP/ZnS nanocrystals

Duchamp M.1, Xi L.2, Lam Y. M.3, Dunin-Borkowski R. E.1, Kardynal B.2
1Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich, Germany, 2Peter Grünberg Institute 9, Forschungszentrum Jülich, Germany, 3Institute of Materials for Electronic Engineering II, RWTH-Aachen, Sommerfeldstr. 24, D-52074 Aachen, Germany
martial.duchamp@gmail.com

We examine InP/ZnS nanocrystals (NCs), with possible core-shell or alloyed structures [1][2]. As InP and ZnS are both cubic and have very similar lattice constants, their compounds are difficult to distinguish using high-resolution transmission electron microscopy (TEM). This information is important, since the presence of a ZnS shell is essential in reducing surface recombination but also prevent oxidation of the InP [3].
Scanning TEM and electron energy-electron spectroscopy (EELS) were performed at 80 kV, in order to quantify the distribution of In- and Zn-containing compounds in individual NCs and their oxidation states after oxidation of the NCs in an oxygen/argon plasma to simulate aging process. Data analysis involved spectral unmixing using vertex component analysis (VCA) [4][5], in order to improve the signal-to-noise ratios of In and Zn elemental maps and to extract spectral signatures at the O K edge.
Figures 1a and b show conventional background-subtracted elemental maps measured for the In M5,4 and Zn L3,2 edges. VCA was applied over the energy range 850-1100 eV. A spectral component corresponding to the Zn L3,2 edge can be identified. The corresponding map (Fig. 1e) is similar to Fig. 1b but less noisy. Surprisingly, an abondance map corresponding to In is also obtained over this energy range, where no In edge is present. This signal originates from the background of the In M3 edge (678 eV) and is similar to Fig. 1a, which was extracted at the In M5,4 edge.
Figure 2 shows results obtained using VCA over the energy range 450-600 eV. Of the three extracted components, component 1 corresponds to an In signal. The abundance map associated with this component is similar to the In maps as a result of the presence of the In M5,4 edge in this energy range. More interestingly, the O K edge shows a shoulder at ~533eV. This shoulder is in a similar position but is less intense than the first peak in an In2O3 reference spectrum, suggesting that the NCs are partially oxidised. Although a clear signature of the oxidation of ZnS is not observed, oxidation of the In could be explained by the presence of either an incomplete ZnS shell or a ZnS layer that oxidized during the plasma treatment.
Our results show that a thin ZnS shell does not protect InP from oxidation sufficiently well for long-term applications. Our use of the VCA algorithm allows the oxidation of In in sub-5-nm InP/ZnS NCs to be identified with much greater confidence than using conventional background-subtraction methods.

[1] H. Borchert et al. Nano Lett. 2 (2002) 151; [2] K. Huang et al. ACSNano 4 (2010) 4799; [3] J. Jasinski et al. Solid State Commun. 141 (2007) 624; [4] M. Duchamp et al. Appl. Phys. Lett. 102 (2013) 133902; [5] C. Boothroyd et al. Ultramicroscopy, in press (2014)

The authors acknowledge financial support from the European Union under the Seventh Framework Programme (project references 312483 - ESTEEM2 and NWs4LIGHT).

Fig. 1: (a, b) Background-subtracted elemental maps corresponding to the In M5,4 and Zn L3,2 edges; (d, e) Corresponding maps obtained by applying VCA over the energy range 850-1100 eV. (c) Spectra extracted from the spectrum image at the positions marked in (a); (f) Spectral components associated with the maps shown in (d, e)

Fig. 2: Spectral components extracted over the energy range 450-600 eV, which includes the In M5,4 and O K edges. The corresponding abundance maps are shown on the right. A reference In2O3 spectrum is also shown. The scale bar is 15 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2715 Investigation of Detailed Monolayer MoS2 Edge Structure and Defect Configuration by Atomic Resolution Scanning Transmission Electron Microscopy

Li K.1, Hong J.2, Jin C.2, Zhang X.3
1Imaging and Characterization Core Lab, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia, 2Department of Materials Science and Engineering, Zhejiang University, China, 3Division of Physical Science, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia
kun.li@kaust.edu.sa

Recent intense research and development activities in graphene have regained strong research interest in its related two dimensional (2D) counterparts, especially BN and transition metal dichalcogenides (TMDCs), with MoS2 under the focus. The idea has been fortified again that in addition to composition and structure dimensionality also plays a very important role in determining the fundamental properties due to quantum confinement effect. These materials possess exotic properties that are absent in their bulk forms. For MoS2, understanding its edge and defect configuration at atomic level is critical for realizing its application potentials as electronic devices and catalysis, as it affects the electronic band structure of MoS2 and the catalytic behavior.

Here we employ a probe Cs corrected Titan scanning transmission electron microscope (STEM) operated at 80 kV to study the defect and edge structure of monolayer MoS2. Z-contrast STEM technique is used to differentiate between Mo and S atom columns, with Mo showing brighter contrast and S showing darker contrast. Vacancies with different size are found in our study, ranging from mono vacancy to large triangular-shaped vacancies. Fig. 1 shows a triangular-shaped vacancy with a lateral length of 3 unit cells, revealing Mo-terminated Klein edge. Mo-terminated zigzag edge is found in a bigger triangular-shaped vacancy with a lateral length of 4 unit cells, as shown in Fig. 2. The results suggest that S atoms are easier than Mo atoms to be removed under electron beam illumination and Mo-terminated edge is the most often found type. We also find in our experiment that when a nano-ribbons is formed, one side of it is Klein edge, and the other side is zigzag, where removed Mo atoms tend to be absorbed. Based on this observation we believe that for catalysis application, zigzag edge should also be the preferred absorption site for noble catalytic atoms. Dislocation pairs are also found in this study with atomic resolution (Fig. 3) and the Burgers vector is also defined (Fig. 4); it is a 60-degree dislocation.

With no doubt the capability of unambiguously identifying defect and edge configuration at atomic level will facilitate process optimization for realizing the promising applications of MoS2 and its related TMDCs.

Fig. 1: Atomic resolution defect configuration of triangular-shaped vacancies with the lateral length of three unit cell and Mo-terminated Klein edge.

Fig. 2: Atomic resolution defect configuration of triangular-shaped vacancies with the lateral length of four unit cell and Mo-terminated zigzag edge

Fig. 3: Dislocation pairs in MoS2 Monolayer.

Fig. 4: Dislocation in Figure 3 with Burgers vector identified
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-2842 Gold repartition at surfaces and interface in silicon nanowires: A TEM / APT confrontation

Grillet N.1, David T.1, Roussel L.1, Neisius T.2, Cabie M.2, Gailhanou M.1, Alfonso C.1, Charaï A.1, El Kousseifi M.1, Hoummada K.1, Descoins M.1, Mangelinck D.1
1Aix-Marseille Université – CNRS, IM2NP, Faculté des Sciences de Jérôme, F-13397 Marseille, France, 2Aix-Marseille Université, CP2M, Faculté des Sciences de Jérôme, F-13397 Marseille, France
nadia.grillet@im2np.fr

The development of new methodologies is needed for precise measurement of concentration and localization of different species insmall objects like nanowires(NWs). Transmission Electron Microscopy (TEM) and Atom Probe Tomography (APT) are two major techniques which give complementary informations at atomic scale.
This work deals with post-growth repartition of the gold used as catalyst during Molecular Beam Epitaxy (MBE) growth of silicon NWs. Electron microscopy analysis was performed using Scanning Transmission Electron Microscopy - High Angle Annular Dark Field (STEM-HAADF), electron tomography, and Energy-dispersive X-ray spectroscopy (EDS) to collect complementary information on morphology, structure and chemistry. Atom probe tomography has ability for both 3D imaging and chemical composition measurements at the atomic scale. The two techniques were used to characterize the same samples containing silicon nanowires deposited by MBE.

We observed in STEM-HAADF that the NWs have a hexagonal (only {112} faces) and/or dodecagonal section ({112} and {110} faces) and present a 'saw-tooth' faceting on one over two {112} faces (cf. Figure 1 a) and b)).
We found that gold clusters are spread on the surfaces of the NWs, but no gold was observed in the NW bulk. Furthermore, the gold coverage is uneven on the different faces of the NW. Its repartition is very homogeneous on the ‘flat’ {112} faces. But, when the {112} saw-tooth faceting is present, the gold spreads preferentially on the {113} facets, rather than on the {111} facets (cf. Figure 1 c)) [1].
A rough estimation considering that gold clusters are hemispherical allowedus to estimate that {113} facets are covered by 3 to 6ML of gold which is largely higher than coverage found in literature [2].

The three dimensional distribution of Au has been determined in the volume and on the surface of the Si NW also by APT. These results show no gold detection in the bulk of the NW (Figure 2), which is in good agreement with previous studies [3]. Moreover, Au clusters detected on the Si-NW surface by APT are in accordance with TEM measurements.

An investigation of the interfacial region between catalyst and silicon nanowire has also been done using EDS mapping and APT. Both techniques indicate that a silicon oxide layer was formed between the gold catalyst and the Si NW. In addition, ATP measurements showed the presence of a SiAu alloy layer containing less than 1% gold underneath the catalyst droplet.

[1] T. David et al., Journal of Crystal Growth, 383, 151-157 (2013)
[2] C. Wiethoff et al., Nano Letters 8, 3065-3068(2008)
[3] J.E. Allen et al., Nature Nanotechnology 3, 168-73 (2008)

Fig. 1: Tomography STEM-HAADF showing the different facets and the repartition of gold on them

Fig. 2: STEM-HAADF / EDS mapping versus APT at the Au/Si interface
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-3052 Structure and Phase Analyses of Nanoparticles using Combined Analysis of TEM scattering patterns

Boullay P.1, Lutterotti L.2, Chateigner D.1
1CRISMAT, CNRS UMR 6508, CAEN, France, 2Department of Industrial Engineering, University of Trento, TRENTO, Italy
philippe.boullay@ensicaen.fr

The development of materials science at the nanoscale questions the characterization techniques on their ability to describe small objects, either individually or as large assemblies. Transmission electron microscopy (TEM) appears as one of the techniques able to provide quantitative results using imaging, spectroscopic or diffraction methods. Aiming the structure, size and phase analysis of nanoparticles, a TEM approach would ideally combine these methods at the nanometer scale but analyses on individual particles are not ideal if one wants a representative statistical analysis.

Another approach would be based on the quantitative analysis of electron diffraction intensities similarly to what is done in X-ray Powder Diffraction (XPD). Selected Area Electron Diffraction patterns of an assembly of nanoparticles exhibit ring patterns analogous to those from XPD, hereafter called Electron Powder Diffraction patterns (EPD). Phase identification and structure refinement of such powder diffraction patterns can be reached by search-match routines followed by Rietveld analysis [1-2] or PDF (Pair Distribution Function) [3-4] methods. Besides the phase identification and structure refinement issue, we will show that the average size and shape of the crystallites (Fig. 1) as well as quantitative texture analysis (Fig. 2) can be obtained from EPD [5]. Using Rietveld analysis within the Combined Analysis methodology, almost routine analyses of nanoparticles in the form of powders and thin films can be achieved. Complementary measurements can be added, for instance Energy Dispersive X-ray Spectroscopy in order to constrain the refinements in cases for which elemental variations are of matter, and PDF, in order to quantify even amorphous structures [3,6].

This reciprocal space approach allows a fast access to statistically meaningful information about the average size and shape of an assembly of nanoparticles (agglomerated or not). It is thus very complementary to direct imaging of isolated nanoparticles. Fast and insensitive to sample drift, this approach shall be advantageously used for gaining quantitative information from in-situ environmental studies of dynamic processes involving nanoparticles.

[1] T.E. Weirich, M. Winterer, S. Seifried, H. Hahn and H. Fuess, Ultramicroscopy 81 (2000) 263.
[2] A.M. Tonejc, I. Djerdj and A. Tonejc, Mat. Sci. Eng. C 19 (2002) 85.
[3] T. Takagi, T. Ohkubo, Y. Hirotsu, B.S. Murty, K. Hono and D. Shindo, App. Phys. Lett. 79 (2001) 485.
[4] A.M.M. Abeykoon, C.D. Malliakas, P. Juhás, E.S. Božin, M.G. Kanatzidis, S.J.L. Billinge, Z. Kristallogr. 227 (2012) 248.
[5] P. Boullay, L. Lutterotti, D. Chateigner and L. Sicard, Acta Cryst. A (2014) in press.
[6] D.J.H. Cockayne and D.R. McKenzie, Acta Cryst. A 44 (1988) 870.

LL and DC warmly thank the Conseil Régional de Basse-Normandie and FEDER for financing LLs' Chair of Excellence at CRISMAT, and the Université de Caen Basse-Normandie for two months as invited professor of LL. PB and DC thanks the project FURNACE funded by the French research agency (contract ANR-11-BS08-0014).

Fig. 1: a) Mn3O4 nanoparticle’s aggregates. Associated EPD in b) and 1D plot in c) representing the full integration along the Debye rings. The profile is fitted (Rw=2.06% and RBragg=1.55%) considering the sample contribution and compared with XPD in d). e) TEM bright field image of isolated particles. f) Average size and shape of the Mn3O4 nanoparticles.

Fig. 2: a) EPD for 2 extreme and 0° sample tilts obtained on a Pt thin film deposited on a Si single crystal substrate. b) corresponding 1D patterns using Dh = 10° and for h = 180°. c) 2D plots for the 35 1D-patterns of each 2D pattern in a). Experimental data (bottom) and fits (up) are represented, Pawley pattern matching. Square root intensity scales.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-3066 Atomic scale studies of individual catalyst nanoparticles with atom probe tomography

Cairney J. M.1, Felfer P. J.1, Eder K.1, Maschmeyer T.2, Masters A.2
1Australian Centre for Microscopy & Microanalysis, The University of Sydney, Sydney, NSW 2006 Australia, 2School of Chemistry, The University of Sydney, Sydney, NSW 2006 Australia
julie.cairney@sydney.edu.au

From sunscreen to optoelectronics, sensors, catalysis and drug delivery, nanometer-scale particles play an important role in a rapidly growing range of applications. An important example of the commercial application of nanoparticles is in the field of catalysis. By maximising the surface area of catalytic metals through the use of nanoparticles, catalytic reactivity can be greatly enhanced and the selectivity strongly influenced. Bi- or multimetallic particles offer even greater scope for fine-tuning.

To better understand their catalytic performance, one must gain a detailed understanding of the size, shape, composition and, most importantly, the arrangement of atoms within and on the surface of the particles. While some atomic scale information on the structure of nanoparticles has long been accessible through electron microscopy [1], identifying the chemical nature and 3D location of the individual atoms remains a challenge using such techniques.

The potential for APT to provide microstructural information for catalysis is well-recognized, and experiments on nanoparticles have proven to be promising, but experimentally challenging [2,3]. Here, we demonstrate how high-resolution atom probe tomography (APT) can be used to quantitatively determine the three-dimensional distribution of atoms within a Au@Ag core-shell nanoparticle with a resolution of +/- 0.5 nm. Specifically, we will describe several major advances in atom probe techniques in recent years that are specifically suited to the study of nanoparticles. These include new specimen preparation techniques that overcome the conventional barrier to the study of nanoparticles by atom probe, and recent advances in the available methods to extract information about the segregation of atoms to three dimensional surfaces that allow mapping of the distribution of the shell species in core-shell particles.

By using these new tools, we reveal that the elements in the Au@Ag nanoparticles are not evenly distributed across the surface and that this distribution is related to the surface morphology and residues from the particle synthesis. Access to this type of information is a revolutionary step forward for the rational design of nanoparticles.

References:

[1] Kiely, C., Electron microscopy: New views of catalysts. Nature Materials, 2010. 9: p. 296-297.

[2] Xiang, Y., et al., Long-chain terminal alcohols through catalytic CO hydrogenation. Journal of the American Chemical Society, 2013. 135(19): p. 7114-7117.

[3] Tedsree, K., et al., Hydrogen production from formic acid decomposition at room temperature using a Ag-Pd core-shell nanocatalyst. Nat. Nano, 2011. 6(5): p. 302-307.

The authors acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Australian Centre of Microscopy & Microanalysis

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-3178 Imaging Nano Segregation in Advanced Pt Alloy Fuel Cell Electrocatalysts

Gan L.1, 3, Heggen M.2, Cui C.1, Rudi S.1, Strasser P.1
1The electrochemical Catalysis, Energy and Materials Science Laboratory, Department of Chemistry, Technical University Berlin, 10623 Berlin, German, 2Ernst Ruska Center for Microscopy and Spectroscopy with Electrons, Forschungszentrum Juelich GmbH, 52425 Juelich, Germany, 3Division of Energy and Environment, Graduate School at Shenzhen, Tsinghua Unviersity, Shenzhen 518055, PR China
lgan.thu@gmail.com

Segregation is an important physical phenomenon in alloy materials and has significant influences on the physical and chemical properties. In particular, segregation at the surface or subsurface can drastically change the molecular adsorption properties of alloy surfaces and thus becomes a promising way to design highly active catalysts.1 Atomic understanding of segregation effect in nanoscale alloy catalyst particles is therefore crucial yet still challenging for future catalyst designs.

In this talk, we will highlight some of our recent works on understanding and controlling nano segregation effect in advanced Pt alloy catalysts for fuel cell technologies.2-5 We demonstrate how alloy composition (Fig. 1), particle size (Fig. 2), and particle shape (Fig.3) can result in different segregation behaviors in Pt alloy nanoparticles and thus drastically influence their catalytic activity and stability. Focus will be placed on the atomic imaging of nano segregation by using state-of-the-art aberration-corrected scanning transmission electron microscopy (STEM) and electron energy loss spectroscopy (EELS) and in situ experiments. In particular, by using STEM-EELS elemental profile/mapping, we revealed novel compositional segregation patterns in PtxNi1-x core-shell nanoparticles, showing an unexpected Ni-segregated inner shells depending on the bulk alloy composition (Fig. 1).2 Furthermore, we discovered a distinctly different compositional segregation in octahedral PtxNi1-x nanoparticles, which featured a surprising Pt segregation at the edges/corners and Ni segregation at the facets (Fig. 3).4 We explored the physical origin for these distinct segregation behaviors and their impact on the catalytic activities and stability for fuel cell reactions. This will be further complemented by in-situ STEM-EELS experiments to study the structural and compositional evolution of Pt alloy nanoparticles during nanoparticle synthesis, post thermal annealing, and solution-phase electrocatalysis, shedding important light for catalyst designs with desired segregation patterns and chemical properties.

References:

(1)Stamenkovic, V. R. et al. Science 2007, 315, 493-497. (2)Gan, L., Heggen, M., Rudi, S. & Strasser, P. Nano Lett 2012, 12, 5423-5430. (3)Gan, L., Heggen, M., O'Malley, R., Theobald, B. & Strasser, P. Nano Lett 2013, 13, 1131-1138. (4)Cui, C., Gan, L., Heggen, M., Rudi, S. & Strasser, P. Nature Mater 2013, 12, 765-771. (5)Gan, L., Cui, C., Rudi, S. & Strasser, P. Top Catal 2014, 57, 236-244.

This work was supported by U.S. DOE EERE award DE-EE0000458 via subcontract through General Motors and by Ernst Ruska Center for Microscopy and Spectroscopy with Electrons, Forschungszentrum Juelich GmbH, Germany. 

Fig. 1: Figure 1. Aberration-corrected STEM-EELS elemental profiles of dealloyed PtNi (a, d), PtNi3 (b, e) and PtNi5 (c, f) core-shell NPs, showing near-surface Ni-rich inner shells.2

Fig. 2: Figure 2. STEM images and EELS line profiles of size-selected spherical PtNi3 catalyst after stability test. Nanoporous particles formed at larger sizes (ca. 10 nm) and, consequently, lower near-surface Ni content as well as larger Pt shell thickness.3

Fig. 3: Figure 3. EELS elemental mapping of octahedral PtNi1.5 nanoparticles along (a) <110> direction and (b) <100> direction, showing that Pt segregated at the edges and corners whereas Ni segregated at the facets. (c) The revealed structural model. 4
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-3257 Atomic Level In-situ Characterization of Metal/TiO2 Photocatalysts under Light Irradiation in Water Vapor

Zhang L.1, Crozier P. A.1
1Arizona State University, Tempe, USA
liuxian.zhang@asu.edu

Photocatalysts have potential applications for solar fuel generation either through water splitting or CO2 reduction. It is now recognized that atomic level in situ observations are critical for understanding the structure-reactivity in photocatalysts in the presence of reactant and product species and during in-situ light illumination. TiO2 is a promising photocatalyst used for self-cleaning, pollutants degradation and water splitting etc. Metal particle co-catalysts such as Pt are coupled to the semiconductor to provide chemically active sites and attract excited electrons preventing charge recombination. Herein we use TiO2 as a model material to develop in situ photocatalytic experimental methodology and explore structure changes of metal/semiconductor photocatalysts. We employ a modified ETEM with a broadband light source to study the behavior of metal particles on TiO2 semiconductor surfaces under photoreaction conditions.
Well defined anatase nanoparticles were prepared following a hydrothermal method. Metal co-catalysts such as Pt, Ag and Cu were loaded onto the anatase nanobars using methods such as dry impregnation, photo-deposition and sputtering. An FEI Tecnai F20 ETEM was modified to allow samples to be illuminated with light with intensity up to 10 suns [1]. In situ analysis was performed tracking structure changes in photocatalytic vapor phase water splitting reactions. Ex-situ experiments were performed to compare or confirm in-situ observations under exposure to a 450W xenon lamp with a mirror reflecting 360nm to 460nm light. TEM images for ex-situ experiments were taken from an FEI aberration corrected Titan TEM.
The initial anatase particles shown in Figure 1a appear crystalline on the surface and the surface is smooth and atomically abrupt. Figure 1b shows a crystal after 40 hrs exposure to water and light without prior exposure to the electron beam. When the titania is exposed to light and water vapor, the initially crystalline surface converts to an amorphous phase one to two monolayers thick [2]. 5% wt Pt particles were loaded onto anatase nanoparticles and well dispersed through proper heat treatment. Figure 2a also shows initial Pt on TiO2 materials. After exposure to a xenon lamp in liquid water for 6hrs, Pt particles show significant sintering as shown in Figure 2b. Pt/TiO2 sample shows significant surface disordering (Figure 2b). In-situ experiments were performed to study the evolution of the Pt sintering, TiO2 surface roughening and the Pt/TiO2 interface changes. Other metal co-catalysts will also be discussed in the presentation.
References:
[1]. Miller, B.K.; Crozier, P.A. Microscopy and Microanalysis 2013, 19, 461-469
[2]. Zhang, L.; Miller, B. and Crozier P. A. Nano Lett. 2013 13 (2), 679–684

The support from US Department of Energy (DE-SC0004954) and the use of ETEM at John M. Cowley Center for HR Microscopy at Arizona State University is gratefully acknowledged.

Fig. 1: a) Initial anatase crystal at 150°C without exposure to water or light. b) After 40 hrs in 1 Torr H2O, 12 hrs light exposure.

Fig. 2: a) Initial 5%wt Pt on anatase particles, b)after ex-situ 6hrs exposure to 360nm-460nm light in liquid water; (with insertions low magnification images).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-3315 Quantitative Z-contrast Imaging of Zeolite-supported Metal Clusters and Single-metal-atom Complexes With Single-Atom Sensitivity

Xu P.1, Yang D.1, Martinez-Macias C.1, Kistler J. D.1, Chotigkrai N.2, Gates B. C.1, Browning N. D.3
1Department of Chemical Engineering and Materials Science, University of California, Davis, Davis, CA 95616, USA, 2Department of Chemical Engineering, Chulalongkorn University, Bangkok 10330, Thailand, 3Fundamental and Computational Science Directorate, Pacific Northwest National Laboratory, Richland, WA 99352, USA
amy.pinghongxu@gmail.com

Supported metal catalysts, in particular zeolites and oxide supported noble metals, are widely applied in industrial processes, such as petroleum refining, automobile exhaust conversion and petrochemical conversion. A crucial challenge in studying these catalysts lies in structural nonuniformity, which hinders the tuning of catalytic properties for control of selectivity and activity. In this work, we report investigation of supported metal catalysts with small, uniform structures on highly crystalline supports to gain a fundamental understanding of supported catalysts. Here we present direct measurements of such catalysts, using aberration-corrected scanning transmission electron microscopy (STEM) at atomic resolution with single atom sensitivity. STEM is well-suited for characterizing these catalysts, as the high atomic number difference between the support and the supported metal provides a strong contrast in the STEM images. Quantitative analysis was performed on metal sizes, shapes and their bonding locations within the cavities of zeolite structures, which is essential in studying structure-property relationship. Our results also demonstrated that STEM technique is a very powerful when complemented by extended X-ray absorption fine structure and infrared spectroscopies for identification of ligands, determination of metal-metal distances and coordination number.

High electron-dose (105-108 e-/A2) STEM imaging was applied for these highly beam sensitive materials, with images taken quickly before significant destruction or modification of of structures. High signal-to-noise ratio for this approach provides the advantage of easy image interpretation. Results of characterization of a range of supported heavy metals, including iridium, platinum and rhodium supported on various zeolites will be presented. Zeolites were chosen as the support material because of their wide applications in industry and their high degrees of crystallinity, which provide well-defined structures for imaging. Our results demonstrated high contrast of the STEM images characterizing these catalysts, which is unattainable from previous studies, as exemplified by Figure 1 showing mononuclear iridium species supported on zeolite Y. Detailed determination of the configuration of the metal species in the structure and the interaction between these metal complexes and the ligands, including the support, will be presented.

This work was supported in part by the United States Department of Energy (DOE) Grant No. DE-3-BDOE797 through the University of California, Davis, the Laboratory Directed Research and Development Program (LDRD): Chemical Imaging Initiative at Pacific Northwest National Laboratory (PNNL), and the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility at PNNL.

Fig. 1: Aberration-corrected HAADF-STEM image of zeolite Y supported iridium species. Bright features encircled are examples of iridium species, mostly mononuclear complexes.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-3345 Microscopic experiments with radial junction solar cells based on silicon nanowires

Fejfar A.1, Hývl H.1, Ledinský M.1, Vetushka A.1, Kočka J.1, Misra S.2, Foldyna M.2, Lin Wei Yu2, Roca i Cabarrocas P.2
1Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 2Laboratoire de Physique des Interfaces et des Couches Minces (LPICM), Ecole Polytechnique, CNRS, Palaiseau, France
fejfar@fzu.cz

Microscopic measurements of Si thin films and nanostructures can provide interesting insights for their applications, e.g., for an operation of corresponding solar cells. This was the case for amorphous and microcrystalline Si films, but also for structures of polycrystalline Si on glass.

Radial junctions based on silicon nanowires (SiNWs) are an example of modern nanostructured solar cell designs with excellent light trapping and efficient photogenerated charge collection. A single pump-down process used to prepare a randomly grown matrix of SiNWs and conformal p-i-n radial junctions led to cells with efficiencies over 8% [1]. Considerable influence of irregularities in SiNWs lengths, orientations, shapes and mutual interaction on the photovoltaic action can be expected. Direct measurement of these effects requires microscopic measurements of photoresponse. This is possible using atomic force microscopy (AFM) with conductive cantilever which serves as a contact to individual radial junctions [2]. At the same time the cantilever can measure the local nanomechanical properties, including local stiffness of the wires, which can only sustain contact forces up to ~1 nN. Resulting conductivity maps show substantial variation of the local electronic properties. The AFM tip cannot reach deeper into the SiNWs matrix and correlation with scanning electron microscopy of the identical nanowires was sought in order to identify the reason for conductivity variations. The results are discussed in terms of random photodiode arrays connected in parallel with overall performance limited by weak diodes.

[1] S. Misra et al., Sol. Energy Mat. Sol Cells. 118 (2013) 90–95.

[2] A. Fejfar et al., Sol. Energy Mat. Sol. Cells. (2013) 228–234.

This work was supported by Czech Science Foundation projects 13-12386S,  and 14-15357S and the LNSM (Laboratory of Nanostructures and Nanomaterials) infrastructure framework LM2011026 supported by Ministry of Education, Youth and Sports of the Czech Republic.

Fig. 1: SEM view of the radial junction solar cells based on Si nanowires (top) and scheme of the conductive AFM characterization (bottom).

Fig. 2: Map of local current observed by C-AFM within 5x5 micrometers area (top) and local current superposed on the topography (bottom).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-1-O-3374 In-situ Observations of Pt Nanoparticle Growth Using Aberration-corrected TEM and Graphene Liquid Cells

Ercius P.1, Yuk J. M.2, 3, 4, Park J.3, Kim K.2 3, Lee J. Y.4, Zettl A.2, 3, Alivisatos A. P.3
1NCEM, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, 2Department of Physics, UC Berkeley, CA 94720, 3Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, 4Department of Materials Science and Engineering, KAIST, Daejeon 305-701, Korea
percius@lbl.gov

TEM aberration correctors now allow for structures to be investigated at atomic resolution with high contrast, which could greatly benefit in-situ observations of physical, chemical and biological phenomena. Recently, in-situ TEM observations of the growth of nanoparticles in liquids revealed new phenomena during the formation of colloidal inorganic particles, but the relatively thick SiN windows and even the liquid material trapped inside degrades TEM resolution and SNR [1]. The growth mechanisms of such materials could greatly benefit from high-resolution characterization although such experiments were difficult due to thick SiN windows. Graphene sheets have been successfully used as a single-atom thick substrate for high-contrast HR-TEM imaging, and its high flexibility, mechanical strength and impermeability allows the encapsulation of liquid under TEM vacuum conditions [2-3]. We introduce a type of liquid cell using graphene sheets to entrap a colloidal growth solution for in-situ HR-TEM imaging.

Graphene liquid cells (GLC) were created by superimposing two graphene sheets grown on separate grids. A Pt growth solution is pipetted on top of the opposing graphene substrates. The Pt growth solution intercalates between the graphene sheets and stays trapped after drying in air. Figure 1 shows a low-magnification TEM image of the encapsulated solution and an illustration of the GLC. For in-situ experiments, pockets of Pt-growth material are first identified using a low electron dose at low magnification. The electron dose is increased to 103 – 104 A/m2 at high magnification to reduce the Pt precursor and begin nanocrystal growth. We observed growth and coalescence of colloidal Pt nanoparticles at atomic resolution at 3.85 fps using TEAM I at 80keV and a Gatan US1000 CCD camera. The combination of a 2 atom thick membrane to contain a small amount of liquid and the chromatic aberration corrector (C-COR) provide unprecedented resolution during Pt nanoparticle growth.

Resolution and SNR are sufficient to image atomic columns, facets and twins of individual nanoparticles in each movie frame. Figure 2A) and B) show particle coalescence resulting in a FCC single crystal and a twinned FCC crystal, respectively. The twin structure remains for the duration of our observations.

Liquid encapsulated between graphene sheets provides an ideal in-situ system to study nanoparticle growth and coalescence with atomic resolution. The technique can be readily applied to study a diverse range of systems in-situ in a HR-TEM.

References:

[1] H. Zheng et al, Science 324, 1309 (2009)

[2] Z. Lee et al., Nano Lett. 9, 3365 (2009)

[3] J.M. Yuk et al., Nano Lett. 11, 3290 (2011)

GLC work supported on DOE contract no. DE-AC02-05CH11231. NCEM is funded by DOE contract no. DE-AC02-05CH11231.

Fig. 1: Low resolution TEM image of a 100 nm diameter liquid pocket and illustrations of a graphene liquid cell.

Fig. 2: Growth of Pt nanoparticles via coalescence along <111> directions imaged by HR-TEM in a GLC with relative times from the start of the reaction. A) Two particles coalesce to form a FCC single crystal. B) Two particles form a twinned nanocrystal as indicated in the included FFT pattern. 2 nm scale bars.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1458 EFFECT OF INTERGROWTH DISTRIBUTION AND PRESENCE OF DEFECTS ON CATALYTIC PERFORMACE OF MFI/MEL MATERIALS

Gomes M. E.1, Imbert F.2, Gonzalez G.1
1Lab. Materiales, Centro de Ing. Materiales y Naonotecnologia, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela, 2Laboratorio de Cinética y Catalisis, Departamento de Química, Facultad de Ciencias, Universidad de Los Andes, Mérida 5101 - Venezuela
megomez@ivic.gob.ve

The impact of zeolites as catalyst for refining processes has been mainly associated to the regular distribution of channels and cavities that determines the distribution of product species on terms of geometry and size. In the search of new structures with new topologies, the controlled synthesis of zeolites with stacking defects forming intergrowth structures looks very promising.
The nature and distribution of intergrowth structures, and recurrently defect structures, like twins, as well as new types of structural imperfections can have a dramatic effect on the catalytic performance of these materials, due to the formation of new cavities in the intersections, resulting in new product distribution.
The structural disorder and its correlation with the catalytic activity have not been very well studied, neither the controlled synthesis of structural disorder. This is the fundamental base for the controlled design of microporous materials of disorder structures.
In the present work, the effect of different of intergrowths domain distribution of MFI/MEL and the presence of defect structures, on the catalytic performance of these materials has been studied using of n-decane hydroconversion as a model reaction.
The synthesis of the intergrowths of MFI/MEL was carried out by the combination of TBA and TPA organic molecules, TBA first added to colloidal silica solution and after 2 h of agitation TPA was incorporated, and hydrothermal treatment was followed at 150C and 90 C, obtaining proportions of 80MFI/20MEL and 70MFI/30MEL respectively.
The amount of intergrowth formation was determined by XRD, by fitting the experimental patterns to simulated patterns of intergrowth structures generated using the software DIFFaX.
The resulting distribution of products for MEL and 70MFI/30MEL and 80MFI/20MEL (Fig.1), clearly indicates that a new configuration of channels and cavities is present, forming larger cavities at the intersections that allows an increased amount of bulky products (di-branched) to be obtained. HRTEM was an essential technique to comprehend this behavior. For the intergrowth materials, a random distribution of intergrowths domains and numerous defects are present: vacancies, pore coalescence, that were responsible for the product distribution obtained in the catalytic reaction. (Fig.2). While the MEL presents more ordered and homogeneous structure (Fig. 3)
In this work, it was shown that the properties of a catalyst are governed by its microstructure and chemistry on an atomic scale, and electron microscopy methods were essential to directly analyze these properties.

Fig. 1: Fig. 1 Distribution of di-branched and mono-branched products reached at maximum isomerization in the catalytic reaction

Fig. 2: Fig. 2 HRTEM image of intergrowth 20%MEL/80%MFI

Fig. 3: Fig. 3 HRTEM image of MEL
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1432 Morphological changes induced by reaction in a RuFe bimetallic catalyst: a BF-TEM and XED-Spectrum Imaging investigation

Teixeira-Neto E.1, Vignado C.2, Jordão E.2, Figueiredo F. C.2, 3, Carvalho W. A.3
1Laboratório de Microscopia Eletrônica, LNNano, CNPEM, C.P. 6192, 13083-970, Campinas - SP, Brazil, 2Faculdade de Engenharia Química, UNICAMP, C.P. 6066, Campinas, SP, 13083-970, Brazil., 3Centro de Ciências Naturais e Humanas, UFABC, Santo André-SP, 09210-170, Brazil.
erico.teixeira.neto@gmail.com

Catalyst deactivation is a major challenge for the catalysis community. The proposed mechanisms of catalyst deactivation include sintering, re-oxidation of metal components and surface reconstruction and mechanical deactivation through attrition. The bimetallic RuFe system has been investigated and employed as an interesting alternative catalyst in many applications. [1]
In this work we show results on the determination of morphological changes of an alloyed (1:1) RuFe/TiO2 (6% m/m) catalyst. This material was prepared by impregnating the TiO2 support with a solution of Ru3+ and Fe3+ and subsequently drying the suspension in a rotatory evaporator. The material was oxidized in ambient atmosphere at 600C for 2 h and then thermally processed in a H2-rich atmosphere at 400C for 1 h. The as prepared catalyst was used in the hydrogenation of dimethyl adipate in a Parr reactor at 250C and 50 atm of H2 for 15h. After the reaction, the catalyst was recovered and analyzed by TEM and XED-SI. The images and spectroscopic information shown here are representative of a detailed investigation of this system.
Figure 1 shows bright field (BF-TEM) images of RuFe particles deposited on the surface of TiO2 support. The as prepared bimetallic nanoparticles appear as dispersed dark hemispheres. A marked morphological change is observed in the catalyst after the reaction: in Fig. 1-A, small dark particles are seen embedded in an irregular shaped gray matrix. The inset in B show fragmented particles in detail.
In Figure 2, BF-TEM images and XED-SI chemical maps of the as prepared catalyst show correlated distributions of Fe and Ru, which is evidence of the formation of a RuFe solid solution. After the reaction, the Fe content is distributed throughout the gray matrix observed in Fig. 1 and Ru is concentrated at positions associated with the small dark gray particles seen in BF.
The decrease in the catalytic performance observed during the reaction can be attributed to the change in the distribution of metallic domains within individual particles. The initial morphology of the hemispherical RuFe solid solution particles changes to Ru-rich particles embedded into a matrix of iron oxide. This morphological description will provide new arguments to the understanding of the observed catalytic performance.

1. Nikolaos E. Tsakoumis, Magnus Rønning, Øyvind Borg, Erling Rytter, Anders Holmen, Catalysis Today 154 (2010) 162-182.

The authors thank Fapesp (2013/11298-0) and LME-LNNano-CNPEM (JEOL JEM 2100).

Fig. 1: BF-TEM images of RuFe particles deposited on the surface of TiO2 support. The as prepared bimetallic nanoparticles appear as dispersed dark hemispheres. A marked morphological change is observed after the reaction: in A, small dark particles are seen embedded in an irregular shaped gray matrix. The inset in B show fragmented particles in detail.

Fig. 2: XED-SI chemical maps of the as prepared catalyst show correlated distributions of Fe and Ru, which is evidence of the formation of a RuFe solid solution. After the reaction, the Fe content is distributed throughout the observed gray matrix (Fig. 1) and Ru is concentrated at positions associated with the small dark gray particles seen in BF.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1438 Plasmonic Photocatalyst Ag/AgCl Nanohybrids on Titanate Thin Film for Photocatalytic Application

Tang Y. X.1, Cheng Z.1, Dong Z. L.1
1School of Materials Science and Engineering, Nanyang Technological University, Singapore
zldong@ntu.edu.sg

Semiconductor photocatalysts have been extensively studied for the removal of organic compounds in waste water using solar energy [1, 2]. In this work, we demonstrate a novel plasmonic photocatalyst silver/silver chloride nanohybrids on the titanate thin film obtained via a facile and cost-effective approach [3, 4], which involves the following steps. Firstly, the sodium titanate thin film is prepared using a traditional hydrothermal method at 200 oC for 6h. Secondly, the Na+ ions in the interlayer of the titanate is replaced by Ag+ ions through an ion-exchange process. Then the obtained silver titanate readily reacts with HCl vapor to form the AgCl particles on the titanate thin films. Finally, the visible-light-driven plasmonic photocatalyst Ag/AgCl/titanate is obtained by partially reducing the Ag+ ions from the AgCl particles with the aid of Xe lamp illumination.

Typical FESEM and TEM images of the titanate film and the AgCl/titanate film are shown in Fig. 1 and Fig. 2 respectively. The as-prepared titanate film shows porous honeycomb-like features (Fig. 1a). Each honeycomb consists of 3~6 sided walls, inside which intertwined titanate nanowires are present. The diameter of a single nanowire is in the range from 40 to 50 nm. The X-ray diffraction pattern from the titanate film is shown in Fig. 1b, and the peaks are indexed as coming from orthorhombic titanate phase Na2Ti2O5. After the reaction with HCl, new peaks corresponding to the cubic AgCl phase are observed. Electron microscopy studies indicate that the dense AgCl nanoparticles are uniformly distributed on the surface of each titanate nanowire without agglomeration (Fig. 1c and 1d), and the particles size is around 50 nm (Fig. 2b). Fig. 3 shows that the as-prepared Ag/AgCl/titanate film photocatalyst exhibites higher activity in the visible region of the solar spectrum for the degradation of phenol solution, while the titanate thin film shows negligible activity for the phenol removal. This room-temperature synthesis route could be easily extended to prepare various solar light responsive semiconductors via metal ion exchange and gas reaction process for photocatalytic applications.

References

[1] X.C. Wang, K. Maeda, A. Thomas, K. Takanabe, G. Xin, J.M. Carlsson, K. Domen, M. Antonietti, Nat. Mater. 8 (2009) 76-80.

[2] X. Chen, S.S. Mao, Chem. Rev. 107 (2007) 2891-2959.

[3] Y. X. Tang, V. P. Subramaniam, T. H. Lau, Y. K. Lai, D. G. Gong, P. D. Kanhere, Y. H. Cheng, Z. Chen, Z. L. Dong, Appl. Catal. B: Environ., 106 (2011) 577.

[4] Y. X. Tang, Z. L. Jiang, J. Y. Deng, D. G. Gong, Y. K. Lai, H. T. Tay, I. T. K. Joo, T. H. Lau, Z. L. Dong, Z. Chen, ACS Appl. Mat. Interfaces, 4(2012) 438.

The authors thank the Environment and Water Industry Programme Office (EWI) under the National Research Foundation of Singapore (grant MEWR651/06/160) for the financial support.

Fig. 1: (a) FESEM image of the as-prepared titanate thin film showing honeycomb-like structure, (b) X-ray diffraction patterns of titanate film and AgCl/titanate film, (c) FESEM image showing the morphologies of the AgCl/titanate film, and (d) FESEM image showing uniform distribution of AgCl nanoparticles on titanate nanowire surface.

Fig. 2: The TEM images of (a) titanate nanowires, and (b) AgCl/titanate nanowires. The samples are obtained from the titanate film and AgCl/titanate film via ultrasonic treatment in water.

Fig. 3: Comparison of photocatalytic activityof titanate film and Ag/AgCl/titanate film samples for the photocatalyticdecomposition of phenol in water under the visible light illumination. Thelight intensity is around 115 mW/cm2.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1442 POLYMORPHS EVOLUTION DURING CRYSTALLIZATION OF BETA ZEOLITE

Sagarzazu A.1, Gonzalez G.1
1Centro de Ing. Materiales y Nanotecnología, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela
asagarza@ivic.gob.ve

Molecular sieves are open-framework materials that can separate a mixture of different molecules on the basis of molecular size and shape. Among them zeolite beta  is     one of the most important with numerous industrial uses as a result of its structure of polymorphs  and a large pore openings of 7 Å.  It is formed by the intergrowth of two or three polymorphs (A, B and C) related by a combination of different stacking planes.  Searching for new framework topologies in such materials, with specific chemical and physical properties, a method to obtain different proportions of polymorphs has been employed in order to control the microstructure and chemistry on an atomic scale, therefore electron microscopy characterization is essential to directly analyze these structures.  Hydrothermal synthesis was used as crystallization method. The syntheses of intergrowths are based on the combination of different concentrations of   the organic template (TEAOH) and different silica/alumina ratios.     High resolution Scanning and Transmission Electron Microscopy, electron diffraction and X-ray powder diffraction techniques have been used for characterizing the structures.  To identified the different polymorphs the Multislice method was used to generate HRTEM images with specific crystallographic orientation for different focal and thickness series, using  the software Cerius2 and  the proportion of intergrowths was calculated by fitting the XRD data to the simulated patterns using DIFFaX,  a software design to calculate stacking defects in zeolites.
The mechanisms behind the formation of different proportions of polymorphs depended strongly on the synthesis parameters studied.  Although, most of the authors reported that the proportion of polymorphs in beta is 60%B-40%A, in the present work it was found this proportion fluctuates with the crystallization time for the different synthesis conditions employed, the lowest (51%B) and the highest (68%B) content of polymorph B was obtained for   SiO2/Al2O3 = 100 and TEA2O/ SiO2 =0.27 and 0.75 respectively. Therefore, it seems that high template content stabilizes polymorph B. Fig. 1 shows   HRTEM images for these samples.
Electron microscopy was an essential technique to understand the mechanism of formation of these materials and to understand their structure. The complementary use of all the microscopy techniques provided a wealth of unique information for the extensive characterization of these solids.

Fig. 1: Fig. 1 HRTEM images for beta zeolites obtained with different synthesis conditions: a., b. SiO2/Al2O3 =100,  TEA2O/ SiO2 =0.27,  2d. c., d. SiO2/Al2O3 =100, TEA2O/ SiO2 =0.75, 12d.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1461 EFFECT OF SYNTHESIS PARAMETERS ON THE MESOPOROUS STRUCTURE OF SBA-15 AND SBA-16

Soto D. A.1, Gomes M. E.1, Gonzalez G.1
1Laboratorio de Materiales. Centro de Ingeniería de Materiales y Nanotecnología, Instituto Venezolano de Investigaciones Científicas. Caracas, Venezuela.
damarysoto@gmail.com

SBA-15 and SBA-16 exhibiting arrangements of mesopores, have received particular attention for applications involving selective host-guest interactions or diffusion of large molecules.
SBA-15, prepared with the triblock copolymer PEO20PPO70PEO20 (Pluronic P123), consists of a mono-dimensional channel system distributed in a two-dimensional hexagonal structure. SBA-16 consists of two non-interpenetrating three dimensional
channel systems with spherical mesoporous cage-like cavities connected through windows.
A good understanding of how the synthesis conditions of these materials affect the meso- and macro-structure characteristics is important for their applications, since it allows to control  their properties.
In the present work the effect of variation of different synthesis conditions in the structure of both materials have been systematically investigated.
The synthesis was carried out in acidic medium from the triblock copolymers surfactants Pluronic F127 (EO106PO70EO106) and Pluronic P123 (PEO20PPO70PEO20) to obtain SBA16 and SBA 15, respectively  and TEOS was used as silica source. The synthesis parameters studied were temperature (70 to 110 ̊C), time (24 to 72 hours) and agitation. 
The synthesis carried out using Pluronic 123, resulted in an increase in pore diameter with temperature and time, from 5.6 nm at 70C, 24h to 8.3 nm at 110C, 72h and therefore  a decreases in mesoporous area and increase in  pore volume. It was observed that the microporosity was lost at high temperatures, and the mesoporous wall thickness decreases.
On the other hand, the synthesis carried out with Pluronic 127 showed a very small variation in  pore size with temperature and time. However, the mesoporous area increased, and the microporosity decreased with temperature showing some disorder on mesoporous arrangement at 100C, as it is observed by HRTEM.
Fig 1 shows HRTEM images of mesoporous materials SBA 15 synthesized at 90 °C, 48h and Fig 2 and 3 SBA 16 synthesized for 48h at 90 and 110 C, respectively, showing the disorder structure obtained at high temperatures.

Saidi Duno, Paola Patete, Edgar Cañizales for TEM facilities, Lisbeth Lozada for TEM sample preparation.

Fig. 1: SBA-15 synthesized with F123 at 90°C, 48h

Fig. 2: SBA 16 synthesized with F127 at 90°C, 48h.

Fig. 3: SBA 16 synthesized with F127 at 110°C, 48h.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1537 Characterization of silica-coated Au/Fe2O3 nanoaggregates

Krumeich F.1, Sotiriou G. A.2,3, Starsich F.2, Pratsinis S. E.2
1Laboratory of Inorganic Chemistry, ETH Zurich, Zurich, Switzerland, 2Particle Technology Laboratory, ETH Zurich, Zurich, Switzerland, 3Department of Environmental Health, Harvard University, Boston, USA
krumeich@inorg.chem.ethz.ch

The plasmonic characteristics of metals like Au or Ag dramatically change with particle size. The increased light absorption of nanoparticles (NPs) moreover depends on the wavelength and is maximized when the electrons in the conduction band are in their resonance state. Relaxation processes turn this oscillation energy into phonons, with an efficiency that depends on various parameters such as the particle size, shape and aggregation [1]. The thereby generated heat can be utilized for various applications, including cancer treatment. If Au NPs are selectively taken up by cancer cells, they can be activated photothermally by laser irradiation and the resulting heat can destroy these cells [2]. Here we report on the electron microscopy characterization of hybrid agglomerates (50 - 100 nm in diameter) consisting of SiO2-coated Fe2O3 and Au nanoparticles that show promising plasmonic and superparamagnetic properties [3]. This hybrid material was synthesized by enclosed flame spray pyrolysis, a very flexible and scalable technology [4].
TEM images (Figure 1) confirm that the Au/Fe2O3 NPs are indeed coated by an amorphous SiO2 shell which is ca. 2.5. nm thick here. The dark disks correspond to Au NPs with diameters between 10 – 40 nm, while the Fe2O3 NPs appear gray similar to the silica layer which encloses both types of NPs. The crystalline Au and Fe2O3 NPs furthermore show some lattice planes. STEM is employed for detailed characterization of these aggregates (Figure 2). In the HAADF-STEM (Z contrast) image, bright disks correspond to the Au NPs whereas faint gray areas indicate the presence of the less heavy scatterers (i. e., Fe2O3 and SiO2), as additionally confirmed by EDXS analysis of small areas (Figure 3a) and EDXS mapping (Figure 3b,c). Note that the crystalline Fe2O3 NPs are also detectable as areas showing lattice fringes in the PC-STEM image (Figure 2b) [5].
These results reveal that the Au and Fe2O3 NPs are predominantly located next to each other forming Janus-, or dumbbell-like nanoaggregates and that they are encapsulates by SiO2. The comprehensive characterization of the aggregates is important as the distance between the Au NPs determines the plasmonic interparticle coupling and this distance can be finely tuned by closely controlling the SiO2 shell thickness [3].

[1] P. K Jain et al. Accounts Chem. Res. 41 (2008) 1578.
[2] L. R. Hirsch et al., Proc. Natl. Acad. Sci. USA, 100 (2003) 3549.
[3] G. A. Sotiriou et al., Adv. Funct. Mater., 2014, http://dx.doi.org/10.1002/adfm.201303416.
[4] A. Teleki et al., Sens. Actuators, B, Chem. 119 (2006) 683; A. Teleki et al., Langmuir 24 (2008) 12553; G. A. Sotiriou et al., Adv. Funct. Mater. 20 (2010) 4250.
[5] F. Krumeich et al., Micron 49 (2013) 1.

Electron microscopy was performed at the electron microscopy center of ETH Zurich (ScopeM).

Fig. 1: TEM images of silica-coated Au-Fe2O3 aggregates revealing the coating of the Au and Fe2O3 NPs by an amorphous silica layer (microscope: Tecnai F30 (FEI), FEG, operated at 300 kV).

Fig. 2: HAADF-STEM (Z contrast) (a) and PC-STEM (phase-contrast) (b) images of the silica-coated Au-Fe2O3 aggregates (microscope: HD2700CS (Hitachi) with probe corrector (CEOS), cold FEG, operated at 200 kV).

Fig. 3: HAADF-STEM images (a,b) with the results of EDXS measurements of the indicated areas in (a) and EDXS elemental mapping (c) of (b). Au: green; Fe: blue; Si: red. (microscope: HD2700CS (Hitachi) with EDX spectrometer (EDAX Gemini system)).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1554 Photo-induced lattice accommodation in Ag/Cu composite nanoparticles

Yasuda H.1
1Research Center for Ultra-High Voltage Electron Microscopy & Graduate School of Engineering, Osaka University, Osaka, Japan
yasuda@uhvem.osaka-u.ac.jp

Nanoparticles exhibit specific structural and optical properties which are different from those of the corresponding bulk materials. The lattice softening is one of the specific properties originated from the shallow interatomic potential. On the other hand, localized plasmon in metallic nanoparticles is recently focused on the optical properties. The electric field induced by the localized plasmon may interact with the lattice vibration and enhance the lattice softening.
We confirmed in our previous research that a lattice accommodation takes place in a two-phase nanoparticle which has a lattice misfit. The lattice accommodation is induced not only on the interface between the two phases but also all over the nanoparticle. In such a lattice-accommodated two-phase nanoparticle, if only one phase is resonantly excited by the localized plasmon using well-defined photo-illumination, that is, the electron-phonon interaction is induced in the only one phase, how will the accommodated lattice behave to photo-illumination ?
In the present work, photo-induced lattice accommodation in Ag/Cu composite nanoparticles has been studies in situ by laser-coupled TEM with a double source evaporator, in order to see an electron-phonon interaction in the nanoparticles.
Fig. 1(a) shows a BFI of Ag/Cu composite nanoparticles and the corresponding DFI taken from Ag 111 reflection. Two kinds of morphologies are observed as shown schematically in the figure. One is core-shell structure (type A), and the other is particle-connected structure with a planer interface (type B). The amount of type A is larger than that of type B. Fig. 1(b) shows electron diffraction profiles from the nanoparticles before, during and after photo-illumination with the energy of 2.3 eV. All the diffractions are identified as Debye-Scherrer rings of the fcc silver and copper. The lattice constant of copper with and without photo-illumination are 0.370 and 0.366 nm, respectively. The changes in the lattice constant take place reversibly. The fact that no changes in the lattice constant are induced by photo-illumination in pure silver or copper nanoparticles denies an effect by the thermal expansion.
It was evident that photo-induced lattice accommodation takes place in Ag/Cu composite nanoparticles. Photo-illumination with the energy of 2.3 eV resonantly enhances the localized plasmon with the energy of approximately 2.0 eV in copper nanoparticles. An enhancement of the local electric field in copper nanoparticles may induce the lattice vibration and the subsequent lattice softening in the copper core region. Consequently, it is considered that the lattice of the silver shell accommodated by the copper core is relaxed to increase toward the lattice constant close to that of pure silver.

Fig. 1: (a)A BFI of Ag/Cu composite nanoparticles and the corresponding DFI taken from Ag 111 reflection. (b)Electron diffraction profiles from the nanoparticles before, during and after photo-illumination.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1562 HAADF-STEM of Road Aged Diesel Oxidation Catalysts

Ward M. R.1, Hyde T.4, Boyes E. D.1,2, Gai P. L.1,3
1Department of Physics and the York Nanocentre, University of York, UK, 2Department of Electronics and the York Nanocentre, University of York, UK, 3Department of Chemistry and the York Nanocentre, University of York, UK, 4Johnson Matthey Technology Centre, Sonning Common, UK
michael.ward@york.ac.uk

Diesel oxidation catalysts (DOCS) reduce the amount of pollutants emitted by diesel automobiles. It is well known that CO, NOx, hydrocarbons and soot are harmful to the environment (1). DOCs generally use nanoscale Pt nanoparticles supported on a γ-Al2O3 wash-coat which is held on a macro-scale monolithic structure in the car’s exhaust. Figure 1 shows a diagram of this type of structure. Pt is expensive and rare so it is a necessity to reduce the amount of Pt used in DOCs but also improve their long-term stability. One solution is to add Pd to Pt (1, 2). In addition to sintering, constituent atomic species in bimetallic nanoparticles can segregate over time. There are no detailed studies into this aging mechanism from real commercial DOCs. Here, we describe the aging mechanisms of a 57,000 km road aged bimetallic-DOC (PtPd-Al2O3) with HAADF-STEM (3).

A double aberration corrected JEOL 2200FS with Thermo Scientific Si(Li) window EDX was used (3). The specimens were prepared by first slicing and crushing monolith channels. The debris from this process was suspended in ethanol before being despotised onto a holey-C film Cu TEM grid (3). A fresh, unused monolith and the 57,000 km aged variant were supplied. Despite being used in a real automobile, there were no major issues with contamination in HAADF-STEM or HRTEM with the aged variant.

HAADF-STEM is ideally suited to image the DOC material due to the large difference in atomic number between the nanoparticles and the support. The nanoparticles were found to have grown by almost 400 % when comparing the aged DOC to the fresh DOC. The nanoparticles in the fresh DOC were 2.50 nm in diameter on average and 11.00 nm in the aged variant. In general, rounded surfaces of the nanoparticles are present in the fresh DOC. In the aged DOC, the rounded surfaces remain in the majority but the proportion of faceted nanoparticles had increased. Furthermore, HAADF-STEM and EDX was able to show that intensity variations in a minority of the nanoparticle images were attributed to the segregation of Pt and Pd (3). Pd was found at the edge of large segregated nanoparticles as predicted by theory (4). In some cases, the Pt/Pd appeared to form bands within the nanoparticles suggesting partial segregation which has not been observed from a commercial DOC before.

References

1. M. V. Twigg, Catal Today 117, 407 (2006).

2. A. Russell, W. S. Epling, Catal Rev 53, 337 (2011).

3. M. R. Ward, T. Hyde, E. D. Boyes, P. L. Gai, Chemcatchem 4, 1622 (Oct, 2012).

4. A. B. Shah et al., Nano Lett 13, 1840 (Apr, 2013).

The authors thank the EPSRC for support from critical mass grant EP/1018058/1.

Fig. 1: Structure of a DOC monolith (a) inside exhaust, (b) magnified view of the monolith channels and (c) magnified view of the wash-coat (Al2O3) and nanoparticles

Fig. 2: Typical HAADF-STEM images of the (a) fresh and (b) road aged DOC showing Pt-Pd nanoparticles
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1586 Characterisation of mesoporous silica nanoparticles for chemotherapeutic applications

Young N. P.1, Huang X.2, Townley H. E.2
1Department of Materials, University of Oxford, Parks Road, Oxford, U.K. , 2Department of Engineering Science, University of Oxford, Oxford, U.K.
neil.young@materials.ox.ac.uk

Mesoporous silica nanoparticles present a wide range of applications, amongst which is an attractive means of delivery for pharmaceuticals within the body. Utilising the high internal surface area, tunable size and low toxicity of these nanomaterials, drugs may be targeted to sites within the body, yielded improvements over conventional treatment methodologies. In this study we have investigated the suitability of a number of different mesoporous silica nanoparticle structures for carrying a drug cargo [1]. Nanoparticles were characterised in terms of their physical parameters; size, surface area, internal pore size and structure. Additionally these were compared to properties specific to successful application in drug delivery; namely the loading and unloading profiles for a model therapeutic, and also the response of nanoparticles to conditions similar to those found inside the body. This data allows an informed decision to be made on the optimum nanoparticle structures required to maximise cargo capacity and optimise temporal control of the unloading. Controlled capping of the pores was also found to improve on the drug delivery capability.

Figure one shows SEM and TEM images of three of the classes of mesoporous silica nanoparticles investigated in the present study. These were named hexagonal mesoporous silica nanoparticles (a,b), blackberry-like mesoporous silica nanoparticles (c,d), and finally chrysanthemum-like mesoporous silica nanoparticles (e,f) on the basis of their structures. Overall the hexagonal particles were found to be ideally suited to drug delivery following confirmation of the properties described above. High-resolution TEM and tilt-series HAADF-STEM were used to fully characterise the internal pore structure and arrangement within these nanoparticles. Importantly this was found to be ordered with pore channels that were continuous throughout the volume of the nanoparticles, contributing to the high cargo carrying potential and efficient unloading profile.

[1] X. Huang, N.P. Young, H.E. Townley, Nanotechnology and nanomaterials 4 (2014) 1. DOI: 10.5772/58290

Fig. 1: A range of mesoporous silica nanoparticles imaged via SEM and TEM. Hexagonal (a) and (b), blackberry-like (c) and (d) and chrysanthemum-like (e) and (f).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1589 The effect of Ce0.8La0.2O1.9 support modifiers on the microstructure and N2O decomposition (de-N2O) performance of γ-Al2O3 supported Ir catalysts

Delimitis A.1, Pachatouridou E.1,3, Papista E.2, Iliopoulou E. F.1, Marnellos G. E.1,2, Konsolakis M.3, Yentekakis I. V.3
1CPERI / CERTH, Thermi, Thessaloniki, Greece, 2University of Western Macedonia, Kozani, Greece, 3Technical University of Crete, Chania, Crete, Greece
andel@cperi.certh.gr

N2O has been widely recognized as a hazardous greenhouse gas exhibiting 300 times higher Global Warming Potential compared to CO2, as well as an ozone layer destruction contributor. One of its major sources is fossil fuels and biomass combustion and, consequently, several methodologies have been considered towards its end-of-pipe emission control. Catalytic decomposition represents the most promising method, due to lower energy demand and cost. Currently, Ir-based catalysts have gained considerable interest as promising alternatives for de-N2O process. Enhancement of the Ir active phase intrinsic features via support-mediated promotional effects comprises the subject of the present study. In particular, the effect of Ce0.8La0.2O1.9-modified γ-Al2O3 support (AlCeLa) on the Ir nanostructural characteristics and its de-N2O activity is investigated, using a combination of electron microscopy (TEM, HRTEM) and image analysis methods.
The morphology of the unmodified 0.5 wt% Ir/γ-Al2O3 sample is depicted in Fig. 1. IrO2 catalyst adopts both a medium-size (up to 70 nm), crystalline rectangular particle morphology (a), as evidenced by the Selected Area Diffraction (SAD) pattern in (b), and a smaller and disordered particles one (c), densely aggregated on top of γ-Al2O3. Supporting Ir on AlCeLa results in the exclusive formation of larger size, highly crystalline IrO2 particles, as illustrated in Fig. 2(a) and (b), although the Ir loading is identical in both catalysts. The particles’ mean size is up to 500 nm in Ir/AlCeLa. Their high crystalline quality is presented in Fig. 3(a), where the edge of a IrO2 particle is shown, viewed along its [001] zone axis. Measurements of the lattice spacings resolved in the image resulted in d110=0.317 nm and d200=0.223 nm, in good agreement with their theoretical values. This is further confirmed by the Geometric Phase Analysis (GPA) results in Fig. 3(b). The strain map reveals a uniform distribution, even at the surface crystal edges, where any contamination by impurity elements or crystal defects formation may be more pronounced. Strain leaps, white arrowed in Fig. 3, were only measured at regions of crystal misorientations due to particle inclination.
The superior structural quality of Ir/AlCeLa catalyst was reflected in its outstanding ~100% and 90% N2O conversion records, in the absence and presence of O2, respectively. This is most probably a result of the trend of oxygen, formed by N2O decomposition, to desorb more easily from highly crystalline, clear IrO2 surfaces rather than from defected cites, mainly present in disordered, poorly crystalline small Ir particles. This inevitably leads to higher N2O decomposition activity in the former case, rendering Ir/AlCeLa a highly efficient de-N2O catalyst.

Financial support by the program “THALES” (MIS 375643), co-financed by the Greek Ministry of Education and Religious Affairs and the European Social Fund is acknowledged.

Fig. 1: (a) and (c) TEM images from the Ir/γ-Al2O3 catalyst, revealing the two distinct morphologies; (b) typical SAD pattern from the area in (a). Reflections attributed to IrO2 are denoted in (b).

Fig. 2: (a) TEM image and (b) [001] SAD pattern of a typical IrO2 particle in the Ir/AlCeLa catalyst. The difference in size and crystallinity is outlined.

Fig. 3: (a) HRTEM image, viewed along [001] and corresponding GPA strain map (b) from the edge of an IrO2 particle in the Ir/AlCeLa catalyst. A uniform distribution of strain is illustrated; peaks are only observed at regions of crystal inclination, as shown by the strain profile inset in (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1619 High resolution HAADF investigation of Ga droplets on Si(001) surfaces

Beyer A.1, Werner K.1, Stolz W.1, Volz K.1
1Philipps-Universität Marburg, Faculty of Physics and Materials Science Center, Marburg, Germany
andreas.beyer@physik.uni-marburg.de

The growth of III/V material on Si substrates opens a wide field of new applications and devices [1]. The initial stages of the nucleation and especially the type of element (III or V) it is started with may have a crucial impact on the interface structure and therefore a device´s performance [2]. In this study Ga was deposited on Si substrates without the presence of group V elements to investigate the processes occurring during these early stages of growth.
The samples were grown via metal organic vapor phase epitaxy in an AIX 200 GFR reactor. To investigate the impact on the morphology two different precursors, triethylgallium and trimethylgallium, were used and the growth temperature was varied between 400 and 500°C. Electron transparent samples were prepared along an <110> axis of the silicon by conventional mechanical grinding and final ion milling in a Gatan PIPS. The samples were characterized in a double C S-corrected JEOL JEM 2200 FS scanning transmission electron microscope (STEM) operating under high angle annular dark field (HAADF) conditions resulting in Z-contrast.
On the surface of the Si Ga droplets form which can be identified in HAADF images by their bright contrast, due to the higher atomic number of Ga with respect to Si (Fig. 1). The number of droplets clearly scales with amount of supplied precursor during the growth. Moreover, the droplets are not only confined to the surface but penetrate into the Si forming a pyramidal structure with boundaries on {111} lattice planes. Complementary energy dispersive X-ray measurements confirm that these pyramids contain Ga. The observed morphology can be explained by the fact that the liquid Ga etches the Si at the growth temperature. By addition of a precursor for group V after the Ga deposition, crystallization of the droplets can be enforced. Therefore, the droplets can serve as nucleation sites for the growth of low dimensional materials like nanowires.
This contribution will show how HAADF STEM can be used to investigate etching processes on an atomic scale.

References

[1] Liebich et al., Appl. Phys. Lett. 99, 071109 (2011).
[2] Volz et al., J. Cryst. Growth 315, 37 (2011).

Funding of the DFG in the framework of GRK 1782 is gratefully acknowledged.

Fig. 1: High resolution HAADF image of Ga droplet formed on the Si surface. The droplet penetrates into the Si and is framed by boundaries on {111} lattice planes which are indicated by broken lines.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1623 Plasmonic properties of hollow AuAg nanostructures by STEM-EELS

Genç A.1, Arenal R.2, 3, Patarroyo J.4, Henrard L.5, Gonzalez E.6, Puntes V.4, 7, 8, Arbiol J.1, 8
1Institut de Ciència de Materials de Barcelona, CSIC, Campus de la UAB, 08193 Bellaterra, Spain., 2ARAID Fondation, 50018 Zaragoza, Aragon, Spain, 3Laboratorio de Microscopias Avanzadas(LMA), Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, 50018 Zaragoza, Spain., 4Catalan Institute of Nanotechnology (ICN), Campus de la UAB, Edifici Q (ETSE), 08193 Bellaterra, Barcelona, Spain, 5Department of Physics, University of Namur, rue de Bruxelles 61, B-5000 Namur, Belgium, 6Instituto Geofísico, Facultad de Ingeniería, Pontificia Universidad Javeriana, 110231, Bogota, Colombia, 7Universitat Autònoma de Barcelona (UAB), Campus de la UAB, 08193 Bellaterra, Barcelona, Spain, 8Institucio Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Catalonia, Spain.
agenc@icmab.es

The surface plasmon resonances are the collective oscillation of the conduction electrons of a metal excited by an electromagnetic radiation. During the last decade, plasmonic properties of metal nanoparticles have been attracted great interest owing to their potential applications in different fields such as electronics, photonics, biotechnology and Raman spectroscopy. Characteristics of the surface plasmon resonances, hence plasmonic properties, are known to be affected by the small modifications in size, shape and composition of the nanostructures, therefore it is essential to be able to directly correlate the surface plasmon resonances with the structural properties at the nanoscale. In this study, we have obtained the in-plane 2D distribution of the surface plasmonic resonances of hollow AuAg nanostructures [1], by means of low loss electron energy loss spectroscopy (EELS) in an aberration corrected scanning transmission electron microscope (STEM), equipped with a monochromator, with sub-eV and sub-nanometer resolutions. The studied complex nanoparticles are nanoengineered from solid Ag cubes to different hollow AuAg nanostructures such as nanoframes and multi walled nanoboxes [1]. We have investigated the local plasmonic property modulations on each nanostructure and correlated them their structural features. We have also correlated the obtained experimental results with models performed in the frame of discrete dipole approximation.

[1] E. González, J. Arbiol, V. F. Puntes, Science, 334, 1377 (2011).

Aziz Genç acknowledges the Ministry of National Education of Turkey for the PhD scholarship. 

Fig. 1: Figure 1: (a) background substracted EEL spectra extracted from the selected areas in the inset EELS SI. (b) is the plasmon energy map between 1.9 and 2.4 eV and (c) shows plasmon intensity maps between 1.8 and 3 eV with 0.2 eV windows (please note that the intensities are normalized for all maps).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1717 Enhanced Phytochemical approach for fabrication of Cobalt Nanoparticles

Debut A.1, Kumar B.1, Cumbal L.1
1Centro de Nanociencia y Nanotecnologia, Universidad de las Fuerzas Armadas ESPE, Av. Gral. Rumiñahui s/n Sangolqui, P.O. BOX 171-5-231B, Ecuador
apdebut@espe.edu.ec

Fabrication of cobalt nanoparticles using Passiflora tripartita var. mollissima fruit extract is an ecofriendly approach; produces various sizes and morphologies, including spherical, hexagonal and triangular. The P. tripartita fruit, known in Ecuador as “taxo & tumbo” belongs to the Passifloraceae plant family comprises around 530 species originated from temperate and tropical South America. Two different sonication conditions were employed for the synthesis of the cobalt nanoparticles and their growth recorded, in order to analyze the effect of the phytochemical synthesis and the ultrasonic irradiation on the morphology and size of the final product. The synthesized nanoparticles were characterized by U.V.-Vis, Dynamic Light Scattering, Transmission Electron Microscopy (TEM) with Selected Area Electron Diffraction (SAED) and X-ray diffraction. TEM analysis showed polydispersed nanoparticles with size ranges from 110 nm to 10 nm at different time interval and ultrasonic irradiation power. The X-ray diffraction analysis revealed the face-centered cubic geometry and SAED confirmed partial crystalline or amorphous nature of cobalt nanoparticles. Infrared spectrum measurements were carried out to hypothesize the possible biomolecules (flavonoid C & O-glycosides) responsible for stabilization the cobalt nanoparticles using P. tripartita. This simple, ecofriendly, and significantly low-cost protocol can be employed at ease and compatibility for pharmaceutical and biomedical applications.

This scientific work has been funded by the Prometeo Project of the National Secretariat of Science, Technology and Innovation (SENESCYT), Ecuador.

Fig. 1: Cobalt nanoparticles at different sonication conditions
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1738 TEM/STEM investigations of silver nanoparticles embedded in titanium oxides for photochromic applications

Pailloux F.1, Diop D. K.1,2, Babonneau D.1, Simonot L.1
1Pprime Institute, UPR 3346 CNRS-Univ. Poitiers, France, 2LHC, UMR CNRS 5516, St Etienne, France
frederic.pailloux@univ-poitiers.fr

Nanocomposite films composed of Ag nanoparticules (NP) within a TiOx matrix present photochromic properties [1]. The permanent or reversible changes of color occurring under illumination by UV/visible lasers rely on the control of the localized surface plasmon resonance (SPR) of Ag NP. They result from the tuning of the NP size/shape distribution through photo-activated redox reactions occurring specifically with the TiOx matrix.

The morphology of Ag:TiOx nanocomposite is investigated by high-angle annular dark-field HAADF-STEM, structural informations are obtained by energy filtered electron diffraction (EFED) coupled with electron energy loss spectroscopy (EELS). Samples grown under different deposition conditions (TiOx thickness, O2 pressure in the chamber, ...) are investigated.

For TiOx grown under a metallic sputtering mode (low oxygen pressure), the STEM images reveal an homogeneous distribution of Ag nanoparticles with a rather large size distribution and various shapes. Their cristallinity is assessed by energy filtered electron diffraction. The porosity of the TiOx matrix is revealed by the HAADF images. Whereas the diffraction pattern of TiOx would suggest an amorphous structure the ELNES recorded on the O K and Ti L23 edges suggest the presence of a short range ordering of theTiO6 octaedra. The change of growing mode (under higher oxygen pressure) for TiOx, produces dramatic changes in the morphology of the nanocomposite: the Ag particles, if still present, have not been clearly resolved.

The influence of the thickness of the TiOx capping layer is investigated as well. It reveals that a threshold thickness exists below which the samples become sensitive to the electron beam, which promote morphological changes of the nanoparticles.

The morphological and structural insights are further compared with in-situ reflectance measurements [2].

References

[1] L. Nadar, N. Destouches, N. Crespo-Monteiro, R. Sayah, F. Vocanson, S. Reynaud, Y. Lefkir, J. Nanopart. Res. 15 (2013) 2048

[2] V. Antad, L. Simonot, D. Babonneau, Nanotechnology 24 (2013) 045606

This work is supported by ANR Photoflex project

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1748 Nano-Branched Free Standing Gold Foils

Rodríguez-González B.1, Vázquez-Vázquez C.2, Ameneiro Prieto O.2, Correa-Duarte M. A.2
1CACTI, University of Vigo, E-36310 Vigo, Spain, 2Department of Physical Chemistry, University of Vigo, E-36310 Vigo, Spain
jbenito@uvigo.es

Herein we present results about the obtaining, characterization and formation mechanism of nano-branched free standing gold foils. Dendritic and nano-branched gold foils have potential applications as SERS active substrates in sensing.[1] Foils were prepared by a facile wet chemical synthesis method using gold salt and a reducing agent derived from the formaldehyde molecule. The overall method is cost-effective and allows for a facile transfer to a wide range of substrates for different sensing applications. As example, we have achieved the transfer of the nano-branched gold foil to paper, glass and silicon wafers.

In Figure 1(A) we show a low magnification TEM image of one of the obtained foils, it is clear the nano-branched structure displayed by the gold foil. This structure looks convenient for the flowing of liquids or gases through the large openings between the branches; this opens a wide field of versatile applications. Figure 1(B) shows the branches in more detail; they are formed by polycrystalline gold with multiple grain boundaries and twinning planes. The presence of those defects is a direct consequence of the foil formation mechanism.

In order to understand the formation mechanism, and the origin of the final structure of the foils, we have conducted electron microscopy studies over samples removed from the reaction vessel at different reaction times. These studies allow us to propose a multi-step formation mechanism. The first step is the reduction of the gold followed by the immediate formation of small gold nanoparticles or clusters in the reaction medium. These nanoparticles migrate to the liquid surface where the foil starts to develop due to an aggregation process. The arrangement in branches and gaps are due to a particular disposition of the particles along the aggregation process.

[1] Y. Wang, M. Becker, L. Wang, J.Liu, R. Scholz, J. Peng, U.h Gösele, S. Christiansen, D. H. Kim, and M. Steinhart, Nano Letters 2009 9 (6), 2384-2389

Fig. 1: (A) Low magnification TEM image of the nano-branched gold foil. (B) TEM image showing the grain boundaries and defects of the branches.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1779 In-situ observation of morphological changes of gold nanorods under near infrared pulsed laser irradiation

Matsumura S.1, Sumimoto N.1, Yamamoto T.1, Yasuda K.1, Niidome Y.2
1Department of Applied Quantum Physics and Nuclear Engineering, Kyushu University, Fukuoka, Japan, 2Department of Chemistry and Bioscience, Kagoshima University, Kagoshima, 890-8580, Japan
syo@nucl.kyushu-u.ac.jp

The controllable optical properties of metal nanoparticles have been an active research field because of their potential technological applications. Gold nanorods are ultrafine particles 20?150 nm in length and 5?20 nm in diameter. Their anisotropic shape gives rise to a surface plasmon (SP) absorption band corresponding to the longitudinal SP mode along the long axis in the near infrared region, in addition to a SP band for the transverse mode in the visible light region. The characteristic wavelength of the former SP mode is controlled by the aspect ratio of the rods. The longitudinal SP band is usually much more pronounced than the transverse SP band, and it is exploited for potential technological applications unique to gold nanorods. When irradiated with pulsed laser light, gold nanorods are deformed into different shapes, such as spheres, Φ-shape and elongated rods. Qualitatively, such deformations are considered to result from heating due to light absorption. However, the deformation behavior of gold nanorods remains largely unknown because most of the experiments have been performed ex-situ in irradiated aqueous solution. Recently, we erected a pulsed laser-light illumination system attached to a high-voltage electron microscope (HVEM) to observe light-induced behaviors of nano objects. In the present study, we observe in-situ the structural transformation in gold nanorods induced by irradiation. The JEM-1300NEF HVEM was operated at an accelerating voltage of 1250 kV, and laser pulses of λ= 1064 nm with 6-8 ns duration were simultaneously illuminated.

　Figure 1 reveals a sequential structural change in gold nanorods irradiated by 0, 1, and 7 laser pulses at 310 J/m2/pulse. One may notice that most of the gold nanorods have transformed their shape after a single laser pulse. However, additional laser pulses induce little further change in the nanorods, as shown in Fig. 1 (c). This attenuation of deformability can be explained in terms of the blue shift of the longitudinal SP band with the decrease of aspect ratio. HRTEM imaging reveals that the outer deformation is accompanied by total atomic restructuring in the nanorod interiors, involving generation and annihilation of planar defects, as shown in Fig. 2.

The present study was partly supported by Gant-in-Aid for Challenging Exploratory Research (#23656387) and for Scientific Research (B) (#25289221) from JSPS.

Fig. 1: In-situ observation of gold nanorods irradiated with laser pulses at 310 J/m2/pulse. Before irradiation (a), after 1 pulse (b), and after 7 pulses (c).

Fig. 2: HRTEM images of a gold nanorod before laser irradiation (a), after exposure to 1 pulse (b) and 2 pulses (c). Laser intensity is 425 J/m2/pulse.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1849 Atomic stoichiometry of the industrial-style Co-promoted MoS2 nanocatalysts

Zhu Y.1, Ramasse Q. M.2, Brorson M.1, Moses P. G.1, Hansen L. P.1, Kisielowski C. F.3, Helveg S.1
1Haldor Topsøe, Nymøllevej 55, DK-2800 Kgs. Lyngby (Denmark), 2SuperSTEM Laboratory, STFC Daresbury, Keckwick Lane, Daresbury WA4 4AD (UK), 3National Center for Electron Microscopy and Joint Center for Artificial Photosynthesis, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94708 (USA)
yuaz@topsoe.dk

The knowledge of the position and the chemical identification of atoms at non-periodic sites in nanostructured catalysts is essential for the understanding of their catalytic functionality and can eventually lead to rational materials design. In the field of oil refining, transport fuels with ultra-low sulfur contents are produced by catalytic hydrodesulfurization (HDS) processes that rely on nanocrystalline MoS2–based catalysts.1 The HDS activity of MoS2 nanocrystals can be significantly promoted by transition metals such as Co and this catalytic activity enhancement is commonly attributed to the so-called “Co-Mo-S” phase, having Co located at the edges of the Mo plane of the MoS2 nanocrystals.2 However, detailed structural information regarding the Co promoter for the industrial catalysts is lacking.3

Recent advances in high-resolution (scanning) transmission electron microscopy ((S)TEM) imaging have opened up the possibility to study industrial-style MoS2 nanocatalysts with atomic-level resolution and sensitivity.4 By means of high-resolution TEM and high-angle annular dark-field (HAADF) STEM, the elemental distribution in unpromoted single-layer MoS2 nanocrystals was resolved5 and allowed for a distinction of the edge terminations.6 A combination of aberration-corrected HAADF imaging and electron energy-loss spectroscopy (EELS) is a promising approach for atomic chemical analysis; however, few characterizations have been possible owning to experimental challenges, such as the fine balance between interpretable signals and electron beam damage.

Here, we employed atomic-resolved HAADF-STEM and EELS at low primary electron energy to obtain the first site-specific identification of single-atom Co promoter and the associated S reconstruction in doped single-layer MoS2 nanocrystals (Fig. 1). Interestingly, single-atom Fe contaminants were unambiguously identified, competing with Co for the same sites at the S-edge. The present analytical capability of pinpointing local stoichiometry atom-by-atom with one atomic number sensitivity could be highly beneficial for improving the accuracy of the knowledge on complex nanostructures.

1 F. Besenbacher, et al., Catalysis Today 130, 86 (2008).

2 H. Topsøe, et al., Hydrotreating Catalysis (Springer, 1996).

3 O. Sorensen, et al., Applied Catalysis 13, 363 (1985).

4 C. O. Girit, et al., Science 323, 1705 (2009).

5 C. Kisielowski, et al., Angewandte Chemie, International Edition 49, 2708 (2010).

6 L. P. Hansen, et al., Angewandte Chemie, International Edition 50, 10153 (2011).

7 Y. Zhu, et al., (2014) submitted.

This work is supported in part by the UK Engineering and Physical Sciences Research Council, the Office of Science, Office of Basic Energy Sciences of the U.S. Department of Energy, the Danish Council for Independent Research (grant HYDECAT) and for Strategic Research (grant CAT-C).

Fig. 1: Fig. 1. a) High-resolution HAADF image of the S-edge of a monolayer Co-Mo-S nanocrystal. Corresponding EEL elemental maps of b) the combination of Mo (blue) and Co (red) and c) of S (yellow). d) Sum of EEL spectra, integrated over a 3 x 3 pixel window (probe size). f) Industrial-style Co-Mo-S ball model, with a side view of the Co-promoted S-edge.7
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1853 Nanostructured cobalt ferrite for gas sensing

Leroux C.1, Madigou V.1, Giorgio S.2, Lopes-Moriyama A. L.3, Pereira de Souza C.3
1University of Toulon, CNRS, La Garde, France, 2Aix Marseille University,CNRS, Luminy, France, 3Universidade Federal do Rio Grande do Norte, Natal, Brazil
leroux@univ-tln.fr

Nowadays, measurement and control systems for pollutant and toxic gas emissions gain increasing importance in the frame of sustainable development. Although gas sensors devices are widely commercialized, they still suffer from drawbacks like lack of selectivity and stability. Parameters like time of reaction, time of recovery, reproducibility, working temperature, should also be considered. To overcome some of these disadvantages, nanostructured materials are investigated. The detection function of the sensing material is dependant of a high surface to volume ratio, but also to the exposed crystallographic facets. Nanoparticles present high surface to volume ratio, but tend to agglomerate. One way to overcome to some extent this phenomenon is to build hierarchical and hollow oxide nanostructures [1]. The transduction function of the sensing material is more linked to the composition and structure. It should be possible to tailor the reactivity and sensitivity of the sensing materials by controlling their composition, their structure, phase, shape, size, and size distribution [2]. Hence, we were interested in studying cobalt ferrites as nanoparticles or thin films for applications in gas sensors. The cobalt ferrite (CoFe2O4) attracts considerable attention due to its good chemical stability, mechanical hardness, magnetic behavior and catalytic activity [3-4].

Octahedron-like nanoparticles of CoFe2O4 were synthesised using a hydrothermal technique. Several microscopy techniques like SEM, conventional TEM coupled with EDS, high resolution TEM, environmental TEM, were carried out in order to understand the mechanisms involved in the growth of the grains and their reaction under gas. The particles have an octahedral shape, with sizes around 20 nm (Figure 1). Samples were observed in a TEM under 1mbar gas pressure and were submitted to H2 -O2 cycles, at ambient temperature. The {111} facets became more rounded under oxygen (Figure 2). Before that, the {100} facets extended which led to truncated octahedra. The same phenomenon was already observed in case of metallic nanoparticles [5].

References

[1] J.-H. Lee, Sensors and Actuators B 140 (2009) 319–336

[2] C. Wang, L. Yin , L. Zhang, D. Xiang and R. Gao, Sensors 2010, 10, 2088-2106

[3] D.S. Mathew, R.S. Juang, Chem. Eng. J. 129 (2007) 51–65.

[4] L. Ajroudi,S. Villain,V. Madigou,N. Mliki,Ch. Leroux, J. Cryst. Growth 312 (2010) 2465–2471.

[5] M. Cabié, S. Giorgio, C.R Henry, M. Rosa Axet, K Philippot, B. Chaudret,J. Phys. Chem. C 114 2160-2163, 2010

This work was done in the general framework of the CAPES COFECUB Ph-C 777-13 and ARCUS PACA BRESIL french-brazilian cooperation projects.

Fig. 1: The same CoFe2O4 nanoparticle viewed along a [110] direction, and viewed along a [111] direction after tilting, along with a drawing of the octahedron projection.

Fig. 2: Evolution of one CoFe2O4 nanoparticle under O2.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1856 Covellite nanocrystals and their evolution by addition of metals atoms: HRTEM and Exit Wave study

Bertoni G.1,2, Riedinger A.1, Xie Y.1, Brescia R.1, Pellegrino T.1, Manna L.1
1Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy, 2CNR-IMEM, Parco Area delle Scienze 37/A, 43124 Parma, Italy
giovanni.bertoni@imem.cnr.it

Copper sulphides (Cu2-xS) and related nanocrystals are promising candidates in optoelectronics device, due to their intrinsic p-type doping and tunability of their band-gap with stoichiometry.
In particular, the covellite structure (CuS) has one third of the Cu atoms in triangular coordination and two thirds of Cu atoms in tetrahedral coordination. At the same time, two thirds of S atoms form disulfide groups and one third are single sulfide ions.(1) At the tetrahedral sites, the Cu atoms are bound to the S atoms of the disulfide bonds. These different sites can be resolved in HRTEM (here we use negative C3 imaging conditions) or Exit Wave reconstructions (EWR) from side views (as [100] or [210] orientations) of Cu1.1S nanocrystals (see Figure 1). Indeed, at opportune values of defocus ΔZ, the S-S disulfide layers connected appear bright in the image, permitting to directly visualize them.
Our group demonstrated how the evolution from Cu1.1S (covellite type) to Cu2S (chalcocite) was accompanied by the red-shifting of the localized surface plasmon resonance (LSPR) generated by free holes generated in the valence band (and with a dominant in-plane mode).(2) The resonance gradually disappears as the Cu2S stoichiometry is reached (i.e. no copper deficiencies left). These states seem then to be linked to the disulfide bonds present of the CuS covellite structure (see Figure 1). In general, we expect by adding Mx+ atoms, the following transformation:

Cu1.1S + γMx+ + γe- → Cu1.1MγS ,

in which S(-1) is reduced to S(-2), possibly by breaking the S-S bonds, and the metal keeps its (x+) oxidation state. In this presentation we focus on Pd(II) doped CuS nanocrystals synthesized by chemical methods. Pd is added by using Palladium(II)-acetylacetonate plus ascorbic acid. We see how by increasing the amount of Pd atoms, the CuS structure is progressively lost, as the number of disulfide layers is gradually reduced, as can be seen from the HRTEM images (see Figure 2). Consequently, the density of holes in the valence band is lowered and the plasmon resonance is consequently red-shifted and reduced.
This is indeed a demonstration of the tunability of the LSPR with the amount of metal atoms in Covellite type nanocrystals.

[1] Pattrick R.A.D. et al. Geochim. Cosmochim. Acta, 1997, 61 (10), pp. 2023–2036
[2] Xie Y. et al. J. Am. Chem. Soc., 2013, 135 (46), pp. 17630–17637

European Union FP7/2007-2013 Grant Agreement 240111 (ERC Grant NANO-ARCH) and European Union FP7 Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3).

Fig. 1: a) EWR phase together with the experimental image (ΔZ = -34 nm, C3 = -26 um) and the simulated image from [210] side view of a CuS nanodisk. At about -30/+20 nm defocus the intensity is transferred to the planes with Cu-S in tetrahedral coordination, giving a direct visualization of S disulfur planes (see the intensity profile in b).

Fig. 2: The nanocrystals maintain their hexagonal shape while adding Pd metal atoms. However, the number of S-S layers is progressively reduced as Pd increases, as well as the LSPR is reduced in intensity and red shifted.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1874 Plasmon Enhanced Fluorescence Imaging on Linear Arrays of Metal Half-Shells

Farcau C.1, 2, Astilean S.1, 2
1Institute for Interdisciplinary Research in Bio-Nano-Sciences, Babes-Bolyai University, Cluj-Napoca, Romania, 2Faculty of Physics, Babes-Bolyai University, Cluj-Napoca, Romania
cosmin.farcau@phys.ubbcluj.ro

Recent studies on the fluorescence emission of fluorophores located nearby metallic
nanostructures allowed the observation of peculiar phenomena such as modification of
radiative transition rates, enhancement of emission quantum yield, or directional emission.
The mentioned effects are induced by coupling between emitter dipole and surface plasmon
polariton excitations. Controlling these interactions with ordered metal nanostructures
bearing well-defined plasmon modes offers the means for advancing applications requiring
e.g., an enhanced emission, improved photostability, or larger FRET distances.

Here we discuss our studies of Surface Enhanced Fluorescence (SEF) on metal-coated
colloidal uni-axial arrays. These hybrid plasmonic-photonic crystals were obtained
by colloidal convective self-assembly (CSA) on DVD templates and metal film evaporation [1].
Their morphology is resolved by electron microscopy and atomic force microscopy (see Figure 1),
while their polarization-sensitive optical response is evidenced by transmission and
reflectivity micro-spectroscopy. SEF of fluorophores adsorbed on top of a spacing layer
are studied by both steady-state and time-resolved fluorescence. Furthermore, fluorescence
lifetime imaging (FLIM) is performed to highlight the correlations between topography / optical
response / SEF. These results on fluorescence emission enhancement, plasmon-controlled emission
polarization, lifetime modification and imaging can be of interest both fundamentally, for
better understanding of plasmon-coupled emission, but also from the application point of view, for the
design of sensors or other light-emitting devices.

[1] V. Saracut, M. Giloan, M. Gabor, S. Astilean, C. Farcau, ACS Appl. Mat. Interf. 2013, 5, 1362.

This work was supported by a grant of the Babes-Bolyai University, under the contract GTC_34057/2013.

Fig. 1: AFM image of the hybrid plasmonic-photonic crystal consisting of gold half-shells onto polystyrene microsphere linear arrays.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1906 EDXS on MoS2-base/Al2O3 HDS catalysts: A chemical distribution study of silicon

Angeles-Chavez C.1, Toledo-Antonio J. A.1, Cortes-Jacome M. A.1
1Mexican Institute of Petroleum, Molecular Engineering, Distrito Federal, MEXICO
cangeles@imp.mx

Electron microscopy (SEM and TEM) is a powerful tool for the characterization of a wide range of solid catalysts. Both microscope types give direct evidence of the morphology, chemical composition and crystalline structure in the different scales (micrometer to nanometer). The improvements of the instruments in the spatial resolution, energy resolution, efficiency of the detectors and data collection, has improved very much the quality of the obtained results and the silicon dispersion on gamma alumina particles used for the preparation of MoS2-based (hydrodesulfurization) HDS catalysts shows these new capabilities.
MoS2-base/Al2O3 HDS catalysts are widely used to remove sulphur from hard-to-desulfurize compounds such as 4,6-dimethyldibenzothiophene. Their catalytic performance is directly related to the dispersion of the MoS2 structure and the current scientific research is focused on achieving higher dispersion of Co-Mo-S active catalytic sites. In this work, we add silicon atoms on the surface of the alumina particles to modify their acidic properties and increase the dispersion of the Co-Mo-S structure. The Si atoms were aggregated to tri-lobular extruded of alumina by an incipient wet process using a silicon solution. Subsequently, the extruded were calcined and characterized by SEM and TEM.
The concentration of O, Al and Si in the sample, obtained by EDXS, was 46.90, 49.74 and 3.36 wt% in average, respectively. This chemical quantification indicates that the Si was integrated as SiO2 in the sample to a concentration around 7.2 %. The silicon permeation in the extruded was revealed by a composition study through the cross section of extruded. The result obtained is shown in Figure 1. A homogeneous concentration of silicon inside the extruded is observed. Therefore, this sample was the strongest candidate to impregnate the active phases (P, Co and Mo). Their dispersion was evidenced by concentration profiles (Figure 1) and chemical mapping, see Figure 2. Homogeneous dispersion of Co and Mo is appreciated in both results. However, the dispersion silicon was heterogeneous. This sample was subsequently sulfided to produce the Co-Mo-S structures. The result obtained is illustrated in Figure 3. MoS2 structures fully dispersed on the Al2O3-7. 2%SiO2 surface in HRTEM images is observed. Therefore, from these first results, the presence of SiO2 on gamma-alumina contributes to the formation of the MoS2 structures. However, still it is necessary improve the spreading of silicon in the extruded.

The authors acknowledge financial support to IMP through project D.00447.

Fig. 1: Concentration profiles in the tri-lobular extruded. Before impregnation (Si graph) and after impregnation (Co, Mo and P graphs).

Fig. 2: Chemical mapping of Si, Co and Mo in the tri-lobular extruded after impregnation.

Fig. 3: HRTEM image showing the MoS2 structures on the Al2O3-7.2%SiO2 surface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1970 High resolution HAADF-STEM imaging of MoS2 nanolayers in industrial hydrotreating catalysts

GAY A. S.1, GIRLEANU M.2, TALEB A. L.1, BAUBET B.1, HUGON A.1, DEVERS E.1, ERSEN O.2
1IFP Energies Nouvelles - Rond point de l'échangeur de Solaize - BP 3 - 69360 Solaize (France), 2IPCMS-UMR 7504 CNRS-Univ. de Strasbourg - 23 rue du Loess - BP 43, 67034 Strasbourg cedex 2 (France)
anne-sophie.gay@ifpen.fr

Current severe environmental legislations constrain a strong decrease of sulfur concentration in fuels. Thus, the improvement of hydrotreating catalysts is of major importance. Co-promoted MoS2 based catalysts supported on alumina are known to be industrially used in the selective gasoline hydrodesulfurization (HDS) process. The challenge is to increase the selectivity of these catalysts. Catalyst performance (in particular selectivity) is suspected to be related to the local structure (ie 2D morphology) of the active phase, composed of MoS2 nanolayers promoted by cobalt. The equilibrium morphology is usually well predicted by theoretical approaches based on density functional theory (DFT) [1,2]. For since, it has also been visualized in model materials supported on gold or graphite by STM [3] and HAADF-STEM [4].
For that study, we observed MoS2 and CoMoS industrial catalysts, supported on delta-alumina by HAADF-STEM using a JEOL TEM 2100F with a Cs-corrected condenser. MoS2 and CoMoS catalysts were prepared by incipient wetness impregnation and sulfided under pure H2S at atmospheric pressure, either at 550°C or 700°C.
In MoS2 catalyst sulfided at 550°C, nanolayers present mainly a truncated triangle shape, in good accordance with DFT predictions [1]. Nevertheless, the morphologies are quite irregular : some nanolayers present a more isotropic shape. Some clusters are also observed. In MoS2 catalyst sulfided at 700°C (Fig 1), nanolayers are larger, well crystallized and morphologies are more homogeneous : mainly truncated triangles and some isotropic multi-facetted slabs.
CoMoS catalyst sulfided at 500°C present mainly hexagonal or irregular shape. No truncated triangle morphology is present. Some clusters are present. In addition, slabs are more stacked and aggregated than in non-promoted catalyst. At higher sulfidation temperature (Fig 2), the morphology of the nanolayers is homogeneous : all slabs are large, well crystallized, isotropic with many edges. No cluster is present. This observation is attributed to a combined effect of temperature and promoter edge decoration impacting the resulting 2D morphologies of CoMoS slabs [2].
In conclusion, this study highlights that HR-HAADF-STEM is a powerful technique to observe MoS2 nanolayers, even supported on alumina in industrial catalysts. In perspective of this work, changes of 2D morphology of nanolayers will be correlated to selectivity measured by catalytic tests.

[1] H. Schweiger, P. Raybaud, G. Kresse, H. Toulhoat. J. Catal. 207, 76-87 (2002).
[2] E. Krebs, B. Silvi, P. Raybaud. Catal. Today 130, 160-169 (2008).
[3] J. V. Lauritsen et al. Journal of Catalysis 197, 1–5 (2001)
[4] L. P. Hansen et al., Angew. Chem. Int. Ed. 2011, 50, 10153-10156

The authors thank P. Raybaud for helpful discussions about DFT.

Fig. 1: MoS2/alumina catalyst sulfided at 700°C under pure H2S

Fig. 2: CoMoS/alumina sulfided at 700°C under pure H2S
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1973 Ti-assisted polarity inversion in ordered GaN nanorods investigated by transmission electron microscopy and density functional theory

Kong X.1, Li H.1,2, Albert S.3, Bengoechea-Encabo A.3, Calleja E.3, Draxl C.2, Trampert A.1
1Paul-Drude-Institut für Festkörperelektronik, Hausvogteiplatz 5–7, D-10117 Berlin, Germany , 2Institut für Physik and IRIS Adlershof, Humboldt-Universität zu Berlin, D-12489 Berlin, Germany, 3Dpto. Ingenieria Electronica, ETSI Telecomunicacion, Universidad Politecnica, Ciudad Universitaria, 28040 Madrid, Spain
x.kong@pdi-berlin.de

GaN nanorods are considered as promising building blocks for the realization of novel high performance light-emitting diodes due to their superior structural perfection. Ordered arrays of uniform GaN nanorods and axial (In,Ga)N/GaN heterostructures were achieved on Ti masked GaN (0001) templates by selective area growth using plasma-assisted molecular beam epitaxy. Here, we will report on the unexpected observation of polarity inversions accidentally found in those nanorods that we have analyzed by convergent beam electron diffraction and electron energy-loss spectroscopy (EELS). The inversion domains (IDs) with diameter of less than 10 nm cross the entire nanorod and originate at the homo-junction bounded by a stacking faults-like planar defect. Lattice imaging based on high-resolution transmission electron microscopy (TEM) is applied to determine an extra (0002) lattice plane in connection with this basal plane ID boundary. Spatially resolved EELS measurements reveal the presence of Ti impurities, which is possibly responsible for the formation of planar defects and the associated polarity inversion. In order to prove this assumption, to clarify the Ti lattice site occupation on the GaN (0002) plane and to explain the polarity inversion effect, we have performed first-principles total-energy calculations within the framework of density functional theory (DFT). They show that Ti monolayer adsorption on the GaN basal plane generates an energetically favorable atomic configuration that contains a planar defect resulting in a polarity inversion. The calculations perfectly match with the TEM observations. The influence of the polarity on the growth of axial (In,Ga)N nanorods is further discussed in detail.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-1999 Systematic comparison of catalytic properties and applicability of d-elemental nanoclusters inside SWNT in-situ and on the atomic scale by means of AC-HRTEM

Zoberbier T.1, Chamberlain T.2, Biskupek J.1, Bichoutskaia E.2, Khlobystov A.2, Kaiser U.1
1Electron Microscopy of Materials Science, Ulm University, Germany, 2School of Chemistry, University of Nottingham, United Kingdom
thilo.zoberbier@uni-ulm.de

Catalysis on the nanoscale plays an important role on the one hand in terms of an enormous increase of efficiency/transformation rate on the other in the formation of defined nanostructures and functionalized materials. In both the morphologic properties of the catalyst plays an important role as well as the metal specific chemical properties. The possibility of controlled regulation and adaption of these parameters will lead to maximum gain and highest selectivity in chemical reactions and tap the full potential of catalysis in industry and the development of novel nanostructures. However neither the catalytic mechanisms are understood on an atomic level nor is there systematic studies on a fundamental base to understand why different metal type deviate in their applicability.

In our experiments we perform atomically resolved in-situ imaging of chemical reactions between d-elemental metals and a carbon environment inside SWNTs by means of aberration-corrected high-resolution transmission electron microscopy (HRTEM). The experiments aim to fundamentally understand and compare the catalytic properties of different catalysts by variation of a large range of transition-metals in equivalent experiments. This enables a detailed study of the processes essentially characterizing the aptitude and applications of the different metals, such as formation of metastable transient structures, formation of ordered carbon networks in different morphologies, annealing and reorganization processes and ability to assimilate carbon source material. Moreover the investigations allow a study of intermediates and the underlying chemical properties of Pi- and Sigma- bonding, metal-cohesive forces and solubility of carbon in metal.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2015 TEM study of TiO2 photocatalyst layers deposited on carbon nanosheet templates by atomic layer deposition

Kurttepeli M.1, Deng S.2, Verbruggen S. W.3, 4, Guzzinati G.1, Cott D. J.5, Lenaerts S.3, Detavernier C.2, Bals S.1, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium, 2Department of Solid State Science, Ghent University, Krijgslaan 281/S1, B-9000 Ghent, Belgium, 3Department of Bio-science Engineering, Sustainable Energy and Air Purification, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium, 4Department of Microbial and Molecular Systems, Center for Surface Chemistry and Catalysis, KU Leuven, Kasteelpark 11 Arenberg 23, B-3001 Heverlee, Belgium, 5Imec, 75, Kapeldreef, B-3001 Leuven, Belgium
mert.kurttepeli@uantwerpen.be

Due to its distinctive physical properties, chemical stability, bio-compatibility, non-toxicity and low cost, titanium dioxide (TiO2) is of great interest for a wide range of applications [1], [2]. The great potential of TiO2 nanostructures is obvious, but the desired physical and chemical properties of the materials will only be reached if a complete understanding of the relation between the activity and the structure of the materials has been obtained. In order to perform a detailed characterization of such nanostructures, transmission electron microscopy (TEM) is an ideal tool. Not only structural, but also chemical and electronic information can nowadays be obtained, even atomic column by atomic column [3], [4]. Nevertheless, one should take into account that conventional TEM images are only two-dimensional (2D) projections of three-dimensional (3D) objects. Therefore, TEM has been expanded to 3D, which is referred to as "electron tomography".
Hereby, we present results from different TEM characterization techniques to investigate the effects of annealing in helium environment on the structure of TiO2 layers deposited onto carbon nanosheets (CNSs) using atomic layer deposition (ALD). Using monochromated STEM-EELS, areas with TiO2 in anatase and amorphous form have been identified. From these maps, it is observed that the coating is mostly in anatase form, and there is only a low amount of amorphous TiO2 after annealing (see Fig 1). The graphite distribution map additionally indicated the presence of graphite throughout the layer. To investigate the 3-D structure of the material, HAADF-STEM electron tomography was applied (see Fig 2). The volume renderings proved both the homogeneity of the ALD coating throughout the CNSs layer, and the porosity of the complete film.
[1] A. Fujishima and K. Honda, Nature 238, 37 - 38 (1972).
[2] A. Kay and M. Grätzel, Solar Energy Materials and Solar Cells 44, (1996).
[3] K. W. Urban, Nature Materials 8, 260 - 262 (2009).
[4] D. A. Muller, Nature Materials 8, 263 - 270 (2009).

The authors acknowledge financial support from European Research Council and Sim-Flanders.

Fig. 1: HAADF-STEM image of the sample. Colored elemental STEM-EELS map with (B) anatase-TiO2 (red) and (D) graphite (green) is embedded. The amorphous-TiO2 elemental map is given in (C).

Fig. 2: Visualizations of the 3-D reconstruction of the sample depicted along different orientations are given in (A) and (B). A slice (orthoslice) through the 3-D reconstruction is presented in (C).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2323 Preparation and characterization of systems with plasmonic metal nanoparticles for fluorescence-lifetime imaging microscopy

Kokoskova M.1, 2, Pavlova E.1, Hromadkova J.1, Slouf M.1, Sloufova I.2, Sutrova V.2, Vlckova B.2, Kapusta P.3, Hof M.3, Michl M.4
1Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic, 2Dept. of Physical and Macromolecular Chemistry, Charles University in Prague, Hlavova 8, 128 40 Prague 2, Czech Republic, 3J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejškova 2155/3, 182 23 Prague 8, Czech Republic, 4Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, V Holešovičkách 2, 180 00 Prague 8, Czech Republic
marketa.kokoskova@natur.cuni.cz

Luminescence of luminophores localized in a close proximity of plasmonic nanoparticles (NPs) such as Au or Ag are known to be completely or at least partially quenched [1]. However, in the case of larger distances from the metal surface a luminescence enhancement may be observed [2]. In order to investigate nanoparticle-luminophore distance effects, we focused our attention on reproducible preparation of homogeneous and well-defined model samples.

We studied systems with gold as well as silver NPs prepared by different techniques. The first set of samples were [substrate–AuNPs–spacer–luminophore] systems differing by AuNPs morphology. The substrates were microscopic cover glasses with constant thickness (specimens for fluorescence lifetime imaging microscopy, FLIM) and carbon-coated copper grids (specimens for TEM, controls). AuNPs were sputter-coated on the substrate; their morphology was controlled by sputtering time and subsequent thermal treatment. The spacer layer was created by thermal evaporation of carbon. The testing luminophores were widely used quantum dots and Ru(II) tris(2,2’-bipyridine); those were both sprayed and drop deposited onto the sample. The second set of samples comprised [substrate–AgNPs–luminophore] systems. In this case the AgNPs were prepared chemically by reduction of silver nitrate by hydroxylamine hydrochloride and in a form of a single aggregate deposited onto microscopic cover glass [3]. The testing luminophore /Ru(II) tris(2,2’-bipyridine) was drop deposited. The luminescent signal was measured immediately after deposition.

The size and the shape of Au and Ag nanoparticles were monitored by TEM and FEGSEM. We demonstrated that the combination of sputter coating and thermal treatment could yield NPs ranging from 5 nm up to several mm. The average size of the AgNPs was ~30 nm. The presence and homogeneity of luminophore on the surface was verified by TEM and EDX. Preliminary FLIM experiments of the systems with AuNPs showed quite inhomogeneous distribution of fluorescence lifetimes. Parallel TEM investigations suggested that the luminophores were deposited in multiple layers. Therefore, the different distances of luminophores from different layers might explain the observed distribution of FLIM signal. In the case of Ag NPs systems and drop deposition of luminophore, the surface-enhanced luminescence was observed.

References: [1] Geddes CD et al., Fluoresc. 2002, 12, 121., [2] Lakowicz JR, Anal. Biochem. 2001, 298, 1., [3] Sutrova, V. bachelor thesis, PřF UK, Praha 2013.

GACR P208/10/0941, TACR TE01020118 and GAUK 558213.

Fig. 1: TEM images of sputtered and thermally treated AuNPs (ts = sputtering time, thermal treatment: 450 °C/15 min).

Fig. 2: Fluorescence lifetime images and elastic scattered light images of AuNP/C/QD 510 system (A, B) and AgNP/Ru(bpy)3 system (C, D).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2031 Strain Concentration in Fivefold Twins

Yu R.1, Wu H.1, Zhu J.1
1Tsinghua University, Beijing, China
ryu@tsinghua.edu.cn

Multiple twinning is widespread in both natural and synthesis matter. The two types of multiple twinning, lamellar and cyclic, have attracted much attention due to their unique structures and properties. Lamellar twinning was shown to give a combination of high strength and toughness in copper [1], and highest creep resistance in titanium aluminide alloys [2]. Cyclic twinning, as another type of multiple twinning, occurs in an even wider range of materials, including not only inorganic small particles and thin films, but also proteins and virus [3]. The fivefold twinning is the most common form for multiple cyclic twinning [3].

The fivefold twinning has also attracted attention from the viewpoint of symmetry, which is an important concept in modern science. In fact, the fivefold rotational symmetry is geometrically forbidden in periodic crystals, although widely found in quasicrystals. Due to the geometrical imcompatibility, the fivefold twins have to be strained relative to the single-crystalline counterpart. Various models for the strain distribution have been proposed, including the linear homogeneous strain, angular and radial homogeneous strain, and the inhomogeneous strain models.

In this work, the atomic structure of the fivefold twins in diamond and silicon have been investigated by combining aberration-corrected transmission electron microscopy and first-principles calculations. In contrast to the strain distribution in metallic systems, which has small inhomogeneity, the strain in the fivefold twins of semiconductors depends significantly on the Pugh’s ratio of shear modulus to bulk modulus. For diamond with very high Pugh’s ratio, the strain is highly concentrated at the twin boundaries. Correspondingly, the frontier orbitals are located at the surfaces, in contrast to the case of silicon, where the frontier orbitals are close to the center.

References:

1. L. Lu et al., Science 304, 422 (2004).

2. F. Appel, and R. Wagner, Mater. Sci. Eng. R. 22, 187 (1998).

3. H. Hofmeister, Cryst. Res. Techno. 33, 3 (1998).

Acknowledgement: This work was supported by National Basic Research Program of China (2011CB606406), NSFC (51071092, 51371102, 11374174, 51390471, 51390475), and Program for New Century Excellent Talents in University. This work used the resources of the Beijing National Center for Electron Microscopy and Shanghai Supercomputer Center.

Fig. 1: (a) Aberration-corrected TEM image of and (b) strain distribution in diamond five-fold twins.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2094 Direct observation of Ti vacancies in Ti0.87O2 nanosheet using low-voltage monochromated TEM

Ohwada M.1, Kimoto K.1, Mizoguchi T.2, Ebina Y.1, Sasaki T.1
1National Institute for Materials Science, 2The University of Tokyo
kimoto.koji@nims.go.jp

Titania nanosheets [1] are two-dimensional single crystals of a titanium oxide with a thickness of one titanium or two oxygen atoms (Fig. 1a), and they show attractive material properties, such as photocatalytic reactions. The titania nanosheets are synthesized from a layered titanate K0.8Ti1.73Li0.27O4 through a soft chemical procedure (i.e., delamination), and the atomic arrangement of Ti-O layers in the parent crystal are basically preserved in the titania nanosheets. The nanosheets have the composition of Ti0.87O2, including cation vacancies at Li-substituted Ti sites of the parent crystal. In general, atomic vacancies affect the stability of crystal structures and material properties; therefore, it is important to reveal the atomic structure around Ti vacancies and their distribution in the nanosheets.

The observation of atomically thin materials requires not only high spatial resolution but also high sensitivity and low irradiation damage. We found that oxide nanosheets are substantially beam-sensitive, in contrast to a graphene and related materials. For instance we reported the topotactic reduction of a Ti0.87O2 nanosheet to Ti2O3 nanosheet [2].

We performed low-voltage and low-dose TEM observation using Titan3 at 80 kV with an image corrector (CEOS, CETCOR) and a monochromator, whose energy spread is 0.1 eV (FWHM). Attainable information limit under this condition was found to be 90 pm [3]. Figure 1b shows a high-resolution TEM image observed under underfocused condition [4]. The TEM image shows several bright areas as indicated by arrows, and we integrated these portions of the TEM image contrast (Fig. 2a). Based on the experimental results we constructed Ti vacancy structure models, and the atomic positions were optimized using first-principles calculation (the CASTEP code) as shown in Fig. 2b. The multislice simulation result based on the model successfully reproduces the experimental result (see Fig. 2c), and we found that the two oxygen atoms near the Ti vacancy are considered to be desorbed during the TEM observation [4].

[1] T. Sasaki, et al., J. Am. Chem. Soc. 118 (1996) 8329. [2] M. Ohwada, et al., J. Phys. Chem. Lett. 2 (2011) 1820. [3] K. Kimoto, et al., Ultramicrosc. 134 (2013) 86. [4] M. Ohwada, et al., Scientific Reports 3 (2013) 2801.

We thank Drs. Nagai, Ishizuka, Inoke, Lazar, Freitag, Sato and Suenaga for invaluable discussions. This work is supported by Nanotechnology Platform of MEXT and Research Acceleration Program of JSPS.

Fig. 1: A crystal structure model of Ti0.87O2 nanosheet (a), and a high-resolution TEM image of a Ti0.87O2 nanosheet. The TEM image is observed under underfocused condition. White arrows in Fig. 1b indicate several bright areas, suggesting Ti vacancies.

Fig. 2: (a) Experimental TEM image which is obtained as an average of the portions in Fig. 1b. (b) The atomic arrangement near the Ti vacancy optimized using the first-principles calculation. (c) The multislice simulation of a TEM image based on the optimized structure model.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2140 Structure of Plasmonic Nanocomposites obtained by Thermal and Laser Annealing of AlN:Ag Multilayers grown by Magnetron Sputtering

Bazioti C.1, Dimitrakopulos G. P.1, Kehagias T.1, Komninou P.1, Siozios A.2, Lidorikis E.2, Koutsogeorgis D. C.3, Patsalas P.1, 2
1Physics Department, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece, 2Department of Materials Science and Engineering, University of Ioannina, GR-45110 Ioannina, Greece, 3School of Science and Technology, Nottingham Trent University, NG11 8NS, Nottingham, United Kingdom
gdim@auth.gr

Localized surface plasmon resonances (LSPR) exhibited by plasmonic nanoparticles (PNPs) depend on PNP size, shape, distribution, and on the refractive index of the surrounding matrix. In this regard, efforts are undertaken to elucidate and control these parameters depending on growth conditions and post-growth treatment.
The structural properties of AlN thin films containing Ag PNPs were studied using TEM/HRTEM methods, and the results were correlated to the optical response. Magnetron sputtering (MS) was employed to deposit initially AlN:Ag multilayers with either amorphous (a-AlN) or nanocrystalline wurtzite-structured matrix (w-AlN) [1]. In one set of samples (series A), laser annealing (LA) using up to 700 mJ/pulse at 193 nm was employed in order to photomodulate the PNPs. In a second sample series (series B), flash thermal annealing (TA) was applied sequentially after MS deposition of each Ag layer, followed by LA in order to tailor the final microstructure.
In sample series A, LA dissolved the multilayer structure up to approximately half of its initial thickness, as shown in Figs. 1(a) and 1(b). This influence was more intense in the a-AlN case. Sample series B comprised just four 3 nm thick Ag interlayers embedded between AlN layers of 12 nm nominal thickness. TA led to complete structural reorganization resulting in dissolution of the layers and to a rather homogenous PNP distribution in the a-AlN case [Fig. 2(a)]. In the w-AlN case, an inhomogeneous PNP distribution was obtained, as larger PNPs were confined into two zones, one close to the substrate and one close to the surface. After LA, the homogenous PNP dispersion was destroyed in the a-AlN case, and larger PNPs were created [Fig. 2(b)]. For the w-AlN matrix, the PNP-zone close to the substrate was not dissolved, but still an improved PNP arrangement was obtained.
PNP enlargement by LA was described as an Ostwald ripening phenomenon. Larger PNPs were found close to the film surface, due to the enhanced Ag surface diffusivity. TA promoted Ag segregation, leading to even larger PNPs. The w-AlN crystallinity appeared almost unaffected, due to the strong ionic character of the atomic bond. Crystallinity was found to limit PNP enlargement as shown in Fig. 3, due to the resistance of the lattice to deformation. Overall it was demonstrated that controlled annealing processes can be employed to modulate the LSPR signal depending on the initial structure of the samples, as well as on the matrix crystallinity.

Work partially supported by the EU FP7 Project ‘SMARTRONICS’, Grant Agreement No 310229.

Fig. 1: Cross sectional bright field (BF) TEM overall images of multilayer a-AlN:Ag nanocomposites. (a) The nanocomposite prior to LA, comprising twenty Ag layers of 3 nm thickness with a 7 nm periodicity. (b) The nanocomposite after LA showing dissolution of the top half layers and ripening of PNPs.

Fig. 2: Cross sectional BF TEM overall images of a-AlN:Ag nanocomposites (a) after combined MS-growth plus sequential TA, and (b) after post-growth LA of the sample.

Fig. 3: HRTEM image showing Ag PNPs embedded in nanocrystalline w-AlN following LA treatment. Ag{111} and AlN{01.0} d-spacings are indicated.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2154 Detection of neuroendocrine tumor markers using nanostructured biosensors based on Au nanoparticle / Au film sandwich architecture

Boca-Farcau S.1, Farcau C.1, Astilean S.1
1Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute on Bio-Nano-Sciences, Babes-Bolyai University, 42 Treboniu Laurian St., 400271 Cluj-Napoca, Romania
sanda_c_boca@yahoo.com

Neuroendocrine tumors as are pheochromocytomas are dangerous tumors that require consideration in a large number of patients. Currently, the biochemical diagnosis of neuroendocrine tumors is based on plasma or urinary measurement of the direct secretory products of the adrenomedullary-sympathetic system or their metabolites, specifically catecholamines or their metanephrine derivatives. However, the techniques used for analysis of plasma free metanephrines, i.e. high-performance liquid chromatography (HPLC) or HPLC coupled with mass-spectrometry, are technically-demanding and time consuming which limit their availability [1]. Nano-biosensors based on colloidal gold or silver nanoparticles have proved their applicability for the accurate detection of tumor markers using Surface-Enhanced Raman Scattering (SERS) technique [2]. Recently special interest has been devoted to a type of biosensing platform made of individual gold nanoparticles (AuNPs) over gold films. This structure showed an increased sensitivity compared to self-assembled Au nanoparticles on other solid substrates [3], which was attributed to the additional electric field enhancement by the electromagnetic coupling between the nanoparticles and their supporting metal films.

In the present work we demonstrate a simple, fast and low-cost method for deposition of Au nanoparticles onto flat Au films with the aim of creating a SERS sensing platform with good enhancement and high signal reproducibility. Gold nanoparticles of tunable size and shape were synthesized by simple or seed mediated growth method. The structure and surface morphology of the nanoparticle-film sandwiched structure was characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), and UV–vis spectroscopy. Methanephrine metabolite was dropped into the sandwich structure and the SERS enhancement as a function of the deposited particle properties was measured using for excitation three laser lines (532, 633 and 785 nm). The obtained results demonstrate that the resultant Au-nanoparticle film exhibit noticeable SERS amplification of the adsorbed metabolite and can be used in the design of efficient, stable SERS-active substrates for the detection and identification of specific tumor markers.

References:

1. M. Procopiou, H. Finney, S.A. Akker, S.L. Chew, W.M. Drake, J. Burrin, A.B. Grossma, Eur J Endocrinol. 2009 ,161,131-40.

2. H. Hwang, H. Chon, J. Choo, J.-K. Park, Anal. Chem., 2010, 82, 7603-7610.

3. C.L. Du, C.J. Du, Y.M. You, C.J. He, J. Luo, D.N. Shi, Plasmonics, 2012, 7,475-478.

This work was financially supported by Babes-Bolyai University, Cluj-Napoca, Romania under the Research Grant for Young Scientists, contract GTC-UBB No. 34056/2013.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2165 Diffusion effects investigations of self-organized Gold nanostructures on Ge(001) surface by Electron Microscopy

Jany B. R.1, Nikiel M.1, Szajna K.1, Indyka P.2, Krok F.1
1Jagiellonian University, Marian Smoluchowski Institute of Physics, Reymonta 4, PL30059 Krakow, Poland, 2Jagiellonian University, Faculty of Chemistry, Ingardena 3, PL30060 Krakow, Poland
benedykt.jany@uj.edu.pl

The self-organized gold nanostructures on Ge(001) surface are currently of special interest due to their applications for mono-molecular electronic devices [1,2]. The understanding of electrical as well as physical properties of the system is of great importance.
The Ge(001) substrate samples were cleaned to achieve atomically flat terraces by low energetic ions bombardment and by annealing. Next, 6 ML of Au was deposited by the Molecular Beam Epitaxy in room temperature. Later, the sample was post-annealed to temperature from 473 K to 770 K. The gold self-organizes to create island structures on Ge surface as depicted in Figure 1.
The morphology of Au/Ge(001) samples was measured for different post-annealing temperatures with SEM FEI Quanta 3D FEG. The island surface density and their sizes were measured providing the information on surface diffusion effects. The autocorrelation analysis shows that there exists preferred island orientation along crystallographic directions on the substrate surface.
Cross sections from the Au/Ge(001) samples were prepared using FIB technique for transmission electron microscopy measurements conducted with TEM FEI Tecnai Osiris 200 kV equipped with Super-X EDX detector. The TEM measurements show that some island are submerged in germanium substrate. The chemical composition of the islands was mapped by the STEM/EDS measurements. This uncovered core/shell structure of the islands, with germanium shell on top. The crystalline nature was first studied by Selected Area Electron Diffraction (SAED) diffraction and Dark Field imaging. Later, detailed investigations were performed by Nano Beam Diffraction (NBD) measurements in STEM micro-Probe. This showed differences in crystalline structure of the islands.
The electron microscopy gives the possibility to fully study the creation dynamics and to completely characterize the fabricated nanostructures. Surface diffusion effects are investigated by the SEM as well as effects of diffusion processes into the bulk Ge crystal are measured by the TEM cross sections. This gives the unique scientific possibilities to fully investigate the evolution of the self-organized systems. The results and used experimental techniques will be discussed.

[1] C. Joachim et. al., Nature 408, 2000
[2] M. Wojtaszek et. al., Advances in Atom and Single Molecule Machines, Vol.1, 2012

The authors gratefully acknowledge financial support from the Polish National Science Center, grant no.DEC-2012/07/B/ST5/00906. The research was carried out with equipment purchased with financial support from the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (Contract No. POIG.02.01.00-12-023/08).

Fig. 1: Self-organized gold island grown on Ge(001) surface, In center: TEM image of cross section through the gold island, Top: SEM secondary electron image shows the gold islands on the germanium surface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2172 Equilibrium shape changes of PtCu alloy nanoparticles across the order-disorder transformation - An in-situ TEM study

Chatterjee D.1, Ravishankar N.1
1Materials Research Centre, Indian Institute of Science, Bangalore, India
dipanwita.chatterjee06@gmail.com

Platinum alloy nanoparticles find application in the field of electrocatalysis and gas phase reaction catalysis. The activity and stability of the catalyst
nanoparticles depend on the exposed crystal facets. Hence, the shape changes of the catalyst nanoparticles affect their surface properties like adsorption of gases and
hence the catalytic activity. Shapes assumed by the particles can be either kinetic or thermodynamic. If the particles are equilibrated under fixed conditions of
temperature, pressure, volume and composition, they attain their thermodynamic or equilibrium shape which is given by the Wulff construction. While the shape changes
with temperature has been studied for monometallic particles, there are very few studies on alloy nanoparticles. In particular, the shape changes associated with
changes in ordering of nanopartciles has not been investigated.

We have chosen a class of bimetallic A50B50 type alloy system which undergoes order to disorder phase transformation with the increase of temperature. The equilibrium
shape changes of the alloy systems having a varied range of heat of mixing across the transition from B2 ordered to A2 disordered structure and L1o ordered to A1
disordered structure have been studied theoretically. Shape change in terms of change in the area of the exposed facet was observed with changing degree of order in
the alloy system (Figure 1).

Experimentally equilibrium shape changes have been observed for PtCu system which undergoes transformation from ordered rhombohedral structure to disordered cubic
structure using in-situ heating techniques in transmission electron microscope (TEM). The system has been designed such that the alloy nanoparticles nucleated on the
MgO cubes appear edge-on on the face of the cube when tilted to its [001] zone axis and the shapes of the equilibrated alloy particles at different temperatures
implying different order parameters have been imaged at high resolution. The facet lengths of the equilibrated particles were observed to change monotonically with
decreasing order parameter that corresponds to the facet area change observed in the theoretical study (Figure 3).
The equilibrium shape change with the degree of order in an alloy system has been observed for the first time and its implication is immense, not only in terms of its
fundamental basis but also in terms of the application of the alloy particles undergoing order-disorder phase transformation where the property of equilibrium shape
change with degree of order could be exploited in the field of catalysis.

Financial support from DST is acknowledged. The electron microscopes are a part of the Advanced Facility for Microscopy and Microanalysis at IISc.

Fig. 1: Figure 1. Theoretically derived equilibrium shapes for B2 ordered to A2 disordered phase transition wherein the relative area of 110 and 100 facets vary with decreasing order parameter. The effect of different heat of mixing is also shown in this figure.

Fig. 2: Figure 2. (a) Low magnification image of MgO cubes with PtCu alloy nanoparticles nucleated on the cubes. (b) and (c) faceted ordered alloy nanoparticles appearing edge-on on MgO substrate.

Fig. 3: Figure 3. PtCu alloy nanoparticle on MgO substrate equilibrated at three different temperatures showing changes in the facet length in two-dimensional projection which translates to facet area change in the three-dimensional particle.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2178 PtBi Alloy Nanoparticles on Nitrogen-Functionalized Reduced Graphitic Oxide Support for Electrocatalysis

Tripathi S.1, Ravishankar N.1
1Materials Research Centre, Indian Institute of Science, Bangalore, India
shalinitripathi2307@gmail.com


The use of Pt-based electrocatalyst for methanol and formic acid oxidation reactions suffers from coarsening and deactivation of catalyst due to adsorption of CO. In this context, alloying Pt with a non-noble element has been shown to reduce the poisoning effect of CO significantly. Also,the presence of a conducting catalyst support can inhibit the catalyst coarsening without compromising the facile electron transfer. In this work, we report a microwave-based wet chemical approach for alloying Pt nanoparticles with Bi on a reduced graphitic oxide (RGO) support, which enhances the stability of the catalyst. Furthermore, we illustrate a way to improve the electron transfer by increasing the conductivity of the support through nitrogen functionalization of RGO. This wet-chemically synthesized graphitic oxide sheets facilitated the doping of the nitrogen at a very low temperature compared to the other reported physical processes, which can be attributed to the numerous localized defects in the GO sheets. Detailed transmission electron microscopy has been applied to understand the underlying mechanism in order to engineer the composition and morphology of the catalyst alloy nanoparticles. Our study also suggests that the alloying happens by nucleation of Bi on pre-formed Pt nanoparticles. Furthermore, the lower melting point of bismuth and its higher diffusivity facilitates the formation of intermetallic PtBi structure at such a low temperature. Thus, a microstructure-based thorough mechanistic understanding of the catalyst fabrication presented in this work imparts a control over shape, size and composition of the catalyst.

TEM facilities provided by Advanced Facility for Microscopy and Microanalysis (AFMM), and XPS facility of CENSE, Indian Institute of Science, Bangalore, India.

Fig. 1: Fig1. (a) LM and (b) HRTEM images of Pt nanoparticles, (c) PtBi alloy nanoparticles on RGO support; (d) SAED showing PtBi phase; (e) BF image of Pt NPs on NGO support; (f) shows the corresponding DF image; (g) LM and (h) HRTEM showing ordered PtBi phase over NGO support

Fig. 2: Fig.2: (a) High resolution XPS spectrum of Pt4f and (b) Bi4f from fabricated catalyst; (c) quantification shows a 1:1 atomic ratio of Bi and Pt

Fig. 3: Fig.3: Schematic showing the MW-based mechanism of selective heterogeneous nucleation for fabrication of alloy catalyst on support
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2194 Nucleation texture of metal nanoparticles on amorphous substrates

Chatterjee D.1, Akash R.1, Kamalnath K.1, Ravishankar N.1
1Materials Research Centre, Indian Institute of Science, Bangalore, India
dipanwita.chatterjee06@gmail.com

Crystals nucleating homogeneously tend to adopt their
equilibrium shapes to minimise the barrier for
nucleation. For heterogeneous nucleation on a substrate,
the Wulff shape of the crystal itself is translated,
rotated and truncated by the substrate. The orientation
and level of truncation determimes the volume of the
so-called Winterbottom shape. We have calculated the
preferred orientation of heterogeneous nucleation on an
amorphous (isotropic) substrate by assuming that the
wetting of the solid nucleus on the substrate is
constant for the different orientations of nucleation.
Under the given conditions, the preferred orientation of
nucleation is the one for which the exposed volume of
the crystal on the substrate is minimum, as for such an
orientation the nucleation barrier is the minimum.

Theoretical calculations for obtaining minimum energy
Winterbottom shapes of nuclei of FCC metal at their
preferred orientations for a range of wetting conditions
have been done and the results are shown in Figure 1.
Experimentally, we have attempted to estimate the
orientation of nucleation of few hundreds of FCC metal
nuclei in order to statistically conclude the preferred
direction of orientation of heterogeneous nucleation on
an amorphous carbon substrate. Precession Electron
Diffraction (PED) technique is being used to scan over
regions containing a good number of nuclei and obtain an
orientation map from which the nucleation orientation of
the metal nuclei is to be determined.

Very fine nuclei of Au or Pt nanoparticles have been
nucleated on functionalized amorphous Carbon coated
Copper grid by microwave reduction of the precursor
salts in ethylene glycol medium. Electron diffraction
pattern obtained from such fine nuclei do not contain
enough number of spots for a reliable indexing of the
pattern using standard diffraction patterns for the
particular metal. So, the orientation map obtained from
the sample has a very low reliability index. For
optimization of conditions to obtain reliable
orientation mapping, PED scan on homogeneously nucleated
Au particles of around 8 nm diameter [inset of Figure 2
(a)] have been carried out and the resulting orientation
map has been shown in Figure 2(b). Here the 8 nm
particles could be resolved properly, as can be seen in
the virtual bright field image of the scanned area in
Figure 2(a) but the reliability index is poor because of
the polycrystalline nature of the Au nanoparticles.
Results on the nucleation texture of different
nanoparticles will be presented with detailed analysis
of the suitable microscopy conditions required for the
same.

Financial support from DST is acknowledged. The electron microscopes are a part of the Advanced Facility for Microscopy and Microanalysis at IISc.

Fig. 1: Figure 1. Preferred orientation of nucleus of FCC crystal heterogeneously nucleating on amorphous substrate at different wetting condition defined by Δs. Δs is related to the difference in the subtrate-vapour and substrate-particle interfacial energy.

Fig. 2: Figure 2. (a) Virtual bright field image of the Au nanoparticles on amorphous Carbon generated after the PED scan, inset showing a low magnification image of the area scanned, (b) Orientation map, different colours designating definite directions of the crystals in the scanned area.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2222 Quantification of PtIr Catalyst Nanoparticles Using ADF STEM

MacArthur K. E.1, Jones L. B.1, Lozano-Perez S.1, Ozkaya D.2, Nellist P. D.1
1Department of Materials Science, University of Oxford, Oxford, UK , 2Johnson-Matthey Technical Centre, Reading, UK
katherine.macarthur@materials.ox.ac.uk

Bimetallic catalyst nanoparticles for hydrogen fuel cell applications contain less Pt and exhibit higher catalytic activity than pure Pt particles.1 We have analysed Pt/Ir alloy particles which have shown improved resistance to CO poisoning. In order to understand these systems further it is necessary to examine their 3-dimensional atomic structure. Quantification of annular dark-field scanning transmission electron microscope images uses atomic resolution images as data sets for extracting composition and thickness information. Calculating the scattering cross-section (CS) of each atomic column provides robustness to many experimental parameters2 providing greater flexibility when imaging such challenging samples.
An automated code3 carries out detector normalisation,4 peak finding, background subtraction and column wise integration making it now possible to analyse and compare many particles. The measured CSs are assigned to atom counts through comparison with a simulation library. Simulations were carried out using the QEP μSTEM software matching experimental conditions of a 300kV microscope,5 with detector angles 34.9-190mrad and a probe convergence angle of 20.2mrad, with 30 phonon configurations. Due to their proximity in atomic number Pt and Ir are indistinguishable below 14 atoms thickness, Figure 1. Above 15 atoms the CS trend of each species begins to diverge; this is also the thickness where the accuracy of the atom-count assignments is greater than ±1 atoms making the error too large for accurate counting. Armed with the number of atoms within each column and their x-y coordinates, we can reconstruct the 3-dimensional structure from a single experimental image by assuming no vacancies and minimising surface steps.
To validate the experimental nanoparticle structure, the theoretical Wulff shape for a Pt/Ir alloy particle, was constructed using the Wulffman code,6 Figure 2, and orientated to a comparable viewing direction. The energies of the different alloy surface facets were assumed to be a linear combination of the pure elements.7 The considerable similarity between experiment and the Wulff shape demonstrates the accuracy of the atom counting results. Deviations can be explained by the quantised nature of such small length scale facets and the surface steps which are thought to be critical for catalytic activity.

1 Z Liu et al, Catalysis Review 55 (2013), p255-88
2 H E et al, Ultramicroscopy 133 (2013), p109-119
3 The Absolute Integrator code is free for academic use from www.lewysjones.com/software/
4 J M LeBeau et al, Nano Letters 10 (2010), p4405-8
5 B D Forbes et al, Physical Review B 82, (2010) 104103
6 A R Roosen et al, Computational Materials Science 11 (1998) p16-26
7 B D Todd and R M Lynden-Bell, Surface Science 281 (1993), p191-206

The research leading to these results has received funding from the European Union Seventh Framework Program under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3), and from the EPSRC (grant number EP/K032518/1)

Fig. 1: Simulated library of the scattering cross-section of a pure Pt or Ir crystal with increasing sample thickness. With only 1 atomic number between Pt and Ir they are indistinguishable below 14 atoms. However, the elements have different channelling lengths; this produces a deviation at much higher atom counts.

Fig. 2: Reconstruction of an experimental Pt/Ir particle, left, and equivalent Wulff plot, right. The Wulff plot has been orientated to similar orientation for comparison, (111) faces are purple (with the close packed hexagonal arrangement in the hard sphere model), (100) faces are blue (with square arrangement), and the (110) faces are red.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2226 Mixed FeOx-CeO2-x nanomaterials for chemical looping characterized by transmission electron microscopy and spatially resolved EELS

Turner S.1, Meledina M.1, Galvita V.2, Poelman H.2, Marin G. B.2, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 2Laboratory for Chemical Technology, Ghent University, Technologiepark 914, 9052 Ghent, Belgium
stuart.turner@uantwerpen.be

Mixed FeOx-CeO2-x nanomaterials are promising candidates for use as oxygen storage materials in the production of H2 by chemical looping. The technology of chemical looping is based on periodic reduction/re-oxidation cycles of metal oxides, designed to convert hydrocarbons to hydrogen with a quality that exceeds the requirements of all types of fuel cells.1,2 In this work, a series of mixed FeOx-CeO2-x with varying Fe/Ce content are characterized using a combination of advanced imaging techniques and spatially resolved EELS, in order to characterize the presence and nature of the constituting components. The oxide materials are studied throughout the oxidation/reduction cycle, paying special attention the morphology and surface features of the FeOx/CeO2-x material.

Low iron content materials (e.g. 5wt.% FeOx/CeO2-x) typically consist of ceria nanoparticles with sizes ranging from approximately 20 to 60 nm. Electron diffraction and imaging show no evidence for the presence of a separate Fe2O3 (or FeOx) phase in this material. The ceria nanoparticles do show the presence of nanometer-sized voids, which have previously been observed in nanosized ceria. Spatially resolved EELS maps show that both voids and ceria surfaces are decorated with isolated Fe atoms, and that the surface atoms of the ceria nanoparticles and the voids are in a reduced state compared to bulk CeO2.3 Particular attention has been paid to possible changes in the oxidation state and clustering of these Fe species upon oxidation and reduction. The high iron content materials consist of α-Fe2O3 nanoparticles decorated by significantly smaller ceria nanoparticles. In these samples, both structural and valency changes at the FeOx/CeO2-x interface upon cycling have been studied in detail.

1) V. Galvita et al., Topics in Catalysis 2011, 54, 907.

2) V. Galvita et al., Ind. Eng. Chem. Res. 2013, 52, 8416

3) S. Turner et al. Nanoscale, 2011, 3, 3385

S.T. gratefully acknowledges financial support from the Fund for Scientific Research Flanders (FWO). V.G. and H.P acknowledge financial support from the 'Long Term Structural Methusalem Funding by the Flemish Government'.

Fig. 1: (a) Overview HAADF-STEM image of a 5wt.% FeOx–CeO2-x sample. (b) HR-HAADF-STEM showing strong faceting and the presence of voids. (c) Overview HAADF-STEM image and corresponding EELS maps: the Fe is enriched at the ceria surface and within the voids.

Fig. 2: (a) High resolution EELS references for Ce4+ and Ce3+. (b) Overview HAADF-STEM image and (c) Ce3+/Ce4+ map showing surface reduction in the ceria nanoparticles. (d) HAADF-STEM image of the surface of a ceria nanoparticle with (e) corresponding EELS spectra from the surface (black spectrum) and near-surface (blue spectrum) regions.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2252 3D STEM of highly anisotropic insertions in nitride nanorods: a challenge to FIB preparation techniques and transmission electron tomography

Niehle M.1, Trampert A.1
1Paul-Drude-Institut für Festkörperelektronik, Berlin, Germany
niehle@pdi-berlin.de

The ongoing request for innovative semiconductor devices for opto-electronics motivates the growth of low-dimensional objects such as nanowires or nanorods. The realization of designed heterostructures  based on axial or radial symmetry depends on the nanorod's shape, i.e. on its surface facets, and growth conditions. The understanding of the physical properties of the resulting low-dimensional heterostructures necessitates the detailed three-dimensional (3D) microstructure information. Consequently, there is a demand to further establish transmission electron tomography as a feasible tool in materials science – especially for nanoscale semiconductor heterostructures – along with the challenging site specific preparation of adequate samples.
The investigation of inclined GaN nanowires grown on a non-polar (11-22) GaN template with (In,Ga)N insertions at the top by scanning transmission electron microscopy (STEM) tomography is presented in this work. The objects' geometrical arrangement (Fig. 1b) requires a sophisticated sample preparation technique in a dual-beam device comprising a scanning electron microscope (SEM) and a focused ion beam (FIB). On the one hand, the technique allows to isolate the target within an electron transparent lamella (Fig. 1a). On the other hand, the positioning of the lamella realized by the incorporated  micromanipulator and the versatile sample stage enables the chemical sensitive high-angle annular dark field (HAADF) STEM imaging along a <11-20> direction (Fig. 1c) that is not straight forwardly available in conventionally prepared samples. The mounting of the sample with its [0001] orientation along the tilt axis will be discussed.
To access the complex morphology (facets, layer thickness, In content) of (In,Ga)N insertions in the GaN based objects, a HAADF STEM tilt series has been acquired over a tilt range of 165°. The 3D reconstruction reveals the shape (Fig. 2a) and the anisotropic occurrence of (In,Ga)N insertions in layers parallel to the facets of the object (Fig. 2b). The isosurface rendered volume shows that the object is limited by the hexagonal m- and rplanes as well as a rough cap parallel to the c-plane. The r-planes close to the substrate normal are only weakly developed whereas the other four are clearly formed. The cross-sections through the reconstructed 3D volume show high abundance of In in red color whereas the parts dominated by green belong to the GaN core and shell.
This study demonstrates the unique access to complex three-dimensional morphological and chemical information of nanoscale semiconductor heterostructures by HAADF STEM tomography. The requirement of a sophisticated sample preparation technique has to be underlined.

We gratefully acknowledge Enrique Calleja Pardo providing samples for the presented investigations.

Fig. 1: (a) The SEM image represents the target object within the lamella suitable for tomography. (b) The schematic of the target object illustrates its special geometry which challenges TEM sample preparation. (c) The HAADF STEM image exhibits the lamella in cross-section. The white arrow in image (a) and (c) marks the object that is presented in Fig 2.

Fig. 2: (a) Isosurface representation of the three-dimensionally reconstructed object along the direction perpendicular to the substrate and the view onto a (1-100) side facet. (b) The cutaway of a cube from the object (schematic) offers the view onto three ortho-slices parallel to low indexed lattice planes providing chemical information.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2257 Characterisation of air and water stable Cobalt nanorods

Marcelot C.1,2, Lentijo-Mozo S.1, Hungria T.1, Gatel C.2, Fazzini P. F.1, Cormary B.1, Tan R.1, Respaud M.1, Soulantica K.1
1Université de Toulouse; INSA, UPS, CNRS, LPCNO 135 avenue de Rangueil, 31077 Toulouse, France., 2Centre d’Elaboration de Matériaux et d’Etudes Structurales (CNRS), 29, rue Jeanne Marvig, 31055 Toulouse, France
cgarcia@insa-toulouse.fr

The synthesis of hybrid nanoobjects containing a metallic magnetic core and a shell constituted by a noble metal is highly desirable because of their potential use in the fields of electronics, optics, catalysis, biology and medicine. In these nanoparticles, the magnetic core provides the possibility to manipulate the nanoparticle by a magnetic field and the noble metal shell offers protection from oxidation, a surface for functionalization by biomolecules and depending on the metal core additional properties (catalytic, plasmonic etc). In this context, Co anisotropic nanoobjects such as nanorods and nanowires are of special interest for applications in which hard magnetic materials are required. However the development of a continuous shell of a noble metal around Co nanoparticles is a challenge due to incomplete covering by the noble metal. We will describe new hybrid Co-metal core-shell nanorods of different shell composition and thicknesses. The growth of a complete shell is accomplished by introduction of a buffer layer between Co and the noble metal, compatible with the two otherwise immiscible materials. The complete shell protects the Co nanorods from oxidation, as demonstrated by HRTEM (Fig.1) and EDS (Fig.2) analysis and corroborated by the magnetic measurements. These results prove that the magnetic properties of Co, which are very sensitive to oxidation, are stable after exposition of the nanorods to the air for several weeks. Furthermore when the metal shell is thick, it can provide oxidation protection of the Co-core in aqueous solutions for prolonged periods of time. After ligand exchange these nanorods can be transferred from organic solvents into aqueous solutions.

The authors thank the the European Commission for the FP7 NAMDIATREAM project (EU NMP4-LA-2010-246479), the Programme Investissements d'Avenir under the program ANR-11-IDEX-0002-02, reference ANR-10-LABX-0037-NEXT".

Fig. 1: Core-shell nanorod HREM

Fig. 2: Core-shell nanorod STEM-EDS
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2261 Chemical Analysis on the Nanometer Scale: Characterization of Copper Nanoparticles by Electron Energy Loss Spectroscopy and Energy Filtered Transmission Electron Microscopy

Schaumberg C. A.1, Wollgarten M.2, Rademann K.1
1Department of Chemistry, Humboldt-Universität zu Berlin, Berlin, Germany, 2Helmholtz-Zentrum Berlin für Materialien und Energie, Berlin, Germany
christian.schaumberg@chemie.hu-berlin.de

A major challenge of modern nanoscience is the need for a detailed knowledge of the chemical composition of novel materials on the nanometer scale. This challenge can be addressed by applying analytical methods to the transmission electron microscopy (TEM). In particular electron energy loss spectroscopy (EELS) opens a wide field of opportunities. Peaks at characteristic core edge energies in EEL spectra of a selected sample area provide information on the presence of certain chemical elements. Beyond that, the fine structure and the chemical shift of the observed core edges provide insights in the composition on an atomic level.[1]
Our work focuses on the characterization of copper nanoparticles generated by pulsed laser ablation of µm-sized powders in organic liquids.[2] The study of different copper precursors points to a reductive step during the synthesis of the copper nanoparticles. In order to investigate the formation mechanism in detail, a profound knowledge of the oxidation state of the copper atoms in the resulting particles is mandatory.
Oxidized copper shows distinct features, so called “white lines”, at the copper L2,3 edge in the EEL spectra. These white lines originate from energy losses through transitions of 2p electrons to empty 3d orbitals. As the 3d orbitals are completely filled for metallic copper the white lines are suppressed. Thus the occurrence of white lines can be used to determine the oxidation state of copper atoms.[3]
With this approach we can show, that the choice of the precursor not only determines the structural by also the chemical properties of the resulting copper nanoparticles. These findings are complemented by elemental maps obtained by energy filtered transmission electron microscopy (EFTEM). The variation of the precursor powder and the investigation of the resulting nanoparticles by EELS and EFTEM leads to a concept for the particle formation mechanism which will be presented.

References

[1] R. F. Egerton, Electron Energy-Loss Spectroscopy in the Electron Microscope, 3rd ed.; Springer: New York, Dordrecht, Heidelberg, London, 2011.
[2] C. A. Schaumberg, M. Wollgarten, K. Rademann, J. Phys. Chem., submitted.
[3] D. Shindo, K. Hiraga, A.-P. Tsai, A. Chiba, J. Electron Microsc., 42, 48-50 (1993).

Fig. 1: TEM images (left) and EEL spectra (right) of nanoparticles generated by laser ablation of CuO powder (top) and Cu3N powder (bottom). The TEM images show the filter entrance aperture used to record the EEL spectra. Thus, the EELS intensity originates solely from the depicted area.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2267 Consequences of gas dynamics on morphology and chemistry during Electron Beam Induced Deposition

Winkler R.1, Fowlkes J.2, Szkudlarek A.3, Melischnig A.1, Utke I.3, Rack P. D.2 4, Plank H.1 5
1Center for Electron Microscopy, Graz, Austria, 2Center for Nanophase Materials Sciences, Oak Ridge, USA, 3Laboratory for Mechanics of Materials and Nanostructures,Thun, Switzerland, 4Department of Materials Science and Engineering, Knoxville, USA, 5Institute for Electron Microscopy and Nanoanalysis, Graz, Austria
robert.winkler@felmi-zfe.at

Focused Electron Beam Induced Deposition (FEBID) is a versatile direct write tool for the fabrication of functional (3D) nanostructures. FEBID uses gaseous precursor which adsorb and diffuse on the surface where they get locally decomposed by a finely focused electron beam. Although many different application concepts have been demonstrates, final applicability depends strongly on predictable morphologies and defined deposit chemistries. Both demands require locally constant precursor coverage which leads to constant ratios between available precursor molecules and potentially dissociation electrons species which is denoted as working regime. What seems to be straightforward turns out to be very complicated when dimensions approaches the nanoscale where local working regimes are influenced by a number of variables like directional gas flux effects, deposit related barriers hindering ideal diffusion or geometrical shadowing effects, comprehensively discussed in this contribution. As starting point it will be demonstrated how patterning directions relates to the directional gas flux, caused by the geometrical arrangement of the gas injection system. It is found that volume growth rates (VGR) can vary by more than 50 % for different patterning orientations which significantly complicates the predictable deposit volumes (Fig. 2). Furthermore, it is shown how the chemistry changes along with the VGR which has strong implications on final functionalities. To demonstrate these effects in a comprehensive way, a new patterning strategy is introduced which visualize morphological and chemical effects within one deposit. Based on these experiments a model is derived which fully explains the observations taking directional adsorption, surface diffusion and local replenishment effects into account as well. The experiments are complemented by finite difference simulations and numeric calculations in well agreement and support the proposed dynamic model of laterally varying working regimes. In order to investigate the tunability of the regime situation, the accessible process parameters during deposition are systematically varied. It is demonstrated how constant working regimes can be established (Fig. 1) which provide both, predictable morphologies and laterally constant chemistries as indispensably required for potential applications. In summary the study demonstrates the nanoscale implications of molecular gas and surface dynamics on final deposit volumes and chemistries. Furthermore, it is also shown how stable conditions can be achieved by a careful setup of the deposition process which is essential for further steps toward industry related FEBID application.

We gratefully thank Prof. Ferdinand Hofer, Roland Schmied, Angelina Orthacker, Martina Dienstleder, Florian Kolb, Barbara Geier and Laura Resch and acknowledge the FFG for financial support.

Fig. 1: 3D AFM height images of FEBID structures fabricated with spiral out patterning strategy and constant electron doses. Unbalanced process parameters (1 ms dwell times) lead to disruption of the morphology (a) in contrast to balanced conditions b) (100 µs dwell times) as effect of indicated directional gas flux component.

Fig. 2: lateral variations of segment heights (red) and chemistry by means of C / Pt ratios (blue) for a disrupted deposit (Fig. 1a). Each patterning point has been patterned only once which demonstrates the strong implications of the directional gas flux effects.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2349 Atomic-scale Defects Leading to Lattice Strain in Single-crystal Ultrafine Gold Nanowires

Kundu P.1,3, Turner S.1, Van Aert S.1, Ravisankar N.2, Van Tendeloo G.1
1Electron Microscopy for Materials Research (EMAT), University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 2Materials Research Center, Indian Institute of Science, Bangalore 560012, India, 3Institute of Bioelectronics (PGI-8), Forschungszentrum Juelich, D-52425 Juelich, Germany
paro.124@gmail.com

Gold nanowires of molecular scale dimension are of fundamental as well as technological interest owing to their tunable electrical transport characteristics leading to ballistic conduction. This implies single electron sensitivity making them potentially active material for catalysis and molecule sensing. This demands a large scale production of the wires in pristine form for applicability and a detailed atomic structure study to interprete their properties different from the bulk. Although the chemical synthesis route has been reported and electrical transport studies have been carried out recently on the single crystal 2 nm gold wires of large aspect ratio (approx. 500 or more), the structural investigation is not done so far. HRTEM combined with image simulation and exit wave reconstruction can provide information on the local atomic structure, however, with aberration corrected microscopes and advanced analytical methods one can analyse the structure with picometer precision. This method is limited to atomically thin samples. Quantitative HAADF-STEM is a technique to analyse the structure of even few tens of nanometer thick samples and it allows us to determine atom positions in the lattice and determine elemental composition of the atomic columns. Aberration corrected electron microscopy, therefore, combined with advanced quantification methods is a state of the art technique to extract information atom-by-atom 1. Here we present our investigation on these ultrafine gold nanowires to determine their atomic structure by low dose aberration corrected high resolution (S)TEM. Quantification reveals patterned strain in the crystals which increases at the surface layer of atoms and that the wires are faceted with irregular atomic scale surface steps 2. These structural aspects can be related to their unique electrical features and makes them potential candidates for catalysis and sensorics. Besides, from the HRSTEM image, atom counts in the atomic columns in viewing direction is obtained and a 3D visualization of the wire atomic structure could also be deduced. Further, we looked into the atom dynamics due to interaction with the electron beam at higher dose which gives an insight to its mechanical behaviour and stability. Figure 1. provides an overview of the lattice strain and the atom counting analysis.


1 G. V. Tendeloo et al. Adv. Mater. 24, 5655-5675 (2012)
2 P. Kundu et al. ACS Nano 8, 599-606 (2014)

S.V.A and S.T. gratefully acknowledge financial support from the FWO. G.V.T. and P.K. acknowledge the ERC Grant N246791-COUNTATOMS. N.R. acknowledges financial support from the Department of Science and Technology (DST).

Fig. 1: (a) Aberration corrected HRTEM of 2 nm thin wire in [11 ̅0] zone (b) magnified view of the selected wire portion analyzed showing displacement of atomic columns (marked by arrows). (c) High resolution HAADF-STEM image (false color) of the wire analyzed for determining the atom counts in the columns in the [11 ̅0] zone direction as in (d).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2351 A Study on Formation and Thermal Stability of Au-silica Hybrid by Electron Tomography

Kundu P.1,3, Heidari H.1, Bals S.1, Ravishankar N.2, Van Tendeloo G.1
1Electron Microscopy for Materials Research (EMAT), University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 2Materials Research Center, Indian Institute of Science, Bangalore 560012, India, 3Institute of Bioelectronics, Forschungszentrum Jülich, D – 52425 Jülich, Germany
paro.124@gmail.com

SiO2 based metal nanoparticle hybrids form an important class of material which finds application in catalysis, sensors and biotechnology. Although several protocols exist for synthesizing these hybrids, the complete understanding of the morphology, composition and distribution of the heterounits in three dimensions is lacking. Conventional imaging techniques like SEM and TEM gives information on the size, external/surface morphology and partially on the shape of the nanostructure, but it can be misleading for detailed understanding of the internal distribution and structural composition of the hybrid with a 3D shape. However, these are important factors governing their functionalities like catalytic activity, stability, sensitivity, plasmonic behavior etc. Also, thermal stability of such hybrids are important to be investigated and only few studies are reported on that. Here we present a simple wet chemical route to obtain extremely stable Au nanoparticles (5 – 10 nm) decorated SiO2 spheres without using any external linkers. We investigated the composition by STEM-EDX and 3D ordering of the heterounits of the hybrid using STEM tomography. It reveals presence of Au nanoparticles exclusively on the surface of the SiO2 spheres and not inside the matrix. The same characterization method has been used for understanding the mechanism of formation of the hybrid, intermediate nanostructures resulted in course of reaction. This study reveals that the hybrid formation is mediated by self-assembling of Au nanoparticles due to the presence of oleyl amine and (3-mercaptopropyl) trimethoxysilane (MPTMS) on the Au surface and a formation mechanism of the hybrid is deduced. Thermal stability test is performed and change in morphology is studied by tomography which reveals an excellent stability of the structure up to 400oC beyond which the Au particles starts migrating from the surface of the SiO2 sphere into the matrix. However, no significant coarsening of the particles are observed. These kind of structural changes can have significant impact on physical properties or their functional behavior related to surface activity. This also implies low mobility of the Au particles on the SiO2 surface which is advantageous for several applications 1. The method being general could be used for making similar SiO2 based hybrid to stabilize metal nanoparticles.


1 P. Kundu et al. Angewandte Chemie Int. Ed. DOI: 10.1002/anie.201309288

Funding from the European Community’s Seventh Framework Program ERC grant N°246791 – COUNTATOMS, COLOURATOMS, as well as from the IAP 7/05 Programme initiated by the Belgian Science Policy Office is acknowledged. Funding from Department of Science and Technology (DST) is also acknowledged.

Fig. 1: A schematic description of formation of the Au-SiO2 hybrid, where the Au nanoparticles anchor only to the surface of SiO2, via Au nanoparticle self-assembly; however, on heating to higher temperature (400 oC) the Au particles migrate inside the matrix but not aggregate on the SiO2 surface. This confirms a good thermal stability of the hybrid.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2352 Growth and packaging of ultrathin Au nanowires for enhanced thermal stability: An in-situ TEM study

Kundu S.1, Ravishankar N.1
1Materials Research Centre, Indian Institute of Science, C.V. Raman Avenue, Bangalore 560012, India
subhodex@gmail.com

Ultrathin Au nanowires are potential candidate for catalysis, sensing, plasmonic and biological applications. Most of these applications require a clean interface for better performance. Fragility on polar solvent cleaning and hydrophobicity due to the associated linkers limit the use of the nanowires in their as-synthesized form. We have developed a strategy for growth of these nanowires directly on substrates (Figure 1) that imparts stability to the wires. The study on growth and mechanism of nanowire formation on substrates has been carried out using electron microscopy (SEM & TEM) and other techniques.
Poor thermal stability limits the use of these nanowires to low temperature applications only. Hence, for high temperature applications proper packaging of the nanowires is required. A simple wet-chemical method has been developed to coat these nanowires with mesoporous SiO2 (Figure-2) and TiO2 coatings. The SiO2 layer thickness could be controlled very easily by this method by varying the reaction time. Coating thickness of a few nanometers could be obtained. In-situ TEM thermal stability studies have been carried out on the SiO2 coated nanowires. Figure 3 shows the TEM images of the nanowires as the temperature is increased over a period of 4-5 hours. Bare nanowires had been drop-casted on the same grid for comparison. The non-coated nanowires (marked in red) break into nanoparticles at very low temperature as shown in the set of images. Coated nanowires became segmented at similar temperatures but the segments show remarkable stability at high temperature (5530C) for long times.

NR acknowledges Department of Science and Technology (DST) India for financial support. The electron microscopes are a part of the Advanced Facility for Microscopy and Microanalysis (AFMM) at the Indian Institute of Science.

Fig. 1: TEM image showing ultrathin Au nanowires grown on Carbon support.

Fig. 2: Au nanowires with a thin layer of SiO2 coated to enhance thermal stabilty. Inset shows a thicker coating of SiO2 on the nanowires.

Fig. 3: In-situ TEM heating experiment reveals that SiO2 coated Au nanowires are stable at a temperature of 5530C whereas the drop-casted bare Au nanowires break (marked in red).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2363 Mechanism of Au2Sx/CdS Nanorod Formation by Cation Exchange

Kundu S.1, Kundu P.2, Tendeloo G. V.2, Ravishankar N.1
1Materials Research Centre, Indian Institute of Science, C.V. Raman Avenue, Bangalore 560012, India, 2Electron Microscopy for Materials Research (EMAT), University of Antwerp, Groenenborgerlaan 171,B- 2020 Antwerp, Belgium
subhodex@gmail.com

Cation exchange is the process by which the cation in a compound is replaced by another cation from a suitable precursor. It is very difficult to replace any cation by Au, since the high electron affinity of Au leads to reduction of the precursor to form metallic Au rather than undergoing cation exchange. The competition between cation-exchange and reduction is not well understood. For the case of Au or other elements, one of the processes may be dominant over the other depending on the choice of system and the experimental conditions. Knowing the criterion and having a rational understanding of the process is essential for rational synthesis of heterostructures. In our study, we show that cation exchange is unexpectedly dominant over reduction for the case of CdS-Au.
Bright-field TEM imaging (Figure 1) reveals the presence of small, faceted particles of Au on the CdS nanorods. However, on careful observation it shows the formation of more particles under the electron beam. When the concentration of the Au precursor is low, most of the Au2Sx (x=1 & 3) formed as a result of cation-exchange is on the surface, which on exposure to the electron beam leads to the formation of faceted Au particles. In the case of a higher precursor concentration, the beam effects are highly accentuated as the Au2Sx is present across the depths of the sample which results in shortening of the nanorods; in some cases along with the formation of Au nanoparticles. Energy dispersive X-Ray mapping in STEM mode (Figure 2) clearly depicts the change taking place due to beam irradiation. The HAADF-STEM image in Figure 3a further shows three different regions of contrast. Careful investigation of the high magnification STEM images (Figure 3b) reveals the presence of the cubic Au2S phase which confirms that cation-exchange indeed takes place under the reaction conditions. Thermodynamic calculations have been carried out to understand the experimental observation that paves the way for better predictability of the viable product for various systems under different reaction conditions.

NR acknowledges the Department of Science and Technology (DST) India for financial support. PK and GVT acknowledge the ERC Advanced Grant COUNTATOMS.

Fig. 1: Bright field TEM image showing Au attached to CdS nanorods.

Fig. 2: More of such Au nanoparticles form under the electron beam as is evident from the HAADF-STEM image and the corresponding EDS map.

Fig. 3: (a) At high magnification we observe three different regions of contrast as marked by the blue dotted line. (b) Atomic resolution imaging clearly shows the Au2S and CdS domains.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2377 Recent advances in Catalysis Research using Electron Microscopy

Wagner J. B.1, Deiana D.1, Chorkendorff I.2, Stephens I.2, Hansen T. W.1
1Center for Electron Nanoscopy, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark, 2Center for Individual Nanoparticle Functionality, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
jakob.wagner@cen.dtu.dk

Electron microscopy provides a highly versatile platform for the characterization of supported metal nanoparticles for heterogeneous catalysis. With both high spatial resolution as well as spectroscopic capabilities, the EM platform can characterize materials in detail. Recent developments include high solid angle EDX detectors, which can rapidly acquire high-resolution elemental maps, and micro electro-mechanical systems (MEMS) based heating holders that can heat samples at very high rates with only little spatial drift. With the addition of environmental capabilities, the microscope can even probe samples under reactive environments.
It is impractical to use all techniques and modification on a single instrument. Hence, in order to obtain the complete picture of catalyst samples, several platforms can be employed.
A recent trend in catalysis is the use of materials that have been engineered at an atomic level. In particular, Density Functional Theory (DFT) can be used to computationally screen for new materials. These are often multi-metal alloys, which add new functionality and can reduce the amount of precious metals. Such samples can be size selectively produced either by physical routes, e.g. time of flight mass selection or chemical synthesis e.g. micelle encapsulation. Whereas these approaches may not be technically applicable for large-scale synthesis, they provide a valuable route for gaining fundamental knowledge.
Here, we show findings from three different systems used in three different reactions. Namely Pt-Y for oxygen electroreduction to H2O, Pd-Hg for electrochemical synthesis of hydrogen peroxide and ruthenium based catalyst used for methanation [1-3]. With these examples, we illustrate two principle points of nanoparticle functionality: composition and shape.
In the case of the bimetallic catalysts, the elemental distribution in the nanoparticles is of fundamental interest: Do they form a core-shell system or do form an evenly distributed mixture/alloy? Using a high solid angle EDX detector, elemental maps can be efficiently collected and the elemental distribution monitored. Such verification is essential to understand the working principle of the catalyst.
Ruthenium nanoclusters can be used for methanation of carbon monoxide, a reaction used to clean up feed gas for e.g. proton exchange fuel cells (PEM). As-synthesized, the Ru particles assumed high surface-area raspberry-like shapes. However, after treatment under conditions relevant for the methanation reaction, the particles adopted more spherical shapes.
[1] F. Masini et al. J. Catal. 308 (2013) 282
[2] S. Siahrostami et al. Nature Materials 12 (2013) 1137
[3] A. Verdaguer-Casadevall et al. Nano Letters 13, dx.doi.org/10.1021/nl500037x

Fig. 1: a) STEM micrograph of a nanoparticle and b-d) corresponding Y, Pt and combined X-Ray elemental maps.

Fig. 2: a) STEM micrograph of a nanoparticle and b-d) corresponding Hg, Pd and combined X-Ray elemental maps.

Fig. 3: Ruthenium nanoparticle imaged under different conditions relevant for the methanation reaction. a) Room temperature, vacuum; b) 427°C, vacuum; c) 427°C, 230 Pa 1:10 CO/H2.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2392 Global-refinement electron exit wave reconstruction from focal series of ceria nanoparticles

Borisenko K. B.1, Young N. P.1, Kirkland A. I.1
1Department of Materials, University of Oxford, Parks Road, Oxford, OX1 3PH, UK
konstantin.borisenko@materials.ox.ac.uk

Nanoparticles play an increasingly important role in catalysis. At the nanoscale, stoichiometry and the thermodynamic stability of different crystallographic facets may be modified as compared to the bulk, which is one of the reasons for their increased importance. Understanding activity and selectivity of nanoparticle catalysts requires detailed understanding of catalytic reactions at the atomic scale. An important step towards this goal is to obtain accurate atomic structures of catalytic nanoparticles and especially of their surface and immediate subsurface regions.

Aberration-corrected high resolution transmission electron microscopy (HRTEM) is a well suited tool for studying atomic structures of such particles. Quantitative analysis of the electron exit wave obtained from the experimental series of images with different focus can in principle provide some additional information on the three-dimensional structure.

In the present work we test an enhanced approach to quantitative exit wave restoration from the focal series of HRTEM images obtained for ceria nanoparticles. The existing linear exit wave restoration codes are based on the assumption that the sample under investigation is a weak-phase object. This approximation applied to a general object can result in an incorrect restoration. A more general approach is to reconstruct the exit wave by minimising the sum of squared differences between the simulated and experimental images where both the amplitude and the phase of the exit wave can be restored accurately. We suggest using the exit wave reconstructed by the linear approach as an initial approximation for this more general reconstruction. The successful restoration using the suggested method is dependent on knowing accurate aberration parameters of the microscope, especially the focus range and the focal step, and accurate image alignment in the experimental focal series. These are not routinely available from the actual experimental conditions. The suggested approach employs refinement of the exit wave together with both the aberration parameters and the image alignment in a single refinement cycle. The resulting exit wave is compared with exit wave obtained by theoretical multislice simulations and with the exit wave obtained by linear restoration software. We also investigate the origin of the increased contrast at the edges of the nanoparticles seen in the reconstructed phase, examining whether it is a consequence of adsorbed light species or a manifestation of electrostatic surface potential.

Financial support from the European Union under the Seventh Framework Program under a contract for an Integrated Infrastructure Initiative (Ref 312483-ESTEEM2) is gratefully acknowledged.

Fig. 1: Global-refinement exit wave restoration algorithm implemented in the present study.

Fig. 2: Reconstructed electron exit wave amplitude a) and phase b) of ceria nanoparticles. Note the bright contrast at the edges of the nanoparticles in the phase image.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2497 TEM and STEM observations of Au/Fe2O3 catalysts

Akita T.1, Maeda Y.1, Kohyama M.1
1National Institute of Advanced Industrial Science and Technology (AIST)
t-akita@aist.go.jp

Gold exhibits characteristic catalytic properties when Au nano-particles are supported on the metal oxides [1,2]. It has been reported that catalytic properties depending on the kind of the metal oxide supports are observed for various catalytic reactions. For example, high catalytic activity is observed in the low temperature CO oxidation when TiO2 was used as support and high catalytic activity for water-gas-shift reaction at low temperature was observed when the CeO2 is used as support [3]. The origin of the catalytic properties of Au catalysts is not clear yet although it is suggested that the interface between the small Au particle and the metal oxide support act as active sites [4,5]. In order to clarify the relation between the fine structure and the catalytic properties at the Au-metal oxide interface, we have carried out the structure analyses on the small Au particle supported on TiO2, NiO and CeO2 with a transmission electron microscopy (TEM) and annular dark field scanning transmission electron microscopy (ADF-STEM) [6,7]. In this experiment, the basic structure of Au nano-particles supported on Fe2O3 was observed in atomic scale by HRTEM and STEM.

Au/Fe2O3 catalysts were prepared by solid grinding method using organogold complex [8] and deposition precipitation (DP) method. γ-Fe2O3 fine particle (Nanophase Tech. Corp.) which has spinel structure was used for support. The catalysts were calcined at 573K for 4 hours in air. The observations were carried out by using aberration corrected TEM/STEM (FEI Titan3 G2 60-300). Accelerating voltage for the observation was 300kV.

Figure 1 shows typical ADF-STEM images of Au/γ-Fe2O3 catalyst prepared by DP method. Small Au particles of approximately 2-10 nm in diameter are deposited on theγ-Fe2O3 support. Theγ-Fe2O3 support crystal exhibit polyhedral shape with low index facets such as {111}, {100}. Figure 2 shows profile-view HRTEM images of Au particles onγ-Fe2O3 (111). The incident electron beam direction was adjusted alongγ-Fe2O3 [1-10] zone axis. Gold particles tend to be deposited on theγ-Fe2O3 surface with the preferential orientation relationships of (111)[1-10]Au//(111)[1-10]γ-Fe2O3 or (111)[-110]Au // (111)[1-10]γ-Fe2O3 for theγ-Fe2O3 (111) surface. The high resolution STEM observations were also carried out for the Au/γ-Fe2O3 interface.

References

[1] M. Haruta et al., Chem. Lett., (1987) 405.

[2] M. Haruta, Catal. Today 36(1997)153.

[3] H. Sakurai et al., Appl. Catal. A: General 291 (2005)179.

[4] T. Fujitani et al., Angew. Chem. Int. Ed. 48(2009) 9515.

[5] T. Fujitani et al., Angew. Chem. Int. Ed. 50(2011)10144.

[6] T. Akita et al., Surf. Interface Anal.40, (2008)1760.

[7] T. Akita et al., J Mater Sci. 43(2008)3917.

[8] T.Ishida et al., Chem. Eur. J. 14 (2008) 8456.

The authors are grateful to Ms. F. Arai, Ms. C. Fukada and Ms. M. Makino for their assistance with sample preparation.

Fig. 1: FIG. 1. Typical ADF-STEM image of Au/γ-Fe2O3 catalyst.

Fig. 2: FIG. 2. HRTEM image of Au onγ-Fe2O3 substrate.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2533 Interconnection of Nanoparticles within 2D Superlattices of PbS/ Oleic Acid Thin Films

Simon P.1, Bahrig L.2, Baburin I. A.2, Formanek P.3, Röder F.4, Sickmann J.4, Lichte H.4, Hickey S. G.2, Eychmüller A.2, Kniep R.1, Rosseeva E.5
1] Max Planck Institute for Chemical Physics of Solids, Nöthnitzer Straße 40, 01187, Dresden,Germany, 2TU Dresden, Physical Chemistry, Bergstrasse 66b, D-01062 Dresden, Germany, 3Leibniz-Institut für Polymerforschung Dresden e.V., Hohe Straße 6, 01069 Dresden, Germany, 4Institute of Structure Physics, Triebenberg Laboratory for High-Resolution Electron Microscopy and Holography, Technical University of Dresden, Zum Triebenberg 50, 01328 Dresden Zaschendorf, Germany, 5University of Konstanz, Physical Chemistry, POB 714, D-78457 Konstanz, Germany
Paul.Simon@cpfs.mpg.de

Ensembles of nanoparticles possess collective properties that are dissimilar to those demonstrated by the individual particles and self-assembly has emerged as a powerful means by which the structure and properties of inorganic nanoparticle arrays can be manipulated.. In order to aid in the resolution of the keenly contested debate between proponents of the fibrillation model and those of the electrostatic forces interaction model the structure formed by monolayers of PbS colloidal nanocrystals was investigated using high-resolution spherical aberration corrected TEM, high-resolution electron holography and energy filtered TEM [1,2]. By employing this suite of techniques it could be observed that the truncated octahedrally shaped nanoparticles form 2D close-packed layers interconnected by organic fibrils of oleic acid which are partially mineralised by PbS. These bridges, whose diameters are between 0.3 and 2 nm, keep the face to face orientation of the nanoparticles fixed, thus preventing them from assuming an arbitrary orientation. The complex and textured structure of the monolayer assembly is caused by the habit of the truncated octahedral PbS nanoparticles bearing angles close to ideal values of 54° and 71° between their {100} and {111} faces. By means of electron holography, approximately 10-15 fibrillar interconnections between neighbouring particles in the as-prepared films have been observed. Each nanoparticle is surrounded by six other individuals. At least two or three organic “linkages” are formed between the particles and connect to a nearest neighbour. Most of the organic connections can be mineralised successively by PbS during careful annealing. By using this bottom-up technique access to length scales of sub-nanometer dimensions, presently not accessible to top-down techniques can be attained. This type of isolated but yet interconnected structure formed by the inorganic bridges, represents an ideal “isolated but connected” structure that preserves the effects of quantum confinement present within the individual nanoparticles whilst at the same time having the potential to provide high electron mobility throughout the extended structure.

[1] P. Simon, E. Rosseeva, I.A. Baburin, L. Liebscher, S.G. Hickey, R. Cardoso-Gil, A. Eychmüller, R. Kniep, W. Carrillo-Cabrera, Angew. Chem. Int. Ed. 2012, 51, 10776-10781.

[2] P. Simon, L. Bahrig, I.A. Baburin, P. Formanek, F. Röder, J. Sickmann, S.G. Hickey, A. Eychmüller, H. Lichte, R. Kniep, E. Rosseeva, Adv. Mater. 2014 DOI: 10.1002/adma.201305667

Fig. 1: 3D representation of the phase image retrieved from the electron hologram. Color code corresponds to 4 nm height from green to blue. The bridging organic fibrils appear yellow.

Fig. 2: 2D representation of phase image. The PbS nanoparticles and the interconnecting sub-nanometer oleic acid fibrils appear bright in the phase image.

Fig. 3: (a) Cs-corrected HR-TEM image of two nanoparticles interconnected by a PbS bridge. The PbS bridge (red arrow) has a diameter of 0.3 nm and a length of 1.5 nm. The periodicity along the bridge corresponds to 0.3 nm which is equivalent to the (200) lattice plane of PbS. (b) Digitally zoomed area.

Fig. 4: Idealized model of isolated but interconnected PbS nanoparticles.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2556 Atomic Resolution Characterization and Dynamics due to Beam Interaction of Ni base Nanoparticles for Energy Devices

Calderon H. A.1, Godinez-Salomon F.2, Solorza-Feria O.2, Specht P.4, Kisielowski C.3
1Dept. Física, ESFM-IPN, Zacatenco D.F. 07338, Mexico, 2Dept. Química, CINVESTAV, Mexico D.F., Mexico, 3JCAP and NCEM, LBNL, Berkeley, CA 94720, U.S.A., 4Dept. Mats. Sci. Eng., UCB, Berkeley, CA 94720
hcalder@esfm.ipn.mx

Ni base nanoparticles (NPs) are characterized under low dose conditions in TEM mode. These nanoparticles are mainly designed to act as catalysts in energy devices. Ni, NiO and Pt@NiO nanoparticles are investigated. Particularly the use of Nio@Pt for solar cells (artificial photosynthesis) is attractive, namely the hydrogen evolution center, while NiO has been tested as a catalyst for the oxygen evolution center. Consequently an atomic characterization of the involved nanocrystals is of particular importance. Here, transmission electron microscopy is used with the objective to determine nature, shape and atomic distribution of Pt for different loadings (0-16 at.%) on a Ni core basis. In all cases the electron dose rate has been kept in the range 20-150 e-/Å2s in order to avoid surface rearrangement by interaction with the electron beam. The TEAM 05 (80 KeV) has been used together with focal series reconstruction (EWR) to recover both phase and amplitude images that provide information of the spacing and the chemical nature of the corresponding atomic columns. Two procedures have been used for synthesis of nanoparticles. One of them produces Ni and the other NiO-NPs. NiO NPs are then covered with different loadings of Pt in order to create incomplete core shell structures but with superior catalytic activity. Figure 1 shows phase images of Ni NPs, their size varies from 1 to 7 nm and can agglomerate most likely due to their magnetic characteristics. The dose rate used to acquire the experimental images is 30 e-/Å2s. Figure 1b shows experimental images of NiO NPs acquired with a dose rate of 120 e-/Å2s, their average size is around 1.5 nm. During processing Pt is deposited on NiO particles and a typical example is given in the phase images shown in Figs. 2a-b, the dose rate is around 55 e-/Å2s and the Pt coverage is nominally 8 at. %. The nanoparticles have mostly irregular shapes. There is a negligible particle transformation due to the weak interaction with the electron beam. These NPs are nevertheless susceptible to alteration in shape and structure as a consequence of electron beam sample interaction. An example is given in the phase images shown Figs. 3 a-c. In these cases, the dose rate has been increased from 55 e-/Å2s (Fig. 3a) to 300 e-/Å2S (Fig 3b) and 1400 e-/Å2s (Fig. 3c). The particle under observation initially losses atoms that apparently redeposit on the carbon support and migrate (partially) to form a new crystal. The selected NP becomes bicrystalline at the end of this experiment that clearly shows the need to use a proper electron dosage for observation and the possible large influence of thermal effects. Phase images have been used for simulation in order to determine the Pt coverage.

CONACYT (FOINST. 75/2012, 129207 and 148304) and IPN (COFAA-SIP) are gratefully acknowledged for financial support.

Fig. 1: Figure 1. (a) Phase image of Ni nanoparticles and (b) Experimental image of NiO nanoparticles at a dose rate of 150 e-/A2s.

Fig. 2: Fig. 2. Phase images of NiO nanoparticles with a Pt coverage of 8 at.%. and taken with a dose rate of 55 e-/Å2s.

Fig. 3: Fig. 3. Phase images of NiO nanoparticle with an 8 at. % Pt coverage. (a) Dose rate of 55 e-/Å2s. (b) Dose rate of 300 e-/Å2s and (e) Dose rate of 1400 e-/Å2s.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2562 Localization microscopy (SPDM) facilitates high precision control of lithographically produced nanostructures

Grab A. L.1, Hagmann M.2, Dahint R.1, Cremer C.2,3
1Angewandte physikalische Chemie, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany, 2Kirchhoff-Institute for Physics, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany , 3Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany
martin.hagmann@kip.uni-heidelberg.de

In numerous fields, the development of innovative technologies requires a refinement and miniaturization of existing systems resulting in an increasing requirement for process friendly quality and dimension control for industry. Localization microscopy (SPDM) provides a precise control of nanostructures, which are indispensable for example for optronics, biosensing applications, manufacturing of electrical elements, biomedical applications, environmental issues, flow profiles in air or water and self-cleaning surfaces.
The principle of this "superresolution microscopy" technique is the use of "point-like" objects carrying different spectral signatures (e.g. fluorescent dyes different in absorption and/or emission spectra; fluorescent dyes with different life-times; time dependence of luminescence, reversible bleaching behaviour, etc.).
Using a lithographic approach, highly regular nanostructures have been generated and marked with Alexa 647 dyes. The spatial organization of the dyes on nanostructured surfaces consisting of interconnected cubes has been averagely localized down to 6 nm using localization microscopy. Herewith we illustrate two aspects: The application potential of localization microscopy as an integrated process for quality control in addition to the absolute spatial calibration of Spectral Precision Distance Microscopy (SPDM).
The findings will be important in the field of product control for industrial applications and long-term fluorescence imaging and calibration for most super-resolution fluorescence microscopes in general. As SPDM improves the optical resolution compared to standard fluorescence microscopy, structure dimensions and the excellent quality of the lithographical grating were resolved beyond the Abbe limit with high precision.

The authors gratefully acknowledge the sample fabrication by the Karlsruhe Nano Micro Facility, especially we like to express our gratitude to A. Nesterov-Müller. All authors thank S. Dithmar for financial support and our dear colleagues Gerrit Best, Sabrina Rossberger, Dr. Udo Birk, Florian Schock and Dr. Fanny Liu. We thank the Boehringer Ingelheim Foundation for generous support.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2567 Mechanical Behavior at the Nanoscale: the benefits of coupling of In situ TEM nano-compression and compression inside a Diamond Anvil Cell

ISSA I.1,2, Calvié E.1, Joly-Pottuz L.1, Rethore J.2, Amodeo J.1, Esnouf C.1, Chevalier J.1, Garnier V.1, Masenelli-Varlot K.1
11Université de Lyon, INSA-Lyon, CNRS, MATEIS, 69621 Villeurbanne, France, 22Université de Lyon, INSA-Lyon, CNRS, LaMCoS, 69621 Villeurbanne, France
inas.issa@insa-lyon.fr

Nanometer sized objects are attracting large attention nowadays due to their breakthrough mechanical properties such as high hardness, crack propagation resistance and high elastic limit in comparison to the bulk of their counterparts [1]. In situ TEM nanoindentation is a particularly well suited technique for the mechanical testing of nano-sized objects. Images of the deforming sample and force-displacement curves can simultaneously be acquired. The challenge remains in the identification of the material mechanical behavior, namely the constitutive law with the intrinsic parameters – Young modulus, yield strength, Poisson ratio – as well as the understanding of the deformation mechanism.In this study, we propose an innovative method for a complete mechanical analysis of nanoparticles in the size range [30 nm-300 nm]. This protocol consists in coupling of in situ TEM nano-compression tests of isolated nanoparticles, image analysis and mechanical simulations. After the experiments, the load–real displacements curves are measured by Digital Image Correlation. Then a constitutive law is obtained through an inverse Finite Elements simulation. The determination of a constitutive law includes the determination of the material intrinsic parameters such as Young modulus, Yield stress, hardening coefficient, and stress at fracture.In this presentation, the method will be presented through the analysis of transition alumina nanoparticles. It will be shown that such ceramic nanoparticles can undergo large plastic deformation, which is not observed in the bulk (Fig1). The parameters of the constitutive law will be discussed in the light of the literature, and especially the work from K. Zeng et al. [1]. They showed that the electron beam, during in situ TEM nano-compression tests of silica nanoparticles, creates structural and bonding defects throughout the entire sample and facilitates the plasticity of the nanoparticles.The deformation mechanisms will be investigated through performed compression experiments in a Diamond Anvil Cell, at room temperature and in the absence of electron beam. We will present the results obtained from HRTEM observations of thin foils extracted from samples compacted at various uniaxial pressures. We will show that plastic deformation occurs also in this case (Fig2). Moreover, the appearance of a nanoparticle preferential orientation will be evidenced (Fig3). This point will be discussed in function of the possible slip systems. Finally, we will demonstrate that such HRTEM analysis gives interesting pieces of information, which permit to better understand how the nanoparticles behave and deform during the in situ experiments.
[1] Kraft et al. Annual Review of Materials Research,2010.
[2] K. Zheng et al. Nature Communications, 2010.

S. Le Floch, D. Machon . Institute ILM, Université Lyon 1 (compaction nano-powder DAC)

Fig. 1: TEM in situ nano-compression force-displacement curve. The simulations using DIC-FE (red) or the analytical method (blue). A good agreement is found for both simulations with the experiment, especially for the DIC-FE method which takes into account the plastic regime, contrary to the analytical method which is valid only in the elastic domain.

Fig. 2: (Left) TEM image revealing the plastic deformation of transition alumina nanoparticle compacted in a DAC at 5 GPa uniaxial pressure. (Right) TEM image of compacted transition alumina in DAC at 20 GPa. It reveals the oriented crystallographic texture with respect to the compression axis.

Fig. 3: (a) HRTEM image of the FIB thin foil of a zone of contact between two alumina nanoparticles. (b) Fourier Transform of the dotted zone of the deformed particle.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2568 In situ TEM Nano-Compression and Mechanical Analysis of MgO

Issa I.1,2, Amodeo J.1, Joly-Pottuz L.1, Réthoré J.2, Esnouf C.1, Garnier J.1, Morthomas J.1, Masenelli-Varlot K.1
1Université de Lyon, INSA-Lyon, CNRS, MATEIS, 69621 Villeurbanne, France, 2Université de Lyon, INSA-Lyon, CNRS, LaMCoS, 69621 Villeurbanne, France
inas.issa@insa-lyon.fr

Nanometer-sized objects are attracting large attention nowadays due to their breakthrough mechanical properties such as high hardness, crack propagation resistance and high elastic limit in comparison of the bulk state of the studied material [1].
Moreover, these nano-objects exhibit large plastic deformation under high load; this was not expected for certain materials and especially for ceramics. Large numbers of studies nowadays are dedicated to plastic deformation of Metals at the nano-scale, and few are reported on ceramics [2, 3].
The origin of this plastic deformation is still not very well defined. Mechanisms proposed are size dependent, and link this behavior to dislocations nucleation at surfaces and slipping on certain planes depending on the crystal orientation with respect to the solicitation direction. Another mechanism proposed is the mechanical twining via full dislocations dissociations into partial Shockley dislocations that glide on a slipping plane (the denser) of the crystal.

A protocol consisting of in situ TEM nano-compression tests of isolated nanoparticles coupled with data processing by Finite Elements and Molecular Dynamics simulations has been developed [2], and applied to the study of spherical alumina nanoparticles. Identification of deformation mechanisms remains quite difficult since the orientation of the nanoparticle on the substrate prior to compression is not controlled.
In this study, we will present in situ TEM nano-compression experiments on MgO nanocubes. The main advantage of studying such nanocubes lies in the fact that their crystallographic orientation with respect to the indenter tip is fully known. It will be shown that MgO can undergo large plastic deformation, more than 50%, without any fracture. Then, we will propose a mechanical behavior law from the analysis of the images and curves followed by Finite Elements simulation. Finally, deformation mechanisms will be identified from the comparison between the contrasts in the images and Molecular Dynamics simulations [4].

[1] Kraft et al. Annual Review of Materials Research (2010) 40:293-317
[2] Calvie et al. Journal of the European Ceramic Society (2012) 32:2067-71
[3] Korte et al. Acta Materialia (2011) 59:7241-54
[4] Amodeo et al. Acta Materialia (2011) 59:2291-2301

Fig. 1: 100 nm edge size, MgO Nanocube before compression in situ in TEM

Fig. 2: 100 nm edge size, MgO Nanocube After compression in situ in TEM

Fig. 3: Stress-strain curve Obtained from Load-Real displacements curve of the nanocube compressed in situ

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2577 Monodisperse embedded nanoparticles derived from an atomic metal-dispersed precursor of layered double hydroxide for architectured carbon nanotube formation

Tian G.1, Zhao M.1, Zhang B.2, Zhang Q.1, Zhang W.3, 4, Huang J.1, Chen T.1, Qian W.1, Su D.2, 3, Wei F.1
1Beijing Key Laboratory of Green Chemical Reaction Engineering and Technology, Department of Chemical Engineering, Tsinghua University, Beijing, China, 2Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, China, 3Department of Inorganic Chemistry, Fritz Haber Institute of the Max Planck Society, Berlin, Germany, 4Department of Energy Conversion and Storage, Technical University of Denmark, Roskilde, Denmark
tian-gl10@mails.tsinghua.edu.cn

Monodisperse metal nanoparticles (NPs) with high activity and selectivity are among the most important catalytic materials. However, the intrinsic process to obtain well-dispersed metal NPs with tunable high density (ranging from 1013 to 1016 m-2) and thermal stability is not yet well understood. Herein, the preparation of metal NPs with tunable areal density from layered double hydroxide (LDH) precursors in which the metal cations were pre-dispersed at an atomic scale was explored. Large quantities of mesopores induced by the Kirkendall effect were formed on the as-calcined layered double oxide (LDO) flakes. The O atoms bonded with Fe3+ cations were easily to be extracted at a temperature higher than 750 oC, which greatly increased the mobility of Fe. Consequently, coalescence of the reduced Fe atoms into large NPs enhanced the Kirkendall effect, leading to the formation of monodisperse embedded Fe NPs on the porous LDO flakes. The flake morphology of LDHs was well preserved, and the areal density of Fe NPs on the LDO flakes can be well controlled through adjusting the Fe content in the LDH precursor. With higher Fe loading, larger Fe NPs with higher areal density were available. When the areal density was increased from 0.039 to 0.55, and to 2.1 × 1015 m-2, the Fe NPs embedded on the LDO flakes exhibited good catalytic performance for the growth of entangled carbon nanotubes (CNTs), aligned CNTs, and double helical CNTs, respectively. This work provides not only new insights on the chemical evolution of monodisperse NPs from an atomic metal-dispersed precursor, but also a general route to obtain tunable NPs as heterogeneous catalysts for chemical and material production.

References:
1. G. L. Tian, M. Q. Zhao, B. S. Zhang, Q. Zhang, W. Zhang, J. Q. Huang, T. C. Chen, W. Z. Qian, D. S. Su and F. Wei, J. Mater. Chem. A, 2014, 2, 1686–1696

The work was supported by the Foundation for the China National Program (No. 2011CB932602) and Natural Scientific Foundation of China (No. 21306102). Bingsen Zhang is supported by the IMR SYNL-T.S. Keˆ Research Fellowship. Bingsen Zhang thanks the financial support provided by the China Postdoctoral Science Foundation (2012M520652).

Fig. 1: STEM image of Fe distributed on LDO flakes.

Fig. 2: (a) Entangled CNTs grown on LDH-I, (b) aligned CNTs grown on LDH-III, and (c) double helical aligned CNTs grown on LDH-V. (d) The phase diagram of CNTs grown on flat/flake substrates with different catalyst densities and sizes.[1]
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2623 Effect of the amount of dopant and the synthesis method on structure and morphology of nanocrystalline Ce1−xRExO2−y

Mendiuk O.1, Kepinski L.1
1Institute of Low Temperature and Structure Research, PAS, Wrocław, Poland
o.mendiuk@int.pan.wroc.pl

Nanocrystalline pure or doped ceria is an important material widely used in various fields of technology, including optics, microelectronics and catalysis. Doping of ceria with transition metal ions enhances its property and improves the thermal stability of nanocrystalline ceria against sintering. It has been established that catalytic activity of ceria nanoparticles depends strongly on their morphology: nanoparticles with cube or rod morphology, exposing {1 0 0} planes at the surface, are desirable for catalytic reactions of CO and soot combustion [1,2].
In this work mixed Ce1−xLnxO2−y (Ln=Gd, Er) oxides were synthesized by the hydrothermal treatment [3,4]. Two modifications of the hydrothermal treatment – classical and microwave assisted – were applied. The effect of the amount of dopant and the synthesis method on the phase composition and morphology of the of lanthanide oxides was studied by SEM-EDS, EBSD, TEM, XRD and Raman spectroscopy.
By classical hydrothermal treatment, for low doping level, nanocubes of the mixed Ce-Ln oxide with fluorite structure and bimodal size distribution (small 5-20 nm and much bigger 50-80 nm) were formed (Fig.1), while at higher doping (x > 0.3 ) rod-like particles of Ln hydroxide were also observed. Using of microwave radiation enabled the synthesis of the nanocubes of the mixed oxides at significantly shorter time, but the resulting materials is different: over broad range of Ln contents (0.05 <x< 0.5) particles with nanorod and nanocube morphology were obtained (Fig.2). TEM show that smallest particles with low doping level, which could not be characterized by SEM, contains mostly regular cube shape particles, though there is a fraction of small particles having rounded corners (Fig.3). SAED pattern contain sharp rings that can be assigned to fluorite structure of ceria. Particle size distribution is very broad and bimodal.
EBSD combined with EDS was used to analyze the structure and composition of unusual, large oxide nanocubes (50 – 80 nm) appearing in the samples (Fig.4). It appeared that the nanocubes of the mixed Ce-Ln oxide have fluorite type structure of CeO2 and are single crystals but not aggregates of smaller crystallites.
[1] X.W. Liu, et.al., J. Am. Chem. Soc. 131 (2009) 3140–3141;
[2] K.B. Zhou, X. Wang, X.M. Sun, Q. Peng, Y.D. Li, J. Catal. 229 (2005) 206–212;
[3] H.X. Mai, L.D. Sun, Y.W. Zhang, R. Si, W. Feng, H.P. Zhang, H.C. Liu, C.H. Yan, J. Phys. Chem. B 109 (2005) 24380–24385.;
[4] Z. Wang, Q. Wang, Y. Liao, G. Shen, X. Gong, N. Han, H. Liu, Y. Chen, ChemPhysChem., 12 (2011) 2763–2770

The authors thank Mrs. E. Bukowska for XRD measurements and Mr. M. Ptak for recording Raman spectra

Fig. 1: SEM image from Ce0.95Er0.05O2−y (classical hydrothermal treatment)

Fig. 2: SEM image from Ce0.95Er0.05O2−y (microwave assisted hydrothermal treatment)

Fig. 3: TEM image and SAED pattern from Ce0.95Er0.05O2−y (classical hydrothermal treatment)

Fig. 4: EBSD indexed pattern from single nanocube of Ce0.95Er0.05O2−y
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2625 Fractal growth of porous Cu2S nano-crystals

Zhu G. Q.1, Wang C. H.1, Shi L.1, Lu W.1, Zhang J. P.1
1Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, China 215125
jpzhang2008@sinano.ac.cn

In this work we report on the structure characteristics of Cu2S nano-crystals and the fractal features in crystal growth.

The Cu2S crystals were produced by using a carbon-coated TEM copper grid added with a few drops of dispersed Graphene loaded with sulfur nano-particles in ethanol. When the solution is dried, a variety of well-constructed tine crystals were observed near the copper bars in microscope, which were mostly dendrites as shown in Figure 1a, similar to the observation by Q Han[1]. The diffraction patterns obtained from different dendrites all showed a 6-fold symmetry, as shown in Fig.1b, which can be indexed with hexagonal Cu2S[2]. No other phases or amorphous copper sulfide, as reported in [1], were observed.

Actually the crystallized dendrites exhibit a porous feature since they are composed of numerous nano-crystals in size of few nanometers, as indicated in dark-field scanning TEM images, an example presented in Figure2a. An interesting question is the diffraction pattern from a large area of a dendrite having a number of branches, see in Fig.1a, show a simple [001] pattern that means all nano-crystals are so well oriented that not only along the C-axis, but also the atomic arrangement of those c-planes are aligned in 3o of rotation with respect to each other, see the Fig.1b. That implies hundreds or even thousands of copper sulfide nano-particles bonded together porously could behave as a single crystal, rather than a randomly arranged one.

Of the well-oriented nano-crystals the produced copper-sulfide crystals present interesting self-similarity morphologies, the dendrites like a leaf, a fern, or even a mountain top, some of them We calculated the fractal dimensions using the box-counting method [3] on the nano-crystals and the Matlab codes designed by San Pedro [4]. The fractal dimension of a typicl Cu2S-dentride, as shown in Figure 3a is 1.8623, while the ideal value is 2.

[References]

[1] Qiaofeng Han, Shanshan Sun, Jiansheng Li and XinWang, Nanotechnology 22 (2011) 155607 .

[2] Cava. R.J., Reidinger. F., Wuensch. B.J., Solid State Ionics, 5 (1981) 501.

[3] G. Hartvigsen, The Analysis of Leaf Shape Using Fractal Geometry. The American Biology Teacher. 62 (2000) 664.

[4] S. San Pedro, Fractal Dimensions of Leaf Shapes, Math 614-Sp2009 Web site: http://www.math.tamu.edu/~mpilant/math614/StudentFinalProjects/SanPedro_Final.pdf

[Acknowledgement]

This project is supported by National Basic Research Program of China (2010CB934700) and National Natural Science Foundation of China (Grant No. 21210004).

Fig. 1: Figure 1. (a) Dendrites observed on Cu-bars of thin carbon film coated copper grids; (b) the corresponding electron diffraction pattern from the selected area circled in (a) indicating a [001] oriented Cu2S and the C-plane of different branches are well-aligned within 3o in rotation respectively.

Fig. 2: Figure 2. An enlarged image from a portion of a dendrite obtained in dark field scanning TEM mode (Z-contrast imaging) exhibits a porous structure composed of nano- Cu2S-crystals (bright dots) in size of 5±1 nm and different size of holes (black dots).

Fig. 3: Figure 3. Cu2 Nano-crystal growth produces a variety of fragmented self-similar shapes similar to leafs, ferns, or other plants, a typical example shown in (a). After the fractal dimension analysis, the grayscale image (a) became a binary image (b) with a threshold of 190, estimated from the slope of the test-fit line as show in (c).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2628 In situ deposited nanocarbon for TEM characterization of zeolite supported metal catalysts

Chu Y.1, Zhang B. S.2, Zhang Q.1, Wang Y.1, Su D. S.2, Wei F.1
1Beijing Key Laboratory of Green Chemical Reaction Engineering and Technology, Department of Chemical Engineering, Tsinghua University, Beijing, China, 2Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, China
zhang-qiang@mails.tsinghua.edu.cn

Zeolite supported metal catalysts are widely used while the electron beam sensitive zeolite makes the characterization of the catalysts using electron microscope (EM) difficult. In this contribution, a sacrificial-zeolite specimen preparation (SZSP) technique is developed for the EM analysis of the catalyst. The metal particles are transferred from the zeolite support to the deposited nanocarbon generated in the metal catalyzed hydrocarbon reaction. SAPO-34 zeolite with Al2O3 binder supported Pt catalyst is employed as the model catalyst. Pt catalyzed propane dehydrogenation reaction is carried out to deposit the nanocarbon overlayer which the Pt particles are transferred to as the new support for EM observation. The original catalyst, the deposited nanocarbon and the Pt particles on the new support are characterized by scanning electron microscope (SEM), transmission electron microscope (TEM-EDXS), thermogravimetry/differential thermal analysis (TG-DTA), Raman spectrometry, scanning transmission electron microscope (STEM-EDXS). The coke deposited on SAPO-34 and Al2O3 are of different morphologies and structures. The as-observed distribution of Pt particles on the new support suggests enrichment of Pt on SAPO-34. The shape and size of the Pt particles as well as the strong Pt-SAPO-34 interaction are directly observed. The shape and size of the Pt particles as well as the mechanism of SMSI between Pt and the original support are directly observed. This offers a novel route to monitor the metal size and the interaction between the metal and support, which shed a light on the mystery science of heterogeneous catalyst and provide new insights on the relationship among the structure, active site, and reactivity.

We thank Ling Hu and Tongwei Wu for their help with the transmission electron microscope and the H2-chemisorption experiment. This work was supported by National Basic Research Program of China (973 Program, 2011CB932602), Research Fund for the Doctoral Program of Higher Education of China (No. 20100002110022) and the IMR SYNL-T.S. Kê Research Fellowship.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2659 Magnesium Oxide Nanoparticles Studied by Electron Microscopy

Gärtnerová V.1, Remiášová J.1, Jäger A.1
1Institute of Physics AS CR - Prague 8 (Czech Republic)
gartner@fzu.cz

Magnesium oxide can be used in a wide range of applications covering, for instance, a catalyst in organic chemistry, an adsorbent for a variety of toxic substances, and as a refractory material. There are many routes for preparation of MgO particles but the smallest crystallite size is usually obtained via sol-gel techniques. Here, we present an innovative method for production of magnesium oxide nanoparticles and their microstructure characterization by scanning and transmission electron microscopy (SEM and TEM). MgO can be prepared via a reaction between magnesium (Mg) and methanol (CH3OH) that can be described as

Mg + 2CH3OH → Mg(OCH3)2 + H2            (1)

where the final products are magnesium methoxide Mg(OCH3)2 and hydrogen H2. This reaction, however, practically does not occur at ambient temperatures and must be accelerated either by catalyst or by heating at higher pressures in reflux apparatus. The most common catalyst used is iodine. Although iodine is essential for nutrition, due to its toxicity in elemental form, higher price and problematic manipulation, this element introduces an obstacle for use. Very recently, it was shown that the reaction (1) can be significantly accelerated by Zn in solid solution of Mg. Final product Mg(OCH3)2 is a valuable precursor for production of nanocrystalline MgO (particle size ~5 nm) by simple thermal decomposition (400°C/2h), see Fig. 1.

Beside SEM and TEM, we employed a set of additional experimental techniques such as, mass spectroscopy combined with thermogravimetry, differential scanning calorimetry (DSC) and BET surface area analysis for thorough characterization of MgO nanoparticles prepared by thermal decomposition of Mg(OCH3)2.

Financial support offered by COST MP1103, MEYS LD13069 and LM2011026 is appreciated.

Fig. 1: Fig.1: SEM image of MgO powder (left) and high resolution TEM image of MgO particles (right).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2662 Structural Characterization of Inclined GaN Nanowires Grown in r-plane Sapphire by MBE

Lotsari A.1, Dimitrakopulos G. P.1, Kehagias T.1, Adikimenakis A.2, Komninou P.1, Georgakilas A.2
1Physics Department, Aristotle University of Thessaloniki, GR-541 24, Thessaloniki, Greece, 2Microelectronics Research Group (MRG), IESL, FORTH, P.O. Box 1385, 71110 Heraklion Crete, Greece; and Physics Department, University of Crete, Heraklion Crete, Greece
komnhnoy@auth.gr

We present a structural characterization of inclined GaN nanowires (NWs) on r-plane sapphire grown by plasma-assisted molecular beam epitaxy (PAMBE) under nitrogen rich conditions and after excessive substrate nitridation. The NW size and density were dependent on the nitridation conditions. Photoluminescence measurements of these NWs showed excellent crystal quality and strong emission even at room temperature.
The NWs were grown along the c-axis and subtended a 61o angle to the r-plane sapphire substrate as shown in the TEM image of Fig.1, where the two growth variants are also observed. A rough and discontinuous nonpolar a-plane GaN thin film was formed between the NWs. By combining TEM observations in cross-section and plan view geometries, the crystallographic model of the NWs was constructed. CBED was employed in order to identify the polarity of the NWs. Using HRTEM, the growth origin of the NWs was elucidated.
It was found that the sapphire nitridation pre-treatment enhances substrate roughness forming a stepped surface which provides facets for the nucleation of semipolar nanocrystals [1,2]. These nanocrystals evolve into NWs under N-rich rich growth conditions. Moiré fringes observed close to the interface confirm the presence of such interfacial areas. Analysis of the Moiré fringes along with Bragg filtering of the HRTEM images were performed for the identification of the NW nucleation sites. Geometrical phase analysis (GPA) was also employed at the points of emanation of the NWs since different phases like AlN formation or sapphire protrusions could promote the initial nucleation of the NWs. It was found that the NWs were either grown on semipolar GaN nanocrystals (as shown in Figures 2 and 3) or directly on sapphire steps. No other phase was identified at the growth origin of the NWs.
HRTEM image analysis was also performed at the grain boundaries between the nonpolar a-plane GaN matrix and the NWs in order to identify the accommodation mechanism between the two orientations. The orientation relationship between the two corresponds to a 90o <1210> rotation which ensures high coincident symmetry. Energetically favourable grain boundaries comprised flat terraces with disconnections being introduced between them in order to accommodate the misfit [3].

[1] J. Smalc-Koziorowska et al., Appl. Phys. Lett. 93, 021910 (2008)
[2] J. Smalc-Koziorowska et al., J. Appl. Phys. 107, 073525 (2010)
[3] J. Kioseoglou et al., J. Appl. Phys. 111, 033507 (2012)

This research has been co-financed by the European Union (European Social Fund - ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: THALES: Reinforcement of the interdisciplinary and/or inter-institutional research and innovation

Fig. 1: CTEM image of the inclined GaN NWs viewed along the [1101]Al2O3 zone axis. Between the NWs, a rough thin film of nonpolar a-plane GaN is formed

Fig. 2: HRTEM image of a NW grown between two nonpolar a-plane GaN crystals (n-GaN). In the point of emanation of the NW a small semipolar crystallite (s-GaN) is identified

Fig. 3: HRTEM image showing the nucleation site of a thin NW. The base of the NW is surrounded by semipolar GaN
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2670 The Reactivity and Structural Dynamics of Supported Metal Nano-Clusters using Electron Microscopy, in situ X-ray Spectroscopy, and Electronic-Structure Theory and Simulations

Yang J. C.1, Johnson D. D.3, Nuzzo R. G.2, Frenkel A. I.4, Ciston J.6, Stach E. A.5, Bonifacio C. S.1, Rehr J.7, Long L.1
1University of Pittsburgh, Pittsburgh, PA, USA, 2University of Illinois at Urbana-Champaign, Urbana, IL, USA, 3The Ames Laboratory (US Department of Energy), Ames, IA, USA, 4Yeshiva University, New York, NY, USA, 5Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, NY, USA, 6National Center of Electron Microscopy, Lawrence Berkeley National Laboratory, Berkeley, CA, USA, 7University of Washington, Seattle, WA, USA
judyyang@pitt.edu

Heterogeneous catalysis, which impacts the worldwide economy and sustainability due to its ubiquitous role in energy production, depends sensitively on the nano-sized 3-dimensional structural habits of nanoparticles (NPs) and their physicochemical structural sensitivity to the environment. Very small metal clusters can exhibit patterns of reactivity and catalytic activity that are dramatically distinct, and sometimes completely opposite, than behaviors seen with larger clusters. It therefore remains a significant need in research to fundamentally understand and predict the local structure and stability of catalytic materials that can be specifically tailored by design and optimized for an application in technology. Our focus is on the development of integrated characterization and modeling tools and their applications appropriate for carrying out detailed studies on metallic nanoscale clusters comprised of a few to as many as 100 metal atoms. Two state of the art methodologies, synchrotron X-ray absorption fine-structure (XAFS) and quantitative scanning transmission electron microscopy (STEM) methodologies are used and specially designed for determining the 3D structure and structural habits, both individually and as an ensemble, critical for understanding metallic nanoclusters. The experimental work is integrated with theoretical calculations. It is now clear that the structural dynamics of small metallic clusters is actually quite complex. For example, we have shown that the structures of Pt NPs may be both ordered and disordered (Fig 1), depending on its size, support and adsorbates where theoretical simulations predicted and corraborated all of the experimental data (Fig 2). While bulk amorphous Pt is unstable, its existence in NPs is a manifestation of their mesoscopic nature. To bridge the theory-experiment gap, we are producing model Pt/γ-Al2O3 systems using oxidation of NiAl(110) to form a thin film of single crystal γ-Al2O3. To bridge the complexity gap, we are developing an universal environmental cell that is compatible currently with synchrotron XAFS and environmental TEM.

Research was supported by Office of Basic Energy Sciences of U.S. Department of Energy (DE-FG02-03ER15476); ETEM was performed at Center for Functional Nanomaterials, Brookhaven National Laboratory (DE-AC02-98CH10886); statistical studies of NP visibility at NCEM (DE-AC02-05CH11231); and computational support by Ames Laboratory (DE-AC02-07CH11358), operated for DOE by Iowa State University.

Fig. 1: FS-HRTEM images and histograms of Pt NP structures on gamma-Al2O3:(a-c) Disordered NPs < 2nm and (d) ordered 1.2 nm NP, magnified in (e) and its FFT in (f). Histogram of ordered and disordered NPs and on g-Al2O3 (g). Fraction of ordered NPs vs. size (h) for g-Al2O3, with a transition zone of 1.1-2.5 nm.

Fig. 2: Relative DFT energy change for Pt37 in different structural motifs and chemical environments. Electron gain (loss) in yellow (red)] of the lowest-energy structures on C and γ-Al2O3 with(out) H are indicated. Spheres of dark (light) blue show the Pt (H) atoms, and magenta show support atoms.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2709 Unexpected hexagonal CeAlO3 phase formation at low temperatures depending on the hydrogen purity.

Malecka M. A.1, Kepinski L.1
1Institute of Low Temperature and Structure Research, Polish Academy of Sciences, P.O. Box 1410, 50-950 Wrocław 2, Poland
m.malecka@int.pan.wroc.pl

Solid state reaction between highly dispersed CeO2 and alumina provides to crystalline CeAlO3 formation. In normal conditions cerium aluminate crystallizes in the cubic crystal system and its structure can be described in space group Pm-3m (221). This presentation reports results of studies on formation and structure of CeAlO3 in CeO2-Al2O3 system (Ce:Al = 1:10). Samples were prepared by simple impregnation technique from aqueous solution of cerium nitrate. As-prepared samples were dried and pre-heated at 550 oC in static air. Next, CeO2-Al2O3 samples were calcined at temperatures from 850 to 1100 oC in hydrogen flow. Morphological and structural changes upon heat treatment in the reducing atmosphere at evaluated temperatures were studied by HRTEM, XRD and XPS methods. Unexpected, hexagonal phase of CeAlO3 was observed on XRD patterns (see fig. 1) for samples after thermal treatment at lower temperatures (~850 oC). Increasing of heating temperature up to 1000-1100 oC provided to conventional cubic CeAlO3 crystallization (see fig. 1). Moreover, new hexagonal phases of CeAlO3 have been found on HRTEM and SAED images recorded for studied samples (see insets in fig. 1). Depending on the hydrogen purity (concentration of residual oxygen), thereby Ce3+/Ce4+ concentration in cerium aluminate structure, two kind of hexagonal CeAlO3 phases could be formed. It would seem that in case heating CeO2-Al2O3 system in hydrogen flow at low purity, part of Ce4+-ions has not been reduced. As it was found on XRD patterns, formed hexagonal phases are differed in a and b lattice parameters, where c parameter remains stable. Additional, clearly visible differ between samples heated at 850 oC in hydrogen flow (pure and oxygen polluted) was in color of both samples. First of them was gray and second one was dirty-yellow, what could be the proof for presence of Ce4+ ions in sample.

This work was financially supported by the National Science Centre Poland (grant UMO-2011/01/B/ST5/06386). The authors thank Mrs. Z. Mazurkiewicz for valuable help with preparation of the samples and Mrs. E. Bukowska for XRD work.

Fig. 1:  XRD patterns and HRTEM images (in sets) obtained for samples heated at 850 oC in hydrogen flow (A) pure, (B) oxygen polluted and (C) at 1100 oC.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2716 On the structures of sequentially deposited Ru-Au bimetallic catalysts for green chemistry

Wang D.1, Villa A.2, Kotula P. G.3, Prati L.2, Kübel C.1
1Institute of Nanotechnology, and Karlsruhe Nano Micro Facility, Karlsruhe Institute of Technology, Hermann-von-Helmholtz Platz 1, 76344, Eggenstein-Leopoldshafen, Germany, 2Dipartimento di Chimica, Università di Milano, via Golgi 19, I-20133 Milano, Italy, 3Materials Characterization Department, Sandia National Laboratories, Albuquerque, NM 87185-0886, USA
di.wang@kit.edu

Aerobic catalytic oxidation has been appreciated in view of its application in green chemistry in contrast to non-catalytic methods. By controlling the synthesis methodology, the structures of nanocatalysts can be designed towards specific activity and selectivity for the reaction of interest. Recent developments in analytic electron microscopy techniques have enabled the structure and chemistry of individual catalyst particles to be resolved at sub-nanometer resolution. In this presentation, we will focus on two activated carbon (AC) supported Ru-Au bimetallic catalysts synthesized by sequential deposition following a two-step procedure [1] to correlate the elemental distributions and surface decoration with the activity and selectivity of the catalysts.

For Ru@(Au/AC), Ru(III) was reduced with H2 at 80 °C in the presence of preformed Au/AC. Au@(Ru/AC) was prepared by depositing PVA stabilized Au nanoparticles onto a commercial Ru/AC. The catalysts were examined in an image aberration corrected FEI Titan 80-300 electron microscope with conventional EDX detector as well as in a probe aberration corrected Titan 80-200 with in column Super-X EDX detector.

Spectrum imaging with probe corrected STEM and Super-X EDX detector offers high spatial resolution and the opportunity to resolve the components by multivariate analysis before changing the structure during electron beam irradiation. In Fig. 1a, components containing Au and Ru are displayed, forming an Au core-Ru shell structure in the case of Ru@(Au/AC). The high resolution HAADF STEM image in Fig. 1b shows two Au particles with Ru clusters (lower intensity) situated on their surface. In the case of Au@(Ru/AC), we obtained a more inhomogeneous distribution, with the presence of small Ru particles and larger bimetallic particles, as seen in Fig. 2b. Interestingly, the bimetallic particles, as shown e.g. in Fig. 2a, is also composed of an Au core and Ru shell. Both catalysts were tested in oxidation of n-octanol in toluene and oxidation of glycerol in water, respectively. Ru@(Au/AC) shows almost no activity in the former case but is highly active for the latter; while Au@(Ru/AC) behaves in the contrary, being very active for oxidation of n-octanol but shows only limited activity in glycerol oxidation. The TEM results and catalytic tests therefore suggests that Ru is the main active phase in oxidation of aliphatic alcohols and the addition of Au has a detrimental effect on the Ru particles on it. But this Au core-Ru shell structure leads to distinctly enhaced activity in oxidation of water soluable and highly hydrophilic polyols [2].

Reference
[1] D. Wang, A. Villa, F. Porta, D. Su, L. Prati, Chem. Commun. (2006) 1956.
[2] L. Prati, F. Porta, D. Wang, A. Villa, Catal. Sci. Technol. 1 (2011) 1624.

Fig. 1: a) Component maps by multivariate analysis, with red for Au and green for Ru, and b) HAADF STEM image of bimetallic particles showing Ru situated on Au core for the Ru@(Au/AC) catalyst.

Fig. 2: a) Components maps by multivariate analysis, with red for Au and green for Ru, and b) HAADF STEM image of segregated small Ru particles together with big bimetallic particles for the Au@(Ru/AC) catalyst.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2739 Substitutional Gold Doping in ZnO Mesocrystals as Outstanding Catalyst for CO Oxidation

Liu M. H.1, Chu M. W.2, Chen Y. W.3, Kuo J. R.3, Mou C. Y.1
1Department of Chemistry and Center of Condensed Matter Science, National Taiwan University, 2Center of Condensed Matter Science and Center for Microscopy and Nano Analysis, National Taiwan University, 3Institute of Atomic and Molecular Sciences, Academia Sinica
mhliu0811@gmail.com

Zinc oxide is a wide band-gap, n-type semiconductor and also an important material in understanding fundamental optical physics. However, only few research groups utilized ZnO material as a support to do the catalysis researches because of its inertness. Herein, we prepare a new type of ZnO material, namely twin-brush ZnO (TB-ZnO) mesocrystals, which were as a novel support for gold. Gold nanoparticles were deposited on TB-ZnO by means of a modified DP method, forming Au/TB-ZnO catalyst. The catalytic activity of Au/TB-ZnO in CO oxidation was examined from -50 oC to 30 oC (Fig. 1), showing an extraordinary performance in comparison with conventional Au/ZnO catalysts.
To unravel the origin of the outstanding catalysis, the catalyst was characterized using aberration-corrected canning transmission electron microscope (Cs-corrected STEM), high-resolution transmission electron microscopy (HRTEM), X-ray absorption spectroscopy (XAS) and FTIR study of adsorbed CO. Through the analysis of Cs-corrected STEM (Fig. 2) and the refined data from EXAFS (not shown here), it is definitely realized that a number of zinc sites of ZnO sub-lattice were substituted by gold. This unexpected phenomenon was also supported by theoretical calculation. With DFT calculations (4×4×3 ZnO model), it is found that when Zn vacancies exist in the TB-ZnO support, gold atoms can not only diffuse into ZnO but tend to aggregate instead of random dispersion (white arrows in Fig. 2). When the catalyst was treated at 200 oC and further underwent a series of CO reactions, the substituted gold would segregate from interior, resulting in the formation of 2 nm AuNPs in size (Fig. 3). For the analysis of CO adsorption on AuNPs, there are three types of gold species on the support surface, including Au0, Au+ and Au3+. It is significant that the substitution of gold into the ZnO lattice was observed for the first time and further contributes an extraordinary activity in CO oxidation. Consequently, through several examination results, it can be realized that outstanding activity is apparently originated from high active gold atoms and ca. 2 nm gold nanoparticles. This synthetic approach of Au/TB-ZnO can open up a new opportunity to design an excellent catalyst with a finely-controlled particle size.

Fig. 1: CO conversion of Au/TB-ZnO catalysts as a function of low-temperature from -50 oC to 30 oC. The activity revealed at -10 oC was 0.13 molCO•(molAu•s)-1.

Fig. 2: Aberration-corrected HAADF-STEM image of O2-pretreated Au/TB-ZnO catalyst at 200 oC. (The insert is the Fourier transformed result from the corresponding image.)

Fig. 3: The HRTEM image from ultramicrotome slice of Au/TB-ZnO with O2 pretreatment underwent a series of CO oxidation reactions ramping from -20 oC to 90 oC.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2816 Hydrothermal synthesis of twin-based multiply branched rutile-type TiO2

Jordan V.1, Podlogar M.1, Rečnik A.1
1Department for Nanostructured Materials, Jožef Stefan Institute, Ljubljana, Slovenia
vanja.jordan@ijs.si

Production of nanostructures, especially 3D branched architectures with controlled morphology and size, has been the focus of research in the recent years. One of such materials that can be grown in complex branched morphologies is rutile (TiO2). While being applicable in variety of applications,1 rutile is known to be prone to twinning, which can be exploited as a basic structural element for growing branched structures.2 Branched structures of rutile-type TiO2 are obtained in an acidic medium with the use of metalorganic Ti-precursor. Several nucleation mechanisms have been proposed in the literature, however the true nature of branching is yet to be explained.3-5 Several synthesis routes have been tested to study twinning of rutile. Following the synthesis pathway suggested by Tomita et al. (2006),3 1st generation of twinning was obtained. Briefly, titanium powder was dissolved in H2O2 and NH3, to produce titanium oxyhydroxide. This was followed by a ligand exchange reaction with glycolic acid, to form Ti-glycolato complex, which was hydrothermally treated at 200 °C for 1-24 hours to obtain nanocrystalline rutile. 2nd generation of twins was obtained by a subsequent hydrothermal treatment of already existing twins by the addition of Ti-butoxide in strongly acidic medium. Another approach, following Zhou et al. (2011),4 yielded 2nd generation of twins in a single synthesis step. Two generations of twinning were obtained through hydrothermal synthesis in acidic medium. As precursor, Ti-butoxide was used and dissolved in 7-10M HCl aqueous solution. The syntheses were conducted at different temperatures and processing times. Morphology and composition of the products were characterized by SEM and TEM. The first route yielded clusters of twinned rutile crystals (Fig. 1). Electron diffraction study of twin relations indicated that the products are composed of (101) and (301) twins, with the characteristic angles of 114° and 55°, respectively.2 The second synthesis route led to formation of complex-branched structures, with abundant twinning and other types of intergrowths (Fig. 2a). Unlike in the first synthesis route, the rutile crystals here are composed of numerous parallel rutile fibers lined along the crystallographic c-axis (Fig. 2b and 2c). In crystals that are oriented along the c-axis inherent porosity can be observed, which might be a consequence of imperfect fiber alignment. Further, the presence of anatase phase, as suggested by several authors,5 could not be confirmed, nevertheless some unidentified reflections that could correspond to this TiO2 phase are observed in electron diffraction patterns (Fig. 2d). Pores and imperfect alignment of the fibers indicate the possible mechanism of rutile formation and branching.

1. C. Cheng, H.J. Fan, Nano Today 7 (2012) 327-343.
2. N. Daneu, H. Schmidt, A. Rečnik, W. Mader, Am. Mineral. 92 (2007) 1789-1799.
3. K. Tomita, V. Petrykin, M. Kobayashi, et al., Angewandte Chemie 45 (2006) 2378-2381.
4. W. Zhou, X. Liu, J. Cui, D. Liu, J. Li, , et al., CrystEngComm 13 (2011) 4557-4563.
5. D. Li, F. Soberanis, J. Fu, et al., Crystal growth & Design 13 (2013) 422-428.

Fig. 1: (a) {101} and {301} twins of rutile obtained from Ti-glycolato complex, (b) TEM image of (301) twin. Fig. 2: (a) Rutiles synthesized from Ti-butoxide. (b) Fibrous rutiles coinciding in twin-type orientations. (c) Close-up of rutile roughly aligned fibers. (d) Single rutile fiber in [001] projection. Diffraction rings mainly correspond to rutile.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2820 Growth mechanism of Ag catalysed InAs nanobelts grown in MOCVD

Xu H.1,3, Gao Q.2, Tan H. H.2, Jagadish C.2, Zou X.3, Zou J.1,4
1Materials Engineering, University of Queensland, 2Department of Electronic Materials Engineering, Australian National University, 3Department of Materials and Environmental Chemistry, Stockholm University, 4Centre for Microscopy and Microanalysis, University of Queensland
h.xu5@uq.edu.au

Zinc-blende structured Au catalysed epitaxial III-V semiconductor nanowires prefer to grow along the [111]B direction featuring approximately hexagonal cross sections with side-wall facets dominated by six {112} planes or six {110} planes. In contrast, only a hand-full of studies in non-Au catalysed 1-D nanostructure growth have been reported up to date. In this study, by using Ag as catalysts, we demonstrate to grow 1-D InAs nanobelts grown along <112>B directions. Through detailed electron microscopy characterizations, the growth mechanism of these InAs nanobelts is explored.

Commercially available 40nm Ag nanoparticles were used to grow epitaxial 1D InAs nanostructures on GaAs (111)B substrate in a MOCVD reactor. The growth was carried out using trimethylindium and arsine as the group III and group V precursors, respectively. A growth temperature of 500°C and a V-III ratio of 2.9 were selected as the key growth parameters.

Fig. 1a is an overview SEM image and shows the general morphology of as-grown Ag-catalyzed 1-D nanostructures. From the enlarged SEM image (Fig. 1b), inclined nanostructures show belt-like morphology. Fig. 1c is a side-view SEM image of a typical nanobelt, from which the inclined angle of the nanobelt is measured as ~70° (when the electron beam is parallel to a <110> direction and perpendicular to the inclined nanobelts), so that its axial direction can be crystallographically determined to be along <112>B directions. It is of interest to note that "steps" can be found on the top facets of the nanobelt, whereas the bottom surface is relatively smooth. Fig. 1d and e are SEM images of a typical nanobelt viewed from top-view and edge-on view, respectively. As can be seen from the top view (Fig. 1d), the nanobelt is tapered. When the nanobelt is viewed along its axial direction (Fig. 1e, ~20° tilt), the sidewall facets of the nanobelt shows a rectangular shape with two short {110} facets and two long facets developed from {111} planes. The top facet contains many surface "steps" consistent with other SEM observations.

By using a number of TEM techniques, the structure, sidewall facets, chemical composition, planner defects and nanobelt/catalyst interface of the as-grown nanobelts are investigated in detail, from which a growth schematic of Ag-catalyzed InAs nanobelt is proposed and shown in Fig. 2. During Ag catalyzed 1-D nanostructure growth, the catalyst promotes two processes: (1) collects group III growth material and (2) transports the growth material to the growth front of the nanostructure and nucleates the growth. It is believed that the formation of the “steps” and the planer defects is closely related to the In concentration in the catalyst during the nanobelt growth.

The Australian National Fabrication Facility and Australian Microscopy and Microanalysis Research Facility are gratefully acknowledged. The Wenner-Gren Foundation and 3DEM NATUR project at MMK, Stockholm University are also gratefully acknowledged.

Fig. 1: (a) SEM overview of the Ag-catalysed InAs nanostructures. (b) SEM image of a typical nanobelt. (c) Side-view of a typical nanobelt. (d) and (e) a typical nanowire viewed when the beam is perpendicular to the substrate and parallel to the nanobelt axial direction.

Fig. 2: Schematic illustration of the “step” and defect formation. Perturbations in In concentration introduces new facets during the nanobelt growth.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2892 Microstructure Study of W1-xMox O3 0.33H2O for Tunable Wavelength Absorption

Arzola-Rubio A.1, Ornelas C.1, Antúnez-Flores W.1, Collins-Martínez V.1, Paraguay-Delgado F.1
1Centro de Investigación en Materiales Avanzados S. C., CIMAV Miguel de Cervantes 120, Chihuahua, Chih. México. CP 31109.Ar
alejandro.arzola@cimav.edu.mx

Hydrated tungsten oxide WO3 0.33H2O has been studied extensively due to its electronic and optoelectronic properties, it has has an enormous potential application ranging from condensed-matter physics to solid-state chemistry [1], such as photo-electrochemical energy conversion, gas sensors, photocatalysis, lithium-ion batteries, solar cells [2]. Tremendous effort has been dedicated to the synthesis, solid solution mechanism and property investigation of W1-xMox O3 0.33H2O over the past years. This material showed improved electrochromic, gas sensing, catalytic, lithium ion transport, and photocatalytic properties [3] when compared with their single oxide WO3 and MoO3. Recently, Zhou et al. were capable of modulate the band gaps of the W1-xMox O3 0.33H2O materials with different Mo/W ratio values [4]. We synthesized a series of W1-xMoxO3 0.33H2O nano/microstructures with controlled stoichiometry (x = 0, 0.25, 0.50, 0.75). With gradual increase of Mo content, we narrowed the band gap from 2.61 to 2.10 eV. This result is better than Zhou et al. but in our case, we use friendly to the environment chemical precursors such as ammonium heptamolybdate and ammonium metatungstate instead of metal powders.
Figure 1a shows a SEM image for orthorhombic WO3 • 0.33H2O, the particles have an average length and wide of 100 and 50 nm respectively. Figure 1b is a bright field TEM image and inset is the SAED pattern for the W75Mo25O3 0.33H2O compound, the diffraction spots were to the orthorhombic structure and it has [3,0,-1] zone axis, which is a single crystal. Figure 1c corresponds to TEM image of solid solution W50Mo50O3 0.33H2O and its corresponding SAED pattern which indexed to orthorhombic structure too, with [2,-1,0] zone axis, this pattern was from a particle labeled with Z1. In the case of this compound, there is different size of particles which measurements are 160nm length and 80nm width. Figure 1d shows SEM image of the compound W25Mo75O3 • 0.33H2O. It can be notice hexagonal flake-like particles with lengths of ∼150nm and widths of ∼70nm.
We were able to measure and characterize our compounds with advanced microscope techniques such as SEM and TEM getting information about structure by SAED patterns where we were capable to index all spots and determine the zone axis and figure out all crystalline path growths of these type materials.

References
1. Zheng HD, Ou JZ, Strano MS, Kaner RB, Mitchell A, Kalantarzadeh K (2011) Adv Funct Mater 21:2175.
2. Turyan I, Krasovec UO, Orel B, Saraidorov T, Reisfeld R, Mandler D (2000) Adv Mater 12:330.
3. Baeck, S. H.; Jaramillo, T. F.; Jeong, D. H.; McFarland, E. W. Chem. Commun. 2004, 390–391.
4. Zhou, L.; Zhu, J.; Yu, M.; Huang, X.; Li, Z.; Wang, Y.; Yu, C. J. Phys. Chem. C 2010, 114, 20947−20954.

Fig. 1: FIG. 1 SEM, TEM and SAED images of W1-xMoxO3 • 0.33H2O (x = 0, 0.25, 0.50, 0.75) nano/microstructures
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2896 Microstructural and Electrical Characterization of Langmuir-Blodgett Films of Ultrathin Semiconductor Nanoheterowires

Ashokkumar A. E.1, Li H.1, Hayat A.1, Dalui A.2, Jafri H.4, Sarma D. D.3, Acharya S.2, Leifer K.1
1Department of Engineering, Applied Materials Science, Uppsala University, Sweden, 2Centre for Advanced Materials, Indian Association for the Cultivation of Science,Kolkata, India, 3Solid State and Structural Chemistry Unit, Indian Institute of Sciences Bangalore, India, 4Electrical Engineering Department, Mirpur University of Science and Technology, Pakistan
anumolea@gmail.com

Semiconductor heterostructures with suitable band alignment which can promote charge separation is interesting for various applications in electronics and optoelectronics. Chemical synthesis methods are developed for obtaining complex nanostructures including semiconductor hybrids of few nanometers sizes leading to quantum confinement and resultant unique properties. The assembly of such structures on substrates as thin films can facilitate its characterization and further applications. In this work, heterostructures consisting of ZnS rods and CdS dots with sequential alignment synthesized by wet chemical synthesis is investigated in this direction. Ultrathin superlattice nanowires of ZnS-CdS were developed into monolayer thin films on SiO2/Si substrates using Langmuir-Blodgett (LB) technique. The various aspects of the assembly are investigated. The morphology of the film and the orientation of the nanowires in the LB film were studied by microscopy techniques including Scanning Electron Microscopy, Transmission Electron Microscopy and Atomic Force Microscopy. Due to the sub 2 nm size of the nanowires, Transmission Electron Microscope is mandatory to observe the alignment of the individual nanowires and also to observe the components of the hetero- nanowire. Transfer of the Langmuir-Blodgett film on to Cu grids facilitates the observation of the film under TEM.
The electrical characterization of such sub 2-nm wires is challenging as the fabrication of electrical contacts is nontrivial. A nanoplatform where the electrodes with a spacing of ~ 50 nm fabricated using lithography and Focussed Ion Beam technique was developed for this purpose. The LB film was deposited onto the substrate with such prefabricated electrodes. The electrical and optical properties of the LB films are presented.
Thus the present work investigates the role of microscopy techniques in characterizing LB films of nanomaterials and also attempts to bridge the gap between wet chemical synthesis of semiconductor nanowires and their device fabrication.

The Swedish Foundation for International Cooperation in Research and Higher Education (STINT) is acknowledged for research grant.

Fig. 1: SEM image of Langmuir-Blodgett film of ZnS-CdS heterowires

Fig. 2: AFM image of Langmuir-Blodgett film of ZnS-CdS on SiO2/Si substrate

Fig. 3: TEM bright field and high resolution images of the ZnS-CdS Langmuir-Blodgett film transferred to Cu grid
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2899 Self-assembled Supraballs by Spherical Confinement

Wang D.1, de Nijs B.1, Dussi S.1, Smallenburg F.1, Filion L.1, Pietra F.2, Meeldijk J. D.3, van Dijk-Moes R.2, van Huis M.1, Vanmaekelbergh D.2, Imhof A.1, Dijkstra M.1, van Blaaderen A.1
1Soft Condensed Matter, Debye Institute for Nanomaterials Science, Utrecht University, Utrecht, The Netherlands, 2Condensed Matter and Interfaces, Debye Institute for Nanomaterials Science, Utrecht University, Utrecht, The Netherlands, 3Electron Microscopy Group, Utrecht University, Utrecht, The Netherlands
d.wang@uu.nl

Colloidal crystalline supraparticles, which are self-assembled from size- and morphology- controlled nanoparticles, can exhibit many different interesting meta-materials properties, while still having a size in the colloidal domain and thus the possibility with a second self-assembly step to form other interesting structures. An example is colloidal crystal lattices with Bragg-reflections for visible light. In research aimed at making colloidal crystalline supraparticles by having monodisperse spherical nano- (and micron-sized) colloids crystallize in slowly evaporating oil emulsion droplets, we discovered icosahedral symmetry in the resulting dried colloidal crystals for particle number less than ~100,000. Subsequent computer simulations confirmed the icosahedral symmetry even in the absence of any attractions and thus are entropically favored over the face-centered-cubic (FCC) crystal structure that is stable in the bulk.1

We also extended the spherical confinement method to a binary particle system and an anisotropic rod-like particle system. For instance, 6.2 nm Au nanocrystals and 22.0 nm FexO/CoFe2O4 nanocrystals were used to synthesize binary supraballs. CdSe/CdS quantum rods can also self-assemble into supraballs. By tuning the concentration of the nanocrystals, supraballs with different structures and sizes can be obtained. After freeze drying, the structure of the supraballs was studied with high-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) and secondary electron scanning transmission electron microscopy (SE STEM). Work is in progress to study the structures of the more complex supraballs by HAADF STEM tomography.

Reference

(1) Bart de Nijs et al. submitted.

Fig. 1: SE STEM image of supraballs with rhombicosidodecahedron structure made from 6.0 nm FexO/CoFe2O4. (Scale bar 50 nm)

Fig. 2: HAADF STEM image of binary supraballs made from 6.2 nm Au and 22.0 nm FexO/CoFe2O4. (Scale bar 50 nm)

Fig. 3: SE STEM image of binary supraballs made from 6.2 nm Au and 22.0 nm FexO/CoFe2O4. (Scale bar 50 nm)

Fig. 4: HAADF STEM image of supraballs made from CdSe/CdS quantum rods. (L=53.2 nm, D=4.1 nm) (Scale bar 50 nm)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2901 On the correct grain size characterization of nanometric polycrystalline materials

Pinto A. L.1, Gama G. R.1, Silva A. M.1
1Centro Brasileiro de Pesquisas Físicas (CBPF) 1
pinto@cbpf.br

There has been an increasing interest in polycrystalline materials with nanometric grain size. These materials may be produced through thin film technology1, electrodeposition or severe plastic deformation. The grain size is the most basic microstructural parameter that is usually described in a sample. Some properties may be predicted directly from the grain size, but as to nanomaterials, they have called for the revision of some concepts2. One of the most common ways of characterizing the grain size at a TEM is through dark field images. As the diffraction contrast obtained in bright field does not allow a proper description of the microstructure, the objective aperture is used for illuminating grains within a determined range of crystallographic orientations given by the size of the objective aperture used. In this work we have used this approach and then we have compared to the microstructures obtained through nanodiffraction mapping. Cu thin films were deposited over an oxidized Si(100) substrate through DC magnetron sputtering with a 5mTorr Ar plasma. The substrates were positioned at 10cm from a Cu target. Four different currents were used for the deposition: 30, 75, 100 and 125mA. Depositon times were calibrated to obtain 100nm thin films. TEM analysis was performed at a Jeol 2100F equipment with Nanomegas ASTAR nanodiffraction mapping system. Dark field images were obtained from each sample, which resulted in the grain mean size of 89, 22, 20 and 15nm respectively for the 30, 75, 100 and 125mA samples. Fig. 1 presents a bright field image and a dark field image from the 100 mA sample. The grain size estimation through dark field images or even from bright field images may lead to serious misinterpretation of the results. Using a 30μm objective aperture the crystal orientation deviation is much greater than the 15o commonly used for defining a grain; on the other side using a 5μm aperture almost only one grain is measured per picture. Using nanodiffraction mapping we have obtained 7, 16, 15 and 17nm as grain mean size, respectively. Fig. 2 presents an orientation map and an Index map from 100mA sample at which it is possible to notice the distinct (100) and (110) textures. This is the reason for the poor distinction between neighbor grains. It is also clear there are some larger grains that seem to have grown at the expense of their smaller neighbors. These larger grains have a different orientation from the predominant texture. The use of nanodiffraction mapping at the TEM not only gives a better evaluation of the grain size but also gives much more information about the microstructure.

References

1 – Hodge, A.M. et al. Materials Science and Engineering A 429 (2006) 272–276.

2 – MEYERS, M. A. et al. JOM, April (2006) 41-48.

The authors thank to CAPES, CNPQ and FINEP for financial support of this work.

Fig. 1: Fig. 1 – (a) Bright field image and (b) dark field image from sample 100 mA obtained with 5 μm objective aperture.

Fig. 2: Fig. 2 – (a) orientation map ((001) – red, (101) – green and (111) – blue color reference), (b) reliability map and index map from 100 mA sample.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-2977 qHRTEM analysis of the (211)B In(Ga)As QDs/GaAs heterostructure

Florini N.1, Kioseoglou J.1, Dimitrakopulos G. P.1, Walther T.2, Hatzopoulos Z.3, Pelekanos N. T.3, Kehagias T.1
1Physics Department, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece, 2Department of Electronic and Electrical Engineering, University of Sheffield, Mappin St, Sheffield S1 3JD, UK, 3Materials Science & Technology and Physics Departments, University of Crete and IESL/FORTH, GR-71003 Heraklion, Greece
kehagias@auth.gr

The nanostructural properties and strain state of In(Ga)As quantum dots (QDs), embedded in GaAs (211)B by plasma-assisted molecular beam epitaxy (PAMBE), such as shape and dimensions, existence of associated dislocations, thickness of the wetting layer and the possibility of interdiffusion or segregation phenomena in the QDs, were investigated by high-resolution and scanning transmission electron microscopy (HRTEM-STEM) methods. HRTEM imaging showed that the wetting layer thickness does not exceed 2 monolayers. Moreover, the embedded QDs are not associated with any linear defects, suggesting fully strained and optically active nanostructures. However, the shape and dimensions of QDs cannot be precisely extracted, due to the dark strain contrast surrounding the QDs. Conversely, STEM annular bright-field (ABF) imaging revealed that In(Ga)As QDs are elongated along the [-111] direction [Fig. 1(a)].

Quantitative measurements of the local strain in the QDs from HRTEM images have been performed, by the geometrical phase analysis (GPA). GPA is used to determine the strain field in a HRTEM image with respect to a reference region. The GPA lattice strain eg = (α – αGaAs)/αGaAs, α being the in-plane or the out-of-plane strained values of In(Ga)As QDs, is defined relative to the unstrained GaAs underlayer corresponding values, which was taken as reference. GPA measurements using a g/2 mask, showed that the in-plane strain approximates 0, implying a fully registered heterostructure at the interface, as also illustrated in the HRTEM images of two individual QDs in Figs. 1(b) and (c). Assuming a biaxial strain state of the QDs, the corresponding GPA strain surface plots of two QDs along the growth direction are also shown. The quantitative analysis of the InxGa(1-x)As QDs on GaAs resulted in chemical composition maps of the investigated QDs. The In content was found to vary from 52% at the base of the QDs to almost 100% at the apex area [Figs. 1(b) and (c)], implying possible Ga segregation in the initial stages of QD growth and formation of an InGaAs alloy. However, since the QDs are entirely embedded in GaAs, possible influence from the matrix cannot be excluded.

Moreover, the samples were analyzed by energy dispersive X-ray (EDX) spectroscopy in order to estimate the chemical composition on the InAs QDs in comparison to the quantitative GPA measurements.

Research co-financed by the European Union (European Social Fund–ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF)–Research Funding Program: THALES, project NANOPHOS.

Fig. 1: (a) ABF STEM image depicting the embedded InAs QDs. (b) & (c) HRTEM images of embedded InAs QD along the [0-11] zon eaxis and the corresponding GPA strain surface plots along the growth direction.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3045 Atomic-scale characterization of light-emitting diodes based on ordered InGaN nanocolumns

Torres-Pardo A.1, 2, Bengoechea-Encabo A.3, Albert S.3, Sánchez-García M. A.3, López-Romero D.3, Gacevic Z.3, Calleja E.3, González-Calbet J. M.1, 4
1Dept.Química Inorgánica, Facultad de Químicas, Universidad Complutense de Madrid, 28040, Madrid, Spain, 2CEI Campus Moncloa, UCM-UPM, Madrid, Spain, 3ISOM-Dept. Ingeniería Electrónica, ETSIT, Universida Politécnica, 28040 Madrid, Spain, 4Centro Nacional de Microscopía Electrónica CNME, 28040 Madrid, Spain
jgcalbet@ucm.es

The potential of InGaN alloys to generate light emission in the UV to IR range makes them an ideal choice for light emitting diodes (LED) covering the whole visible range and beyond. LEDs based on self-assembled nanocolumns (NCs) with InGaN/GaN disks constitute an alternative to conventional LED planar devices which major limitation is a strong reduction in efficiency at high current injection [1]. However, the efficiency and reliability of LEDs based on self-assembled NCs are hindered by a strong dispersion of electrical characteristics among individual nanoLED. Polychromatic emission derives from an inhomogeneous distribution of indium concentration, changes in the NCs geometry and the inherent tendency of InGaN alloys to develop composition fluctuations as a function of the polarity of the growth crystallographic planes [2]. The recent development of selective area growth of NCs by molecular beam epitaxy has allowed the achieving of highly homogeneous and controllable GaN/InGaN NCs with improved crystalline quality and higher control over the indium distribution [3].

In this work, we present results on the characterization of blue, green and yellow LEDs based on ordered NCs with InGaN active layers (figure 1). The detailed structural characterization of the nanostructures has been performed by scanning transmission electron microscopy (STEM) carried out on an aberration-corrected JEOL-JEMARM200 microscope [4]. High crystal quality of the NCs is set by the analysis of atomically-resolved high angle annular dark field (HAADF) images, while the polarity determination of the semiconductor NCs is followed by locating the nitrogen atomic columns in annular bright field (ABF) images (figure 2). The indium distribution within the InGaN disks is studied by EDS elemental mapping, confirming homogeneity of the InGaN layers. The optical response is evaluated from the analysis of electroluminescence spectra.

[1] E. Kioupakis, P. Rinke, K. T. Delaney, C. G. Van de Walle, Appl. Phys. Lett. 98, (2011), 161107.

[2] A. L. Bavencove, G. Tourbot, J. Garcia, Y. Desieres, P. Gilet, F. Levy, B. Andre, B. Gayral, B. Daudin, and L. S. Dang, Nanotechnology,22, (2011), 345705.

[3] S. Albert, A. Bengoechea-Encabo, M. A. Sanchez-Garcia, X. Kong, A. Trampert, E. Calleja, Nanotechnology 24, (2013), 175303.

[4] Y. Li, L. Zhang, A. Torres-Pardo, J.M. González-Calbet, Y. Ma, P. Oleynikov, O.Terasaki, S. Asahina, M. Shima, D. Cha, L. Zhao, K. Takanabe, J. Kubota, K. Domen, Nature Communications, 4, (2013), 2566.

Authors acknowledge financial support by the Spanish projects MAT2011-23068 and CSD2009-00013. Research by A.T.P. has been supported by PICATA postdoctoral fellowship CEI Moncloa.

Fig. 1: (a) Cross-sectional SEM images of a representative sample. (b) Top view SEM picture. (c) Low magnification TEM image of GaN/InGaN nanocolumn.

Fig. 2: (a) Schematic draw of [010] GaN structure with wurzite-type structure. (b) Atomically resolved High Angle annular Dark Field (HAADF) image and (c) corresponding Annular Bright Field (ABF) image revealing the Ga and N atomic columns on the wurzite-type structure.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3074 In-situ monitoring gas-solid reaction in nanoparticles by a static nanoreactor in the TEM

Wu M.1, Shen C.1, Zandbergen H.1
1Kavli Institute of NanoScience, HREM, Delft University of Technology, Delft, The Netherlands
m.y.wu@tudelft.nl

There is a big interest in realizing TEM experiments beyond the ~ 20 mbar pressure regime that can be achieved by ETEM. This is possible using a nanoreactor concept, in which the gas is enclosed along the beam direction by two very thin membranes of for instance SiN (1). With this approach Yokosawa (2) showed that pressures up to 4.5 bar are obtainable. An obvious question in this approach is what kind of resolution can be obtained, given that the resolution limit is now no longer set by the electron microscopes, provided these are equipped with aberration correctors. In the approaches reported by Creemer and Yokosawa gas tubes embedded in the TEM holder were used to lead gas to the nanoreactor and this requires a sophisticated gas supply system. Such a system of gas inlet and outlet and gas regulation could be hazardous for both the TEM and the TEM-user if there exists a leakage.
Here we present a new type of gas holder, which we will call a static gas holder, which has also two windows but no dynamic gas supply system (figure 1). Instead, the holder has a separable tip, which contains an airtight chamber that can store gas with volume of 1.5 to 10 cubic millimeter. Gas is loaded in or pumped out through a valve in the tip (see figure 1). Similar to the dynamic nanoreactor, it consists of two silicon chips, which have a low stress 400 nm thick SiN membrane of for instance 400 um x 400 um. One of the two membranes contains a Pt heater spiral and both of the membranes contain with 5-20 small thin SiN membranes “windows” with thickness of 10-20 nm. The windows of the top and bottom chip have to be aligned to be overlapping, such that a sample on top of one of the windows can be investigated by transmission electron microscopy. In this system, the temperature can be changed within a second over for instance 100°C with low specimen drift. Since the gas volume is very small, no harm to the gun part of the TEM when there is a sudden release of all gas inside the nanoreactor and the tip. An obstacle for high-resolution imaging can be the contamination in the system, which can originate from sample, chips, gases, O-rings etc. We demonstrate that when contamination is minimized, the resolution of the system can reach the resolution limitation of the microscope at gas pressures of e.g. oxygen of at least 0.6 bar (figure 2 and figure 3).

1. J.F. Creemer, S. Helveg, G.H. Hoveling, S. Ullmann, A.M. Molenbroek, P.M. Sarro, H.W. Zandbergen, Ultramicroscopy 108 (2008) 993– 998.
2. Tadahiro Yokosawa, Tuncay Alan, Gregory Pandraud, Bernard Dam, and Henny Zandbergen Ultramicroscopy 112(2012)47–52.

This work is supported by ERC NEMinTEM Project 267922.

Fig. 1:  (a) Image of the static gas holder and an enlarged view of the tip part. (b) Cross sectional sketch of the holder tip disassembled and assembled onto the holder.

Fig. 2: (a) PdOx nanoparticles in O2 with pressure of 0.645 bar at 500 °C. (b) FFT of image (a), the white circle is 1 Å. The triangle indicates a diffraction spot with a d-spacing of 0.88 Å.

Fig. 3: Pd nanoparticles in H2 with a pressure of 0.52 bar at 200 °C. (b) FFT of image (a), the white circle is 1 Å. The triangle indicates a diffraction spot with a d-spacing of 0.85 Å.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3055 Atomic resolution imaging of SiOx quantum dots in diamond by TEM/STEM

Sung K. T.1, Wei L. L.1, Chiu K. A.1, Chang L.1
1Department of Materials Science and Engineering, National Chiao Tung University, Hsinchu, Taiwan
lichang@cc.nctu.edu.tw

Si-based quantum dots (QDs) have received intensive studied in the past decade because of their light emitting properties. Most Si-based QDs are fabricated to be embedded in silicon dioxide. Here we show that amorphous SiOx QDs can be embedded within single crystalline diamond. A SiOx film was deposited on a 2 mm (111) single crystal diamond substrate by sputtering. The sample was then treated with hydrogen plasma to form QDs, followed by microwave plasma chemical vapor deposition (CVD) of homoepitaxial diamond. Cross-sectional TEM specimens in diamond <110> orientation were prepared by focused ion beam. TEM/STEM observation was carried out in a JEOL ARM200F microscope with STEM annular dark field (ADF) image resolution of ~ 0.8 Å.

Figures 1(a) and (b) show typical STEM BF and ADF images, respectively, in which Si-based QDs are seen in dark and bright contrast. The size of the QDs is ~ 2-6 nm. The QDs covered with CVD diamond can be observed in the HRTEM image (Fig. 2 ). It can be seen that lattice fringes continuously cross over the areas of the QDs, illustrating diamond homoepitaxy. The atomically resolved STEM-ADF image in Fig. 3 shows a QD (~ 2 nm size) in very bright contrast superimposed with diamond atomic columns, indicating that the QD is amorphous and embedded in single crystalline diamond. Furthermore, only Si, O, and C peaks are detected in x-ray EDS measurements on those QDs, suggesting that the QDs are SiOx (x ~ 0.6). Also, photoluminescence measurements show light emitting wavelength at a peak > 520 nm.

The work was supported by National Science Council, Taiwan, R.O.C. under Contract No. 101-2221-E-009-049-MY3.

Fig. 1: (a) STEM-BF and (b) STEM-ADF images obtained from an interfacial region.

Fig. 2: HRTEM image showing diamond lattice fringes.

Fig. 3: STEM-ADF image in atomic resolution showing SiOx QDs in diamond.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3065 Atomic surface diffusion on Pt nanoparticles quantified by high-resolution transmission electron microscopy

Schneider S.1,2, Surrey A.1,2, Pohl D.1, Schultz L.1,2, Rellinghaus B.1
1IFW Dresden, Institute for Metallic Materials, Dresden, Germany, 2TU Dresden, Institute of Condensed Matter Physics, Dresden, Germany
sebastian.schneider@ifw-dresden.de

The continuing development of aberration-corrected high-resolution transmission electron microscopy (HRTEM) led to the possibility to study the structure of specimens at the atomic scale in great detail and with highest precision [1]. Besides the determination of these static structural information, dynamic changes of the specimen, e.g., due to the impact of the imaging electron beam, are often observed in (scanning) TEM [2], in particular when working at high doses that are frequently mandatory to investigate the structural and chemical properties. Such dynamic phenomena which are mostly related to unwanted radiation damage [3], may also occur spontaneously without the electron beam and are most easily observable on surfaces. It is, however, still an open question whether the observed atomic surface diffusion, which is the main underlying mechanism of most of these processes, can be solely ascribed to the physical properties of the material, or if and to which extent it is rather promoted by the impact of the imaging electron beam.
Recently, a HRTEM method was introduced that allows for the quantitative estimation of the surface diffusion coefficient, and it was shown that this method can be used to quantify the surface self-diffusion on Au nanoparticles [4]. The method is based on the analysis of temporal fluctuations in the occupancy of surface atomic columns.
Thus in the present study, the motion of atoms at the surfaces of Pt nanoparticles is characterized by means of aberration-corrected HRTEM with the resolution of individual atomic columns. Fig. 1 shows six out of 31 example images of a Pt nanoparticle on a holey amorphous carbon film supported by a copper grid. It can be seen that during the time delay of 0.8 s between the acquisition of two images, some atom columns are emptied while other neighboring columns are filled. Even though atoms are indistinguishable and only a two-dimensional projection of the three dimensional diffusion can be registered in the TEM, the applied method is capable of a quantitative estimation of the diffusion coefficient from the temporal sequence of HRTEM images.
The coefficient of the surface self-diffusion of Pt as derived with this novel approach turns out to be in very good agreement with the results of both experimental [5] and theoretical [6] studies.

[1] Urban, K.W., Science 321 (2008), p. 506 - 510.
[2] Bals, S. et al., Nature Communications 3 (2012), 897.
[3] Egerton, R. et al., Micron 35 (2004), p. 399 - 409.
[4] Surrey, A. et al., Nano Letters 12 (2012), p. 6071 - 6077.
[5] Bassett, D., Webber, P., Surface Science 70 (1978), p. 520 - 531.
[6] Feibelman, P.J., Physical Review Letters 81 (1998), p. 168 - 171.

Fig. 1: Temporal series of images of a Pt particle. The delay between two subsequent images is 0.8 s. “o”/”e” = occupied/empty atomic column. From left to right, the culumn occupation changes as follows: turquois: o, o, o, o, e, e; white: o, o, e, e, e, e; green: e, e, o, o, e, e; blue: o, o, e, e, o, e; yellow: o, o, o, o, o, e; red: e, o, e, o, e, o.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3070 Analytical TEM study of Au-Ag bimetallic catalysts prepared by solid grinding method

Akita T.1, Maeda Y.1, Kohyama M.1
1Research Institute for Ubiquitous Energy Devices, National Institute of Advanced Industrial Science and Technology (AIST)
t-akita@aist.go.jp

Gold catalysts exhibit high catalytic activity for low temperature CO oxidation [1]. It has been reported that active site is interface between Au and metal oxide supports [2]. Activation of oxygen at the interface seems to be important step for the CO oxidation reaction. While CO molecules are adsorbed on Au surface, the oxygen seems to be activated at the interface between Au and metal oxide support. Details of mechanism of whole reaction is not clarified yet. On the other hand, addition of Ag also improves the activity of Au catalysts [3,4]. It is significant to investigate the mechanism of improvement of the activity by adding Ag in order to study the general mechanism of oxygen activation for low temperature oxidation. In this study, Au-Ag bimetallic catalyst was prepared by solid grinding method [5], and fine structure of Au-Ag bimetallic catalysts was investigated by aberration corrected TEM/STEM. The distribution of Au and Ag in one nanoparticle was also investigated by EDS.

Au-Ag bimetallic catalysts was prepared by simultaneous solid grinding method using Me2Au (acac: acetylacetonate) and Ag (acac). Au and Ag precursor and supports was physically mixed for 20min in Ar atmosphere. Subsequently, the catalysts were calcined at 300°C for 4 hours in air. Catalytic activity was measured by using a fixed bed reactor and a standard gas containing 1 vol.% CO in air. The structure of Au-Ag bimetallic catalysts was observed by aberration corrected TEM/STEM (FEI Titan3 G2 60-300). EDS measurement was carried out by high sensitive EDS system, Super-X (Bruker) equipped with 4-silicon drift detectors (SDD).

Figure 1 indicates catalytic activity for CO oxidation of Au/SiO2 and Au-Ag/SiO2 catalysts. It is clearly confirmed that the catalytic activity of Au/SiO2 is improved by addition of Ag. This effect is prominent for inert supports such as SiO2 and Al2O3. Figure 2 shows ADF-STEM image of Au-Ag/SiO2 catalyst. Nanoparticles with the diameter of 2-10nm are well dispersed on the SiO2 support by solid grinding method. Elemental maps by EDS were carried out and both Au and Ag signal was detected from most nanoparticles. This is indicating that the Au-Ag bimetallic nanoparticles are formed by simple mixing of individual organic complexes of Au and Ag by solid grinding method.

References

1. M. Haruta, T. Kobayashi, H. Sano , N.Yamada, Chem. Lett. (1987) 405.

2. T. Fujitani, I. Nakamura, Angew. Chem. Int. Edit. 50 (2011) 10144.

3. A.Q. Wang, J.H. Liu, S.D. Lin, T.S. Lin, C.Y. Mou, J. Catal. 233 (2005) 186.

4. Y. Iizuka, T. Miyamae, T. Miura, M. Okumura, M. Daté, M. Haruta, J. Catal. 262 (2009) 280.

5. T. Ishida, M. Nagaoka, T. Akita, M. Haruta, Chem. Eur. J. 14 (2008) 8456.

The authors are grateful to Ms. F. Arai, Ms. C. Fukada and Ms. M. Makino for helpful work on the preparation of catalysts and measurements of catalytic activity.

Fig. 1: Catalytic activity for CO oxidation of Au/SiO2 and Au-Ag/SiO2 catalysts. SV: 20000mLh-1g-cat.-1

Fig. 2: ADF-STEM image of Au-Ag/SiO2 catalyst.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3091 Structural properties of GaN/AlN/GaN core-double shell nanowires

Koukoula T.1, Kehagias T.1, Kioseoglou J.1, Eftychis S.2, Kruse J.2, Georgakilas A.2, Karakostas T.1, Komninou P.1
1Physics Department, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece, 2Microelectronics Research Group, Physics Department, University of Crete, P.O. Box 2208, GR-71003 Heraklion, and IESL/FORTH, P.O. Box 1385, GR-71110 Heraklion, Crete, Greece
komnhnoy@auth.gr

Core-shell nanowires (NWs) are an ingenious discovery of interfacial nanoengineering that comprises structural characteristics, which cannot be reproduced by any kind of epitaxial growth. This stems from the fact that core-shell NWs tolerate a larger lattice mismatch without interfacial defects than other heterostructures, thus offering a wider area of band-gaps with several potential optoelectronic applications. It is established that wurtzite NWs grown along the polar direction are bounded by the {10-10} m-planes. As a result, core-shell NWs comprise non-polar {10-10} interfaces between the core and the shell.
The structural properties of core-double shell (GaN/AlN/GaN) NWs, grown by plasma-assisted molecular beam epitaxy (PAMBE) on Si(111), were explored by transmission electron microscopy (TEM) methods. GaN NWs were grown on a thin AlN nucleation layer (3 nm) for 3h, with intermediate AlN spacers (10-15 nm thick) deposited at 1h and 2h growth time [Fig. 1(a)]. High-resolution TEM (HRTEM) imaging revealed the core-double shell morphology with an AlN shell of 0.7-1 nm thick, and a GaN shell varying from 1.6 to 2.7 nm [Fig. 1(b)]. Line profiles of HRTEM images along the growth axis showed that this particular configuration imposes the c-lattice constant of the AlN shell to be adapted to the c-lattice constant of the GaN core. Therefore, a full elastic accommodation of the AlN on GaN is established, considering the absence of misfit dislocations (MDs) from the interface.
The strain state of NWs was evaluated by geometrical phase analysis (GPA). A gradual relaxation of the AlN spacers was observed from the GaN/AlN interfaces towards the center of the spacer for the a-lattice parameter as illustrated in Fig. 1(c), without the presence of MDs. The corresponding FFT is shown in Fig. 1(e). The GPA strain map of the AlN spacer/GaN along the growth direction and the corresponding line profile are shown in Figs. 1(d&f). Regarding the AlN shell, the GPA lattice strain along the growth direction was estimated near zero, verifying the HRTEM observations on the full lattice registration. Considering the very small thickness of the shell, the average in-plane lattice constant approximates pseudomorphic growth. This implies that the AlN shell deviates from the biaxial strain state. Moreover, the GaN shell exhibits the relaxed lattice constant values in both in-plane and out-of-plane directions. It seems that the core-shell configuration of the NWs induces strain fields, which may be exploited in band-gap engineering.

This research has been co-financed by the European Union (European Social Fund - ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: THALES: Reinforcement of the interdisciplinary and/or inter-institutional research and innovation.

Fig. 1: (a)TEM image illustrating the NWs morphology along with their schematic model. Black arrows denote the AlN spacers (b) HRTEM image depicting the double shell-core configuration (c) In-plane GPA phase image of the AlN/GaN and (e) the corresponding FFT (d)&(f) GPA strain map of the AlN/GaN along the growth direction and the corresponding line profile
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3092 Surface dependent structure of GaN nanowires spontaneously grown on Si

Koukoula T.1, Kehagias T.1, Kioseoglou J.1, Eftychis S.2, Kruse J.2, Georgakilas A.2, Komninou P.1
1Physics Department, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece, 2Microelectronics Research Group, IESL, FORTH, P.O. Box 1385, GR-71110 Heraklion, and Physics Department, University of Crete, P.O. Box 2208, GR-71003 Heraklion, Greece
tkouk@auth.gr

Self-assembled GaN nanowires (NWs) were grown by plasma-assisted molecular beam epitaxy (PAMBE) on Si(111) substrates. Treatment of the substrate surface is critical for NWs growth, as well as their morphological features and crystal quality, and hence their optical properties. To this end, a transmission electron microscopy (TEM) study was performed, to compare the spontaneous nucleation of GaN NWs, when they grow on bare Si, with or without nitridation of the surface, and when they grow on top of an AlN nucleation layer (NL) of varying thickness.
Direct GaN growth on bare Si without nitridation resulted in a high density of tilted GaN NWs, grown on a thin amorphous SixNy layer due to the inevitable interaction of the active N species with the Si surface. NW tilting is attributed to the roughness of the SixNy layer, following the roughness of a stepped Si surface. Indeed, high-resolution TEM (HRTEM) images revealed that tilted NWs nucleated on SixNy just over Si surface steps. When the surface was intentionally nitridated, prior to NWs growth, axial alignment of NWs substantially improved, owing to the formation of a thicker SixNy at the GaN/Si interface, which accommodated any Si surface steps (Fig. 1). Moreover, wurtzite GaN crystalline remnants detected on SixNy might have functioned as potential NW nuclei at the onset of NW growth. Besides the axial inclination of GaN NWs from the growth direction, plan-view observations showed, occasionally, a ~3o in-plane rotation between GaN and Si. We constructed the interfacial atomistic models of a GaN NW epitaxially grown on Si, and a NW where the (0001) planes of GaN were rotated about 3o relative to the (111) planes of Si. In both cases, the GaN/Si superlattice unit cell exhibits a hexagonal shape.
In order to optimize the Si surface treatment, an AlN NL with thickness ranging from 2 nm to 20 nm was used, either as-grown or annealed under active N, i.e., nitridated. In contrast to the previous cases, the amorphous SixNy layer was eliminated from the interface allowing improved alignment and crystal quality of the GaN NWs. When using a 2 nm thick AlN nitridated NL, GaN islands appeared along with GaN NWs (Fig. 2). Conversely, a compact faceted GaN layer with sparse GaN NWs was observed over a 20 nm thick AlN NL. The latter also emerged when the 2 nm AlN NL was not nitridated, however in this case a significantly higher density of NWs was observed (Fig. 3). Therefore, at the initial stages of NL growth, AlN forms 3D islands, which during annealing evolve into a compact 2D AlN NL affecting the morphology of the NWs and the GaN faceted domains.

Research co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: THALES, project “NanoWire”.

Fig. 1: (a) TEM image, near the [11-20]GaN zone axis (z.a.), showing the morphology of GaN NWs grown on nitridated Si. (b) HRTEM image of the GaN/Si interface, along the [ 11-20]GaN z.a., with GaN crystalline remnants (black arrows) on top of the amorphous SixNy layer.

Fig. 2: (a) TEM image, off the [11-20]GaN z.a., depicting the improved alignment of NWs, when they grow on top of 2 nm AlN nitridated NL. (b) HRTEM image of the GaN/AlN/Si interface, along the [11-20]GaN z. a., showing the solid AlN NL.

Fig. 3: (a) TEM image, near the [11-20]GaN z.a., depicting a compact faceted GaN layer when the 2 nm AlN NL was not nitridated. (b) HRTEM image, along the [11-20]GaN z.a., showing the formation of AlN 3D islands.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3101 Analytical characterization of bimetallic gold-ruthenium catalysts supported on ceria zirconia mixed oxides

Chinchilla L. E.1, Olmos C.1, Blanco G.1, Kurttepeli M.1, Bals S.1, Van Tendeloo G.2, Villa A.2, Prati L.2, Calvino J. J.3, Chen X.1, Hungría A. B.1
1Dpto. de C.M.I.M.Q.I.,Universidad de Cádiz, Spain, 2EMAT,University of Antwerp, Belgium, 3Dipartimento di Chimica,Universita’ degli Studi di Milano, Italy
ana.hungria@uca.es

In the present work, HAADF STEM and XEDS studies were performed in a TITAN 80-50 and a JEOL 2010F microscopes on a series of supported AuRu bimetallic catalysts. In microscopy studies of such complex systems, conclusions are usually drawn about the composition of the particles having analyzed a limited number of them, but rarely estimations are done to know whether the set of chosen particles are representative of the catalyst at a macroscopic level. The results have highlighted the difficulty to characterize the composition of bimetallic systems with particles of varying sizes and metal contents. Despite the technical issues related to STEM mode accompanied with XEDS of very small particles, at least 80 particles of each catalyst were analyzed (Fig 1). All catalysts presented a large fraction of monometallic particles, and a variable amount of particles displaying XEDS signals corresponding to the two metals. XEDS analyses showed that the particles within the small size range were predominantly Ru rich, larger particles were found to be Au and also entities containing both Au and Ru were detected. Based on the models reported by Van Hardeveld et al. [Surf. Sci. 1969, (2), 189] for fcc (Au) and hcp (Ru) crystallites, the precise relationship between the particle size and the total amounts of atoms was calculated. We estimated the number of Au and Ru atoms in each particle present in the particle size-composition diagrams (Fig 1). For 1:2AuRuCZ, instead of the atomic ratio 1Au:2.4Ru measured by ICP, a ratio 1Au:0.25Ru was estimated. The discrepancy may result from the lack of representativeness of microanalysis of individual particles, which caused underestimations of the amount of smaller particles, richer in Ru than the larger (> 5 nm) particles. This hypothesis is supported by the results of XPS analysis showing a relationship 1Au:14Ru corresponding to a presence on the surface of the catalyst of a large number small particles of Ru versus a more aggregated state of Au, given that studies by HAADF-STEM electron tomography have excluded encapsulation phenomena of the metallic phase on the support. An unbiased confirmation of the presence and weight of each particle type can be performed by high resolution XEDS maps (Fig 2), where an aggregate containing a large number of small particles of Ru and an only Au particle of about 30 nm can be seen. In summary, bimetallic AuRu catalysts can be rather non-uniform and can show variation in particle size and composition, due to limited miscibility of the metal components. Catalytic tests showed that bimetallic catalysts were more active than pure Au and Ru catalysts for octanol oxidation, suggesting that Au-Ru interaction, albeit limited, increase the specific activity with respect to the pure components.

Funding from Junta de Andalucía (FQM-3994) and EU FP7 Program (Grant Agreement 312483-ESTEEM2) is gratefully acknowledged. X.C. and A.B.H. thank Ramon y Cajal Program. L.E.C. thank Armand Béché

Fig. 1: Particle size-composition diagrams and relative contribution by particles at a given metal composition for AuRuCZ catalysts

Fig. 2: HAADF-STEM image and XEDS elemental distribution maps (Ce-L, Ru-K, Zr-L and Au-L) recorded on the 1:2 AuRuCZ catalysts. Also is presented an overlay map of the Au, Ru and Ce chemical distribution
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3116 Preparation and characterization of Zn (II) complex, [Zn(bipy)2(C6H5)2CHCO2)](ClO4)(bipy), -SLNs formulation

Dikmen G.1, Kani I.2
1Eskişehir Osmangazi University of Eskişehir, Central Research Laboratory, 2Anadolu University of Eskişehir, Department of Chemistry, Eskişehir, Turkey
gokhandikmen@anadolu.edu.tr

Metal organic frameworks offer diverse chemistry as metal-medicine. In these complexes, the metal serves to coordinate the organic ligands. The direct use of metal complexes sometimes is restricted due to lethal side effects. To overcome their disadvantages, solid lipid nanoparticles (SLNs) have been introduced as an alternative drug delivery systems. They carry anticancer compounds with different physiochemical characteristics, higher drug stability, improved pharmacokinetics and controlled drug release. In this study, we synthesized bimetallic Zn(II) complex, [Zn(bipy)2(C6H5)2CHCO2)](ClO4)(bipy), with the reaction of bifunctional 2,2′-bipyridine (bipy) and diphenyl acetic acid (C6H5)2CHCO2H). SLNs formulation prepared by hot homogenization methods and characterized by Zeta Sizer, NMR (Nuclear Magnetic Resonance) and SEM (Scanning Electron Microscopy). In conclusion, SLNs of Zn(II) complex have good stability at -16.5 mV and average particle size around 230 nm. We determined chemical structure as 3D by using XRD. In addition, NMR spectras were carried out tween 80, complex and complex loaded SLN formulations and these spectra compared with each other. According to NMR spectra, both difference in chemical shifts and new peaks were not observed for complex loaded SLN and plasebo SLN. Moreover,  the particle size of Zn (II) complex-SLN formulations was also supported by using SEM. In general, the Zn (II) complex-SLN formulations were spherical shape and uniform in particle size. 

Keywords: Zn, Solid lipid nanoparticles (SLN), Scanning Electron Microscopy (SEM), XRD.

Fig. 1: Figure 1. Sem photo of Zn(II) complex-SLNs formulation.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3118 Characterization and spectral measurement of light emission from individual Au nanoparticles using scanning tunnelling microscopy

Nepijko S. A.1, Chernenkaya A.1, Medjanik K.1, Chernov S. V.1, Yarmak A. V.2, Odnodvorets L. V.2, Schulze W.3, Schönhense G.1
1Institute of Physics, University of Mainz, Staudingerweg 7, 55128 Mainz, Germany, 2Sumy State University, Rimsky-Korsakov Str. 2, 40007 Sumy, C.I.S./Ukraine, 3Fritz Haber Institute of the Max Planck Society, Faradayweg 4-6, 14195 Berlin, Germany
nepijko@uni-mainz.de

The light emission spectra of individual Au nanoparticles induced by a scanning tunnelling microscope (STM) have been investigated. At the same time the nanoparticles were characterized by STM measurements. Two-dimensional ensembles of tunnel-coupled Au nanoparticles were prepared by thermal evaporation onto a native oxide silicon wafer in ultrahigh vacuum (10-10 mbar). We present the experimental evidence of photon emission from Au nanoparticles excited with W tip in the tunnel (tip voltage lower than 5 V) and field emission modes (tip voltage higher than 5 V). In the first case the tunnel current has inelastic component that is used for the tip-induced plasmon excitation. The photon emission that corresponds to them is characterized by a maximum at 1.62 eV. In the second case the photon emission spectrum is more complicated. The photon emission spectrum for Au nanoparticle obtained after subtraction of photon emission from the substrate (the native oxide silicon wafer) is characterized by peaks at 2.22 and 1.45 eV connected with the Mie plasmon and the density of unoccupied states above the Fermi level, relatively. The low-energy peak at 1.45 eV has not been discussed in literature. It was more pronounced then in other publications most likely due to more blunt W tip in our experiment and consequently larger applied voltage (the Au nanoparticle size was a few nanometers in all cases). The use of an STM in the field emission mode with the light signal detection allows implementing of low-energy electron-photon spectroscopy (inverse photoemission spectroscopy).

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3142 A complete TEM study of microstructural changes within bifunctional refining catalysts at different stages of their preparation

Moldovan S.1, Grillet N.2, 1, Florea I.3, 1, Danilina N.4, Minoux D.4, Ersen O.1
1Institut de Physique et Chimie des Materiaux de Strasbourg, UMR 7504 CNRS, 23 rue du Loess, 67034 Strasbourg, France, 2Institut Materiaux Microelectronique Nanosciences de Provence, UMR 7334 CNRS, Campus de St Jérôme, 13397 Marseille, France, 3Ecole Polytechnique, Route de Saclay, 91128 Palaiseau, France, 4Total Petrochemicals Research Feluy, Zoning Industriel, Zone C, 7181 Feluy, Belgium
simona.moldovan@ipcms.unistra.fr

Owing to their exceptional properties the bifunctional catalysts are widely employed for processing of heavy oils and are generally composed of an active cracking matrix, a binder and an active (de)hydrogenation function. The actual study focuses on the morphological evolution of bifunctional refining catalysts consisting of a zeolite, an alumina binder and metal sulfides. Three general steps schematized in Figure 1 are considered for the preparation of the catalysts: the extrusion of the zeolite by using alumina as binder, impregnation with Ni and Mo salts and sulfidation to obtain the active metal sulfide phase. The morphology of the zeolite grains is originally marked by spherical mesopores and channels, as well as micropores. The extrusion with alumina does not change fundamentally the zeolite 3D porous structure, but fixes the alumina grains on the zeolite crystals surfaces. The needle-like shaped geometry of the alumina grains contribute to the built-up of a novel porous structure on the zeolite grain surface, without a considerable modification of the accessibility to the zeolite porous structure. The impregnation with Ni and Mo salts leads to a new nanometric architecture, such that one identifies large and small nanoparticles (NPs) within both the zeolite grain and the embedding alumina matrix. A combined analysis evidenced that on the zeolite grains, the small NPs (mean size: 5 nm) are Ni-dominated, whilst the bigger NPs are Mo-rich. One cannot exclude that Mo can appear as small particles and/or as atomic clusters. The zeolite grain morphology and pores size delimit the NPs location: the small NPs will access the micropores, whereas the big ones will be placed exclusively on the mesopores and the channels rims. Though the Mo was predominantly found in the large particles, one cannot exclude that Mo-rich NPs, atoms and/or atomic clusters would penetrate the zeolite grain anchored to the micropores rims. Most of particles deposited on the alumina grains are in Mo, but small amounts of Ni have been also identified. The sulfidation treatment is expected to induce at a structural level the formation of metal sulfide slabs. Indeed, they have been identified mostly on the alumina grains, but also within the zeolite pores inner rims. These finding backups our observations on the presence of Mo NPs in the zeolite pore after impregnation. The electron tomography stressed the slabs location as against the zeolite pores: the metal sulfide slabs do not locate only on the inner mesopores, but also decorate the micropores rims. New 3D core-shell structures were identified in the inner zeolite mesopores: the core is most probably the Ni sulfide, whereas the shell is constituted by MoS2 slabs disposed around the core but not always in contact with it.

Fig. 1: Figure 1. General steps followed for preparation of bifunctional refining catalysts.

Fig. 2: Figure 2. Micrographs acquired under different modes showing the evolution of the zeolite with the preparation steps: A. Slice and model of the zeolite; B. EFTEM image and slice of the extruded zeolite; C. Slices of the TEM and STEM volumes of the impregnated specimen; D. STEM-HAADF micrograph and section corresponding to sulfided specimen.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3159 Wurtzite ZnO/Zinc Blende ZnS Coaxial Heterojunctions and Hollow Zinc Blende ZnS Nanotubes: Synthesis, Structural Characterization and Optical Property

Huang X.1,2, Willinger M. G.2, Fan H.1, Xie Z. L.2, Wang L.1, Hoffmann A. K.2, Lee C. S.3, Meng X. M.1
1Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, P. R. China, 2Department of Inorganic Chemistry, Fritz Haber Institute of the Max Planck Society, Faradayweg 4-6, 14195 Berlin, Germany, 3Center of Super-Diamond and Advanced Films (COSDAF) & Department of Physics and Materials Science, City University of Hong Kong, Hong Kong SAR, P. R. China
xinghuang@fhi-berlin.mpg.de

Synthesis of ZnO/ZnS heterostructures in thermodynamic conditions generally results in the wurtzite (WZ) structure of the ZnS component because its WZ phase is thermodynamically more stable than its zinc blende (ZB) phase. In this report, we demonstrate for the first time the preparation of ZnO/ZnS coaxial nanocables composed of single crystalline ZB structured ZnS epitaxially grown on the WZ ZnO nanorod via a two-step thermal evaporation method. The deposition temperature is believed to play a crucial role in determining the crystalline phase of ZnS. Through a systematical structural analysis, the ZnO core and the ZnS shell are found to have an orientation relation of (0002) WZ ZnO//(002) ZB ZnS and [01-10] WZ ZnO//[2-20] ZB ZnS. Observation of the coaxial nanocables in cross-section reveals the formation of voids between the ZnO core and ZnS shell during the coating process, which is probably associated with the nanoscale Kirkendall effect known to result in porosity. Furthermore, by immersing the ZnO/ZnS nanocable heterojunctions in an acetic acid solution to etch away the inner ZnO cores, hexagonal shaped ZnS nanotubes orientated along the [001] direction of ZB structure were also achieved for the first time. Finally, the optical property of the hollow ZnS tubes was investigated. It was found that the tubes can give a strong green emission which may originate from some self-activated centers, vacancy states, interstitial states or structural defects. However, for those tubes with residual ZnO located on tops, they showed much lower emission intensity due to the type-II band alignment of ZnO/ZnS heterojunction that can efficiently decrease the recombination of the electron-hole pairs in both ZnO and ZnS. Our study gives some insights on the controlled fabrication of 1D semiconductors with desired morphology, structure and composition and the as-synthesized WZ ZnO/ZB ZnS coaxial nanocables and ZB ZnS nanotubes provide ideal candidates for the study of optoelectronics of II-VI semiconductors at the nanoscale.

We thank the financial support from the “Strategic Priority Research Program" of the Chinese Academy of Sciences (XDA09040203).

Fig. 1:  (a) TEM image of the nanocables’ cross-sections; (b) HRTEM image and (c) reconstructed structure from (b), cyan: ZnO, red: ZnS; (d) FFT of (b); (e-f) Enlarged HRTEM image recorded from regions of i and ii of (b); (g) HAADF image as well as elemental mapping ; (h-i) EDX data corresponding to spots A and B, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3199 Self-assembled catalyst promotion by overgrowth of layered ZnO in industrial Cu/ZnO/Al2O3 catalysts

Lunkenbein T.1, Schumann J.1, Behrens M.1, Schlögl R.1, Willinger M. G.1
1Fritz-Haber-Institute of the Max-Planck-Society
lunkenbein@fhi-berlin.mpg.de

Methanol is one of the most important petrochemical molecules. It is considered as a prospective sustainable synthetic fuel obtained by the catalytic hydrogenation of CO2.[1] Industrial relevant catalysts are mainly composed of Cu (>50 mol%)/ZnO in combination with a structural promoter, such as Al2O3 (<10 %).[2] The presence of ZnO drastically increases the intrinsic activity of Cu-based catalysts. This Cu-ZnO synergy can be explained by the appearance of strong metal support interaction (SMSI) upon reduction in hydrogen.[3] The nature of the SMSI effect is versatile and can be expressed by electronic or morphological changes. In the former case an electron transfer from the support to the metal can occur, whereas for the latter situation a migration of the partially reduced oxide over the metal particle arises.
For model systems this migration has already been proposed in one of the early studies of Cu-ZnO synergy[4] and has recently been identified as metastable graphitic ZnO by IR measurements.[5]
Here we present experimental evidence for the presence of this metastable graphitic ZnO overlayer on Cu nanoparticles in an industrially relevant Cu/ZnO/Al2O3 catalyst for methanol synthesis. Direct structural imaging and elemental mapping in the transmission electron microscope show the formation of a layered ZnO overgrowth during reduction (Fig 1).
The results demonstrate a step further towards a complete understanding of the synergistic effects in Cu-ZnO based catalyst for methanol synthesis.


References

[1] G. A. Olah, Angew. Chem. Int. Ed. 2005, 44, 2636-2639.

[2] M. Behrens, S. Zander, P. Kurr, N. Jacobsen, J. Senker, G. Koch, T. Ressler, R. W. Fischer, R. Schlögl, J. Am. Chem. Soc. 2013, 135, 6061-6068.

[3] a) S. J. Tauster, S. C. Fung, R. T. K. Baker, J. A. Horsley, Science 1981, 211, 1121-1125; b) S. J. Tauster, S. C. Fung, R. L. Garten, J. Am. Chem. Soc. 1978, 100, 170-175.

[4] J. C. Frost, Nature 1988, 334, 577-580.

[5] V. Schott, H. Oberhofer, A. Birkner, M. Xu, Y. Wang, M. Muhler, K. Reuter, C. Wöll, Angew. Chem. Int. Ed. 2013, 52, 11925-11929.

Fig. 1: A) Representative TEM micrograph of the reduced catalyst that shows the graphitic overlayer. B) The corresponding energy filtered TEM map of the O K edge indicates a core-shell structure. C) Scanning TEM image of Cu/ZnO/Al2O3. The inset denotes electron energy loss spectra of the Cu L2,3 and Zn L2,3 edge of one single Cu nanoparticle (see ROI).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3206 In-situ electrical characterization of single ZnO nanowires

Fontana M.1, 2, Chiodoni A.1, Bejtka K.1, Jasmin A.1, 2, Porro S.1, Pirri C. F.1, 2
1Istituto Italiano di Tecnologia, Center for Space Human Robotics, Torino, Italy, 2Dipartimento di Scienza Applicata e Tecnologia, Politecnico di Torino, Torino, Italy
marco.fontana@iit.it

Among the different ZnO nanostructures, nanowires (NWs) are of particular interest for applications: they are envisioned as possible future building blocks for nanoelectronics [1] due to their well-defined geometry and possibly enhanced electronic transport properties, coupled with size-dependent piezoelectric response [2]. It is of great importance to understand how the electronic transport behavior of the NWs is influenced by morphology and crystalline structure on one side, and by the particular approach adopted for the implementation of electrical contacts at the nanoscale on the other side. Electrical measurements on single NWs have been reported in literature, with resistivity values ranging over many orders of magnitude ([3,4]), depending on the synthesis techniques, morphology, crystalline structure and defects, type of electrical nanocontacts, ambient conditions and experimental set-up. Therefore, work still needs to be done in order to gain fundamental understanding of the electrical properties of single ZnO NWs and their relationship with synthesis and structure.
In this work we report on the structural, morphological and electrical characterizations of ZnO NWs by means of Field Emission Scanning Electron Microscopy (FESEM), Transmission Electron Microscopy (TEM) and two-probe I-V measurements performed in-situ by the dual-beam FIB-FESEM system. The ZnO NWs were grown by low-pressure chemical vapor deposition (LPCVD) on Si wafers. The morphology of the samples was initially characterized by FESEM: the aspect ratio and the homogeneity of the cross-section along the whole NWs were evaluated. High-resolution TEM characterization shows that the NWs are wurtzite single crystals and they are oriented along the [001] crystalline direction. In order to perform electrical measurements, the NWs were detached from the Si substrates, ultrasonically dispersed in ethanol and then transferred onto SiO2 substrates for the subsequent preparation of metallic contacts. FIB-induced deposition of Pt pads from a gas precursor (methylcyclopentadienyl-trimethyl platinum) was carried out and two-probe measurements were performed in the dual-beam chamber by direct contact of two micromanipulators on the deposited Pt contacts. In the case of NWs with suitable length (> 4 µm), the deposition of more than two contacts was considered to perform transmission line measurements in order to gain information about the contact resistance.

1. Z.L. Wang, Material Science and Engineering R 64 (2009), 33-71
2. H.D. Espinosa et al., Advanced Materials 24 (2012), 4656-4675
3. E. Schlenker et al., Nanotechnology 19 (2008), 365707
4. Jr-Hau He et al., Nanoscale 4 (2012), 3399

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3233 New understanding of the atomic structure and nonstoichiometry of Sc-doped TiO2 photocatalysts by using aberration corrected STEM ARM200CF microscope and EELS spectroscopy

Bakardjieva S.1, Klie R.2, Paulauskas T.2, Philips P.2, Bezdička P.1, Šubrt J.1, Janoš P.3, Gartnerova v.4, Jager A.4
1IIC AS CR v.v.i., 250 68 Rez, Czech Republic, 2UIC Chicago, 845 W Taylor Street, 60607 Chicago, Illinois, USA, 3UJEP, Králova Výšina 3132/7, Ústí nad Labem, 400 96, Czech Republic, 4IP ASCR v.v.i., Na Slovance 1999/2, 182 21 Praha, Czech Republic
snejana@iic.cas.cz

The studies on scandium doped TiO2 photocatalyst nanocrystallites are still limited in the iterature. We select Sc-doped process as an effective method to control the surface structure of TiO2 by generation of defects into TiO2 lattice. Characterization of structures at atomic resolution was performed by aberration-corrected JEM-ARM200CF microscope allows for 68 pm spatial resolution. Z-contrast STEM images were collected using HAADF detector. EELS was acquire atomic-scale maps of the chemical composition and assess the local bonding and Ti valence. This is the first example of a Ti-Sc-O system demonstrated that the substitution of Sc for Ti results in changes in photocatalytic activity due to the preferred occupancy of Sc atoms and its effects on the anatase lattice. The changes of both the lattice parameters and surface morphology are related to the chemical bonding between Ti and Sc cations and oxygen atoms, and species formed at the surface with respect to oxygen deficiencies.The highest activity was observed in TiO2-Sc 4.18 at.%. The photocatalytic activity of the Sc doped TiO2 strongly depends on Sc concentration and particle size of both the dopant ion and TiO2 matrix. A general view of the Sc-doped anatase is shown in Fig. 1. The well-known spherical morphology of TiO2 is established. Detailed atomic scale analysis performed with STEM-HAADF detector shows composites with core-shell structure (Fig.2a). High resolution Z-contrast images demonstrates that the scandium ion dispersed into TiO2 nanoparticle and its concentration in the shell regions is higher and formed scandium-rich regions where Sc3+ replace Ti3+ and mixed oxygen vacancy generated composite (Ti3+xTi4+1-x)O2-x is developed. Therefore, the Sc ion can be caused defects by introducing oxygen vacancies in the lattice of TiO2. Two different crystallographic regions can be recognized based on the intensity and the atomic column symmetry. The border region has much larger intensity. This contrast is explained mainly by the effect of the strain field presented in the TiO2@Sc interface (Fig.2b,c and Fig. 3.). HAADF find out that the lattice parameters of the doped TiO2 samples begin to be larger than pure TiO2. We suppose that during nano-crystal growth, the method by which solutions of reacting components are mixed and the intensity of their stirring can influence the precipitation and the physical characteristics of the product. The precipitation of Sc-doped TiO2 results from mixing of two liquids on microscale level. The mixture consists of entirely segregated parts with different local concentration. In such an way a molecular diffusion occurs to form crystals where the inner TiO2 core has a different composition from the outer shell

The authors thank Project EnviMod UJEP for fanantial support and Dr. Stengl team for synthesis of samples.

Fig. 1: HRTM and HAADF images of Sc doped TiO2

Fig. 2: Atomi scale HAADF images of TiO2@Sc core-shell and interfaces

Fig. 3: ABF images and EELS of grain boundaries

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3270 Oxidation study of silver nanoparticles

BAZAN-DIAZ L. S.1, HERRERA-BECERRA R.2
1PCeIM, UNAM, 2INSTITUTO DE FISICA, UNAM
bazanlulu@gmail.com

During the last few years has been a great interest to develop new functional nanomaterials for diverse applications, including areas such as semiconductors, optical devices, photo-electrochemistry, among others [1]. It is well known that the formation of nanoparticles is highly sensitive to different the reaction conditions. Particularly, the control of shape and size of the nanoparticles are parameters of major importance for the different applications where they are used. One of the main physicochemical parameters that control the final size and shape of nanoparticles is the pH [2], resulting in the formation of Ag nanoparticles of different morphologies depending on precursors concentration at different pH values.

Silver nanoparticles (AgNP) have been subject of several studies due to their diverse applications; however, it has been observed that when AgNP are synthesized in aqueous solutions they might suffer oxidation that could derive in loss of activity[3].

In this work, we studied the oxidation of AgNP obtained in aqueous solutions, by employing a so-called "green" method for the production of nanoparticles, with especial focus on the characterization of forms and structures of AgNP. The method employed here is at room temperature, using synthetic tannins as principal reducing reagents and AgNO3 as precursor in aqueous solution. During the reactions, the pH was modified by using NaOH solutions at low concentrations, obtaining different sizes and shapes of AgNP. The mixtures were subjected to ultrasonic treatment, centrifugation and drying. This straightforward method has proven its effectiveness in the reduction of metal nanoparticles on earlier work by our group [4].

It was observed that exist a critical stable size, after it AgNP have tendency to oxidize. Advanced analytical electron microscopy characterization will be employed to determinate the critical size and the final structure, shape, atomic arrangement and chemical composition of the AgNP produced by this method.

References:

[1] L.A.Botello et al., Ingenierías, Octubre – Diciembre 2007, Vol. X, N°. 37
[2] Fang Liao, Zhou Feng, Xing-QiHu, Ionics 17, (2011)81-86.
[3] Chung-Ming Li, I.M. Robertson, M.L. Jenkins, J.L. Hutchison, R.C. Doole, Micron 36 (2005)9-15.
[4] Herrera-Becerra, R. et al; 2010. Appl. Phys. A, 100 (2), 453-459.

The authors acknowledge the financial support from DGAPA with grant PAPPIT IN105112 and from the Graduate Program PCeIM, UNAM.Our gratitude to Roberto Hernández and Cristina Zorrilla for the technical support given.Authors thank access to the facilities of the Kleberg Advanced Microscopy Center and the Research Centers in Minority Institutions (RCMI) at The University of Texas at San Antonio, USA.

Fig. 1:
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3275 In-situ Electron beam induced transformation of boehmite to gamma alumina

Ozkaya D.1
1Johnson Matthey Technology Centre, Reading UK
ozkayad@matthey.com

Gamma alumina is widely used as a support material for industrial catalysts such as refining petrochemicals or Fischer-Tropsch processes. Boehmite, Aluminum oxihydroxide (AlOOH), is the precursor to Gamma-alumina (γ-Al2O3) in the manufacturing process[1].The transformation from boehmite to gamma alumina is critical to the understanding of the structure of gamma alumina, thus has been widely studied [2]. The removal of water from the structure creates a metastable gamma phase with a structure that gets classified as tetragonal or cubic depending on the choice of unit cell [2]. The changes to the structure and surface area during this transformation are still not very well understood. One of the problems with studying boehmite using transmission electron microscopy (TEM) is the electron beam sensitivity of boehmite. Boehmite transforms to gamma alumina if observed at ambient temperatures in the TEM even under low beam dose conditions. Here, Cryo-TEM has been used to slow down the transformation under the beam in-situ as the sample de-hydrates and forms gamma alumina. The main aim of the study was to observe changes in the shape and form of the nanocrystalline material after it has transformed to gamma alumina. The properties of gamma alumina is what determines the diffusion rates and sintering behaviour of Platinum group metals (PGM’s)in most of the current auto catalysts. The study was carried out using a Gatan Cryo holder at -184C in a FEI Tecnai F20 Twin lens configuration S/TEM. The boehmite sample was dispersed on a holey carbon coated Cu-grid and put into the cryo-holder and the microscope before cooling. Some ice formation was observed at some parts of the sample as boehmite itself contains some excess water. A sequence of images was acquired as function of time together with diffraction patterns to confirm the structural transformation. A sequence of diffraction patterns diffraction patterns of boehmite and gamma alumina are shown in Figure 1 where the rings formed have been identified using JEMS software. The difference is quite stark in the way the pattern goes as the boehmite is orthorhombic and gamma alumina is pseudo cubic or tetragonal structure. The characterisation of the diffractions patterns shown together with the calculated spacings on the diffraction patterns in figure 1.

Figure 2 shows the sequence of in-situ images from boehmite to gamma alumina, the only driving force for the transformation being the electron beam. Images were acquired in sequence together with the diffraction patterns at regular intervals. The images shows that the shape of the crystallites remain the same after the transformations has taken place. However there is a slight, in shrinkage in the size of the particles around 10-15% after the transformation.

I would like to thank Stephen Spratt from Johnson Matthey Technology Centre for help during cryo experiments

Fig. 1: Figure 1. Series of diffraction patterns at different time intervals showing the transformation from boehmite to gamma alumina under the electron beam. The calculated diffraction patterns from boehmite and gamma alumina are shown for comparison

Fig. 2: Figure 2. Series of in-situ images taken at various time intervals from the same area that diffraction patterns were taken in figure. Transformation from boehmite to gamma alumina transformation does not cause drastic change in the crystal shape but a 10-15% reduction in size.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3278 TEM Characterization of Core/Shell (FexOy/Au) nanoparticles

Mendoza Cruz R.1, Herrera Becerra R.2
1PCeIM-Insititute of Physics, UNAM, 2Institute of Physics, UNAM.
rumec21@gmail.com

In the last years it has been much interest on the synthesis of bimetallic or core/shell nanoparticles since these systems present novel properties different from the monometallic particles of the same elements. Even more, the properties can be tuned by varying the way of mixing the elements. In systems where a magnetic core as iron oxide is covered by a noble metal shell (Ag, Au or Pt), the magnetic properties and the efficient adsorption of molecules and optical properties of the shell can be exploited for numerous applications in fields as biomedicine, biotechnology or catalysis. Also, a metallic shell can modify the surface of the magnetic cores and provide them of extra stability against aggregation or oxidation. However, to achieve novel characteristics, control of the size and thickness of the shell must be reached, since all of these could generate loss of their intrinsic properties.

Many reports on the synthesis of iron oxide core and gold shell can be found and several methods have been reported to control the nanoparticle size and the phase. Nevertheless, few works have been developed on the structure and shape of the magnetic seeds, and the final structure and morphology of this core/shell system. The study of the chemical composition, internal structure, size and morphology is of great interest given that the properties of these systems depend on these parameters. For instance, the interphase effect between the two materials can yield to an enhancement of the catalytic activity or in the magnetic moment of the total particle [1,2].

In this work, a tannin-assisted method for the synthesis of core/shell nanoparticles (iron oxide/gold) is carried out. This method has been effective for the production of iron oxide particles with a narrow size distribution [3]. The final structure and morphology are determined by high resolution transmission electron microscopy. This work attempt to contribute to the understanding of all these factors have on the novel properties of oxide core/metal shell nanoparticles.

References:

[1] L. Guczi, et al. Topics Catal. (2006) Vol. 39, Nos. 3-4, 137-142.
[2] S. Banerjee, et al. J. Appl. Phys. (2011) 109, 123902.
[3] Herrera-Becerra, R et al. Appl Phys A (2010) 100, 453-459.

The authors acknowledge the financial support from DGAPA with grant PAPPIT IN105112 and from the Graduate Program PCeIM, UNAM.
Our gratitude to Roberto Hernández and Cristina Zorrilla for the technical support given.
Authors thank access to the facilities of the Kleberg Advanced Microscopy Center and the Research Centers in Minority Institutions (RCMI) at The University of Texas at San Antonio, USA.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3293 The InAs Islands Morphology and Structure Dependence With the FIB Ion Dose Used to Induce Its Localized Growth.

Ribeiro Andrade R.1,6, Miquita D. R.1, Vasconcelos T. L.2, Kawabata R.4,6, Malachias A.5, Pires M. P.3,6, Souza P. L.4,6, Rodrigues W. N.5,6
1Centro de Microscopia, UFMG, Belo Horizonte, MG, Brazil, 2Divisão de Metrologia de Materiais, INMETRO, Duque de Caxias, Brazil, 3Instituto de Física, UFRJ,Rio de Janeiro, Brazil, 4LabSem/CETUC, PUC-Rio, Rio de Janeiro, Brazil, 5Departamento de Física, ICEx, UFMG, Belo Horizonte, MG, Brazil, 6DISSE – Instituto Nacional de Ciência e Tecnologia de Nanodispositivos Semicondutores, CNPq/MCT, Brazil
rodriban@gmail.com

Semiconductor quantum dots have optoelectronic properties that attract and justify the efforts of many research teams around the world. However the production of high efficiency optoelectronic devices depends on a fine control of the morphology, density, size, shape, uniformity and chemical composition of these nanostructures. Several techniques to induce a localized growth of QDs can be combined with modern growth techniques in an attempt to control the precursor’s nucleation on the substrate surface. One of techniques employed in this process is the Focused Ion Beam Microscopy (FIB) [1,2]. In this technique a gallium ion beam is used to create localized surface defects that become nucleation sites for the localized growth of quantum dots. In this work we used this technique to create an array of holes on InP(001) surface to serve as diffusion barrier increasing the nucleation rate located during InAs grown by Metal Organic Vapor Epitaxy. The doses used were 3.7x1015, 5.6x1015 and 1.3x1016 Ga+/cm2. Islands were grown for two sub-monolayer coverages, occurring mostly in clusters in the inner surfaces of the FIB produced cavities. For low doses templates the nanostructures are mainly coherent. For high doses the islands are mostly incoherent and numerous. A simple model correlating the surface potential of the template with the net adatom flow to the cavities is presented. Two regimes were identified, coarsening and coalescence when low doses were applied, and incoherent growth when high doses were used.

This work was supported by Programa Institutos Nacionais de Ciência e Tecnologia, CNPq/MCT, CAPES, FAPEMIG and FAPERJ. We would like to thank the UFMG Microscopy Center as well the Brazilian National Light Synchrotron Laboratory for the technical and financial support.

Fig. 1: Figure (a) shows a schematic diagram of the adatoms diffusion process into the cavities and islands growth. (b) Analysis of islands density on an InP modified substrate as a function of ion dose, indicating the presence of two distinct growth regimes.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3304 Long-range chemical orders in gold-palladium nanoalloys studied by aberration-corrected TEM

Nelayah J.1, Nguyen N. T.1, Alloyeau D.1, Wang G.1, Ricolleau C.1
1 Laboratoire Matériaux et Phénomènes Quantiques, Université Paris Diderot/CNRS, UMR 7162, Bâtiment Condorcet, 4 rue Elsa Morante, 75205 Paris Cedex 13, France
jaysen.nelayah@univ-paris-diderot.fr

Gold-palladium nanoparticles (Au-Pd NPs) are of great industrial and scientific interests as they are promising catalysts for many oxidation and hydrogenation reactions. Catalytic activity of Au-Pd NPs can be fine-tuned by controlling the ratio and arrangement of the surface atoms at their surface. As in the bulk phase, Au and Pd atoms can be mixed in any proportion in nanoalloys to form chemically-disordered face-centered cubic (fcc) structures. Aside from the fcc structures, electron diffraction studies of epitaxially-grown Au-Pd thin films have also hinted at the existence of long-range chemical orders of L10 type (around composition AuPd) and of L12 types (around compositions Au3Pd and AuPd3). However, no long-range chemical-order has ever been observed in Au-Pd NPs. In this contribution, we report the first observation of long-range chemical orders in Au-Pd NPs using an aberration-corrected transmission electron microscope (TEM).

Monodispersed Au-Pd NPs under 10 nm were grown by pulsed laser deposition in vacuum on either freshly cleaved NaCl(001) single crystals or amorphous carbon films of standard TEM grids. Material transfer to the support and precise control of particle composition was achieved by alternately ablating two ultra pure Au and Pd targets. The NPs were annealed in vacuum at temperatures superior to 400°C. Epitaxially-grown NPs were transferred to standard TEM grids prior to annealing. The structure of the as-grown and annealed NPs was studied by high-resolution imaging on a JEM-ARM 200F TEM.

Fig. 1 shows a corrected TEM image (UHRTEM) of an epitaxially-grown Au-Pd NP in closed [001] orientation. Due to the rapid kinetics of the deposition process, the structure of all as-grown NPs was in a non-equilibrium state disordered fcc type. Transition to the equilibrium structure occurred during high temperature annealing. Extensive UHRTEM studies of Au-Pd NPs of various compositions annealed in different conditions (support, temperature, time) showed that both L10 and L12 chemical order are stable in the Au-rich NPs at temperatures as high as 600°C. UHRTEM images of a L10- and a L12- chemically-ordered NPs in closed [001] orientation are shown in Fig. 2 and 3 respectively. Besides chemical ordering, particle ripening also occurred during thermal annealing. Single particles X-ray analysis in TEM scanning mode demonstrates clearly that crystal growth proceeds through Ostwald ripening phenomenon. Due to the difference in evaporation rate of Au and Pd atoms from the NPs during this ripening process, the chemical composition of the NPs became size-dependent after annealing processes with a systematic enrichment in Pd as particle size increases.

The authors acknowledge the support of the French Agence Nationale de la Recherche (ANR) under reference ANR-11-BS10-009. We are also grateful to Région Ile-de-France for convention SESAME E1845 for the support of the JEOL ARM 200F electron microscope installed at Paris Diderot University.

Fig. 1: UHRTEM image of an as-grown Au-Pd NP composed of 62 % at Au viewed close to the [001] direction. The fast fourier transform of the image is displayed in insert. It shows that the structure of the NP is of disordered fcc type.

Fig. 2: UHRTEM image of a L1o chemically-ordered NP obtained after annealing the sample in Fig 1 at 600°C in vacuum during 10h. Chemical ordered is evidenced through the appearance of superlattice structures in the fast-fourier transform of the image.

Fig. 3: Image of a L12 chemically-ordered NP obtained after annealing at 500°C in vacuum during 8h a sample with composition comparable that of Fig 1.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3307 Ex-situ and In-situ Analysis of MoVTeNb Oxide by An Aberration-Corrected Scanning Transmission Electron Microscope

Xu P.1, Sanchez-Sanchez M.2, Browning N. D.3, Lercher J. A.2,3
1Department of Chemical Engineering and Materials Science, University of California, Davis, Davis, USA, 2Department of Chemistry and Catalysis Research Center, Technische Universitat Munchen, 85748 Munich, Germany, 3Physical Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, WA 99352, USA
amy.pinghongxu@gmail.com

Short chain olefins, especially ethylene and propylene are in very high demand worldwide making them important industrial raw materials. Two-phase MoVTeNb oxide, has shown great promise in oxidative dehydrogenation (ODH) of converting inexpensive light alkanes into these olefins. In this work, we report an identification of the nature of crystalline termination of the catalytically relevant M1 phase at atomic scale using an aberration-corrected scanning transmission electron microscope (STEM). Figure 1 shows a typical M1 phase particle viewed in the crystal growth direction (the [001] orientation). It is clearly observed that at standard conditions no amorphous overlayer is formed on the external surface of the particles. Based on the analysis of over 50 particles, it is shown that the lateral surfaces of these rods are faceted and the most preferential lateral facets have been determined to be {010}, {120} and {210} (as shown in Figure 1 B-D for STEM images and Figure 1 E-G for crystallographic models). These results indicate that morphology of the M1 phase particles might have a large impact on catalytic activity of this system. Additionally, electron energy loss spectroscopy and energy dispersive X-ray spectroscopy has been applied to determine the vanadium distribution on the surface, which is believed to play an important role in catalytic performance of the M1 phase in ODH.

Reaction conditions affect the surfaces of the M1 phase, which makes it essential to perform the in situ experimental observations for a fundamental understanding of the catalytic performance of this material. Direct imaging of structural changes in the M1 phase was performed under an oxidative atmosphere in an environmental TEM. Tellurium units were observed to disappear from hexagonal channels when heated to 350 °C in 10 mbar oxygen/argon (23%/77%), while the crystal framework remained unaffected. Further in-situ experiments using a much higher partial pressure (above 1 bar) are underway to investigate the relationship of structural changes to catalytic performance of this system under realistic reaction conditions.

This work was supported in part by the United States Department of Energy (DOE) Grant No. DE-3-BDOE797 through the University of California, Davis, the Laboratory Directed Research and Development Program (LDRD): Chemical Imaging Initiative at Pacific Northwest National Laboratory (PNNL), and the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility at PNNL.

Fig. 1: (A) Unprocessed STEM image showing crystalline and faceted surface of M1 phase. (B-D) Magnified view of rectangular areas 1-3 in (A), showing different configurations of facets {010}, {120} and {210}, respectively. (E-G) Rendering of the crystallographic model of the three facets shown in (B-D).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3369 Transformation of CdSe/Cu3P/CdSe Nanocrystal Heterostructures upon Thermal Annealing

Falqui A.1,2, Genovese A.1, Casu A.1, De Trizio L.1, Manna L.1
1Nanochemistry, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy, 2Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST) – Thuwal 23955-6900, Kingdom of Saudi Arabia
andrea.falqui@gmail.com

Thermal annealing in conjunction with electron irradiation can also be exploited to create new nanostructures. One key point of nanocrystal (NC) heterostructures tranformation is that the relative thermal or irradiation stability/reactivity of a certain material domain in the heterostructure can be much different from that of the same domain if it were the only constituent of the NC. This is due to its proximity to domains of other materials that can elicit concomitant processes such as diffusion of chemicals, alloying, charge transfer and others. We have studied the in-situ phase transformation of CdSe/Cu3P/CdSe NCs. Starting from hexagonal Cu3P nano-platelets, and working at higher temperatures, we have obtained sandwich-shaped nanoparticles consisting of one top and one bottom layer of CdSe encasing each original Cu3P platelets according an epitaxial-relationship (Figure 1 a-d). When these sandwich-like heterostructures were annealed under high vacuum (pressure »10-4 Pa) up to 450°C, sublimation of P and Cd species with concomitant interdiffusion of Cu and Se species were observed by in-situ high resolution TEM (HRTEM) and energy filtered TEM (EFTEM) analyses. These processes transformed the pristine sandwiches, triggering the complete evolution of each original heterostructure into single NCs that were thoroughly characterized by fcc lattice, with d-spacings compatible with those of fcc Cu2Se (Figure 1 e-f). Under the same conditions the single domains, i.e the pristine (uncoated) Cu3P platelets and isolated CdSe NCs were stable, no transformation occurred. Therefore, the thermal instability of these heterostructures under high vacuum might be explained by the preferential diffusion of Cu species from Cu3P cores into CdSe domains through epitaxial related interfaces. The Cu interdiffusion triggered sublimation of Cd, as well as out-diffusion of P species and the partial dissolution of NCs together with the overall transformation of the sandwiches into Cu2Se single NCs (Figure 2). Therefore, one further development in this direction could be that of creating plasmonic micro- and nanostructures “on demand”, for example, by annealing with a laser individual or groups of CdSe/Cu3P/CdSe NCs deposited on a substrate [1].

[1]. De Trizio et al. ACS Nano, 5, (2013), 3997.

The authors acknowledge financial support from European Union through the FP7 starting ERC grant NANO-ARCH (contract no. 240111). We thank Marijn van Huis and Anil Yalcin for many useful discussions.

Fig. 1: a) CdSe/Cu3P/CdSe view. b) HAADF STEM image and EDX line profile c) HRTEM image of epitaxial interfaces. d) FFT patterns of CdSe (blue) and Cu3P (red) showing lattice similarities; habit schema. e) HRTEM (RT @ 300°C) of single NC showing CdSe/Cu3P/CdSe structure. f) HRTEM (400°C) of the same NC completely converted into fcc Cu2Se.

Fig. 2: Elastic filtered zero loss images (a-c) and the corresponding EFTEM elemental maps (d-f) of several NCs observed at room temperature (RT), 400° and 450°C displaying Cu diffusion. Elemental maps of P (132 eV, green,) and Cu (931 eV, red). Zero loss image at 450°C shows sublimation morphologies. Scale bars 50 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3378 Formation of NiSi2 nanoparticles epitaxially embedded in silicon nanowires

Panciera F.1,2, Chou Y. C.2,3,4, Reuter M. C.2, Hofmann S.1, Ross F. M.2
1Department of Engineering, University of Cambridge, Cambridge, UK, 2IBM Research Division, T. J. Watson Research Center, Yorktown Heights, NY, USA, 3Department of Electrophysics, National Chiao Tung University, Hsinchu city, Taiwan , 4Center for Functional Nanomaterials, Brookhaven National Laboratory, Upton, NY, USA
fp301@cam.ac.uk

Silicon nanowires (SiNWs) have a variety of applications including field-effect transistors (FETs), energy storage, and chemical and biological sensors. These applications generally require electrical contact to be made with the nanowires. For this reason, metal silicides formed by solid state reactions with Si nanowires have attracted great attention in the last few years. However, silicides also have prospects in applications such as spintronics, sensors and energy storage devices at the nano-scale.

We developed a novel method to synthetize silicon/silicide heterostructures by introducing silicide nanoparticles inside silicon nanowires. We found that the Au droplets, which originally catalysed the formation of the SiNWs themselves, can also act as a catalyst for the Ni-Si reaction. This catalytic reaction leads to immediate formation of NiSi2, even at low temperature. To demonstrate this process, we first synthesise SiNWs with a radius between 20 and 50 nm and the usual growth direction, (111). Synthesis took place in an ultrahigh vacuum transmission electron microscope using the vapour-liquid-solid method with Au as the catalyst and disilane as the precursor gas. After the wires were formed, a Ni layer was then deposited by electron beam evaporation at room temperature, without breaking the vacuum. During annealing, as shown in Figure 1, an octahedral silicide particle formed within the liquid AuSi droplet. Measurements of crystal structure allow this to be identified as the NiSi2 phase. This silicide nanoparticle first formed and moved around in the liquid droplet and only later on became attached to the AuSi/Si(111) interface, forming an epitaxial contact (Figure 1a). Once the NW growth was restarted by flowing the disilane precursor gas, the silicide particle became incorporated into the NW forming a Si-silicide heterostructure in which a nanoscale silicide region is embedded epitaxially within the nanowire (Figure 1b, d). By repeating the process, we can embed multiple silicide particles within a single nanowire. We will present video-rate imaging allowing measurements of the nucleation, growth and incorporation of the silicide (Figure 1c) and discuss the mechanism and kinetics. We will then show that the silicide growth mechanism we have demonstrated here for Ni deposited on SiNW applies to other systems as well and can be exploited to introduce nanosized particles in various kinds of nanowires. We will finally discuss the potential applications of these new heterostructures for future nanowire-based devices.

Fig. 1: (a) TEM images recorded at 400°C after deposition of 1nm of Ni onto a Si nanowire. (b) TEM images recorded at 500°C and 1×10-5 Torr Si2H6. The incorporation of the silicide inside the Si matrix is visible. (c) The plot of length vs. time for the nanowire in (b). (d)  A higher magnification image of a NiSi2 particle showing its octahedral shape.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3404 Lanthanum Nanoparticles: Synthesis and Structural Properties

Schabes-Retchkiman P. S.1, Romero-Ibarrra Josue E.1
1Instituto de Fisica, UNAM, Mexico, DF, Mexico
pabloschabes@yahoo.com.mx

In the past decade, a lot of attention has been given for the development of novel strategies for the synthesis of different kinds of nano-objects. Most of the current strategies usually use physical or chemical principles to develop nano-objects with multiple applications. One such system is the Lanthanum (La) system, with notorious catalytic properties.
Lanthanum has diverse properties derived from the structure of the atom: óptical, magnétic, catalytic, superconductors, etc. (in particular, new nanometric scale properties; Reactivity, cathodos in SOFC, etc.) These type of metals are usually synthesized as oxides (La2O3) In this work we have synthesized metallic Lanthanum (Lazero) and La2O3,by bio-reduction with plants.

In these work we show that through a bioreduction method [1] we have been able to produce small Lanthanum nanoparticles, (2-5 nm) and La nanorods, using two different reductor-plants i.e. alfalafa and Callistemon citrinus,  which is interesting because it constitutes a pest. In essence the reaction that seems to take place in both plants is the following acting the water-soluble tannins remaining from the plant treatment:
 

The controlling parameter is the pH of the solution in which the reactions take place [1].

Characterization: Electron microscopy characterization included transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM) characterization, EELS (electron energy-loss spectroscopy) HAADF (High Angle-Annular Dark Field). The samples were prepared by placing a drop of the solution on carbon-coated copper grids and allowed to dry. Electron microscopy was performed in a JEOL JEM-2010F FasTem Microscope at IFUNAM, equipped with EDS and EELS analysis. High resolution images were obtained under many different conditions and the images analyzed by obtaining digital diffractograms by FFT (Fast Fourier Transform)

The synthesized La0 nanoparticles with average diameter of 3 nm can be observed in figure 1. The formation of the nanophases can be appreciated in the HAADF images, particles corresponded to metallic Lanthanum. Some of the particles were oxides as well, some clustered. Rods were also observed. The main difference between the two synthesis was that with the callistemon the particle density was smaller than for alfalfa, which might be due to the breaking of cell walls having very different energies.


Conclusion: The pH of the solution constitutes a parameter of easy control, that defines the size and morphology of the LaN (particles or rods or wires) .

For pH 13, nanorods of metallic character were obtained.

As a final thought, we have to remark that these synthesis methods are simple and self-sustainable.

We are indebted to the personnel at LACMIF, IFUNAM, particularly Mr Roberto Hernandez.

Fig. 1: HAADF of Lanthanum nanoparticles pH10

Fig. 2: Typical morphologies observed for pH10.

Fig. 3: Metallic La particles

Fig. 4: Metallic La, Larger particle.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3412 Electron Beam Nanosculpting and Controlled Morphological Transformation of Kirkendall Oxide Nanochannels

Molina-Luna L.1, Abdel-Aziz A. A.2, 4, Buffière M.3, Tessier P. Y.4, Du K.5, Choi C. H.5, Kleebe H. J.1, Konstantinidis S.2, Bittencourt C.2, Snyders R.2, 6
1Department of Material- and Geosciences, Technische Universität Darmstadt, Germany, 2Chimie des Interactions Plasma-Surface (ChIPS), CIRMAP, Research Institute for Materials Science and Engineering, University of Mons, Belgium, 3imec, Heverlee,Belgium, 4Institut des Matériaux Jean Rouxel, Université de Nantes, France, 5Department of Mechanical Engineering, Stevens Institute of Technology, Hoboken, New Jersey, United States, 6Materia Nova Research Center, Mons, Belgium
molina@geo.tu-darmstadt.de

The nanomanipulation of metal nanoparticles inside oxide nano-tubes, synthesized by means of the Kirkendall effect, is demonstrated. In this strategy, a focused electron beam, extracted from a transmission electron microscope source, is used to site-selectively heat the oxide material in order to generate and steer a metal ion diffusion flux inside the nanochannels. The metal ion flux generated inside the tube is a consequence of the reduction of the oxide phase occurring upon exposure to the e-beam. We further show that the directional migration of the metal ions inside the nanotubes can be achieved by locally tuning the chemistry and the morphology of the channel at the nanoscale. This allows sculpting organized metal nanoparticles inside the nanotubes with various sizes, shapes, and periodicities. This strategy is based on the control of the thermally activated local diffusion of Cu ions inside the nanotube using an e-beam extracted from a TEM source. The migration of Cu ions was found to be governed by a surface diffusion mechanism occurring on the innerwalls of the nanotube. This nanomanipulation technique is very promising since it enables creating unique nanostructures that, at present, cannot be produced by an alternative classical synthesis route. Aditionally, temperature dependent in-situ TEM experiments were carried out yielding a controlled morphological transformation of the Kirkendall oxide nanochannels.


References

El Mel A-A, Molina-Luna L, Buffière M, Tessier P Y, Du K, Choi C-H, Kleebe H-J, Konstantinidis S, Bittencourt C, Snyders R. Electron Beam Nanosculpting of Kirkendall Oxide Nanochannels. ACS Nano, 2014, 8 (2), pp 1854–1861.

This work was funded in part by the Directorate of Research in Wallonia, under the scope of the ERA-NET MATERA Programme and by the COST Action MP0901. The French Community of Belgium is acknowledged through the “Cold Plasma” project. The TEM´s employed for this work were partially funded by the German Research Foundation (DFG).

Fig. 1: Morphological evolution of an oxide nanotube upon exposure to an electron beam. (a) TEM image of the as-grown oxide nanotube. (b-e) formation of Cu nanoparticles inside the oxide nanotube upon exposure of several regions to the e-beam for different subsequent shots. The time of each shot was 2 s. Scale bar:100 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3414 Novel M1/M2 heterostructure in Mo-V-M-Ta (M = Te or Sb) complex oxide catalyst revealed by aberration corrected HAADF STEM

He Q.1, Woo J.2, Guliants V. V.2, Borisevich A.1
1Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA, 2School of Energy, Environment, Biological and Medical Engineering, University of Cincinnati, Cincinnati, Ohio, USA
heq1@ornl.gov

Direct interpretability and atomic number (Z) sensitivity of HAADF combined with sub-Å resolution makes it the techniques of choice for uncovering atomic-scale underpinnings of materials behavior[1]. For Mo-V-M-Ta (M = Te or Sb) catalysts, HAADF has revealed local cation distribution[2], structure and chemistry of the crystal defects[3], polar domain structure[4], surface[5] and other features that helped to understand their high performance in propane (amm)oxidation[6]. Previous works showed that the M1 phase (Fig 1a) is the active phase, while another phase in the system, namely M2 (Fig 1b), has a distinctive synergistic effect with the M1 by improving the selectivity. Aiming to understand and further exploit this synergistic effect, we studied a series of M2 phase catalysts by HAADF imaging in an aberration corrected STEM.

Our results show that the M2 phase in Mo-V-Te-Ta system, unlike other reported M2 phases[4], has unusual microstructure: the side planes of the M2 phase crystals are decorated with pentagons (e.g. Mo6O21), the building blocks for M1 phase (Figure 2). These pentagons appear to contain heavy cations (e.g. Ta) in higher concentration than the bulk of the material, showing brighter HAADF contrast in the center. Two types of the interface between the pentagon units and the hexagon units in the M2 phase matrix are identified. Interestingly, monolayer pentagon coverage is dominantly found associated with the type I interface (Figs. 2b,c), where multilayer coverage is always found with the type II interface (Figs. 2d,e). In a M2 phase in a related Mo-V-Sb-Ta system, surface pentagon layers were also observed. In some areas, surface pentagon layers serve as seeds for the M1 phase attached to M2 surface. To our best knowledge, this is the first example of the intergrowth of these two very distinct structures.

Our observations suggest that the pentagon layers come from a self-assembly process of the preformed pentagon units during the synthesis.[7] The presence of Ta, a sub-group V element, is found crucial for this intergrowth. Comparing samples with and without Ta in composition clearly suggests that Ta stabilized the pentagon units during synthesis. Correlation of the microstructure with the catalytic performance will also be discussed. We believe that this work will pave the way for development of novel catalysts with M1/M2 heterostructures that can improve the catalytic properties by maximizing the synergistic effect.

[1] Pennycook, SJ et al., Philos. Trans. Roy. Soc. A(2009)
[2] Yu, JJ et al., Catal. Commun.(2012)
[3] Pyrz, WD et al., Chem. Mater.(2010)
[4] Zhu, YH et al., Chem. Mater.(2012)
[5] Zhu, Y et al., Angew. Chem. Int. Ed.(2012)
[6] Shiju, NR et al., Appl. Catal., A(2009)
[7] Sadakane, M et al., Angew. Chem. Int. Ed.(2009)

The MSE Division, US DOE; through a user project in ORNL’s CNMS, sponsored by the SUF Division, Office of BES, US DOE; The CSGB Division, Office of BES, US DOE.

Fig. 1: Polyhedral models of the ab planes of (a) the M1 and (b) the M2 phases in molybdenum vanadate oxide catalysts (e.g. MoVTeTa Oxide). Pentagon units, heptagonal channels, hexagonal channels and unit cells are highlighted in blue, yellow, red and black respectively. Sparsely occupied Te sites in heptagonal channels are omitted for clarity.

Fig. 2: (a) A HAADF image of the pentagon decorated M2 phase particle. (b,c)&(d,e) magnified views and proposed models of Types I (purple) and II (green) interfaces between the pentagon units (blue) and the hexagon units (red), respectively. Their representative units are highlighted in black. Heptagonal channels are highlighted in yellow.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3415 Synthesis and Electron Microscopy Characterization of Non-stoichiometric Tin Oxide Structures

Orlandi M. O.1, Suman P. H.1, Barbosa M. S.1, Longo E.1, Varela J. A.1
1Institute of Chemistry, Sao Paulo State University, Brazil
orlandi@iq.unesp.br

Nanomaterials have attracted the attention of researchers in the recent past due to their interesting properties. Moreover, there is a wide applicability of these materials in several areas of knowledge, for example, chemicals sensors, solar cells and microelectronic devices. However, in order to use these materials in devices it is very important to have a deep morphological and structural characterization of materials and electron microscopy techniques are important tools for these characterizations.

In this work, we used a carbothermal evaporation method to obtain tin oxide structures. By controlling the oxygen amount inside the furnace it was possible to grow structures in different oxidation states; i.e. SnO2, Sn3O4 and SnO. The samples were characterized by scanning and transmission electron microscopy, X-ray diffraction and electrical measurements. SEM characterization showed that SnO material is composed by nanobelts with spheres at their extremities and micro discs (which can be separated by sedimentation) while Sn3O4 and SnO2 materials are composed of nanobelts. TEM and SAD characterization showed that all synthesized materials are single-crystalline and SAD patterns in different zone axis enabled the determination of the growth direction of each material. Based on these results, it was possible to propose that SnO nanobelts grow following a self-catalytic vapor-liquid-solid (VLS) mechanism and Sn3O4 and SnO2 nanobelts grow by a vapor-solid (VS) process. The growth mechanism of discs are not completely known, but is related to the solidification of SnO vapor. While SnO2 is a well-known material in literature, there are only few works on SnO and Sn3O4 structures. Based on it we also studied the sensor response of materials to reducing and oxidizing gases and it was possible to correlate the sensor mechanism of materials with the exposed planes of structures.

We would like to thank the funding agencies FAPESP and CNPq for supporting this work.

Fig. 1: SEM image of SnO nanobelts. In the inset there is a TEM image of a single belt.

Fig. 2: SEM image of SnO microdiscs. In the inset there a SEM image of a single disc.

Fig. 3: a) SEM general view of Sn3O4 nanobelts. b-d) Details of Sn3O4 nanobelts.

Fig. 4: a) SEM general view of SnO2 nanobelts. b-d) Details of SnO2 nanobelts.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3419 Characteristaion of local surface plasmon resonance modes of metallic nanostructures with cathodoluminescence and electron energy loss spectroscopy in the (S)TEM

Stowe D. J.1, Wilkinson N.1
1Gatan Inc, R&D Headquarters, Pleasanton, CA, USA
dstowe@gatan.com

There is significant interest in understanding and controlling surface plasmon resonance modes of metallic nanostructures for use in chemical sensing and nano-optics applications. The optical effects of local surface plasmon resonance (LSPR) modes are only observable at a length scale below the diffraction limit of far field optical experiments meaning that alternative techniques are needed to study individual nanostructures. A focussed electron beam of a (scanning) transmission electron microscope (S)TEM can be used to generate local surface plasmon resonance (LSPR) modes; cathodoluminescence (CL) and electron energy loss spectroscopy techniques can be used to observe the generation and propagation of LSPRs with high spatial (<1nm) and spectral resolution (<2meV) and allow direct correlation with particle morphology. Here we report the use of CL and EELS in the characterisation of gold and silver nanoparticles using the Gatan Vulcan CL detector and Gatan GIF Quantum EEL spectrometer.
Figure 1 shows luminescence patterns of gold prisms acquired by (S)TEM-CL. Gold prisms of different size reveal very different LSPR patterns; a gold prism with sides of 250nm shows strong emission at 1.80eV (660nm) at the three corners (Figure 3c) whereas an 800nm particle shows luminescence dominated by a strong central LSPR node (1.53eV, 803nm) with other lower intensity LSPR modes along the edges (1.43 and 1.70eV, 870 and 705nm).
Light emitted from the specimen in the forward and backwards directions (in the direction of, and in the opposite direction to the fast electron) was measured (simultaneously) enabling differences in the intensity and spectral characteristics to be determined. A simple model system of a 120nm diameter sliver nanosphere was investigated. Preferential emission of the quadrapole LSPR mode was observed in the forward scattered direction whereas the dipole LSPR mode was preferentially emitted in the backward scattered direction, in agreement with Mie theory.

The authors would like to thank Dr M Bosman and Dr Ziyou Li for the provision of specimens and Dr G. Fern and Dr. D McComb for access to microscope facilities.

Fig. 1: Figure 1. (a) and (b) bright field STEM image of a two gold prisms (250 and 800nm sides respectively); (c) and (d) panchromatic STEM-CL images reveal ‘bright’ local surface plasmon resonance modes (e) and (f) CL spectra acquired from selected positions. 
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3425 ELECTRON MICROSCOPY STUDIES OF 1D TiO2 NANOSTRUCTURES WITH LOW IMPROVED PHOTOCATALYTIC ACTIVITY

Acosta D.1, Cabrera J.2, Rodríguez J.2, Candal R.3, Alarcón H.2, López A.2, Arenas J.1
1Instituto de Física, UNAM, México D.F., 2Universidad Nacional de Ingeniería, Lima, Perú, 3Universidad de Buenos Aires, Argentina
jarenas@fisica.unam.mx

Nanowire/nanorods TiO2 nanostructures (NS) of approximately 8 nm in diameter and around 1000 nm long were synthesized by alkaline hydrothermal treatment of sol-gel made TiO2 or P-25 TiO2. Anatase like 1D TiO2 NS were obtained in both cases. The 1D NS made using seeds from Sol Gel TiO2 nanopowders turn on rod-like NS and presents lower surface area than the NS made from commercial TiO2 P-25 (97 y 279 m2/g, respectively).In both cases, the 1D NS showed lower photocatalytic activity than P25 nanoparticles. However, the rod-like NS obtained from TiO2 Sol Gel seeds displayed slightly higher efficiency than the original seeds. Despite the higher surface area shown by the NS, the photocatalytic efficiency did not improve with respect to their precursor seeds. This phenomenon can be associated with the lower crystallinity of 1D TiO2 in both materials.
From SEM and TEM micrographs of sol-gel TiO2 synthesized nanoparticles, large and compact aggregates (Figure 1a) with radius around 7 nm are detected. After 18 h of exposing the TiO2 sol-gel made seeds, to alkaline hydrothermal treatment, the NPs, turned to tube-like NS (Figure 1b) with inner and external diameter in average of approximately 5.6 and 8 nm respectively. After the hydrothermal treatment the samples were acid treated to exchange Na+ by H+; it was observed the tubular structure was conserved in spite of the acid treatment. Figure 1c displays SEM and TEM micrographs of TiO2 NS obtained by alkaline hydrothermal treatment of P-25 seeds during 18 or 24 h, followed by acid exchange and annealing at 400ºC. The 1D structures are preserved. As in the previous case, tubes were obtained by both hydrothermal treatment time (18 and 24 h). But in this case the tubular structure remains after annealing process. The images clearly shows that as consequence of the annealing the tube like NS turned into short rod-like particles. These results suggest that during the annealing the structure of the tubes collapsed, cutting the tubes in smaller pieces but preserving in part their original morphology.
The growth mechanism of 1D TiO2 NS synthesized by alkaline hydrothermal method from TiO2 nanoparticles is still under discussion. It has been suggested that it take place by the rolling of hydrogen titanate laminar structures during the ion exchange step (Kasuga, 1998).Other authors, suggest the 1D structure formation during the treatment of TiO2 in NaOH aqueous solution (Zhao, 2010). Our results seem to be in agreement with the last authors because the tubes were well formed, as it is observed in Figure 1b, before the application of acid treatment .

The financial support of CONCYTEC and Alianza del Pacifico program to J.Cabrera is here recognized. Also we thanks to Roberto Hernandez for technical help and the financial support of DGAPA-UNAM IN 105541 project to Dr. D. Acosta laboratory work.

Fig. 1: Figure 1. a) Electron micrographs of TiO2 1-D NS obtained from sol-gel and hydrothermally treated for 18 h and after acid treatment. b) 1-D NS from sol-gel nanoparticles, hydrothermally treated and after annealing at 400°C. c) TiO2 NS by alkaline hydrothermal treatment of P-25 seeds during 18 hrs, after acid treatment and annealed at 400°C.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3432 Lutetium Oxide Nanoparticles: Synthesis by a green method.

Schabes -Retchkiman P. S.1
1Instituto de Fisica, UNAM, Mexico City, Mexico
pabloschabes@yahoo.com.mx

The nanostructured systems present promising properties , optical, magnetic and catalyic properties. [1]. In particular a search for new designs at the nanometric scale has increased during the later years and its uses diversified.the interest in metallic oxides is due to applications in catalysis optical and electronic devices  [2-5]. Lutetium oxide has uses in laser crystals optical devices and ceramics, because of the opossibility of obtaining with high purity it is considered as target for Xray emission because of its high density.

In general this compound is used as a dopant in the formation of granate for laser crystals. Since the oxides . are electrically non-conductive but by developing certain structures the electrical conductivity can be achieved and used in the cathode of Fuell-Cells and Oxygen generation systems. Being an anhydrous compound of basic character, it allows redox reactions, which helps in wáter electrolysis. Furthermore, since it is an insoluble compound, and very stable it can be used in the fabrication of cathodes of light ceramic structure in fuel-cells saving on weight. For the use of this oxide three important parameters are looked after: size, structure and elementary composition.

Lutetium Oxide nanoparticles were obtained in a high yield by means of biosynthesis with alfalfa. The morphology and crystal structure were characterized by high resolution transmission electron microscopy, EELS and Z-contrast microscopy. It is shown that the synthesis method employed produces small Lutetium Oxide nanoparticles, (2-5 nm), Lu2O3, as characterized from the optical diffractograms of individual particles.

The synthesis of  Lu2O3 nanoparticles was done by the bioreduction method similar to the one proposed by G. Canizal [6], for gold nanoparticles . The synthesis was designed in principle to obtain nanoparticles of metallic Lu, but due to the characteristics of Lu the metallic oxide was obtained. Figure 1 shows size distribution function for Lutetium NP. Figure 2 shows pH7 HAADF of Lutetium NPs, Figure 3 presents HRTEM of Lu oxide particles, showing some have coalesced for pH7. Conclusion; The bioreduction method has been ueful to produce Lu2O3 NP of quantum size character.
1. H. S. Nalwa, Handbook of Nanostrucutred Materials and Nanotechnology, Academic Press, San Diego, CA (2000).
2. M. L. Wu, D. H. Chen and T. C. Huang, J. Colloid Interf. Sci. 243, 102 (2001).
3. B. Veisz, L. Toth, D. Teschner, Z. Paal, N. Gyorffy, U. Wild and R. Schlogl, J. Mol. Catal. A-Chem. 238, 56 (2005).
4. M. O. Nutt, J. B. Hughes and M. S. Wong, Environ. Sci. Technol. 39, 1346 (2005).
5. R. Narayanan and M. A. El-Sayed, J. Phys. Chem. B 109, 12663 (2005).
6. G. Canizal, J. A. Ascencio, J. Gardea-Torresday, M. Jose´-Yacaman, J. Nanopart. Res. (2001) 475.

The authors thank Ms Indira Blanco, Mr. L. Rendón Vázquez and the LACMIF-UNAM personnel for technical help. This research has been partially supported by DGAPA-UNAM project # . IN120006

Fig. 1: Size distribution function for Lu Oxide NPs

Fig. 2: HAADF showing Lutetium Nanoparticles distinct contrast.

Fig. 3: Lutetium oxide nanoparticles, typical shapes and structures
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3446 Structure and Morphology Study of Pure and Mixed ZnO and ZnO2 Nanoparticles

Paraguay-Delgado F.2, Roman E.1, Gómez M. M.1, Solís J. L.1, Antunez-Flores W.2
1Universidad Nacional de Ingeniería, Faculty of Science, Av. Túpac Amaru 210, Lima 25, Perú, 2Centro de Investigación en Materiales Avanzados S. C., CIMAV, Miguel de Cervantes 120, CP 31109 Chihuahua, Chih. México.
francisco.paraguay@cimav.edu.mx

Synthesis of ZnO2 nanoparticles was performed via a sol–gel technique assisted with UV irradiation. One gram of zinc acetate dehydrate, Zn(CH3COO)2.2H2O, was dissolved under vigorous stirring in a mixture of 50 ml distilled water and 5 ml of 30% H2O2. The resulting solution was then irradiated with a 300W Ultra-Vitalux lamp (Osram), positioned 10 cm above the solution, for 30 min at ambient temperature. This procedure resulted in the formation of a white zinc peroxide colloidal suspension. The ZnO2 nanoparticles were precipitated by centrifugation. The precipitate was then washed using distilled water until a pH of 8 was reached. Finally the resultant white solid was dried at 80 °C for 12 h, similar to follow in the reference [1]. The resultant powder was annealed between 100 and 220°C for 1 h in an oven with air atmosphere. The morphology, structure and domain size of the nanoparticles were determined by X-ray diffraction, and scanning transmission electron microscopy. By X-ray diffraction, all patterns can be indexed to the zinc peroxide phase for samples prepared up to 120°C. For a sample prepared at 160°C we had a mixture of ZnO2 and ZnO, while for particles treated at 220°C all the material was pure ZnO.
Micrographs shows STEM images for zinc oxide and zinc peroxide nanoparticles. Fig 1 shows rounded ZnO particles, with an average grain size of 18±5 nm. The inset displays that the ZnO d-space was 2.8 Å. Fig 2 shows an image of ZnO and ZnO2 mixture, in the inset figure can be appreciated rund conglomerated particles. There are two types of particles, the bigger ones belong to ZnO and the smaller ones belong to ZnO2. The information of the atomic columns acquired by HAADF detector indicated that ZnO d-spaces were between 2.8 Å and 2.6 Å. This parameter must be connected to synthesis conditions of the material. In any case the average diameter size was 145 ± 55 nm.
Figures 3 and 4 belong to images of pure ZnO2 particles acquired by HAADF and BF detectors respectively. At low magnification can be observed spherical shapes with broad size dispersion between 40 and 287 nm. The average diameter was 130±64 nm. At higher magnification these conglomerates displays small grains (≈ 5 nm). Figure 1d confirmed that each small grain had d-space values which belong to ZnO2.
Using electron microscope techniques we have studied in detail the morphology and the structure of ZnO nanoparticles, ZnO2 nanoparticles and a mixture of both. The ZnO2 nanoparticle are of great interest, because they had interesting microbiological characteristics [2].

References
[1] R Colonia, J L Solís and M Gómez, Adv. Mat. Sci.: Nanotechnol 5 (2014) 015008 (4pp).
[2] R Colonia, V. Martinez, J. L. Solís and M. M.Gómez, Rev. Soc. Quim. Peru 79(2)2013 126 (10pp)

Thanks to P. Pisa and E. Torres for their technical help, at nanotech Cimav.

Fig. 1: STEM image by HAADF and BF images for ZnO particles, inset image show d space of ZnO.

Fig. 2: ZnO-ZnO2 particles, d-sapace belongs to ZnO.

Fig. 3: ZnO2 particles conglomerate in a spherical shape, inset can be notice the nanoparticles.

Fig. 4: BF image showing d-spaces of ZnO2.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3458 Blue luminescence related to stacking faults in ZnO:Ag

Tsiaoussis I.1, Potin V.2, Bourgeois S.2, Khranovskyy V.3, Eriksson M.3, Yakimova R.3
1Department of Physics, Solid State Physics Section, Aristotle University Thessaloniki,, 2I.C.B. - Laboratoire Interdisciplinaire Carnot de Bourgogne, UMR 6303 CNRS - Université de Bourgogne 21078 DIJON CEDEX, France , 3Department of Physics, Chemistry and Biology (IFM), Linköping University, 58183, Linköping, Sweden
tsiaous@auth.gr

Ag was used as catalyst for growth of vertical ZnO nanorods. Moreover, Ag also acts as an amphoteric dopant, existing both on substitutional Zn sites and in the interstitial sites, as substitutional acts as acceptor. Therefore, the effects of Ag on the optical properties of ZnO is of importance. We have grown the ZnO microrods by MOCVD with Ag catalyst on Si (100) substrates at Ts = 773 K. The elongated quasi single crystal structures were observed to have corrugated side facets.

The presence of SFs affects the luminescence properties of ZnO by creating additional peak at 3.321 eV (at 4 K). This peak co-exists with the common donor bound excitonic (D0X) and free excitonic recombination’s peaks as well as their respective phonon replicas (Fig.1). Furthermore, the SFs related peak is stable at least up to 350 K, providing splitting of the near band edge emission of ZnO into two peaks: FX emission appears at 375 nm and this of SFs at 386 nm (Fig.2). Therefore, visualizing of the two emissions was performed by CL mapping: the two types of emission are spatially resolved. These radiative recombination processes are under investigation by time-resolved PL. It is proposed, that at high Ag concentrations, the SFs formation is favored.

High concentrations of basal SFs were found to be responsible for the surface corrugation. A TEM study of the cross-section of ZnO/Ag/Si has revealed additional unusual contrast in bright field mode. The featured lines were located parallel to the substrate plane, e.g. perpendicular to the c-axis of the NR. Presuming that the reason for this is the extended defects, we have studied individual NRs by HRTEM. A number of basal plane [0001] stacking faults were observed, penetrating the NRs perpendicular to its c-axis (Fig. 3). BSFs were found to be quasiperiodically inserted every 5 - 10 nm along the NRs. It has to be mentioned that SFs are observed in both types of NRs. We attribute the appearance of BSFs as due to the Ag dopants.

Theoretical study in wurtzite GaN by Schmidt et al. [T. Schmidt et al. Phys. Rev. B 65 033205 (2002)] has shown that it is energetically favourable for Mg atom to reside at some distance from the fault plane. We believe, that in our case Ag atoms behave similarly, and their availability in the proximity of SFs transform them to a radiative recombination centre, existing up to room temperature.

We acknowledge the Linköping Linnaeus Initiative for Novel Functional Materials (LiLi-NFM) for supporting this work. Dr. Ioannis Tsiaoussis would like to thank Dr. Valerie Potin for enabling the TEM experiments at the University of Burgundy.

Fig. 1: LT PL spectrum (4 – 100 K) of ZnO:Ag microrods (the SEM image is as inset)

Fig. 2: The micro-PL spectra taken along the microrod: the transition of the PL intensity from UV to Blue emission is shown. The light emissions are visualized by CL mapping (inset).

Fig. 3: High concentration of basal SFs were found to be responsible for the surface corrugation as it is seen in the HRTEM image.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3482 Study of contact behaviour of TiO2 nanoparticle agglomerates by means of AFM and in-situ TEM

Salameh S.1, Mädler L.1, Seo J. W.2
1Foundation Institute of Materials Science (IWT), Department of Production Engineering, University of Bremen, Bremen, Germany, 2Department of Materials Engineering, KU Leuven, Leuven, Belgium
maria.seo@mtm.kuleuven.be

Adhesion forces between individual nanoparticles play an important role in many different processes such as fluidization, agglomeration and coating. Currently, adhesion between particles is interpreted in terms of continuum models that are able to take into account the effects of capillary forces, surface roughness and electrostatics. This approach is generally suitable for particle sizes in the micrometer range. However, for smaller particles with characteristic sizes in the range of 10 nm, more subtle effects beyond continuum theories can influence and even dominate the adhesion behavior. We have studied adhesion forces and contact behaviour of TiO2 nanoparticles with a diameter in the range of about 10 nm. These nanoparticles were produced in a flame spray reactor using the liquid precursor consisting of 0.5 molar Ti(IV) isopropoxide in xylene. Inside a TEM, we studied stretching and de-agglomeration behaviour of TiO2 nanoparticle agglomerates using an AFM/TEM holder. These in-situ observations were correlated with the force measurements obtained from AFM force spectroscopy. To be precise, the AFM data were based on the statistical analysis of the force peaks measured in repeated approaching/retracting loops of an AFM cantilever into a film of nanoparticle agglomerates. The in-situ TEM data revealed sliding and rolling events first leading to local rearrangements in the film structure when subjected to tensile load, prior to its final rupture caused by the reversible detaching of individual nanoparticles. The associated contact force of about 2.5 nN is in quantitative agreement with the results of Molecular Dynamics simulations of the particle-particle detachment [1]. Our results indicate that the contact forces are dominated by the structure of water layers adsorbed on the particles’ surfaces at ambient conditions. This leads to non-monotonous force-displacement curves that can be explained only in part by classic capillary effects, and highlight the importance of considering explicitly the molecular nature of the adsorbates [1].

We also studied the size dependent contact behavior of nanoparticle agglomerates by using four different size-fractionated agglomerates, with median values in the range of 78 to 161 nm. Force-distance curves of AFM as well as in-situ TEM observations show that the length of the chains and the amount of rearrangements depend on the agglomerate sizes. Larger agglomerates require more work for their aggregate rearrangement before the final breakage is induced [2].

[1] S. Salameh , J. Schneider, Jens Laube, A. Alessandrini, P. Facci, J. W. Seo, L. Colombi Ciacchi and L. Mädler, Langmuir 28 (2012), p. 11457. Langmuir 28 (2012), p. 11457.

[2] Salameh, R. Scholz, J.W. Seo and L. Mädler, Powder Technology 256 (2014) p. 345.

We acknowledge the Flemish Hercules Stichting (HER08/25), KU Leuven STRT1/08/025 and DFG for funding this project SPP 1486 under grants MA 3333/3 and CO 1043/3.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3503 Imaging and microanalysis of plasmonic Ga nanoparticles

Suvorova A. A.1, Losurdo M.2, Brown A. S.3, Rubanov S.4, Bruno G.2
1Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Perth, Australia, 2Institute of Inorganic Methodologies and Plasmas at National Council of Research (CNR), Bari, Italy, 3Electrical and Computer Engineering Department, Duke University, Durham, North Carolina 27705, United States, 4Electron Microscope Unit, Bio21 Institute, University of Melbourne, Melbourne, Victoria 3010, Australia
alexandra.suvorova@uwa.edu.au

Plasmonic nanoparticles (NPs) are of considerable interest due to plasmon tunability and potential applications as biosensors, photonic, optoelectronic and photovoltaic devices. These applications rely on the fabrication of metallic NPs on technologically important substrates and on the possibility to control the surface plasmon resonance (SPR) properties. The ability to create tunable (from the UV to the visible) plasmonic nanosystems using Ga NPs is differentiating gallium from the commonly used noble (gold and silver) metals. In our previous work, we have demonstrated the efficiency of Ga NP-based platforms in localized surface plasmon resonances (LSPR) tunable over the UV to the near IR spectral range1-3.

Here we describe a range of imaging and microanalysis electron microscopy techniques that are highly suitable for the study of the Ga-based plasmonic nanosystems. Ga nanoparticles were deposited onto sapphire, silicon, glass and graphene substrates in a Veeco GEN II molecular beam epitaxial system under ultrahigh vacuum conditions at room temperature with a constant Ga flux. TEM cross-sectional samples were prepared by the FEI Nova dual beam focused ion beam (FIB) system. A range of microanalytical electron microscopy techniques can be used to characterise the NPs at the nanoscale level. Scanning electron microscopy (SEM) imaging has been applied to study morphology and growth dependent modifications of the Ga NPs. Transmission electron microscopy (TEM) and associated analytical tools have been used to determine the structural and compositional properties of the nanostructures at a subnanometer scale. High resolution imaging revealed crystalline core/ amorphous shell structure for Ga NPs grown on sapphire and amorphous  Ga structure for Ga NPs grown on other substrates. Energy-filtered imaging  showed compositional uniformity of the Ga core and the presence of oxide layer on the NPs surface. Low-loss imaging confirms the presence of Ga, with particle contrast being maximised close to the Ga plasmon energy (13.8eV). The Ga plasmon signal is significantly higher for the crystalline core of the Ga NPs. In summary, a range of electron microscopy techniques can be used to identify and characterise Ga NPs at the nanoscale level. Such information is important for understanding structural and optical properties of Ga-based nanosystems.

1Wu, P. C.; Kim, T. H.; Brown, A. S.; Losurdo, M.; Bruno, G.; Everitt, H. O. Appl.Phys. Lett. 2007, 90, 103119.

2Yi, C.; Kim, T. H.; Jiao, W.; Yang, Y.; Lazarides, A.; Hingerl, K.;Bruno, G.; Brown, A. S.; Losurdo, M. N. Small 2012, 8, 2721–2730.

3M Losurdo, C Yi, A Suvorova, S Rubanov, T-Ho Kim, M M. Giangregorio, W Jiao, I Bergmair, G Bruno, A. S. Brown ACS Nano 2014 in press

Fig. 1: SEM image of Ga NPs grown on sapphire.

Fig. 2: TEM image showing the Ga NPs in cross-section.

Fig. 3: EFTEM imaging: zero-loss image of  Ga NPs.

Fig. 4: Low loss imaging of Ga NPs showing core/shell structure
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3507 STEM Electron Diffraction and High Resolution Used in the Determination of Thiolated Gold Clusters

Ponce A.1, Bahena Uribe D.1, Tlahuice Flores A.1, Santiago U.1, Whetten R.1, Jose Yacaman M.1
1University of Texas at San Antonio
arturo.ponce@utsa.edu

Gold nanoparticles protected by thiolate ligands have attracted extensive research activity because of their enhanced optical, electrochemical, and other application-related properties. The desired physicochemical properties are strongly dependent upon size, composition, and structure. It is therefore critical for improved materials design to correlate the cluster structure and bonding with the properties sought for applications. However, the determination of atomic structures of nanomaterials is a challenging task even for composition elucidated gold−thiolate nanoclusters where gold cores span various symmetries. Determination of the total structure of molecular nanocrystals is an outstanding experimental challenge that has been met, in only a few cases, by single-crystal X-ray diffraction. Described here is an alternative approach that is of most general applicability and does not require the fabrication of a single crystal. The method is based on rapid, time-resolved nanobeam electron diffraction (NBD) combined with high-angle annular dark field scanning/transmission electron microscopy (HAADF-STEM) images in a probe corrected STEM microscope, operated at reduced voltages. In the current presentation, we will show the new structures of Au130 and Au144 thiolated clusters explored in a combined experiment-theory approach [1-2]. A full map in reciprocal space has been simulated and compared with the experimental patterns using STEM diffraction as well as atomically resolved images obtained through aberration-corrected STEM-HAADF images. The nanobeam diffraction (NBD) through the STEM imaging mode is controlled by the condenser lens system. The combination of probe-corrected STEM imaging and quasi-parallel beam diffraction (D-STEM) is obtained by positioning the beam in the STEM image at a single nanoparticle using the Digiscan control. The scan is stopped and positioned arbitrarily at a xy position on the screen. Subsequently, the electron diffraction pattern is recorded using a digital charge couple device (CCD) camera. D-STEM mode works in the diffraction plane, the overlapping of the convergent disks is optimized by a compensation of the last condenser lens (C3) and the use of the adaptor lens (ADL) at the hexapole coils of the CEOS corrector.

[1] A. Tlahuice-Flores, U. Santiago, D. Bahena, E. Vinogradova, C.V. Conroy, T. Ahuja, S.B.H. Bach, A. Ponce, G. Wang, M. Jose-Yacaman and R.L. Whetten, J. Phys. Chem. A, 2013, 117 (40), pp 10470–10476
[2] D. Bahena, N. U. Santiago, A. Tlahuice, A. Ponce, S.B.H. Bach, B. Yoon, R.L. Whetten, U. Landman and M. Jose-Yacaman, J. Phys. Chem. Lett., 2013, 4 (6) , 975-981.

This project was supported by grants from the National Center for Research Resources (5 G12RR013646-12) and the National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health. The authors would also like to acknowledge the NSF PREM # DMR 0934218.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-3512 Electron beam induced surface modification of semiconductor nanowires in a chlorine environment - A new route to electrical tailoring of nanodevices

Wanzenboeck H. D.1, Mika J.1, Shawrav M. M.1, Gavagnin M.1, Ismail B.1, Zeiner C.1, Lugstein A.1, Stoeger-Pollach M.1, Bertagnolli E.1
1Vienna University of Technology, A-1040 Vienna, Austria
heinz.wanzenboeck@tuwien.ac.at

Scanning electron microscopy is not only a high-resolution imaging technique for nanocharacterisation of materials, but the focused beam of electrons can also be used for inducing chemical reactions in the nanometer-regime. For focused electron beam induced processing (FEBIP) precursor gas is introduced into the vacuum chamber and the electrons interact with precursors adsorbed on the sample surface. It has already been demonstrated, that metalorganic precursors lead to deposition of materials including noble metals such as Pt or Au as well as magnetic metals such as Fe or Co.

We have recently introduced a controlled etching process that is sustained by the irradiating electron beam. With the semiconductors Si and Ge we have not observed spontaneous etching, while material could be etched in the areas exposed to the electron beam. A clean vacuum chamber is a prerequisite for this process and has been achieved with an in-situ ozone cleaning procedure of the chamber.

In this work we report on the controlled etching of Si-nanowires and of Ge-nanowires. Nanowires themselves are smart nanomaterials with very promising characteristics and may be used for nanoelectronic devices, innovative sensor concepts and for photovoltaics applications. Focused electron beam induced etching (FEBIE) offers a further alternative to modify these nanomaterials in-situ in a SEM. With dynamic experiments in the SEM we have investigated the chemical reactions on the nanoscale. The material modification with regard to, its composition and its electrical has been investigated.

The custom-designed tailoring of electrical properties of nanowires is essential for the development of new devices. FEBIE is a versatile approach for trimming of Si-nanowires as the low-energy electrons inflict no significant crystallographic damage and cause no contamination of the silicon nanowire. This in-situ preparation allows to keep specimens as close as possible to their native state.

With chlorine as etch gas even without geometrical thinning of nanowires the short-term irradiation was observed to result in a change of electrical properties towards a diode-like characteristics. The effect of electron exposure under the presence of molecular chlorine was investigated. Additional to structural studies also an electrical characterisation of contacted Si-nanowires and a TEM nanostructure analysis was performed.

FEBIE has been established as a novel approach that allows for tailoring of material properties by controlled in-situ modification of nano-scaled materials. Potential future applications of FEBIE to design and to develop of new nanomaterials for sensor applications and for photonics applications will be discussed.

We acknowledge financial support by the Austrian Science Fund (FWF) under project P24093. TEM analyses were carried out at the University Service Centre for Transmission Electron Microscopy, Vienna University of Technology.

Fig. 1:  Scanning Electron Microscope LEO 1530 VP with the custom-built gas injection system for chlorine

Fig. 2: Schematic illustration of nanowire modification by focused electron beam induced etching.

Fig. 3: Left: Setup for the electrical measurement of the semiconductor nanowire. The top image shows a SEM image in top view Right: Electrical behavior of the Si-nanowire. The I-V-curve shows the electrical properties before and after FEBIE modification in a chlorine environment.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5699 Microscopic and spectroscopic techniques as useful tools in the construction of nanobiosensors based on carbon nanotubes

Guadarrama L.1, Chanona J.1, Hernandez H.1, Manzo A.2, Martínez A.3, Calderon G.1, Suarez E.1
1Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Naciona, 2Escuela Superior de Ingeniería Química e Industrias Extractivas, Instituto Politécnico Nacional, 3Centro de Investigación en Computación, Instituto Politécnico Nacional
jorge_chanona@hotmail.com

A biosensor is a device, which converts a biological response between a target analyte and a bioreceptor into an electrical signal. The bioreceptor can be a microorganism, organelle, cell, enzyme, antibody, nucleic acid etc. All these kind of sensors can exploit the advantages from high surface-to-volume-ratio property of nanomaterials. Carbon nanotubes (CNT) present outstanding electrical and chemical properties and could interact with organic and inorganic compounds therefore can be functionalized with supramolecular complex. The morphology and quality of CNT can be determined by the use of transmission (TEM) and scanning (SEM) electron microscopy. Raman and X-ray photoelectronic (XPS) spectroscopy allow identify the type of CNT and verify the chemicals modifications produced on CNT during the functionalization process. These techniques provide useful information about the biosensor construction process like homogeneity of the CNT network or wide of the layer. This project presents the microscopic and spectroscopic characterization of multi-walled CNT simultaneously purified and functionalized through an acid treatment with HNO3-H2SO4 with the objective of make them more reactive trough the formation of acid carboxylic groups on the CNT and then use it as support for amyloglucosidase (AMG) as a probe molecule and check if the enzyme is still active. All CNT were analyse using SEM, TEM, Raman and XPS. Raman spectra allow observe how the acid treatment removes impurities of the CNT (Fig 1). Chemical functionalization with carboxylic groups is evidenced by XPS spectra showing a peak at 288.5 eV characteristic of carboxylic groups and a shoulder at 287 eV possibly associated with peptide bonds (Fig 2). By the TEM micrographs (Fig 3) it is possible to observe the enzyme onto CNT contrasted with uranyl acetate (1%). The results of enzymatic assay prove that the AMG preserve 50 % of its activity compared against native enzyme. The double-layer capacitance, obtained from the current versus potential characteristics at different scan rates (mV/s), was also obtained for the CNT in each step of functionalization and finally the CNT/AMG system was tested in optimal conditions for the AMG catalyze the substrate. The different responses in the electrochemical capacitance and the results of enzymatic kinetics provide evidence of an adequate functionalization of CNT for their use as electrochemical biosensor. Microscopic and spectroscopic techniques prove that are not only useful but necessary tools for biosensors construction.

CONACyT and PIFI-IPN for the scholarship. Financial through the projects 20131864, 20130333, 20140387 and Red-SIP-NTC-FET at IPN-Mexico and from CONACyT 161793, 133102. The TEM images were obtained with the project supported by a grant from the National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health.

Fig. 1: Raman spectra for A) raw carbon CNT and B) purified CNT

Fig. 2: XPS spectra for raw CNT, purified CNT and system CNT-AMG. Inset: zoom in the range 286-291 eV.

Fig. 3: TEM image of a CNT covered with AMG and contrasted with uranyl acetate at 1%
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5750 A Comprehensive study of the nucleation and growth mechanism of Au anisotropic nanoparticles: From Seeds to Bipyramids

Ihiawakrim D.1, Ersen O.1, Hirlimann C.1, Florea I.2, Treguer-Delapierre M.3, Majimel J.3, Spuch-Calvar M.3
1IPCMS, CNRS-Université de Strasbourg, Strasbourg, France, 2LPICM, CNRS-Ecole Polytechnique, Palaiseau, France, 3ICMCB, CNRS-Université de Bordeaux, Pessac, France
dris.ihiawakrim@ipcms.unistra.fr

In the framework of the development of nano-sized materials with new optical properties induced by the size effect or by a specific morphology, the study of noble metal nanoparticles takes nowadays a prominent position, due in particular to their plasmonic properties. Attention has been paid to the quest for a synthesis method able to provide Au nanostructures with precise shape and crystallographic orientation. Thus, it was demonstrated that by using a seed mediated technique [1] and tuning the Au seeds concentration that play the role of nucleation centers, one can synthesize Au bipyramids (BPs) with various aspect ratios inducing various symmetries in the basal plane. From a fundamental point of view, it is expected that changing the morphology of these NPs may induce modifications of their optical properties. To synthetize Au BPs with controlled aspect ratios a good understanding of the nucleation and growth mechanisms is needed. The goal of this work is to perform a comprehensive analysis based on an approach combining modern TEM techniques: conventional TEM imaging mode, HR imaging using both TEM and STEM HAADF modes [2] and STEM-HAADF electron tomography. This type of analysis provides reliable information regarding the morphology and the crystallographic structure of both Au seeds and Au BP and thus allows elaborating reliable hypothesis on the nucleation and growth processes of these anisotropic NPs. We present here a detailed study performed on Au NPs presenting aspect ratios of 2, 3, and 5. An icosahedral (penta-twinned decahedron) shape of Au seeds NPs presenting a 4 nm size was firstly evidenced (Fig. 1). In a second step, a detailed HRTEM analysis on Au BP allowed us to directly observe the highly stepped nature of the BP surface constituted by {151} lateral facets (Fig. 2). Finally, the analysis of the reconstructed volumes obtained by electron tomography showed that the bipyramidal morphology is preserve for all the studied nanoparticles (Fig. 3a). However, some differences between them can be observed regarding the shape of their tips, the symmetry of the equatorial plane and the characteristics of the steps present on the surface (Fig. 3b). Particularly, the larger the BP is, the sharper the tips and higher the surface steps are. In addition, the analysis of the transversal sections for each volume showed that the symmetry of the equatorial plane changes from a hexagonal one for high aspect ratios to a pentagonal one for low ratios.

References:

[1] Liu, M.;, Guyot-Sionnest, P.J., J. Phys. Chem. B, 2005, p 22192.

[2] Burgin, J. et al. Nanoscale 2012 p.1299.

The authors gratefully acknowledge funding from the ANR under Grant number ANR-BLANSIMI10-LS-100617-15-01.

Fig. 1: (a) HR-STEM HAADF image of a 4 nm Au seed NP; (b) Projection at 0° extracted from the tilt series used to reconstruct the volume of an area containing several Au seeds NP;(c) XY slice through the reconstructed sub-volume of an individual Au seed NP evidencing its icosahedral morphology .

Fig. 2: (a), (b) High Resolution TEM micrographs of an Au bipyramid showing the crystallographic nature of the stepped lateral facets; (c) Schematical representation illustrating the oriented assembling of Au seeds icosahedrons.

Fig. 3: (a) 3D Models of Au bipyramids with three various aspect ratios obtained by electron tomography; (b) Illustration of the presence of steps on the external surface of the BP.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5771 Application of EFTEM and EELS for investigations of electronic and structural properties of nanostructures

Sobczak K.1, Borysiuk J.1, Li T.1, Dabrowski J.1, Dluzewski P.1
11Institute of Physics PAS al. Lotników 32/46 ,PL 02668 Warsaw
ksobczak@ifpan.edu.pl

Transmission electron microscope offer wide range of measurement possibilities like high efficient electron energy loss spectroscopy (EELS) and energy filtered transmission electron microscopy (EFTEM)[1]. That allows, among others, the measurement of surface plasmons resonance (SPR) and in the case of structures with the size below tens nanometers – Localized Surface Plasmons Resonance (LSPR)[2].
The phenomena of electron energy loss can be exploited in both scanning and imaging working mode of TEM. In the scanning mode a spectrum is recorded at a given beam position and therefore the spatial resolution is determined by beam size and specimen thickness, which indicate a volume from which the spectrum is collected. The energy resolution depends on monochromaticity of the incident electron beam and the quality of a spectrometer. Nowadays  the energy resolution of monochromator is 0,2 eV at a 300 keV and about 0,15 eV at a 80 keV. STEM mode connection with such a good energy resolution allows the measurement of the plasmon resonance eve n in nanostructures with dimensions less than 5 nm.
EFTEM technique is to form an image with electrons within a certain kinetic energy range. The EFTEM standard procedure for elemental mapping is based on recording three images: two pre-edge images with electron energy loss window before an absorption edge and one post-edge image with energy window after the absorption edge. The element mapping is obtained by the post-edge image after removing a background extrapolated from two pre-edge images. The intensity of the resulting image is proportional to the concentration of the element for which the absorption edge was used. Fig. 1 and 2 shows the application of the EFTEM elements mapping procedure in the case of AlN/GaN heterostructure.
EFTEM is very useful method to mapping of the distribution of elements in the investigating sample. Fig.3 and 4. present maps obtained by EFTEM method for Ag nanoparticles. An interesting application of EFTEM is mapping of electronic properties with a use of plasmon absorption [3]. It is well known that plasmon excitation energy depends on the mobility and density of charge carriers. The energy of plasmon’s peak is correlated with local structure and therefore gives information about relation between electronic properties and structural defects.


[1] F.J. Garcia de Abajo. Optical excitations in electron microscopy Rev. Mod. Phys. 2010;82:209-275
[2] JA. Scholl, AL. Koh, JA. Dionne. Quantum plasmon resonances of  individual metallic nanoparticles Nature 2012;483:421-428
[3] J. Nelayah, M. Kociak, O. Stephan, FJG. de Abajo, M. Tence, L. Henrard, D. Taverna, I. Pastoriza-Santos, LM. Liz-Marzan, Ch. Colliex. Mapping surface plasmons on a single metallic nanoparticle. Nature Physics 2007;3:348-353

The project was financed by The National Science Centre Nr DEC-2012/05/N/ST3/03163, DEC-2011/03/B/ST5/02698 and No. POIG.02.01-00-14-032/08.

Fig. 1: a) HRTEM image of GaN dopped with Al

Fig. 2: b) Mapping of the Al by EFTEM method.

Fig. 3:  a) HRTEM image of silver nanoparticles

Fig. 4: b) Mapping of N line of the Ag using The EFTEM method.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5778 The use of Microscopy in the development of certified reference materials for nanotechnology

Gerganova T.1, Roebben G.1, Kestens V.1, Kerckhove G.1, Vissers R.2, Boydens E.2, Held A.1, Emons H.1
1European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, 2440 Geel, Belgium, 2Umicore Group R&D, Analytical Laboratory, Watertorenstraat, B- 2250 Olen, Belgium
Tsvetelina-Ivanova.Gerganova@ec.europa.eu

The use of Microscopy in the development of certified reference materials for nanotechnology

Authors: Tsvetelina Gerganova1, Gert Roebben1, Vikram Kestens1, Giovani Kerckhove1, Rita Vissers2, Eddy Boydens2, Andrea Held1, Hendrik Emons1

1) European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, 2440 Geel, Belgium

2) Umicore Group R&D, Analytical Laboratory, Watertorenstraat, B- 2250 Olen, Belgium                       

Abstract: Nanotechnology is an enabling technology, which has the potential to greatly improve many areas of human life through newly developed nanomaterial-based products. The safe use of such products requires an understanding of the possible release of nanoparticles from such products, and an assessment of the potential hazards resulting from the exposure to these particles. This understanding and assessment must be underpinned by accurate measurement data of both the chemical and physical properties of nanoparticles.

Amongst others, size and shape have been identified as important properties of nanoparticles. In this context microscopy is a powerful technique to investigate and to quantitatively assess both metrics. The accuracy of the results obtained with microscopy techniques can only be guaranteed if the measurements are performed in a metrologically sound manner. Certified reference materials (CRMs) are indispensable tools for method performance verification and validation, as they enable a quantitative assessment of the method trueness and the uncertainty of measurement results. This work discusses the value and limitations of different microscopy techniques in the development of CRMs for use in the size and shape measurements of nanoparticles. 

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5796 3D structural & chemical analysis of ternary PtRh/SnO2 catalysts for Ethanol Oxidation in Direct Ethanol Fuel Cells

Parlinska M.1,2, Li M.3, Kowal A.4,5,6,7
1Facility for Electron Microscopy & Sample Preparation, Univ. of Rzeszow, Poland, 2Int. Centre of Electron Microscopy for Materials Science, Dept. of Physical & Powder Metallurgy, Faculty of Metal Engineering & Industrial Computer Science, AGH University of Science and Technology, Krakow, Poland , 3Dept of Chemistry, Brookhaven National Laboratory, Upton, New York 11973, United States, 4Dept of Mat. & Natural Sci., Univ. of Rzeszow, Poland, 5Center for Synthesis and Char. of Nanomaterials, Univ. of East Sarajevo, Bosnia & Herzegovina, 6Central Lab. of Batteries & Cells, Forteczna 12, Poznan, Poland , 7Elcatak, ul. Pod Sikornikiem 8, Krakow, Poland
nckowal@cyf-kr.edu.pl

One possible solution for new, efficient, and environmentally-friendly technologiy for transforming chemical energy into electricity are fuel cells [1]. Ethanol seems to be an ideal fuel, as it is a non-toxic liquid and can be produced cheaply and efficiently from grasses. The best performance in ethanol oxidation reaction, was obtained for PtRh/SnO2 nanoparticles designed and synthesized by the Adzic group [2-5]. Additives such as F or Sb can be added into SnO2 in order to enhance its catalytic activity [6]. Structural and chemical investigations of the nanocatalysts with different Pt:Rh:Sn ratio were performed by TEM Osiris FEI operating at 200 kV and equipped with Super-EDX. Fig. 1(a) shows a STEM HAADF image of the PtRh/SnO2 catalyst with a Pt:Rh:Sn ratio = 1:1/3:1, which is relatively homogeneously distributed on the Vulcan carbon substrate. Unfortunately, a direct distinction between the tin oxide and PtRh particles is not possible, Fig. 1(b). Individual particles with a size from 2-10 nm are observed. Fig.2 shows the HAADF image of the PtRh/SnO2 particles with quantified EDX maps of Pt, Rh and Sn. Individual Pt particles of 2-5nm in the map are distinguishable, what is not the case for the SnO2 particles. The Rh signal is rather weak and is located in the same areas as Pt. The SnO2 particle size starts from 4-5 nm for individual particles. In the sum EDX map of Pt, Rh and Sn, it can be clearly seen, that the SnO2 particles are not completely coated by PtRh particles, therefore areas with pure tin are visible (blue in Fig. 3). The Pt:Rh ratio determined by EDX in this sample is 3:1. The right image in Fig. 3 shows the HRTEM image of the PtRh/SnO2 particles, which are visible as dark dots on the amorphous, circular Vulcan carbon substrate.

[1] V.S. Bagotsky, Fuel Cells: Problems and Solutions 2nd Ed.; John Wiley & Sons: Hoboken, New Jersey, 2012 pp. 3-5
[2] A. Kowal, M. Li, M. Shao, K. Sasaki, M.B. Vukmirovic, J. Zhang, N. S. Marinkovic, P. Liu, A. Frenkel, R. R. Adzic, Nature Materials, 2009, 9, 325-330.
[3] A. Kowal, S.Lj. Gojković, K.-S. Lee, P. Olszewski, Y.-E. Sung, Electrochem. Comm. 2009, 11, 724-727.
[4] R. Adzic, A. Kowal, (Brookhaven National Laboratory), Patent Application Publication, Pub. No. US2009/0068505 A1 (Mar. 12, 2009).
[5] M. Li, A. Kowal, K. Sasaki, N. Marinkovic, D. Su, E. Korach, P. Liu, R. Adzic, Electrochima Acta, 2010, 55, 4331-4338.
[6] M. Parlinska-Wojtan, R. Sowa, M. Pokora, A. Martyła, K. S. Lee and A. Kowal, Surf. & Interfaces Anal. Published on-line 15 Feb. 2014, DOI: 10.1002/sia.5384

Fig. 1: HAADF STEM images of the PtRh/SnO2 catalyst deposited on the Vulcan carbon substrate: (a) overview image showing a uniform distribution of the catalyst nanoparticles; (b) magnified view – the PtRh and tin oxide particles are not directly distinguishable in the HAADF detector.

Fig. 2: (left to right) STEM HAADF image of the PtRh/SnO2 particles and the corresponding quantified EDX maps of Pt, Rh and Sn.

Fig. 3: left image: EDX map of the catalyst showing the distribution of Pt, Rh and Sn in the sample; right image: HRTEM image of the PtRh/SnO2 (dark dots) particles on amorphous carbon.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5798 Ozone decomposition over supported Ni/Pd catalysts synthesized by extractive-pyrolytic method

Batakliev T.1, Georgiev V.1, Serga V.2, Anachkov M.1, Rakovsky S.1
1Institute of Catalysis, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria, 2Riga Technical University, Institute of Inorganic Chemistry, 2169 Riga, Latvia
todor@ic.bas.bg

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5805 Silver coated perlite catalyst for ground-level ozone degradation

Georgiev V.1, Blaskov V.2, Stambolova I.2, Batakliev T.1, Shipochka M.2, Vassilev S.2, Anachkov M.1, Eliyas A.1, Rakovsky S.1
1Institute of Catalysis, Sofia, Bulgaria 1, 2Institute of General and Inorganic Chemistry, Sofia, Bulgaria
vlado@ic.bas.bg

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5831 Benefit of HRTEM to explain the catalytic behavior of gold catalysts on Co- and Fe-doped ceria supports

Petrova P.1, Zanella R.2, Ivanov I.1, Pantaleo G.3, Venezia A M.3, Ilieva L.1
1Institute of Catalysis, Bulgarian Academy of Sciences, Sofia, Bulgaria, 2Centro de Ciencias Aplicadas y Desarrollo Tecnológico Universidad Nacional Autónoma de México, 3Istituto per lo Studio di Materiali Nanostrutturati, CNR, I- 90146 Palermo, Italy
petia@ic.bas.bg

The dispersion of gold is one of the key factors for the high catalytic performance of gold based catalysts. Nanosized gold catalysts supported on Co- and Fe-modified ceria were studied in important for environmental point of view processes: i) complete benzene oxidation (CBO) over gold (3wt%) catalysts on Co-doped ceria supports (5, 10 and 15 wt% Co3O4) prepared by mechanochemical mixing (MM); ii) CO-free hydrogen production for fuel cells application via WGS and PROX over gold (3wt%) catalysts on Fe-doped ceria supports (5, 10 and 20 wt% Fe2O3) prepared by mechanochemical mixing (MM) or impregnation (IM). The catalysts were characterized by different methods (XRD, XPS, TPR). Gold dispersion was evaluated by means of high resolution transmission electron microscopy (HRTEM) and high angle annular dark field (HAADF) measurements. The study is focused on the relationship between gold dispersion and catalytic activity, depending on the method of supports preparation and the dopant amount.


Very high catalytic activity in CBO was observed over gold catalyst on MM prepared ceria with 10 wt% Co3O4. It was significantly higher compared to 5 wt% or 15 wt% dopant. The HRTEM/HAADF results revealed that the doping with 10 wt.% Co3O4 was favorable for the highest gold dispersion: the highest part of very small particles (0.5 nm) and the lowest amount of bigger particles (3.5 nm and above) can be seen for Au10CoCeMM catalyst (fig. 1). It correlates with the highest reducibility and the highest oxidation activity in CBO.

The MM or IM preparation methods of Fe-doped ceria supports of gold catalysts affected in different way the catalytic behavior in the WGS and PROX reactions. Gold catalysts on IM supports exhibited WGS activity lower than that of gold/ceria. Significantly better WGS performance was demonstrated using MM. The observed differences were explained mainly by the differences in gold dispersion determined by the preparation method (considering the features of the multicomponent supports as well): HRTEM/HAADF results in accordance with XRD and XPS data showed higher gold dispersion in the case of supports prepared by MM. The gold dispersion is not such a key factor for PROX because the variations in the CO conversion as well as the selectivity dependence on the preparation method were not very substantial.


Important role for PROX at realistic conditions with CO2 and water in the gas feed played the formed using IM method nanosized hematite particles. This Fe-phase was evidenced by detailed analysis of HRTEM images in agreement with the Mössbauer results at LNT. Covering the ceria grains it leads to lower surface basicity and by this way they could improve the resistance toward CO2 deactivation.

This work was supported by ESF (Grant BG051PO001-3.3.06-0050).

Fig. 1: Catalytic activity, HRTEM/HAADF images and size distribution histograms of gold paricles for the Au5CoCeMM, Au10CoCeMM and Au15CoCeMM catalysts
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5833 Combining electrons and ions: a (S)TEM-FIB microscopy application to unveil the final surface structure of a washcoated monolithic catalyst

Hernández-Garrido J. C.1, Gaona D.1, Gómez D. M.1, Vidal H.1, Gatica J. M.1, Sanz O.2, Rebled J. M.3, Peiró F.4, Calvino J. J.1
1Universidad de Cádiz, Cádiz, Spain, 2Universidad del País Vasco, San Sebastián, Spain, 3Institut de Ciència de Materials - CSIC, Barcelona, Spain, 4Universitat de Barcelona, Barcelona, Spain
jcarlos.hernandez@uca.es

The use of structured materials consisting of catalyst-coated honeycomb-type monoliths offers some advantages in comparison with the powder catalysts: e.g. higher exposed surface area and/or improvement of the active site-reactant contact. The dip-coating is the most extended procedure to load the powdered catalyst onto the monolith. Although the structural and chemical characterization at the nanometer scale of powder catalysts by (scanning) transmission electron microscopy, (S)TEM, techniques is in most cases well established, the characterization at such scale of catalytic devices, as it is the case of coated monoliths, poses currently a few challenges, some of them related to basic strategies to obtain representative information from the observations and others related to the sample preparation steps. In any case the approach is mandatory to determine the influence of the preparation procedure used to load the monolith on the final structure of both the active catalyst powder and of the coating itself.

There are several techniques to characterize devices at the micrometer scale but they present some disadvantages related to get a sample without mechanical damages. More recently, Focused ion beam (FIB) offers a solution of this particular (S)TEM characterization challenge, because it allows extracting precisely positioned, nanometer-sized sections of this type of devices, suitable for high resolution studies by different (S)TEM techniques. In this contribution we report how the combination of scanning electron microcopy (SEM)+(S)TEM + FIB studies yields valuable information from specific areas of a Co3O4/La-modified-CeO2 catalyst deposited by means of washcoat techniques on honeycomb cordierite (400 cells/in2) and Fe-C-based monoliths, designed for catalytic combustion of volatile organic compounds.

X-EDS maps in STEM-mode provided detailed information about the spatial distribution of these components. Remarkable, a Co3O4 layer was detected the external surface of the washcoat surrounding an inner core made up of an ensemble of the nanosized CeO2 support crystallites, suggesting that during the washcoating process the two components of the initial active catalysts, a ceria-supported cobalt oxide, segregate in space giving rise to a stratified structure in the coating layer of the catalytic device, far too different from the initial powder in which the two components were intimately mixed. These findings were confirmed in both cases, ceramic or metallic monolithic-supports. We remark the potential of the combined FIB-STEM characterization to give detailed important information about the surface of the monolith, which it is not possible to obtain by macroscopic characterization techniques and even by Electron Microscopy techniques.

Fig. 1: Catalyst-coated ceramic (left) and metallic (right) honeycomb-type monoliths.

Fig. 2: SEM images recorded at the different steps of the preparation of the FIB sample of the monolith washcoated. Adapted from Hernandez-Garrido et al. J Phys Chem C, 117(25):13028. Adapted with permission.

Fig. 3: X-EDS element (Co, Ce and Al) distribution maps recorded on the catalysts powder (a), the waschcoating suspension (b) and the final FIB sample from the ceraminc monolith. Adapted from Hernandez-Garrido et al. J Phys Chem C, 117(25):13028. Adapted with permission.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5885 TiO2 nanoparticles obtained by laser ablation in water: influence of pulse energy and duration on the crystalline phase

Canton P.1, Giorgetti E.2, Marsili P.2, Muniz-Miranda M.3, Vergari C.4, Giammanco F.4
1Department of Molecular Sciences and Nanosystems, University of Venezia, Mestre, 30170, Italy cantonpa@unive.it, 2Istituto dei Sistemi Complessi - CNR, Via Madonna del Piano 10, Sesto Fiorentino (Firenze), Italy, 3Department of Chemistry “U. Schiff”, University of Firenze, Firenze, Italy , 4Department of Physics "E. Fermi", University of Pisa, Pisa, Italy.
cantonpa@unive.it

Pulsed laser ablation (PLAL) can be used for production of stable and unprotected TiO2 nanoparticles (NPs) in pure solvents [1]. In general, rutile or anatase phase are required, depending on the applications, the first one being more attractive in the development of pigments and the second one more appropriate in photocatalysis. However, in spite of the large amount of work described in the literature, the control of the crystalline phase of the obtained samples is still a challenging tasks.
For this purpose, we performed a thorough characterization of the ablation of a Ti target in deionized water, by using the 1064 nm fundamental wavelength of a ns or a ps Nd:YAG laser and by tuning the energy per pulse and the fluence on target. We analyzed the colloids by UV-vis and Raman spectroscopy and by SAED, NBD, HRTEM and found some experimental rules which allow the control of the different phases of the oxide. According to Raman tests, we obtained the characteristic bands at 440 and 605 cm-1 of rutile NPs with ns pulses and prevalently rutile or anatase NPs with ps pulses, being anatase (395, 506 and 625 cm-1) more abundant when ps ablation is carried out with high energy pulses (see Fig. 1, 2 showing BF and NBD of three nanoparticles evidencing crystalline and amorphous structures).
The previous experimental results were compared with a theoretical model, which gives a detailed description of the ablation process during the laser pulse and the subsequent time-space evolution of key parameters of yields, i.e. pressure and temperature, in the surrounding solvent. By using simplified rate equations and phase diagrams of Ti oxides, the model not only allows to explain the observed energy and pulse-width dependence of TiO2 crystalline phase, but also provides a guide to choose the experimental parameters required to isolate the different crystalline species .

References.
[1] J. S. Golightly and A. W. Castleman, Jr.; J. Phys. Chem. B 110 (2006) pp. 19979-19984

Fig. 1: BF of 8mJ ps ablation specimen

Fig. 2: NBD of the three particles shown in figure 1
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5946 Self-assembly of nanoparticles into 3D supraparticles

Kister T.1, Kraus T.1
1INM – Leibniz Institute for New Materials, Saarbruecken, Germany
thomas.kister@inm-gmbh.de

In the last decade, nanoparticles (NPs) have been applied in optics, magnetics and electronics. For example, fluorescent semiconductor NPs and metal NPs are used as markers for cells. Uniform NPs have been shown to self-assemble into regular structure like 2D or 3D superlattices. Depending on conditions such as solvent and temperature, the resulting superlattices can have different crystalline structures [1]. Supraparticles (SP) are self-assembled 3D clusters of NPs which are stably dispersed in a solvent. They can be produced using oil-in-water emulsions. Nanoparticles are confined inside the dispersed nonpolar phase. Upon evaporation of the oil phase, NPs arrange into SPs [2]. Their exact arrangement depends strongly on the surfactant that stabilizes the emulsion [3]. The SP retain most properties of the NPs; coupling between the closely packed NPs leads to plasmon shifts and energy transfer. SPs can also be formed from dispersions containing different NPs. The resulting SPs combine the properties of its constituents. For example, binary SP from gold and cadmium selenide (CdSe) NPs exhibit optical plasmon absorption due to the gold NPs and fluorescence due to the CdSe quantum dots.
Electron microscopy is the only available technique that provides sufficient resolution to study shape and structure of the SPs. It can resolve the particles’ arrangement inside the SP. SPs containing two types of NPs may be binary crystals, random mixtures of the constituent or Janus-like structures. Electron microscopy resolves such differences. Figure 1 shows a TEM picture of a SP produced from gold NPs. The structure of the SPs is similar to that of minimum energy particle arrangements known as Lennard–Jones clusters. We found that SPs from monodisperse gold or silver NP tend to exhibit such cluster-like structures. CdSe NPs and mixtures of CdSe and gold NPs exclusively assembled into SPs with glassy structures (Fig. 2).
So far, the structure of SPs was estimated from their 2D projections obtained from TEM. Currently, electron tomography measurements are performed to reconstruct precise 3D NP arrangements. Projections of a single SP are recorded at different angles in the range of -45° to 45°. We employ the “BART” and the “SIRT” to reconstruct a 3D model in order to quantitatively characterize the inner part of SPs. The main technical difficulty is posed by the dense gold NP cores. Under certain angles, the low-density spacing between the cores is sufficient for electrons to penetrate the entire SP. We will exploit this property to improve reconstruction.

1. E. Shevchenko et al. J. Am. Chem. Soc., 2006, 128, 3620−3637
2. J. Lacava et al. Nano Letters, 2012. 12(6), 3279-3282
3. J. Lacava et al. Soft Matter, 2014, 10, 1696-1704

Fig. 1: Ordered supraparticles from gold nanoparticles

Fig. 2: Binary supraparticles from gold and cadmium selenide
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-5995 SEM, HRTEM and HAADF analysis of nickel-doped ceria nanorods obtained by hydrothermal method

Romero-Núñez A.1, Hernández-Cristobal O.1, Díaz G.1
1Instituto de Física, Universidad Nacional Autónoma de México, México
araromero@fisica.unam.mx

A series of NixCe(1-x)O2 nanorods with different nickel contents were synthesized via a simple hydrothermal method. The aim of this work is to simultaneously control the composition and morphology of cubic ceria (CeO2) structure. These concepts are important defining catalytic properties of CeO2 or any other material [1]. Even though it is already known that composition and morphology are critical to improve catalytic properties a simultaneous control over such factors has been barely approached. Tuning the morphology into one-dimensional shapes leads a preferential exposure of reactive facets which improve catalytic performance [2]. SEM and TEM images of a NixCe(1-x)O2 nanorods sample are shown in figure 1a and 1b respectively. A one dimensional rod-like morphology, which is expected to exhibit {110} and {110} reactive ceria planes, is directly observed. In figure 1c HRTEM analysis of the same sample shows a [110] direction growth that corroborates the preferential exposure of {110} and {110} surface planes. Figure 2 show the STEM analysis of NixCe(1-x)O2 nanorods. HAADF image, figure 2a, also confirms the rod-like morphology. Cerium and nickel EDS elemental mapping, figure 2a and 2b, show a dispersed and homogeneous distribution of Ni species in the ceria host structure. Ni species distribution is a critical factor for catalytic properties as selectivity, activity and stability [3]. Catalytic performance for CO oxidization is superior in the doped NixCe(1-x)O2 nanorods samples than in undoped ceria nanorods. This is in agreement with the extrinsic formation of defects, which is inherent of the formation of the solid solution, and with the high dispersion of Ni that was corroborated by EDS mapping analysis. Nickel-doping and one-dimensional morphology are tuned together for the first time on ceria structure showing good catalytic properties. A full understanding of catalytic performance could only achieved with the careful structure analysis provided by microscopic techniques. EELS analysis is currently in progress and will be also presented to complement the present work.

[1] Li T, Xiang G, Zhuang J, Wang X. Chem Commun 2011;47:6060–2.
[2] Nolan M, Parker SC, Watson GW. Surf Sci 2005;595:223–32.
[3] Barrio L, Kubacka A, Zhou G, et al. J Phys Chem C 2010;114:12689–97.

Authors want to thank financial support of CONACyT-176509, PAPIIT-IN107512, PAEP-PCeIM and CONACyT-BN-332648, and technical support of Antonio Gómez-Cortés, Roberto Hernández, Manuel Aguilar and Antonio Morales.

Fig. 1: Electron microscopy characterization of NixCe(1-x)O2-NR. 1D rod-like morphology of samples is confirmed by (a) SEM and (b) TEM images. (c) HRTEM image view along [110] in which interplanar distances and angles corroborate the [110] growth direction.

Fig. 2: STEM analysis of NixCe(1-x)O2 nanorods (a) HAADF image, EDS spectroscopic mapping of (b) Cerium and (c) Nickel.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-6007 Microstructure of Pt nanoparticles deposited on stainless steel in simulated boiling water reactor environment

Veleva L. V.1, Grundler P. V.1, Ramar A.2, Ritter S.1
1Paul Scherrer Institut, Nuclear Energy and Safety Research Department, 5232 Villigen PSI, Switzerland, 2GE-Global Research, EPIP zone, Bangalore-560037, INDIA
lyubomira.veleva@psi.ch

In boiling water reactors (BWR) radiolysis products of water (O2/H2O2) generate a highly oxidising environment which may result in an increased susceptibility to stress corrosion cracking (SCC) of reactor components. The technology of online noble metal chemical addition (OLNC) was developed by General Electric, where noble metal compounds are injected into reactor feed water of a BWR to mitigate SCC on internals and recirculation systems. Upon injection into the hot water (220-288°C), Na2Pt (OH)6 decomposes to form Pt nanoparticles. These nanoparticles deposit on all water wetted reactor components, and in presence of H2, catalyse the reduction of O2/H2O2, thus decreasing the electrochemical corrosion potential.

In order to assess this SCC mitigation technique, the Pt particle distribution and deposition behaviour on stainless steel coupon specimens, exposed to simulated BWR water conditions, using a sophisticated high-temperature water loop, has been investigated in detail at PSI. The Pt treated stainless steel coupons were first studied by field emission gun SEM. To better understand the catalytic behaviour and the bonding of the Pt particles to the oxide film, the microstructure and morphology of single Pt nanoparticles was studied by STEM and TEM techniques, including EDS. TEM specimens were prepared by using a replica technique, allowing the removal of Pt particles together with some of oxide crystals from the outer part of the oxide layer.

Preliminary results have shown that the Pt particles are homogeneously distributed on the surface of the oxide layer, with sizes in the nanometric range (Figure 1). It has been observed that the Pt particles precipitate in various locations on the oxide surface, including different crystals, edges, and facets (Figure 2). STEM and TEM high resolution observations confirmed that the Pt particles (Figures 3 and 4), have a crystalline structure and different shapes: round shape without or with facets (Figure 3), or rhomboidal shape (Figure 4). Edges, steps, corners and twin boundaries are often sites of high catalytic activity; therefore particles rich in such features are likely to be more efficient catalysts.

The difference in the observed Pt particles including shape, size and orientation to the oxide matrix could indicate various mechanisms involved in the nucleation of the Pt precipitates, as well as their binding to the oxide surface.

The financial support by ENSI, the contributions of the nuclear power plants KKL, KKM, and the microscopy centre of ETHZ to this work, are gratefully acknowledged.

Fig. 1: SEM BSE image of Pt nanoparticles precipitates on the oxide surface

Fig. 2: STEM HAADF Z-contrast image of Pt nanoparticles on an oxide crystal, occupying different facets, edges and corners.

Fig. 3: STEM bright field image of two Pt nanoparticles, one with facets oriented in [111] direction and the second one with a round shape and no visible facets.

Fig. 4: TEM bright filed image of a Pt nanoparticle with a rhomboidal shape, attached to the oxide matrix and oriented in [111] direction.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-6011 Electrochemically deposited CeO2-NiO powders for solid oxide fuel cells: an in situ transmission electron microscopy study

Catalano M.1, Taurino A.1, Zhu J.2, Crozier P. A.3, Mele C.4, Bozzini B.4
1IMM-CNR, Via Monteroni, 73100 Lecce, Italy, 2Center for Solid State Science, ASU, Tempe, AZ 85287, USA, 3School for Engineering of Matter, Transport and Energy, ASU, Tempe, AZ 85287-6106, 4Dip. Ing. Innovazione, Università Salento, via Monteroni, 73100 Lecce, Italy
massimo.catalano@le.imm.cnr.it

Chemical and structural modifications of electrochemically deposited cermet precursor of Ni/NiO/CeO2, for the realization of solid oxides fuel cells (SOFC), were studied by conventional and in situ transmission electron microscopy (TEM). These cells are devices for effective conversion of chemical energy into electricity and heat, with low environmental impact. Cermets offer high ionic and electronic conductivity and high reforming and electrocatalytic activity. There is considerable interest in lowering the operating temperature of such devices, and doped cerias represent one possible materials choice. Doping ceria with oxides of lanthanides (Dy and Tb) improves the ionic and electronic conductivity and also increases the electrocatalytic activity of cermets [1-2]. In this work, the following CeO2-NiO samples were studied: (i) pure, (ii) Dy-doped, (iii) Tb-doped and (iv) co-doped with Dy and Tb. High spatial resolution in situ TEM techniques were used to monitor the changes in chemical/physical properties of these materials at the nanoscale [3], during thermal treatments employed to fabricate active cermets. High spatial resolution TEM observations were performed in a JEOL 2010F, an aberration corrected ARM200F and an environmental FEI Tecnai F20 transmission electron microscopes, with the combined use of TEM analytical techniques. The materials were initially analyzed in their as deposited form, then after ex situ heat treatments at 600°C for 1 hour in a furnace. After assessing the general modifications of the materials, they were subjected to in situ cycles of aging (temperature up to 700°C for maximum 300 minutes in an oxygen atmosphere) and the changes of their structural properties were monitored. Fig. 1(a) shows a typical high resolution image obtained from the pure and untreated sample, consisting of CeO2 nanograins (size < 5nm) apparently laying over a polycrystalline NiO layer, with much larger grain size. The insets show the Fast Fourier Transform (FFT) from the areas marked in the figure. CeO2 in the cubic phase was detected; evidence for the rhombohedral NiO phase was found. Upon ex situ heat treatment, coarsening of the grains occurs, the CeO2 ones reaching sizes up to 10-20 nm. Large NiO crystals can be observed, as shown in figure 1(b). Doping results in an amorphization of the samples. Fig. 1 shows the images from the co-doped sample, before (c) and after (d) ex situ annealing. The images of the same sample after the in situ treatment are shown ((e) and (f)). The details of the ex situ and in situ treatments will be compared and discussed.

[1] B. Bozzini et al., Electrochem. Commun. 24 (2012) 104

[2] C. Mele and B. Bozzini, Energies 5 (2012) 5363

[3] R. Wang , P. A. Crozier and R. Sharma, J. Phys. Chem. C 113 (2009) 5700

We acknowledge support from US NSF DMR-1308085 and the use of the electron microscope at the LeRoy Eyring Center for Solid State Science at ASU.

Fig. 1: High resolution images of: pure untreated a) and treated b) samples and FFTs from marked areas, with zone axis identification and attribution to the CeO2 (1, 2….labels) and NiO (1’, 2’….labels); co-doped untreated c) and treated d) samples and relevant diffraction patterns; co-doped sample untreated e) and in situ treated d) at 700°C for 170 min
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-6019 The influence of oxygen on the metal seeded growth of SnO2 nanowires

Krekeler T.1, Mader W.2
1Technische Universität Hamburg-Harburg, Hamburg, Germany, 2Rheinsche Friedrich-Wilhelms-Universität Bonn, Bonn, Germany
krekeler@uni-bonn.de

Tin dioxide (SnO2) is the most common sensor material for detection of reducing gases like CO, H2, CH4 etc. [1]. Many contributions about gas sensors based on SnO2 nanowires have been published over the last decade, showing the great potential of nanostructured sensor materials [2]. One versatile method for the synthesis of nanowires is the well investigated vapor-liquid-solid (VLS) mechanism with gold particles as a catalyst [3]. The exact growth mechanism of metal oxide nanowires is however still a matter of discussion because of the insolubility of oxygen in gold.

In this work we describe the synthesis of SnO2 nanowires by MOCVD technique and determine the influence of oxygen on the nanowire growth by methods of electron microscopy. The nanowires are grown via a reaction of TMT (Sn(CH3)4) with oxygen (O2) on fused silica substrates with gold particles as a seed. Synthesis using optimized reaction parameters (t = 10 min, T = 800 °C, p = 1 Pa) and a TMT:O2 molar ratio of 1:35 yields SnO2 nanowires of 3 µm length and 30 nm width. The nanowires are terminated by a facetted particle with corresponding width (Fig. 1a). The growth direction was determined to be <101> of the cassiterite modification of SnO2 from HRTEM (Fig. 1b-d). EDS measurements show that the particles are tin-free gold particles (Fig. 2).
If the TMT:O2 ratio is raised to 1:1.3 by keeping the TMT flow rate constant and reducing the oxygen flow rate, the growth speed strongly increases by a factor of 15. The terminating particles are now of a round shape and about twice the diameter of the nanowires (Fig. 3). EDS measurements show significant amounts of tin in the gold particle, indicating the formation of a Sn-Au alloy.

The relationship between growth speed and TMT:O2 ratio is supposed to be tied to the state of aggregation of the catalytic particle. A high TMT:O2 ratio allows for accumulation of tin in the particle, leading to a liquefied particle and therefore higher surface diffusion rates to the particle-nanowire interface resulting in faster growth. A low TMT:O2 ratio averts the accumulation of tin in the catalytic particle and liquefaction of the particle. This leads to lower diffusion rates of the reactants and thus slower growth of the nanowire.

These results clearly demonstrate that, in contrast to the classic VLS-mechanism, growth of SnO2 nanowires can occur without liquefaction of the catalyst particle. Therefore the growth mechanism of metal oxide nanowires can be better described as a surface mediated "metal seeded growth" [4].

1. J. Watson, Sens. Actuators (1984), 5, 29-42.
2. B. Wang, J. Phys. Chem. C (2008), 12, 6643-6647.
3. R.S. Wagner, W.C. Ellis, Appl. Phys. Lett. (1964), 4, 89.
4. W. Mader, Cryst. Growth Des. (2013), 13, 572−580.

All research leading to this contribution has been done at the Rheinische Friedrich-Wilhelms-Universität Bonn.

Fig. 1: (a) TEM BF-image of SnO2 nanowire, (b) closeup of the interface between catalyst particle and nanowire, (c) closeup of the tetragonal packed tin cations in SnO2, (d) FT of c. Zone axis determined to [111], growth axis to <101>.

Fig. 2: TEM BF-image and EDS-Spectrum of facetted Au particle on SnO2 nanowire. Red asterisk marks origin of EDS-Spectrum.

Fig. 3: TEM BF-image and EDS-Spectrum of round shaped Sn-Au particle on SnO2 nanowire. Red asterisk marks origin of EDS-Spectrum.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-1-P-6029 Synthesis and Heterostructures of Metal Dichalcogenides Monolayer

Lee Y.1
1National Tsing Hua University
yhlee.mse@mx.nthu.edu.tw

Recently, monolayers of layered transition metal dichalcogenides (TMDc), such as MX2 (M = Mo, W and X = S, Se, Te), have been reported to exhibit excellent optoelectronic performances and diverse interesting properties. Monolayers in this class of materials offered a burgeoning field in fundamental physics, energy harvesting, electronics and optoelectronics. However, growth mechanisms and transfer of CVD-TMD monolayers remain challenge issues.[1~3] Hence, a feasible synthetic process and transfer techniques to overcome the challenges are essential. Here, we demonstrate the growth of high-quality TMD monolayers using chemical vapor deposition (CVD) with seeding promoter of aromatic molecules. The growth of monolayer TMD single crystals is achieved on various surfaces and a possible growth mechanism of the seed-activated growth would be presented.

We would like to demonstrate some techniques in transferring the TMD monolayers to diverse surfaces, Some characterization techniques and applications of vdw heterostructures were presented, which may which may stimulate the progress on diverse hybrid structures with TMDc monolayers.

Reference

[1] Yi-Hsien Lee, et al., Adv. Mater., 24 (17), p.2320-2325 (2012)

[2] Yi-Hsien Lee, et al. Nano Lett., 13 (4), 1852–1857 (2013)

[3] Xi-Ling, Yi-Hsien Lee*, et al., Nano Lett., 14 (2), p.464–472 (2014)

[4] Lili Yu, Yi-Hsien Lee, X. Ling, E. Santos, Y.C. Shin, Y. Lin, M. Dubey, E. Kaxiras, J. Kong, H. Wang, T. Palacios, Nano Lett, 14 (6), p.3055-3063 (2014)

[5] Xin-Quan Zhang et al, (in preparation)

We thank the Ministry of Science and Technology of the Republic of China (103-2112-M-007-001-MY3) for partial support of this research.

↑ Go to the Table of content ↑

MS-2. Carbon-based nanomaterials, nanotubes, fullerenes, graphenes

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-2-IN-2482 Irradiation-induced Modifications and Beam-driven Dynamics in Low-dimensional Materials

Meyer J. C.1, Eder F. R.1, Mangler C.1, Kaiser U.2, Kotakoski J.1
1University of Vienna, Physics department, Vienna, Austria, 2University of Ulm, Central Facility for Electron Microscopy, Ulm, Germany
jannik.meyer@univie.ac.at

Irradiation-induced phenomena open a plethora of pathways for material modifications far beyond the thermal equilibrium and which are beyond the reach of direct synthesis. By using electron beams, such modifications can be induced and simultaneously observed at the atomic level. Moreover, in the analysis of 2-D materials, the position of every atom (rather than the atomic column) can be discerned in a high-resolution image.

For example, the introduction of multi-vacancy defects in graphene can be readily observed under 100kV aberration-corrected HRTEM [1], and the combination of vacancy creation and bond rotations can be used to convert graphene into two-dimensional amorphous carbon [1,2]. Atom loss can be directly counted in the images as a function of dose and in this way we have measured the knock-on sputtering cross sections for carbon atoms in graphene [3]. Recently, we have carried out a statistical analysis of the 2-D amorphous carbon (or carbon glass) structures with variable degree of disorder [4], enabled by an automated image analysis. For the first time, this provides atomic configurations for a continuous transition from a crystalline to an amorphous state, which were used for a statistical analysis based on experimentally obtained atomic coordinates.

At lower energies, the formation of defects in the pristine lattice is less likely, but existing defects convert from one configuration to another and migrate under the beam. Fig. 1 shows results from a 60kV STEM experiment under ultra-high vacuum conditions where double vacancies in graphene are extraordinarily stable; meaning that they neither convert into higher-numbered vacancies nor trap carbon and convert back to a pristine lattice, for long sequences of images. Nevertheless, the defects rapidly move via beam-driven bond rotations [5].

Although beam-driven dynamics are useful to modify materials under direct observation, these effects are also a major obstacle for the analysis of the pristine state of the sample. Using simulated data, we have shown a new approach to extract information from very low dose exposures [6], which will be discussed in the second part of the presentation. If this can be achieved also with experimental data, it may provide a novel route to circumvent the limitations of radiation damage in the analysis of materials.

[1] J. Kotakoski et al., Phys. Rev. Lett. 106 (2011), p. 105505. [2] J. Kotakoski et al., Phys. Rev. B. 83 (2011), p. 245420. [3] J. C. Meyer et al., Phys. Rev. Lett. 108 (2012), p. 196102. [4] F. Eder et al., Scientific Reports, in press (2014).  [5] J. Kotakoski et al., submitted (2014). [6] J. C. Meyer et al., Ultramicroscopy in press (DOI: 10.1016/j.ultramic.2013.11.010)

Austrian Science Fund (FWF: P25721-N20, M1481-N20 and I1283-N20), European Research Council (ERC) project PICOMAT, German Ministry of Science (DFG), Ministry of Research and the Arts (MWK) of the State of Baden-Wuertternberg within the SALVE project, Computational time from the Vienna Scientific Cluster.

Fig. 1: (a-d) Four subsequent frames of a di-vacancy defect. The transition from (a) to (b) requires at least four bond rotations (indicated by arrows) while only one bond rotation is sufficient from (c) to (d). (e-h) partial STEM images of different di-vacancy configurations, indicating that a transformation has occurred during the scan.

Fig. 2: (a) Simulated STEM data for infinite dose, and (b) processed for 500 electrons per square Angstrom. (c) Maximum-likelihood reconstruction from a larger area of low-dose data [6].
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-2-IN-2939 Defect structure and dynamics in two dimensional crystals under in situ heating and biasing conditions

Alem N.1
1Materials Science and Engineering Department, Center for Two Dimensional and Layered Materials, Penn State University, University Park, PA, USA
nua10@psu.edu

The past decade has seen incredible progress in the ability to isolate and manipulate two dimensional (2D) crystals. Such crystals are made of a network of atoms with strong bonds in the crystal plane, and much weaker out-of-plane van der Waals bonds. Due to this unique structure and dimensionality, charge carriers can be confined in two dimensions resulting in peculiar physical, chemical, and electronic properties. Such unexplored properties can be controlled and tuned through defects, step edges, interfaces, and grain boundaries. In this study, ultra-high resolution aberration-corrected electron microscopy is used to investigate the chemical and atomic structure of the edges, defects and grain boundaries in atomically thin two dimensional crystals, i.e. graphene, hexagonal boron nitride (h-BN) and tungsten disulfide (WS2). In addition, we use ultra-high resolution TEM to probe the structure of defects, edges, grain boundaries and their stability and dynamics under in situ thermal and electrical conditions [1-2]. Using high resolution electron microscopy imaging coupled with exit wave reconstruction technique, we have observed reconstruction of the edges in graphene into an armchair structure and formation and growth of 5-5-8 line defects originating from the holes in a monolayer graphene under joule heating conditions [1]. Fig. 1 shows an example of a hole in graphene under such conditions and formation of a line defect originating from the hole. In contrast to graphene, we observe holes with a zigzag structure in a monolayer of hexagonal boron nitride (Fig. 2) under in situ heating conditions. Unusual defect structures, such as 5-7 defects, are formed in h-BN and along the grain boundaries at high temperatures, although their formation is not expected in hexagonal boron nitride [2]. This talk will also address the stability and migration dynamics of grain boundaries in a monolayer WS2 as opposed to defect dynamics in graphene [3].

References:

[1] J. H. Chen, G. Autès, N. Alem, F. Gargiulo, A. Gautam, M. Linck, C. Kisielowski, O. V. Yazyev, S. G. Louie and A. Zettl, Controlled Growth of a Line Defect in Graphene and Implications for Gate-Tunable Valley Filtering, PRB, In press.

[2] A. L. Gibb, N. Alem, J.H. Chen, J. K. Erickson, J. Ciston, A. Gautam, M. Linck, and A. Zettl, Atomic Resolution Transmission Electron Microscopy of Grain Boundaries in Chemical Vapor Deposition Hexagonal Boron Nitride, JACS, 135 (18), (2013) 6758–6761.

[3] A. Azizi, X. Zou, P. Ercius, Z. Zhang, A. L. Elías, N. Perea-López, M. Terrones, B. I. Yakobson, and N. Alem, Dislocation and Grain Boundary Migration in 2D Transition Metal Dichalcogenides, submitted.

This work was performed at the physics department at University of California Berkeley, as well as the Materials Science and Engineering Department and the Center for Two Dimensional and Layered Materials at Penn State University. TEAM microscope at the National Center for Electron Microscopy at Lawrence Berkeley National Lab was used for this study.

Fig. 1: HREM image of a hole in graphene film reconstructed to maintain armchair edges under joule heating conditions. A 5-5-8 line defect (shown with arrow) originating from the hole is formed and extended into the defect free region of the film. The atoms are white. Scale bar is 1 nm.

Fig. 2: HREM image of a monolayer of hexagonal boron nitride under in situ heating at 450 °C. Holes are mostly observed with zigzag structure, while mono-vacancies are considered the main defects at this temperature. The atoms are white. The scale bar is 1 nm.
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-2-IN-3128 TEM and cathodoluminescence with nanometric spatial resolution on BN nanostructures

Pierret A.1, 2, Schué L.1, 3, Fossard F.1, Moldovan S.4, Ersen O.4, Ducastelle F.1, Barjon J.3, Loiseau A.1
1LEM, ONERA-CNRS, Châtillon, France, 2CEA-CNRS-UJF group , 3GEMaC, UVSQ-CNRS, Versailles, France, 4IPCMS, Univ. Strasbourg-CNRS, Strasbourg, France
annick.loiseau@onera.fr

Hexagonal boron nitride (h-BN) is a wide band gap semiconductor (6.4eV), which can be synthesized, as its carbon analog graphite, as bulk crystallites, nanotubes and nanosheets. Investigation of their optoelectronic properties is made difficult because of the paucity of high quality samples and suitable investigation tools. These structures meet nevertheless a growing interest for deep UV LED and graphene engineering. A deeper understanding of the interplay between the structural and luminescence properties of different BN structures and how these properties can be further exploited for their characterization are therefore highly needed.

Such studies are now possible thanks to the recent development of dedicated photoluminescence (PL) and cathodoluminescence (CL) experiments running at 4K and adapted to the detection in the far UV range (up to 6eV) [1]. We can also combine various TEM techniques and CL experiments in a FEG-SEM with a spatial resolution of 3nm on the same nano-object. With these tools, we investigated the structure and luminescence of various structures, from high quality crystals [2], exfoliated nanosheets to multi-wall nanotubes [3].

As a result, BN materials present original optical properties, governed by excitonic effects in the 5.5–6eV energy range. Two kinds of excitonic luminescence have been identified and are called S and D lines [4]. As revealed from CL-TEM analyses, D lines are issued from defective areas (Fig 1), so that D/S ratio can be used as a qualification parameter of the defect densitiy [5]. This procedure has been applied to understand the first luminescence studies of few layers individual BN flakes [5].

Concerning nanotubes, CL images reveal that the luminescence in the 5.5–6eV energy range is strongly inhomogenous and oscillating. Thanks to a deep investigation combining different TEM techniques, we have shown that the tubes display a complex twisted faceted structure and that the twist period is correlated with the luminescence oscillations (Fig 2). Furthermore, we could show that excitons, responsible for the spectacular localization of the luminescence, are trapped to specific defects, twisted along with the faceting structure.

Finally, low-loss EELS providing an alternative approach to the nature of electronic excitations [6], we will show how it is an efficient tool to investigate the local structure and optical properties with an energy resolution below 100meV of different BN layers and nanotubes.

[1] P. Jaffrennou et al., PRB 77 (2008) 235422

[2] T. Taniguchi et al., J. Cryst. Growth 303 (2007) 525

[3] C. Tang et al., Chem. Commun. 12 (2002) 1290

[4] K. Watanabe et al., PRB 79 (2009) 193104

[5] A. Pierret et al., PRB 89 (2014) 035414

[6] R. Arenal et al., PRL 95 (2005) 127601

The research leading to these results has received funding from the European Union Seventh Framework Programme under grant agreement n°604391 Graphene Flagship. D. Golberg, T. Taniguchi and K. Watanabe from NIMS Japan are warmly acknowledged for providing samples.

Fig. 1: (a) SEM image of a h-BN crystallite; (b), (c) Corresponding CL images recorded (b) on the main S line (S3-S4), and (c) on the main D line (D4). (d) Map of the D/S ratio. (e) CL spectra recorded in the areas #1 (grain boundary) and #2 (middle of the grain), indicated in (a).

Fig. 2: Images of a BN nanotube: (a) 3nm spatially resolved CL image recorded at 5.49 eV (226 nm); (b), (c) Corresponding TEM images in (b) bright-field mode, and (c) dark-field mode on the (100) reflection. (d) Heptagonal tube cross-section obtained by tomography experiment. (e) Structure of the tube as deduced from (b-d) images.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-1592 Illuminating the electronic properties of individual carbon nanotubes with photons and electrons

Rossouw D.1,3, Bugnet M.1, Najafi E.2, Lee V.1, Hitchcock A. P.1, Botton G. A.1
1McMaster University, Hamilton, Canada, 2California Institute of Technology, California, USA, 3Present address: University of Cambridge, Cambridge, United Kingdom
dr418@cam.ac.uk

Carbon nanotubes (CNTs) exhibit unique physicochemical properties that have led to their use in a variety of novel materials science applications. Despite rapid progress in the theoretical and experimental investigation of CNTs, techniques capable of studying the structural and electronic properties of individual tubes are limited. Here, the spectral signature of carbon is used to identify the electronic character of individual single-walled CNTs. In addition, a newly built laser-TEM system is used to study light-induced structural and electronic distortions in individual CNTs.
Using high-resolution EELS, we differentiate metallic and semiconducting SWCNTs based on the fine structure of the recorded carbon K edge [1]. While the overall features in the C-K edge are similar for metallic and semiconducting tubes, differences are observed in the fine structure of the π* peak between 284 and 286 eV (Fig. 1); semiconducting nanotubes have a shoulder to the left of the π* peak, metallic to the right. Results from scanning transmission X-ray microscopy performed on the same electronically pure SWCNTs are in good agreement with EELS and are of comparable spectral resolution. The quality of the EEL spectra of individual SWCNTs opens up the possibility to probe the electronic state of single-SWCNT devices.
The study of light driven electronic and structural changes in matter is fundamental to understanding materials properties and performance. While ultra-fast and time-resolved experiments provide unique information based on measurements from very short-time intervals, not much is known on the steady-state response of nanomaterials to an intense continuous beam of light [2]. To address this, a unique system has been built to deliver a focused and continuous laser spot coincident with the electron beam inside a TEM. The laser-TEM system allows the study of structural and electronic modifications in nanomaterials under intense light irradiation. Structural and electronic distortions in individual CNTs have been studied in-situ [3]. When illuminated, a multi-walled CNT expands radially with coupled changes in its σ* conduction band (Fig. 2), as well as in its π* plasmon spectral band. Such observations may aid our understanding of the unique photoconductivity and luminescence properties of CNTs.

[1] D. Rossouw, G.A Botton, E. Najafi, V. Lee, A.P. Hitchcock. ACS Nano, 6, 10965 (2012).
[2] A. Howie. The European Physical Journal - Applied Physics, 54, 33502 2011.
[3] D. Rossouw, M. Bugnet, and G.A. Botton. Physical Review B, 87, 125403 2013.

D.R. acknowledges support from the University of Cambridge and the Royal Society in the form of a Newton International Fellowship.

Fig. 1: Differentiating between individual metallic (a) and semiconducting (b) SWCNTs by their EELS carbon K edge (c).

Fig. 2: (a) The boxed region of a multi-walled CNT extending over a hole in the support grid selected for analysis (b) contains 12 tubules and is free of any obvious defects. (c) Changes in the C-K edge of the tube during laser illumination are strongest in the vicinity of the σ* peak and are fully reversible.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-1827 Nanometric resolved cathodoluminescence on few layers h-BN flakes

Bourrellier R.1, Amato M.2, Meuret S.1, Tizei L.1, Giorgetti C.2, Gloter A.1, Heggie M.3, March K.1, Stephan O.1, Reining L.2, Kociak M.1, Zobelli A.1
1Laboratoire de Physique des Solides, Univ. Paris-Sud, CNRS UMR 8502, F-91405, Orsay, France, 2Laboratoire des Solides Irradies, Ecole Polytechnique, Route de Saclay, F-91128 Palaiseau and European Theoretical Spectroscopy Facility (ETSF), France, 3Department of Chemistry, University of Surrey, Guildford GU2 7XH, United Kingdom
romain.bourrellier@u-psud.fr

Within the latest years number of layered materials at reduced dimensions have demonstrated remarkable optical properties. However most studies focused on perfect system and the role of defects as optical active centers remain unexplored. Hexagonal boron nitride(h-BN) is one of the most promising candidates for light emitting devices in the far UV, presenting a single strong excitonic emission at 5.8 eV. However, a single line appears only in pure monocrystals, obtained through complex process[1]. Common h-BN samples present more complex emission spectra that have been attributed to the presence of structural defects. Despite a large number of experimental studies up to now it was not possible to attribute specific emission features to well identify defective structures.
Here we address this fundamental questions by adopting a theoretical and experimental approach combining few nanometer resolved Cathodoluminescence (CL) techniques with high resolution TEM images and state of the art quantum mechanical simulations.
Recently, the Orsay team has developed a CL detection system integrated within a STEM[2]. This unique experimental set up is now able to provide full emission spectra with a resolution as low as few tens of meV associated with an electron probe size of 1nm. A CL spectrum-image can thus be recorded in parallel with an HAADF image.Nanometric resolved CL on few-layer chemically exfoliated h-BN crystals have shown that emission spectra are inhomogeneous within individual flakes. Emission peaks close to the free exciton appear in extended regions. Complementary investigations through high resolution TEM allow to associate these emission lines with extended crystal deformation such as stacking faults and folds of the planes[3].
By means of ab-initio calculations in the framework of Many Body Perturbation Theory (GW+BSE) we provide an in-depth description of the electronic structure and spectroscopic response of bulk hexagonal boron nitride in the presence of extended morphological modifications. In particular we show that, in a good agreement with the experimental results, additional excitons are associated to local symmetry changes occurring at crystal stacking faults.
Additional features appearing within the band gap present a high spatial localization, typically less than 100 nm, and thus they can be related to individual point defects. When addressed individually through a highly focused electron probe they might have a single photon emitter quantum character. This hypothesis has been recently confirmed by experiments combining our CL system with an Hanbury Brown and Twiss interferometer.

[1] K. Watanabe et al, Nat. Mater. 3 (2004) p. 404
[2] L. Zagonel et al Nano Lett. 11 (2011) p. 56
[3] R Bourrellier et al, arXiv:cond-mat/1401.1948 (2014)

Fig. 1: a Bright field and b dark field images of an individual BN flake. c Overall emission spectrum of the flake andindividual spectra taken at specific probe positions indicated in panel b. d-h Emission maps for individual emission peak.Intensity is normalized independently within each individual map
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-1850 Atomic scale investigations of electron beam sensitive molecules embedded in carbon nanotubes

Biskupek J.1, Chamberlain T. W.2, Skowron S. T.2, Bayliss P. A.2, Bichoutskaja E.2, Khlobystov A. N.2, Kaiser U.2
11. University of Ulm, Central Facility of Electron Microscopy, Electron Microscopy Group of Materials Science, Albert Einstein Allee 11, D-89069 Ulm, Germany, 2School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom
johannes.biskupek@uni-ulm.de

Aberration corrected high resolution transmission electron microscopy (AC-HRTEM) at conventional accelerating voltages of 200 or 300 kV allows atomic structural investigations with sub-Ångstrøm point resolution. Specimens made of light atoms such as carbon nanostructures or Li-based materials are easily subjected to knock-on damage at 200 keV and higher energies. The nowadays state-of-the-art is to operate the CS-corrected microscopes at 80 keV energies to lower the damage of carbon nanostructures as demonstrated for graphene and carbon nanotubes below the knock-on threshold. Molecular organic structures including fullerenes (e.g. C60), tetrathiafulvalene (TTF, sulphur rich molecule) or coronene can be embedded into carbon nanotubes [1, 2] or sandwiched between graphene sheets [2] not to only act as the host “sample holder” but also to minimize charging effects. However, the typical energies of 80 keV used for AC-HRTEM investigations are eventually still too high and may damage and modify the delicate embedded molecules, especially if they contain hydrogen atoms in their structure. Therefore, the investigation of molecular organic structures in their pristine states is still a challenge.

We explore the possibilities of reduction of electron energies to 40 keV or even 20 keV to enhance the stability of molecules. The low electron energies require the correction of 5th order geometric aberrations and the correction of chromatic aberrations to compensate for the higher elctron wavelength.

We also study the advantages of dedicated low-dose techniques to minimize beam damage. Also modifications of the nanostructures prior to the actual imaging process are applied to enhance the stability against the electron irradiation.

Fig.1 shows an example HRTEM images of C60 molecules at different stages of electron irradiation. At 80 kV already a relatively small accumulated dose of 5×107 e-/nm2 is sufficient to form first dimers of C60 . However, no visible changes of the structure of C60 molecules are visible at 40 kV after irradiation with the same accumulated electron dose. A coalescence of the C60 molecules is clearly visible at a electron dose of 2×108 e-/nm2 and 80 kV irradiation. It requires almost two orders of magnitude higher dose (40 times) to initiate observable coalescence of the C60 molecules at 40 kV

[1] A. Chuvilin et al., Nature Materials 10 (2011) 687

[2] T. W. Chamberlain et al., ACS Nano 6, (2012) 3943

[3] G. Algara-Siller et al., APL 103 (2013) 203107

We acknowledge financial support from the German Research Foundation (DFG) and the Ministry of Science, Research and Arts (MWK) of the state Baden-Wurttemberg within the Sub-Ångstrøm Low-Voltage Electron Microscopy project (SALVE). We are grateful to P. Hartel and M. Linck (CEOS company) for assisting CC/CS corrected HRTEM experiments.

Fig. 1: HRTEM images of C60 molecules in single-walled carbon nanotubes imaged at 80 kV (left, CS-corrected) and 40 kV (right, CC/CS-corrected). At 40 kV it requires an almost two orders of magnitude increase in dose (40 times) to get first indications of coalescence and rupturing of the C60 molecules.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-1977 In-situ electron mciroscopy of carbon atom chains

Banhart F.1, La Torre A.1, Cretu O.2
1Institut de Physique et Chimie des Matériaux, University of Strasbourg, 23 rue du Loess, 67034 Strasbourg, France, 2National Institute of Advanced Industrial Science and Technology, Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan
florian.banhart@ipcms.unistra.fr

Carbon chains are sp-hybridized strings of carbon atoms; they may may be considered as the elements of a one-dimensional phase of carbon. Atomic carbon chains have been proposed since a long time until they were observed by electron microscopy. According to theory, the chains may be bonded by either alternating single/triple carbon-carbon bonds (polyyne) or by double bonds throughout the chain (cumulene). Their electrical and mechanical properties and their stability have been subject of many theoretical studies; however, no experimental information has been available. Now, by using an STM stage (Nanofactory) in a TEM in an in-situ study, carbon atom chains have not only been made but also characterized electrically [1]. The chains were obtained by establishing a contact between a metallic tip and graphene ribbons. Retracting the tip while an electrical current flowed through the contact led to the unraveling of carbon atoms from the graphene ribbons. Figure 1 shows the simplified principle of the experiment and figure 2 the development of a typical carbon chain, spanning here between two graphene filaments. The electrical conductivity of the chains could be measured in such a way and was found to be much lower than predicted for ideal chains. Figure 3 shows the measured current-voltage characteristics of a chain. Theory predicts that strain in the chains determines their conductivity in a decisive way. Indeed, carbon chains are always under varying non-zero strain that transforms their atomic structure from cumulene to polyyne, thus inducing a tunable band gap. The modified electronic structure and the characteristics of the contact to the graphitic periphery explain the conductivity of the locally constrained carbon chains. New experiments show the local chemistry and the bonding at contacts between metals and carbon chains as well as characteristic current-voltage curves, depending on the type of contact. Dedicated experiments show qualitatively that the chains have an outstanding mechanical strength, in accordance with theory. The results show a perspective toward the synthesis of carbon chains and their application as the smallest possible interconnects or even as one-dimensional semiconducting devices.

[1]  O. Cretu, A. R. Botello-Mendez, I. Janowska, C. Pham-Huu, J.-C. Charlier and F. Banhart, Nano Lett. 13, 3487 (2013)

Funding by the French Agence Nationale de Recherche (projects NANOCONTACTS, NT09 507527 and NANOCELLS, ANR12 BS1000401) is gratefully acknowledged.

Fig. 1: Unraveling an atomic carbon chain from a graphene ribbon by passing a current through the junction and retracting the STM tip.

Fig. 2: Formation of a carbon chain between two graphene ribbons (FLG). The time scale as well as the length of the chain are indicated. In (f) the chain is broken.

Fig. 3: Carbon chain (arrowed) and its current-voltage characteristics.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-2002 Measuring the temperature dependence of the Debye–Waller Factor in Graphene using electron diffraction techniques.

Allen C. S.1, Fan Y.1, Liberti E.1, Kim J.1, Warner J. H.1, Kirkland A. I.1
1Department of Materials, University of Oxford, OX1 3PH. UK.
christopher.allen@materials.ox.ac.uk

Within the thermodynamic limit lattice vibrations in a two dimensional (2D) crystal should destroy any long range order. As such prior to the isolation of monolayer graphene in 2004, 2D crystals were thought impossible to realise.[1] In order to explain the surprising stability of 2D crystals it is necessary to establish a detailed understanding of their lattice vibrations and how they might be affected by the properties of the crystal, for example domain size or defect density.
To date there have been many theoretical predictions of the phonon band structure of graphene with supporting experimental evidence from Raman spectroscopy measurements.[2] Recently the mean-square displacement (or Debye-Waller factor) of graphene atoms from their equilibrium lattice position has been measured from electron diffraction patterns.[3]
Using in-situ heating and cooling TEM holders we have recorded diffraction patterns from single crystals of mono-layer graphene at varying tilt angles and temperatures ranging from 100K – 1500K (figure 1). Careful analysis of these diffraction patterns has allowed us to extract values for the in-plane mean square displacement (Debye-Waller factor) of atoms over the whole temperature range. By studying the tilt dependence of the diffraction spot intensity we have also measured the out of plane atomic displacements relating to the flexural phonon modes of the graphene lattice.
We compare our results to theoretical predictions for the Debye-Waller factor based on calculations of the phonon-dispersion relation of graphene and comment on the validity of these models over the temperature range investigated.

[1] L.D. Landau et al. Statistical Physics . Part I (Butterworth-Heinemann, Amsterdam, 2003)
[2] E. Pop et al. MRS Bull. 37, 12, 1273-1281 (2012)
[3] B. Shevitski et al. Phys. Rev. B. 87,045417, (2013)

The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative¬I3).
References

Fig. 1: Figure 1. a. Selected area diffraction pattern of mono-layer graphene taken at room temperature and zero tilt. b. Real space TEM image of the graphene sample imaged through the selected area aperture used for diffraction.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-2010 XEDS-STEM tomography of multilayer nanotubes for 3D chemical characterization

Kurttepeli M.1, Bladt E.1, Deng S.2, Cott D. J.3, Detavernier C.2, Bals S.1, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium, 2Department of Solid State Science, Ghent University, Krijgslaan 281/S1, B-9000 Ghent, Belgium, 3Imec, 75, Kapeldreef, B-3001 Leuven, Belgium
mert.kurttepeli@uantwerpen.be

During the last decade, there has been an increasing demand on the 3D characterization of materials, which led to the development of different electron tomography techniques. BFTEM and HAADF-STEM based electron tomography are among those that are commonly performed in materials science. However, these techniques are mainly used to obtain the 3D morphologies of the nanostructures rather than the chemical information. Through the 3D composition mapping using STEM coupled with the X-ray energy dispersive spectrometry (XEDS) via symmetrically arranged XEDS detector design, it is now possible to resolve the 3D distribution of elements in nanoscale materials and to elucidate the 3D chemical information in a large field of view of the TEM sample [1].
We present the application of the XEDS-STEM tomography technique for 3D chemical imaging of nanoscale materials. We performed this technique to investigate the 3D chemical distribution of titanium dioxide (TiO2) and vanadium oxide (VOx) coated carbon nanotubes (CNT). Figure 1 and 2 show the results of 3D tomography applied to CNT-TiO2-VOx-TiO2 using both HAADF STEM and XEDS-STEM techniques. The comparison of simultaneously acquired HAADF-STEM and XEDS-STEM tomography results shows that XEDS-STEM tomography succeeds to provide 3D chemical information of the material in addition to the 3D morphology, in spite of the low, neighboring atomic numbers of Ti, V and C. As presented in Fig 2, XEDS-STEM tomography resolves the individual Ti, V and C containing layers and reveals that the coating of CNT by TiO2-VOx-TiO2 was uniform and conformal. One important advantage of XEDS-STEM tomography is the decreased electron beam induced damage in the TEM samples. With the improved XEDS detectors which give higher collection efficiencies, the specimen damage is minimized significantly [1]. This enabled the precise 3D nanoscale chemical characterization of fine structures as in our case without the shape and size changes of the object during acquisition. Through XEDS-STEM tomography technique it is therefore possible to resolve 3D compositional variations at nanoscale with high accuracy.

[1] A. Genc, L. Kovarik, M. Gu, H. Cheng, P. Plachinda, L. Pullan, B. Freitag and C. Wang, Ultramicroscopy 131, 24 - 32 (2013).

The authors acknowledge financial support from European Research Council and Sim-Flanders.

Fig. 1: Volume rendered 3D visualizations of HAADF-STEM reconstruction, revealing only the 3D morphology.

Fig. 2: Volume rendered composite 3D visualizations of XEDS reconstructions, revealing the 3D elemental distribution of Ti (assigned green), V (assigned red) and C (assigned blue).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-2037 Local boron environment in B-doped diamond films studied by advanced TEM and spatially resolved EELS

Turner S.1, Lu Y.1, Idrissi H.1, Janssens S. D.2,3, Haenen K.2,3, Sartori A. F.4, Schreck M.4, Verbeeck J.1, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 2Institute for Materials Research (IMO), Hasselt University, Wetenschapspark 1, B-3590 Diepenbeek, Belgium , 3IMOMEC, IMEC vzw, Wetenschapspark 1, B-3590 Diepenbeek, Belgium, 4Universität Augsburg, Institut für Physik, D-86135 Augsburg, Germany
stuart.turner@uantwerpen.be

Diamond is an attractive material for many technological applications, because of its extreme hardness, chemically inert surfaces, high Young’s modulus and large band gap of 5.5 eV. One of the most commonplace synthesis methods for nanocrystalline diamond (NCD) and epitaxial, single crystal thin films is microwave plasma assisted chemical vapour deposition (MPCVD). Many of the technological applications of diamond require specific semiconducting properties of the material and therefore doping is necessary. The most effective doping with p-type character is obtained by inserting boron in-situ during the growth process. Boron doping of diamond allows from mild p-type character for low [B] to a metallic regime and also superconducting properties at liquid helium temperatures for very high [B].1

However, much debate surrounds the question of the position and coordination of the B dopants in the diamond, especially in defective regions of the material. As B doping leads to an increase in defects in diamond grains and films upon growth, it is plausible that the boron dopants are preferentially embedded in defective regions.

In this work, conducting films of B-doped nanocrystalline diamond and single crystal diamond grown by MPCVD have been investigated in both plan-view and cross-section orientation by a combination of aberration-corrected (scanning) transmission electron microscopy (HR-ADF-STEM) and spatially resolved electron energy-loss spectroscopy (STEM-EELS) performed on a state-of-the-art aberration corrected instrument. Using these tools, the B concentration, distribution and the local B environment in this type of thin nanocrystalline diamond films have been determined.

Concentrations of ~1 at.% of boron are found to be embedded within the pristine diamond lattice. Boron distribution maps however clearly reveal a preferential enrichment of boron at defective areas like twin boundaries, incoherent defects and even dislocations in diamond thin films. Inspection of the EELS fine structure reveals a distinct difference in coordination of the B dopants in “pristine” diamond areas and in defective regions, identified through comparison of the experimental EELS fine structure to density functional theory (DFT) calculated fine structure signatures.2,3,4

1) Ekimov E.A. et al. (2004) Nature, 428, 542-545

2) Turner S. et al. (2012) Nanoscale, 4, 5960-5964

3) Lu Y.-G. et al. (2012) Applied Physics Letters, 101, 041907

4) Lu Y.-G. et al. (2013) Applied Physics Letters, 103, 032105

S.T. gratefully acknowledges financial support from the Fund for Scientific Research Flanders (FWO).

Fig. 1: B:NCD film. (a)&(b) Overview ADF-STEM images. (c) Image of a single defected diamond grain. (d)&(e) Survey image and quantitative B distribution map. B is clearly enriched at defects. (f) B-K edge fine structure from a diamond (black) and defect region (red). (g) C-K edge fine structure from a diamond (black) and defect region (red).

Fig. 2: Epitaxial B:diamond thin film. (a) ADF-STEM image of the thin film on a diamond substrate. A high density of dislocations is present in the film. (b)&(c) ADF image of a single dislocation and B map. B is enriched at the dislocations. (d) B-K edge from a pristine film region (black), a dislocation-rich region (red) and an etch pit (green).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-2104 The making and electrical biasing of graphene nanoribbon-based devices inside the TEM

Rodríguez-Manzo J. A.1, Puster M.1, Qi Z. J.1, Balan A.1, Charlie Johnson A. T.1, Drndić M.1
1Department of Physics and Astronomy, University of Pennsylvania, Philadelphia, USA
rjulio@sas.upenn.edu

The band gap of graphene nanoribbons (GNR) makes them suitable candidates for sensor devices. Their dimensions (one-atom thickness and widths of tens of nanometers or less) make it difficult to experimentally correlate their electrical properties with changes in their width, edge structure or defect concentration. In this context, we discuss two examples in which GNR-based devices were characterized and also modified within a TEM with the electron beam while an electrical bias was applied.

In the first example, we describe how to fabricate GNR-nanopore devices, which are promising candidates for next-generation DNA sequencing, with the converged electron beam of a TEM [1]. Such devices normally comprise a 2-10 nm diameter pore formed with the beam at the edge or in the center of a 100 nm-wide GNR on a 50 nm-thick silicon nitride membrane. We discuss the changes on GNR conductance when such devices are irradiated with a 200 keV beam and the differences between irradiating with a homogenous (TEM mode) versus a scanned condensed beam (STEM mode). By minimizing the electron dose at 200 kV in STEM mode we were able to prevent electron beam-induced damage and make nanopores in highly conducting GNR. The resulting devices, with unchanged resistances after nanopore formation, can sustain micro ampere currents at low voltages (∼ 50 mV) in buffered electrolyte solution and exhibit high sensitivity, with a large relative change of resistance upon changes of gate voltage, similar to pristine GNR without nanopores (see Figure 1).

It is a truism that before characterizing GNR one must fabricate them, and this is a challenge by itself. In the second example, we describe how to use the condensed beam of a TEM with corrected spherical-aberrations to sputter carbon atoms from predefined areas in electrically-connected free-standing graphene sheets to obtain GNR with sub-10 nm widths [2]. This approach allows us to correlate the lattice and edge structure of sub-10 nm wide GNR with their electrical properties (see Figure 2).

These two examples illustrate the advantages of combining standard TEM observation of GNR-based devices with their electrical biasing as well as the challenges involved in this type of in situ TEM experiment, where chips fabricated with standard lithographic techniques are coupled to TEM sample holders through electrical contacts.

[1] Towards sensitive graphene nanoribbon-nanopore devices by preventing electron beam induced damage. M. Puster, J. A. Rodríguez-Manzo, A. Balan, M. Drndić, ACS Nano 7, 11283 (2013). [2] Correlating atomic structure and transport in suspended graphene nanoribbons. Z. J. Qi, J. A. Rodríguez-Manzo, Andrés R. Botello-Méndez, et al. Submitted for publication (2014).

This work was supported by NIH Grant R21HG004767 and NBIC through NSF NSEC DMR08-32802. We acknowledge use of TEM facilities at Pennsylvania and Rutgers Universities. JZQ and CJ acknowledge SRC contract #2011-IN-2229, NSF AIR Program ENG-1312202. Part of this work was done at the CFN in BNL, supported by the U.S. DOE, Contract No. DE-AC02-98CH10886 (FEI-Titan ACTEM through proposal 31972).

Fig. 1: (a) Chip-carrier and (b) detail of 200 um-wide SiNx window containing 4 GNR. (c) GNR-nanopore device. (d) HAADF STEM image of a nanopore next to a GNR. (e) HAADF STEM image of a 100 nm-wide GNR. Inset: positioning of the beam at the edge of the GNR with a precision of ~ 4 nm. (f) Resistance of a GNR during nanopore formation.

Fig. 2: (a) Sample holder with mounted chip and (b) detail of 500 nm-wide free-standing GNR. (c) Reduction of GNR width by carbon sputtering with the condensed electron beam. (d) HRTEM image of biased sub-10 nm GNR. (e) Resistance changes as a function of GNR width reduction.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-2415 Evidence of re-crystallization in graphene probed by atomic scale imaging

Okuno H.1, Tyurnina A.2, Pochet P.1, Dijon J.2
1CEA-Grenoble, INAC/SP2M, Greoble, France, 2CEA-Greboble, LITEN/DTNM, Grenoble, France
hanako.okuno@cea.fr

Graphene shows great potential for future nanoelectronics due to its extraordinary electronic properties and structure-engineerable nature. Recently CVD based graphene production technology has given insights to the possibility of large scale application. Since the CVD grown films are typically polycrystalline with numerous grain boundaries (GBs), it is important to characterize and control grain size and GBs, generally believed to limit the transport properties of graphene film. In this work, we report on a peculiar grain evolution occurring during the growth process. This evolution is revealed by means of Raman spectroscopy that gives access to a statistically characterization of the graphene structure (grain size, presence of defects etc.). Besides, we need some other complementary techniques to fully understand the detailed atomic structures. This is done by means of atomic-resolution TEM. Indeed, following the invention of aberration corrector (AC), HRTEM direct imaging has become possible on one atom thick layer of carbon. This technique allows us to analyse the detailed atomic structures of and around the GBs together with fundamental information of each grain. In this work, graphene continuous films synthesized on platinum substrate with a specific configuration of CVD set-up are atomically characterized using AC-HRTEM imaging. Fig. 1 shows Raman spectra with corresponding HR-TEM images of our samples at different stages of the growth process. The evolution of both orientation and size of the grains is observed during the process. Nanometer size grains already connected with various orientations (see FFT on the inset) at stage I are further re-oriented and merged together along some pre-dominant directions in stage II and finally form large single crystal domains in the last stage. In stage II, two neighboring grains are typically aligned zig-zag to armchair at the boundary (Fig. 2a), which might be a low-energy crystallographic configuration. Slightly misoriented neighboring domains are connected with the presence of some dislocations (Fig. 2b). A lot of small domains are observed enclosed within larger ones (Fig. 2c) with the same zig-zag to armchair misorientation. Generally in CVD processes, the orientation and achievable size of grains are determined at the early nucleation stage. In our case, the small grains already form a continuous film at the early stage and further transform to low defective large crystals. We infer that such large scale re-crystallization of graphene is enhanced thanks to the platinum substrate. The latter hypothesis is supported density functional theory (DFT) calculation.
[1] O. Lehtinen et al., Nature Communications, DOI : 10.1038 /ncomms3098 (2013)

Fig. 1: Raman spectra of graphene continuous films at different stages of the synthesis process. HR-TEM images (a-c) correspond to typical atomic structures of stage I, II and III, respectively. Fourier transform shown on insets is taken from 50x50nm2 area of each sample. Scale bar is 2 nm.

Fig. 2: The upper panels show low-pass Fourier filtered images and the lower panels with maximum filtering [1]. Typical structures observed at stage II of (a) boundary between grains aligned zigzag to armchair, (b) dislocation between two grains with slight angle mismatch and (c) small domains inside another large grain. Scale bar is 1 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-2445 Atomic Resolution Structure Study of Fluorinated Graphene by Phase Restoration of Focal Series of Images

J Kashtiban R.1, Dyson M. A.1, Raveendran-Nair R.2, Zan R.4, Bangert U.3, Sloan J.1, Geim A. K.2
1University of Warwick, Coventry, UK, 2University of Manchester, Manchester, Uk, 3University of Limerick, 4Niğde University, Niğde, Turkey
r.jalilikashtiban@warwick.ac.uk

One approach towards band gap engineering of the graphene(Gr) which is scalable and cost effective is the chemical modification route to produce 2D derivatives of wonder material of suitable quality for monolayer devices although few such phases exist. GO and (CH)n are disordered or unstable while stoichiometric fluorographene(FG) exhibits significant corrugation. Fluorination of a Gr sheet results in FG, a Gr derivative with each fluorine atom connected to one carbon atom at the basal plane of Gr by a stronger SP3 bonding. Chair-C2F is a highly ordered material that demonstrates selective alternating fluorination and presents with an undistorted 2D morphology in contrast with stoichiometric but corrugated CF with the consequence that the former is a potentially much more tractable material for 2D device fabrication [1].A structural and morphological study of this material by means of atomic resolution TEM has yet not been reported. Exit wave restoration(EWR) by means of focal series of HRTEM images [2] can be performed to recover phase at the exit plane of the sample. Here we reveal, by atomic resolution EWR for the first time , that chair-C2F is a stable Gr derivative and demonstrates long-range order limited only by the size of a functionalized domain. Monolayer C2F was produced by partially fluorinating a suspended CVD grown Gr sample using direct fluorination method [3]. Focal series of images of Gr and chair-C2F were obtained at 80 kV in an aberration-corrected TEM instrument. EWR images reveal that imaged single carbon atoms and carbon-fluorine pairs in chair-C2F alternate strictly over domain sizes of at least 150 nm2 with electron diffraction indicating ordered domains up to ~0.16 μm2. Our results[4] also indicate that, within an ordered domain, functionalization occurs on one side only as theory predicts[1].

Figure 1 EWR and SIM EWRs of pristine Gr and monolayer chair C2F and figure 2 is a demonstration of long-range order within a 64 nm2 domain of C2F.

Refrences:

[1] Şahin, H, et al., Phys. Rev. B, 83 (2011)
[2] Coene, W.M.J., et al., Ultramicroscopy 64(1996) 109
[3] Nair, R.R., et al., Small, 6(2010) 2877
[4] Kashtiban et al., submitted.

We thank the EPSRC for funding through a studentship for M. A. D. and for a P. D. R. A. Fellowship for R. J. K. and additional support provided by the Warwick Centre for Analytical Science (EP/F034210/1).

Fig. 1: Figure 1 a-b, Graphene(Gr) and C2F models.c-d, Equivalent domains of EXP. and SIM. phase for Gr and chair-C2F respectively. e,f line profiles for the EXP and SIM phase for Gr and chair-C2F.g overlaid full plots of the EXP and SIM phase contrast for Gr, respectively.h overlaid full plots of the EXP and SIM phase contrast for chair-C2F, respectively.

Fig. 2: EWR image of a 250 nm2 sheet of highly ordered sheet C2F with an unrippled 64 nm2 domain highlighted. b,  64 nm2 domain in a exhibiting a high degree of order (scale bar = 2 nm). c, Surface plot from b in which the orange-yellow apexes correspond to ordered –CF< units. d, Line profiles (I-III) obtained through either >C–CF< or >C–CF< dumbbells.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-2630 Unstacked double-layer templated graphene for high-rate lithium-sulfur batteries

Zhao M. Q.1, Zhang Q.1, Huang J. Q.1, Tian G. L.1, Nie J. Q.1, Peng H. J.1, Wei F.1
1Beijing Key Laboratory of Green Chemical Reaction Engineering and Technology, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
zhang-qiang@mails.tsinghua.edu.cn

Preventing the stacking of graphene is essential to exploiting its full potential in energy-storage applications. The introduction of spacers into graphene layers always results in a change in the intrinsic properties of graphene and/or induces complexity at the interfaces. Here, we show the synthesis of an intrinsically unstacked double-layer templated graphene via template-directed chemical vapor deposition. The as-obtained graphene is composed of two unstacked graphene layers separated by a large amount of mesosized protuberances and can be used for high-power lithium-sulfur batteries with excellent high-rate performance. Even after 1000 cycles, high reversible capacities of ca. 530 and 380 mA h g-1 are retained at 5 and 10 C, respectively. The preparation of the unstacked DTG is scalable and serves as a general strategy for the fabrication of a broad class of electrode materials for supercapacitors and lithium-ion, lithium-sulfur, and lithium-air batteries. We expect these DTG materials to have potential applications in the areas of environmental protection, nanocomposites, electronic devices, and healthcare because of their intrinsic large surface area, extraordinary thermal and electric conductivity, robust 3D scaffold, tunable surface chemistry, and biocompatible interface. Because unstacked layered nanostructures are not limited to graphene, we foresee a new branch of chemistry evolving in the stabilization of nanostructures through 3D topological porous systems.

References: Zhao MQ, Zhang Q, Huang JQ, Tian GL, Nie JQ, Peng HJ, Wei F. Nature Communications 2014, 5, 3410

We acknowledge the FEI company and Carl Zeiss Microscopy for their technical assistance. This work was supported by the National Basic Research Program of China (973 Program, 2011CB932602) and the Natural Scientific Foundation of China (No. 21306102).

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-2634 Oxidation resistance of reactive atoms in graphene

Chisholm M. F.1, Duscher G.2, Windl W.3
1Oak Ridge National Laboratory, Oak Ridge, TN, USA, 2University of Tennessee, Knoxville, TN, USA, 3The Ohio State University, Columbus, OH, USA
chisholmmf@ornl.gov

Carbon layers down to a thickness of a single layer have been known to form on metal surfaces for more than 50 years. Graphene on metal surfaces is also known to lead to catalytic deactivation.[1,2] However, it has not yet been recognized that graphene can also offer a level of oxidation protection to individual atoms or small clusters of atoms in/on the graphene layer. Here we will show using annular dark field imaging in a scanning transmission electron microscope and electron energy-loss spectroscopy (EELS) that Si and Fe atoms incorporated in a graphene layer are not oxidized. We further combine the microscopy data with first-principles density-functional calculations of the reaction of the impurity with graphene and the impurity with oxygen. Interestingly, our density-functional theory calculations explain these observations are due to preferential bonding of O to non-incorporated atoms and H-passivation effects.

Figure 1 shows annular dark-field images of a single Si atom segregated to a carbon vacancy and a single Fe atom segregated to a carbon divacancy. Si has also been found in graphene divacancies, but Fe has not been imaged in graphene single vacancy sites. The observed intensities are consistent with there being just a single impurity atom incorporated in the graphene defects, i.e. no additional oxygen. EELS confirms the identification of these impurity atoms as Si and Fe. The spectrum from a Si atom (Fig. 2c) shows an edge onset of about 102eV which is less than the oxide value and close to the edge onset for Si in of SiC. No oxidation features are apparent in the Si-L2,3 edge and no O-K edge was detectable in the spectrum. The core loss spectrum from a Fe atom (Fig. 2f) not only confirms the identification it also indicates that the Fe atom is not oxidized. The ratio of the Fe L3/L2 edges is lower than any seen for the various forms of Fe oxide. There have been no previously published experimental or theoretical studies on the oxidation resistance of iron or silicon incorporated in graphene to our knowledge. This is a potentially important discovery. Improved resistance to oxidation has important consequences for some catalytic reactions and small devices based on single atoms or small clusters of non-noble metals. Graphene as a substrate appears to protect single atoms and small clusters of atoms from oxidation without completely isolating them.[3]

References

1. S. Hagstrom, H.B. Lyon, G.A. Somorjai, Phys. Rev. Lett. 15 491 (1965).

2. R. Schlogl in Handbook of Heterogeneous Catalysis, vol. 1 G. Ertl, H. Knozinger, F. Schuth, J. Weitkamp (eds.) Wiley-VCH Weinheim 2008 p. 357.

3. M.F. Chisholm, G. Duscher, W. Windl, Nano Lett. 12 4651 (2012).

This research was supported by the Materials Sciences and Engineering Division of the Office of Basic Energy Sciences, U.S. Dept. of Energy and by NSF Award Number DMR-0925529 and the Center for Emergent Materials at The Ohio State University, a NSF MRSEC (Grant DMR-0820414). W.W. acknowledges support from the Ohio Supercomputer Center under project PAS0072.

Fig. 1: ADF images of graphene. The as recorded data (a,b) were corrected to remove noise and probe tail effects (c,d). Images a and c show a Si atom in a C vacancy site. Images b and d show an Fe atom in a C divacancy site. The scale mark on each image corresponds to 0.2 nm. Taken from Ref. 3.

Fig. 2: EEL spectrum image data from single atoms incorporated in graphene. ADF image of a Si atom in graphene (a), Si composition map (b) and spectrum obtained over the Si atom (c). ADF image of an Fe atom (d), Fe composition map (e) and the spectrum obtained over the Fe atom (f). The scale marks correspond to 0.2 nm. Taken from Ref. 3.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-2651 Dislocations in bilayer graphene — Materials science meets physics

Butz B.1, Dolle C.1, Niekiel F.1, Weber K.2, Waldmann D.3, Weber H. B.3, Meyer B.2, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany, 2Interdisciplinary Center for Molecular Materials (ICMM) and Computer Chemistry Center (CCC), Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany, 3Chair for Applied Physics, Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany
benjamin.butz@ww.uni-erlangen.de

Dislocations represent one of the most fascinating and fundamental concepts in materials science. First and foremost, they are the main carriers of plastic deformation in crystalline materials. Furthermore, they can strongly affect the local electronic and optical properties of semiconductors and ionic crystals. In materials with small dimensions they experience extensive image forces, which attract them to the surface in order to release strain energy. However, in layered crystals like graphite dislocation movement is mainly restricted to the basal plane. Thus the dislocations cannot escape enabling their confinement in crystals as thin as only two monolayers. To explore the nature of dislocations under such extreme boundary conditions, the material of choice is bilayer graphene, the thinnest imaginable quasi-2D crystal, in which such linear defects can be confined. Homogeneous and robust graphene membranes (Figure 1a, b) derived from high-quality epitaxial graphene on SiC [1] provide an ideal platform for their investigation.

Here we report on the direct observation of basal-plane partial dislocations (Burgers vector 1/3<1-100>) in freestanding bilayer graphene (Fig. 1a, b) by transmission electron microscopy and their detailed investigation by diffraction contrast analysis (Figure 1c, Burgers vector analysis 2c) and atomistic simulations (Figure 2a, b, and e) [2]. Our investigation reveals striking size effects. First, the absence of stacking fault energy, a unique property of bilayer graphene, leads to a characteristic dislocation pattern (Figure 1c, center), which corresponds to an alternating AB ↔ BA change of the stacking order (Figure 1c, right). Most importantly, our experiments in combination with atomistic simulations reveal a pronounced buckling of the bilayer graphene membrane (Figure 2a-d), which directly results from accommodation of strain (Figure 2e). In fact, the buckling completely changes the strain state of the bilayer graphene and is of key importance for the electronic properties. Our findings will significantly contribute to the future understanding of the structural, mechanical and electronic properties of bilayer and few-layer graphene.

[1] D. Waldmann, B. Butz, S. Bauer, J.M. Englert, J. Jobst, K. Ullmann, F. Fromm, M. Ammon, M. Enzelberger, A. Hirsch, S. Maier, P. Schmuki, T. Seyller, E. Spiecker, H.B. Weber, ACS Nano 7 (2013) 4441-4448

[2] B. Butz, C. Dolle, F. Niekiel, K. Weber, D. Waldmann, H.B. Weber, B. Meyer, E. Spiecker, Nature 505 (2014) 533-537

We thank J. Müller, E. Bitzek and A. Kohlmeyer for discussion and P. Schmuki for use of equipment. The research was supported by the DFG (SFB953, Cluster of Excellence EXC 315).

Fig. 1: a) Graphene membranes on SiC. b) One membrane at higher magnification: number of layers indicated. c) Series of bright-field (BF) and dark-field (DF) TEM images of same area. {11-20} images show pronounced contrast due to the presence of partial dislocations (dark lines), while {2-200} images depict respective changes of stacking sequence AB ↔ BA.

Fig. 2: a) Membrane topography and b) side-/top-view of pair of partial dislocations (change of stacking sequence enlarged shown). c) Burgers vector analysis using {11-20} DF images. d) Validation of Burgers vector analysis and atomistic model by DF-image simulation. e) Atomistic-strain distributions and derived disregistry/Burgers vector distributions.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-2924 Thermal contact resistance between multiwalled carbon nanotubes and supporting substrates measured using electron thermal microscopy

Nilsson H.1, Voskanian N.1, Cumings J.1
1Department of Material Science and Engineering University of Maryland, College Park
hnilsson@umd.edu

Multiwalled carbon nanotubes (MWCNTs) have a high intrinsic thermal conductivity which makes them a promising material for heat management in nanoscale electronic devices. However, experimental results have shown that the total conductance is strongly limited by microscopic thermal resistances. These include the contact resistance between the nanotube and the substrate it rests upon, as well as the contact resistance with metal contacts. Due to characterization difficulties, exact values of the contact resistances have not been determined. In fact, literature estimates vary greatly. Some of these characterization difficulties include spatial resolution and the inability to separate resistance values within the system. An in-situ TEM technique developed by our group, called Electron Thermal Microscopy (EThM) allows us to obtain thermal maps with spatial resolution on the order of 10s of nanometers. The technique uses a specialized holder to locally heat an individual nanotube either directly by biasing or passively by a connected palladium heater wire. Indium nanoislands deposited on the backside of the sample membrane act as local temperature probes; their phase transition from solid to liquid at 156 degrees Celsius can easily be seen with dark field TEM imaging. Previous results using this technique have determined that the contact resistance with the silicon nitride is at least 250 Km/W (K. H. Baloch, N. Voskanian, M. Bronsgeest, and J. Cumings, "Remote Joule heating by a carbon nanotube," Nature Nanotechnology, 2012.). Here we present new device geometries featuring slits in the membranes to control the spread of heat through the membrane. This allows us to separate the heat transferred to the substrate via the MWCNT and via metal contacts. With more precise determination of the contact resistances it will be possible to get a better understanding of the thermal transport physics of MWCNTs.

Fig. 1: Schematic showing sample device geometry. A nanotube is heated passively via a palladium heater wire. Both sides of the tube are anchored with palladium pads. The slit through the membrane underneath one side of the nanotube is used to control heat transport across the membrane so that the contact resistance can be determined.

Fig. 2: Dark field TEM image demonstrating the melting of indium nanoislands when current is passed through a simple palladium heater wire.

Fig. 3: TEM image showing a palladium heater wire patterned on top of a nanotube.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-3121 Imaging functional groups in graphene oxide at atomic resolution

Moreno M. S.1, Boothroyd C. B.2, Duchamp M.2, Kóvacs A.2, Monge N.3, Morales G. M.3, Barbero C. A.3, Dunin-Borkowski R. E.2
1Centro Atómico Bariloche, 8400 - S.C. de Bariloche, Argentina, 2ER-C and PGI-5, Forschungszentrum Jülich, D-52425 Jülich, Germany, 3Universidad Nacional de Río Cuarto, X5804BYA Río Cuarto, Argentina
meketo@gmail.com

Graphene oxide is a form of graphene with its surface modified by the addition of functional groups such as carboxylic groups, ketones and hydroxyl. The structure and distribution of the functional groups depends on the synthesis method used and they affect its chemical, electrical and mechanical properties. Of particular interest is the chemical composition and spatial distribution of these functional groups. Direct identification of the groups by high-resolution imaging is not possible at present, but they can be made visible by bonding heavy atoms, such as Ba, to selected groups and imaging the distribution of the heavy atoms.
High-resolution images of Ba doped graphene oxide taken at 80kV in a Cs and Cc corrected transmission electron microscope (TEM) show the structure of the graphene oxide with minimal electron beam damage but are not able to identify the Ba atoms. We attempted to use energy-filtered electron microscopy to image the Ba M5 and M4 edges at 781eV but radiation damage over the long exposures required prevented location of the Ba atoms to better than a few nm. Compositions measured using energy loss spectroscopy (EELS) revealed a progressive loss of O with increasing temperature and that 1% O is retained at 800°C.
High-resolution scanning transmission electron microscopy (STEM) at room temperature proved to be impossible due to contamination from migration of the functional groups on the graphene surface. Contamination was overcome by imaging with the graphene oxide above 400°C in a stable heating holder.
Simultaneous STEM energy-loss spectrum images (SI) and high-angle annular dark-field (HAADF) and bright-field (BF) images acquired at a specimen temperature of 800°C allowed us to correlate the location of the Ba atoms with features in the high-angle dark-field images at atomic resolution.
Simultaneously acquired HAADF and BF-STEM images (Fig. 1) show bright and dark dots at the same positions. These bright dots are identified as Ba atoms by EELS as shown in Fig. 2a. The corresponding elemental maps extracted from the SI are shown in Fig. 2b-d. The maps also show that Ca and O occur together and that Ba is not associated with a significant concentration of O. The positions of Ba atoms attached to functional groups on graphene oxide can therefore be mapped with atomic spatial resolution by using a combination of STEM and TEM techniques. The fact that Ca was observed to correlate strongly with O suggests that it could be used as a marker for the positions of the O-containing groups.

We acknowledge support from the European Union under the FP7 and a contract for an Integrated Infrastructure Initiative (ESTEEM2) and CONICET (Argentina) with a CONICET-DFG grant.

Fig. 1: Simultaneously acquired (a) HAADF and (b) bright-field STEM images at a specimen temperature of 800°C after recording the spectrum image (Fig.2), whose approximate area is marked by the box in (a). The bright dots in the HAADF image (a) correspond to the dark dots in the bright-field STEM image (b).

Fig. 2: (a) EEL spectrum extracted from a 3x3 pixel (0.25x0.25 nm) part of the spectrum image centred on a single Ba atom marked by the arrow in (b). (b-d) Elemental maps of the Ba M4,5, O K and Ca L2,3 edges obtained from the SI (200x200 spectra, dwell time 0.02s per spectrum, after VCA processing).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-3205 Graphene Nanoribbons with atomically well-defined edges through Scanning Transmission Electron Microscopy

Vicarelli L.1, Xu Q.1, Zandbergen H. W.1
1Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands
l.vicarelli@tudelft.nl

We recently demonstrated a controllable and reproducible method to obtain suspended monolayer graphene nanoribbons with atomically defined edge shape [1]. Our method exploits the electron-beam of a Scanning Transmission Electron Microscope (accelerated at 300 kV) to create vacancies in the lattice by knock-on damage and pattern graphene in any designed shape.

The small beam spot size (0.1 nm) enables close-to-atomic cutting precision, while heating graphene at 600o C during the patterning process avoids formation of beam-induced Carbon deposition and allows self-repair of the graphene lattice. Self-repair mechanism is essential to obtain well-defined (zig-zag or armchair) edge shape and, if the electron beam dose is lowered, to perform non-destructive imaging of the graphene nanoribbons.

Drawing the electron-beam path with a software script, we were able to obtain reproducible graphene nanoribbons with a minimum width of 2 nm and defined edges (see Fig. 1 and 2). In order to unravel some of the predicted properties of these graphene nanoribbons, we are currently exploring their transport properties through in-situ electrical measurement inside a Transmission Electron Microscope.

Early results show that large-area suspended graphene is stable over gaps of ~1 μm size. Both CVD grown and exfoliated graphene have been used. Performing 2 wire measurements, we saw that contact resistance between graphene and gold contact pads has a non-neglectable influence on the measurements, although it can be greatly reduced with in-situ thermal annealing above 300°C.

References:
[1] Q.Xu, M. Wu, G. F. Schneider, L. Houben, S.K. Malladi, C. Dekker, E. Yucelen, R.E. Dunin-Borkowski, and H.W. Zandbergen, ACS Nano 2013 7 (2), 1566-1572

The research leading to these results has received funding from the European Research Council, ERC Project n. 267922

We thank Kavli NanoLab Delft for the support provided in the fabrication of our NEMS devices. 

Fig. 1: Annular dark-field STEM image of a nanoribbon array, illustrating the reproducibility of the patterning. These four patterns were created using a script-controlled electron beam.

Fig. 2: HRTEM image of a nanoribbon in monolayer graphene sculpted at 300 kV and 600o C and imaged at 80 kV and 600o C. The yellow line indicates a zig-zag edge. An atom structure model for zig-zag edge is given as inset in the figure.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-3438 3D insight on the catalytic nanostructuration of few-layer graphene

Melinte G.1, 2, Janowska I.2, Baaziz W.2, Florea I.1, Moldovan S.1, Arenal R.3, 4, Wisnet A.5, Scheu C.5, Begin-Colin S.1, Begin D.2, Pham-Huu C.2, Ersen O.1
1Institut de Physique et Chimie des Matériaux de Strasbourg, CNRS-Université de Strasbourg, 23, rue du Loess, 67037 Strasbourg, France , 2Institut de Chimie et Procédés pour l’Energie, l’Environnement et la Santé, CNRS, ECPM, Université de Strasbourg, 25, rue Becquerel, 67087 Strasbourg, France, 3Laboratorio de Microscopias Avanzadas, Instituto de Nanociencia de Aragon, Universidad de Zaragoza, 50018 Zaragoza, Spain, 4ARAID Fundation, Calle Mariano de Luna, 50018 Zaragoza, Spain, 5Department of Chemistry and Center for NanoScience, Ludwig Maximilians University, Butenandtstr. 11, 81377 Munich, Germany
georgian.melinte@ipcms.unistra.fr

The catalytic cutting of few-layer graphene (FLG) is attracting nowadays an increase attention due to its potential applications in both the field of catalysis and graphene nanoribbons (GNRs) fabrication.[1,2] The nanopatterning of FLG sheets with open and subsurface channels develops during a chemical reaction between the metal nanoparticles (NPs) and the carbon substrate under a hot gaseous atmosphere (H2, O2).[3,4] The use of the FLG powder in the field of catalysis is limited by the restacking process which significantly decreases its surface accessibility. By channeling the FLG sheets the restacking effect is significantly reduced. This is due to the creation of a porous network which will not only increase the surface accessibility but also will create a network of defects that will further serve as anchorage sites for the surface decoration. Moreover, the nanopatterning of FLG has the potential of creating nanosheets with well-defined shapes and edge configurations which can be transformed in single-layer GNRs by simple techniques as for instance the chemical exfoliation.
To characterize the channeling process and the obtained nanostructures we used an initial system consisting in Fe3O4 NPs dispersed on FLG sheets. The FLG/Fe3O4 composite has undergone a heating treatment in a H2 atmosphere. HR-TEM shows that the well-defined channels are not randomly oriented but follow specific crystallographic directions i.e. <11-20> and <10-10> (Figure 1), leading to the formation of two types of edge morphologies, zigzag and armchair, respectively. The electron tomography analyses reveal interesting features on both the nanopattering mechanisms and properties of the nanostructured FLG sheets. Figure 2 indicates the effect of a topographical step-up of the FLG sheet . Accordingly, one observes that after interaction the cutting direction remains unchanged but the depth of the open-surface channel is changing proportional with the height of the step-up. Figure 3 displays the impact of a step-up event with the height larger than the size of the NP. As previously, the cutting direction remains unmodified and the result is the formation of subsurface channel. When topographic step-down events are encountered by the active NPs, the particle either stops or changes the direction.
[1] Mei-xian Wang et al., J. Phys. Chem. Lett., 4, 1484−1488 (2013).
[2] Ci Lijie et al., Nano Res. 1, 116-122 (2008).
[3] Tim J. Booth et.al, Nano Lett. 11, 2689–2692 (2011).
[4] Datta S. et. al Nano Lett. 8(7), 1912-1915 (2008).

Fig. 1: HR-TEM images illustrating the preferential crystallographic orientation of the tranches with a zigzag (a) and armchair (b) edge morphology (scale bars 5 nm).

Fig. 2: a) TEM projection of a selected channel (scale bar 50 nm). b) XY slice through the reconstructed volume. c) XZ slices through the selected channel at the positions numbered in Figure 1a. d) YZ slice taken through a region indicated in Figure 1a with a yellow dotted line. Scale bars 20 nm.

Fig. 3: a) TEM projection of two subsurface channels (scale bar 50 nm). b) XY slice through the reconstructed volume (scale bar 20 nm). c) and d) YZ slices taken at the position indicated in Figure 1a with the yellow line (scale bar 20 nm).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-2-O-3480 Plasmon Tailoring in Graphene through Lattice Impurities and Ad-Atoms

Pan C. T.1, Pierce W.2, Boothroyd C.3, Ramasse Q.4, Kepaptsoglou D.4, Bangert U.5
1School of Physics and Astronomy, The University of Manchester, Manchester M13 9PL, UK, 2School of Materials, The University of Manchester, Manchester M13 9PL, UK, 3Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Gruenberg Institute, Juelich Research Centre, D-52425 Juelich, Germany, 4SuperSTEM Laboratory, STFC Daresbury Campus, Daresbury WA4 4AD, UK, 5Department of Physics and Energy, University of Limerick, Limerick, Ireland
Ursel.Bangert@ul.ie

Although there is considerable documentation on efforts to tailor and employ plasmons to merge photonics and electronics, and use surface plasmons for subwavelength optics [1,2] and enhancement of the photovoltaic conversion efficiency, especially by making use of the surface plasmons at metal nano-clusters [3], little has been reported on single atom plasmon effects [4]. Graphene’s potential for terahertz nano-scale plasmonic devices, has so far only been realised via gating and patterning [5,6]. However, defects in the graphitic plane, including vacancies and dopant atoms, can intrinsically alter the electronic structure and hence lead to effects such as plasmon enhancement and change of the plasmon energy in the uv/vis region, a phenomenon that can be exploited for coupling with light.

We present observations, using high resolution (S)TEM in combination with electron energy loss spectroscopy and energy filtered imaging, of the effects of single or few-atom impurities on plasmons in the uv/pi-plasmon energy regime in graphene. We accompany the experiments by WIEN calculations, which reveal new transitions in graphene for various metal ad-atoms species (Ti, Pd) and also for Si (fig. 1) and substitutional dopants such as B and N: a peak at around 1-2 eV is introduced which is not present in energy loss spectra of pristine graphene. Both, position and intensity of this peak change according to doping/dosing levels. The increase of the latter shifts this peak towards the uv regime (3eV). These transitions are mostly ascribed to single particle (SPE) and intraband excitations or to SPE-π plasmon coupling and not to the creation of new plasmon peaks in the graphene-dopant system. The same applies to defect and edge-states. Our experimental observations are in general support of the above predicted additional absorption features in the uv. More so, we observe intensity enhancement around metal atoms (e.g., Pd) at graphene edges (fig. 2), which we also find, although to a much lesser extent, at pristine graphene edges. This intensity increase does, however, not arise from new spectral features and is ascribed to the enhancement of intrinsic low loss features of graphene, where metal atoms/ defects act as atomic antennae, due to donation of d-electrons, in the case of transition metals. The efficiency of this process appears to vary with the transition metal, and seems to be high for, e.g., Pd.

[1] W L Barnes et al, Nature , 424 (2003) 824-830

[2] S A. Maier, H A Atwater, J. Appl. Phys 98 (2005) 011101

[3] J Nelayah, et al Nature Physics, 3 (2007) 348-352

[4] W Zhou et al, Nature Nanotech, 7 (2012), 161-165

[5] T J Echtermeyer et al, Nature Comms (2011), DOI: 10.1038/ncomms1464

[6] L Ju et al Nature Nanotech, DOI: 10.1038/NNANO (2011) 146

Fig. 1: Simulated in-plane (left) and out-of-plane (right) EEL spectra of a single (a) Au, (b) Pd, (c) Cr, (d) Ti and (e) Si adatom on (solid curves) and spectra after the carbon atoms of the graphene are removed (red dashed curves). Spectra are shifted along the Y axis and are all on the same scale. The two bottom spectra are of pristine graphene.

Fig. 2: Images from an EFTEM image series obtained in a monochromated triple -corrected Titan-PICO: a) enhancement at 3.5-4 eV and b) depletion at 5-5.5 eV of the loss intensity at a hole in graphene with Pd deposit, c) HREM image prior to the EFTEM series with magnified boxed area, showing Pd atoms (arrowed) decorating the edge of the hole.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-1483 Structural Analysis of Electron-Beam-Irradiated C60 Single Crystal Films Using High-Resolution Transmission Electron Microscopy and Electron Diffraction

Masuda H.1, Yasuda H.2, Onoe J.1
1Tokyo Institute of Technology, 2Osaka University
hidmasuda@nr.titech.ac.jp

  We have found that one-dimensional (1D) uneven peanut-shaped C60 polymer is formed by electron-beam (EB) irradiation of a pristine C60 film [1], and exhibits novel physical properties arising from 1D metal, such as the geometric curvature effects on the Tomonaga-Luttinger liquid states [2]. For the polymer structure, in situ infrared (IR) spectra and density-functional calculations suggested the 1D polymer has a cross-linked structure (Fig. 1(c)) roughly close to that of the P08 C120 isomer (Fig. 1(b)) obtained from the general Stone-Wales transformation [3]. Although the previous results indicated the polymer to have 1D peanut-shaped structure, we have examined the structure of the 1D polymer formed from a C60 single crystal (SC) film more precisely, using HRTEM and electron diffraction (ED).
  The 1D C60 polymer film was formed on a mica substrate by EB irradiation of a pristine C60 SC film in an UHV chamber. After confirmed that all C60 molecules were polymerized to form the 1D polymer using in situ IR spectroscopy, we ripped the film off the mica and mounted it on a Cu mesh in air, and observed the film by TEM.
  Figure 2 shows HRTEM images and ED patterns of the pristine and the EB-irradiated C60 films. The C60 film is a FCC SC with [111] orientation, which contains twins as stacking faults on (111), and shows weak spots E1 and E2 as 1/3 and 2/3 of 422 series, respectively. The EB-irradiated C60 SC film shows three new features. Firstly, E1 and E2 become intense, indicating symmetry reduction and FCC transferred to HCP. Secondary, spots of 220 series become doublet. Since the corresponding distance of these spots is 5.0 Å and 4.6 Å, respectively, the intermolecular distance (di) between adjacent C60 molecules is estimated to be 10.0 Å and 9.3 Å for each. Finally, each spot becomes an arc-like stretched line of ca. 9.2°. This arises from a slight loss of the orientation. These results show the asymmetric shrinkage of crystal structure along one given direction.

  C60 FCC structure changes to 1D polymer BCO (Fig. 1(d)). Furthermore, judging from the intense E1 spot, BCO changes to HCP-m (Fig. 1(e)). Figs. 2(e, f) show the simulated ED patterns of BCO and HCP-m based 1D C60 polymer model (the di of 9.3 Å) with 3-fold symmetry derived from three possible polymerization directions on (111) of FCC C60 SC film, using QSTEM code [4]. Since each pattern well reproduces the experimental ED pattern, a mixed stacking model of BCO and HCP-m 1D C60 polymer structures is suitable for the EB-irradiated C60 SC film [5].
[1] J. Onoe et al., Appl. Phys. Lett. 82, 595 (2003).
[2] J. Onoe et al., Europhys. Lett. 98, 27001 (2012).
[3] A. Takashima et al., J. Phys. D: Appl. Phys. 45, 485302 (2012).
[4] C. Koch, Ph.D. thesis (2002).
[5] H. Masuda et al., to be submitted.

This work was supported by “Advanced Characterization Nanotechnology Platform (MEXT)” at Osaka University and by the collaborative research fund of J-Power.

Fig. 1: Molecular models of C60 (a), P08 C120 isomer (b), 1D C60 polymer (c). The unit cell of 1D C60 polymer with body-centered orthorhombic structure (BCO) (d), and hexagonal closed-packed-based monoclinic structure (HCP-m).

Fig. 2: Experimental HRTEM images (insets: FFT patterns) and ED patterns of pristine C60 SC (a, c), EB-irradiated C60 SC (b, d), simulated ED patterns using 1D C60 polymer model with intermolecular distances of 9.3 Å in polymer direction (e, f).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-1536 Mapping electronic states in Graphene

Löffler S.1,2, Pardini L.3, Hambach R.4, Kaiser U.4, Schattschneider P.1,2, Draxl C.3
1Institute of Solid State Physics, Vienna University of Technology, Austria, 2University Service Centre for Transmission Electron Microscopy, Vienna University of Technology, Austria, 3Department of Physics, Humboldt University Berlin, Germany, 4Electron Microscopy Group of Materials Science, Ulm University, Germany
stefan.loeffler@tuwien.ac.at

Graphene and similar carbon-based materials are currently the focus of much research. In particular, their peculiar electronic properties are arousing a lot of interest. At the same time, the question of the influence of defects – such as vacancies or dopant atoms – is of particular practical importance. Recently, it has been reported that different dopant atom configurations change the charge distribution [1] and, therefore, give rise to different electron energy-loss spectrometry (EELS) signals [2]. Likewise, introducing vacancies changes the local crystal structure [3] and, hence, also the local charge distribution and EELS signal.

This gives rise to the hope to map the electronic states using energy-filtered transmission electron microscopy (EFTEM) [4]. In this work, we present predictions regarding the possibility of direct mapping of electronic states in both ideal, pristine Graphene and Graphene with defects using EFTEM. To that end, calculations of the electronic states of Graphene, with and without defects, were carried out using the full-potential all-electron density functional theory (DFT) package "exciting" [5]. Subsequently, its output was used to calculate the inelastic scattering kernels which, combined with elastic scattering calculations, ultimately result in EFTEM images.

The EFTEM images were calculated for a variety of acceleration voltages and lens aberration functions to simulate realistic conditions, as well as investigate the optimal experimental conditions. Fig. 1 shows EFTEM simulations for Graphene with and without core-hole effects included in the DFT calculations. It is clearly visible that this greatly alters the expected EFTEM images. Fig. 2 goes a step further and shows the images to be expected when mapping a vacancy. This demonstrates that EFTEM at high spatial resolution could become an invaluable tool for the study of electronic states in carbon-based materials.

[1] Meyer et al., Nat Mater 10 (2011) 209
[2] Zhou et al., PRL 109 (2012) 206803
[3] Meyer et al., Nano Lett 8 (2008) 3582
[4] Löffler et al., Ultramicroscopy 131 (2013) 39
[5] http://exciting-code.org/

The authors acknowledge financial support by the FWF (I543-N20), the DPG and the MWK Baden-Württemberg.

Fig. 1: Simulated EFTEM images for 40 keV electrons for pristine multi-layer Graphene without (left) and with (middle) core-hole effects under ideal imaging conditions. In both cases, an energy loss of 6.5 eV above the C K-edge onset was used. Additionally, the partial density of states is shown (right).

Fig. 2: Simulated EFTEM images for Graphene with a vacancy. The images show π* (left) and σ* (right) states. The circle marks the vacancy. Both images were simulated for a beam energy of 80 keV and ideal imaging conditions.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-1730 In-Situ Environmental TEM Observation of the Formation of Defects in Growing Carbon Nanotubes

Yoshida H.1, Takeda S.1
1The Institute of Scientific and Industrial Research, Osaka University
h-yoshida@sanken.osaka-u.ac.jp

As-grown carbon nanotubes (CNTs) generally have various grown-in defects, such as vacancies, pentagon-heptagon pairs, bending, and irregular interlayer spacing. It is well known that the electronic and mechanical properties of CNTs are affected by these grown-in defects. An understanding of the formation mechanism of the CNT grown-in defects is required for the growth of defect-free CNTs exhibiting ideal properties and CNTs with intentionally induced defects exhibiting modified properties. Recent environmental transmission electron microscope (ETEM) [1] observations of chemical vapor deposition (CVD) growth of CNTs have provided us with knowledge of the growth mechanism. We have clarified that CNTs grow from structurally fluctuating iron carbide Fe3C and iron molybdenum carbide (Fe,Mo)23C6 nanoparticles [2-4]. However, in situ studies on the formation of defects in growing CNTs are limited. In this study, we have elucidated the origin of grown-in defects in CNTs, such as bending, irregular interlayer spacing, change in the diameter, and change in the number of graphitic layers, by in situ atomic-scale ETEM observations of the CVD growth of CNTs [5].

Figure 1 shows the growth of a CNT with a drastic disorder of the interlayer spacing. We also observe large changes in the CNT diameter during growth as shown in Fig. 2. Our ETEM observations clearly demonstrate that deformation of the nanoparticle catalysts during CNT growth triggers the formation of these grown-in defects [5]. The small deformation of nanoparticle catalysts at the interface with CNTs gives rise to the formation of bends and disorder of the interlayer spacing (Fig. 1) in CNTs. The changes in the diameter (Fig. 2) and number of graphitic layers in CNTs are caused by the large protrusion on and shrink deformations of nanoparticle catalysts. Based on the ETEM observations, we will propose the formation mechanism of grown-in defects in CNTs.

References

[1] S. Takeda and H. Yoshida, Microscopy, 62 (2013) 193.

[2] H. Yoshida, S. Takeda, T. Uchiyama, H. Kohno, and Y. Homma, Nano Lett., 8 (2008) 2082.

[3] H. Yoshida T. Shimizu, T. Uchiyama, H. Kohno, Y. Homma, and S. Takeda, Nano Lett., 9 (2009) 3810.

[4] H. Yoshida, H. Kohno, and S. Takeda, Micron, 43 (2012) 1176.

[5] H. Yoshida and S. Takeda, Carbon, 70 (2014) 266.

This work was supported by JSPS KAKENHI Grant Number 24710117.

Fig. 1: Drastic disorder in the interlayer spacing of graphitic layers in a growing CNT from a nanoparticle catalyst [5]. The observation time is shown in the images.

Fig. 2: Large Change in the diameter of a growing CNT from a nanoparticle catalyst [5]. The lattice images in the nanoparticle catalyst corresponds to a (Fe,Mo)23C6-type structure [3,4]. The observation time is shown in the images.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-1745 Electron microscopic evidence for a tribologically induced phase transformation as the origin of wear in diamond

Zhang X.1, Schneider R.1, Müller E.1, Mee M.2, Meier S.2, Gumbsch P.1, 2, Gerthsen D.1
1Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany, 2Fraunhofer Institute for Mechanics of Materials IWM, Freiburg, Germany
xinyi.zhang2@kit.edu

The origin of wear and the low friction coefficient of diamond is still an intensely debated problem in tribology. Here we study coarse-grained diamond films, deposited by plasma-enhanced chemical vapor deposition, which were tribologically loaded on a ring-on-ring tribometer against a similar diamond counterpart. The microstructure of worn and unworn regions of the diamond film was studied by transmission and scanning electron microscopy. Amorphous carbon (a-C) layers are observed on both as-deposited and on tribologically tested diamond, but differ significantly as far as thickness and morphology are concerned. The a-C layer with a thickness of up to several 100 nm on as-deposited diamond is attributed to the plasma deposition process. For the tribologically tested region of the film, the TEM images (Fig. 1) demonstrate that the µm-sized grains at the rough original diamond surface are almost completely flattened indicating that a significant amount of material must have been removed including the residual a-C layer from the deposition process. In contrast to the as-deposited a-C residue, the tribo-induced a-C layer is comparably uniform with a thickness below 100 nm. The TEM sample from the wear track prepared by conventional techniques (Fig. 2) confirms the findings of the FIB-prepared sample. A few of the TEM samples containing a tribo-induced a-C layer contain grain boundaries of the underlying polycrystalline diamond in the electron transparent region. It is found that the thickness of the a-C layer changes quite abruptly on grains with different crystallographic orientations (white arrow in Fig. 2). Fig. 3 clearly shows that the interface between the crystalline diamond and the tribo-induced amorphous a-C layer is not crystallographically flat but displays a nm-scale roughness. The anisotropic phase transformation and the small roughness of the interface are regarded as evidence for an atom-by-atom wear process. Quantitative electron energy loss spectroscopy of the C-K ionization edge, performed in a transmission electron microscope, reveals the transition from sp3-hybridized C-atoms in diamond to a high fraction (65 %) of sp2-hybridized C-atoms in the tribo-induced a-C layer within a region of less than 5 nm thickness.

XZ acknowledges funding from China Scholarship Council (CSC) (No. 2010606030). PG acknowledges support from Deutsche Forschungsgemeinschaft DFG (project grant Gu 367/30).

Fig. 1: Overview TEM image of a cross-section FIB-lamella taken from the wear-track region.

Fig. 2: Overview TEM image of a conventional cross-section TEM sample prepared from the wear-track region.

Fig. 3: HRTEM image of the interface region between crystalline diamond and the tribo-induced amorphous carbon layer with the diamond oriented along the [111] zone axis. The approximate position of the interface is marked by a dashed line.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-1752 Atom-by-atom STEM EELS investigation of n- and p- doped graphene.

Kepaptsoglou D. M.1, Seabourne C. R.2, Hardcastle T.2, Nicholls R.3, Pierce W.4, Zan R.4, Bangert U.4, Scott A. J.2, Ramasse Q. M.1
1SuperSTEM Laboratory, STFC Daresbury Campus, United Kingdom, 2Institute for Materials Research, SPEME, University of Leeds, Leeds, United Kingdom, 3Department of Materials, University of Oxford, Oxford, United Kingdom, 4School of Materials, University of Manchester, Manchester, M13 9PL, United Kingdom
dmkepap@superstem.org

Graphene, or the miracle material as it has become known, has promised to revolutionize the world of electronics by replacing Si-based technology [1,2]. Graphene however is an excellent conductor – often described as a ‘zero bandgap’ semiconductor, an attribute which so far limits its widespread application in devices. Among various solutions to tailor its properties for practical implementation, the introduction of dopants in the graphene lattice is predicted to have a drastic effect on graphene's band structure [3], such as the opening of an optical bandgap or an increase in charge carrier density resulting in n- or p-type doping, with carrier concentrations allowing practical transistor applications. The introduction of dopants such as N in graphene is most commonly achieved during the chemical growth process, with varying levels of success regarding the purity of the samples, which often contain contaminants, defects and secondary impurities. We have recently demonstrated an alternative, cleaner method by successfully doping freestanding single layer graphene with N and B through low energy ion implantation [4], achieving retention levels of the order of ~1%.

In this work we use STEM-based spectroscopy [5,6], to study the impact of single N or B dopant atoms on the electronic structure of the graphene membrane. Z-contrast imaging and atomically resolved electron energy loss spectroscopy were performed in a Nion UltraSTEM100 dedicated STEM instrument and were used to unambiguously identify single dopant atoms (fig. 1) and to determine the doping levels as a function of ion implantation energy and flux. Furthermore, the electronic structure modifications due to the presence of these dopant B or N atoms are strikingly demonstrated by a clear signature in the near-edge fine structure of the B and N EELS K edges but also that of C K edge of neighboring C atoms (fig. 1). Ab initio calculations are used to simulate experimental spectra (fig. 2) and to rationalize the experimental observations, thus providing further insight into the nature of bonding around the foreign species.

[1]A. K. Geim et al., Nat Mater 6, 183 (2007).
[2]K. Kim et al., Nature 479, 338 (2011).
[3]S. Casolo et al., Nanostructured Mater 115, 1 (2010).
[4]U. Bangert et al., Nano Lett 13, 4902 (2013).
[5]Q. M. Ramasse et al., Nano Lett 13, 4989 (2013).
[6]R. J. Nicholls et al., ACS Nano 7, 7145 (2013).

SuperSTEM is the UK National Facility for Aberration-Corrected STEM and is funded by the UK Engineering and Physical Sciences Research Council (EPSRC)

Fig. 1: HAADF STEM images of a) N and b) B implanted graphene showing single N and B substitutions in the graphene lattice, respectively. The images were low-pass filtered and colorised for clarity.

Fig. 2: EEL spectra from a) N and b) B implanted graphene samples, showing the near edge fine structure of the C K, N K edges and B K and C K edges of the N- and B-doped sample, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-1807 Water-soluble multi-layered graphene nanosheets via high temperature acidic treatment of graphite oxide

Gevko P. N.1, Tur V. A.1, Okotrun A. V.1, Bulusheva L. G.1
1Nikolaev Institute of Inorganic Chemistry SB RAS, Novosibirsk, Russia
paul@niic.nsc.ru

In recent years graphene has attracted wide attention from the scientific community. Its exceptional conductivity, high specific surface area and mechanical strength can be used in the energy storage devices (such as supercapacitors, lithium-ion batteries), composites, sensors, as well as makes it promising as a substrate for the deposition of various particles (metal oxides and sulfides , metal nanoparticles, conductive polymers, etc.)
For some applications, it is necessary to obtain stable solutions (or dispersions) of graphene. From the viewpoint of practical application, aqueous solutions of graphene have great advantages compared with the organic solvents and the possibility of obtaining of these solutions is actively investigated recently.
Previously we have proposed the method of high-temperature treatment of graphite oxide in concentrated sulfuric acid. It was shown that such treatment leads to the formation of the product which contains a large number of holes in carbon layers and called as a «perforated graphite». It was noted that during the process the color of the liquid phase is changed to brown. Subsequent study of the composition of this phase showed that it is, in fact, a solution of multi-layered graphene nanosheets (MGNS). In this work, we perform the further development of method of water-soluble graphene nanoparticles obtaining in order to increase the yield of MGNS, as well as the comprehensive investigation of these particles. It was shown that treatment of graphite oxide in a concentrated sulfuric acid at a temperature of 200°C leads to the formation of MGNS with in-plane size of ~300 nm and small amount of oxygen in its structure in a form of various functional groups. To improve the yield of MGNS the mixture of concentrated sulfuric/nitric acids was used. It was find that initial graphite oxide almost totally decomposed with the formation of MGNS, but the resultant product has a larger amount of oxygen in its structure.
Thus, the present study demonstrates a simple high temperature acidic treatment of graphite oxide as a method of obtaining of easy water-soluble MGNS as well as data of comprehensive study of these nanosheets.

Fig. 1: AFM-images of MGNS on silicon substrate
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-1822 Relations between deficiencies in CVD deposited graphene and the lattice defects of the Ni (111) substrate

Fogarassy Z.1, Rümmeli M. H.2, 3, Gorantla S.4, Bachmatiuk A.2, 3, 4, Dobrik G.1, Kamarás K.5, Biró L. P.1, Havancsák K.6, Lábár J. L.1
1Hungarian Academy of Sciences, Research Centre for Natural Sciences, Institute for Technical Physics and Materials Science, Hungary, 2IBS Center for Integrated Nanostructure Physics, Institute for Basic Science (IBS), Republic of Korea, 3Department of Energy Science, Department of Physics, Republic of Korea, 4Leibniz Institute for Solid State Materials Research Dresden, Germany, 5Hungarian Academy of Sciences, Wigner Research Centre for Physics, Institute for Solid State Physics and Optics, Hungary, 6Department of Materials Physics, Eötvös Loránd University, Hungary
fogarassy@mfa.kfki.hu

In this work graphene layers were investigated that were deposited by chemical vapor deposition (CVD) on nickel (111) thin film substrates. The Ni (111) thin substrates themselves were grown previously on bulk sapphire (0001) substrates at 550°C with 33nm/min. growth velocity. The nickel layer grew epitaxially on the sapphire, as it is shown by the diffraction in figure 1. The Ni (111) planes were parallel to the sapphire (0001) planes. Twin boundaries were formed in the nickel layers. At some places grains were rotated with 30° about an axis perpendicular to the sapphire surface.
The graphene deposited on the Ni (111) was investigated by TEM, Cs-corrected HRTEM, STM, AFM and by SEM / EBSD. According to [1] above 600°C, attempts to grow graphene on Ni (111) only resulted in the appearance of multi-layer turbostratic graphite. Here we show how the orientation of the first graphene layer and the appearance of additional turbostratic layers are related to the quality and orientation of the Ni (111) substrate. The nickel substrates were annealed in hydrogen before the graphene deposition. The graphene was deposited at atmospheric pressure from a mixture of argon, methane and hydrogen gases. The sample was cooled in argon and hydrogen gas mixture. Large area continuous single-layer graphene formed on top of nickel (111). Above it both another turbostratic graphene partial layer (Fig. 2), and graphite flakes were formed at certain locations. Such flakes are seen in the SEM image of figure 3.a. The continuous single-layer graphene formed epitaxially on the nickel (111) and the epitaxial relation was corroborated by both STM and TEM analyzes.
The growth of the thinner and smaller flakes was suppressed by changing the gas concentration. SEM and EBSD studies showed that the thicker flakes grew above the incoherent twin boundaries and high-energy (30°) grain boundaries of nickel.
Our results show that the first atomic layer grew as a continuous and epitaxial graphene layer on nickel (111). The additional local turbostratic layers and graphite flakes grew above the incoherent twin boundaries or high-energy boundaries in the nickel substrate. No thicker flakes were observed above coherent twin boundaries.
[1] Patera L.L. et al., ACS Nano 7 (9) 7901–7912 (2013)

Fig. 1: SAED diffraction pattern recorded from the nickel and sapphire interface. The nickel layer grew epitaxially on the sapphire.

Fig. 2: Cs-corrected HRTEM image recorded from a local turbostratic graphene flake over the large area continuous graphene layer.

Fig. 3: Figure a) SEM image. The dark patches of different gray shades are from an additional turbostratic graphene layer and graphite flakes. Figure b) EBSD orientation map. The straight lines mark locations where twin boundaries reach the surface of nickel.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-1848 Observation of Nanobubbles on Graphene with Atomic Force Microscopy

Ko H. C.1, Yang C. W.1, Hsu W. H.1, Hwang I. S.1
1Institute of Physics, Academia Sinica, Nankang, Taipei, 105, Taiwan, R.O.C.
hsienchenko@gmail.com

For the last 14 years, a variety of experimental studies have revealed the existence of nanobubbles at liquid/solid surface. Nanobubbles have attracted much scientific interest because of several highly disputed properties and many potential applications in fields from surface to nanofluidics [1]. Previous studies suggested that nanobubbles prefer to form on hydrophobic surfaces [2]. In this work, we prepare a substrate of a flat hydrophilic substrate with a small area covered by a hydrophobic material. The purpose is to see the preference of nanobubble formation on such an inhomogeneous substrate in water. Muscovite mica is a hydrophilic substrate that is strongly attracted to water, but graphene interacts weakly with water. Here we prepare a mica substrate with a small patch of mechanical exfoliated graphene layers. We inject water supersaturated with air on this sample and image the solid/water interface with atomic force microscopy (AFM). Figure 1a shows a height image taken with PeakForce mode. A high density of nanobubbles forms on graphene, but none is seen on the mica. Figure 1b is a higher-resolution image, which shows that nanobubbles can form on graphene of different thicknesses. Our observations demonstrate that the surface hydrophobicity has significant effect on nanobubble formation. Further study may help understanding the accumulation of gases at solid-water interfaces.

References

[1] Attard, P. Langmuir 12, 1693-1695 (1996)

[2] Agrawal, A. Nano Lett. 5, 1751-1756 (2005)

We thank support for this work from National Science Council of ROC (NSC96-2628-M-001-010-MY3 and NSC99-2112-M-001-029-MY3) and Academia Sinica

Fig. 1:  (a) Height image of graphene deposited on a mica surface in air-super-saturated water. (b) A higher-resolution image taken in the outlined region in (a).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-1947 Pt-terminating Carbyne Observed by Aberration-Corrected TEM

Kano E.1, 2, Hashimoto A.2, Takeguchi M.2
1University of Tsukuba, Tsukuba, Japan, 2National Institute for Materials Science, Tsukuba, Japan
KANOU.Emi@nims.go.jp

  Carbyne is a one-dimensional (1D) single atomic linear chain, composed of sp-hybridized carbon atoms. Some low-dimensional carbon allotropes, such as zero-dimensional fullerenes, quasi-one-dimensional (quasi-1D) carbon nanotubes, and two-dimensional graphene, have recently been discovered and have attracted worldwide attention for their unique properties. Theoretical studies have predicted that carbynes have more remarkable properties than the other low-dimensional materials. However, these properties have not yet been proven experimentally because of the difficulties encountered in production and during observation.
  Here, we report a novel, reproducible method of carbyne formation using Pt atoms on graphene. The formation and dynamics of carbynes were observed on an atomic scale by aberration-corrected TEM (JEM-ARM200F, JEOL, Japan) operating at an accelerating voltage of 80 kV. The samples were obtained by transferring monolayer graphene membranes onto in situ heating chips (E-chips for Aduro, Protochips, USA). Pt was deposited by a plasma sputtering system. We observed independent Pt atoms that appeared on a clean graphene surface. Using independent Pt atoms is an important key to produce and stabilize carbyne chains. We observed the migrations of Pt and C atoms on the graphene surface at 400 °C with an in situ heating holder (Aduro heating holder, Protochips, USA).
  Figures 1A–C show TEM images of carbyne formation. Three Pt atoms captured some carbon atoms, resulting in the formation of a C-shape chain (Fig. 1A). The Pt atoms and the chain moved around freely for 82 s, and then the chain suddenly turned into a straight chain (Fig. 1C). Both ends of the chain were terminated by Pt atoms, and the chain remained motionless for more than 20 s. From Fig. 1C, the length between the Pt atoms at both ends was measured to be approximately 1.5 nm. It corresponds to a model composed of 11 carbons with two Pt atoms (Fig. 1D). Fig. 1E shows a simulated image using this model.

A part of this work was supported by “Nanotechnology Platform Project” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Fig. 1: Figure 1. (A to C) TEM images of carbyne chain, which exhibited a variety of ringed, curved, straight shapes. Scale bars are 1 nm. (D) Atomic model of straight carbyne. (E) Simulated TEM image.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-1987 Active role of carbon during the formation of porous cerium oxide layer used as catalyst in fuel cell

Lavkova J.1,2, Khalakhan I.1, Chundak M.1, Vorokhta M.1, Potin V.2, Matolinova I.1, Matolin V.1
1Department of Surface and Plasma Science, Charles University, Czech Republic, 2Laboratoire Interdisciplinaire Carnot de Bourgogne, Université de Bourgogne, France
vpotin@u-bourgogne.fr

The global trend in renewable energy investment is developing the new energetic system using hydrogen; well know as a hydrogen economy. The key devices seem to be the fuel cells (FC) that convert chemical energy from hydrogen or hydrocarbons into kinetic or electrical energy. The most critical component of the FC is catalyst. The versatile element in catalysis is platinum (Pt) that efficiently mediating a multitude of chemical reaction. Unfortunately, Pt is rare element and its high price, exceeded that of gold, limits large-scale applications. Therefore, not surprisingly, the goal of reducing the amount of Pt is the major driving force in the catalysis research.

The Pt - CeO2 porous layers have been reported to be significantly active catalysts for CO oxidation, hydrogen production and oxidation of ethanol. Thin – film technology permits to produce large variety of hetero-materials with different composition (low concentration of platinum) and morphology (porous structure) of layers. The knowledge of the materials structure is fundamental for the best understanding of their physical and chemical properties.

The key role of carbon in the porosity creation of the catalyst layer is presented. The morphology of the CeO2 films prepared by magnetron sputtering on graphite foil was investigated by using microscopy tools – the Atomic Force Microscopy (AFM), the Scanning Electron Microscopy (SEM) and the Transmission Electron Microscopy (TEM). These studies show modification of carbon, confirmed by the Energy-Dispersed X-ray Spectroscopy (EDX) – see Fig. 1. The formation of cerium carbides crystals on the catalyst-substrate interface was observed using the High-Resolution TEM (HR-TEM). Moreover, the reduction of cerium as a result of the interaction with the carbon support was obtained by spectroscopies – the X-ray Photoelectron Spectroscopy (XPS) and the Electron-Energy Loss Spectroscopy (EELS). Finally, the structural model of the system is designed.

The authors acknowledge the support by the Czech Science Foundation under grant No. 13-10396S, ANR within IMAGINOXE project (ANR-11-JS10-001) and by EU FP-7-NMP-2012 project “chipCAT” under contract No. 310191. J.L. is grateful to the Conseil Regional de Bourgogne (PARI ONOV 2012).

Fig. 1: The modification of carbon after deposition of 20nm thick CeO2 layer on graphite foil – a) the material contrast obtained by the Scanning Transmission Electron Microscopy (STEM), b) the element map obtained by EDX.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2025 Dynamic Motion of Ru-Polyoxometalate Ions (POMs) on Functionalized Few-Layer Graphene

Ke X.1, Turner S.1, Quintana M.2, Hadad C.3, Montellano-Lopez A.3, Carraro M.4, Sartorel A.4, Bonchio M.4, Praro M.3, Bittencourt C.5, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium, 2Instituto de Fisica, Universidad Autonoma de San Luis PotoSi, Manuel Nava 6, Zona Universitaria, 78290 San Luis Potosi, SLP, Mexico, 3CENMAT, INSTM, UdR di Triest, Dipartimento di Scienze Chimich e Farmaceutich, University of Trieste, Piazzale Europa 1, I-34127 Trieste, Italy, 4ITM-CNR, Department of Chemical Sciences, Unviersity of Padova, Via F. Marzolo 1, 35131 Padova, Italy, 5ChiPS,, Unviersity of Mons, Rue du Par 20, B-7000 Mons, Belgium
xiaoxing.ke@uantwerpen.be

Recent advances in state-of-art aberration-corrected transmission electron microscopy (AC-TEM) have demonstrated its power in resolving the atomic structure of nanomaterials and nanohybrids down to the limit. Particularly, the interfacial structures of nanohybrids have strong influence on the properties and performances of the materials and thus need to be understood at atomic level. Aberration-corrected imaging shows enhanced resolution and improved signal-to-noise ratio, which largely benefits the straightforward interpretation at nanohybrids interface, particularly for soft 2D nanomaterials. An example on the interface study of a water oxidation catalyst functionalized on graphene surface is demonstrated at ultra-high resolution in this abstract.

The as-studied water oxidation catalyst, a tetraruthenate oxo-cluster ([Ru4(H2O)4(μ-O)4(μ-OH)2(γ-SiW10O36)2]10-, referred to as polyoxometalate (POM) ions in this text, has been recently discovered to hold promising application in water splitting. The effective grafting of the Ru4POM catalysts on a conductive substrate is therefore crucial in order to promote its further application in nanodevices on the large scale. Few layer graphene (FLG) is a best candidate due to its superior electric and mechanic properties such as high carrier mobility etc. Exploring the behaviour of Ru4POM on graphene supports is therefore one of the key issues in further applications, where the selection of functional group is important in tailoring its performance. Thus in this study we demonstrate how the interactions between Ru4POM molecules and supporting graphene layers can be studied by aberration corrected transmission electron microscopy (AC-TEM) at low voltage (80kV). Under the 80 kV irradiation of the electron beam the Ru4POM demonstrates dynamic motion on the graphene surface. The motion of the Ru4POM is captured as a series of images and is shown to vary as a function of time under certain constraints in the Ru4POM rotation. The frequency and amplitude of rotation is found to be related to the nature of the functional group used, including a polyamine dendron and a N,N,N-trimethyl benzenaminium moiety in our study. Distortions in the Ru4POM structure are revealed as well, suggesting that the ions can stand instantaneous structural changes without losing their integrity. The stability of the Ru4POM-graphene hybrid during the imaging corroborates the long-term robustness of the material applied to multielectronic catalytic processes.

X. Ke and G. Van Tendeloo acknowledge funding from the European Research Council under the FP7 ERC Grant Nº 246791–COUNTATOMS.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2040 Spatial distribution of particles in the graphene-nanoparticles system

Mantlikova A.1, Pacakova B.1, Kalbac M.2, Repko A.3, Vejpravova J.1
1Institute of Physics of the ASCR, v.v.i., Na Slovance 2, Prague 8, 182 21, Czech Republic, 2J. Heyrovský Institute of Physical Chemistry of the ASCR, v.v.i., Doleškova 3, Prague 8, 182 23 Czech Republic, 3Faculty of Science, Charles University in Prague, Albertov 6, Prague 2, 128 43, Czech Republic
mantlikova@fzu.cz

Graphene (GN) has been in the focus of intensive research in material science and nanotechnology in recent years due to its unique electrical, optical, thermal and mechanical properties. Creation of the graphene-nanoparticles (GN-NPs) system could lead to the improvement of the physical properties of GN due to the change of its topography via creation of wrinkles.

We have focused on the basic characterization of the GN-NPs systems, especially on the study of the NP spatial distribution on the substrate, which has a significant influence on the GN wrinkling. The samples possessing different concentration of the CoFe2O4 NPs (6 nm) on the Si/SiO2 substrate covered by the GN layer were characterized by High Resolution Scanning Electron Microscopy (HRSEM) and Atomic Force Microscopy (AFM). Different concentration of the NPs for individual samples was confirmed both by the HRSEM and AFM measurements and creation of the GN wrinkles around the NPs below the GN monolayer and their dependence on the NP concentration has been observed (Fig. 1).

The real NP spatial distribution determined by the HRSEM and AFM was compared with that one obtained from simulation in Matlab as follows: the NPs were randomly distributed inside the square box of 1 µm edge length, divided to the regular lattice with inter-node distances equal to the NP diameter for prevention of the NP overlap. The mean interparticle distances were calculated in both cases (real and simulated NP spatial distribution) for the nearest neighbors using the triangulation procedure. The results of both NP spatial distributions clearly demonstrate decrease of the interparticle distances (Fig. 2). Moreover, the resulting interparticle distances obtained from the simulation correspond very well to those obtained from the real positions of NPs determined by the AFM, showing that the NPs are randomly distributed on the surface and the influence of the substrate corrugations on the NP distribution is negligible.

This work was supported by Czech grant agency, project number P204/10/1677.

Fig. 1: The SEM image of the GN-NPs sample with high (a) and low (b) concentration of NPs. The GN wrinkles are clearly visible on both images, the border between the GN and substrate could be found on right image.

Fig. 2: The concentration, c dependence of interparticle distance, d for GN-NPs samples (a). The simulated NPs spatial distribution for the least (1:10000) concentrated (b) and the most (1:1000) concentrated (c) samples.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2084 Projected potential of graphene estimated by off-axis electron holography

Geiger D.1, Biskupek J.1, Algara-Siller G.1, Kaiser U.1
1Electron Microscopy Group of Materials Science, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany
dorin.geiger@uni-ulm.de

Graphene [1] is one of the most investigated 2D-structures in the last decade. Some efforts were undertaken [2,3,4] to determine the projected potential (PP) of graphene for different layer thicknesses. Off-axis electron holography (EH) [5] allows to recover numerically the image wave and, for known residual aberrations of the TEM, to reconstruct the object exit wave. The accuracy is significantly increased by using a Cs-corrected TEM [6,7].
CVD graphene transferred to holy carbon was cleaned using active carbon or Al2O3 powder at ~200°C at the sample surface [8] (fig.1a). Because of the quite long time needed for TEM-adjustment in EH-mode, searching for adequate sample locations and positioning in EH, new contamination appears (fig. 1b,c). Consequently, the graphene layers are often not perfectly clean and the PP is generally higher than the ideal values, cleanliness of the graphene layers proves out to be essential for accurate results.
The illumination of our Cs-corrected FEI Titan 80-300 TEM with rotatable Möllenstedt biprism, was optimized for EH at 80kV [2], where beam damage of graphene is reduced. Taking the C1–C3 condenser lens setting [9], the elliptical illumination could be optimized and using a reduced extraction voltage by ~2kV, a decrease of the electron energy distribution was achieved. Finally the hologram contrast could be noteworthy improved [2]. The experiments till now show, presumably due to contamination, a quite large dispersion and a tendency to higher values than the calculated ones. Using the independent atom model (IAM) and/or the density functional theory (DFT), image simulations and calculations of the PP, were made using the programs QSTEM [C. Koch] and JEMS [P. Stadelmann].
To characterize the local thickness and the PP of graphene, we took holograms in high-resolution TEM. The analysis of the profile lines in the reconstructed object phase allows the determination of the local thickness variations (fig. 2). Contamination and the EH-restriction, to use only object areas next to vacuum, make difficult to find large ideal uniform sample areas. Phase jumps, related to thickness variations, show for most of the results up to now, phase shifts of <0.08 rad at a phase detection limit per single hologram of ~2π/70. To conclude our studies, additional results with better statistics will follow.

1. A. K. Geim, K. S. Novoselov, Nature Materials 6 (2007) p.183.
2. D. Geiger et al., MS.7.P199,  3. F. Börrnert et al., IM.5.P110,  4. L. Ortolani et al., MS.7.P211, Proc. MC 2013.   5. H. Lichte, UM 20 (1986), p. 293.    6. M. Haider et al., UM 75 (1998) p. 53. 

7. D. Geiger et al., Micr. Microanal. 14 (2008), p. 68.  8. G. Algara-Siller et al., submitted. 

9. F. Genz et al., IM.2.P046, Proc. MC 2013.

This work was supported by the DFG (German Research Foundation) and the Ministry of Science, Research and the Arts (MWK) of Baden-Württemberg in the frame of the Sub-Angstrom Low-Voltage Electron microscopy (SALVE) project.

Fig. 1: Specimen contamination state immediately after insertion in the TEM (a), about three hours later (b) and at the end of the TEM-session (c).

Fig. 2: Reconstructed phase image from the object exit wave of graphene layers at vacuum, showing some contamination (a) with the phase profile along the arrow (b). Holograms were taken with Cs-corrected FEI Titan 80-300 TEM at 80kV in HRTEM mode.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2099 Microstructure investigation of Ru/CNT hybrid synthesized by different methods

Zhang B.1, Pan X.1, Su D.1
1Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, China
bszhang@imr.ac.cn

Ruthenium (Ru) as catalysts or functional additive has drawn considerable interest, owing to its fascinating unique properties and amazingly high efficiency in various reactions.[1-2] The shape, size distribution and spatial dispersion of supported Ru nanoparticles (NPs) could significantly contribute to the final performance of catalysts, especially for many structure-sensitive heterogeneous reactions. For the purpose of simultaneously achieving excellent catalytic activity and high stability for practical applications, there have been many attempts to explore preparation routes, such as impregnation, the polyol process, metal organic chemical vapor deposition, deposition–precipitation, microwave irradiation, and so on. However, there are few reports about comparing the structures of supported Ru nanocomposite synthesized by these methods systematically. Herein, we selected Ru supported on carbon nanotube (Ru/CNT) as model hybrid material, and used advanced electron microscopy to reveal the detailed structural features of Ru/CNTs prepared by difference methods, such as the local and surface structures, particles size distribution (PSD), and thermal stability. For instance, Ru NPs supported on oxygen functionalized CNTs (Ru/OCNTs) synthesized by ethylene glycol (EG) reduction and conventional impregnation (IP) methods have been compared.[3] Transmission electron microscopy (TEM), scanning TEM (STEM), and X-ray diffraction (XRD) characterizations reveal that the sample prepared by EG reduction method with ultra-small size and highly dispersed Ru NPs onto OCNTs, is superior to that obtained by conventional impregnation method (Fig. 1). The result of Ru NPs evolution process investigated by in situ heating TEM (Fig. 2) indicates that the Ru/OCNTs prepared by EG reduction method has good thermal stability, which may lengthen catalyst service life availably. In addition, we also studied the structural rearrangements of Ru NPs supported on CNTs from twinned Ru NPs into Ru single nanocrystals under the microwave irradiation by aberration-corrected electron microscope.[1] Our work can be expected as an important reference for the design and fabrication of ultra-small metal NPs with optimal morphologies and high thermal stability for a variety of chemical reactions.

Reference
[1] B. Zhang, X. Ni, W. Zhang, L. Shao, Q. Zhang, F. Girgsdies, C. Liang, R. Schlögl, D.S. Su, Chem. Commun. 2011, 47, 10716-10718.
[2] X. Ni, B. Zhang, C. Li, M. Pang, D.S. Su, C.T. Williams, C. Liang, Catal. Commun. 2012, 24, 65-69.
[3] X. Pan, B. Zhang, B. Zhong, J. Wang, D.S. Su, Chem. Commun. 2014, in press (DOI: 10.1039/C3CC48710E).

We gratefully acknowledge the financial support provided by NSFC of China (21203215, 21133010, 51221264, 21261160487), MOST (2011CBA00504), Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA09030103) and the China Postdoctoral Science Foundation (2012M520652).

Fig. 1: HAADF-STEM images of Ru/OCNTs-IP (a) and Ru/OCNTs-EG (b). The insets in (a) and (b) are the corresponding histograms of PSD.[3]

Fig. 2: Typical TEM images of the Ru/OCNTs-EG sample treated using an in situ heating TEM holder at RT (a), 350 oC (b), and 600 oC (c) and the HAADF-STEM image after heating (d). Inset in Fig. 2d is the corresponding histograms of PSD.[3]
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2102 Formation mechanism and bending properties of carbon nanotetrahedron/nanoribbon structures

Kohno H.1
1Kochi University of Technology
hideokohno@gmail.com

A carbon nanoribbon is formed when a carbon nanotube flattens in one direction. We have found that a switching in the flattening direction results in the formation of a carbon nanotetrahedron in the middle of a carbon nanoribbon [1] (Fig. 1). Our TEM and SEM observations suggest a model of its formation mechanism as follows. When a carbon nanotube is expelled from an Fe catalyst nanoparticle, the tube is forced to flatten, and there are two preferable directions of flattening, which we call the origami mechanism. When one direction is dominant, a nanoribbon is formed, while a nanotetrahedron is formed when a switching of the flattening direction occurs (see Ref. 1 for more details).

To reveal bending properties of our carbon nanotetrahedron/nanoribbon structures, they were examined using a micromanipulator in a TEM and their bending behavior was observed in-situ [2]. We have found that a nanotetrahedron/nanoribbon structure bent at a nanotetrahedron/nanoribbon junction, and that the bending was reversible and repeatable. The nanotetrahedron/nanoribbon structures kept their shape during being bent and did not expand to take a tubular form. Our results show that the nanotetrahedron/nanoribbon structures have excellent durability against bending. The nanotetrahedron/nanoribbon structures can be bent at nanotetrahedron/nanoribbon junctions sharply and do not break, therefore we expect that the nanotetrahedron/nanoribbon structures can be used for three-dimensional wiring.

[1] Hideo Kohno, Takuya Komine, Takayuki Hasegawa, Hirohiko Niioka, and Satoshi Ichikawa, Nanoscale 5 (2013) 570.
[2] Hideo Kohno and Yusuke Masuda, to be submitted.

This work was supported in part by Adaptable and Seamless Technology Transfer Program through Target-driven R&D, Japan Science and Technology Agency.

Fig. 1: TEM images and schematic illustration of carbon nanotetrahedron/nanoribbon structures (from Ref. 1).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2174 A new structural model for Graphene Oxide and Reduced Graphene Oxide as revealed by core EELS and DFT

Tararan A.1, Zobelli A.1, Benito A.2, Maser W.2, Stéphan O.1
1Laboratoire de Physique des Solides, Univ. Paris-Sud, CNRS UMR 8502, F-91405, Orsay, France, 2Department of Chemical Processes and Nanotechnology, Instituto de Carboquímica ICB-CSIC, C/Miguel Luesma Castán 4, E-50018 Zaragoza, Spain
anna.tararan@u-psud.fr

Graphite oxide (GO) is known since the middle of the XIX century. In the latest years it has attracted a renewed interest as a precursor for a cheap large-scale production of graphene. Indeed, GO conserves graphite layered structure with an expanded interlayer distance that facilitates exfoliation. A subsequent reduction yields a material whose properties are very similar to those of graphene but strongly depend on the local structure and stoichiometry. However, many questions remain still open about GO and reduced GO (RGO) chemical homogeneity and the functional groups effectively present.
In previous spectroscopy studies the oxygen content in GO ranges from 22% to 32%. However, TEM images revealed that GO is very inhomogeneous at the nanometer scale. Still, no spatially resolved spectroscopic studies have yet been reported and only average evaluations are provided in literature.1 EELS in a STEM could give access to the suitable scale but GO and RGO are highly sensitive to irradiation.
In this study we overcame this limitation by adopting an experimental set up combining a liquid nitrogen cooling system at the sample stage, a low accelerated electrons beam (60 keV) and a liquid nitrogen cooled CCD camera with a low read-out noise of three counts r.m.s. and a negligible dark count noise. Hyperspectral core EELS images have been acquired in a low dose mode (order of 105 e-nm-2) at a 10 nm spatial resolution.2
Chemical maps for GO and RGO (see figure) show regions within individual flakes with different oxidation levels. Whereas oxygen rates averaged over the whole area are in agreement with literature, we observe that the oxygen content can locally rise up to 60%.
Lower oxidized GO regions present a fine structure at the carbon K-edge similar to amorphous carbon, while highly oxidized regions show specific core EELS signatures. RGO samples display the well-known fine structure profile of graphite, proving an excellent restoration of the carbon network. Nevertheless regions characterized by residual oxygen exhibit an additional sharp peak.
These results have been combined with complementary DFT analysis of formation and binding energies for different oxygen functional groups and concentrations and EELS spectra simulations. This allowed us to provide a new structural model compatible with our experimental findings. We suggest a full functionalization with hydroxyl groups in the strongly oxidized regions, while in lower oxidized regions also epoxide groups are expected.

1K. A. Mkhoyan, A. W. Contryman, J. Silcox, D. A. Stewart, G. Eda, C. Mattevi, S. Miller, and M. Chhowalla, Nano Lett. 9, 1058 (2009).

2M. M. v. Schooneveld, A. Gloter, O. Stéphan, L. F. Zagonel, R. Koole, A. Meijerink, W. J. M. Mulder, and F. M. F. d. Groot, Nat Nano 5, 538 (2010).

The authors acknowledge support from the European Union in Seventh Framework Programme under Grant Agreement No. 312483 (ESTEEM2).

Fig. 1: EELS hyperspectral analysis of Graphene Oxide and Reduced Graphene Oxide: oxygen concentration maps, associated histograms and carbon K-shell EELS edges extracted from the selected regions.
Fig. 2:
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2219 Surface formation of electrospun carbon nanofiber mats controlled by HRSEM

Zhigalina V. G.1, Ponomarev I. I.2, Razorenov D. Y.2, Ponomarev I. I.2
1Shubnikov Institute of Crystallography RAS, Moscow, Russia, 2Nesmeyanov Institute of Organoelement Compounds RAS, Moscow, Russia
v.zhigalina@gmail.com

Hydrogen fuel cells will play the leading role among alternative energy sources in the XXI century. Electrocatalytic and gas diffusion layers of a fuel cell consist of electroconductive materials and metal layers, which are the most critical components. Carbon nanofiber nonwoven materials are the most promising materials owing to their thermal and chemical resistance, higher sorption capacity, electrical conductivity and mechanical properties. Their properties determine the most important fields of their application: for purifying various liquid and gaseous media, as a reinforcing filler in composites for accumulating gaseous or condensed substances and also for creating catalysts with higher activity, selectivity and stability. Polyacrylonitrile is the most promising and the most used polymer for producing carbon nanofiber nonwoven materials. Recently electrospinning has been used to create new materials for various alternative power supplies [1]. By this method highly porous fiber mats can be molded from solutions of polymers. Due to various additives the properties and characteristics of the produced materials can differ widely.
The main problems are to improve the porosity of the carbon nanofiber mats, to reduce their electroresistivity and to decrease the precious catalytic metal concentration. The aim of the present work is to investigate the influence of different treatments on the morphology, metal particle distribution and surface structure of electrospun polyacrylonitrile mats.
The surface investigation of obtained mats was performed by a high resolution scanning electron microscopy (HRSEM) in a FEI Quanta 250 FEG and a FEI Helios 600 DualBeamTM with EDX analysis.
The obtained electrospun polyacrylonitrile fiber mats were 10-100 μm thick, which depends on the molding conditions and treatment temperature with the fiber diameters in the range of 50-400 nm. Most of the fibers had a characteristic diameter of 100-150 nm and a length of several tens of microns [2]. These mats with a smooth surface are shown in Fig. 1. High temperature annealing (at 1200 and additional 2800 oC) and chemical treatment (by polyvinylpyrrolidone and polyimide) led to significant changes in the morphology, length and surface condition (Fig. 2). The chemical treatment was performed for a better deposition of Pt particles because of the formation of cavities on the fibers’ surface [3]. As a result, a thick Pt nanoparticles coating was formed on their surface (Fig. 3).

1. Dong Z, Kennedy SJ, Wu Y Journal of Power Sources 2011 196 4886
2. Ponomarev II et al. Doklady Physical Chemistry 2013 448(6) 670
3. Zhigalina VG et al. Nanomaterials and Nanostructures - XXI Century 2012 4 36

The investigation was supported by RFBR grant № ofi-m-11-03-12115.

Fig. 1: Nontreated electrospun carbon fiber mats.

Fig. 2: Carbon fibers with a damaged surface after annealing at 1200 and 2800 oC.

Fig. 3: HRSEM image of carbon fibers coated by Pt (a) and corresponding EDX spectrum (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2376 Low-Energy Electron Diffractive Imaging of Graphene Based on a Single-Atom Electron Source

Hsu W. H.1, 2, Chang W. T.1, Lin C. Y.1, 3, Chen Y. S.1, Lai W. C.1, 3, Hwang I. S.1, 2
1Institute of Physics, Academia Sinica, Nankang, Taipei, Taiwan, 2Department of Materials Science and Engineering, National Tsing Hua University, Hsinchu, Taiwan, 3Department of Physics, National Taiwan University, Taipei, Taiwan
hsuwh@phys.sinica.edu.tw

    A single-atom tip (SAT) can be an ideal field emitter of electron beams. It has been shown that noble-metal covered W(111) SATs can be reliably prepared1,2 with several controllable profiles3. The growth of the faceted pyramidal tips is a thermodynamic process. These SATs are both physically and chemically stable and can be regenerated through a simple annealing in vacuum, ensuring a long operation lifetime. Both the brightness and spatial coherency of these single-atom electron sources are orders of magnitude better than those of the state-of-the-art electron sources used in current electron microscopes4.

    We have built a low-energy electron point projection microscope (PPM) combined with a retractable micro-channel plate detector (MCP), housed in an ultra-high vacuum (UHV) chamber, to image nano-objects. A schematic is displayed in Fig. 1. The PPM is a shadow microscope where an object is placed between the electron point source and a detector screen. The magnification of the image depends on the tip-sample distance (d) and the sample-detector distance (D). We record the high resolution bright-field image when D is large. On the other hand, the diffraction patterns of the object at large angles can be obtained when D is small.

    With the advantages of the high stability of single-atom electron source and high contrast due to low energy of the source, some characteristics on graphene can easily be observed. Fig. 2b shows ribbon-like patterns in each diffraction disk, which are also visible in the bright-field image in Fig. 2a. Figs. 3a, b, and c show the bright-field images of graphene with the same beam energy of 270 eV at 0 second, 75 seconds, and 150 seconds, respectively. Both growth (labeled with yellow arrows) and migration (labeled with red arrows) of particles can be observed in a continuous-time imaging.

Reference

1 H.-S. Kuo, I.-S. Hwang, T.-Y. Fu, J.-Y. Wu, C.-C. Chang, and T.T. Tsong , Nano Lett. 4(12), 2379 (2004).

2 H.-S. Kuo, I.-S. Hwang, T.-Y. Fu, Y.-C. Lin, C.-C. Chang, and T. T. Tsong, Jap. J. Appl. Phys. 45, 8972 (2006).

3 W.-T. Chang, I.-S. Hwang, M.-T. Chang, C.-Y. Lin, W.-H. Hsu, and J.-L. Hou, Rev. Sci. Instrum. 83, 083704 (2012).

4 C.-C. Chang, H.-S. Kuo, I.-S. Hwang, and T. T. Tsong, Nanotechnology 20, 115401(2009).

We acknowledge the financial support from Academia Sinica and National Science Council.

Fig. 1: Schematic of our electron point projection microscope with a retractable MCP. The single-atom tip is driven by a X-Y-Z piezo-manipulator for approaching and scanning of the tip to the sample. The magnification of the bright field image is M = (D + d)/d.

Fig. 2: (a) Bright-field image of suspended graphene sheet, recorded with 500 eV and sample-detector distance D = 130 mm. (b) Diffraction pattern of the same sample recorded with 480 eV and sample-detector distance D = 35mm.

Fig. 3: Bright-field images of graphene recorded with 270 eV at (a) t = 0 s, (b) t =75 s, and (c) t = 150 s.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2386 Implantation and atomic scale characterization of self-interstitials in free standing graphene

Lehtinen O.1, Vats N.1, Algara-Siller G.1, Knyrim P.1, Kaiser U.1
1Ulm University, Materials Science Electron Microscopy
ute.kaiser@uni-ulm.de

A surplus density of carbon atoms is introduced into free-standing graphene by means of low-energy implantation [1]. The implantation is conducted using an evaporating carbon coating apparatus, designed for depositing thin layers of amorphous carbon on non-conducting specimens for electron microscopy. By careful tuning of the deposition parameters, a low enough density of extra atoms is reached in order to produce isolated self-interstitial dimers in graphene. The structure of these defects is imaged at the atomic scale, employing aberration corrected high resolution transmission electron microscopy. The earlier predicted, completely sp2-hybridized structural configurations of ad-dimers in graphene [2,3] are experimentally verified. Additionally larger aggregates of extra atoms and edge dislocation dipoles incorporated in the graphene lattice are observed, and based on atomistic modeling, such structures are determined to be energetically favourable arrangements for the extra atoms. All of the adatom structures are predicted to strongly buckle out-of-plane. Such blister-like structures can be expected to have higher reactivity than pristine graphene, which can be advantageous when functionalization of graphene is desired. Further on, defect structures containing surpulus carbon atoms have been predicted to have exciting electronic and magnetic properties [4], and our experiment demonstrates that such structures can, in fact, be fabricated.

[1] Lehtinen, O., Vats, N., Algara-Siller, G., Knyrim, P. and Kaiser, U., (2014), in review

[2] Lusk, M. T. and Carr, L. D., Phys. Rev. Lett. 100 (2008) 175503.

[3] Lusk, M. T., Wu, D. T. and Carr, L. D., Phys. Rev. B 81 (2010) 155444.

[4] Lehtinen, P. O., Foster, A. S., Ayuela, A., Krasheninnikov, A. V., Nordlund, K. abd Nieminen, R. M., Phys. Rev. Lett. 91 (2003) 017202.

[5] Lehtinen, O., Kurasch, S., Krasheninnikov, A. V. and Kaiser, U., Nat. Commun. 4 (2013) 2098

This work was supported by the DFG (German Research Foundation) and the Ministry of Science, Research and the Arts (MWK) of Baden-Württemberg in the frame of the Sub-Angstrom Low-Voltage Electron microscopy (SALVE) project and by the DFG through the TR21 project.

Fig. 1: Self-interstitial dimers in graphene at 80 kV AC-HRTEM. First column raw images of inverse Stone-Thrower-Wales defect and its two polymorphs. Second column: same images after maximum filtering [5], Third column: wire frame models of the defects. Fourth column: relaxed atomic structures. Scale-bar 1 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2423 Topologically induced dielectric response in multilayer graphene nanocones

Hage F. S.1, 2, 3, Kepaptsoglou D. M.1, Ramasse Q. M.1, Seabourne C. R.4, Scott A. J.4, Prytz Ø.3, Gunnæs A. E.3, Helgesen G.2, 3, Brydson R.4
1SuperSTEM Laboratory, SciTech Daresbury, Daresbury, U.K , 2Physics Department, Institute for Energy Technology, Kjeller, Norway, 3Department of Physics, University of Oslo, Oslo, Norway., 4Institute for Materials Research, SPEME, University of Leeds, Leeds, U.K
fshage@superstem.org

Among the multitude of known carbon nanostructures, graphene nanocones are quite unique. These multilayer cones are characterised by macroscopic apex angles (0˚, 112.9˚, 84.6˚, 60˚, 38.9˚ and 19.2˚), which correspond to the incorporation of zero to five pentagons (P=0-5) in a graphene sheet [1] (where P=0 corresponds to the case of a flat disc). Due to their topology, graphene cones are ideal for investigating the effect of pentagonal defects on local valence electron structure in multilayer carbon nanostructures. This was done here by recording the dielectric response at cone apices by means of valence electron energy loss spectroscopy in the aberration corrected dedicated scanning transmission electron microscope.

Fig. 1a shows a distinct feature in the loss spectrum at 1.5 eV, originating from the tip of a two pentagon cone (P=2, fig. 1b). From ab inito simulations this feature was attributed to the presence of the pentagonal defects themselves. This was explained by pentagons inducing topology specific localised low energy states, where the 1.5 eV feature arises as a sum over interband transitions involving such states. Upon extension, this indicates that multilayer graphene cones should show great promise as field emitters [2].

Localisation of collective modes was investigated by the momentum dependence (i.e. dispersion) of their associated loss peaks: where a vanishingly small dispersion corresponds to a localised state. Fig. 2a shows the π plasmon dispersion from the tip of multilayer cone with five pentagons at its apex (P=5, fig. 2b) compared to that of a flat multilayer discs (P=0). While a parabolic dispersion indicates significant ‘graphite-like’ plasmon propagation in the disc, the vanishing plasmon dispersion in the P=5 cone indicates a high degree of confinement at its apex [3]. The observed slightly negative cone plasmon dispersions will also be discussed. All data were acquired with a Nion UltraSTEM100 operated at 60kV, and all computational modelling was carried out using the CASTEP DFT code at the University of Leeds ARC1 facility.

[1] A. Krishnan et al., Nature, 388 (1997) 451
[2] F.S. Hage et al., Nanoscale, 6 (2014) 1833
[3] F.S. Hage et al., Physical Review B, 88 (2013) 155408

SuperSTEM is the national facility for aberration corrected STEM, supported by the UK Engineering and Physical Sciences Research Council. This work was supported by the Research Council of Norway under Contract No. 191621/V30.

Fig. 1: Figure 1 (a) Valence electron energy loss spectrum originating from the tip of the (P=2) cone shown in the high angle annular dark field (HAADF) image in (b).

Fig. 2: Figure 2 (a) Dispersions of the π plasmon of a cone with five pentagons at the apex (P=5) and a flat disc (P=0). (b) HAADF image of a five pentagon cone (P=5).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2542 Atomic resolution of nanocrystalline Ge and SbTe encapsulated inside carbon nanotubes

Marks S.1, Kashtiban R.1, Sloan J.1
1University of Warwick
s.r.marks@warwick.ac.uk

We have the ability to image individual atoms using high resolution transmission electron microscopy (HRTEM) yet for most materials this has only been achieved whilst looking at a section of a larger system. There has been relatively little work investigating nanocrystalline systems that have been formed inside extremely small growth spaces[1]. This is of interest due to the possibility that with this bottom up growth method we may see the formation of new structures[2].
Melt filling is a technique that allows us to encapsulate materials inside ≈1 nm carbon nanotubes (CNTs) therefore allowing us to image molecules formed inside confined spaces. This is achieved by grinding a combination of CNTs and the desired material. It is then heated to above its melting temperature, cooled and annealed, repeatedly, inside a furnace before being dispersed onto a lacy carbon grid. The dispersed filled carbon nanotubes were then imaged using a JEOL ARM200F TEM/STEM at 80kV accelerating voltage, a HRTEM is required as individual atomic resolution is essential. The CNTs were commercially acquired and had a range of diameters from 1.1 – 1.6 nm. Two systems were investigated, Germanium (Ge) and Antimony Telluride (SbTe). Once successfully filled the systems were then recreated using Crystalmaker and simulated using SimulaTEM in an attempt to reproduce the achieved image.
Ge was successfully filled into CNTs but rendered a small filling ratio due to the Ge reacting with the lab glass used during the melting phase. Even though the filling ratio was low, filled CNTs were captured with high resolution. The Ge was found to be growing in the [010] crystal orientation relative to the bulk with lattice spacings that are consistent with bulk Ge. The system captured in Figure 1 has been recreated and simulated to prove it has formed from bulk Ge. Streaking is visible on the atomic spots of Ge, this is due to the CNT being tilted rather than exactly perpendicular to the beam.
SbTe also successfully filled into the CNTs but again with a low filling ratio. Two different systems were observed within the SbTe sample; the first a 4 atom motif and the second a 5 atom (Figure 2). This is of high interest as a single atom variation will affect the quantum structure of the system. Both systems were recreated and found to have lattice spacing similar to that of the bulk with growth once again in the [010] direction, this is due to SbTe and Ge both having a FCC crystal structure.

[1] J Sloan et al. "Two layer 4: 4 co-ordinated KI crystals grown within single walled carbon nanotubes." Chemical Physics Letters 329 (2000): 61-65.
[2]R Carter et al. "Correlation of structural and electronic properties in a new low-dimensional form of mercury telluride." Physical review letters 96 (2006): 215501.

Fig. 1: Crystallographic simulation of Ge encapsulated inside a CNT and an image of the encapsulated Ge with an overlay of the simulated system.

Fig. 2: Crystallographic simulations of encapsulated SbTe, below is the captured structure with the simulation superimposed. Both 5 layer and 4 layer SbTe systems are included.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2592 Structural transformations in electrospun Pt-decorated carbon nanofibers

Zhigalina O. M.1, Zhigalina V. G.1, Ponomarev I. I.2, Razorenov D. Y.2, Ponomarev I. I.2, Kiselev N. A.1
1Shubnikov Institute of Crystallography RAS, Moscow, Russia, 2Nesmeyanov Institute of Organoelement Compounds RAS, Moscow, Russia
zhigal@crys.ras.ru

It is a well-known fact that good support material must have a high surface area which disperses the nanoparticles over, be porous enough to transmit gas flow and have a good electrical conductivity. Therefore nowadays carbon nanofibers (CNFs) are considered to be very promising support materials [1]. Recently electrospinning, a simple, inexpensive technique, has attracted significant attention in the preparation of CNFs [2]. Thin catalyst layers and their supporting substrates are the most critical components and up to now the problem of their optimisation is far from trivial.
In this investigation we have studied a possibility of electrospun CNFs carbonization by different methods such as high temperature annealing, chemical treatment and carbonization initialized in the metal particles presence. Pt-decorated CNFs were obtained using different catalytical coating and treatment techniques. To improve the CNF porosity and the process of catalytic nanoparticles deposition the CNFs were obtained on a base of polyvinylpyrrolidone (PVP) and polyimide (PI) polymer mixture and annealed at T = 1200 and 2800 °С [3].
The specimens for TEM investigations were prepared by the dispersion of CNF mats in acetone using an ultrasonic bath to get single fibers and to separate the bundles. These suspensions were dropped onto the Cu lacey carbon grids. The samples structure was characterized by a high resolution transmission electron microscopy (HRTEM) in a FEI Tecnai G2 30ST with SAED, EDX analysis and HAADF STEM detector and a FEI Titan 80-300 with probe Cs corrector at an accelerating voltage of 300 kV.
It was shown that PVP/PI treatment with annealing at 250 оС or at 1200 оС stimulated the fiber carbonization process without changes to their morphology or surface destruction, but the carbonization was incomplete. High temperature annealing at Т = 1200 и 2800 оС led to full fiber carbonization. In the structure of fibers the straight graphene planes were mainly observed (Fig. 1). In the case of combined high temperature (1200 оС) and chemical (PVP+PI) treatment the carbon nanofibers consisted of “curly” graphene planes within the whole fiber space (Fig. 2). In this case for the fibers carbonized in the Fe particles presence the same structure was revealed as well. As a result of this process the fibers’ surface became porous which promoted platinum nanoparticles to form a thick layer on the fibers’ surface (Fig. 3-4).

The investigation was partially carried out using IC RAS Research Centre and NRC “Kurchatov institute” equipment and supported by RFBR grant ofi-m-11-03-12115.

Fig. 1: HRTEM image of a carbonized nanofiber with straight graphene planes.

Fig. 2: TEM image of a carbonized nanofiber with “curly” graphene planes.

Fig. 3: TEM image of CNFs covered by Pt nanoparticles.

Fig. 4: HRTEM image of Pt nanocrystals.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2607 Nitrogen-Doped Graphene/Carbon Nanotube Hybrids: In-Situ Formation on Bifunctional Catalysts and Their Superior Electrocatalytic Activity for Oxygen Evolution/Reduction Reaction

Tian G.1, Zhao M.1, Yu D.2, Kong X.1, Huang J.1, Zhang Q.1, Wei F.1
1Beijing Key Laboratory of Green Chemical Reaction Engineering and Technology Department of Chemical Engineering, Tsinghua University, Beijing, China, 2School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore
tian-gl10@mails.tsinghua.edu.cn

There is a growing interest in oxygen electrode catalysts for oxygen reduction reaction (ORR) and oxygen evolution reaction (OER), as they play a key role in a wide range of renewable energy technologies such as fuel cells, metal-air batteries, and water splitting. Nevertheless, the development of highly-active bifunctional catalyst at low cost for both ORR and OER still remains a huge challenge. Herein, we report a new N-doped graphene/single-walled carbon nanotube (SWCNT) hybrid (NGSH) material as an efficient metal-free bifunctional electrocatalyst for both ORR and OER. NGSHs were fabricated by in situ doping during chemical vapor deposition growth on layered double hydroxide derived bifunctional catalysts. Our one-step approach not only provides simultaneous growth of graphene and SWCNTs, leading to the formation of three dimensional interconnected network, but also brings the intrinsic dispersion of graphene and carbon nanotubes and the dispersion of N-containing functional groups within a highly conductive scaffold. Thus, the NGSHs possess a large specific surface area of 812.9 m2 g-1 and high electrical conductivity of 53.8 S cm-1. Despite of relatively low nitrogen content (0.53 at%), the NGSHs demonstrate a high ORR activity, much superior to two constituent components and even comparable to the commercial 20 wt% Pt/C catalysts with much better durability and resistance to crossover effect. The same hybrid material also presents high catalytic activity towards OER, rendering them high-performance cheap catalysts for both ORR and OER. Our result opens up new avenues for energy conversion technologies based on earth-abundant, scalable, metal-free catalysts.

This work was supported by National Basic Research Program of China (973 Program, 2011CB932602) and Natural Scientific Foundation of China (No. 21306102).

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2694 TEM Characterization of ALD-grown TiO2 on CNT

Zhang Y.1, Utke I.2, Erni R.1
1Electron Microscopy Center, EMPA, Swiss Federal Laboratories for Materials Science and Technology, Überlandstrasse 129, CH-8600 Dübendorf, Switzerland, 2Laboratory of Mechanics of Materials and Nanostructure, EMPA, Swiss Federal Laboratories for Materials Science and Technology, Feuerwerkstrasse 39, CH-3602 Thun, Switzerland
yucheng.zhang@empa.ch

A thriving field in nanotechnology is to develop synergetic functions of nano-materials by taking full advantages of unique properties of each component. In this context, combining TiO2 nano-crystals and carbon nanotubes (CNTs) offers enhanced photo-sensitivity and improved photo-catalysis efficiency, which is crucial to achieving sustainable energy and preventing environment pollution and hence has aroused a tremendous research interest. Despite progress in synthesis and performance of the material system, further research is required to understand some fundamental aspects of the material system, such as how TiO2 nucleates and grows on CNTs, and what is the bonding at the TiO2-CNT interface. Answers to these questions also help to design nano-composites based on CNTs and metal/metal-oxides with novel functionalities.
In this work an atomic layer deposition (ALD) technique has been adopted to grow TiO2 nano-particles on multiwall-CNTs (MW-CNTs). Control of the crystallinity, particle size and morphology of TiO2 can be obtained through deposition parameters adopted in ALD and a surface pre-treatment of MW-CNTs using O2 plasma. Transmission electron microscopy (TEM) has been very useful to characterize the ensemble structurally, chemically and electronically. In particular, electron energy loss spectroscopy (EELS) in the scanning TEM (STEM) mode has been employed to study C-K and Ti-L2,3 edge fine structures in TiO2, CNTs and their interface, in order to shed light on the mechanism of nucleation and growth of TiO2 on CNTs, as well as the interfacial bonding of the ensemble.

The author Dr. Y Zhang would like to thank Marie Curie Cofund action for financial support

Fig. 1: TEM micrographs of TiO2 deposited on CNT at 200°C for various ALD numbers of cycles: (a), (c) and (e) are after 20, 200 and 750 cycles respectively, without plasma pre-treatment; (b), (d) and (f) are 20, 200 and 750 cycles respectively, with the CNT subjected to O2 plasma pre-treatment. The insets show the corresponding diffraction patterns.

Fig. 2: Core-loss EELS spectra show the Ti_L2,3 and O_K edges of TiO2 on CNT after 20 ALD cycles with and without O2 plasma pre-treatment. Difference in the near edge fine strucuture indicates different crystallinity of TiO2.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2699 Multi-scale investigations of nitrogen doping in graphene

Alloyeau D.1, Riccardi E.1, Lagoutte J.1, Ricolleau C.1, Wang G.1, Gallais Y.1
1Laboratoire Matériaux et Phénomènes Quantiques, Université Paris Diderot - CNRS, Paris, France
damien.alloyeau@univ-paris-diderot.fr

The electronic, thermal, and mechanical properties of graphene are exceptionally sensitive to lattice imperfections, surface functionalization and doping. Therefore, atomic scale structural and electronic investigations in this material are critically important for understanding these properties. Graphene samples produced by CVD method were doped with nitrogen by plasma exposure. We have exploited the complementarities of aberration-corrected TEM, Scanning Tunnelling Microscopy (STM) and micro-Raman spectroscopy to investigate the link between the structural and the electronic properties of N-doped graphene. Our experimental protocol allows applying these characterization techniques on the same samples in order to reduce the gap between micro and atomic scales investigation.

STM and HRTEM were used to characterize the nitrogen-induced single-point defects in graphene and the charge redistribution due to chemical bonding. As previously reported [1], the charge redistribution due to the insertion of nitrogen atoms in graphene that is easily detected by STM, allows the detection of such a low-contrast defect by HRTEM (Fig. 1). Our study highlights two important structural information about N-doped graphene and doping process. At first, Cu-supported graphene during the plasma exposure are more likely to be N-doped than suspended graphene. Secondly, the high variability of the C/N ratio on the same graphene sample reveals that nitrogen doping is not spatially homogeneous. This latter result pushed us to combine HRTEM and micro-Raman investigations on same micron-large areas of the samples, in order to provide a deeper understanding of the Raman spectrum as a function of the structure (holes, number of layers) and the Nitrogen doping rate of graphene (Fig. 2). 

[1] Meyer et al. Nature materials, 10, 209 (2011)

We are grateful to Region Ile-de-France for convention SESAME E1845, for the support of the JEOL ARM 200F electron microscope installed at the Paris Diderot University.

Fig. 1: HRTEM image of single layer graphene before nitrogen doping (a). Atomic scale analysis of nitrogen insertion in graphene: the charge redistribution due to chemical bonding is observed by aberration-corrected TEM (b) and STM (simulation of the structure and charge distribution in insert) (c).

Fig. 2: Raman spectra of the G-band optical phonon in two different spots of a Nitrogen-doped suspended graphene sample: the shift of the phonon peak energy (ωG) and its broadening show a significant variation of the chemical potential due to different Nitrogen doping.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2786 Bilayer graphene structures formed by passage of current through graphite: HRTEM and HAADF-STEM studies

Harris P.1, Slater T.2, Haigh S.2, Hage F.3, Kepaptsoglou D.3, Ramasse Q.3
1Electron Microscopy Laboratory, J.J. Thomson Building, University of Reading, Reading, RG6 6AF, UK, 2School of Materials, The University of Manchester, Manchester, M13 9PL, UK, 3SuperSTEM Laboratory, SciTech Daresbury, Keckwick Lane, Daresbury, WA4 4AD, UK
thomas.slater-5@postgrad.manchester.ac.uk

The subject of this paper is a new form of carbon which can be formed by passing an electric current through graphite [1,2]. This new carbon apparently consists of hollow graphitic shells bounded by curved and faceted planes, typically made up of two graphene layers. We describe studies of this carbon using high resolution transmission electron microscopy (HRTEM) and high-angle annular dark-field scanning transmission electron microscope imaging (HAADF-STEM). These studies appear to confirm that the bilayer graphene structures are 3-dimensional.
The carbon was prepared in an arc-evaporator which is normally used for coating specimens for scanning electron microscopy. Following evaporation, a small deposit was formed in the area where the two graphite electrodes made contact, and it was this deposit which contained the “transformed” carbon.
Some conventional TEM images of the transformed carbon are shown in Fig. 1. In the low magnification image (Fig. 1(a)), it can be seen that the outline of the structure is much more irregular than in normal graphite, with many curved and unusually-shaped features. Higher magnifications images, such as Fig. 1(b), show that the transformed carbon consists largely of bilayer graphene.
In order to determine the 3-dimensional shapes of the graphene structures we have used HAADF-STEM imaging. Both individual images and tilt sequences have been analysed. Individual HAADF-STEM images were recorded on an aberration-corrected Nion UltraSTEM100, operated at 60kV. Figure 2(a) shows a HAADF-STEM image of a region in which a nanotube is joined to a larger bilayer structure. The contrast in this image, in combination with a quantitative analysis of the near edge fine structure of the C K EELS edge [4], indicate that the edges of the structure are highly curved. This is consistent with the 3-dimensionality of this material.
Tilt series were recorded using an FEI Titan microscope operated at 80kV. A typical tilt sequence is shown in Fig. 2(b). This appears to show a 3-dimensional particle with the shape of a flattened cone.
Structural transformation of graphite as a result of the passage of an electric current has been observed by other groups [e.g. 5,6]. These groups have discussed the process in terms of the sublimation and edge reconstruction of flat graphene. However, as argued here, there are good reasons for believing that the transformed carbon is in fact 3-dimensional. If this is correct, this new carbon may have a number of possible applications, for example in supercapacitors.

[1] PJF Harris, J. Phys.: Condens. Matter 21 (2009), 355009.
[2] PJF Harris, Carbon 50 (2012) p.3195.
[3] PJF Harris et al., in preparation.
[4] XT Jia et al., Science 323 (2009) p.1701.
[5] JY Huang et al., PNAS 106 (2009) p. 10103.

The authors gratefully acknowledge funding from EPSRC, HM Government and the USA Defense Threat Reduction Agency.

Fig. 1: Conventional HRTEM images showing structure of carbon following passage of current.

Fig. 2: HAADF-STEM images of structures in transformed carbon. (a) Image showing junction between bilayer nanotube and larger region, (b) tilt sequence of approximately conical structure.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2825 Band Gap Expansion and Low-Voltage Induced Crystal Oscillation in Low-Dimensional Tin Selenide Crystals

Sloan J.1, Carter R.2, Suyetin M.3, Dyson M. A.1, Trewhitt H.1, Liu Z.4, Suenaga K.4, Giusca G.5, Kashtiban R. J.1, Bell G.1, Bichoutskaia E.3
1Department of Physics, University of Warwick, Coventry, CV4 7AL, UK, 2Department of Materials, University of Oxford, South Parks Road, OX1 3PH, UK, 3School of Chemistry, University of Nottingham, Nottingham, NG7 2RD, UK, 4National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8565, Japan, 5National Physical Laboratory, Teddington, TW11 0LW, UK
j.sloan@warwick.ac.uk

SnSe forms all-surface two atom-thick low dimensional crystals when encapsulated within single walled nanotubes (SWNTs) with diameters < 1.4 nm. Density Functional Theory (DFT) studies indicate that low-dimensional SnSe crystals typically undergo band-gap expansion. In slightly wider diameter SWNTs (~1.4-1.6 nm), we observe that three atom thick low dimensional SnSe crystals undergo a previously unobserved form of a shear inversion phase change resulting in two discrete strain states in a section of curved nanotube (not shown here). Under low-voltage (i.e. 80-100 kV) imaging conditions in a transmission electron microscope, encapsulated SnSe crystals undergo longitudinal and rotational oscillations, possibly as a result of the increase in the inelastic scattering cross-section of the sample at those voltages. Initial AC-TEM images were obtained at 100 kV using a JEOL 2010F transmission electron microscope fitted with a CEOS aberration corrector for which Cs was tuned to ~0.001 mm. Additional AC-TEM images were obtained at 80 kV were obtained on a JEOL JEM-ARM200F fitetd with an imaging corrector for which Cs was tuned to ~0.001 mm. 

Inside the narrower 1-1.4 nm SWNTs, we observed bilayer 2×2 SnSe nanocrystals (Fig. 1) and obtained images effectively viewed parallel to <001> relative to an ideal 2x2 rocksalt structure fragment. Systematic measurements of the lateral spacings of these encapsulated SnSe nanorods (Fig. 1(b)) relative to the centre point of the SWNT wall indicate that the obtained microstructure is undistorted and does not deviate significantly from the idealised 2x2 structure. A 2x2x6 atomic layer Sn12Se12 cluster based on average lattice spacings from the AC-TEM images (Fig. 1(b)) was DFT optimised and the resulting model used to generate a mutlislice image simulation from this model embedded in a (9,9) SWNT (Fig. 1(c)). DFT computed densities of states (DOS) for both bulk and 2x2 SnSe nanocrystals revealed that the latter have an expanded band gap of ca. 1.41 eV relative to the corresponding bulk bandgap of the rocksalt form (i.e. 0.68 eV). We had intended to perform higher resolution studies on the embedded SnSe nanocrystals using exit wave reconstruction from focal series of images but found that at 80 and 100 kV accelerating voltages, the embedded SnSe nanocrystals oscillate inside the encapsulating SWNTs (Fig. 2(a) and (b)).    

[1] Carter et al. Dalton Trans. 2014, published online DOI: 10.1039/c4dt00185k. 

J.S. and R.J.K. acknowledge the Warwick Centre for Analytical Science (EP/F034210/1). Z.L. and K.S. acknowledge JST-CREST and MEXT (19054017). E.B. acknowledges an ERC Starting (Consolidator) Grant, an EPSRC Career Acceleration Fellowship, and an EPSRC Research Leaders Award (EP/G005060).

Fig. 1: (a) AC-TEM image of (2 × 2)SnSe@SWNT. (b) Enlargement with dots indicating the centres of the Sn–Se columns and SWNT wall. (c) Second enlargement from (a) with overlaid multislice image simulation. (d) Side-on view and (b) end-on view of the experimental structure model.

Fig. 2: (a) This sequence of images obtained over ~12s at reveals two different modes of oscillation of 2 × 2 SnSe in a ~1 nm diameter SWNT. (b) Simulations and models of different rotational states of a 2 × 2 SnSe fragment in an (8,8) SWNT relative to an “ideal” <100> orientation (i.e. bottom right).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2850 Influence of charge carriers density on flexural phonon spectrum in graphene measured by electron diffraction

Kirilenko D. A.1
1Ioffe Institute
zumsisai@gmail.com

Graphene is a specific form of matter – an electronic membrane [1,2,3]. It is known that free-standing graphene undergoes severe corrugation [4]. And the main reason for this is flexural phonons. At the same time, structural fluctuations in graphene are influenced by charge carriers, which are easily generated in gapless graphene. It gives rise to a complicated phenomenon, so that electronic properties of graphene are defined by the interplay between charge carries and lattice distortions. This interplay enormously complicates the theory of transport in graphene [5]. For instance, full calculations of thermal dependence of graphene’s resistivity are still to be completed [6].

Previously, a technique for measuring of the flexural phonon spectrum basic parameters in free-standing graphene was presented [7], which was later expanded to measuring of the full spectrum profile. The technique uses electron diffraction obtained in transmission electron microscope (TEM) to scan the reciprocal lattice of graphene that gives information on its structural distortions. The feature of the electron diffraction imaging is that provides information on rapidly varying structural distortions, what is inaccessible by most of other techniques. It is remarkable, that electron beam of TEM does generate charge carriers in graphene. Variation of the electron beam intensity changes the generated charge carriers density. This allows measuring the changes of the flexural phonons spectrum related to the influence of charge carriers.

A significant dependence of the small-wavevector (long undulations) part of the flexural phonons spectrum has been found. Whereas, the large-wavevector part seems to avoid the charge carriers influence. In the accessible range of electron beam densities and, thus, generated charge carriers densities, the amplitude in the left part of the spectrum is being successively suppressed with increasing charge carriers density. That is, graphene becomes noticeably smoother at large scale. It must influence graphene’s resitivity at increased densities (or bias voltage in graphene-based devices).

Finally, influence of the charge carriers density on corrugation of suspended graphene has been measured and degree of the specific electron-phonon coupling estimated.

1. E.-A. Kim and A. H. Castro Neto, Europhysics Letters 84 (2008), 57007.
2. D. Gazit, Phys. Rev. B 80 (2009), 161406(R).
3. P. San-Jose, J. Gonzalez and F. Guinea, Phys. Rev. Lett. 106 (2011), 045502.
4. J.C. Meyer, A.K. Geim et al., Nature 446 (2007), p. 60.
5. M. Gibertini, A. Tomadin et al., Phys. Rev. B 85 (2012), 201405(R)
6. S. Das Sarma, S. Adam et al., Rev. Mod. Phys. 83 (2011), p. 407.
7. D.A. Kirilenko, A.T. Dideykin and G. Van Tendeloo, Phys. Rev. B. 84 (2011), 235417.

This work was supported by Russian Foundation for Basic Research.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2878 Imaging and Spectroscopy of Graphene/Hexagonal Boron Nitride Lateral Heterostructure Interfaces

Basile L.1,2, Liu L.3, Gu G.3, Vlassiouk I.2, Lupini A. R.2, Unocic R. R.2, Idrobo J. C.2
1Escuela Politécnica Nacional, Quito, Ecuador, 2Oak Ridge National Laboratory, Oak Ridge, USA, 3The University of Tennessee, Knoxville, USA.
lbasilec@gmail.com

Boundaries or in-plane interfaces in two-dimensional (2D) materials will play a critical role in future device applications. For example, electronic and mechanical properties are affected by structure, chemistry, morphology, and location of a grain boundary [1,2]. By using microscopic tools, we recently demonstrated lateral coherence in an in-plane heterostructure of graphene and hexagonal boron nitride (BN) on samples staying on the growth substrate [3]. An atomic-resolution STEM can be an ideal tool to image the presumably atomically sharp graphene-BN interface, but contaminations introduced during the transfer process hinder its direct observation.

In this study, we examined the contaminant-covered graphene-BN boundary using EELS in an aberration-corrected STEM, Nion UltraSTEM 100, equipped with a cold field emission electron source, a corrector of third and fifth order aberrations, and a Gatan Enfina spectrometer [4]. To avoid graphene and BN knock-on damage we operated the microscope at an acceleration voltage of 60 kV. A convergence semi-angle of 30 mrad, and 54 to 200 mrad collection semi-angles were used to obtain the medium angle annular dark field (MAADF) images. The EEL spectrum maps were collected with an energy resolution of ~350 meV.

Fig. 1(A) shows an experimental MAADF image of the graphene-BN boundary. Fast Fourier Transform of the areas shown in (A) indicates that the BN is aligned with the graphene monolayer. Evidence of a sharp interface is provided by the chemical map shown in (E), where the boron K-edge clearly defines a sharp graphene-BN interface.

Fig. 2 shows intensity profiles along the yellow lines of Fig 1. The left panel of Fig 2 shows that the graphene-BN boundary is composed of monolayer graphene and monolayer BN. The right panel shows a transition width of 0.5 nm between graphene and BN as determined from the boron K-edge signal. The transition width is defined from 25% to 75% of the values of the boron K-edge signal across the graphene-BN interface.

Direct observation of a boundary at atomic resolution requires a reliable method to free the graphene-BN interface of contaminants. We will discuss our current efforts on removing contaminants by in-situ annealing, thus revealing the buried graphene/BN interface. Our preliminary results indicate that regions of thousands of nanometer squares of clean graphene are produced during in-situ annealing. The method opens the door for the study of the long-range structure of 2D lateral heterostructure interfaces at the atomic scale.
[1] Adam W. Tsen, et al., Science 336 (2012), 1143
[2] Gwan-Hyoung Lee, et al., Science 340 (2013), 1073
[3] L Liu et al, Science 343 (2014), 163
[4] OL Krivanek et al, Ultramicroscopy 108 (2008), 179

National Secretariat of Higher Education, Science, Technology and Innovation of Ecuador (LB), NSF, the Defense Advanced Research Projects Agency (LL & GG), Office of Basic Energy Sciences, U.S. Department of Energy (DOE) (ARL), Center for Nanophase Materials Sciences, Office of Basic Energy Sciences, U. S. DOE (RRU, JCI).

Fig. 1: (A) MAADF image of a buried graphene-BN boundary. (B) and (C) fast Fourier transforms of the highlighted areas shown in (A). (D) and (E) simultaneously acquired STEM image and EEL spectrum map of the region shown in (A), respectively. The blue/white dashed lines indicate the graphene-BN boundary. Scale bars are 5 nm. Adapted from Ref. [3].

Fig. 2: (A) Intensity profile along the yellow line in Fig. 1(A). The intensity profile indicates that the boundary is composed of monolayer graphene and BN. (B) Boron K-edge intensity profile along the yellow line in Fig. 1(E). The boundary is sharp with a transition width of 0.5 nm. The spatial resolution is 0.5 nm. Adapted from Ref. [3].
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2888 Wetting behavior of ionic liquid on a carbon nanotube

Imadate K.1, Hirahara K.1
1Osaka University, Osaka, Japan
hirahara@mech.eng.osaka-u.ac.jp

It is interesting question how a single nanomaterial such as carbon nanotube gets wetting by liquid. Wettability of materials is generally determined by the balance of interface tensions acting at air-liquid, liquid-solid, solid-air boundaries, but nanometer-scale morphologies of solid surfaces often cause anomalous wetting behavior. Regarding carbon nanotubes, they have extremely high curvature surfaces due to their cylindrical shapes with nanometer scale diameters. In this study, we investigated wetting behavior on a single CNT by in-situ electron microscopy. Prior to the experiment, CNT probe was prepared by using TEM-STM holder for nanomanipulation in a transmission electron microscope (TEM, JEM-2500SE) at 90kV acceleration voltage. A multi walled CNT was attached to the tip of cantilever probe used for scanning probe microscopy. On the other hand, ionic liquid was used as a liquid specimen, since it is rather stable in vacuo due to extremely low evaporation pressure. It was supported as the liquid level was parallel to the incident beam direction on a specimen stage of nanomanipulator. Tip of the CNT was then approached to the ionic liquid from the normal direction to the liquid level, and the series of images were recorded as movies at the moment when the tip touched to the ionic liquid. As the result, meniscus formed at the contact region, and a thin film with 3nm thickness simultaneously formed to cover entire the CNT. The contact angle measured at the meniscus was almost zero. These results indicate that CNT shows autophobic wetting, although macroscale droplet on a plane graphite surface shows about 25˚ contact angle. In addition, similar experiments were performed in a scanning electron microscope, and attractive wetting forces were measured on the basis of Wilhelmy method for CNTs with 5~15 nm diameters. Measured values indicated a tendency to be greater than the expected values from Wilhelmy equation representing the correlation of the force, tube diameter and surface tension. Instead, fitted curve to the experimental data showed the increment of effective diameter of cylindrical sample. The corrected value was 2.84nm, which is consistent to the thickness of liquid film formed on CNT. Accordingly, the wetting behavior observed in the present study can be explained by considering that the liquid film acts as a part of a solid cylinder, which suggests a possibility that liquid molecules are rather strongly constrained on the CNT surface.

Ionic liquid used in this study was provided by T. Tsuda in Osaka University.

Fig. 1: SEM images of a CNT probe before and after contacting to the ionic liquid. We can see that the CNT is pulled into the ionic liquid due to the attractive wetting force. For this case, the force was measured by 1.2nN.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2898 Measurement of the tensile force applied to a carbon nanotube during the axial shrinking deformation

Hirahara K.1, Nishiyama Y.1
1Osaka University, Osaka, Japan
hirahara@mech.eng.osaka-u.ac.jp

We have studied on deformation process of a bridged carbon nanotube (CNT) during Joule heating by in-situ transmission electron microscopy (TEM). Many papers reported that a CNT got thin or cut at the central portion due to or cutting and reconnecting of bonds or sublimation of carbon atoms during the Joule heating [1~4]. In this study, another type of deformation was observed, namely shrinking deformation along the axial direction of the CNT. We found that such a shrinking was observed when the CNT was bridged between rather frexible electrodes, namely the CNT could change its length during the heating. This result suggested that these deformation process strongly depended on how release the tensile stress applied to the CNTs caused by sublimation of carbon atoms during the Joule heating. Therefore we measured the stress loaded to the CNT during the Joule heating. For the measurement, a cantilevered probe for scanning probe microscopy was used as the flexible electrode, which spring constant was 0.02~0.41 N/m. A single or double wall CNT is bridged between the cantilevered probe and a Pt/Si substrate by operating a nanomanipulation holder (TEM-STM system, nanofactory) in TEM (JEM-2500SE, 90kV). Current is then applied to the CNT, and its deformation process was recorded. When the CNT began shrinking, cantilevered probe underwent deflection and made balance to the tensile force, so that the tensile force applied to the CNT were able to be measured by monitoring the deflection of cantilevered probe. In this system, tensile stress applied to the CNT gets increase as the degree of shrinking increased, and the CNT finally cut or detached from the electrode. Experimental results revealed that the shrinking deformation of CNTs occurred with loading tensile stress under 1.9 N/m2. It is also suggested that such a shrinking deformation was promoted when topological defects formed; carbon atoms may selectively evaporate from such defective site.

[1] H. Maruyama, et al., Appl. Phys. Ex. 3, (2010) 025101.
[2] T. D. Yuzvinsky, et al., Nano Lett. 6 (2006) pp. 2718-2722.
[3] J. Y. Huang, et al., Nature 439 (2006) pp. 281.
[4] K. Hirahara et al., Appl. Phys. Lett. 97 (2010) 051905.

Fig. 1: A series of TEM images showing shrinking deformation of a carbon nanotube. We can see that the cantilevered probe underwent deflection by pulling. Initial length of  this nanotube was 239nm, and shrank about 100 nm at the botom image.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2925 Probing the dynamics of structure defects and chemical dopants in monybdenum disulfide monolayer at elevated temperature by Cs-corrected STEM

Jin C.1, Lv D.1, Hong J.1
1State Key Laboratory of Silicon Materials, Key Laboratory of Advanced Materials and Applications for Batteries of Zhejiang Province, Department of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang, PR China.
chhjin@zju.edu.cn

As a representative family member of the emerging two-dimensional transition metal dichalcogenides (TMDCs), atomically thin molybdenum disulfide has attracted intensive research efforts owing to its unique structural and electronic properties that has promised a wide application in future nanoelectronic and optoelectronic devices [1-3]. Since defects plays an important role on tailoring the physical and chemical properties of any semiconductors, molybdenum disulfide is not an exception. Therefore it is very important to resolve the structure defects and figure out the impact of these defects on the physical properties of molybdenum disulfide. [4-6]

In this talk, we will present our latest progress on studying the atomic defects and dopants in molybdenum disulfide monolayers by aberration-corrected STEM. Furthermore, with the assistance of MEMS-heating technique, the adsorption, migration and coalescence of structural defects at elevated temperatures can be directly observed in situ. The absorption sites, diffusion pathways and the associated activation energy are experimentally determined experimentally, which are further supported by the DFT calculations.

Reference:

[1] K. F. Mak, C. Lee, J. Hone, J. Shan, and T. F. Heinz, Physical Review Letters 105, 136805 (2010).

[2] A. Splendiani et al., Nano Letters 10, 1271 (2010).

[3] Q. H. Wang, K. Kalantar-Zadeh, A. Kis, J. N. Coleman, and M. S. Strano, Nature Nanotechnology 7, 699 (2012).

[4] P. Komsa, J. Kotakoski, S. Kurasch, O. Lehtinen, U. Kaiser, and A. V. Krasheninnikov, Physical Review Letters 109, 035503 (2012).

[5] A. M. van der Zande et al., , P. S. Huang et al., Nature Materials 12, 554 (2013).

[6] W. Zhou et al., Nano Letters 13, 2615 (2013).

The work on microscopy is done in the EM Center of ZJU. This work is financially supported by the NSFC (51222202,), the National Basic Research Program of China (2014CB932500), the Program for Innovative Research Team in University of Ministry of Education of China (IRT13037) and the Fundamental Research Funds for the Central Universities (2014XZZX003-07).

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2934 Probing band structures of atomically thin molybdenum disulfide by EELS

Hong J.1, Li K.2, Jin C.1, Zhang X.2, Yuan J.3, Zhang Z.1
1State Key Laboratory of Silicon Materials, Key Laboratory of Advanced Materials and Applications for Batteries of Zhejiang Province, Department of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, P. R. China., 2Advanced Nanofabrication, Imaging and Characterization Core Lab, King Abdullah University of Science and Technology (KAUST), Thuwal 239955, Kingdom of Saudi Arabia, 3Department of Physics, University of York, Heslington, York, YO10 5DD, United Kingdom
jinhuahong436@gmail.com

In recent years, semiconducting MoS2 has attracted much public attention because of its hexagonal structure, proper bandgap (1.3~1.8eV) and potential application in nanoelectronics and valleytronics. Currently a clear and complete picture of bandgap transition (<2eV), higher interband transition (~5eV) and plasmon resonance (~23eV) associated with the thickness-dependent electronic structure is still lacking. In this talk, we will present the EELS study on the electronic structures of atomically thin MoS2.
        We use a spherical aberration corrected TEM (FEI Titan Cube) to conduct angle resolved EELS measurement. This microscope is equipped with a monochromator providing an energy resolution of 0.14eV which help us resolve fine structures of low loss EEL spectrum and obtain band structure of MoS2. A transition from indirect to direct gap is illustrated as the thickness decreases down to monolayer. Other strong interband transition peaked at 3.1eV and 4.5eV and high-energy π+σ Plasmon excitation at 23eV are also presented as a function of thickness and momentum transfer q. These excitations (not easily accessible by conventional optical characterization) in atomically thin MoS2 are reported for the first time. Their energy redshift with the decreasing thickness and monotonically-increasing linewidth dispersion with q indicate the spilling-out effect and Landau damping, respectively, in this low dimensional electron gas system. Our investigation provides a successful paradigm to depict the electronic structures of any other novel transition metal dichalcogenides (TMDs).

This work on microscopy was carried out in the Imaging and Characterization Core Lab of KAUST in Saudi Arabia. This work is financially supported by the National Science Foundation of China (51222202,), the National Basic Research Program of China (2014CB932500), the Program for Innovative Research Team in University of Ministry of Education of China (IRT13037).

Fig. 1: Figure 1 (a) The primitive cell of monolayer MoS2 with lattice basic vector a1 and a2. Purple atom: Mo; yellow: S2. (b) Reciprocal lattice (electron diffraction pattern)  and first Brillouin zone. (c) The scattering geometry of angle resolved EELS . (d) Corresponding angle-resolved spectrum profile with qy along ΓM direction in (c). 
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-2938 HRTEM studies of Bamboo-like nanotubes found in the carbonaceous chondrite Allende meteorite.

Rendon L.1, Cocho G.1, Cruz H.1, Ortega F.2, Reyes M.2, Buhse T.3, Garibay V.4, Santiago P.1
1Instituto de Física, Universidad Nacional Autónoma de México; Circuito de la Investigación Científica s/n. Ciudad Universitaria. C.P. 04510, México D.F, 2Instituto de Geología, Universidad Nacional Autónoma de México. Circuito de la Investigación Científica s/n. Ciudad Universitaria. C.P. 04510, México D.F., 3Facultad de Ciencias, Universidad Autónoma del Estado de Morelos. Av. Universidad 1001, Col. Chamilpa, 62209 Cuernavaca, Morelos, México., 4Instituto Mexicano del Petróleo. Eje Central Lázaro Cárdenas Norte 152 Col. San Bartolo Atepehuacan, C.P 07730, México.
paty@fisica.unam.mx

In February 8, 1969 a large carbonaceous chondrite meteorite fell in Allende Chihuahua, Mexico. Carbonaceous chondrites meteorites are very important because of their organic compounds and peculiar composition. Allende meteorite has large and abundant chondrules (mm-sized) in olivine matrix, large refractory inclusions, a low degree of aqueous alteration and graphitized carbon. Its large carbon content has represented an interesting source to the study of evolution and lineage of carbon chemistry, from nebular to current ages and has been related with prebiotic Earth, because its collisions and impacts with early Earth formation represent an organics extraterrestrial input. The organic composition in carbonaceous chondrites is diverse, and it is possible to mention as example kerogenic material macromolecular, sugar alcohols, ketones, amines, and amino acids. The nanostructures in this type of meteorites have been identified as fullerenes, carbon onions and the possible presence of carbon nanotubes (CNTs) has been suggested since 2006. Actually, inorganic serpentine nanotubes were described by Zega and co-workers in meteoritic matrix of carbonaceous chondrites, ranking in ~20nm diameter tube.

At high temperatures, carbon precursors are decomposed or evaporated and then condensed to build the sp2 graphite networks of CNTs. High temperatures are normally obtained from external heating, which is highly energy-consumed. Theoretically, such a problem can be solved by employing hugely exothermic reaction systems like the conditions of the early sun.

The sample was obtained from the collection of the Geology Institute, at the Autonomous National University of Mexico. In order to avoid the contamination of the meteorite sample, we drilled a hole with a steel laboratory spatula in the small piece of the meteorite. The powder obtained was supported in a microscopy glass slide and grinded with a second slide. The powder was supported in an electron microscopy grid to be observed in a JEM-2200FS TEM.
By HRTEM we observed bamboo-carbon nanotubes (BCNTs) in the meteorite sample as well as polyhedrical carbon structures. BCNTs can be thought as coaxial graphene sheets built of sp2 bonded. The tubes are concentric and coaxial. They also are highly defective and several bounds are broken, this fact promotes active bonds to act as chiral templates of other organic molecules.

Authors acknowledge the financial support from DGAPA-UNAM, through grant IN113411.

Fig. 1: Figure 1. a) BCNTs of about 20 nm wide. A highly defective structure is shown in the tubes indicating a chirality. b) A closer view of the coaxial structures.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-3037 Topography mapping of ultrathin layered crystals

Dolle C.1, Niekiel F.1, Mittelberger A.1, Butz B.1, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Friedrich-Alexander-Universität Erlangen-Nürnberg
christian.dolle@ww.uni-erlangen.de

Quasi-2D crystalline materials are widely investigated to explore their optical, electronic and mechanical properties. The most prominent examples in this class of materials are graphene, hBN, and recently dichalcogenites, e.g. MoS2.

The third dimension of those crystals may not be neglected since freestanding membranes are thermodynamically forced to form intrinsic ripples. Moreover, the resulting topography of such materials is expected to have a severe influence on their properties. For example, strain, which can alter the band structure, is caused by any change of the surface inclination. In graphene corrugations have already been confirmed by Meyer et al. applying electron diffraction [1]. Inspired by that study we developed a method to determine the topography of freestanding membranes by diffraction-contrast TEM imaging. The procedure is based on dark field (DF) tilt series using at least two independent g-vectors oriented perpendicular to the respective tilt axis. In those DF images, the measured intensity directly depends on the local excitation condition and thus on the local inclination of the membrane. By tilting, the reciprocal lattice rod (relrod) is scanned simultaneously in each sub-region of the DF images. To determine the inclination of each sub-region with respect to the specific g-vector, the maximum of the tilt-angle dependent intensity distribution is fitted. This is done for two different tilt series and the obtained data are used to calculate the absolute inclination of each sub-region and thus to determine the membrane topography.

We applied the procedure to freestanding membranes from high-quality epitaxial graphene on SiC [2]. Fig. 2a) depicts a representative {11-20} DFTEM image of such a few-layer graphene membrane. The local mean image intensity represents the number of graphene layers as proven by rocking curves. The sharp dark lines in the DF image are due to basal-plane partial dislocations, which have an additional impact on the local topography [3]. To demonstrate the strongly different intensity distributions along different directions Fig. 2e)-f) show exemplary DF images at 0 tilt for 3 independent {11-20} directions. It can be recognized that, while the wavy topography leads to strong, almost parallel contrast variations in the (11-20) and (1-210) images, the (2-1-10) DF image (with g perpendicular to the wave-direction) is less influenced.

While in the used example the basal-plane partial dislocations have a severe influence on the topography of the material, it will be shown that even the choice of the TEM support has a strong impact on the topography of defect-free membranes.

1Meyer et al., Solid State Commun. 2007, 143, 101

2Waldmann et al., ACS Nano 2013, 7, 4441

3Butz et al., Nature 2014, 505, 533

We acknowledge financial support by the Cluster of Excellence: Engineering of Advanced Materials and SFB 953: Synthetic Carbon Allotropes.

Fig. 1: a) Model of inclined membrane: Inclinations non-parallel to the used g-vector show intensity variations as indicated by the dark and light gray areas, b) Ewald sphere construction, c), d) enlargement for almost flat and strongly inclined membrane area

Fig. 2: a) Graphene membrane with 2-, 3- and 4-layer areas (scale bar 500 nm), b)-d) rocking curves extracted from the areas indicated (2, 3, 4 layers), e)-f) 3-layer graphene DF images obtained with the 3 indicated g-vectors, dotted line shows tilt axis orthogonal to the reflection used for imaging.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-3075 Graphene-based materials and breast cancer cells

Ponticelli G. S.1, Colone M.1, Rago I.2, Sarto M. S.2, Stringaro A.1
1Italian National Institute of Health, 2Research Center on Nanotechnology Applied to Engineering Sapienza University of Rome
gennaro.ponticelli@guest.iss.it

Recent discoveries on graphene, a two-dimensional, crystalline allotrope of carbon, stimulated research on related structures, such as Graphite NanoPlatelets (GNPs), a 1-15 nm thick flake, constituted of 3-48 layers of graphene, obtained starting from Intercalated Graphite Compounds (GIC) via thermochemical exfoliation. These novel nanomaterials are providing fascinating opportunities for biotechnological development because of their unique structures, properties and possible applications.
Graphene and its derivatives are promising candidates for important biomedical applications because of their versatility. Due to the expanding applications of nanotechnology, human and environmental exposures to graphene-based nanomaterials are likely to increase in the future. However, the prospective use of graphene-based materials in a biological context requires a detailed comprehension of their toxicity.
Herein, we report on the interaction of stable and evenly dispersed exfoliated GNPs obtained using an ultrasonic bath for different times (30 min, 50 min and 70 min) with human breast adenocarcinoma cells (SKBR3 and MDA-MB-231) for 24 h. Biocompatibility of nanoplatelets has been evaluated by MTT (Fig. 1) while cell viability has been detected using Trypan Blue assays (Fig. 2). GNPs particles were more cytotoxic in SKBR3 than MDA-MB-231 cells suggesting a cell phenotype-dependent effect.
Furthermore, light microscopy observations (Fig. 3 and 4) and scanning electron microscopy analysis (data not shown) were used to gain understand on the mechanism of cell-nanoplatelets interaction. The bright-field images showed GNPs particles on SKBR3 and MDA-MB-231 cellular surfaces (see arrows).
Our results lead us to expect that efforts with interdisciplinary approaches among chemistry, biology, and engineering will accelerate mechanistic understanding of graphene-based platforms for bio and nanomedicine applications.

Fig. 1: GNPs (30 min, 50 min and 70 min) biocompatibility on SKBR3 and MDA-MB-231 cell lines by MTT test after 24 hrs of incubation.

Fig. 2: SKBR3 and MDA-MB-231 cell viability evaluation by Trypan blue assay after incubation with GNPs (30 min, 50 min and 70 min) for 24 hours.

Fig. 3: Bright-field microscopy image of SKBR3-nanoplatelets interaction. Cells were incubated for 24 hours with GNPs (arrows).

Fig. 4: Bright-field microscopy image of MDA-MB-231-nanoplatelets interaction. Cells were incubated for 24 hours with GNPs (arrows).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-3097 TEM and EELS studies of structures obtained under the different conditions of the thermobaric treatment of C60+CS2

Tyukalova E. V.1,2, Perezhogin I. A.1, Kulnitskiy B. A.1,2, Blank V. D.1,2, Popov M. Y.1,2, Alekseev M. V.1,2
1Technological Institute for Superhard and Novel Carbon Materials, Troitsk, Moscow, Russian Federation , 2Moscow Institute of Physics and Technology State University, Dolgoprudny, Moscow Region, Russian Federation
elizavetatyukalova@gmail.com

In our work we carry out the transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) studies of the samples obtained in a series of experiments on the thermobaric treatment of C60 with addition of CS2 in a diamond anvil cell with shear deformation ability. The main goal of the research is to obtain and characterize the new structures based on C60, because C60 is a very promising precursor for production of superhard materials. TEM study was done by a JEM-2010 microscope with GIF Quantum attachment for EELS. Features of radial distribution of pressure and shear in diamond anvil cell resulted in the coexistence of two types of structures in the samples: the crystalline and disordered phases. The crystalline phase represents itself slightly distorted fragments of the original face centered cubic (FCC) lattice fullerene (fig. 1). Apparently, these distortions appear due to the polymerization of C60 molecules.
Exact structure of disordered phases (fig. 2) has not been established by us, but according to our high-resolution images, it has inherited some elements of symmetry from the FCC lattice of fullerenes. The interplanar spacing in fig. 2 is about 0.35 nm, while the fragments of the “lattice” are deformed and disoriented one relatively to another. The microdiffraction and Fourier analysis have shown that two systems of fringes seen in fig. 2 intersect composing different angles in a range from 70° to 85°.
Figure 3 shows EELS spectra of the obtained specimens. The spectrum of C60 obtained under the pressure of 12-17GPa with shear deformation (fig. 3, (a)) correspond to the structures shown in fig. 1, and it is very close to the spectrum of the FCC lattice of the original C60 (fig. 3, (b)). The spectrum taken from the sample obtained at 25-30 GPa with shear deformation (fig. 3, (c)) and that from the sample obtained under 5 GPa at a temperature of 973° C (fig. 3, (d)) without shear, are almost identical.
At the same time the peaks of the all spectra in fig. 3 have the same positions, but the relative intensities of these peaks in (c) and (d) is different from those in (a) and (b). For example, the initial fullerene in (a) (and sample in (b)) has an absolute maximum at 300 eV, while in (c) and (d) it is about 292 eV. According to the literature data the small peaks at about 287eV correspond to the presence of molecular C60. Therefore, basing on our EELS data, we assume that the molecular C60 is present in the structures of both types in our samples, but the structure shown in fig. 2 is significantly different from the traditional fullerene FCC lattice.

Fig. 1: HRTEM image of the distorted FCC lattice of fullerene [110] zone axis. The interplanar spacing is slightly distorted in different areas, and the angle between planes in such fragments is not always exactly 70.5°.

Fig. 2: HRTEM image of the disordered phase. The interplanar spacing is about 0.346 nm, and the angles between the intersecting planes differ within the range from 70° to 85°.

Fig. 3: EELS spectra of: a) sample obtained at 12-17 GPa b) initial fullerene; c) sample obtained at 25-30 GPa with shear deformation; d) sample obtained at 5 GPa at temperature of 973° C
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-3149 Nitrogen Doped Single-Walled Carbon Nanotubes: Experimental and Theoretical Atomic-Resolved EELS Studies

Arenal R.1, 2, March K.3, Ewels C. P.4, Rocquefelte X.4, Kociak M.3, Loiseau A.5, Stephan O.3
1Lab. Microscopias Avanzadas (LMA), Inst. Nanociencia Aragon (INA), U. Zaragoza, Spain., 2ARAID Fundation, Zaragoza, Spain., 3Lab. Physique Solides (LPS), CNRS-U. Paris Sud, Orsay, France., 4Institut Matériaux Jean Rouxel (IMN), CNRS-U. Nantes, Nantes, France., 5Lab. Etude Microstructures (LEM), CNRS-ONERA, Châtillon, France.
arenal@unizar.es

Having access to the chemical environment at the atomic level of a dopant in a nanostructure is crucial for the understanding of its properties. A very good example in this context is the case of notably nitrogen-doped carbon nanotubes (CNx-NT) because their properties are significantly affected by the atomic arrangement of the dopant atoms in such nanostructures [1-4]. Thus the knowledge of this information requires precision measurements, combining high spatial resolution and high spectroscopic sensitivity. In order to achieve these goals, we have developed, for the first time, atomically-resolved EELS allowing us to detect individual N dopants in single-walled (SW) carbon nanotubes. These results have been compared with first principles calculations.

The STEM-EELS-experiments were performed in a NION UltraSTEM 200, operated at 60 kV. In parallel, HRTEM imaging studies have been performed using an imaging aberration-corrected FEI Titan-Cube microscope working at 80 kV.

Figure 1 displays a HAADF image of a CNx-SWNT where an EEL spectrum-image (SI) has been recorded in the red marked area of the image. Three single EEL spectra, extracted from this spectrum-image, in the marked positions/pixels of Fig. 1 (b) (spectra labelled (i), (ii) and (iii)), the 4th spectrum is the sum of (i) and (ii). The C-K edge is visible in the three spectra. In only two of the spectra of the whole dataset (1755 spectra), the nitrogen signal is also detectable. The nitrogen 1s (N1s) ELNES, expanded in Fig. 1 (c), show a strong peak at ~401 eV, with very little signal at energies above this. Comparing the spectra to density functional theory (DFT) ELNES calculations of possible single nitrogen defects, there is excellent agreement with the spectrum for substitutional nitrogen (Fig. 1 (c)(iii) and atomic model, Fig. 1(d)) across the range of π* and σ* bands. We have also investigated other more complex configurations that we will present and discuss in this contribution [5]. In summary, these studies elucidate a crucial question concerning the nature of the nitrogen atomic configuration of CNx-NTs. In fact, this detailed knowledge of how nitrogen atoms are incorporated in the carbon lattice as well as precisely control of their incorporation are required for the use of these NTs for future technological applications.

[1] R. Arenal, X. Blase, A. Loiseau, Advances in Physics 59, 101 (2010).
[2] P. Ayala, R. Arenal, A. Rubio, A. Loiseau, T. Pichler, Rev. Mod. Phys. 82, 1843 (2010).
[3] P. Ayala, R. Arenal, M. Rummeli, A. Rubio, T. Pichler, Carbon 48, 575 (2010).
[4] C.P. Ewels, M. Glerup, J. Nanosci. Nanotech. 5, 1345 (2005).
[5] R. Arenal, K. March, C.P. Ewels, et al., submitted.

The research leading to these results has received funding from the EU 7th Framework Program under Grant Agreement 312483-ESTEEM2 (I3) and from the French CNRS (FR3507).

Fig. 1: Fig. 1 (a) HAADF image of a CNx-SWNT. An EELS-SI has been recorded in the red area. (b) Selection of EEL spectra extracted from the SI, pixels outlined in the inset HAADF image acquired simultaneously with the SI. Each curve corresponds to a single spectrum from the SI, except the black, which is a sum of previous ones.

Fig. 2: Fig. 2(a) Simulated N1s ELNES (iii) & N partial DOS calculations ((i)purple=pz π*-states, (ii)green=px-y σ*-states) for substitutional N, compared to the experimental spectrum (iv) (Fig. 1(b-iii)). These simulations allow unambiguous assignment of the peak at ~401eV to a substitutional configuration shown in the DFT optimized structure, Fig. 2(b).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-3164 STEM and EELS investigation of graphene nanoribbon epitaxially grown over SiC

Gloter A.1, Palacio I.2, Celis A.3, Nair M.2, Zobelli A.1, Sicot M.4, Malterre D.4, Nevius M. S.5, Berger C.5, de Heer W. A.5, Conrad E. W.5, Taleb-Ibrahimi A.1, Tejeda A.1,2
1Laboratoire de Physique des Solides, Université Paris-Sud, CNRS, UMR 8502, F-91405 Orsay Cedex, France, 2UR1 CNRS/Synchrotron SOLEIL, Saint-Aubin, 91192 Gif sur Yvette, France, 3Synchrotron SOLEIL, L’Orme des Merisiers, Saint-Aubin, 91192 Gif sur Yvette, France, 4Université de Lorraine, UMR CNRS 7198, Institut Jean Lamour, BP 70239, F-54506 Vandoeuvre-lès-Nancy, France, 5School of Physics, The Georgia Institute of Technology, Atlanta, Georgia 30332-0430, USA
gloter@lps.u-psud.fr

Graphene nanoribbons grown on the (1-10n) and (-110n) facets of SiC have demonstrated exceptional electronic properties as ballistic transport along their long direction and a band gap in the small direction [1]. In order to understand these electronic properties, we have performed (S)TEM (HAADF, LAADF, EELS) investigation in combination with STM and ARPES measurements. The (S)TEM have been performed on X-section sample as it can be schematically seen in the figure 1. Using Cs corrected STEM at 60 keV voltage, the structural aspect of the graphene can be maintained for high resolution investigation and EELS spectromicroscopy (Figure 2). These electronic properties (i.e. linear dispersion, no gap and Dirac point at the Fermi level) are precisely observed by angle-resolved photoemission on these ribbons at the (1-107) facet [2] and this will be discussed in term of curvature effect, quantum confinement or presence of sp3 bonding with respect to the STEM-EELS investigation [3].

[1] "Exceptional ballistic transport in epitaxial graphene nanoribbons," J. Baringhaus, M. Ruan, F. Edler, A. Tejeda, M. Sicot, A. Taleb-Ibrahimi, A.-P. Li, Z. Jiang, E.H. Conrad, C. Berger, C. Tegenkamp, and W.A. de Heer, Nature 506, 349 (2014).
[2] “A wide band gap metal-semiconductor-metal nanostructure made entirely from graphene”
J. Hicks, A. Tejeda, A. A. Taleb-Ibrahimi, M.S. M.S. Nevius, F. F. Wang, K. K. Shepperd, J. J. Palmer, F. Bertran, P. Le Fèvre, J. Kunc, W.A. de Heer, C. Berger, E.H. Conrad, Nature Physics 9, 49 (2013).
[3] “The origin of the gap in armchair sidewall nanoribbons: a structural study” I. Palacio, A. Celis, A. Gloter, A. Zobelli, M. Sicot, D. Malterre, M.S. Nevius, C. Berger, W.A. de Heer, E.W. Conrad, A. Taleb-Ibrahimi and A. Tejeda, in preparation.

Fig. 1: Figure 1. General overview of graphene nanoribbons grown on SiC and SiC facets (sidewall ribbons). a) Scheme of the localization of the ribbons on the samples. b) STM image showing the regions with [0001] normal. Plateaus width of 50 nm c) Cross sectional TEM image of the array of ribbons in another sample. Plateaus width of 300 nm.

Fig. 2: Figure 2. STEM-HAADF view of a graphene-SiC interface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-3218 TEM electron diffraction analysis of few-layer Black Phosphorus

Vicarelli L.1, Castellanos-Gomez A.1, Van der Zant H. S.1, Zandbergen H. W.1
1Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands
l.vicarelli@tudelft.nl

Black phosphorus (BP) is an allotrope of Phosphorus characterized by a layered structure. It has been recently shown [1] that, similarly to graphene, it can be mechanically exfoliated to isolate atomically thin layers which have very interesting electrical and photonic properties. Single-layer BP is in fact an intrinsic semiconductor with a direct bandgap (~2 eV) and it has been employed in the fabrication of field-effect transistors with large current on-off ratios and high mobilities (100-3000 cm2/Vs) [1].

Given the rising interest in this layered material, an extensive TEM analysis of few-layer BP was performed [2].
We have investigated the Electron Diffraction (ED) pattern of few-layer black phosphorus transferred on a holey Silicon Nitride membrane with 1 µm holes diameter (see Figure 1(a)). An HRTEM image from a multilayer area of the sample is shown in Figure 1(b). The uniformity in this image indicates that the lattice contains no extended defects (single vacancies cannot be detected). We found that electron diffraction patterns depend on the number of layers and thus ED can be employed to determine the thickness of the BP flakes. We simulated electron diffraction patterns finding that the ratio between the 101 and 200 reflections depends on the number of black phosphorus: in particular this ratio is > 1 for single layer BP and decreases rapidly with the number of layers. The table shown in Figure 2 summarizes the simulated 101/200 intensity ratios for different number of layers, together with the experimental data acquired. Figure 3(a) and 3(b) show an ED taken from a thin region and a thick region of the flake, with 101/200 intensity ratios of 0.4 and 0.01, respectively.
We also noticed the presence of “forbidden” reflections (h+l = 2n+1) in the thin sample, which was not accounted in our simulations. This could be explained by the presence of adatoms on the surface of the black phosphorus layer or a slight distortion of the lattice.

References:
[1] Li, L.; Yu, Y.; Ye, G. J.; Ge, Q.; Ou, X.; Wu, H.; Feng, D.; Chen, X. H.; Zhang, Y.
Preprint at arXiv:1401.4117 (2014)

[2] “Isolation and characterization of few-layer black phosphorus”, Castellanos-Gomez, Andres; Vicarelli, Leonardo; Prada, Elsa; Island, Joshua O.; Narasimha-Acharya, K. L.; Blanter, Sofya I.; Groenendijk, Dirk J.; Buscema, Michele; Steele, Gary A.; Alvarez, J. V.; Zandbergen, Henny W.; Palacios, J. J.; van der Zant, Herre S. J. Preprint at arXiv 1403.0499 (2014)

The research leading to these results has received funding from the European Research Council, ERC Project n. 267922

Fig. 1: (a) Optical image of a black phosphorus flake transferred onto a holey silicon nitride membrane. (b) High resolution transmission electron microscopy image of the multilayered region of the flake (~ 13-21 layers).

Fig. 2: Thickness dependence of the electron diffraction patterns. We display the thickness dependence of the intensity ratio between the 101 and 200 reflections. The experimental data acquired on two spots of the thin flake and one spot of the thicker area has been included for comparison.

Fig. 3: (a) and (b) are the electron diffraction patterns acquired with a 400 nm spot on the thin (~ 2 layers) and on the thick (~ 13-21 layers) region of the flake, respectively.
Fig. 4:
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-3242 In situ growth of layered carbon

Kling J.1, Hansen T. W.1, Wagner J. B.1
1Center for Electron Nanoscopy (DTU Cen), Technical University of Denmark, Kgs. Lyngby, Denmark
jenk@cen.dtu.dk

Nanostructured carbon materials are predicted to play a major role in future electronic applications. Cheaper and smaller components with improved or new functionality and lower power consumption are necessary, where conventional materials reach their limitations. Layered carbon materials, such as graphene or multilayer graphene, can be used for extremely compact devices with outstanding performance [1],[2]. A cheap way to synthesize such materials on a large scale is chemical vapor deposition (CVD) growth on catalysts like copper or nickel [3],[4]. However, the understanding and control of such growth processes are still in their infancy.

Here we present in situ transmission electron microscopy (TEM) experiments in a FEI Titan 80-300 Environmental TEM (ETEM) for studying the growth of layered carbon materials on Ni and Cu catalysts. The ETEM allows imaging with controlled gas environments around the sample up to a few mbar. In combination with a MEMS-based heating holder, growth of layered carbon materials is systematically studied at the atomic level using various carbon sources and growth temperature.

As an example, growth of few layer graphene from C2H2 on a Ni catalyst is shown in Fig. 1-4. NiO particles in the size range up to a few hundred nm are reduced in the microscope under H2 at 500-600°C in order to form a catalytically active Ni surface. Introducing C2H2 at about 650°C leads to growth of layered carbon (Fig. 1-4). By following the appearance of carbon layers, the growth rate dependence on various parameters can be determined directly from the ETEM observations.

[1] K. S. Novoselov, S. V. Morozov, T. M. G. Mohinddin, L. a. Ponomarenko, D. C. Elias, R. Yang, I. I. Barbolina, P. Blake, T. J. Booth, D. Jiang, J. Giesbers, E. W. Hill, and a. K. Geim, Phys. Status Solidi 244, 4106 (2007).
[2] F. Schwierz, Proc. IEEE 101, 1567 (2013).
[3] X. Li, W. Cai, J. An, S. Kim, J. Nah, D. Yang, R. Piner, A. Velamakanni, I. Jung, E. Tutuc, S. K. Banerjee, L. Colombo, and R. S. Ruoff, Science 324, 1312 (2009).
[4] X. Li, W. Cai, L. Colombo, and R. S. Ruoff, Nano Lett. 9, 4268 (2009).

Financial support of the 7th Framework project “GRAFOL” is gratefully acknowledged. The A.P. Møller and Chastine Mc-Kinney Møller Foundation is acknowledged for their contribution toward the establishment of the Center for Electron Nanoscopy in the Technical University of Denmark. Thanks to Søren B. Simonsen and Quentin Jeangros for providing the NiO samples.

Fig. 1: Three layers grown shortly after introduction of C2H2.

Fig. 2: Multiple layers grown 79.2s after Fig. 1; the arrow marks next growing layer close to the metal particle surface.

Fig. 3: Multiple layers grown 80s after Fig. 1; the arrows mark next growing layers close to the metal particle surface.

Fig. 4: Multiple layers grown 80.8s after Fig. 1; the arrows mark next growing layers close to the metal particle surface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-3370 Novel 3-dimensional nanocomposite of covalently interconnected multiwalled carbon nanotubes using Silicon as an atomic welder

Pulickal Rajukumar L.1, Belmonte M.2, Roman B.2, Slimak J.1, Elías A. L.1, Cruz-Silva E.2, Perea-López N.1, Morelos-Gómez A.3, Terrones H.4, Miranzo P.2, Terrones M.1,3
1The Pennsylvania State University, University Park, United States, 2 Institute of Ceramics and Glass (ICV-CSIC), Madrid, Spain, 3Shinshu University, Nagano, Japan, 4Rennselaer Polytechnic Institute, Troy, United States
lzp130@psu.edu

There is a growing interest in synthesizing three-dimensional (3-D) carbon nanotube structures with multi-functional characteristics. Here, we report the fabrication of a novel composite material consisting of 3-D interconnected multi-walled carbon nanotubes (MWNTs) with Silicon Carbide (SiC) nano- and micro-particles. The materials were synthesized by a two-step process involving the chemical coating of MWNTs with Silicon oxide, followed by Spark Plasma Sintering (SPS). SPS enables the use of high temperatures and pressures that are required for the carbothermal reduction of silica and for the densification of the material into a 3-D composite block. Covalent interconnections of MWNTs are facilitated by a carbon diffusion process resulting in silicon carbide formation as silica coated MWNTs are subjected to high temperatures. The presence of SiC in the sintered composite has been confirmed through Raman spectroscopy, which shows the characteristic peak close to 800 cm-1 and also Energy Filtered Transmission Electron Microscopy maps. X-ray Diffraction, Scanning Electron Microscopy, Energy Dispersive X-Ray Spectroscopy and High Resolution Transmission Electron Microscopy have also been used to characterize the produced material. Interestingly, the thermal property measurements of the sintered composite reveal a high thermal conductivity value (16.72 W/mK) for the material. From the electrical point of view, a 3-D variable range hopping (VRH) electron hopping was observed in the composite.

Fig. 1: High Resolution Transmission Electron Microscopy images of SiC/MWNT composite prepared by Spark Plasma Sintering.

Fig. 2: (a) Raman spectrum of the SiC/MWNT sample showing characteristic D, G and G' peaks for MWNTs and the SiC peak at 800 cm-1. (b) X-Ray diffraction data of SiC/MWNT composite. (c) Raman mapping of G peak position and (d) SiC peak  position within a 30 µm x 30µm area.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-3372 Imaging of carbon nanostructures by low energy STEM below 5 keV

Pokorná Z.1, Knápek A.1, Jašek O.2, Prášek J.3, Majzlíková P.3
1Institute of Scientific Instruments of the ASCR, v. v. i., Královopolská 147, Brno, Czech Republic, 2Masaryk University, CEITEC - Central European Institute of Technology, Kamenice 753/5, 625 00 Brno, Czech Republic, 3Brno University of Technology, CEITEC - Central European Institute of Technology, Technická 3058/10, 616 00 Brno, Czech Republic
zuzana.pokorna@isibrno.cz

Our work deals with the imaging of nanostructures composed of light biogenic elements, such as carbon nanotubes, by low energy scanning transmission electron microscopy (STEM). Compared to imaging at the voltages commonly used for TEM and STEM, low energy electrons seem very promising in terms of specimen damage that is caused by a number of elastic and inelastic collisions [1]. In carbonaceous materials, the most problematic is probably the knock-on damage, where the structure can be impaired by carbon atom displacement. To avoid this problem with structures composed of light elements, a reduction in beam voltage going down to 5 keV has recently been proposed [2]. The range below 5 keV has not been explored yet for this purpose, although electron scattering in matter is lower for these energies, which allows achieving a higher spatial resolution [3]. We aim to demonstrate that additional reduction of incident electron energy may yield interesting contrast features.

We used a FEI Magellan 400L microscope capable of high resolution imaging even at low and very low incident electron energies, equipped with a multi-segment, retractable STEM detector. Carbon specimens were prepared e.g. by depositing a solution of commercial Sigma Aldrich nanotubes, with dimethylformamide used as a solvent, on Agar S147 holey carbon mesh grids. Contrast features were recorded by secondary electron (SE), bright field (BF) and dark field (DF) detectors, including high-angle annular dark field (HAADF).

We have studied the aspects influencing the image information, such as incident electron energy, electron dose, sample thickness, the presence of the ubiquitous hydrocarbon contamination layer and other. The results were also tested using Monte Carlo simulations.

References:

[1] INADA, H., et al. Atomic imaging using secondary electrons in a scanning transmission electron microscope: experimental observations and possible mechanisms. Ultramicroscopy, 2011, 111.7: 865-876.

[2] BEYER, Y.; BEANLAND, R.; MIDGLEY, P. A. Low voltage STEM imaging of multi-walled carbon nanotubes. Micron, 2012, 43.2: 428-434.

[3] MÜLLEROVÁ, I.; FRANK, L. Scanning low-energy electron microscopy. Advances in imaging and electron physics, 2003, 128: 310-445.

The financial support of the Czech Ministry of Education, Youth and Sports through projects LO1212 and CZ.1.05/1.1.00/02.0068, and of the Academy of Sciences of the Czech Republic through projects AVČR L100651304 and AVČR L100651402, is acknowledged.

Fig. 1: Secondary electron image of carbon nanotubes at 2 keV incident energy.

Fig. 2: Bright field STEM image of carbon nanotubes at 2 keV incident energy showing several thicknesses of multi-wall nanotubes, including embedded catalytic metal particles.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5812 Morphology of oxidized graphite observed in cross section using FIB, TEM and ESEM

Weisbecker P.1, Delehouze A.1, Rebillat F.1, Epherre J. F.1, Vignoles G.1
1Laboratoire des Composites ThermoStructuraux (LCTS), Université Bordeaux 1, CNRS, CEA, 3 allée La Boëtie, Pessac, F33600, France
weisbecker@lcts.u-bordeaux1.fr

The question of pit shape and growth kinetics during oxidation of graphite by O2 has been the subject of numerous experimental and modeling studies [1]. Hughes an Thomas [2] had evidenced hexagonal pit shapes in natural graphite around 1050°C and have claimed that a transition could exist between hexagons with zig-zag and armchair edges, moreover they have shown that under pure O2, at temperatures ranging between 700°C and 870°C, pits are composed of {11-2l} faces, thus with carbon atoms in the so called armchair configuration. The zig-zag configuration, with {10-1l} faces, can be obtained when the reaction is catalyzed and when pits are formed in presence of H2O [3] or pure H2.

The recent development of graphene technology has led to researches focused on the control of the pits and of the edges morphologies of graphene layers [4]. It is known that the oxidation of a single graphene sheet is different from the oxidation of the graphene sheets stacked in a graphite-like crystallographic ordering [3], however only few information is available concerning the oxidation process along the c-axis.

In this study HOPG samples were oxidized, at 650°C, in a TGA furnace and in the chamber of an ESEM, at 550°C. Various pits with a hexagonal shape were observed and cross sections were obtained using a FIB as shown in figure 1.

TEM observations of the morphologies of etch pits obtained following both protocols are shown in figure 2. The armchair configuration is obtained, as expected, in the TGA experiment, while the zig-zag configuration is obtained inside the ESEM.

Etch pits with armchair configuration exhibit large and well-defined hexagons with numerous steps ranging from 2 to 30 nm in height, and a quasi-vertical inclination. On the other hand, pits with zig-zag configuration are deeper, and their step edges follow the {10-11} planes, i.e. with an angle of ~73°. The striking difference between pits obtained by pure O2 oxidation in these distinct experiments could be due to the existence of some catalytic process taking part in the ESEM experiment. Indeed, there is no guarantee that the ESEM chamber contains only pure O2: traces of ionized species and radicals, as well as of pollutants (like metal atoms or ions) can be present.

References

[1] Delehouzé A. & al. Temperature induced transition from hexagonal to circular pits in graphite oxidation by O2. Appl. Physc. Lett. 99, 044102 (2011)

[2] Hughes EEG, Thomas JM. Topography of Oxidized Graphite Crystals. Nature 1962; 193(4818):838−40.

[3] Yang RT. Etch-decoration electron microscopy studies of the gas-carbon reactions. Chemistry and Physics of Carbon, vol. 19. New York:Dekker; 1984 p. 163-210.

[4] Dobrik & al. Selective etching of armchair edges in graphite (2013) Carbon, 56, pp. 332-338.

Fig. 1: etch pits obtained at 650°C after 100 min oxidation (TGA experiment) and corresponding SEM image of the FIB cross section.

Fig. 2: etch pits observed in TEM bright field mode with the armchair configuration (top), 650°C, TGA furnace, and with the zig-zag configuration (bottom), 550°C, ESEM oxidation.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-3464 Dopants as Morphology Promoters: A Fundamental Study of the Role of Boron and Sulfur in the Formation of MWCNT Junctions

Elías A. L.1, McCreary A.1, Pulickal Rajukumar L.1, Audiffred M.1,2, Cruz-Silva E.1, Reddy A. L.3, Kalaga K.3, Perea-López N.1, Feng S.1, Cruz Viana Neto B.1, Swanson D.1,4, Gutiérrez H. R.5, Vajtai R.3, Meunier V.6, Sumpter B. G.7, Ajayan P. M.3, Terrones H.6, Terrones M.1,8
1Department of Physics and Center for Two Dimensional and Layered Materials, The Pennsylvania State University, University Park, PA 16802, USA, 2Research Center for Functional Materials and Nanomolecular Science, Jacobs University Bremen, Campus Ring 1, D-28759 Bremen, Germany, 3Department of Mechanical Engineering & Materials Science, Rice University, Houston, Texas 77005, USA, 4Chemistry and Physics Departments, Augustana College, Sioux Falls, SD 57197, USA, 5Department of Physics & Astronomy 102, Natural Science Building, University of Louisville, Louisville, KY 40292, USA, 6Department of Physics, Applied Physics and Astronomy, Rensselaer Polytechnic Institute, Troy, NY 12180, USA, 7Center for Nanophase Materials Science, and Computer Science and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA, 8Research Center for Exotic Nanocarbons (JST), Shinshu University, Wakasato 4-17-1, Nagano, 380-8553, Japan
adm273@psu.edu

Doping of carbon nanotubes has proven to be an invaluable tool to modify their physical and chemical properties [1]. In this work, we demonstrate that by using minute amounts of both boron and sulfur as codopants it is possible to synthesize 3-Dimensional arrays consisting of carbon nanotube interconnections. The materials were produced using a spray-assisted chemical vapor deposition experiment, in which small amounts of boron and sulfur sources were added to a solution containing a carbon source and an iron-based catalyst. The resulting structures consisted of nano- and micron-size fibers decorated with radially grown multi-walled carbon nanotubes resembling "nanotentacles." These novel structures were extensively characterized by SEM, HRTEM, and XRD. Spectroscopic techniques have also been used to determine the doping amount and binding environment, such as EELS, XPS, and Raman Spectroscopy. Quantification of EELS spectra shows that boron has a higher concentration on the tips of the radially grown nanotubes. Ab initio calculations were performed in order to understand the role of boron and sulfur as promoters of the negative curvature regions that are necessary to form covalent nanotube junctions. Our calculations show both boron and sulfur incorporation in the graphitic nanotube lattice is more stable when located in curved areas of the nanotube, with boron having a marked preference for negative curvature while sulfur is stable in both negatively and positively curved areas. Due to their high surface area, these synthesized structures have also been tested as electrodes in Li-ion batteries and supercapacitors. Finally, we will demonstrate that these tentacles behave as excellent electron field emitters, maintaining high currents over time at low operating voltages.

[1] B. Sumpter, et al. Int. J. Quant. Chem., Vol 109, 97-118 (2009)


This work is supported by the U.S. Air Force Office of Scientific Research MURI grant FA9550-12-1-0035

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5760 Controlled deposition of iron based nanoparticles on few-layer graphene

Melinte G.1, Liu X.1, Janowska I.2, Baaziz W.2, Moldovan S.1, Begin-Colin S.1, Pham-Huu C.2, Ersen O.1
1Institut de Physique et Chimie des Matériaux de Strasbourg, CNRS-Université de Strasbourg, 23, rue du Loess, 67037 Strasbourg, France, 2Institut de Chimie et Procédés pour l’Energie, l’Environnement et la Santé, CNRS, ECPM, Université de Strasbourg, 25, rue Becquerel, 67087 Strasbourg, France
georgian.melinte@ipcms.unistra.fr

The last decades witness a continuous “miniaturizing” trend that is principally projected in the field of applied electronics. Owing to the development of nanotechnologies, the industry is nowadays capable to produce materials and structures with well-defined sizes, geometries and morphologies. The carbon-based structures like graphene, carbon nanotubes (CNTs) and nanofibers (CNFs) are promising candidates for the development of nanodevices [1,2], which strongly demand for a close control of materials properties at the nanoscale. The scanning tunneling microscope (STM) can be employed to move single atoms with incredible precision [3], but this approach is not adapted for assembling nanodevices with thousands of atoms. The use of CNTs as “nanopipetts” able to transport femtograms of mass to predefined spots can be envisaged [4], as approach based on the Joule assisted electromigration phenomenon, when a high current pass through a metal phase encapsulated inside a CNT.

We propose here a highly precise method for delivering nanoparticles to the graphene and few-layer graphene edges and surfaces using a CNT filled with Fe3-xO4 NPs as a nanopipette. The experiment is realized inside a transmission electron microscope (TEM) by using a STM-TEM holder allowing high precision sub-nanometer movement and high voltage supply. Figure 1 shows several nanoparticles deposited on the surface of the FLG sheet with a radial distribution relative to the CNT/FLG contact point. Figure 2 displays series of a more complex experiment: control the NPs deposition at the FLG edge in time, together with the image of the initial and the final system. The in-situ TEM observation of the experiment has made possible a real time analysis of the structural and chemical properties of both the NPs and the supporting CNT.

1. A. K. Geim, Graphene: Status and Prospects, Science 324, 1530 (2009);

2. R. H. Baughman et al., Carbon Nanotubes--the Route Toward Applications, Science 297, 787 (2002);

3. J. A. Stroscio, D. M. Eigler, Atomic and molecular manipulation with the scanning tunneling microscope, Science, 254, 1319, (1991);

4. K. Svensson, H. Olin, and E. Olsson, Nanopipettes for metal transport, Physical Review Letters, 93 (14), (2004).

Fig. 1: Electron migration with a radial displacement of iron  nanoparticles on the surface of few-layer.

Fig. 2: Electron migration based deposition of iron nanoparticles on the edge of few-layer graphene.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5767 Setting up a Nanolab inside a TEM for 2D Materials research

Sun L.1
1SEU-FEI Nano-Pico Center, Key Laboratory of MEMS of Ministry of Education, Southeast University
slt@seu.edu.cn

With the continuous improvement of in situ techniques inside transmission electron microscope (TEM), the capabilities of TEM extend beyond structurual characterization to high-precision nanofabrication and property measurement. Based on the idea of "setting up a nanolab inside a TEM", we present our recent progress in 2D Materials research including in situ growth, nanofabrication with atomic resolution, in situ property characterization, nanodevice construction and possible applications(e.g. a 5nm-diameter hole on graphene for third-generation gene sequencing, the spongy graphene as an ultra-efficient sorbent for oils and organic solvents, etc.).Fig. 1 shows in situ nanofabrication of suspended molybdenum sulfide sub-nanometer ribbons with uniform width of 0.35 nm from monolayer MoS2 by electron beam irradiation. The mechanism of electron-beam induced high-resolution nanofabrication was also discussed.

References:

[1]L. Sun, F. Banhart, et. al., Science 312, 1199 (2006)

[2]J.R-Manzo, M. Terrones, et.al., Nature Nanotechnology 2, 307 (2007)

[3] L. Sun, A. Krasheninnikov,et.al., Physical Review Letters 101, 156101(2008)

[4] H. Bi, X. Xie, et al., Advanced Functional Materials 22, 4421 (2012)

[5] H. Bi, K. Yin, et al., Advanced Materials 24, 5124 (2012)

[6] Q. Liu, J. Sun, et al., Advanced Materials 25, 165 (2013)

[7] X. Liu, T. Xu, et al., Nature Communications 4, 1776 (2013)

[8] H. Qiu, T. Xu, et al., Nature Communications 4, 2642 (2013)

[9] X. Li, X. Pan, et al., Nature Communications 5, 3688 (2014)

This work was supported by the National Basic Research Program of China under grant Nos. 2011CB707601, the National Natural Science Foundation of China under grant Nos. 61274114, 113279028, 51201032 and 51071044, the Key Grant Project of Chinese Ministry of Education under No 311019, and the Natural Science Foundation of Jiangsu Province under grant Nos. BK2011592, BK2012024.

Fig. 1: In situ fabrication of suspended molybdenum sulfide sub-nanometer ribbons with uniform width of 0.35 nm (possible narrowest molybdenum sulfide nanoribbon) from monolayer MoS2 by electron irradiation.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5789 Quantitative Atom-by-Atom Strain Analysis on Carbon-Supported Platinum Clusters for Fuel CellApplications

DAIO T.1,2, STAYKOV A.3, LYTH S. M.3, LIU J.3, GUO L.3, TANAKA M.4, SASAKI K.1,2,3,5
1International Hydrogen Research Center , 2Department of Mechanical Engineering, 3International Institute for Carbon-Neutral Energy Research, 4Department of Materials Science and Engineering, 5Next-Generation Fuel Cell Research Center of Kyushu university,Fukuoka,Japan
daio.takeshi.900@m.kyushu-u.ac.jp

Precious-metal nanoparticles are well known as good candidates for electrocatalysis, for example in fuel cell applications.One of the most important objectives for using precious metal electrocatalysts is how to reduce loading whilst also increasing electrochemical activity. Generally,this is achieved by minimizing the cluster diameter to increase electrochemical surface area (ECSA), whilst at the same time decreasing the required amount of catalyst. On the other hand, small particle diameters (e.g. clusters containing less than 300 atoms) are thought to have very different behavior from larger, bulk-like particles. Substrate and relaxation effects have been explored by experimental approach and computer simulation. [1,2] However, to the best of our knowledge, microscopy and quantitative analysis of such effects are scarce.

Pt clusters on carbon black were prepared. Cs-corrected STEM analysis and quantitative measurement was applied to predict changes in catalytic activity. We used a JEOL ARM200F equipped with a Cs-corrector and Cold FEG. From atomic resolution STEM images of the Pt clusters, we can see direct evidence of strain. We attempt to quantify this strain by compensating from the crystal model and the measured lattice spacing. At first, attempted strain mapping. We selected a constant area of the Pt cluster from phase mapping, generated from the FFT peak with an applied Gaussian filter. Our atom by atom inspection using geometrical phase analysis revealed the strain and relaxation of the surface area quantitatively. If strain effects catalytic activity, this can explain why we have a decrease in activity below a 3 nm Pt cluster size. In addition, this work may be able to predict what type and cluster size of catalyst could be good candidates for practical fuel cells.

[1] Stamenkovic, Vojislav, et al. "Changing the activity of electrocatalysts for oxygen reduction by tuning the surface electronic structure." Angewandte Chemie 118.18 (2006): 2963-2967.
[2] Power, Timothy D., and David S. Sholl. "Effects of surface relaxation on enantiospecific adsorption on naturally chiral Pt surfaces." Topics in catalysis 18.3-4 (2002): 201-208.

The authors gratefully acknowledge the Center of Innovation.The International Institute for Carbon-Neutral Energy Research was established by World Premier International Research Center Initiative (WPI), MEXT, Japan.References

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5802 A comparative study of quick and simple methods for thickness measurement of graphene by transmission electron microscopy

Pettersson H.1, Coleman J. N.1, Nicolosi V.1
1Trinity College Dublin, Dublin, Ireland
pettersh@tcd.ie

At the present, graphene – a single sheet of carbon – has become one of the most studied and is one of most promising materials of tomorrow. The challenge is the production of graphene is to optimize and control the synthesis conditions [1,2]. Most of the present production methods multiple layers of graphene are obtained.


There are numerous of methods in determining the thickness by transmission electron microscopy (TEM)[3-6]. The most common way seems to visually count the layers by in a bright field (BF) TEM or a dark field (DF) scanning TEM images, however, this is not trivial since it is difficult to have a single layer reference. Diffraction can be used to determine single and double layers [7]. Though, to find an area of the flake that is not folded contains other features that corrupt the interpretation of the diffraction pattern. Also some methods are time consuming due to slow data acquisition and post-processing.

In our work we used a FEI Titan 80-300 TEM equipped with EDX and EELS, operated at 80kV for the determination of the graphene thickness. In figure 1 one can see a BF-TEM, a DF-STEM, an EFTEM and an EFTEM Plasmon thickness maps in which one the thickness can be determine by the intensities of the images. Intensity measurements using about 300 pixels were performed on the two EFTEM techniques on to find the number of layers–thickness dependence, viewed in figure 1e. It can also be observed that the graphene edge structures changed under the electron beam so the plateaus changed shape over time.

[1] M. Lotya et al., J. Am. Chem. Soc. 131 (2009) 3611.
[2] Y. Hernandez et al., Nat. Nanotechnol. 3 (2008) 563.
[3] M.H. Gass et al., Nat. Nonotechnol. 3 (2008) 676.
[4] M. Boese et al., Microsc. Microanal. 16 (Suppl 2), (2010) 1540
[5] D. B. Williams and C. B. Carter, Transmission Electron Microscopy: A Textbook for Materials Science, Springer, New York, 1996
[6] S. Akhtar et al., arXiv:1210.2307v1 [cond-mat.mtrl-sci]
[7] J. Meyer et al., Nature 446 (2007) 60

Fig. 1: a) BF-TEM b) DF-STEM, C) EFTEM Thickness map and d) Plasmon map (10-40 eV). Measured areas marked in the Plasmon map e) Plot of the measured thicknesses relative to the DF-STEM
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5842 Investigation of graphite/carbon spiral nanoribbons using FeCl3–CuCl2–graphite intercalation compounds as precursors

Yang L.1, Cao L. Y.1, Liu H. B.1, Chen J. H.1
1Hunan University, Changsha, China
hunanyangli@aliyun.com

The study of carbonaceous material with spiral morphologies is of great interest in recent years due to their extraordinary mechanical, electrical and field emission properties. Among the variety of methods to synthesize such special carbon materials, chemical vapor deposition (CVD) is the most popular one. And the typical requirements include: (i) an appropriate carbon source, (ii) growth catalysts such as Fe, Co, Ni, (iii) promoter elements such as P, S and (iv) inducer metals such as Cu, Sn, In. Graphite intercalation compounds (GICs) are formed by the insertion of atomic or molecular layers of a different chemical species (usually called intercalant) between graphene layers. The GICs can be used to catalyzer for many matters because that the intercalant will then deintercalate through the edges or other defective sites of the graphite crystal when the temperature is above 80℃.
In this paper, FeCl3-CuCl2-graphite intercalation compounds (GICs) were applied to synthesize graphite/carbon spiral nanoribbons (G/CSNRs) by chemical vapor deposition (CVD) of acetylene and hydrogen. The G/CSNRs were characterized by XRD, SEM and Raman spectra. The XRD patterns confirmed the G/CSNRs did contain graphite. The as-grown CSNRs were thin and twisted, owning a width of 92 nm, at hread pitch of 56 nm and a length in micrometer scale. The Raman spectra showed the imperfect crystallinity of CSNRs. The SEM images showed that a kind of sheet catalyzer particle was formation and they could catalytic synthesis two morphology carbon fibres respectively at their flank and surface. During this CVD process, FeCl3-CuCl2-GICs acted as providers of graphite substrates as well as catalyst carriers. The CVD method mediated by GICs also provided possibilities to design and prepare a variety of hybrid carbon structures.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5890 Laser-Induced Effects in Monolayer Graphene

Mirzayev R.1,2, Herziger F.1, Poliani E.1, Mangler C.2, Meyer J. C.2, Maultzsch J.1
1Institut für Festkörperphysik, Technische Universität Berlin, Berlin, Germany, 2Physics Department, University of Vienna , Vienna, Austria
rasim.mirzayev@univie.ac.at

Tuning the properties of graphene still presents a major challenge in graphene study. This challenge is evident in electronic applications that require high precision tasks.
In this study we present the laser-induced effects in single-layer graphene. Mechanically exfoliated monolayer graphene samples deposited on Si/SiO2 substrates were irradiated with 532 nm green laser at 35 mW power for about 10 minutes under ambient conditions. The irradiated samples were characterized by time-resolved Raman spectroscopy, Raman mapping and Atomic Force Microscopy. Subsequently, the samples were transferred to Quantifoil TEM grids and the structure was investigated by aberration-corrected scanning transmission electron microscopy (STEM).
Our results show that the laser irradiation has locally modified the graphene surface and structure. The time-dependent Raman spectra of graphene undergo dramatic changes during the laser irradiation. However some of these changes disappear in time. Eventually we observe upshift in G mode position, slight increase in D mode intensity, decrease in 2D mode intensity and broadening of its line width. However Raman mapping images show significant D mode intensity.
There are no observable changes in the optical images after the laser treatment whereas in the AFM images certain structures are observed on modified regions.
We explain our results in terms of defect formation by breaking of the sp2 C-C bonds, and formation of an additional layer of material on top of the graphene. This new layer is highly sensitive to electron irradiation and its nature remains to be clarified in more detail.
The results also show that the chemical reactivity of monolayer graphene is enhanced as a result of laser treatment.
The approach can further be utilized for local modification of properties of graphene as well as patterning it by laser-beam irradiation.

Fig. 1: AFM images after laser irradiation. a) Four modified spots on the monolayer graphene under the laser beam. b) Magnified image of (a). c) Height profile along the line shown in (b).

Fig. 2: STEM images of laser irradiated graphene. a) STEM image of the new layer on top of the graphene. b) and c) show the sensitivity of this new layer to electron beam.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5856 The structure of 1D TbBrx crystals inside the SWCNTs.

Kiselev N. A.1, Kumskov A. S.1, Eliseev A. A.2, Vasiliev A. L.1,3, Zhigalina V. G.1, Sloan J.4
1Institute of Crystallography RAS, Moscow, 119333, Russia, 2Department of Materials Science, Moscow State University, Moscow, 119992, Russia, 3NRC , 4Department of Physics, University of Warwick, Coventry, Warwickshire CV47AL, UK
a.kumskov@gmail.com

The 1DTbBrx@SWCNT meta-nanotubes are obtained using a capillary technique and investigated by HRTEM and HAADF STEM in JEOL ARM 200F at 80 kV. Raman spectroscopy is performed in Ranishow Invia Raman microscope. Four versions of 1D TbBrx crystal structure are proposed.
The first type of the structure is characterized by a rhombic unit cell (Pmmm). An <001> crystal axis coincides with the nanotube axis. In this case a Br/Tb ratio is 3.25. A HRTEM image, the model and corresponding image simulation in a (110) projection were shown in Fig. 1.
The second type of the structure is characterized by “rhomboid” defects observed in the (110) projection of 1D crystal when a set of micrographs was taken (Fig. 2). It is suggested that due to electron beam heating Br atoms located in a center of Br (Tb) tetrahedrons are lost. As a result covalent bonds of Tb-Br atoms were replaced by Tb-Tb metallic bonds. This is accompanied by a displacement of Tb atoms towards the center of the tetrahedrons. The Br/Tb ratio is 2.66.
The third type of structure is observed in the HAADF STEM images (Fig. 3). In this case the “rhomboid” defect is observed in each unit cell and the Br/Tb ratio is 2.5.
The fourth structure resemble the third one, but in it additionally four bromide atoms are lost. The Br/Tb ratio is 2 in this case.
The existence of a structural variety for 1D TbBrx crystals can be explained by different oxidation degree of Tb atoms.
In a G-region of Raman spectra of metananotubes a significant peaks shift towards higher frequencies spectrum area is observed in the range from 4 to 7 cm-1 for s-SWCNTs and from 7 to 17 cm-1 for m-SWCNTs. This effect can indicate a higher influence of intercalated nanocrystals on the electronic structure of metallic nanotubes. Furthermore a value of the observed G-mode shift depends on a chemical nature of intercalated halogenide and appears to have maximum values for bromides. According to literature data the G-mode shift towards the region of high oscillation energies can correspond to an electron charge transfer from the SWCNT walls to the nanocrystals (acceptor doping of SWCNTs).

The work is supported by Russian Science Foundation grant #14-13-00747

Fig. 1: HRTEM image of 1DTbBrx@SWCNT. A model of a first type of structure in a (110) projection and a corresponding image simulation (d1=3.4 Å and d2=3.8 Å).

Fig. 2: HRTEM image of a second type of 1DTbBrx@SWCNT structure. The “rhomboid” defects are arrowed.

Fig. 3: STEM HAADF image of a third type of 1DTbBrx@SWCNT structure and its image simulation.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5880 Computational Study on Structural Changes of Carbon Nanomaterials under Electron Irradiation

Yasuda M.1, Yamamoto M.1, Kawata H.1, Hirai Y.1
1Osaka Prefecture University, Sakai, Japan
yasuda@pe.osakafu-u.ac.jp

   A transmission electron microscope observation is indispensable in the characterization of the nanomaterials. However, the electron irradiation causes serious structural changes of the materials. Therefore, the understanding of the electron irradiation effects takes on a growing importance in the recent progress of nanoscience. In the present work, we study the structural changes of carbon nanomaterials under electron irradiation with molecular dynamics (MD) simulation.
   The simulation model is shown in Fig. 1. The motions of carbon atoms in graphene are calculated with the MD simulation. The interaction between an incident electron and a carbon atom is introduced by means of Monte Carlo method. The collision atom is randomly selected. The type of collisions is stochastically determined using collision cross sections.
   When an elastic collision occurs, the momentum transfer from an electron to a carbon atom is calculated by the binary collision theory. When valence electron ionization occurs, a bond breaking is expressed by introducing repulsive forces around the ionized atom. When inner shell electron excitation occurs, two bond breakings are introduced considering Auger effect.
   Figure 2 shows the variation of the potential energy of graphene before and after knock-on and Stone-Wales (SW) defect formation by electron collision at 200 kV obtained by the present simulation. Although the defect formation energies of both defects are close, the potential energy of SW defect is smaller than that of knock-on defect. Therefore, SW defect is more stable than knock-on defect.
   Figure 3 shows the transformation process of the knock-on defect in graphene by annealing at 1500 K. Snapshot of the structural change and the variation of the potential energy are shown. The knock-on defect transfers to a 5-9 or a 5-5 defects by annealing. The defect moves in graphene by switching the structure between 5-9 and 5-5 defects. When the defect arrives at the edge, the transformation of the defect stops and the potential energy largely decreases.
   Various examples of the structural change of carbon nanomaterials obtained by the simulation are shown in the presentation.

This work was supported by JSPS KAKENHI Grant Number 25249052.

Fig. 1: Present simulation model. The interaction between an electron and a carbon atom is modeled by the Monte Carlo method and the motions of target atoms are traced with the molecular dynamics simulation.

Fig. 2: The variation of the potential energy of graphene before and after the (a) knock-on and (b) Stone-Wales defect formation by electron collision.

Fig. 3: Transformation of knock-on defect in graphene by annealing. (a) Snapshot of the structural change and (b) the variation of the potential energy.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5944 Structural investigation of single layer Ti3C2 MXene sheets

Karlsson L. H.1, Persson P. O.1
1Electron Microscopy of Materials Group, Thin Film Physics Division, Department of Physics, Chemistry and Biology (IFM), Linköping University, Sweden
likar@ifm.liu.se

2D materials are of great interest due to their unique properties, and granted the Nobel Prize recently [1,2]. Among 2D materials like Graphene, MoS2, BN, WS2, MXene is one of the most recent additions, exfoliated from the various MAX-phases. The MAX-phases consist of an M-element (transition metal), A-phase (usually an A-group element, commonly groups 13 and 14) and X-phase (C or N), in the form Mn+1AXn where n = 1, 2 or 3 [2]. These materials show unique properties, such as toughness, high temperature and corrosion resistance [3,4]. Exfoliating the MAX phase Ti3AlC2 in hydroflouric acid or ammonium bifluoride, NH4HF2, causes the A element to leave the chemical stable Ti3C2, which then form a layered structure of 2D crystals [5]. The 2D material, MXene, show unique properties as well, such as high conductivity and ductility [2]. The surface chemistry of the material depends on whether the surface is terminated by fluorine or hydroxyl groups [2].
In this contribution, low-kV (60 kV) high-resolution scanning transmission electron microscopy has been applied to investigate the atomic arrangement of single and double layer Ti3C2 MXene sheets in plan-view. Previous TEM investigations have shown that MXene nanosheets are a few nm thick and consist of Ti, C, O and H in a layered structure [2]. However, the exact position of the various elements has not been confirmed. In this study, the positions of the elements have been identified, while also the surface/edge termination and point defects has been investigated. In addition, the elemental properties have been investigating, addressing surface related groups which remain after the etching process.

References:
[1] K. S. Novoselov, et al, Science 306 (2004) p.666
[2] M. Naguib et al, Adv. Mater 23 (2011) p.4248
[3] M. Radovic and M. W. Barsoum, Am. Cer. Doc. Bull. 92 (2013) p.20
[4] P. Eklund et al, Thin Solid Films 518 (2010) p.1851
[5] J. Halim et al, Chemistry of Materials 26 (2014) p.2374

The Swedish Research Council (VR) is gratefully acknowledged for funding. The Knut and Alice Wallenberg (KAW) Foundation is gratefully acknowledged for the Ultra Electron Microscope Laboratory in Linköping. Jun Lu and Michel W. Barsoum are acknowledged for providing the MXene samples.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5981 Low Loss and Core Loss Angular Resolved EELS of hexagonal Boron Nitride

Fossard F.1, Schué L.1, Ducastelle F.1, Loiseau A.1
1LEM, ONERA - CNRS, 29 avenue de la Division Leclerc, Châtillon, France
frederic.fossard@onera.fr

Energy loss function and spectroscopies of an anisotropic crystal are highly orientation dependant and their measurements require to study their anisotropy behaviour. On one hand, X-rays measurements offer the finest energetic and angular resolution but a limited spatial resolution and require synchrotron facilities. On the other hand, electron energy losses (EELs) are highly related to both energy and momentum transferred to the material. Most of the time, EEL spectra are integrating a solid angle around the incident beam direction. Nevertheless, it has been demonstrated that core-loss EELS can be performed in order to obtain angular information in anisotropic materials[1].

Among anisotropic materials, hexagonal Boron Nitride (h-BN) presents several peculiar properties. This layered material has strong in-plane ionic bonds forming an hexagonal lattice whereas layers are weakly bound by Van Der Waals forces. Moreover, it is a large bandgap semiconductor (6.4 eV), which exhibits a rich footprint in the low energy loss region (<50 eV). In this paper, we aim at understanding the angular dependence of the core and low losses and rely them to our actual knowledge on the structural and optical properties of hexagonal Boron Nitride[2].

We used a Libra 200 equipped with an electrostatic monochromator operating at 80 kV. The achievable energetic resolution is 100 meV. We operate in the reciprocal space. Thanks to the in-column filter, energy loss signal is recorded as a function of both the energy and the momentum in a datacube, with the resolution of the exit slit of the filter. In order to achieve a better energetic resolution, we placed a dedicated slit in the entrance of the filter to select a specific crystallographic direction in the reciprocal space and disperse it perpendicularly (Fig 1). In this way, we can link the losses to their angular dependence[3].
Angular Resolved EELS has been performed on foils cut in a HPHT h-BN single crystal along crystallographic orientations using a focused ion beam. As an example, we present the diffraction pattern recorded at the energy of B-K core loss edge (192 eV) in a foil oriented along the [100] zone axis (Fig.2) and the w-q map extracted from the datacube along the (00l) q-direction (Fig.3) near the B-K edge. The difference in the dispersion of the excitation to π* and σ* states is dramatically revealed. We proceeded in the same way in the low loss region and we show that by gathering spectra in the high symmetry directions, we can probe the whole Brillouin zone and represent the plasmon dispersion as a function of the q momentum transferred to the h-BN layers.

[1] R. Arenal et al, APL 90 (2007) 204105
[2] A. Pierret et al., Phys. Rev. B 89 (2014) 035414
[3] P. Wachsmuth et al., Phys. Rev. B 88, 075433 (2013)

The research leading to these results has received partial funding from the European Union Seventh Framework Programme under grant agreement n°604391 Graphene Flagship
Authors want to thank Gerd Benner for experimental support when implementing w-q maps on the LIBRA.

Fig. 1: Left: Principle of the Energy dispersion in the reciprocal space used for recording w-q mapping in the Libra 200 (courtesy G. Benner). Right: Diffraction pattern of a BN foil oriented along the [100] zone axis.

Fig. 2: Filtered diffraction pattern at 191 eV (B-K edge) of the [100] zone axis. White rectangle delimitates the area of interest for angular resolved EELS.

Fig. 3: Fig. 3: angle-resolved EELS of the Fig. 2 rectangular region.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5983 CVD synthesis and characterization of hexagonal Boron Nitride thin films

Andrieux A.1, Dorval N.2, Fossard F.1, Schué L.1, 3, Lavenus P.2, Loiseau A.1
1LEM, ONERA - CNRS, 29 avenue de la Division Leclerc, Cha^tillon, France, 2DMPH, ONERA, Chemin de la Hunière, Palaiseau, France, 3GEMAC, 45 avenue des Etats-Unis UVSQ, Versailles, France
frederic.fossard@onera.fr

Among the new class of 2D materials, hexagonal boron nitride (hBN) is a graphene analog but with very complementary properties. Indeed, this layered material, made of a stacking of planar BN hexagonal networks, is a semiconductor with a large band- gap (~ 6 eV). hBN layers seem to be an excellent 2D dielectric candidate to serve as a graphene substrate in electronic devices or to built heterostructures and to encapsulate graphene for preserving it from its environment [1]. However, synthesis of high- quality and large-area h-BN with controllable number of layers remains a great challenge and thus the implementation of h-BN/graphene hybrid structures is still limited.
To this aim, we have developed a specific route to the synthesis of ultra thin hBN films (1 – 15 layers) based on a Low Pressure Chemical Vapor Deposition (LPCVD) technique, using borazine (B3N3H6) as precursor and metallic foils (Cu, Ni) as substrates. As integration of hBN in devices requires an in-depth knowledge of its structure and properties, we focus particular emphasis to the sample characterization by cross-checking various and complementary spectroscopic and imaging tools, that we present in this work. Samples are studied in their native state or transferred on appropriate substrates for specific studies.
First insight on the structural quality in the macroscopic scale, is provided by inspecting the FWHM width of the main Raman active mode (E2g mode at 1367 cm-1). As shown in Fig.1, the values measured for the layers grown on Ni (13 to 20 cm-1) are close to that of BN single crystals, which serve as reference samples [2].
Second, electron energy loss spectroscopy (EELS) is extensively used as it is capable of both performing a local chemical analysis and probing bonding arrangement and defects. A typical core-loss spectrum is displayed in Fig.2, showing B-K edge. Close inspection of the fine structures near the edges, with reference to recent ab initio calculations [3], indicates that the layers are mainly stacked as in the bulk material. High resolution imaging confirms this view but also reveals that at terraces, upper layers can rotate as in the example in Fig.3.
Finally, Low loss – EELS is used for inspecting the loss function with angular and momentum resolution and to get information on the electronic structure, complementary to cathodoluminescence measurements performed in a dedicated MEB-FEG [4].


[1] C.R. Dean et al., Nat. Nanotechnol. 5 (2010), 722.
[2] Kubota et al., Science 317, 932 (2007).
[3] N. L. McDouglass et al., Microsc. Microanal. (2014)
[4] A. Pierret et al., Phys. Rev. B 89 (2014) 035414

The research leading to these results has received partial funding from the European Union Seventh Framework Programme under grant agreement n°604391 Authors thank ONERA for its Financial support (PRF Graphene), J.-C. Daux, J.-F. Justin, B. Passilly (ONERA), I. Stenger (U. Versailles) and G. Wang (U. Paris 7) for their technical assistance.

Fig. 1: Raman spectrum of LPCVD made BN layers on Ni foils (dark line) shown in insert and of a HPHT single crystal [2] (red line).

Fig. 2: Electron energy core loss spectrum recorded at B-K edge on LPCVD made BN layers with a TEM Libra 200, operated at 80 keV equipped with an electrostatic monochromator and an in column energy filter; dashed lines indicate the energies of the σ* fine structures.

Fig. 3: HRTEM image of LPCVD made BN layers on Ni foils recorded on a TEM ARM 200 CC with a Cs objective corrector. The 1 and 2 squares indicate the area where the FFT patterns have been calculated (right). In square 1, all layers have the same orientation whereas the area 2 displays additional layers twisted by 22° with respect to the underlying ones.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-5999 Counting the number of layer of graphene flakes embedded in polymer by STEM-EELS

Prestat E.1, Shin Y.1, Gorgojo P.1, Zhou K. G.1, Schroeder S.1, Althumayri K.1, Harrison W.1, Budd P.1, Casiraghi C.1, Haigh S. J.1
1University of Manchester, Manchester, UK
sarah.haigh@manchester.ac.uk

Polymers of intrinsic microporosity (PIMs) exhibit molecular sieve behaviour as a consequence of their rigid, contorted macromolecular backbones.[1]The archetypal membrane-forming PIM shows exceptional promise for membrane processes such as organophilic pervaporation, solvent-resistant nanofiltration and gas and vapour separations. PIM-1 defines the 2008 upper bound of performance for important gas pairs such as CO2/N2.[2] Nanocomposite or “mixed matrix” membranes comprising PIMs with suitable nanofillers offer the potential for even better combinations of selectivity and permeability, together with resistance to ageing effects over the period of use. The incorporation of graphene or functionalized graphene platelets may modify membrane performance in a number of ways, depending on their size, distribution and functionality.
PIM-graphene composite solutions have been used to prepare thin composite membrane films by spin coating onto glass substrates. The films have been characterized using Transmission electron microscope (TEM) imaging, Raman spectroscopy and X-rays Photoelectron Spectrosocopy. TEM imaging was performed using a FEI Tecnai F30 S/TEM at 300 kV and a probe-corrected FEI Titan 80-200 G2 ChemiSTEM at 200 kV fitted with a GIF Quantum for electron energy loss spectroscopy (EELS) spectral imaging. TEM specimens have been prepared by transferring the spin-coated membranes onto a holey carbon grid.
Fig. 1 shows a scanning TEM (STEM) images acquired using a low angle annular dark field (ADF) detector. Optimisation of the specimen preparation and imaging conditions allows imaging of the PIMs nanoporous structureat high resolution as well as revealing the distribution of the graphene flakes within the PIM matirx at low magnification, as shown in Fig. 1a and b, respectively.
It is interesting to determine the number of graphene layers for flakes within the PIM but diffraction analysis is complicated by the strong amorphous background generated by the PIM matrix. Here we show that EELS provides an alternative method with which to characterize the thickness of graphene flakes within a polymeric matrix. Due to the anisotropy of the bonds in graphene and the isotropy of the bonds in the polymer, the sensitivity of EELS to the anisotropy of carbon[3] provides insight in the number of layer of the graphene flakes embedded in the polymer (Fig. 2d). Fig. 2b and c show the π* and σ* map of the graphene flake displayed in Fig. 2a. Thicker graphene flakes provide a higher σ*/π* bond ratio enabling EELS to quantify the number of graphene layers for an individual flake.

[1] McKeown and Budd, Macromolecules 43 (2010), p. 5163.
[2] L M Robeson, J. Membr. Sci. 320 (2008), p. 390.
[3] G A Botton, J. Electron. Spectrosc. Relat. Phenom. 143 (2005), p. 129.

Funding from the Engineering and Physical Sciences Research Council (EPSRC, UK) under the Graphene Membranes project and from the Defense Threat Reduction Agency (DTRA, US) is gratefully acknowledged.

Fig. 1: (a) STEM-ADF images of polymer of intrinsic microporosity (PIM) and (b) overview of graphene flakes distribution embedded in PIM.

Fig. 2: (a) STEM-ADF image of a multilayer graphene flake embedded in PIM. (b) σ* and (c) π* map obtained from the EELS C-K edge.(d) Background subtracted EEL spectra corresponding to the regions of the flake shown in (a).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-2-P-6064 Nanopeapod-like structures of cobalt nanoparticles casted inside multi-walled carbon nanotubes

Moldovan S.1, Florea I.2, Melinte G.1, Baaziz W.3, Pham-Huu C.3, Ersen O.1
1Institut de Physique et Chimie des Matériaux de Strasbourg (IPCMS), UMR 7504 CNRS – Université de Strasbourg, 23 rue du Loess, BP43, 67034 Strasbourg Cedex 2, France, 2Laboratoire de Physique des Interfaces et des Couches Minces (LPICM), École Polytechnique - CNRS, Route de Saclay, Bâtiment 408, 91128 Palaiseau Cedex, France, 3Institut de Chimie et Procédés pour l’Energie, l’Environnement et la Santé (ICPEES), UMR 7515 CNRS - Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg-Cedex 02, France
simona.moldovan@ipcms.unistra.fr

Faceted cobalt-based nanoparticles (NPs) with high density and narrow size distribution (50 ± 5 nm) were casted inside the channels of multiwalled carbon nanotubes (CNTs) through thermal decomposition of cobalt stearate in the presence of oleic acid as surfactant and the adequately defunctionalized CNTs. This high rate of filling of CNTs by NPs is mainly due to the confinement effect of CNTs, which act as nanoreactors for the complex decomposition of cobalt [1]. To assess the structural, chemical and morphological characteristics of NPs, crucial requirements for understanding their magnetic behaviour, we have used an approach combining several advanced TEM-based techniques, such as high resolution, EELS spectroscopy, electron tomography and in-situ TEM.

The cobalt-based nanoparticles with octahedral shapes and well-defined facets exhibit morphologies fundamentally different from the classical rounded or needle-like shaped particles, as obtained without the confinement effect. The quantitative application of electron tomography in the STEM mode allowed us to identify the octahedral morphology of cobalt-based nanoparticles casted inside the channels of the CNTs. In addition, a porous structure marked by the presence of pseudo-fractures within the unique nanocrystalline network has been identified by a complete HR-STEM analysis. The degree of oxidation of NPs filling the CNTs considerably decreases as compared with the NPs synthesised independently, as higher amounts of oxygen have been detected on the outer NPs than within the confined ones. The EELS analysis performed on several representative particles show that the inner NPs are principally oxidized close to their external surface and internal voids. After reduction, the NPs morphology changes abruptly from an octahedral shape marked by inner pseudo-fractures to irregular assemblies consisting in Co NPs with mean sizes of 1-2 nm separated by pores. The pores are completely closed and the structure becomes compact in the high temperature range staring from 600°C, whereas in the case of the non-reduced system, the CoO NPs compact at about 400°C.

↑ Go to the Table of content ↑

MS-3. Thin films, coatings and surfaces

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-3-IN-1741 Applications of Quantitative STEM

Hwang J.1, Zhang J. Y.1, Stemmer S.1
1University of California, Santa Barbara
stemmer@mrl.ucsb.edu

High-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM) is highly sensitive to the type and number of atoms in the atomic columns of a sample. Image contrast in HAADF-STEM agrees quantitatively with image simulations [1]. An important complementary method in STEM is position averaged convergent beam electron diffraction (PACBED), which is highly sensitive to information that cannot easily be obtained from HAADF-STEM images, such as small displacements of atom or tilts of oxygen octahedra in perovskite materials [2, 3]. In this presentation, we will discuss our recent [4, 5] work in applications of quantitative HAADF-STEM and PACBED to the characterization of materials and interfaces. Our first example concerns the determination of the three-dimensional location of individual Gd dopant atoms in SrTiO3 [4]. The method is based on using quantitative comparisons of experimental and calculated image intensities. Quantitative measures of the error and a criterion for the dopant visibility were established using an undoped SrTiO3 sample. The overall dopant concentration measured from atom column intensities agrees quantitatively with Hall carrier density measurements. The method is applied to analyze the 3D arrangement of dopants within small clusters containing 4-5 Gd atoms. Our second example discusses the correlation between oxygen octahedral tilts, A-site cation displacements, magnetism and metal insulator transitions in perovskite superlattices and quantum well structures. We show that PACBED in combination with HAADF-STEM imaging can be used to obtain independent information on oxygen octahedral tilts and A-site cation displacements and thus provides insights into strong electron correlation physics [5]. [1] J. M. LeBeau, S. D. Findlay, L. J. Allen, S. Stemmer, Phys. Rev. Lett. 100, 206101 (2008). [2] J. M. LeBeau, A. J. D’Alfonso, N. J. Wright, L. J. Allen, S. Stemmer, Appl. Phys. Lett. 98, 052904 (2011) [3] J. Hwang, J. Y. Zhang, J. Son, and S. Stemmer, Appl. Phys. Lett. 100, 191909 (2012). [4] J. Hwang, J. Y. Zhang, A. J. D'Alfonso, L. J. Allen, and S. Stemmer, Phys. Rev. Lett. 111, 266101 (2013). [5] J. Y. Zhang, J. Hwang, S. Raghavan, and S. Stemmer, Phys. Rev. Lett. 110, 256401 (2013); J. Y. Zhang, C. A. Jackson, S. Raghavan, J. Hwang, and S. Stemmer, Phys. Rev. B 88, 121104(R) (2013).

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-3-IN-3132 Energy Dispersive X-ray spectrometry for thin films and interfaces microanalysis: an application to the γ'/γ'' interface in Inconel 718.

Buffat P. A.1, 2, Czyrska-Filemonowicz A.2
1Centre Interdisciplinaire de Microscopie Electronique, Ecole Polytechnique Fédérale de Lausanne, station 12, 1015 Lausanne, Switzerland, 2International Centre of Electron Microscopy for Materials Science, AGH University of Science and Technology, 30-059 Krakow, Poland
philippe.buffat@epfl.ch

Silicon Drift Detectors (SDD) became available in the last few years for the TEM with much higher count rate capability than the traditional Lithium Drifted Silicon (Si-Li) detectors. In parallel high brightness cold field emission or Schottky electron guns and spherical aberration correctors were added to the STEM probe forming optics to enhance the X-ray generation while bringing the spatial resolution at the single atom column level. Lastly using detectors diodes as large as 100mm2 or fitting several smaller diodes around the sample [1] increases the X-ray collection angle from some 0.1sr up to 0.7 or even 1sr and removing their window improves the sensitivity for low energy X-rays by more than 2 times. With the present analysis software, side entry detectors with a single large diode offer the best quantification accuracy for tilted samples. The multiple diode design is particularly suited for investigation of layered structure cross-sections at no tilt or tomography with a partial compensation of the collection efficiency between opposite diodes up to about 30° tilt. However, it suffers from an undefined take-off angle when the sample is tilted leading to unreliable quantitative analysis.


Inconel 718 – a Ni-base superalloy - contains γ' and γ'' nanoprecipitates buried in a γ matrix (Fig. 1). Observation of the distribution of each phase at intermediate magnification is neither possible by Dark Field TEM [2] nor High Annular Angular Dark Field (HAADF/ STEM) [3]. However the high content of Al and Nb in γ' and γ'', respectively, brings contrast between the phases in EDS maps (Fig. 2). At the atom resolution level, EDS mapping reveal the projected positions of atom species integrated along the electron path across the thin sample. Figure 3 shows element maps of a γ'/γ'' interface. Both phases are separated by a pure Ni monolayer and Nb is present in γ' that was not expected from the traditional model. Line scan across the interface suggests an Al enrichment in the last layer in γ'. This map was acquired in a FEI Titan 3 60-300 in 330s at 200kV with a 220pA probe current. A longer acquisition time would have lead to unacceptable beam damage.
Comparing these EDS results with those of Atom Probe Tomography (APT) [4] shows the complementarity of the lateral and depth resolution of the two techniques and leads to similar concentration estimation in precipitates.

References
[1] P. Schlossmacher et al., Microscopy and Analysis 24 7 (2010) ppS5-S8 (EU).
[2] B. Dubiel et al., J. Microsc. 236/2 (2009) pp149-157
[3] P.A. Buffat et al., Scripta Mater. (submitted).
[4] W.T. Geng, Phys. Rev. B76 (2007) 224102

Part of the study was performed within ESTEEM2 project; financial support from the European Union under FP7 contract for an Integrated Infrastructure Initiative, reference 312483 ESTEEM2 is kindly acknowledged.

Fig. 1: Left: High Angular Annular Dark Field (HAADF) HRSTEM view of a [010] γ''/ γ' co-precipitates with defects at the interface and dispersed in the γ' phase. Right: structure models of the γ matrix, γ' and γ'' phases.

Fig. 2: Left: distribution of phases in the matrix revealed by EDX-STEM mapping: Al(red)+Nb(blue) are characteristic of γ' and γ'' precipitates respectively. Right: composition line scan profiles across precipitates. Caution: precipitates are buried in the matrix and at.% compositions given by the software do not represent those of any pure phase.

Fig. 3: EDX raw counts maps at the γ' / γ'' interface with atom column resolution. To reduce the statistical noise, equivalent unit cells were averaged along the horizontal direction. The interface row is pure Ni. Nb is also present in γ'. (Al, Ti, Nb) and (Nb, Ti) columns face each other across the interface with the shortest possible distance.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-1735 Multilayered Cr(Al)N/SiOx Nanocomposite Coatings Prepared by Differential Pumping Cosputtering

Kawasaki M.1, Nose M.2, Onishi I.3, Shiojiri M.4
1JEOL USA Inc., Peabody, MA, USA , 2University of Toyama, Toyama, Japan , 3JEOL Ltd., Tokyo, Japan, 4Kyoto Institute of Technology, Kyoto, Japan
kawasaki@jeol.com

Nanocomposite films such as TiN/Si3N4 and CrN/AlN have attracted attention as new coating materials. Nose et al. developed a differential pumping cosputtering (DPCS) system1. We have been studying the process and mechanism of film growth in the DPCS system2,3. The DPCS system has two chambers for RF sputtering of different materials and a substrate holder rotating1. Cr50Al50 and SiO2 targets were set in chambers I and II, respectively, and Si wafers heated at 250oC were used as the substrates. First, three depositions were successively performed on the substrate by sputtering in chamber I with different gas flows. They are the transition layers.2,3 Next, the main deposition for composite Cr(Al)N/SiOx was carried out on the transition layers rotated at various speeds for 660 min by operating both chamber I and chamber II. Analytical electron microscopy was performed with a JEM-2800 using a JEOL 100 mm2 SDD for EDS and also a JEOL ARM200F. Fig. 1 show HAADF STEM, EELS, and EDS intensity profiles through the Si substrate A, transition layers B, C, D, and E, and the composite layer F, deposited at a substrate rotation speed of 1 rpm.2 Figs. 2a and 2b show HAADF images of a cross section normal to the substrate surface in layer F and Figs. 2b and 2c show HR-TEM images of a cross section parallel to the substrate. Layer F was a nanocomposite film having the multilayered structure of Cr(Al)N crystalline layers 1.6 nm thick and two-dimensionally dispersed amorphous (a-) SiOx nanoparticles with sizes of 1 nm or less, which are enclosed with the Cr(Al)N crystals. Indentation hardness, measured using a nanoindentation system (Fischerscope, H100C-XYp), revealed that the hardness increases with substrate rotation speed, and also increases with addition of SiOx until a maximum for about 20 vol. % SiOx and then decreases with more addition. Fast rotation and low oxide fraction would make a-SiOx particles smaller, resulting in the formation of Cr(Al)N crystals including very fine a-SiOx particles with small number density. These fine a-SiOx particles can work as obstacles for the lattice deformation of the Cr(Al)N crystals and make the composite films harder, accordingly. 1M. Nose et al., J. Vac. Sci. Technol. A30, (2012) 011502. 2M. Kawasaki et al., ACS Appl. Mater. Interfaces 5, (2013) 3833. 3M. Kawasaki et al., Appl. Phys. Lett. 103, (2013) 201913.

Fig. 1: HAADF image and HAADF, EELS and EDS intensity profiles along the green line in the Cr(Al)N/38 vol. % SiOx nanocomposite coating F and the transition layers B,C,D, and E, which were sputter-deposited on Si substrate A rotating at a rotation speed of 1 rpm.

Fig. 2: Fine structure of layer F. (a) HAADF STEM image of a cross section normal to the substrate. (b) Enlarged image of a part of (a). (c) HR-TEM image of a cross section parallel to the substrate. (d) Enlarged image of a part of (c). Circles indicate a-SiOx particles.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-1891 Solid-state Dewetting of Thin Films Studied by in situ Transmission Electron Microscopy

Niekiel F.1, Kraschewski S. M.1, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), University Erlangen-Nürnberg, Erlangen, Germany
florian.niekiel@ww.uni-erlangen.de

A major issue of thin films is their instability against dewetting at elevated temperatures resulting from the energetically unfavorable configuration in comparison to a set of droplets or particles. This phenomenon can occur at temperatures well below the melting temperature of the bulk material and is denominated as solid-state dewetting [1].
Two different views on solid-state dewetting have developed from applications: Dewetting of mainly metallic or semiconducting thin films poses a degradation mechanism on today's electronics, magnetics and optics applications. It can occur at elevated temperatures in application or even during fabrication causing critical failure. On the contrary controlled dewetting has lately been employed to fabricate ordered arrays of nanoparticles. Different approaches were developed to influence order, shape and spacing, e.g. by the use of structured substrates.
In both cases a thorough understanding of the underlying mechanism of solid-state dewetting is necessary. Surface self-diffusion has generally been accepted to be the main transport mechanism [1], but a recent work showed the importance of grain boundary diffusion and arose doubt whether this generalization can be made [2]. A common way to hinder solid-state dewetting is the use of alloyed thin films. It is however poorly understood, how this influences the mechanism giving rise to the higher stability [3].
In this work we apply advanced in situ transmission electron microscopy (TEM) techniques to study the phenomenon of solid-state dewetting. Au thin films on silicon nitride substrates have been chosen as model system. A DENSsolutions sample heating system is used for in situ heating experiments capable of heat treatments of up to 800 °C at very low drift rates.
Fig. 1 exemplarily shows ADF-STEM images of such an experiment. On the left the as deposited Au thin film is shown, whereas the image on the right shows the thin film after 50 min at 300 °C. The phenomenon of solid-state dewetting is clearly visible. It has to be mentioned, that the as deposited Au thin film was not continuous but already featured voids. This has been exploited to study directly the process of void growth separated from the process of void nucleation.
Fig. 2 underlines the solid-state character of the observed process. It shows subsequent HRTEM images of a bridge between two Au islands at 300 °C at an advanced stage of the dewetting process. The retraction of a surface step can be observed, while the permanent observation of lattice fringes shows the solid-state character of the material.

1. Thompson, Annu. Rev. Mater. Res. 42 (2012), pp. 399-434
2. Kovalenko et al., Acta Mater. 61 (2013), pp. 3148-3156
3. Müller et al., J. Appl. Phys. 113 (2013), 094301

The authors gratefully acknowledge financial support by the German Research Foundation (DFG) via research training group 1896 and the cluster of excellence EXC 315. DENSsolutions is acknowledged for providing the sample heating system.

Fig. 1: ADF-STEM image of (left) as deposited discontinuous Au thin film on silicon nitride membrane, (right) after annealing at 300 °C for 50 min in the TEM.

Fig. 2: Subsequent HRTEM images of Au thin film on silicon nitride membrane at 300 °C at advanced stage of dewetting. The times indicated are relative times between the images. Red arrow highlights retraction of a surface step.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-1956 Quantitative structural and chemical investigation of amorphous and metastable crystalline phase-change alloy thin films by Cs-corrected STEM

Ross U.1, Lotnyk A.1, Thelander E.1, Rauschenbach B.1
1Leibniz Institute of Surface Modification, Leipzig, Germany
ulrich.ross@iom-leipzig.de

Phase-change composites are of interest as active components in next generation electronics phase-change random access memory (PCRAM) [1]. They display a rapid, reversible transition in resistance and reflectivity states due to their unique crystallization behaviour from amorphous to metastable crystalline phase, as well as a low threshold to re-amorphization. The atomic structure transition is therefore closely linked to those electronic and optical properties making the material class useful for data retention.
We have applied the analytical capabilities of a state-of-the-art probe Cs-corrected FEI Titan3 G2 60-300 TEM to the investigation of the atomic structure and phase transition in various thin film samples deposited by pulsed laser deposition [2]. The chosen material systems of ternary compounds along the (GeTe)x-(Sb2Te3)1-x pseudobinary line are well established in applications and widely used as test cases for phase change behaviour. The investigations encompassed samples deposited at various temperatures onto a number of single-crystalline substrates, as well as a range of sample treatments applied in order to induce the phase transitions of interest. In particular, we have performed a detailed study of the crystallization behaviour in GeSb2Te4 and Ge2Sb2Te5. In addition, as-grown textured and epitaxial metastable thin films were investigated with sub-angstrom resolution by comparison with quantitative STEM image simulations performed with the xHREM/STEM simulation software package [3].
The resulting HRSTEM images as well as STEM-EDX maps from the fourfold super-X EDX detector array (see Fig.1) and EELS spectrum analysis allow us to shed further light onto the functional characteristics of these highly beam-sensitive materials. The investigation of fast epitaxial growth onto oriented substrates in particular reveals the formation of defect networks in the metastable phase (see Fig.2) and may offer the potential for the development of phase change thin film structures with improved switching behaviour.

[1] Raoux, S.; Welnic, W.; Lelmini, D.; Chem. Rev. 2010, 110, 240-267.
[2] Lu, H.; Rauschenbach, B. et al.; Adv. Funct. Mater. 2013, 23, 3621–3627.
[3] Ross, U.; Lotnyk, A.; Thelander, E.; Rauschenbach, B.; Appl. Phys. Lett. submitted 02/2014

The financial support of the European Union and the Free State of Saxony (LenA project; project no. 100074065) is gratefully acknowledged.
Keywords: GST, phase-change material, PCRAM, Cs-corrected, HRSTEM, EDX, EELS

Fig. 1: (a) HAADF-STEM overview image of a laser-irradiated GeSb2Te4 layer and (b) corresponding STEM-EDX map quantification results for characterization of laser-crystallization behaviour.

Fig. 2: HR-STEM images of metastable lattice in a textured Ge2Sb2Te5 layer and corresponding image simulations (insets). (a) Defect-free lattice with randomly distributed vacancies, (b) vacancy layered structure, (c) antisite boundary.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-2017 Determination of InAs/AlSb interfaces composition in multilayer systems using HRTEM and STEM-HAADF techniques.

Nicolai J.1, Gatel C.1, Warot-Fonrose B.1, Teissier R.2, Baranov A.2, Magen C.3, Ponchet A.1
1CEMES CNRS-UPR 8011, Université de Toulouse, 31055 Toulouse, France, 2IES CNRS-UMR 5214, 34095 Montpellier, France, 3Laboratorio de Microscopías Avanzadas - Instituto de Nanociencia de Aragón (LMA-INA), Departamento de Física de la Materia Condensada, Universidad de Zaragoza, and Fundación ARAID, 50018 Zaragoza, Spain
julien.nicolai@cemes.fr

InAs/AlSb multilayers grown on (001) InAs substrate are currently developed for short wavelength quantum cascade lasers (2-5 µm). The operation of these devices strongly depends on the properties of the interfaces, which are very complex due to the change of both group III and group V elements. Two different extreme interfaces can thus be envisaged: AlAs or InSb interfaces, where very important strain effects (respectively -7% and 7%) are expected.
The aim of this work is to characterize interface properties as a function of the growth sequences. Especially, we tried to grow three different kinds of interface: spontaneous, AlAs forced and InSb forced using dedicated growth sequences. The samples have been grown by molecular beam epitaxy at 450°C.
The interfacial strain state has been characterized by HRTEM analysis using the Geometrical Phase Analysis (GPA) method. Information about the tensile or compressive character of the interfacial stress can be achieved. This strain state is related to the chemical composition of the interfaces themselves but several chemical compositions can correspond to the averaged strain value. The chemical composition of these interfaces has also been investigated by aberration corrected HAADF-STEM. Several chemical compositions can fit the HAADF intensity profile. The combination of HRTEM and HAADF allowed for discriminating more precisely the interface composition. Then the results are discussed considering the physical elementary mechanisms of epitaxial growth.
We used this analytical method to study the interface formation in relation with the growth sequence. We showed that spontaneously, AlAs type interface with a moderate tensile stress is formed on both, AlSb on InAs and InAs on AlSb interfaces (cf. Fig1; Fig.2). We assume that this kind of interface is favored due to its high thermal stability and energy bond, which leads to the most stable configuration. When forced interfaces are tentatively introduced, interfaces with a strong tensile stress (AlAs type) are achieved at both interfaces. Interfaces with a strong compressive stress (InSb type) can be achieved at AlSb on InAs interface while it is not possible at InAs on AlSb interfaces (cf. Fig.3). We show that these configurations can be explained using simples rules as: the heaviest element of one column can segregate versus the lightest element of the same column, V column element can desorbs while III column element cannot, the chemical bond with the highest thermal stability is favored.


Reference
[1] J. Nicolaï et al., Elastic strain at interfaces in InAs/AlSb multilayers for quantum cascade lasers, Applied Physics Letters. 104, 031907, (2014)

The authors acknowledge the ESTEEM2 project for this support. This work is supported by the French national project NAIADE (ANR-11-BS10-017).

Fig. 1: (a) HREM image of InAs-AlSb superlattice along the [1–10] zone axis with spontaneous interfaces; (b) eyy maps and profiles determined from the geometrical phase analysis of the HREM image (0.8 nm spatial resolution).

Fig. 2: HAADF image of the sample showed on Fig.1 and intensity profile along the growth direction.

Fig. 3: HAADF image of a sample with ‘forced’ interfaces and intensity profile along the growth direction. Interfaces 1 and 2 are, respectively, AlAs and InSb ‘forced’ type.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-2224 Exploring the polarity in oxide thin films

Peters J.1, Beanland R.1, Alexe M.1, Sanchez A. M.1
1University of Warwick, Coventry, UK
j.j.p.peters@warwick.ac.uk

Ferroelectric materials are of major importance in electromechanical and electronic devices including microactuators, high density memories and field effect transistors. The most widely used material, lead zirconium titanate, Pb[ZrxTi1-x]O3 or PZT, combines a high dielectric constant with large pyroelectric and piezoelectric coefficients. The breaking of symmetry, which gives the material its ferroelectric properties, occurs on a sub-unit cell level and usually results in several equivalent orientations and a polydomain structure. The high quality of single crystal thin films grown by pulsed laser deposition (PLD) allows detailed study of the structure, nucleation and growth of these polarization domains.

The boundaries between different domains have to both maintain crystal continuity and accommodate the change of polarization. They have properties different to the domains on either side [1,2] and there is great interest in measuring and understanding the relationship between microstructure, strain, and polarization that takes place in these regions. This can only be done with characterization at the atomic level.

We investigate the local polarity at ferroelectric boundaries in PLD-deposited PZT using transmission electron microscopy (TEM) and high-resolution annular dark field scanning TEM (ADF-STEM). We have developed a modified peak pairs (PP) algorithm to measure the distortions in different sublattices, used to measure the  cation displacements in the structure. The direction of the polarization can be also identified using “digital” electron diffraction [3].

We use tetragonal PZT (x = 0.2), a=0.3905 nm, c=0.4141 nm grown on (001) SrRuO3 (SRO). Most of the PZT layer consists of c-domains, with the inclusion of immobile a-domains lying on planes at 45° to the surface. Interfacial dislocations allow strain relaxation between the PZT and SRO. The 90° rotatation of the polarization at the c:a domain walls is known to inhibit the motion of 180° domain walls [4]. This phenomena reduces the switchable polarization and limits the performance of any device that uses this effect.

Our images reveal the presence of thin a-domains that start as a point at the SRO:PZT interface, rapidly spreading out to a fixed width (<50nm) and continue to the surface. Fig. 1a shows an ADF-STEM images in an area of the PZT film containing an a domain. Strain maps produced by GPA indicate a displacement within the domain consistent with the tetragonal distortion of the material (Fig. 1b). The modified PP algorithm reveals the polarization of the material by mapping the relative displacement of the Zr/Ti in each unit cell (Fig. 2). Digital large-angle convergent beam diffraction patterns show the change in polarity as a difference in the 002 and 00‾2 patterns (Fig. 3).

Fig. 1: (a) ADF-STEM image of the PZT thin film containing an domain. (b) Shear component of the GP analysis. (c) ADF images superimposed with the shear component

Fig. 2: Polarization vector map at the a/c-domain boundary. Scale bar, 2nm

Fig. 3: Digital large-angle convergent beam diffraction patterns on (a) c-domain and (b) a-domain showing the change in polarity
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-2228 Electron energy loss spectroscopy of structure, coordination and interface states of Al/SiO2 interfaces in Al/AlOx/Al Josephson junctions

Zeng L. J.1, Nik S.1, Löffler M.1, Olsson E.1, Greibe T.2, Wilson C. M.2, Delsing P.2
1Department of Applied Physics, Chalmers University of Technology, Gothenburg, Sweden, 2Microtechnology and Nanoscience, Chalmers University of Technology, Gothenburg, Sweden
lunjie@chalmers.se

Al/AlOx/Al Josephson junctions fabricated on SiO2/Si substrate are used as building blocks in superconducting devices such as superconducting quantum bits (qubits) and single electron transistors [1]. The interface between the junctions and the dielectric substrates can accommodate defect energy states that will destroy the coherent quantum states in the superconducting circuits [2]. This phenomenon is called decoherence and becomes the limitation for the future application of the superconducting devices. To understand the origin of decoherence, it is of great importance to study the detailed structure of this interface.

Spatially resolved electron energy loss spectroscopy (EELS) measurements were carried out in a Titan 80-300 transmission electron microscope equipped with monochromator and probe Cs corrector. Both low-loss and core-loss EELS investigations were employed.

The low-loss EELS spectra acquired at the Al/SiO2 interface in Josephson junctions are shown in Fig. 1. Two distinct peaks positioned at ~4.5 eV and ~6.9 eV are present within the band gap of SiO2 in the spectra series. The intensity and position of the peaks vary as a function of the distance to the interface. By simulation of the low-loss EELS based on semi-classical model [3], it is found that the interface plasma peak at Al/SiO2 is expected at ~7.5 eV. From STEM-EELS line profiles of Al-L23 and Si-L23 energy loss near edge structure (ELNES) acquired at the interface region (Fig. 2), it is evident that alumina and silicon are present at the interface as a result of solid-state reaction between the aluminum and silicon dioxide [4]. The existence of alumina and silicon at the interface explains the discrepancy between the experimental data and simulated low-loss EELS results. Moreover, the Al-L23 ELNES shows that alumina formed at the interface has aluminum atoms with an octahedral coordination rather than tetrahedral, which is the most common type of structure of amorphous alumina [5]. By combining the observations from the core-loss and low-loss EELS investigation, we also propose that the signal at around 4.5 eV (the middle of the band gap of SiO2) is correlated to gap states resulting from the presence of elemental Si in the SiO2 substrate.

References:

[1] M. Gurvitch, M. A. Washington, and H. A. Huggins, Appl. Phys. Lett. 42 (1983) 472.

[2] J. M. Martinis, K. B. Cooper, R. McDermott et al., Phys. Rev. Lett. 95 (2005) 210503.

[3] P. Moreau, N. Brun, C. A. Walsh, et al., Phy. Rev. B 56 (1997) 6774.

[4] L. J. Zeng , T. Greibe , S. Nik , C. M. Wilson , P. Delsing and Eva Olsson, J. Appl. Phys. 113 143905 (2013).

[5] D. Bouchet, and C. Colliex, Ultramicroscopy 96 139 (2003).

The authors thank Swedish Foundation for Strategic Research, the Swedish Research Council, the Knut and Alice Wallenberg Foundation and Chalmers Nanotechnology Center for financial support.

Fig. 1: Low-loss EEL spectra acquired at the Al/SiO2 interface in Al/AlOx/Al junctions. The signals within the bandgap of SiO2 (~8.9 eV) are clearly visible.

Fig. 2: STEM-EELS line profile across Al/SiO2 interface in Al/AlOx/Al junction, showing Al-L and Si-L edges. Colors are used to highlight characteristic spectra at four different positions.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-2652 Oxygen Octahedral Tilts: The Origin of Ferromagnetism at Insulating LaMnO3 and SrTiO3 Interfaces.

Roldan M. A.1,2, Salafranca J.1,2, Siemons W.2, Pennycook S. J.3, Varela M.2,1, Christen H. M.2
1Universidad Complutense de Madrid, 2Oak Ridge National Laboratory, 3The University of Tennessee
marolgu@gmail.com

Complex oxide superlattices exhibit many unusual and interesting properties, such as the 2D electron gas found at insulating LaAlO3/SrTiO3 interfaces, and it is usually hard to foretell their macroscopic behavior based only on their constituents properties1. One fact that is well known is that the strong electron-lattice coupling that exists in ABO3 perovskite-type materials results in lattice distortions that play a very important role in the physical properties2. The stress promoted by the lattice mismatch in epitaxial heterostructures can be used to tune the structure near the interfaces, such as the oxygen octahedral rotations, which can provoke important changes in the electronic properties3.

Here we report the presence of an insulating ferromagnetic interface between paramagnetic SrTiO3 (STO) and antiferromagnetic LaMnO3 (LMO) in superlattices grown by pulsed-laser-deposition. In our samples, SQUID magnetometry and polarized neutron reflectometry measurements show that LMO exhibits a ferromagnetic ordering, with the local magnetization enhanced near the interfaces within a region approximately three unit cells thick. In order to analyze the structure, chemistry and electronic properties of these interfaces, we have carried out a study in an aberration corrected STEM, combining annular bright field (ABF) imaging4 with EELS and theoretical calculations.

EELS show the absence of significant charge transfer between neighboring Mn and Ti ions across the interface. ABF images are used to locate oxygen columns and hence, investigation of oxygen octahedral tilts. Fig. 1 shows an ABF image of the whole LMO thin film sandwiched in between STO layers acquired in a Nion UltraSTEM200 operated at 200 kV. Fig. 1 shows the result of Fourier filtering (FFT) the image in 1. In the inset of Fig. 1 an enlarged view of the filtered ABF image is shown. The O columns and the octahedral tilts across the LMO layer can be clearly observed. In order to quantify these tilts, we measure the difference in vertical coordinates between adjacent O columns. The geometry of octahedral rotations in the middle of the LMO layers is consistent with the octahedral tilts reported in the LMO bulk. However, near the interfaces, these rotations are suppressed. We will discuss these findings and relate them to the macroscopic properties of the layers. Density-functional calculations will be used to establish the connection between structural distortions and the enhanced magnetism measured.

References:

1. E. Dagotto, and Y. Tokura, MRS Bull. 33 (2008) p. 1037.

2. A. Vailionis et. al., Phy. Rev. Let. 83 (2011) 064101.

3. A.Y.Borisevich et. al., Phy. Rev. Let. 105 (2010) 087204.

4. S. D. Findlay et al., Appl. Phy. Exp. 3 (2010) 116603.

Research supported by the U.S. Department of Energy, Basic Energy Sciences, Materials Sciences and Engineering Division (MV, ARL, SJP, WS, HC), by the European Research Council Starting Investigator Award “STEMOX 239739” (MR), and by Juan de la Cierva Program (JS).

Fig. 1: High-resolution (left) raw ABF and (right) FFT filtered images of a LMO thin film sandwiched in between STO layers. The interfaces are marked with yellow dashed lines. A green arrow marks the growth direction. The scale bars are 2 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-2944 Synthesis and Characterisation of Boron Nitride

McCulloch D. G.1, McDougall N. L.1, Partridge J. G.1, Lau D. W.1, Nicholls R. J.2
1Physics, School Applied Sciences, RMIT University Melbourne, VIC 3000, 2Department of Materials, University of Oxford, Parks Rd, Oxford, Oxfordshire, OX1 3PH, UK
dougal.mcculloch@rmit.edu.au

Boron nitride (BN) is unknown in nature and only became available commercially in the latter half of the 20th century. It is analogous to carbon in having both a cubic (diamond-like) phase (cBN) and a hexagonal (graphite-like) phase (hBN) [1]. cBN is second only to diamond in hardness, has a wide band-gap, good thermal conductivity and unlike diamond, can be doped both p- and n-type, making it an excellent candidate for use in high-power electronic applications. hBN is a good electrical insulator, has high thermal and chemical stability and its wide band-gap has been recently exploited in the fabrication of compact deep-ultraviolet light emitting devices.

The aim of this work is to prepare novel forms of BN as thin films using advanced plasma synthesis methods. The microstructure of BN materials can be difficult to characterise using conventional methods due to the presence of disorder and the range of complex bonding configurations that can be formed. X-ray absorption spectroscopy (XAS) and electron energy loss spectroscopy (EELS) are complimentary methods which can be used to provide insights into the local bonding environments in materials. However, the interpretation of the near edge structure (NES) which occurs on the characteristic absorption edges is not always straight forward and theoretical modelling is often employed.

Figure 1 compares the B and N K-edges for cBN calculated using CASTEP (v6.1)[2] with EELS and XAS experimental data. On-the-fly generated ultrasoft pseudopotentials were used which enabled the inclusion of core holes [3]. The analysis tool OptaDOS [4] was used to calculate the NES, including lifetime-broadening effects. The best fit to experimental data was obtained using a partial core-hole (also know as a “Slater transition state”[5]). Figure 2 shows the corresponding results for hBN. Again the partial core hole (in this case 0.75 of a 1s electron) provides the best fit with experiment, although the overall agreement is not as good for hBN as for cBN. This work paves the way for the microstructure and bonding in more complex forms of BN to be characterised using a combination of experimental and theoretical NES.

[1] “Synthesis and Properties of Boron Nitride” (Mat. Sci. For., 54-55), eds J.J.Pouch & S.A.Alterovitz, trans tech publication (1991).
[2] S.J. Clark, M.D. Segall, C.J. Pickard, P.J. Hasnip, M.I.J. Probert, K. Refson and M.C. Payne, Zeitschrift für Kristallographie, 220, 567-570 (2005).
[3] S.-P. Gao, C.J., Pickard, A.Perlov and V.Milman, Journal of Physics: Condensed Matter, 21, 104203 (2009).
[4] R. J., Nicholls, A. J., Morris, C. J. Pickard and J. R. Yates, J. Phys.: Conf. Ser. 371 012062 (2012).
[5] A.T., Paxton, A.J., Craven, , J.M., Gregg and D.W. McComb, Journal of Microscopy, 210, 35-44 (2003).

The authors acknowledge the facilities provided by the RMIT Microscopy & Microanalysis Facility and the Australian Synchrotron. The authors also gratefully acknowledge support provided by the Australian Research Council (ARC) and the Australian Academy of Sciences.

Fig. 1: The calculated B and N k edges for cBN compared to EELS and XAS experimental spectra with no core-hole (top), a partial core hole (0.5 of a 1s electron) (middle) and a full core-hole (bottom).

Fig. 2: The calculated B and N k edges for hBN compared to EELS and XAS experimental spectra with no core-hole (top), a partial core hole (0.75 of a 1s electron) (middle) and a full core-hole (bottom).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-3058 Study on the atomic and electronic structure in metal nitride films using advanced TEM

Zhang Z. L.1, Gao Z. W.1, Daniel R.2, Mitterer C.2, Dehm G.1,3
1Erich Schmid Institute of Materials Science, Austrian Academy of Sciences, Leoben, Austria 1, 2Department of Physical Metallurgy and Materials Testing, Montanuniversität Leoben, Austria 2, 3Max-Planck-Institut für Eisenforschung, Düsseldorf, Germany
zaoli.zhang@oeaw.ac.at

Transition metal nitrides have found wide-spread applications in the cutting- and machining-tool industry due to their extreme hardness, thermal stability and resistance to corrosion. The increasing demand of these nitrides requires an in-depth understanding of their structures at the atomic level. This has led to some experimental and theoretical research [1-6]. The films used in this study were deposited by reactive direct current magnetron sputtering of a Cr/V/Ti metal target in an Ar+N2 atmosphere at a constant total pressure of 1 Pa, a target power of 6 kW, and a temperature of 350°C. A TEM/STEM JEOL 2100F operated at 200 kV and equipped with an image-side CS-corrector  was utilized.

We will present some recent results on the atomic and electronic structures of metal nitride thin films (CrN, VN and TiN) on MgO and Al2O3 substrates (Fig.1 and Fig. 2) using advanced TEM techniques, such as CS-corrected HRTEM/STEM, EELS/EDXS, quantitative atomic measurement and electron diffraction analysis as well as theoretical calculations. The atomic and electronic structures of interfaces are analysed and experimental and theoretical results compared in order to unveil. interface induced phenomena between the nitride films and the oxide substrates [2,3].

Particularly, the study on the effect of N defects in a CrN film has led to some interesting conclusions. Ordered nitrogen (N) vacancies were often found to cluster at the {111} planes. Combining independent image analysis, such as atomic displacement/strain measurement using geometrical phase analysis, and spectrum analysis by examining the low loss and core loss, fine structure analysis, some generalized conclusions are drawn: (i) a relationship between the lattice constant and N vacancy concentration in CrN is established [5], (ii) the change of ionicity of the CrN crystal with the N vacancy concentration is shown; (iii) a relation between electronic structure change and elastic deformation in CrN films has been experimentally derived, revealing that the elastic deformation in CrN may lead to a noticeable change in the fine structure of Cr-L2,3 edge, i.e. L3/L2 ratio.

The effect of randomly distributed defects in the films has been explored in a quantitative way using quantitative electron diffraction, combined with HRTEM and EELS analysis. Quantitative electron diffraction analysis reveals that the intensity ratios of (111), and (200) reflections (I111/I200) sensibly varies with the defect densities. Some quantitative relations are established.

[1]. R. Daniel et al, Acta Materialia 58(2010), p. 2621.

[2]. Z. L. Zhang, et al Physical. Review B 82(R)(2010) 060103-4

[3]. Z. L. Zhang, et al Journal of Applied Physics, 110(2011) 043524-4

[4]. Z.L. Zhang et al Physical Review B 87 (2013)014104.

Gabriele Moser and Herwig Felber are gratefully acknowledged for their help with sample preparation, thanks are given to Dr. Hong Li for ab-initio calculations

Fig. 1: Figure1. HRTEM image of the CrN/Cr interface, a defective layer between Cr and CrN originated from the ordered N vacancy.

Fig. 2: Figure 2.  The anisotropic distribution of strain in the defective layer (exx).

Fig. 3: Diffraction pattern along [112] CrN and [0001] Al2O3 zone axis

Fig. 4: HRTEM image along [1-10] CrN and [01-10]Al2O3
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-3069 Nanostructure of Magnetron Sputter Deposited Superhard Ti1-xSixN Thin Films

Lu J.1, Greczynski G.1, Persson P.1, Jensen J.1, Petrov I.1, 2, Greene J. E.1, 2, 3, Hultman L.1
1Thin Film Physics Division, Department of Physics (IFM), Linköping University, SE-581 83 Linköping, Sweden, 2Materials Science Department and Frederick Seitz Materials Research Laboratory, University of Illinois, Urbana, Illinois 61801, 3Department of Physics, University of Illinois, Urbana, Illinois 61801, USA
junlu@ifm.liu.se

Magnetron sputter deposited Ti1-xSixN thin films exhibit superhardness (>40 GPa). SiNx plays an important role in the hardness enhancement.1-3 XRD results show that the metastable ternary compound still retains the NaCl-structure of TiN. It has been speculated that Si either substitutes at cation site or segregates to boundaries. In the second case, Si locates at boundaries between TiN crystallites to form an atomic-layer thick semi-coherent cubic SiNx tissue phase that eventually becomes several atomic layer thick amorphous a-SiNz phase.4 In this work, HRSTEM and EELS were used to study Ti1-xSixN nanostructure at atomic scale. Ti1-xSixN films were grown at 500 °C in mixed Ar/N2 atmospheres by hybrid high-power pulse/dc magnetron co-sputtering (HIPIMS/DCMS).5 Cross-sectional and plan-view TEM specimens were prepared using conventional method. FEI Tecnai UT operated at 200 kV and FEI Titan double Cs corrected instruments were used for BF-TEM images, SAED characterization, and EELS mapping. Figs. 1(a-b) are plan-view and cross-sectional BF-TEM images showing a columnar structure with an average column diameter of ~ 15 nm. Each column consists of bundles of 2~3 nm fibers. All the fibers in one column have the same crystal orientation and the overall film exhibits a strong 002 fiber texture as shown by the SAED patterns in the inserts of Figs. 1(a-b). HRTEM image in Fig. 1(c) reveals crystalline fibers surrounded by amorphous/crystalline boundaries, dependent on boundary thickness. EELS mappings in Figs. 2(a-b) show that the boundaries are rich in Si and N. This nanostructure character is similar to that of high-temperature-annealed arc evaporated Ti1-xSixNy coatings and the proposed nanostructure model of TiN nanocrystallites encapsulated by an amorphous Si3N4 tissue phase.6,7 The EELS spectra in Fig. 2(c) show that the difference of the N K peaks in TiN grains and in SiNz boundaries.

References:
1.    S. Veprek and S. Reiprich, Thin Solid Films (1995) 268, 64.
2.    H. Söderberg, J.M. Molina-Aldereguia, L. Hultman, and M. Oden, J. Appl. Phys. (2005) 97, 114327.
3.    A. Flink, T. Larsson, J. Sjölen, L. Karlsson, and L. Hultman, Surf. Coat. Technol. (2005) 200, 1535.
4.    L. Hultman, Bareno J, Flink A, Söderberg H, Larsson K, Petrova V, Oden M, Greene JE and Petrov I, Phys. Rev. B 75 (2007) 155437.
5.    G. Greczynski, J. Lu, J. Jensen, I. Petrov, J.E. Greene, S. Bolz, W. Kölker, Ch. Schiffers, O. Lemmer and L. Hultman, J. Vac. Sci. Technol. A 30 (2012) 061504-1.
6.    A. Flink, M. Beckers, J. Sjölen, T. Larsson, S. Braun, L. Karlsson and L. Hultman, J. Mater. Res. (2009) 24, 2483.
7.    S. Vepřek, S. Reiprich, L. Shizhi, Appl. Phys. Lett. (1995) 66, 2640.

The authors acknowledge the Knut and Alice Wallenberg Foundation support to the Ultra-Electron Microscopy Laboratory at Linköping University operated by the Thin Film Physics Division.

Fig. 1: (a) plan-view BF-TEM image, (b) cross-sectional BF-TEM image, (c) HRTEM image.

Fig. 2: EELS mapping of (a) Si+Ti, (b) Si+N, (c) EELS spectra from fibers and boundaries.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-3410 Toward 3D mapping of the oxygen octahedral rotations in perovskite thin films unit cell by unit cell

He Q.1, Ishikawa R.1, Lupini A. R.1, Liang Q.2, Biegalski M. D.2, Borisevich A.1
1Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA, 2The Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
heq1@ornl.gov

Oxygen octahedral rotations (OOR) in perovskites couple strongly to properties, providing opportunities for creating novel functional materials.[1] Epitaxial thin films is a natural playground for controlling OOR since the film can be tuned through the elastic, electrical, and chemical interactions with the substrate. It is therefore critical to characterize OOR in thin films. Conventional methods either lack of spatial resolution (e.g. X-ray and Neutron diffraction[2]), unable to provide full 3D information (e.g. direct oxygen column imaging in electron microscopes[3-5]), or are not suitable for unit-cell-resolved measurements (e.g. electron diffraction[6-7]). The recently introduced position averaged converged beam electron diffraction (PACBED)[8] can in principle provide full tilt information and approach unit cell resolution. It, however, requires extensive simulations, which become especially prohibitive for complex systems with several competing phenomena, since each sample parameter such as thickness, polarization, strain, and tilt, adds an extra dimension to the simulation phase space.

In this work, we propose a simpler strategy for characterizing OOR using direct oxygen imaging in STEM, using CaTiO3 as an example. Instead of looking along the in-phase rotating axis [100]pc, we obtain ABF STEM images from the [110]pc zone, along which O atoms in one unit cell form more than one column. Because of that, the shapes of those O columns within one unit cell and their symmetry relations to adjacent unit cells contain information about the OOR system. The phase of the OOR tilts in all three directions can be determined unit cell by unit cell using this approach (Fig.1).

Frozen phonon multislice image simulations[9] are then used to investigate the range of imaging and specimen conditions over which this method can be applied. The contrast for the ABF mode is found to be robust within a ±2 nm defocus range and not sensitive to specimen thickness up to 40 nm (Fig.2), consistent with the previous work.[10] Other factors such as sample mistilt will be also discussed. Finally, different OOR systems with different rotation angles are studied to estimate the detection range with given microscope conditions. The prospects for using this technique on superlattices of materials with distinct tilt systems will also be discussed.

[1] Rondinelli, JM et al., Adv Mater (2012)
[2] Johnson-Wilke RL et al., PRB (2013)
[3] Kirkland, AI et al., Ultramicroscopy (2007)
[4] Kim YM et al., Adv Mater 25 (2013)
[5] Okunishi, E et al., Micron (2012)
[6] Levin I et al., Adv Funct Mater (2012)
[7] Woodward, DI et al., Acta Crystallogr B (2005)
[8] J. Hwang et al., PRB (2013)
[9] Kirkland EJ, Advanced Computing in Electron Microscopy
[10] Findlay, SD et al., Ultramicroscopy (2010)

Acknowledgement MSE Division, US DOE, a user project supported by ORNL’s CNMS, sponsored by the SUF Division, Office of BES, US DOE. R.I. is supported by JSPS Fellowship.

Fig. 1: (a) Experimental ABF image of CTO on LSAT viewed along [110]pc zone axis. (b) Simulated ABF images of CTO models with different OOR systems. Note that a mirror plane (with respect to O columns only) is present for in-phase out-of-plane rotation (c+) and absent for out-of-phase (c-). This can be used to identify the CTO film shown in (a) as c-.

Fig. 2: Simulated defocus-thickness map of ABF imaging of CTO (a-b+c-) viewed along the 110 direction, using the microscope parameters of the UltraSTEM200 at ORNL. The probe size (FWHM) of 0.5A is used. The visibility of the fine structure at the O columns is robust within ±2 nm of focus and is not sensitive to specimen thickness up to 40 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-3-O-3483 STEM and EELS study of Graphene/Bi2Se3 Interface

Kepaptsoglou D.1, Gilks D.2, Lari L.2,3, Ramasse Q.1, Weinert M.4, Li L.4, Lazarov V. K.2
1SuperSTEM Laboratory, STFC Daresbury Campus, Warrington, WA4 4AD, UK, 2Department of Physics, University of York, Heslington, York, YO10 5DD, UK, 3York JEOL Nanocentre, University of York, Heslington, York, YO10 5BR, UK, 4University of Wisconsin Milwaukee, Milwaukee, 53211, WI, USA
vlado.lazarov@york.ac.uk

Bi2Se3 is a 3D topological insulator (TI) that has attracted a lot of research due to exotic properties associated with topologically protected helical two-dimensional surface states and one-dimensional states associated with bulk line defects such as dislocations. Recently it has been shown that when Bi2Se3 is grown either on epitaxial graphene or self-standing graphene flakes [1,2], a rich grain structure of film is developed due to the spiral nature growth of the film. The grain boundaries and growth screw dislocations in this system provide a ground for new physics that can be accessed by combination of scanning tunnelling microscopy and transmission electron microscopy techniques such as STEM-HAADF and EELS. In this work we investigate the nature of the graphene/Bi2Se3 interface in order to understand the complex epitaxy between the film and substrate which ultimately determine the structure and functional properties of the Bi2Se3 surface topological states.


We have grown Bi2Se3 films on both epitaxial graphene/SiC(0001) at 275-325 °C and chemical vapor deposition produced graphene flakes. We have studied their surface states by STM, and interface structure and electronic properties by cross-sectional TEM/STEM and EELS, respectively, by using aberration corrected 200kV JEOL 2200FS and NEON 100 at 100kV.


Fig. 1 shows the interface region of SiC/graphene/Bi2Se3. Due to the Z-contrast sensitivity in the HAADF imaging, we can clearly identify the Bi, Se and Si columns. The dark region between the SiC and Bi2Se3 corresponds to the graphene monolayer which is clearly seen in BF-STEM imaging condition (not shown here). Fig. 2 is EELS spectra from the interface region following the C edge from the substrate through the C layer between the SiC substrate and first Se atomic plane from the Bi2Se3. By quantifying the σ*/π* ratio we found that the C layer at the interface is behaving like self-standing graphene monolayer. This indicates that the bonding between Se and graphene has Van der Waals nature. Such weak bonding would be the key factor for the multiple epitaxial relations which leads to both low and high angle boundaries observed in Bi2Se3 thin films when grown on graphene substrate [1,2].

1. Liu, Y., et al., Charging Dirac States at Antiphase Domain Boundaries in the Three-Dimensional Topological Insulator Bi2Se3. Physical Review Letters, 2013. 110(18): p. 186804.
2. Liu, Y., et al., Tuning Dirac states by strain in the topological insulator Bi2Se3. Nat Phys, 2014. advance online publication.

This research was funded by EPSRC research grants EP/K013114/1 and EP/K032852/1.

Fig. 1: HAADF-STEM image of the interfacial region of SiC/graphene/Bi2Se3. Green solid circles represent Bi, and blue Se atomic columns.

Fig. 2: a) HAADF STEM survey image showing the Bi2Se3 film and SiC substrate, b,c) Si L2,3 and C K EELS elemental maps from the area across the Bi2Se3/SiC interface marked in a. d) Selected C K EELS spectra from the interface region and e)σ*/π* ratio of the C K edge at the Bi2Se3/SiC interface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1406 TEM characterisation of PVD Cu/W nanoscale multilayers

Karlík M.1, Callisti M.2, Polcar T.1,2
1Czech Technical University in Prague, Czech Republic, 2University of Southampton, United Kingdom
Miroslav.Karlik@fjfi.cvut.cz

Nanoscale metallic multilayers (NMMs) have been the subject of an increasing number of studies due to their exceptional mechanical properties. Their unique properties are a result of the high density of interfaces, which change the conventional mechanisms of plastic deformation when the individual layer thickness is below 100 nm. The strength of NMMs depends on the modulation periodicity and several mechanisms have been proposed to explain their ultra-high strength, such as: (1) coherency strain hardening, (2) structure barrier strengthening, (3) modulus mismatch, and (4) intermixing at the interface.

Three sets of Cu/W multilayers about 1 μm thick were deposited with different modulation periodicity (Ʌ = 5/5 nm, 15/15 nm and 30/30 nm) on single crystal (100) Si wafers by a balanced magnetron sputtering apparatus using 2’’ targets of W (99.99% purity) and Cu (99.99% purity). Cross-sectional samples were cut from 3 mm x 5 mm sandwich, mechanically thinned to 20 μm, dimple polished, and ion-beam milled in Gatan PIPS 691 device. Their observation was carried out at 200 kV using FEI Tecnai G2 F20 XT microscope.

The layering structure was well defined in all cases, but the layers, that were initially flat close to the substrate, quickly became wavy as deposition progressed. The waviness in the layers triggered earlier (after the 2nd layer) for Cu/W 5/5 nm than for Cu/W 30/30 nm (after the 5th layer). The transition from planar to wavy layers seemed to be a consequence of the cumulative layer waviness developed in the multilayers and the shadowing effects inherent to sputtering processes. As a result, the multilayers developed a columnar structure, with a column width between 20 and 100 nm, and a high porosity level at the columnar boundaries. The layer waviness amplitude was large enough to break up the layers for the 5/5 nm multilayer, as shown in Fig. 1, but not for the 30/30 nm multilayer (Fig. 2). The selected area electron diffraction (SAED) patterns (inserts in Figs. 1, 2) indicate that Cu and W layers display a nanocrystalline structure, with no clear preferred growth orientation. The W diffraction rings were practically continuous, while the Cu rings were formed by discrete spots. Therefore, the W nanograins were on average much finer than the Cu ones.

From high magnification phase contrast micrographs it can be seen that that Cu layers in the 5/5 nm multilayer are formed of grains 5 nm thick and about 15 nm wide (Fig. 3) and that Cu/W interfaces are smoother than W/Cu, which are rather rugged. Fig. 4 shows local continuity of atomic planes (coherence) across Cu/W interface in the 30/30 nm multilayer and deformed atomic planes with edge dislocations in the W layer.

This investigation was supported by the EC grant RADINTERFACES 263273. TEM observation at the Institute of Physics AS CR, Prague was financed under the project MEYS LM2011026.

Fig. 1: STEM – HAADF micrograph of the 5/5 nm W/Cu multilayer and corresponding SAED pattern.

Fig. 2: STEM – HAADF micrograph of the 30/30 nm W/Cu multilayer and corresponding SAED pattern.

Fig. 3: Cu/W interfaces in the 5/5 nm multilayer are smoother, the W/Cu more rugged.

Fig. 4: Deformed atomic planes in W close to the Cu-W 30/30 nm interface; the arrows point out edge dislocations.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1527 Crystallization of Fe60Cr8Nb8B24 amorphous alloys and coatings

Koga G. Y.1, Gallego J.2, Melle A. K.1, Yavari A. R.3, Bolfarini C.1, Kiminami C. S.1, Botta W. J.1
1Federal University of São Carlos, São Carlos, SP, Brazil, 2São Paulo State University, Ilha Solteira, SP, Brazil, 3Institut National Polytechnique, Grenoble, France
wjbotta@ufscar.br

Fe-based amorphous alloys have an attractive combination of high mechanical strength, high wear resistance and good corrosion properties. Alloys of this family can be produced in the amorphous state by different rapid solidification techniques, and in the present work we studied the crystallization aspects of two types of materials: (a) amorphous Fe60Cr8Nb8B24 (at.%) ribbons produced by melt-spinning and (b) partially amorphous coating produced by high velocity oxygen fuel (HVOF). The amorphous Fe60Cr8Nb8B24 ribbons were produced by melt spinning with copper wheel rotating at a speed of 50ms-1 in an argon atmosphere. Annealing treatments were conducted at different temperatures, during short time (60 s) under controlled atmosphere. Coatings were produced by HVOF over a X80 pipe steel substrate using atomized and further milled powders with particle sizes in the range of 20 to 45 µm.
TEM observations of the amorphous and crystallized ribbons and of the HVOF coating were carried out in a FEI Tecnai G2 200kV equipment, after the usual thinning by ion milling. XRD patterns indicated the as-spun ribbons to be fully amorphous. No diffraction peak due to crystalline phase was also seen in the ribbons annealed at 450 ºC and 550 ºC, but a clear peak was recognized for the sample annealed at 640 ºC. The coatings obtained by HVOF showed high fraction of amorphous phases and the presence of different crystalline phases.

Figure 1 shows STEM bright field micrograph of the amorphous ribbon after annealing at 640 ºC. The sample was fully crystallized although the annealing temperature was slightly lower than the crystallization peak temperature observed by DSC. Figure 2 shows STEM dark field micrograph of the coating layer; as in the ribbons, the crystals are in the nanometric size range and as indicated by XRD and DSC a high fraction of amorphous phase is still present. Figures 3 (a) and (b) show, respectively, the selected area electron diffraction patterns (SAEDP) of the crystallized ribbon and the coating layer. The figure compares both patterns and index the phases as ferrite, FeB and Fe3B in the ribbon and as ferrite and Fe2B in the coating layer. In conclusion, TEM observations of crystallized and partially crystallized Fe60Cr8Nb8B24 alloys obtained by two rapid solidification processes suggest different sequence of phases formation from the melt and from the amorphous solid.

The authors gratefully acknowledge the financial support of the Brazilian institutions FAPESP, CNPq, CAPES and FINEP and Dr. R. Schulz from HydroQuebec, Canada for HVOF processing.

Fig. 1: STEM bright field micrograph of the amorphous ribbon after annealing at 640 ºC.

Fig. 2: STEM dark field micrograph of the coating layer with nanometric size range crystals.

Fig. 3: (a) SAEDP of the crystallized Fe60Cr8Nb8B24 ribbon; (b) SAEDP of the coating layer produce by HVOF from Fe60Cr8Nb8B24 powders. Both patterns indicate the phases associated with the diffraction rings.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1660 Ta based thin films on Ti and SS316L substrates for biomedical applications

Jara A.1, 2, Fréty N.3, Gonzalez G.1, 2
1Escuela de Física, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela., 2Centro de Ingeniería de Materiales y Nanotecnología, Laboratorio de Materiales, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela., 3Institut Charles Gerhardt, UMR 5253 CNRS-UM2-ENSCM-UM, Université Montpellier II, Montpellier, France.
angelicajara13@gmail.com

In this work we discuss the synthesis and characterization of Ta, TaN and TaN/Ta thin films deposited on Ti and SS316L as novel biomaterials to enhance implant bone biocompatibility. An RF sputtering system (13.56 MHz-80W) was used for thin film deposition. Ta thin films were deposited under Ar atmosphere with a pressure of 6.5 Pa and TaN thin films were deposited using Ar/N2 atmosphere with a N2 partial pressure of 2%. In order to evaluate the bioactivity of the thin films a biomimetic method has been used. This consisted in the immersion of the coated materials in simulated body fluid (SBF), an enriched SBF in Ca and PO4 (1.5 Ca and 1.5 PO4) were used.

Thin Films were evaluated by High Resolution Scanning Electron Microscopy (HRSEM) and by Atomic Force Microscopy (AFM). The transverse sections of the deposited Ta, TaN and Ta/TaN films on Si, showed the typical columnar growth of PVD deposition techniques; a layer thickness of 300 nm each was obtained. The planar views of the bilayer Ta/TaN coatings deposited on Ti and on SS316L are shown in Fig. 1 and 2. It can be observed that the microstructure of the bilayer Ta/TaN is denser on Ti than on SS316L. The TaN is deposited first on the metal substrate and then the Ta layer is deposited on top to form the bilayer, therefore the microstructure of the first layer is affected by the nature of the substrate and probably directs the growing of the second layer in a different way. The grain size of Ta in the bilayer on Ti (60 nm) is smaller than on SS316L (120 nm).

The immersion of the different materials in SBF from one to six weeks resulted in a coating of HA crystals, with increased thickness depending on the immersion period. Fig. 3 presents AFM images of TaN on SS316L, after immersion in SBF 1 week and 3 weeks. It is observed that the samples essentially consist of homogeneous granular structures covering the surface, the average roughness of the surface decreases with immersion time in SBF.

Fig. 1: HRSEM micrograph planar view Ta/TaN/Ti

Fig. 2: HRSEM micrograph planar view Ta/TaN/SS316L

Fig. 3: AFM topographic image of TaN/SS316L A) TaN/SS316L Coating, B) TaN/SS316L Coating one week in SBF and C) TaN/SS316L Coating three weeks in SBF.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1678 TEM study of a sol-gel alumina coating on K44X steel after high temperature treatment

Dörfel I.1, Nofz M.1, Sojref R.1, Geipel C.2
11BAM Federal Institute for Materials Research and Testing, Berlin, Germany, 21BAM Federal Institute for Materials Research and Testing, Berlin, Germany
ilona.doerfel@bam.de

A possibility to increase application temperature, application range and lifetime of metallic materials is the protection by coatings. Alumina coatings are one kind of potential solutions and especially their production via the sol-gel-route requires only little effort in surface pretreatment, which can be beneficial for future commercial applications. Several substrates of steel or Ni-base superalloys were coated and tested at different treatment temperatures, times, and atmospheres concerning oxidation protection and related microstructure. Substrates with lower chromium content (9-12 %) like steel X20CrMoV12-1 for instance, were investigated before. Here an ethanolic Boehmite sol is deposited on chromium rich steel (up to 19 % Cr) K44X (X2CrMoTi18-2) by dip coating in two different coating thicknesses: i) 770 nm and ii) 1760 nm. Both samples were heat treated for 30 minutes at 600°C to form the alumina coating and for 500 hours at 900°C in air as oxidation protection test.

The samples for the TEM investigations were prepared as cross sections normal to the coating surfaces by FIB technique in a Quanta 3D instrument (FEI). The TEM investigations were performed by means of an analytical STEM JEM 2200FS (JEOL) at 200 keV. Beside TEM and STEM imaging methods, HREM investigations, energy filtered TEM and nano-beam electron diffraction as well as EDX investigations were accomplished.

At both samples the oxide layers are thicker than the initial coatings. This is caused by diffusion processes of alloying elements from the steel into the coating. Mainly chromium and manganese diffused and formed spinel. Both samples show similar microstructure and elemental distribution, being reflected as diffusion fronts. Independent of the initial coating thickness four areas, differing in composition can be distinguished (Fig. 1, 2). Near the interfaces thin regions with high C and Si content occurred, here small crystallites, containing Cr, Fe O, Si are embedded in amorphous material. Some sections of these zones are delaminated from the steel substrates. Subsequently Cr rich zones exist, followed by Cr-Mn-rich areas with conspicuous large grains in the µm range. At the coating surfaces zones of δ- Al2O3 complete the layer systems. Between the Cr-Mn-rich and the Al2O3-areas delamination appeared again.

The microstructure of alumina coatings on K44X steel differs from that on substrates with lower chromium content and the coating did not reach the expected oxidation protection. The TEM results contribute to the understanding of this behavior and to the development of modified coatings which can improve the oxidation protection of chromium rich substrates for long term applications at high temperatures.

 

Fig. 1: Fig. 1: STEM bright field image

Fig. 2: Fig. 2: EDX elemental maps showing substrate and coating regions
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1736 Thin Films of Thermally Evaporated Au/Atomic Layer Deposited TiO2 on Glass Substrates

Kawasaki M.1, Chen M. J.2, Yang J. R.2, Chiou W. A.3, Shiojiri M.4
1JEOL USA Inc., Peabody, MA , USA , 2National Taiwan University., Taipei , ROC, 3University of Maryland, College Park, MD, USA , 4Kyoto Institute of Technology, Kyoto, Japan
kawasaki@jeol.com

Lin et al. characterized Au/TiO2 films by surface enhanced Raman scattering, observing red-shift in the extinction spectrum with increasing TiO2 film thickness1. We investigate a similar Au/TiO2 structure by analytical electron microscopy. A TiO2 film was deposited on an optical microscope glass slide at 200°C, by 417 cycles of atomic layer deposition (ALD) using tetrakisdimethylamido titanium and H2O1. An Au layer ~3 nm thick was then deposited on the TiO2 film surface by thermal evaporation. The specimen was reinforced with carbon and Pt layers. Analytical EM was performed using a JEM-2100F UHR STEM. TEM images (Figs. 1a and 1b) reveal that the Au film was composed of round particles with diameters of ~15 nm or less2. The thickness of TiO2 layer was 11 nm though the nominal thickness was 19 nm. On this glass substrate, the sticking probability for the TiO2 ALD was poor and the TiO2 layer was amorphous. Fig. 1c illustrates t/λ, where t is the local specimen thickness and λ is the average mean free path of inelastic scattering of electrons. Using t/λ = log (IT /I0), the map was evaluated from Figs. 1d and 1e. This thin specimen with a mean thickness of t < 0.31λ warrants the accuracy of the EELS analysis. Composite EFTEM map (Fig. 1f) indicates the TiO2 layer and the reinforcement of C. Figs. 2b-2f show EDS analysis of the Au/TiO2 microstructure in HAADF image (Fig. 2a). Figs. 2b and 2e show that the substrate was common soda lime glass containing Ca atoms. Fig. 2f indicates the TiO2 layer. However, the EDS maps in Figs. 2b, 2c, and 2e exhibit artifact contrasts (in blue circles) due to the higher background of X-rays stimulated by strong emissions form Au particle. EELS maps in Figs. 2g-2l do not exhibit any artifact in the gold region, confirming the existence of TiO2 layer. Ca atoms could diffuse into an area in less than a few nanometers from the substrate surface. When the amorphous TiO2 layer was thin, voids on its surface might been occupied by diffused Ca atoms, and the deposited Au atoms migrated easily on the surface and formed more Au nuclei, accordingly. This explains the difference in growth behavior among Au films reported in ref. (1). 1M.C. Lin et al., Appl. Phys. Lett. 101, (2012) 023112. 2M. Kawasaki et al., Appl. Phys. Lett. 102, (2013) 091603.

Fig. 1: Au/TiO2 structure on the glass sheet. Conventional (a) and HR-TEM images (b), EFTEM thickness map (t/λ) (c), Zero-loss (0 5 eV) (d) and conventional EFTEM images (e), and composite EDS map (f).

Fig. 2: HAADF image (a), EDS (b-f) and EELS maps (g-j), O-K map with energy-loss near-edge structure for Ti-O (k), and composite EELS map (Ca:red, Ti:green and C:blue).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1806 Structural Investigation of ZnO:Al Films Deposited on the Si Substrates by Radio Frequency Magnetron Sputtering

Chen Y. Y.1, Yang J. R.1, Cheng S. L.2, Shiojiri M.3
1Department of Materials Science and Engineering, National Taiwan University, Taipei, 10617, Taiwan, ROC, 2Department of Chemical and Materials Science Engineering, National Central University, Taoyuan, 32001, Taiwan, ROC, 3Professor Emeritus of Kyoto Institute of Technology, 1-297 Wakiyama, Kyoto 618-0091, Japan
d98527006@ntu.edu.tw

Transparent conducting oxide (TCO) films are widely applied in optical and electronic devices. Among these TCO materials, ZnO:Al has attracted much attention because it is an abundant, inexpensive, non-toxic and environmentally friendly raw material with high crystallinity and good conductivity that is easy to prepare. In this study, ZnO:Al films 400 nm thick were prepared on (100) Si substrates by magnetron sputtering at room temperature and no bias was applied on the substrate. The present samples were not treated with post-deposition annealing. From energy dispersive X-ray spectroscopy and transmission electron microscopy (TEM), seen in Figures 1 and 2, the resulting film consisted of three layers: an amorphous silicon oxide layer, a crystalline Si layer including a small amount of Zn, and the ZnO:Al main film. It revealed that in the initial stage of the deposition, an amorphous silicon oxide layer about 4 nm thick formed from damage to the Si substrate due to sputtered particle bombardment and the incorporation of Si atoms with oxygen. Then a crystalline Si (Zn) layer about 30 nm thick grew on the silicon oxide layer by co-deposition of Si atoms sputtered away from the substrate with Zn atoms from the target. When the deposited film grew over the critical thickness of 30 nm, Si atoms were no longer ejected from the substrate. Finally, a ZnO:Al film with columnar grains normal to the substrate surface was deposited on the Si (Zn) layer. The sputtered particle bombardment greatly influenced the structure of the object films. The (0001) lattice fringes of the ZnO:Al film were observed in high-resolution TEM images, seen in Figure 3, and the forbidden 0001 reflection spots in electron diffraction patterns were attributed to double diffraction. It should be emphasized that the energetic particles and/or ions bombarding the substrate, in particular at an early stage of deposition, form layers in unexpected phases on the substrate, which may greatly influence the optical and electrical properties of the fabricated ZnO:Al films.

References
1 S. Fernández, O. de Abril, F.B. Naranjo, J.J. Gandía, Sol. Energy Mater. Sol. Cells 95, (2011) 2281.
2 J.K. Jeong, Semicond. Sci. Technol. 26, (2011) 034008.
3 T.W. Kuo, S.X. Lin, Y.Y. Hung, J.H. Horng, M.P. Houng, IEEE Photonics Technol. Lett. 23, (2011) 362.

This work was supported by the National Science Council (NSC), Taiwan, under Contract No. NSC-99-2221-E-002-060-MY3. The authors are graceful to Mr. Hsueh-Ren Chen for the high resolution TEM support.

Fig. 1: (a) TEM image of ZnO:Al film deposited on the (001) Si substrate by RF magnetron sputtering. (b) EDS from the dotted circle area in (a). (c) HAADF STEM image of the ZnO:Al/Si. (d) EDS line-scan profiles along the line indicated in (c). An interface layer with brighter contrast in (a) and with darker contrast in (c) was formed.

Fig. 2: (a) HR-TEM image of the interface layer/Si substrate. (b) HR-TEM image of the ZnO:Al film/interface layer. (c)-(f) The corresponding FFT images to the images in areas A-D, respectively.

Fig. 3: (a) and (c) HR-TEM images of different areas in the ZnO:Al film. (b) and (d) the corresponding FFT images to the images in (a) and (d), respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1809 Direct observation of functional layer structure for reverse osmosismembrane by scanning electron microscopy

Miao X. P.1, Huang W. Q.1
1SINOPEC Beijing Research Institute of Chemical Industry，Beijing 100013, China
miaoxiaopei.bjhy@sinopec.com

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1818 Structural characterization of thin films of BiFeO3(BFO)/Ca0.96Ce0.04MnO3(CCMO)//YAlO3(YAO) by transmission electron microscopy

Saidi w.1, Pailloux F.1, Pacaud J.1, Bibes M.2, Barthélémy A.2
1Institut Pprime, UPR CNRS 3346, University of Poitiers, 11 Ave P et M Curie, 86962 Chasseneuil, France, 2Unité Mixte de physique CNRS/Thalès, 91767 Palaiseau, France
wajdi.saidi@univ-poitiers.fr

The last fifty years, numerous studies have been performed on perovskites. This crystal structure common to many oxides, has a wide variety of properties. Among them BFO is one of the most promising multiferroics for applications since both ferroelectric and magnetic orders coexist at room temperature [1, 2].
We study thin films of multiferroic oxide BFO (thickness of 95 nm) epitaxied on the substrate (001) YAO by pulsed laser deposition with a buffer layer of about 25 nm CCMO. The crystal structure of the BFO bulk is described by rhombohedral space group R3c at room temperature with the lattice parameters in the pseudo-cubic lattice a = b = c = 3.94678 Å.
In a previous study, it has been shown that BFO thin film can adopt two different structures. The first exhibits a giant tetragonal-like (T-like) c/a ratio around 1.27 and the second exhibits a small c/a ratio around 1.07 called pseudo-rhombohedral (R-like] [3].
We present here high resolution images and diffraction patterns for cross-section sample. The high resolution images allow identifying the phases present in the different layers of the sample. The diffraction patterns obtained suggest that the T-like phase corresponds to the monoclinic phase Cm exhibiting c/a ratio of about 1.27 and R-like phase is the rhombohedral R3c slightly distorted with a c/a ratio of 1.07.
A HRTEM image of the BFO thin film deposited on CCMO/(001)YAO is shown in Figure 1. Its Fourier Transforms (FT) is shown in Figure 2, indexed in the pseudo-cubic system. In order to analyze the splitting of the diffraction spots observed along the growth direction, a Digital Dark Field (DDF) image was calculated from the four families of spots in the FT. Results is displayed in Figure 3, showing the spatial distribution of the different grains. The spots 1, 2, 3 and 4 can be identified by CCMO/YAO, T-like BFO, R-like BFO (3) and R-like BFO (4).
For the measurement of disorientation between regions we have used the Geometrical Phase-shift Analysis (GPA). Figure 4 displays phase-shift image of 001 spot with the reference in CCMO. The c axis of T-like BFO is tilted about 0.3° from growth direction, on the other side the c axis of R-like BFO (3) and (4) are tilted about 1° and 3.3° from growth direction, respectively.
By using image processing, we can locate spatially the different orientations grains in the image and thus determine the relationship between the crystallographic phases present. This is particularly advantageous in systems where phase transitions guided by the constraints epitaxy occur.

[1] G. Catalan, J. F. Scott. Adv. Mater. 21, 2463 (2009)
[2] R. Ramesh, N. A. Spaldin. Nat. Mater. 6, 21-29 (2007)
[3] R. J. Zeches, M. D. Rossell, J. X. Zhang. Science 326, 977 (2009).

The authors gratefully acknowledge the region Poitou-Charentes for the financial support and the Thales group who provided us with samples.

Fig. 1: HRTEM image of the BFO thin film (thickness about 94 nm) deposited on CCMO (25 nm)/(001) YAO

Fig. 2: Fourier transform of HRTEM

Fig. 3: RGB reconstruction from Digital Dark Field images of 001 spot

Fig. 4: Phase-shift image of 001 spot
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1855 APT versus STEM EDX 3D mapping of transition metal nitride thin films

Parlinska-Wojtan M.1, Sowa R.1
1Facility for Electron Microscopy & Sample Preparation, Faculty of Mathematics & Natural Sciences, University of Rzeszow, Rzeszow, Poland
bpparlin@cyf-kr.edu.pl

Me-N thin films exhibiting a nanocomposite (NC) structure are well known to achieve extremely high values up to 60 MPa [1-2]. According to literature, the nanocomposite structure consists of crystalline MeN (Me = Ti, Cr, Al etc.) nanograins with sizes from 5-20 nm surrounded by an amorphous SiNx matrix. Two aspects of the nanocomposite structure are important: 1. the unambiguous observation by TEM of the two differently structured phases due to the sample thickness superior to the grain size; 2. the structure on the atomic level of interfaces between the crystalline, facetted grains and the amorphous matrix. From the literature review, it is not clear, to which extent the atomic structure of the interfaces between the crystalline grains and the amorphous matrix influences the hardness of the NC coating. There are two contradictory theories. The first claims, that the matrix surrounding the crystalline Me-N grains should be amorphous, beginning from the first layer [3,4]. Conversely, the second theory explains the high hardness by the growth around the crystalline grains of an epitaxial, crystalline layer of the matrix having a thickness of 1-2 monolayers. The aim of this study was to image in 3D the distribution of chemically different phases of the NC structure. For this purpose Atom Probe Tomography (APT) and HAADF Tomography combined with EDX mapping were applied. The APT technique permits reconstructing in three dimensions the chemical composition of a sample with a nearly atomic resolution. The HAADF imagining allows for tomography with a certain chemical contrast, however simultaneous collection of EDX maps will provide more chemical information for reconstruction. This is possible on Tecnai Osiris TEM operating at 200kV equipped with an ultrafast Super EDX system. The sample, grown on a Si substrate, was a TiSiN coating having a fine-grained structure with three amorphous SiN layers having thicknesses of 5 and 10nm. First a pillar with a length of 1200nm and a diameter of 200nm was cut by FIB, Fig. 1(a). The next step was to shape a 800nm long needle and diameter below 100nm transparent to electrons. HAADF and BF STEM were used to evaluate the needle cut form the TiSiN coating for tomography. The chemical layout of the sample was verified by EDX mapping, Fig.2. STEM tomography was compared with the results obtained by APT, exhibiting much higher resolution. Thus 3D chemical imaging may shed some light into the origin of the high hardness in nanocomposites.

1. W.D. Sproul, Science 273 (1996) 889

2. S. Veprek, J. Vac. Sci. Technol. A 17(5) (1999) 2401

3. S. Veprek et.al. Surf. & Coat. Technol. 201(13) p.6064 (2007)

4. S. Veprek, et.al. Surf. & Coat. Technol. 133-134, p.152 (2000)

5. L. Hultman, et. al. Phys. Rev. B, 75(15) p.155437 (2007)

Project UDA-RPPK.01.03.00-18-052/12-00 is acknowledged.

Fig. 1: (a) HAADF STEM image of a pillar cut from a TiSiN coating with 3 SiN amorphous layers grown on a Si substrate; (b) BF STEM image of the TiSiN finegrained structure of the needle shaed by FIB.

Fig. 2: (a) HAADF STEM image of the bottom part of the pillar with the corresponding EDX maps of: (b) Titanium, (c) Silicon; (d) Nitrogen.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1890 Microstructural characterization of Fe-based amorphous coatings produced by HVOF

Gallego J.1, Berger J. E.2, Coimbrão D. D.2, Schulz R.3, Savoie S.3, Bolfarini C.2, Kiminami C. S.2, Botta W. J.2
1São Paulo State University - UNESP, Department of Mechanical Engineering, Av. Brasil Centro, 56, CEP 15385-000, Ilha Solteira/SP, Brazil, 2Federal University of São Carlos - UFSCar, Department of Materials Engineering, Rod. Washington Luis, km 235, CEP 13565-905, São Carlos/SP, Brazil, 3Hydro-Quebec Research Institute, 1800 Boul. Lionel Boulet, Varennes (QC) J3X 1S1, Canada
gallego@dem.feis.unesp.br

Modification with B addition, of a commercial super duplex stainless steel can result in an amorphous alloys which present excellent corrosion and wear resistance. One optimized alloy composition for easy glass forming ability is Fe53Cr22Ni5.6B19 (%at). Amorphous overspray powders of this composition were obtained from spray forming process; further high-energy ball milling resulted in particle size range adequate for the high velocity oxygen fuel (HVOF) processing. HVOF was carried out on low carbon steel substrate to produce a coating with approximately 100 μm of thickness. The coating layer was separated from the substrate to allow preparation of thin foils, which was carried out by ion milling. TEM observations were carried out in a FEI Tecnai G2 200kV equipment and a Philips CM120, operated at 120kV.

Figure 1 shows STEM dark field micrograph of the coating with clear suggestion that the HVOF processing was able to produce a predominantly amorphous layer. Such information was already obtained from X ray diffraction (XRD) patterns and calorimetry measurements by DSC. Figure 1 also shows round features which were associated with the porosity from the HVOF processing and primary crystals from the solidification of the liquid, with sizes, in most of the cases, in the range of 50 - 100 nm.

Figure 2 shows in greater details, again in a STEM dark field micrograph, the morphology and nucleation aspects associated with the crystals. New crystals are clearly nucleating either by direct contact with the existing crystals or in the regions nearby. Figure 3 (a) shows a TEM picture of one of the largest crystal, and corresponding selected area electron diffraction pattern (SAEDP) which included the crystal and the surrounding matrix. The SAEDP clearly indicates an amorphous halo corresponding to the matrix and crystalline pattern which was indexed as the (Fe,Cr)2B tetragonal phase.

Figure 3 (b) shows EDS spectra corresponding to the points marked A (in the matrix) and B (in the particle), respectively. The difference between the two spectra is basically associated with the Ni content, which is high in the matrix and very low in the particle, consistent with the boride phase identified by electron diffraction. Thus, the (Fe,Cr)2B phase is the first phase that forms from the liquid for the Fe53Cr22Ni5.6B19 alloy.

The authors gratefully acknowledge the financial support of the Brazilian institutions FAPESP, CNPq, CAPES and FINEP.

Fig. 1: STEM dark field micrograph of the HVOF coating with predominance of amorphous phase.

Fig. 2: STEM dark field micrograph showing the morphology and nucleation aspects of the crystals forming during HVOF processing.

Fig. 3: (a) TEM micrograph of a (Fe,Cr)2B crystal and its respective SAEDP. (b) EDS spectrum corresponding to the point marked A in the matrix and EDS spectrum corresponding to the point marked B in the particle.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1933 Direct observation of phase changes of MgZnO thin film in in-situ heating TEM study

YOO S. J.1, LEE J. H.1, KIM C. Y.1, KIM H. S.2, KIM J. G.1
1Division of Electron Microscopic Research, Korea Basic Science Institute (KBSI), 169-148 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Korea, 2Department of nano semiconductor engineering, Korea Maritime & Ocean University, 727 Taejong-ro, Yeongdo-Gu, Busan 606-791 Korea
sjyoo78@kbsi.re.kr

Mg element has been reported as a good candidate for the band gap engineering of ZnO because of the similar ionic radius of the Zn2+ and Mg2+, which does not result in a significant misfit strain in the MgZnO/ZnO heterostructure[1]. For this reason, MgZnO thin films have also become the subject of major scientific researches as more attentions have been focused on ZnO.
The properties of the MgZnO thin films are extremely sensitive to its crystal perfection, which is decisively depended not only on growth processes but on post heat treatments such as thermal annealing[2]. Although the annealing is an important way to improve the crystal quality and electrical properties of the MgZnO thin films[3], the experimental reports of the thin films concerned with the crystal structures during the annealing treatment relatively have been less compared to ZnO.
Our work, therefore, employed the in situ heating TEM study to directly observe the change of the crystal structures of the MgZnO thin films deposited on Si substrates.
The results showed that the deposited MgZnO thin film had the layer of nano sized grains between the disordered columnar grains and Si substrate at RT (Fig.1a). This layer was analyzed that the cubic and hexagonal crystal structures were coexisted together based on the measurement of d-spacing values from the FFT pattern image (Fig.1b).
In the in situ heating TEM study, it was found that the change of crystal structures was not occurred up to at 400℃. However, the disappearance of the hexagonal structure was observed at 500℃ in the layer according to the d-spacing values measured from the FFT pattern image (Fig.2b) of the HRTEM image (Fig.2a).
Additionally, the core loss EELS spectra of Zn L-edge and Mg K-edge obtained at the RT (Fig2.c) and elevated temperature of 500 ℃ (Fig.2d) were showed the decreased intensity of Zn L-edge meant the fewer amounts of Zn atoms compared to the RT one.
This phase change process could be caused by the evaporation of Zn atoms in the MgZnO alloy system under high vacuum condition of the specimen chamber of the TEM. The Zn atoms could be a first evaporation element rather than the Mg atoms because the bond enthalpy of MgO is stronger than the one of ZnO[4].
On the basis of our experimental results, the phase change of MgZnO thin film was directly observed by the continuous thermal annealing method in the in situ heating TEM study. This phenomenon is prominent to enhance the crystallinity and control the microstructure of the MgZnO thin film with adjusting the thermal annealing temperature.
[1] Z. Vashaei et al., J.Appl.Phys. 98 (2005) 054911
[2] J. Li et al., J.Cryst.Growth. 314 (2011) 136
[3] X. Zhang et al., Adv.Mat.Res. Vols. 562-564 (2012) 142
[4] S. Lien et al., J.Phys.D:Appl.Phys. 46 (2013) 075202

This work was supported by KBSI (Korea Basic Science Institute) grant to J.-G. Kim (D34804) and NEW & Renewable Energy R&D program (20113020030020) under the Ministry of Knowledge Economy, Republic of Korea.

Fig. 1: Fig. 1. A HRTEM image taken at RT in the region of nano sized grains (a) with the FFT pattern image (b).

Fig. 2: Fig. 2. A HRTEM image taken at 500 ℃ in the region of nano sized grains (a) with the FFT pattern image (b) and core loss EELS spectra of Zn L-edge and Mg K-edge taken at RT (c) and 500 ℃ (d).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1989 Cristallographic structure and oxydation state of cerium in Pt-doped cerium oxide thin films deposited by CVD

Simon P.1, Zanfoni N.1, Avril L.1, Imhoff L.1, Domenichini B.1, Potin V.1
1Laboratoire Interdisciplinaire Carnot de Bourgogne, Dijon, France
valerie.potin@u-bourgogne.fr

Noble metals supported on rare earth metal oxides are able to be active catalysts for fuel cells applications [1,2]. In the past decade, cerium oxide CeO2 has attracted considerable research interest and several studies have shown its potential as a conversion catalyst when combined with Pt, Pd or Au [3,4].

For CeO2/Pt system, the ability of ceria to store and transport oxygen, associated with a valence change from Ce3+ to Ce4+ and the presence of Pt2+/4+ species makes this material very promising [5,6].

In this study, we report TEM investigations on ceria and Pt-doped ceria thin films deposited by CVD on different substrates (Figure 1). SEM and STEM experiments reveal different morphology and porosity depending on the substrate. The crystallographic structure of CeOx layers has been studied by mean of high resolution TEM and shows the presence of different cerium oxides and carbides. Moreover, EELS experiments were performed to determine the cerium oxidation state throughout the layer, which can be related to the CeM5/CeM4 areas ratio [7].

1. Trovarelli, A. Catalytic Properties of Ceria and CeO2 -Containing Materials. Catal. Rev. 38, 439–520 (1996).

2. Fu, Q., Weber, A. & Flytzani-stephanopoulos, M. Nanostructured Au – CeO2 catalysts for low-temperature water – gas shift. Catal. Letters 77, 87–95 (2001).

3. Matolín, V. et al. Platinum-doped CeO2 thin film catalysts prepared by magnetron sputtering. Langmuir 26, 12824–31 (2010).

4. Sahibzada, M., Steele, B. C. H., Zheng, K., Rudkin, R. A. & Metcalfe, I. S. Development of solid oxide fuel cells based on a Ce(Gd)O2-X electrolyte film for intermediate temperature operation. Catal. Today 38, 459–466 (1997).

5. Bunluesin, T., Gorte, R. J. & Graham, G. W. CO oxidation for the characterization of reducibility in oxygen storage components of three-way automotive catalysts. Appl. Catal. B Environ. 14, 105–115 (1997).

6. Vaclavu, M., Matolínová, I., Myslivecek, J., Fiala, R. & Matolín, V. Anode Material for Hydrogen Polymer Membrane Fuel Cell: Pt–CeO2 RF-Sputtered Thin Films. J. Electrochem. Soc. 156, B938 (2009).

7. Garvie, L. A. J. & Buseck, P. R. Determination of Ce4+/Ce3+ in electron-beam-damaged CeO2 by electron energy-loss spectroscopy. J. Phys. Chem. Solids 60, 1943–1947 (2000).

This research is supported by ANR within IMAGINOXE project (ANR-11-JS10-001) and EU within FP-7-NMP-2012 project chipCAT under Contract No. 310191.

Fig. 1: Morphology of crystallized CeOx later deposited by CVD on silicon substrate. The layer exhibits a grain growth with a lot of intergrain porosity.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-1990 Relationships between elaboration conditions, structural parameters and electrical properties in metal oxides nanometric periodic multilayers

Potin V.1, Cacucci A.1, Martin N.2
1Laboratoire Interdisciplinaire Carnot de Bourgogne, Dijon, France, 2Institut FEMTO-ST, Besançon, France
valerie.potin@u-bourgogne.fr

Ti/TiOx multilayered thin films have been deposited by DC reactive sputtering using the reactive gas pulsing process (RGPP). It is implemented to produce regular alternations of metal-oxide compounds at the nanometric scale. Structure and growth have been investigated by High Resolution Transmission Electron Microscopy (HRTEM), Scanning Transmission Electron Microscopy (STEM) Energy Dispersive X-rays Spectroscopy (EDX) and Energy Electron Loss Spectroscopy (EELS). Regularity of titanium -based alternations, quality of interfaces as well as oxygen diffusion through the multilayered structure have been examined taking into account the reactivity of oxygen towards titanium. Electrical measurements have been also carried out with the van der Pauw method to determine resistivity changes with temperature.

CTEM has been performed to determine the thickness of the periodic layers from 6 to 40 nm (Fig. 1a). HRTEM experiments have been carried out to study the atomic structure of the periodic layers (Fig. 1b). The study of HRTEM images has allowed determining a growth model with the following series: (rutile-)TiO2, fcc-TiO and α-Ti (Fig. 2). This result has been confirmed by SAED experiments. Chemical information was obtained from the core-loss EELS and EDX spectra. Core-loss study was particularly performed for TiOx samples to quantify the elemental composition from the Ti-L2,3 and O-K edges. The systematic presence of oxygen has been pointed out in the rich-metal sub-layer, corresponding to the TiO phase already pointed out by HRTEM.

The knowledge of the structural parameters has allowed determining a first relation between the elaboration conditions (control of the pressure value) and the structural parameters (Fig. 2). Electrical and structural results have also been related to propose a law linking the resistivity values to the structural parameters as total thickness etot, metal λmet and oxide λox layers thickness and metal elemental composition CTi (Fig. 3).

This research is supported by ANR within IMAGINOXE project (ANR-11-JS10-001).

Fig. 1: Fig. 1: CTEM (a) and HRTEM (b) cross-section images of the periodic Ti/TiOx multilayers

Fig. 2: Fig. 2: Scheme of the relations between the elaboration conditions and the Ti/TiOx growth model

Fig. 3: Fig. 3: Evolution of the electrical resistivity r with the different structural parameters
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2059 Microstructural Analysis of Self-Organized Nanolamellae in Polycrystalline TiAlN Coatings

Matko I.1, Todt J.2, Sartory B.3, Pitonak R.4, Keckes J.2
1Institute of Physics, Slovak Academy of Sciences, Bratislava, Slovakia, 2Department of Materials Physics, Montanuniversität Leoben, Austria, 3Materials Center Leoben Forschung GmbH, Leoben, Austria, 4Böhlerit GmbH & Co KG, Kapfenberg, Austria
igor.matko@savba.sk

TixAl1-xN hard protective coatings used for high-speed cutting and machining applications are expected to withstand a broad range of thermal, mechanical and chemical loads. The constantly increasing demands combined with the further development of deposition recipes stimulate the development of novel protective coating designs with even better performance.
TiAlN films prepared using physical vapour deposition exhibit polycrystalline structure consisting of nano-sized grains of cubic TiN and AlN, where the latter may further transform into stable and softer wurtzite after thermal treatment at about 800-900°C. Recently a novel self-organized Ti0.05Al0.95N coating was developed which is based on alternation of cubic TiN and hexagonal AlN nanolamellae and which exhibit very good performance. In this work, the microstructural analysis of the novel Ti0.05Al0.95N coating is reported.
Ti0.05Al0.95N was deposited using chemical vapour deposition in a Bernex medium temperature MT-CVD-300 reactor on Si(100) substrates. The films were then characterized using X-ray diffraction and transmission electron microscopy (TEM). A cross-sectional TEM samples were prepared using a dual beam Carl Zeiss Auriga workstation. Analytical high-resolution transmission microscopy was performed using JEOL 2100F microscope operated at 200 kV, equipped by image-side Cs-corrector, Gatan imaging filter (Tridiem) and delivered the atomic resolution of better than 1.4 Å.
High-resolution TEM image recorded using negative Cs imaging condition and images obtained in scanning mode by high angle angular dark field detector (HAADF) sensitive to Z contrast are presented here. As-deposited films exhibit high content of self-organized nanolamellae structure consisting of periodically alternating cubic (c) TiN and wurtzite (w) AlN sublayers (Fig.1.,2.). A high-resolution TEM pattern indicates relatively thick lamella with lattice fringes of various orientations and lattice spacing up to ~2.7 Å which can be well attributed to wurtzite AlN and give an evidence of various crystallographic orientations within the thick lamellae. In thin lamellae mainly lattice fringes with lattice spacing up to ~2.1 Å in directions perpendicular or parallel to lamella interfaces can be observed and they can be attributed to cubic TiN. In both types of lamellae, a very high concentration of structural defects like dislocations and lattice distortions can be observed. The periodicity of AlN and TiN layers is about 13 nm.
After an annealing of the film to 1373 K, this microstructure is transformed into larger grains of chemically inhomogeneous cubic structure (Fig.3.).

Fig. 1: Representative high-resolution TEM pattern from c-TiN / w-AlN lamellae with a periodicity of about 13 nm for as-deposited film.

Fig. 2: HAADF image of the structure for as-deposited film.

Fig. 3: HAADF image of the structure for the film after annealing to 1373 K.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2082 Microscopic study of TiC/ amorphous C thin films

Oláh N.1, Illés L.1, Sulyok A.1, Menyhárd M.1, Kaptay G.2, Balázsi K.1
1Thin Film Physics Department, Institute for Technical Physics and Materials Science, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary, 2Bay Zoltán Nonprofit Ltd. for Applied Research, Institute for Logistics and Production Engineering (BAY-LOGI), Miskolc, Hungary
olah.nikolett@ttk.mta.hu

The combination process of Ti and C is relatively easy and cheap, and at the same time TiC possesses good mechanical properties. The formation of TiC based surface coating has a passivation effect to titanium implant.


In this work, structural and phase changes depending to the Ti content in the films were studied. 135 nm thin nanocomposite thin films were deposited by DC magnetron sputtering on Si/SiO2 substrates in argon atmosphere at room temperature and 0.25 Pa. The input power of carbon target was constant (150 W), while the input power of titanium target was changed between 5 and 70 W. All films were characterized by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and High Resolution Electron Microscopy (HREM). The elemental composition of the films was measured by Energy Dispersive Spectroscopy (EDS) using TEM equipped with a NORAN cooled Ge detector. X-ray Photoelectron Spectroscopy (XPS) was used for matrix characterization. In all cases, XPS and TEM observations confirmed the two main phase; amorphous carbon + carbide phase until 30 W of Ti power and graphitized carbon + carbide phase above 30 W of Ti power. The average size of crystallites increased with higher Ti magnetron power between 5 and 40 nm. In the case of 60 W and more Ti magnetron power, the thickness of amorphous carbon is minimal. Comparing our previous and current results with a semi-empirical equation on the average atomic fraction of Ti showed a good agreement. All the Ti atoms reaching the target surface are bound to TiC during the deposition process.

This work was supported by OTKA Postdoctoral grant Nr. PD 101453, the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. The research leading to this result has received funding for European Community Seven Framework Programme FP7/2007-2013 under grant agreement Nr. 602398 (HypOrth). Nikolett Oláh thanks to FIKU.

Fig. 1: Figure 1. Cross-sectional HREM image of TiC/ a:C nanocomposite films prepared at 50 W of Ti power.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2093 Metal-insulator transitions in extreme electron density SrTiO3 quantum wells

Zhang J. Y.1, Hwang J.1, Jackson C. A.1, Raghavan S.1, Stemmer S.1
1Materials Department, University of California, Santa Barbara, California, 93106-5050, USA
zhangj23@gmail.com

The advancement of high quality thin film growth by molecular beam epitaxy allows unparalleled control in tailoring the unique properties of complex oxides, and also gives rise to new “emergent” phenomena not present in either of the bulk substituents. For example, the formation of an interfacial high-density 2-dimensional electron gas (2DEG) in the SrTiO3/RTiO3 system (where R is a rare-earth, such as Gd or Sm) provides an ideal test base for studying strong electron correlation effects and exploring new physical states in a model system where correlations effects are not present in the bulk [1].

In this presentation, we present recent results exploring the interplay between electron-electron interactions and electron-lattice interactions in thin SrTiO3 quantum wells grown between GdTiO3 and SmTiO3 (Figure 1 [2]). These quantum wells contain a confined 2DEG, which resides in the d-bands of the SrTiO3, with varying 3D electron densities depending on the thickness of the SrTiO3. Using atomic resolution scanning transmission electron microscopy, we demonstrate an intrinsic structural transition in the thinnest quantum wells grown between GdTiO3 by measuring rare-earth cation displacements, which are directly related to the octahedral rotations in the structure [3]. These displacements occur only in the thinnest films (1 and 2 SrO layers) grown between GdTiO3, and occur precisely at a metal-to-insulator transition. Quantum wells grown between SmTiO3 showed much smaller displacements, and transport measurements showed metallic behavior down to the extreme of a single SrO layer. These structural measurements agree closely with calculated structures from density functional theory, which highlight the importance of octahedral rotations in promoting the insulating state, and offer convincing evidence for Mott-Hubbard physics in a material that is a band insulator in the bulk.

Just as importantly, the previous results provide valuable insight into the structural accommodation and changes that occur at the heterointerface of the component materials. Using these principles, we can thereby control the octahedral rotations - which drive many of the unique electrical and magnetic properties - of GdTiO3 by growing varying thickness GdTiO3 between SrTiO3 [3]. Using position averaged convergent beam electron diffraction, we show that indeed the octahedral rotations change as a function of film thickness, and can therefore be controlled by proper choice of heterojunction material and film thickness (Figure 2).

We thank Leon Balents and Ru Chen for DFT calculations and many helpful discussions. This work makes use of facilities at the California NanoSystems Institute and the UCSB Materials Resarch Laboratory.

Fig. 1: Angle difference from 180° of 3 succesive cations in each AO plane and corresponding normalized HAADF intensities for regions containing 2 SrO and 5 SrO layers. SrO layers are highlighted in gold. Dashed lines serve as guides to mark structural distortions (or lack of) in the SrTiO3 wells. Dashed boxes indicate atomic planes of similar intensity.

Fig. 2: Experimental and simulated PACBED patterns of GdTiO3 and SrTiO3. White labels indicate TEM fol thickness and black ones the GdTiO3 layer thickness. Simulated patterns use the expected octahedral tilts from measured cation displacements. The top half of simulated patterns include Gaussian convolution to account for detector point spread function.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2205 Structural features contributing to electrical resistivity in Cu-Mn alloy films

Misják F.1, Nagy K. H.1, Lobotka P.2, Radnóczi G.1
1Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary, 2Institute of Electrical Engineering, Slovak Academy of Sciences, Bratislava, Slovak Republic
misjak.fanni@ttk.mta.hu

The fast development of the electronic industry raises the need of development faster and smaller devices. Consequently, the elements of the integrated devices become smaller and smaller. This, however, puts stringent demands on the contacts and interconnects. Around the presently used copper contacts a barrier layer must be manufactured which prevents the interdiffusion of copper into the dielectric materials. Utilization of self-organized barrier layers can provide a new solution to this problem. Cu-Mn alloy films as barrier layers appear a promising material for future technologies.

The aim of this research is to build a comprehensive view of the phases, structures and morphologies occurring in the Cu-Mn thin film system as well as their scattering mechanisms giving different contribution to the electrical resistivity of the films.

Eleven films, 1 μm thick, were grown at room temperature by DC magnetron sputtering covering the whole concentration interval. Then the electrical resistivity was measured as the function of composition by van der Pauw method. The results were correlated with the films structure studied by TEM.

The electrical resistivity of the films varied between 1.7 and 205 μΩcm and except of two concentration intervals it showed linear dependence on Mn concentration. Utilizing these results the whole alloy region was divided into five parts. Three single-phase regions were identified. Below 20 at% Mn content an fcc solid solution of Mn in Cu exists, in the 40-70 at% Mn interval the structure is an amorphous alloy, and finally above 80 at% Mn an α-Mn based solid solution forms. Two-phase regions exist in the concentration regions between the single-phase ones: at Mn concentrations between 20 and 40 at% the fcc solid solution and amorphous phases are present, while in the interval 70-80 at% Mn the amorphous phase and the α-Mn based solid solution keep balance with each other (Fig. 1.).

The resistivity measurements and the structural information obtained by TEM were used for modelling the conduction mechanisms in the films. In the model, the experimental resistivity was described as the sum of the resistivity due to different scattering mechanisms (Mathiessen rule). As a result, we could conclude, that in the fcc solid solution region and in the amorphous structures the resistivity is influenced by thermal and solute (Nordheim) scattering. In the two-phase regions and in the Mn-based solid solution region in addition to the above scattering mechanisms the grain boundary scattering contributes significantly. In the concentration regions, where the contribution of solute scattering is important Moiij correlation (high resistivity and small negative TCR) was also observed. (Fig. 2.)

The authors acknowledge the financial support of OTKA-K81808, F. Misják the János Bolyai Research Scholarship of the Hungarian Academy of Sciences, P. Lobotka the APVV-0593-11.

Fig. 1: Cross-sectional TEM image of Cu-Mn sample containing 80 at% Mn. The reversal of contrast between under- (a) and over focus (b) images suggests the presence of a second, less dense phase in the grain boundaries.

Fig. 2: Experimental, thermal and solute scattering (a) and the ratio of solute and thermal (ρ0) to measured film resistivity ρg (b) as the function of Mn content. Grain boundary scattering is measurably contributing to the film resistivity where ρ0/ρg is below 1.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2213 Relation between surface and internal structure of Cu-Mn thin films

Nagy K. H.1, Misják F.1, Czigány Z.1, Radnóczi G.1
1Research Centre for Natural Sciences, Budapest, Hungary
nagyk@mfa.kfki.hu

In semiconductor industry with the scaling down of ultra large integrated circuits the idea of self-forming barriers emerged. Cu-Mn alloys have been found a perspective binary alloy system for this purpose. For barrier layers beside excellent diffusion barrier property various other requirements exist such as low resistivity and good adhesion strength. Since surface morphology has considerable effect on both properties the investigation of the interrelation between surface and internal structure is of outmost importance.
The aim of this research is to systematically study the effect of microstructure on the surface morphology as a function of composition in a thin film system. For this purpose TEM, SEM and AFM techniques were used for determining the morphology, phases and surface roughness of the films.
Cu-Mn films of 50 nm thickness were co-deposited by DC magnetron sputtering. For TEM investigations TEM grids coated with carbon film were used while for surface characterisations films were grown on Si/SiO2 substrate. We used combinatorial method to obtain a comprehensive view of properties in the whole composition range of the alloy. Compositional gradient within one sample was achieved by shadowing atomic fluxes using a special experimental setup (Fig.1). The composition gradient was verified by EDS line scan. The size of coherently scattering regions interpreted as grain size was determined from line broadening of electron diffraction peaks.
The combinatorial method makes possible to map the morphological and phase properties in continuously changing composition gradient films and to conclude how these characteristics are interrelated with the structure and growth mechanisms of the film.
We could establish that with varying composition the phase state can change from one phase to two phase material and from crystalline to amorphous. This brought about smoother films for two phase films, when crystal growth during film development was limited by the formation of a second phase. The quantitative relation between lateral and vertical surface features and internal morphologic parameters is evaluated. The variation of internal structure is in accordance with the variation of surface roughness and morphology (Fig. 2). Grain size has strong correlation with the roughness of the films; smaller grains result in smoother surfaces (Fig. 3). Consequently, the lowest roughness values were measured in the amorphous region. The contribution of surface morphology and roughness in the electrical properties will also be discussed in accordance with the phase and internal structure of the films.

The authors acknowledge the financial support of OTKA-K81808. F. Misják and Zs. Czigány acknowledge the support of János Bolyai Research Scholarship of the Hungarian Academy of Sciences.

Fig. 1: Combinatorial experimental setup.

Fig. 2: Microstructure and surface morphology of Cu-Mn alloy films as a function of Mn content as observed by TEM (upper row) and AFM (lower row).

Fig. 3: The relation between grain size and surface roughness as a function of Mn content.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2230 In situ investigation of Ni induced crystallization in amorphous Si thin films

Radnóczi G. Z.1, Dodony E.1, Battistig G.1, Pécz B.1, Vouroutzis N.2, Stoemenos J.2, Frangis N.2
1Research Centre for Natural Sciences, Institute for Technical Physics and Materials Science, Konkoly-Thege Miklós u. 29-33. Budapest, Hungary H-1121, 2Solid State Physics Section, Department of Physics, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
gy.radn@mfa.kfki.hu

The well-known Metal Induced Lateral Crystallization (MILC) process [1] is investigated in-situ in transmission electron microscope with a heating sample holder. Model structures were grown onto thermal oxide coated Si substrates consisting of a CVD grown amorphous silicon (a-Si) layer, a silicon oxide (SiOx) mask layer and a thin nickel layer as shown in Fig 1. Windows (10x10 µm2 and 100x10 µm2) were opened in the SiOx mask layer where the Ni came in contact with the amorphous Si layer. Ni and amorphous Si thicknesses were selected such that all the Ni could be accommodated in the underlying Si during nickel-silicide (NiSi2) formation.
Cross sectional TEM specimens of the relevant sample areas near the window edge were prepared with the Focussed Ion Beam (FIB) technique and heat treated in the microscope. The initial state of the specimen before the heating experiment is shown in Fig 2.
Gradual nickel-silicide formation starting from the interface was observed in-situ at temperatures as low as 250°C. A sequence of images taken at different stages of the NiSi2 formation is shown in Fig 3. Further increasing of the temperature caused the whole Ni amount in the window area to diffuse into the a-Si layer and the lateral crystallization to start originating from newly formed NiSi2 seeds. It has been shown earlier [2] that the Ni-MILC phenomenon is based on the formation of NiSi2 precipitates and their subsequent migration through a-Si film, leaving a trail of crystalline Si grown on the lattice-matched silicide, NiSi2, (misfit only 0.4% to Si). At higher temperatures up to 650°C the lateral crystallization process was observed and investigated with analytical tools to prove the migration of NiSi2 phase.

References
1 S. Y. Yoon, S. J. Park, K. H. Kim, J. Jang and C. O. Kim, J. Appl. Phys. 87, 609 (2000).
2 C. Hayzelden and J. L. Batstone, J. Appl. Phys. 73, 8279 (1993)

This research was financially supported in the frame of a Hungarian-Greek bilateral scientific collaboration (project codes TET-10-1-2011-0570 for Hungary and HUN92 for Greece).

Fig. 1: Construction of the model structure and in-situ MILC experiment.

Fig. 2: Initial state of the FIB prepared TEM specimen before annealing.

Fig. 3: Stages of NiSi2 formation at T=250°C. Nucleation of the silicide phase starts at several points of the Ni/Si interface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2290 Heterogeneous nucleation and growth of CrSi2 in sputtered thin films of silicon/silicon nitride

Nguyen P. D.1, Gunnæs A. E.1, Sunding M. F.1, Olsen A.1
1Department of Physics, University of Oslo, P.O. Box 1048 Blindern, NO-0316 Oslo, Norway
danpn@fys.uio.no

The structure and properties of Si nanocrystals (NCs) have been the subject of intense research for a wide range of applications [1]. One of several methods used for forming the NCs is using magnetron sputtering technique to grow multilayer thin films of Si/Si3N4 [2]. In the present work, the technique was used to grow thin films of alternating Si and Si3N4 layers on single crystal Si (100) substrates. An important factor affecting the quality and properties of the films is the presence of undesirable contaminations. The origin of such contamination can result from the deposition system itself, e.g. the target clamp rings. It was found that during one of our deposition experiment, sputtering also occurred from the stainless steel target clamp rings. Motivation for our work are to identify the contaminations, which were present in the sputtered film, and find a solution for eliminating the contaminations. The Fe and Cr contaminations impurity source in the sputtered film was determined by using advanced techniques in transmission electron microscopy and a desired thin film structure with impurity-free composition was produced successfully after modifying the deposition system. Interestingly, the contamination in the film resulted in nucleation and growth of CrSi2 at the film/Si substrate interface. The structures and mechanisms of heterogeneous nucleation and growth of CrSi2 during the sputtering and thermal annealing processes have been investigated. The crystal structure and orientation relation of CrSi2 precipitations were studied thoroughly by using high-resolution imaging and electron diffraction in transmission electron microscopy. Two distinct different orientation relations have been found: CrSi2 [100](001)||Si [112]( ) and CrSi2 (001)||Si (110), where the latter has not been reported in the literature earlier [3]. These orientation relations can provide important information for the study of CrSi2 system, which is promising for thermoelectric applications and optoelectronic devices.


[1] L. Pavesi, R. Turan, Silicon Nanocrystals, Wiley-VCH Verlag GmbH & Co. KGaA, 2010, p. 1.


[2] G. Conibeer, Silicon Nanocrystals, Wiley-VCH Verlag GmbH & Co. KGaA, 2010, p. 555.

Fig. 1: Fig. 1. (a) Cross-sectional TEM image of as-deposited Si/Si3N4 multilayer film on Si (100) substrates, (b) schematic structure of the contamination free film, and (c) stainless steelclamp ring used in the magnetron during the deposition.

Fig. 2: Fig. 2. (a) Cross-sectional TEMimage and (b)–(d) elementalmaps of Fe, Cr and N, respectively, in the same area. The NCs at the film/substrate interface are Cr-rich, while Fe-rich NCs are distributed in the nitride matrix.

Fig. 3: Fig. 3. (a) HRTEM image of three CrSi2 NCs orientate differently to the Si (100) substrate in areas 2, 3 and 4. The overlap between CrSi2 and Si gives rise to Moiré effects for the part of the crystals below the Si/film interface (area 5). Image was recorded in Si . (b) Diffractograms from FFTs acquired from the corresponding areas shown in (a).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2303 In situ crystallization of Cu-Mn amorphous alloy films

Radnóczi G.1, Nagy K.1, Tóth-Kiss R. J.1, Misják F.1
1Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1525 Budapest, P.O. Box 49, Hungary
radnoczi.gyorgy@ttk.mta.hu

Cu-Mn alloy films are investigated due their possible application in contact and interconnect layers of integrated systems. On the other hand, they are showing peculiarities of phase formation during growth. The most apparent one is the formation of amorphous films in the 40-70 at% Mn region. This presents interest from the point of the formation of amorphous metals by intermixing through condensation from vapour phase, the role of the entropy term in the free enthalpy of the metastable alloy and the kinetic factors in its transformations and stability. To achieve a direct view on the processes in amorphous Cu-Mn alloy films we used in-situ electron microscopy by heating the samples in the TEM. By this technique we investigated the thermal stability of the amorphous phase and its crystallization process. We investigated also the possibility of phase transformations at different temperatures.

50 nm thick films were co-deposited by DC magnetron sputtering at room temperature onto thin carbon films as substrate, supported by Mo microgrids. The composition was varied by regulating the power of the magnetrons. Two samples were designed for measurements having 50 and 70 at% Mn content. The in-situ annealing was carried out in the heating stage of a CM20 electron microscope operated at 200 kV. The temperature was increased in 50 oC intervals, holding the temperature constant for 3 minutes in each step. Electron diffraction patterns were evaluated by the ProcessDiffraction program.

The initial amorphous structure of the film of 50 at% Mn is shown in Fig. 1. The temperature of crystallization depended on composition. For the sample of 70 at% Mn it started at 200 oC and for sample of 50 at% Mn it started at 300 oC. The crystallization process is rapidly consuming all the available amorphous phase and forming a nano-grain (about 10 nm) polycrystalline film in both samples. The forming alloy phases (αMn and γMn) are corresponding to the phases expected from the equilibrium phase diagram (Fig. 2 and Fig. 3). In addition the formation of some fcc MnO occurs.

Increasing the temperature until 500oC causes little change in the structure. Above 500oC grain growth starts and reaches about 50-100 nm at 600oC. Some changes in the phase composition also occur. The Mn content of the Cu based fcc γMn phase decreases with increasing temperature as measured from the change of the lattice parameter (Vegards low). The relative amount of the αMn, γMn and MnO phases also changes with increasing temperature. This, however, is strongly influenced by the consumption of Mn in the MnO formation process.

The authors acknowledge the financial support of the Hungarian Academy of Sciences under the grant OTKA-K81808 and by the János Bolyai Research Scholarship (F. Misják).

Fig. 1: TEM image and electron diffraction intensity profile of amorphous Cu-Mn alloy film of 50 at% Mn content. The cursors in the diffraction profile designate the possible crystalline phases.

Fig. 2: TEM image and electron diffraction intensity profile of Cu-Mn alloy film of 50 at% Mn content after annealing at 300oC for 3 min. The cursors in the diffraction profile designate the observed crystalline phases.

Fig. 3: TEM image and electron diffraction intensity profile of Cu-Mn alloy film of 70 at% Mn content after annealing at 300oC for 3 min. The cursors in the diffraction profile designate the observed crystalline phases.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2312 Preparation of superhydrophilic grid-like mesoporous titania films via structural transformation

Feng Z. D.1, Zhou H.1, Wang C.1, Li S. W.1, Xu B. B.1, Luo X. T.1
1College of Materials, Xiamen University, Xiamen, China
zdfeng@xmu.edu.cn

Recently, periodic mesoporous titania thin films (MTFs) with 3D open-pore structure are actively studied in the self-cleaning fields due to their high surface area and open pores at the surface.

In this study thin titania film with accessible and grid-like porosity was formed via structural transformation from a largepore (~10 nm) 3D hexagonal (P63/mmc) mesoporous titania thin film. The intermediate 3D hexagonal mesoporous titania thin film (MTF) was synthesized using a tetrabutyl titanate (TBT)–P123–BuOH–HCl system by the combination of dip-coating and evaporation-induced self-assembly (EISA). The mesostructures and crystalline phase of the MTFs were characterized, respectively, by scanning electron microscopy (SEM), transmission electron microscopy (TEM), the small-angle X-ray diffraction (SAXD) as well as the wide-angle X-ray powder diffraction (WAXD). The MTF calcined at 350 °C exhibited an ordered honeycomb arrangement over the entire top surface and an ABAB stacking sequence in the cross-section. After being calcined at 450 °C, the 3D hexagonal mesostructure was transformed to a grid-like mesostructure with quasi-perpendicular porosity through sintering–diffusion and pore merging along the c-axis. The accessibility of this grid-like structure estimated by the adsorption of TIRON (disodium 1,2-dihydroxybenzene, 3,5-disulfonate) was higher than that of intermediate structure. The results of pencil hardness test and tape test indicated that these thin films were mechanically robust and exhibit excellent adhesion onto the substrates, which were basic requirements in actual applications.

An interesting finding was that, after structural transformation, the surface wettability changed from hydrophilic to superhydrophilic even without UV irradiation. This phenomenon could be properly explained by the increased accessibility and surface roughness of grid-like structure.

This work was supported by the National High Technology Research and Development Program of China (no. 2009AA03Z327).

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2354 TEM study of nanostructured Cu-Cr alloys

Harzer T. P.1, Raghavan R.1, Djaziri S.1, Dehm G.1
1Max-Planck-Institut für Eisenforschung GmbH, Düsseldorf, Germany
harzer@mpie.de

Non-equilibrium composites have gained a lot of attention in recent years since there is an increased demand for new materials in engineering applications with superior properties like high strength, electromigration resistance as well as an improved oxidation and corrosion resistance. A lot of research has been devoted to metastable binary alloys composed of Cu and transition metals like Mo, W and Cr, since the equilibrium phase diagrams of Cu and these elements show little or no solid solubility. In order to produce nanostructured alloys, non-equilibrium growth conditions are needed. Therefore, vapor deposition techniques, such as molecular beam epitaxy, are the methods of choice.

This study focuses on metastable Cu-Cr alloys, since the two elements show very limited solid solubility and the system possesses a positive heat of formation. The composite films with nominal film thicknesses of 300 nm were grown via co-evaporation of the constituent elements using a molecular beam epitaxy system operated under ultra-high vacuum conditions.

Films over a wide compositional range were synthesized, stretching from almost pure Cu to almost pure Cr films. XRD measurements combined with selected area electron diffraction measurements in a transmission electron microscope showed three phase regimes. For alloys with a Cr concentration up to ~15 at.%, a single phase fcc composite film is formed (Fig.1a). For Cr concentrations higher than 15 at.%, the equilibrium two phase structure of fcc Cu and bcc Cr phase is observed (Fig.1b) whereas the composite films with Cr concentrations higher than ~33 at.% Cr exhibit a transition to a single phase bcc composite (Fig.1c and d). In addition, EDS-mapping revealed a fairly homogenious distribution of elements forming a solid solution within all the composite films (Fig.2) and conventional TEM showed a tremendous reduction in grain size from ~100 nm for low Cr concentration films to ~30 nm for films with high Cr concentration.

Amongst other mechanical tests, nanoindentation experiments were performed on the composite films in order to determine the relationship of microstructural evolution and mechanical properties. For an increasing Cr amount within the alloys, the observed film hardness values ranged from ~5 GPa to ~12 GPa. This enhancement in hardness will be discussed in terms of different strengthening mechanisms like solid solution hardening and Hall-Petch strengthening.

Special thanks to Gerhard Bialkowski for his assistance in thin film production and Heidi Bögershausen for performing nanoindentation experiments.

Fig. 1: Selected area electron diffraction patterns (inverted contrast) of 4 Cu-Cr thin film composites with (a) 5 at.% Cr, (b) 15 at.% Cr, (c) 33 at.% Cr and (d) 64at.% Cr.

Fig. 2: BF TEM images and corresponding EDS-maps of 3 Cu-Cr thin film composites (Cu Kα signal in yellow and Cr Kα signal in green) with (a) 5 at.% Cr, (b) 15 at.% Cr, (c) and (c) 64at.% Cr.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2381 Ag induced low-dimentional structures on the Si(110) surface

Mantsevich V. N.1, Oreshkin A. I.1, Savinov S. V.1, Oreshkin S. I.2
1Moscow State University, Moscow, Russia , 2Sternberg Astronomical Institute, Moscow, Russia
vmantsev@spmlab.phys.msu.ru

Metal adsorption on Si(110) surface received significantly less attention in comparison with other low-index Si surfaces [Si(111) and Si(100)}. This is due to the complicated surface reconstruction and complexity of big surface domains preparation. The increasing interest to this surface analysis is caused not only from fundamental point of view [1] but also by possible applications of Si(110) surface for electronic circuits. Anisotropic structure of Si(110) surface can be used for nanowires growth. P-doped (110) oriented Si samples with the help of Ni-free instrument were mounted on tantalum sample holder, degased at 600C during 24 hours and processed with argon-ion-sputtering. In the final stage of surface preparation the samples were flashed at 1200C. The Ag/Si(110) evaporation was perfomed using a Knudsen-cell type of evaporator. As it was shown previously [2], the initial sites of Ag adsorption on Si(110)-16x2 surface are situated along terrace's borders. It was shown that it should be about 13-14 Ag atoms in the unit cell of 16x2-Si(110)-Ag structure at 0.21 ML Ag surface coverage. With annealing temperature increasing up to 630C we observed new atomic phase (4,6)x(-3,1) on Si(110) surface. Experimental results are presented in Fig.1 (a,b). Uniformly distributed terraces separated by dark rows are visible on the STM images. The surface profile height measured across the STM image changes in the range of 0.1 nm what is smaller than the step's height (0.19 nm) of 16x2-Si(110) surface. So the observed pattern is caused by the surface electronic effect. For correct interpretation of obtained results, STM image was superimposed on unreconstructed Si(110) surface (Fig.2). It's clear that the unit cell of experimentally observed structure is (4,6)x(-3,1). In the surface area restricted by unit cell sizes there are 43 atoms of substrate. Therefore should be 43x0.21=9 Ag atoms in unit cell of structure induced by silver adsorption at 0.21 monolayer coverage. It's resonable to suggest that Ag atoms saturated the Si(110) dangling bonds to minimize an adsorption energy (Fig.3). Based on this fact it's possible to build a model of (4,6)x(-3,1) surface reconstruction (Fig.4). Dotted line shows surface cell corresponding to structure (4,6)x(-3,1) of clean Si(110) surface. A solid line indicates unit cell of Si(110)-Ag-(4,6)x(-3,1) after Ag adsorption. As can be seen from Fig.4 there are 10 atoms of Ag in unit cell of Si(110)-Ag-(4,6)x(-3,1) and  it is in good accordance with experimental results. The appearence of Ag-Si(110)-4x1 surface structure with increasing of Ag coverage up to 0.42 ML and 490C-550C anneleaning temperatures was also demonstrated.

1 B.Z. Olshanetsky et.al., Surf. Sci., 67, 581 (1977)2 N.S. Maslova et. al., JETP Letters, 84, 320 (2006).

This work has been supported by RFBR grants and by the Ministry of education grant for Young Scientists

Fig. 1: STM image of Si(110)-Ag-(4,6)x(-},1) reconstruction, (a) U=-0.6 V, I=51 pA; (b) U=0.6 V, I=51 pA

Fig. 2: STM image of Si(110)-Ag-(4,6)x(-3,1) reconstruction superimposed on unreconstructed Si(100) surface.

Fig. 3: Structural model for surface reconstruction Si(110)-Ag-(4,6)x(-3,1)

Fig. 4: An adsorption site of individual Ag atom on Si(110) surface
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2390 Microscopy of nanocrystalline diamond films

Tóth L.1, Pécz B.1, Rossi S.2, Alomari M.2, Kohn E.2, Anaya J.3, Kuball M.3
1Research Centre for Natural Sciences, HAS, Budapest, Hungary, 2Ulm University, Ulm, Germany, 3Centre for Device Thermography and Reliability, University of Bristol, Bristol, United Kingdom
toth.lajos@ttk.mta.hu

With the use of novel high power and high frequency electronic devices proper extraction of dissipated heat became a major limiting factor hindering further miniaturization. It becomes necessary to deal with the thermal management of such devices and design suitable heat sinks from materials of superb heat conduction. In this respect, diamond is regarded as the ideal heat-spreading material owing to its exceptional thermal conductivity k ~ 2200 Wm-1K-1. However, CVD-deposited diamond films used in most applications have a polycrystalline microstructure which reflects in an anisotropic, thickness-dependent thermal conductivity, whose value increases steadily departing from the small-grained/amorphous nucleation region towards the film’s surface. In order to optimize the thermal properties of the near-to-interface nucleation region it is crucial to understand the correlation between the growth parameters and film microstructure in the earliest stages of film deposition.

Transmission electron microscopy (TEM) offers the unique possibility to study this nucleation layer at the sub-nanometer scale, thus allowing to precisely ascertaining how its composition, structure and thickness are influenced by the choice of the growth parameters.

In this work, nanocrystalline diamond (NCD) thin films were deposited at 760 °C in a hot filament CVD (HFCVD) reactor onto single crystalline silicon substrates nucleated by means of a bias enhanced nucleation (BEN) technique. The growth parameters have been tuned to minimize the nucleation layer thickness and to promote the development of a strong columnar texture in the film. For thinning preparation to the TEM study we applied both FIB technique (with 30 kV Ga+ ions) and standard ion milling (with 10 kV Ar+ ions, followed by low energy milling). The samples were investigated in a JEOL 3010 high resolution TEM (at 300 kV).

The cross sectional TEM images of the interface region shows a transition zone between the diamond film and the Si substrate (Fig. 1). Its thickness is dependent on the deposition parameters but it looks as to contain nanocrystalline grains and amorphous phases as well. With high resolution electron microscopy both diamond and cubic SiC grains of a few nanometer sizes could be identified in this transition region (Fig. 2). In some samples also graphite-related phases (e.g. nano-onions with characteristic inter-shell distance of 0.35 nm) could be observed (Fig. 3). The diamond film itself was found polycrystalline with columnar microstructure. The orientation distribution was more or less random, due to the relatively low deposition temperature.

This work was supported by the Office of Naval Research Global (ONRG) and the Defense Advanced Research Project Agency (DARPA) (Award Number N62909-13-1-N210). Any opinions, findings, conclusions and recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the ONRG or DARPA.

The support of the Hungarian OTKA grant (K108869) is acknowledged.

Fig. 1: Fig. 1 Cross sectional TEM image of the interface between the substrate and the diamond film. The electron diffraction on the inset shows more or less randomly oriented poly-diamond on the single crystal silicon substrate.

Fig. 2: Fig. 2 The transition zone contains a few nm size grains of both SiC and diamond phases.

Fig. 3: Fig. 3 Spherical non-crystalline nanoparticle (nano-onion) found in the transition zone.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2393 Phase Control of Transition-Metal Oxide Films through Interfacial Octahedral Connections

Aso R.1,2, Kan D.1, Shimakawa Y.1,3, Kurata H.1,3
1Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan, 2The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan, 3Japan Science and Technology Agency, CREST, Uji, Kyoto 611-0011, Japan
aso@eels.kuicr.kyoto-u.ac.jp

The octahedral distortions in perovskite transition-metal oxide thin films strongly correlate with the functional properties. The control of such octahedral distortions is crucial for designed fabrication of oxide heterointerfaces with desired properties. However, identification of the controlling parameter for the octahedral distortions associated with the oxygen displacements has remained elusive because of the experimental difficulties in observing oxygen atoms.
In this work, we investigate SrRuO3 (SRO) thin films grown on the GdScO3 (GSO) substrates using high-angle annular dark-field (HAADF) and annular bright-field (ABF) imaging in aberration-corrected scanning transmission electron microscopy (STEM) (JEM-9980TKP1). We found that while the SRO films below 16 nm thickness have the monoclinic structure with the bulk-equivalent tilted octahedra, the SRO films above 16 nm have the tetragonal one with negligibly tilted octahedra [1,2]. We further reveal that the in-plane displacement of the oxygen atoms shared between the RuO6 and ScO6 octahedra at the interface for the monoclinic SRO is larger than that for the tetragonal SRO. The correlation between the interfacial oxygen displacement and the SRO structural phase raises the possibility for controlling film phases by interface engineering of oxygen displacements.
To demonstrate this, we inserted the one unit-cell-thick BaTiO3 (BTO) layer between the 10 nm-thick SRO film and GSO substrate. The bulk BTO with relatively large A-site cation (Ba) has non-tilted octahedra, allowing for minimizing the in-plane oxygen displacements at the interface and thus for blocking the octahedral tilt propagation from the GSO substrate to the SRO film. Figure 1 shows HAADF-STEM image and its intensity profiles in the SRO/BTO/GSO heterostructure, confirming the coherent growth with the chemically sharp interface across the entire heterostructure. The ABF-STEM analyses shown in Fig. 2 reveal that the octahedral tilts in the GSO substrate are drastically suppressed in the BTO layer and that the octahedral tilts in the SRO film are negligibly small. This indicates that while the 10 nm-thick SRO films directly grown on the GSO substrate have the monoclinic structure, the SRO film on the BTO/GSO has the tetragonal one, which is stabilized by the suppression of interfacial oxygen displacements. The results demonstrated that the film structure can be manipulated by adjusting the oxygen displacements through the interfacial octahedral connections.
[1] R. Aso, D. Kan, Y. Shimakawa, H. Kurata, Sci. Rep. 2013, 3, 2214.
[2] D. Kan, R. Aso, H. Kurata, Y. Shimakawa, Adv. Funct. Mater. 2013, 23, 1129.

This work was partially supported by a Grant-in-Aid for Scientific Research (Grant No. 24760009), and a grant for the Joint Project of Chemical Synthesis Core Research Institutions from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The work was also supported by the Japan Science and Technology Agency, CREST.

Fig. 1: (a) HAADF-STEM image and (b) HAADF intensity profiles of the A- and B-site cations in the SrRuO3/BaTiO3/GdScO3 heterostructure. The orange dashed lines also indicate the heterointerfaces.

Fig. 2: (a) ABF-STEM image, (b) octahedral tilt angle θ, and (c) in-plane displacements Δx of oxygen atoms in the SrRuO3/BaTiO3/GdScO3 heterostructure. The green and purple dotted lines represent the bulk counterparts of SrRuO3 and GdScO3, respectively. The orange dashed lines also indicate the heterointerfaces.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2424 Physicochemical issues of hardening mechanisms in Me-N – based nancomposite coatings

Sowa R.1, Arabasz S.2, Parlinska-Wojtan M.1
1Facility for Electron Microscopy & Sample Preparation, University of Rzeszow, Rzeszow, Poland, 2LabSoft SA, Warszawa, Poland
sowa.roman89@gmail.com

The investigated material is a ZrN-SiN thin film with different amount of the SiN phase. This type of structure ensures high hardness of up to 60 GPa. The addition of elements such as X = Al, Cr or O causing an improvement of the oxidation resistance. The aim of this study is to determine the effect of the structure of interfaces between the crystalline ZrXN grains and the amorphous SiN matrix at the atomic level as well as of the chemical composition on the hardness of the deposited coatings. The nanocomposite is a 3D structure, thus even in a TEM lamellas as thin as 50 nm several ZrXN grains will overlap. Thus unambiguous imaging and interpretation of the interface structure between the crystalline grains and the amorphous matrix is impossible. Therefore we proposed to grow a model multilayer system consisting of single-crystalline layers of ZrXN separated by amorphous layer of SiN with thickness corresponding to 1,…,9 monolayers, Fig 1. These multilayers allowed to visualize the atomic structure of the interfaces between the ZrXN and the SiN phases. It was also possible to determine from which thickness the SiN layer really grows amorphous. This 2-D system was chosen to test the premises for different models of superhardening effects observed in nanocomposite systems. The high hardness of MeN-XN layers is related to their nanocomposite structure. A review of the literature is not clear about the effect of the interfaces between the crystalline grains and the amorphous matrix on the hardness. There are two contradictory theories explaining the high hardness. The first one states that the XN matrix around the crystalline MeN grains should be amorphous starting from the first monolayer [1]. The second theory explains the high hardness by forming epitaxial crystallized XN matrix having a thickness of 1-2 monolayers around crystal grains of MeN phase [2]. The obtained structural results for the multilayer model system will be extrapolated to the 3D structure of nanocomposites.

Multilayers coatings were deposited by magnetron sputtering on single crystalline substrates. The identification of the average grain size for different contents of XN phase in nanocomposites was made by XRD. The microstructural investigation was performed using transmission electron microscopy and scanning transmission electron microscopy on a Tecnai Osiris. The chemical analysis was accomplished by energy dispersive X-ray, and the TEM samples, were prepared by mechanical grinding and as a final step the samples were ion milled.

References

[1] S. Veprek and M G J Veprek-Heijman. Surf. & Coat. Technol. 201(13) p. 6064 (2007).

[2] L. Hultman, J Bareno, A Flink, H Soederberg, K Larsson, V Petrova, M Oden, J E Greene and I Petrov, Phys. Rev. B, Vol. 75, No. 15, p. 155437 (2007).

Fig. 1: Model multilayer system consisting of single or polycrystalline layers of ZrXN separated by amorphous layer of SiN.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2496 Spatially resolved EELS to probe the pore structure of porous coatings grown by magnetron sputtering

Lacroix B.1, Godinho V.1, Fernández A.1
1Instituto de Ciencia de Materiales de Sevilla (CSIC - Univ.Sevilla), Seville, Spain
bertrand.lacroix@icmse.csic.es

Over the past years, the synthesis of porous coatings has been largely investigated since it was demonstrated the possibility to tune the materials properties, conferring them high potentialities over a wide range of applications such as in optics, microelectronics, chemical sensing or mechanics.
In a previous study, a new bottom-up method using magnetron sputtering was proposed to grow amorphous silicon-based coatings containing closed porosity fully compatible with the opto- and micro-electronic technologies [1,2].
Recently, we presented a method to determine the atomic density and the pressure of the deposition gas helium trapped inside the nanopores of silicon coatings, using spatially resolved electron energy loss spectroscopy (EELS) experiments in scanning transmission electron microscopy [3]. In that case, pressure up to the GPa range was measured inside the pores.
Based on these results, the study is now extended to other type of materials like cobalt or tungsten films grown using similar conditions in helium atmosphere in order to verify the transferability of the elaboration method and to compare the He trapping in various materials. Our preliminary data obtained on the cobalt coatings confirm that helium remains trapped inside the cavities after the growth, since the EELS spectrum recorded in a pore region exhibits a sharp peak around 23 eV on top of the Co plasmon which corresponds to the He-K edge (Fig. 1). The methods used to process the spectrum image data in order to extract the He signal from the low-loss background and to determine its state inside the pores (density, pressure) will be presented here for the different materials studied.
The methodology to produce coatings with closed porosity was also validated in the case of reactive sputtering deposition and porous silicon oxynitride (SiON) layers with closed pores filled with molecular nitrogen are investigated here. The elemental maps presented in Fig. 2 show a strong accumulation of nitrogen in the pores regions, with the signature of molecular nitrogen in EELS (not shown). However, in that case, the zero-loss peak used as reference cannot be recorded simultaneously with N K-edge since the latter appears in the high-loss range (around 400 eV) which complicates the measurement of the nitrogen state inside the pores. Moreover, the N signal coming from the pores needs to be separated from the SiON matrix contribution. Further details about the data analysis, following the method described in [4] for the (Ga,Fe)N system, will be discussed.

[1] V. Godinho et al., Nanotechnology, 24 (2013), 275604.
[2] V. Godinho et al., Microporous and Mesoporous Materials, 149 (2012), 142.
[3] R. Schierholz et al., submitted.
[4] A. Kovacs, et al., Journal of Applied Physics, 114 (2013), 033530.

This work was supported by the EU 7FP (project Al-NanoFunc CT-REGPOT-2011-1-285895, http://www.al-nanofunc.eu/), the CSIC (project 201060E102), the Spanish Ministry MINECO (projects CSD2008–00023 and CTQ2012-32519) and Junta de Andalucía (TEP217 and PE2012-TEP862).

Fig. 1: (a) HAADF map recorded over a set of pores in the Co coating. (b) Low-loss EELS spectra recorded outside and inside one pore (see positions of the squares in Fig. 1(a)): in the pore region, a sharp and intense peak around 23 eV corresponding to the He-K edge appears on top of the Co plasmon.

Fig. 2: (a) HAADF map recorded over a set of pores in the SiON coating. The elemental maps of the (b) Si K-edge, (c) O K-edge, and (d) N K-edge evidence the accumulation of nitrogen in the pore regions.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2505 Effects of Sodium based electrolytic on microstructure and protective properties of Al2O3 coatings formed on pure aluminum alloy substrates by plasma electrolytic oxidation

Ayday A.1, Durman M.1
1Sakarya University, Faculty of Engineering, Department of Metallurgical and Materials Engineering, Sakarya, 54187, Turkey
aayday@sakarya.edu.tr

The plasma electrolytic oxidation (PEO) process is one of several techniques for fabricating thick metal-oxide coatings on light alloys materials such as aluminum, titanium, and magnesium. The technique involves anodic oxidation of the metal surface in an aqueous electrolytic solution together with localized plasma discharge, which occurs when the electrolytic voltage exceeds the critical polarization potential of the metals [1-3].
Al2O3 ceramic coatings are potentially very effective in developing hard, wear-resistant surfaces, nevertheless, previous treatments and coatings applied to aluminum alloys, for example, by traditional processes such as hard anodizing or thermal spraying, have suffered from the low load support from the underlying material and insufficient adhesion, which reduces their durability [3-5].
In this present work, various concentrations of Na2SiO3 as a special additive, were added to the 2,5 g/l KOH - 2 g/l KF electrolyte system and the effects of Na2SiO3 concentrations varying from 0 to 10 g/l on the morphologies, phase compositions of pure Al with different PEO coatings were studied. The microstructure, phase composition, micro-hardness of Al2O3 ceramic coatings were investigated using X-ray diffraction, scanning electron microscopy and microhardness test.
The samples were coated for 60 min by applying 20 A/dm2 current density, 400 V and Na2SiO3 as electrolyte. Three various concentrations of Na2SiO3, 0, 5 and 10 g/l were used to produce different PEO coatings and the coatings were named as A, B and C code, respectively.
The SEM images of different samples exhibit similar surface morphology, but we can see that the surface is a little coarse and porous for sample A (Fig. 1). From Fig. 2, we can see that the size of pores of sample C decreased obviously and the surface is denser in comparison with sample A. The thickness of the film is change between 30 and 40 μm after PEO treatment. The thickness of film increases with applied voltage increasing the electrolytic solution concentration. According to the XRD patterns, B and C include two crystal phases of γ-Al2O3 and α-Al2O3 after PEO treatment. The average surface microhardness of the pure Al substrates was 120HV0,05. After PEO the microhardness of the coatings increased average to 1600HV0,05.

References
[1] Gu W.C., Lv G.H., Chen H., Chen G.L., Feng W.R., Yang S.Z., Materials Science and Engineering A, 447, 158–162, 2007.
[2] Tillous E.K., Toll-Duchanoy T., Bauer-Grosse E., Surface&Coatings Technology, 203, 1850–1855, 2009.
[3] Zhenga H.Y., Wanga Y.K., Lib B.S., Han G.R., Materials Letters, 59, 139–142, 2005.
[4] Oh Y.J., Mun, J., Kim J.H., Surface & Coatings Technology, 204, 141–148, 2009.
[5] Xin S.G., Song L.X., Zhao R.G., Hu X.F., Surface&Coatings Technology, 199, 184– 188, 2005.

The authors thank Sakarya University, Faculty of Engineering, Department of Metallurgical and Materials Engineering for performing XRD and SEM studies. This work was supported by Sakarya University project (No. 2013-50-01-004).

Fig. 1: SEM micrographs of A Code Sample

Fig. 2: SEM micrographs of C Code Sample
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2517 Effect of the Li+ co-doping on the morphological characteristics of Y2O3: Er3+ films deposited by ultrasonic spray pyrolysis.

Meza-Rocha A. N.1, Soto A. B.1, Huerta E. F.2, Falcony C.1
1Departamento de Física, Centro de Investigación y de Estudios Avanzados del IPN., Av. IPN 2508, México DF, 07360 México., 2Programa de Doctorado en Nanociencias y Nanotecnología, CINVESTAV IPN., Av. IPN 2508, México DF, 07360 México.
asoto@fis.cinvestav.mx

Er3+ doped Y2O3 films have attracted the attention for applications such as optical amplifier, active waveguide, bioimaging applications among others [1,2]. However, the Er3+ emission is often very weak due to the forbiddance of the intra f-transitions [1], which represent a drawback for real applications. Recently, it has been observed that the incorporation of Li+ in small amounts improve considerably the Er3+ emission [1,3]. It has been suggested that the improvement of the Er3+ emission by the Li+ co-doping is due to distortion of the crystalline field and changes on the surface morphology [3].

In this work, the effect of the Li+ co-doping on the luminescent and morphological characteristics of Y2O3: Er3+ is reported. The films were deposited by ultrasonic spray pyrolysis at 500°C without a post-thermal annealing. The thickness was kept around of 100 nm. The Er3+ content was fixed at 1.5 at% in solution. The Li+ content was varied from 1 to 4 at% in solution.

SEM analysis (fig 1) reveals that the surface morphology for films without Li+ is composed by compacted cubic-looking grains with a size in the range of 15-29 nm. As Li+ is added the grains become less compacted up to 2 at% of Li+ with a size of 15-40 nm. For Li+ doping higher than 2 at%, the grains size decrease from 15 to 30 nm and become more compacted as in the case of 0 at% of Li+. It was observed that the Li+ co-doping improves the Er3+ emission by a factor of 4-5 times (fig 2) with the addition of 2 at% of Li+. The emission tends to quench for Li+ co-doping higher than 2 at%. The great improvement of the Er3+ emission is attributed with the distortion of the crystal field around of Er3+ which allows the otherwise intra f-transitions [3], and the reduction of the internal reflection associated with the increase of the grains. The quench effect might associate with the incorporations of large amounts of Li+ ions by creating large amounts of oxygen vacancies reducing the films crystallinity and the grains size.

References

[1] Ting Fan, Qinyuan Zhang and Zhonghong Jiang, J. Opt. 13 (2011) 015001.

[2] Nallusamy Venkatachalam, Tomoyoshi Yamano, Eva Hemmer, Hiroshi Hyodo, Hidehiro Kishimoto, and Kohei Soga, J. Am. Ceram. Soc., 96 (9) (2013)2759.

[3] A. N. Meza-Rocha, E. F. Huerta, E. Zaleta-Alejandre, Z. Rivera-Álvarez, C. Falcony, Journal of Luminescence 141 (2013) 173.

Fig. 1: Figure 1. Typical SEM images of Y2O3: Er3+ doped with (a) 0, (b) 1 (c) 2, (d) 3 and (e) 4 at of Li+.

Fig. 2: Figure 2. Emission spectrum under an excitation of 207 nm for different content of Li+.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2558 Co-terminated structure of the spinel Co3O4(001) surface confirmed by high-resolution annular dark field scanning transmission electron microscopy.

Kitta M.1, Akita T.1, Kohyama M.1
1Research Institute for Ubiquitous Energy Devices, National Institute of Advanced Industrial Science and Technology, (AIST)
m-kitta@aist.go.jp

Spinel Co3O4 is widely known as an active catalyst of CO oxidation reaction with 100% of conversion rate at -80℃ [1]. This high catalytic activity should be dominated by the interaction between gas molecules [2, 3] (CO, H2O) and Co3O4 surfaces, and it was reported that active Co3+ species should play a key role of the reaction [4]. Therefore the atomic scale investigation of the Co3O4 surface structure is essential to discuss the reaction models.
The Co3O4 (111) surface structure was investigated by LEED, STM and DFT calculations, and a Co-terminated configuration on the oxygen sub-surface layer was proposed as a reasonable structure [5]. However, despite frequently observed facets of the crystal [6], the Co3O4(001) surface has been little discussed. Here, we studied the Co3O4(001) surface by ADF-STEM (annular dark field scanning transmission electron microscopy) imaging (Cs-corrected TEM, TITAN3TM at 300 kV) and confirmed the Co-terminated structure.
The Co3O4(110) specimen was prepared by calcination (1023 K for 12 h in the air) of a thinned CoO(110) wafer (purchased by Crystal Base). In the inset of Fig. 1, the morphology of the specimen is shown by the low magnification image. Clear facets are confirmed as (1-11) and (001) edges, respectively. Fig. 1 shows the ADF-STEM image with atomic resolution, acquired from the square area of the inset, where we can see Co columns of Co3O4 [110]. Some bright spots at the top of the (001) surface were confirmed to have the same atomic periodicity as the Co columns to occupy the oxygen-tetrahedral sites, which means that the Co3O4(001) surface has a Co-terminated structure, similar to the Fe3O4(001) surface [7, 8].
The surface model is summarized in Fig. 2. The top-most Co2+ is located on the oxygen bridge site of the (001) surface. The (1×1) surface unit cell shown in black solid lines in the top view model is composed of one Co2+, two Co3+, and four O2-, which fulfill the stoichiometric surface. Therefore we suggest that the (001) surface is terminated by Co2+, and there is no need of major surface reconstructions such as cation defection or oxygen adatoms. Of course, we should discuss the stability and reactivity of this surface structure, and they will be revealed by the DFT calculations in the future.

[1] Yu, Y. et. al., J. Catal 267 (2009) 121-128
[2] Petitto, A. C. et. al., J. Mol. Catal. A Chemical 281 (2008) 49-58
[3] Xu, X. L. et. al., Surf. Sci. 605 (2011) 1962-1967
[4] Xie, X. et. al., Nature 458 (2009) 746-749
[5] Heinz, K. et. al., J. Phys. Condens. Matter 25 (2013) 173001
[6] Xiao, X. et. al., Adv. Mater. 24 (2012) 5762-5766
[7] Seoighe, C. et. al., Surf. Sci. 440 (1999) 116-124
[8] Rustad, J. R. Surf. Sci. 432 (1999) L583-L588

The authors thanks to C. Fukada and M. Makino for sample preparation.

Fig. 1: Fig. 1 ADF-STEM image of Co3O4(001) edge. The color spheres represent O2- (red), Co3+ of oxygen octahedral sites (dark blue) and Co2+ of oxygen tetrahedral sites (light blue). The arrows of light blue color show the atomic column positions of top-most Co2+. The interatomic distance of Co2+-Co2+ is estimated as about 0.56 nm.

Fig. 2: Fig. 2 Summarized model of the Co3O4(001) surface. The observed atomic distance of top Co2+-Co2+ by ADF-STEM (0.56 nm) is in good agreement with the value of 0.571 nm estimated by the simple crystal model.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2642 Nanostructure and strain properties of InAs QDs grown on (211)B GaAs surface

Florini N.1, Dimitrakopulos G. P.1, Kioseoglou J.1, Hatzopoulos Z.2, Pelekanos N. T.2, Kehagias T.1
1Physics Department, Aristotle University of Thessaloniki, GR-54624 Thessaloniki, Greece, 2Materials Science & Technology and Physics Departments, University of Crete and IESL/FORTH, GR-71003 Heraklion, Greece
nflori@physics.auth.gr

Piezoelectric InAs quantum dots (QDs) were grown on (211)B GaAs surface, by plasma-assisted molecular beam epitaxy (PAMBE). The heterostructure was characterized by high-resolution transmission electron microscopy (HRTEM) methods [Fig. 1(a)]. Combining crystallographic data from plan-view and cross-sectional observations, we came up with the 3D shape model of the QDs depicted in Fig. 1(b). QDs present an asymmetric truncated pyramidal faceted configuration comprising the {100}, {110} and {21l} facets, l varying from 2 to 4. Thus, the pyramid [-111] to [0-11] base-aspect-ratio (BAR) depends on the l index of the inclined {21l} planes, since it influences the out-of-plane angle of the pyramid base tip. Typical BAR values between 1.2 and 1.4 were determined for the InAs QDs. Moreover, the height of the QDs depends both on the l value and the level of truncation of the apex of the pyramid, which is variable (5-15 nm).

The degree of plastic relaxation of the InAs QDs on GaAs was estimated by Moiré fringe analysis, fast Fourier transform (FFT) of HRTEM images, and geometrical phase analysis (GPA). It was found that the QDs are rather relaxed due to the presence of misfit dislocations (MDs) at the InAs/GaAs interface [Fig 2(a)]. An average value of 6% for the in-plane lattice strain was estimated near the interfacial region. It is noticed, from GPA analysis, that there is an increase of the lattice strain up to 7.1% close to the apex of the QDs, suggesting that in that area there is negligible residual elastic strain [(Fig. 2(b)]. The lattice constant increases towards the top of the QDs due to the foil thickness effect and thus, the smaller QDs exhibit the lowest strain. It is clear that strain inside the InAs QDs shows evidence of an anisotropic behavior.

The formation of InAs QDs on (211)B GaAs surface was explored in terms of atomistic reconstructions of the energetically favorable configurations in conjunction with HRTEM results. The pyramidal faceted reconstructions of (211)B GaAs, composed of the {100}, {110} and {113} facets, are considered as the most stable configuration, while another less stable reconstruction is formed by the {100}, {110} and {214} surfaces, which is shown in Fig. 3. However, in addition to the {214} surfaces, the {21l} surfaces with l = 2, 3 seem to be experimentally plausible. Since the (211)B surface is faceted, diffusion barriers are much higher with respect to flat surfaces and hence, In adatoms are trapped at the edges of the individual surfaces making the formation of InAs QDs on them favorable with respect to other cleaved flat surfaces (e.g. the {111} surface).

Research co-financed by the European Union (European Social Fund–ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF)–Research Funding Program: THALES, project “NANOPHOS”.

Fig. 1: (a) Plan-view HRTEM image of an InAs QD viewed along the [211] zone axis. Moiré fringes arise due to overlapping of the InAs and GaAs lattices. (b) 3D shape model of the QDs with the l index of the {21l} facets being 3.

Fig. 2: (a) Cross-sectional HRTEM image of an InAs QD viewed along the [0-11] zone axis superimposed with the corresponding GPA strain map. (b) Line profiles of the average strain from the areas marked in (a) by dashed lines, along the [211] growth direction.

Fig. 3: Atomistic model of the faceted GaAs (211)B pyramidal surface shape using the method of infinite triangular prism structures.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2749 TEM characterization of electrochemically deposited Fe- Pd films

Damm C.1, Konczak C.1, Schlörb H.1, Haehnel V.1, Schultz L.1, Sturm T.1, Pöhl A.1, Rellinghaus B.1
1IFW Dresden, Institute for Metallic Materials, Helmholtzstrasse 20, D-01069 Dresden, Germany
c.damm@ifw-dresden.de

Fe-Pd alloys (Pd ~30 at.%) show a martensitic transformation [[1]] and both a thermal [2] and magnetic shape memory effect [3]. This makes the material promising for sensor or fast actuator applications in nanosystems .

The electrochemical deposition of Fe and Pd with a highly stable complexed electrolyte allow it to obtain low dimensional, complicated structures [4]. Deposited Fe70Pd30 samples show a nanocrystalline bcc structure. Heat treatment at 800°C for 10min led to a complete transformation to fcc . This is required for the martensitic transformation [5]. This suggests an impact of the complexing agent amount on the phase structure of the deposits. To generate the fcc structure directly during deposition, detailed investigations on the impact of the complexing agent were performed.

Electrodeposition is performed in a standard three electrode arrangement . The electrolyte contained 0,01 M Pd(NH3)4Cl2, 0,025 M Fe2(SO4)3•9H2O and 0,3 M (NH4)2SO4. To avoid the formation of Fe-hydroxides, the iron ions were complexed by sulfosalicylic acid (C7H6O6S•2H2O; SSC) , varied in concentration of 0,05 and 0,15M. The pH was adjusted to 5 by using H2SO4. The depositions were performed under a deposition potential of E = ‑1,1 VSCE for 10 minutes at room temperature and characterized with respect to composition, structure and microstructure.

X-Ray diffraction indicates a dependence of the Fe-Pd phase structure on the Fe3+/SSC ratio in the electrolyte. A shift in phase structure is accompanied by a change in Fe-Pd composition of ≈10 at.%. The desired composition of Fe70Pd30 is achieved for a slight excess of SSC (Fe3+/SSC ratio of 1:1,2). TEM investigations on a FIB lamellae of this particular Fe70Pd30 sample show a homogenous microstructure without defects consisting of elongated nanocrystals (6…44nm) in bcc structure [Fig. 1] with a preferred <110> orientation. No hints for the presence of a fcc phase have been found.. Fast Fourier Transformation filtered HRTEM images confirm the results [Fig. 2]. Three individually recorded SAD patterns along the layer cross section from the substrate to the surface point out no structure changes inside of the Fe-Pd layer [Fig. 3].

STEM line scans were acquired [Fig. 4]. A constant composition of 70± 5at.% Fe was observed over the complete film thickness except the region in close proximity to the Au interface where 80 at.% Fe were found.

[1] R. Hultgren, C. Zapffe, Nature 142 (1938) 395

[2] T. Sohmura, R. Oshima, F.E. Fujita, Sc. Metall. Mater. 14 (1980) 855

[3] R.D. James, M. Wuttig, Phil. Mag. A 77(5) (1998) 1273

[4] V. Haehnel, S. Fähler, L. Schultz, H. Schlörb, Electrochem. Comm. 12 (2010) 1116

[5] H. Schlörb, M. Uhlemann, V. Haehnel, D. Iselt, A. Gebert, Z. Phys. Chem. 227 (2013) 1071

Fig. 1: TEM bright field image of elongated crystals and a) corresponding SAD (FePd, bcc) and b) nanodiffraction of a separate crystal (FePd, bcc, zone axis [111]

Fig. 2: HRTEM image and corresponding FFT in the layer: FePd, bcc, d110 = 0,210nm

Fig. 3: TEM bright field image of the Fe-Pd layer and corresponding SAD in different regions showing the same bcc structure and preferred orientation

Fig. 4: STEM- linescan over the Fe- Pd layer: Increase of the Fe- content at the interface to Au
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2826 TEM characterization of H2-seclective Pd-based membranes

Gan Y.1, Walmsley J. C.1,2, Holmestad R.1, Peters T. A.3, Stange M.3
1Department of Physics, NTNU, 7491 Trondheim, Norway, 2SINTEF Materials and Chemistry, N-7465 Trondheim, Norway, 3SINTEF Materials and Chemistry, N-0314 Oslo, Norway
yanjie.gan@ntnu.no

Palladium (Pd) and Pd-based membranes have been widely studied for several decades due to their unique hydrogen separation capabilities, and have a large market potential for production of high-purity H2 fuel for application in e.g. power generation with CO2 capture, fuel cells or chemical industry. The most common membrane compositions are pure Pd, and alloys of Pd-Ag, Pd-Cu and Pd-Au, where the alloying element first of all increases H2 permeability or reduces the H2 flux inhibition due to CO and H2S.

An understanding of the impact of segregation and grain growth is crucial to design Pd-alloys for various applications. In the current study, the effects of selected treatments, like H2 permeation and H2S exposure, on free-standing Pd-alloy samples are characterized using JEOL 2100F operated at 200kV. The SINTEF-patented two-step technique of magnetron sputtering was used to prepare Pd-alloy samples. In this study, the Pd81Cu18Ag1 alloy, relevant for the development for highly permeable sulfur-tolerant Pd-Cu alloy membranes, has been used as model system.

Depending on the information sought, the samples can be studied in TEM in plan-view or in cross-section geometry. Plan-view observation is mainly used to study surface morphology, crystal orientation and chemistry. Compared with the plan-view, cross-section observation provides more information from real-space structures, including defects and interfaces with resolution down to atomic scale.

TEM characterization shows that the substrate-interface of the membrane is smoother than growth-interface. In the as-grown membrane, the grains at or near the substrate-interface are tiny and start to be orientated up at about 50nm from the surface, most grains are distinctly columnar after a growth thickness of about 100nm. The grains at the growth-interface are columnar and become much larger with 50nm in average size. The grain structure of the sputtered films thus reflects the growth process. Under the sputtering conditions applied, the nucleation density on the Si wafer substrate is high, and the smooth nature of the wafer surface also affects the resulting roughness on the substrate side. As more material is deposited, some grains grow while others are terminated and covered. The result is a grain size gradient throughout the film, with elongated grains of preferential orientation along the [111] direction. After a H2 permeation test the grains grew at both the substrate-interface and the growth-interface to about 100nm. The growth at the substrate interface was most significant. Some voids less than 10nm along the grain boundaries have also been observed. These voids do, however, not contribute to unselective leakage flux, but still, the long-term effect on stability requires further investigation.

The financial support from the Research Council of Norway (RCN) through the CLIMIT program (Contract No. 215666/E20) is gratefully acknowledged.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2989 Role of defects in the formation of two-dimensional electron gas at LaAlO3/SrTiO3 interfaces

Zhang Y.1,2, Bark C. W.3, Zhou H.4, Ryu S.3, Eom C. B.3, Zou X. D.2, Pan X. Q.1
1Department of Materials Science & Engineering, University of Michigan, Ann Arbor, MI, USA, 2Department of Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden, 3Department of Materials Science and Engineering, University of Wisconsin-Madison, Madison, WI, USA, 4Advanced Photon Source, Argonne National Laboratory, Argonne, IL, USA
yi.zhang@mmk.su.se

The 2DEG at the complex oxide heterointerface between LAO and STO was found to have high carrier mobility and high sheet carrier density at room temperature, both several orders of magnitude larger than at the heterointerfaces of III-V-based semiconductors [1]. Much experimental and theoretical work has been done to investigate the fundamental origins of these electronic properties [2, 3]. However, there is still no single mechanism that can explain the formation of the metallic state which is consistent with all observed experimental details. Here, we present a study of the effects of annealing on the LAO/STO 2DEG interface using atomic resolution STEM and EELS.
Five unit cells (u.c.) of LAO films were grown epitaxially on a TiO2-terminated (001) STO single crystal substrates. Two samples were annealed in oxygen atmosphere at 400 ºC and 600 ºC, respectively. One highly-conducting film left in the as-grown condition for comparison. The film annealed at 400 ºC is insulating, whereas the one annealed at 600 ºC show 2DEG behaviour.
Atomic-resolution EELS mapping was performed at the interface of the 600 ºC annealed LAO/STO film (Fig. 1). The La M4,5 edges map show a gradually decreasing intensity in the STO and drops to zero at a depth of about 5 u.c. below the interface. The Ti L2,3 edges map show strong Ti signals in the LAO film, indicating Ti diffusion and its displacement of Al on the B-site.
EELS line scans were collected across the LAO/STO interfaces of both three films. Elemental distribution profiles are shown in Fig. 2, along with HAADF images of the regions from which they were acquired. The Ti penetrating slightly farther in the LAO film annealed at 600 ºC than in 400 ºC annealed and as-grown films. La diffused into the STO substrate to a depth of nearly 4 u.c. in the as-grown and 400 ºC annealed films and 5 u.c. in the 2DEG film. The spatial distribution of the Ti3+ and Ti4+ signals across the LAO/STO interfaces are shown in Fig. 3. The insulating film has a relatively low concentration of Ti3+, contained to the 4 u.c. adjacent to the interface with LAO. In comparison, the conducting films show a high concentration of Ti3+ through the entirety of the LAO as well as the top 5 u.c. in STO substrate. The high concentration of Ti3+ in the film, which can be attributed to the presence of diffused La and oxygen vacancies, creates the conditions necessary for the formation of a 2DEG [4].
In conclusion, we provide a detailed TEM analysis of the origins and the annealing effect of 2DEGs at the LAO/STO interface. We found that the mixed Ti valence is strongly related with the free carrier density, which is result from the competition between the La substitution and refill of oxygen vacancies.

The work was supported by the AFOSR under Grant No. FA9550-10-1-0524 and by Penn State MRSEC under Grant No. MRSEC DMR-0820404.

Fig. 1: Atomic-resolution EELS mapping was performed from the marked area in (a). (b)-(d) are the elemental maps of the O K, La M4,5 and Ti L2,3 edges. (e) is the colorized EELS elemental map of the O, La and Ti.

Fig. 2: (a), (b) and (c) are the elemental distribution profiles of as-grown, 400 ºC annealed and 600 ºC films. (c), (d) are the spatial distribution of Ti3+ and Ti4+ in these two films.

Fig. 3: (a), (b) and (c) are the spatial distribution of Ti3+ and Ti4+ in the as-grown, 400 ºC annealed and 600 ºC films.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2855 CoxFe3-xO4 films for sensor applications

MADIGOU V.1,2, VILLAIN s.1,2, BENDAHAN M.1,3, ARAB M.1,2, BERNARDINI S.1,3, LEROUX C.1,2
1IM2NP UMR CNRS 7334 , 2Université de Toulon, BP20132, 83957 La Garde Cedex, France, 3Aix Marseille Université, Av.Escadrille Normandie Niemen, 13397 Marseille Cedex, France
madigou@univ-tln.fr

Spinel ferrites are important technological materials due to their magnetic properties [1]. During last decade spinel ferrites have been studied as semiconducting gas sensors [2-3]. The spinel structure is cubic with the general formula MxFe3-xO4 where M is a divalent metallic ion; depending on the composition of the metallic cation, ferrites exhibit n or p type conductivity. In this work, we have studied the cobalt ferrites CoxFe3-xO4. Previously, we have synthetized, by a new one pot solvo-thermal method, small and highly crystallized nanoparticles of cobalt ferrites [4]. We have showed that the semi-conducting behaviour of the particles changes with the cobalt content [5]. Hence, we were interested in studying cobalt ferrites as thin films for applications in gas sensors. Thin films of CoxFe3-xO4 were realized by spin-coating on Si substrate with Pt interdigitated electrodes. The precursor solution was the same as for the synthesis of nanoparticles (cobalt and iron acetylacetonates). Undecanoïc acid was added to the benzyl alcohol in order to improve the solubility of the acetylacetonates. After deposition, the films were annealed at 500°C in air during 2 hours. The obtained films are homogenous in morphology and in chemical composition; they show a nanostructuration of grains with a mean size of 9.5 nm for x=1 (Fig.1) and 8 nm for x=1.8 (Fig.2). The crystallographic structure was verified by electron diffraction, the pattern is indexed in the expected spinel structure (Fig.3). Figure 4 shows grains of a film for x=1.8, the mean grain size measured is about 6.5 nm which is consistent with the value deduced from the SEM observations. The electrical measurements were carried out under reducing gas (NH3) and oxidizing gas (NO2). Under NH3, for x=1 the film is n-type semi conductor and for x=1.8 is p-type semi conductor. Under NO2, the first results were obtained for x=1.8: the electrical resistance decreases which is a typical response of a p-type semi conductor. These results are very promising and particularly, the electrical response is meaningful under low concentration of gas (10 ppm of NO2).

[1] M. Sugimoto, J. Am. Ceram. Soc. 82 (1999) 269.

[2] Z.Sun, L. Liu, D. Z. Jia, W.Pan, Sensors and actuators B, 125 (2007) 144.

[3] Y.-L. Liu, Z.-M. Liu, Y. Yang, H.-F. Yang, G.-L. Shen, R.-Q. Yu, Sensors and Actuators B, 107 (2005) 600.

[4] L. Ajroudi, V. Madigou, S. Villain, N. Mliki, Ch. Leroux J. of Crystal Growth 312 (2010) 2465.

[5] Ch. Leroux, M. Bendahan, V. Madigou, L. Ajroudi, N. Mikli, Sensors and transducers in press (april 2014).

Fig. 1: SEM image of a CoFe2O4 film (mean size particles 9.5 nm)

Fig. 2: SEM image of a Co1.8Fe1.2O4 film(mean size particles 8 nm)

Fig. 3: Electron diffraction pattern of the particles of a Co1.8Fe1.2O4 film (spinel structure)

Fig. 4: TEM image of a Co1.8Fe1.2O4 film (nanoparticles are well crystallized)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2956 Microstructural analysis of metal oxide coatings synthesised using novel energetic deposition methods

Murdoch B. J.1, Mayes E. L.1, Field M. R.2, Partridge J. G.1, McCulloch D. G.1,2
1School of Applied Sciences, RMIT University, GPO Box 2476, Melbourne, VIC 3000, Australia, 2RMIT Microscopy and Microanalysis Facility, RMIT University, GPO Box 2476, Melbourne, VIC 3000, Australia
matthew.field@rmit.edu.au

The possibilities for new metal oxide based materials is forever growing with the introduction of novel deposition methods which allow precise control of the deposition parameters and the ability to dope in order to tailor properties. The conditions used for the deposition of these coatings has an influence on the microstructure which in turn plays an important role in determining physical properties, such as the optical transmission and electrical conductivity. In addition, for many metal oxide materials the structure-property relationship is not well understood. In this work, filtered cathodic arc (FCVA), DC magnetron sputtering (DCMS) and high power impulse magnetron sputtering (HiPIMS) were utilised to reactively grow metal oxide coatings (HfO2 and ZnO) within an oxygen atmosphere.

FCVA deposition is a scalable energetic growth technique which allows for the synthesis of nanoscale coatings with tuneable properties. In this technique, a conductive target material (in this case, our metal) is ablated with a low voltage/high current electron flux. The metal ions are directed through a magnetic double bend towards the substrate through an oxygen environment. FCVA utilises a fully ionised plasma in which the energy of deposition can be controlled by applying an electrical bias to the substrate, heating or by modifying the processing pressure [1]. Thin film coatings grown using FCVA have been shown to have a low rms roughness and a high density [1], which is ideal for device applications. DCMS (low energy neutrals) and HiPIMS (high plasma density) were also selected to grow coatings. In conventional DCMS, inert gas ions (such as argon) are accelerated towards a negatively biased target material. When the target is sputtered, the target material is ejected and forms a thin film coating on the substrate placed nearby within the vacuum. HIPIMS is a technique based on magnetron sputtering, but unlike magnetron sputtering, HIPIMS, uses extremely high power density pulses, achieving a greater ionisation of the sputtered material during deposition [2,3].

The authors gratefully acknowledge the Australian research council (ARC) for funding. They would also like to thank the RMIT microscopy and microanalysis facility and the Australian Synchrotron for assistance with micro-characterisation.

Fig. 1: Cross sectional TEM bright field image of a Zn1-xMgxO film with compositional variations through the thickness.

Fig. 2: Cross sectional TEM dark field image of the same region from figure 1 highlighting cubic MgO (100) planes.

Fig. 3: EELS areal density line scan of the marked region in figure 1, taken from the Zn and Mg core loss edges.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-2988 Hydrogen exposured to microstructure of Er2O3 coating layer prepared by MOCVD process

Shinkawa T.1, Hisinuma Y.2, Tanaka T.2, Muroga T.2, Mikmekova S.3, Sunada S.4, Ikeno S.5, Matsuda K.4
1Graduate School of Science and Engineering for Education, University of Toyama, 2National Institute of Fusion Science; 322-6, 3Institute of the Scientific Instruments of the ASCR, 4Graduate School of Science and Engineering for Research, University of Toyama, 5Hokuriku Polytechnic College
m1371518@ems.u-toyama.ac.jp

In breeding blanket system of nuclear fusion reactor needs to development advanced type of coating to leak control of tritium and reducing magneto hydrodynamic (MHD) pressure drop. In breeding blanket system, material needs to fulfill five conditions. 1. Not break down at high temperature. 2. Low reactivity with Li as a coolant. 3. High electrical resistivity. 4. High permeation control of tritium. 5. High electrically insulating coating of 2µm or more. It has been reported that Er2O3 is excellent electrical resistance and a permeation control effect from various ceramic materials[1]. Hishinuma et. al. succeeded in forming Er2O3 film by metal organic chemical vapor deposition (MOCVD) process as a new technology for large area coating on broad and complicated shaped components[2]. MOCVD process is a concise procedure to form homogeneous and large area coating layer synthesized from a metal organic complex. Two stainless steel 316 (SUS316) disk plates were used in this work. SUS316 plates were coated with Er2O3 film using MOCVD process at 773K for 2 hrs. One was performed permeation of hydrogen with irradiation of the gamma ray after Er2O3 coating, and the other was hydrogen permeation test. XRD analysis with θ-2θ scan mode were carried out on a Philips X'pert system diffractometer using Cu Kα X-Ray irradiation. Scanning Electron Microscope (SEM) operating at 20keV with EDS and SLEEM mode were carried out on a HITACHI S-3500H. TEM sample for cross sectional observation were prepared by Focus Ion Beam (FIB) method (FB-2100, HITACHI) operating at 40keV using gallium ion. Transmission electron microscope (TEM) was carried out on a TOPCON EM-002B operating at 200keV with EDS. Fig. 1 shows the surface morphology of Er2O3 film before hydrogen permeation test and that surface had granular structure. Crystallines on the surface had various size and they look like line up by vapor flow. Fig. 2 shows TEM bright field image obtained for the sample prepared from central part of Fig. 1 by FIB method. There is no gap between Er2O3 and SUS substrate. Columnar grains of Er2O3 were corresponding to SEM image of granular structure in Fig1. SAED patterns obtained from Er2O3 of the sample before hydrogen permeation test was indexed as [001] Er2O3. It can be understood that the growth direction (upper-bottom direction in Fig.2) of Er2O3 film was [110] by analysis of this SAED pattern.

[1] B.A.Pint , P.F.Tortorelli , A.Jankowski , J.Hayes , T.Muroga , A.Suzuki , O.I.Yeliseyeva , V.M.Chernov : J.Nucl.Mater 329-333 (2004) 119-124

[2] Yoshimitsu Hishinuma , Tsutomu Tanaka , Teruya Tanaka , Takuya Nagasaka , Yuzo Tasaki , Akio Sagara , Takeo Muroga : Fusion Engineering and Design 86 (2011) 2530-253

Fig. 1: SEM images of surface Er2O3 film before hydrogen permeation test

Fig. 2: Cross-sectional TEM bright field image of Er2O3 film before hydrogen permeation test
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3010 Microstructure and strain of InGaAs epilayers grown by MOVPE on electrochemically prepared porous GaAs substrates

Dimitrakopulos G. P.1, Bazioti C.1, Grym J.2, Gladkov P.2, Hulicius E.3, Pangrác J.3, Pacherová O.3, Komninou P.1
1Physics Department, Aristotle University of Thessaloniki, GR 54124, Thessaloniki, Greece, 2Institute of Photonics and Electronics AS CR, v.v.i., Chaberska 57, 18251 Praha 8, Czech Republic, 3Institute of Physics AS CR, v.v.i. Cukrovarnicka 10, 16200 Praha 6, Czech Republic
kbazio@physics.auth.gr

The heteroepitaxy of InGaAs on porous (001)-oriented GaAs substrates is studied. It is shown that such substrates can behave in a compliant manner, so that early localization of the elastic strain due to the structural mismatch can be delayed or even suppressed, leading to an increase of the critical thickness.
InxGa1-xAs epilayers with nominal indium contents up to 20% were deposited by metalorganic vapour phase epitaxy (MOVPE) on porous GaAs substrates. The porous GaAs was prepared electrochemically using a fluoride-iodide aqueous electrolyte [1]. This process introduced a high degree of internal nanoscale porosity below a space charge layer, concurrent with low surface roughness and low surface pore density (Fig. 1). Transmission electron microscopy (TEM) and high resolution TEM (HRTEM) observations, together with high resolution x-ray diffraction (HRXRD) were employed in order to study the strain relaxation in the films. Photoluminescence (PL) measurements were employed in order to correlate the structural parameters to the indium content.
Reduced densities of misfit dislocations (MDs) were observed for all samples at the InGaAs/GaAs interfaces compared to heterostructures grown on nonporous GaAs substrates under identical conditions, as shown in Fig. 2 [2]. Cross sectional TEM showed that the MD array was located at a distance of ~30 nm from the level of the internal pores. The MDs were introduced by glide mainly from the substrate side due to nucleation on the pore surfaces, and were of 60o type (Fig. 3). The dislocations often glided in a dissociated form which was attributed to Cottrell atmospheres impeding their motion. Lattice constant measurements by selected area electron diffraction and HRXRD showed that the films that were deposited on porous GaAs retained a significantly higher amount of elastic strain compared those grown on nonporous GaAs. The residual strain did not comply to the plane stress condition since the strain of the film along the growth direction was smaller than anticipated. This behaviour was attributed to the upper part of the GaAs substrate becoming compliant to the misfit with increasing epilayer thickness.

[1] J. Grym et al., Phys. Stat. Sol. C 9, 1531 (2012)
[2] G. P. Dimitrakopulos et al., Appl. Surface Sci., in press (2014).

Work supported under the 2011-2013 Greece-Czech bilateral R&D collaboration project “III-V semiconductor heterostructures/nanostructures towards innovative electronic and photonic applications”, co-financed by the GSRT and the European Union (European Social Fund – ESF).

Fig. 1: Cross sectional TEM (XTEM) image along the [110] direction of the InGaAs/porous GaAs heterostructure, showing the substrate pore channels along <111> directions.

Fig. 2: Two-beam bright field XTEM images of 100 nm thick In0.2Ga0.8As/GaAs heterostructures grown (a) on nonporous GaAs and (b) on porous GaAs substrates. A MD array is visible in (a) whereas only few dislocations are discernible in (b). Both images were taken near the [110] zone axis with g 2-20.

Fig. 3: (a) HRTEM image of 60o dissociated glissile dislocations at the vicinity of a pore. (b) Nucleation of a 60o dislocation at a pore surface. Both images were recorded along the [110] zone axis.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3020 Direct imaging of atomic structure of TiN/MgO(001) interface by Cs-corrected STEM

Wei L. L.1, Do H.1, Chang L.1
1Department of Materials Science and Engineering, National Chiao Tung University
linlung.mse00g@nctu.edu.tw

Thin films of TiN have been applied widely for its good electrical property and chemical stability. High-quality TiN can make further understanding of its properties, particularly its relationship with oxides. Here we characterize the TiN/MgO interface using STEM annual dark field (ADF) and annual bright field (ABF) images and make a comparison of both imaging contrast for the different elements. Though ADF images can easily provide direct visualization of atomic column positions of heavy atoms, for light elements it is usually invisible even at high resolution. Recently, ABF imaging technique has been proved to be useful for observations of atomic positions of light elements in single crystals.

ADF/ABF imaging was performed in a Cs-corrected JEOL ARM200F with a Schottky gun at 200kV with 0.08nm probe. TiN thin films were grown on MgO substrate by pulsed laser deposition method. The full-width at half-maximum of X-ray rocking curve (002) is about 60 arcsec, implying that TiN films deposited on MgO are of good quality. Cross-sectional STEM specimens were prepared by tripod polishing method, followed by Ar-ion milling at 4⁰ and 3-4 kV.

In our observations, all ADF images show that atomic positions of Ti and Mg exhibit strong bright contrast, O in weak bright contrast, whereas the contrast of N atoms is hardly observed, as shown in Fig. 1. The contrast can be further improved as shown in the filtered images (the insets in Fig. 1) in which O positions are clearly visible while N ones are still barely seen. Also, it is difficult to see the contrast difference of O from N at the interfacial region. The ABF image in Fig. 2 clearly reveals that atomic columns of Ti, N, Mg, and O as dark spots where the darkness depends on their atomic number. Also the visibility of all atomic positions can be significantly increased in the filtered ABF images, but it remains difficult to identify exact atomic species at the interface because the difference in the intensity of light element atoms is too low. Interestingly, the ABF contrast shows additional dark spots in MgO where exist no atomic columns between Mg along <112>, and similar contrast is also seen in some TiN regions. The causes for such contrast may need further investigations. Both ADF and ABF images show that the TiN/MgO interface is almost fully coherent with epitaxial relationships of (001)TiN//(001)MgO and [1-10]TiN//[1-10]MgO because of a small lattice mismatch (δ=0.466%) between MgO and TiN. The arrangements of Mg, O, Ti and N atomic columns can be seen in order. Moreover, both images indicate that the ionic bonding sequences of cations and anions along [001] direction through the TiN/MgO interface is maintained without any interruption from MgO to TiN, i.e., Mg-O-Ti-N and O-Mg-N-Ti atomistic bonding.

The work was supported by National Science Council, Taiwan, R.O.C. under Contract No. 101-2221-E-009-049-MY3.

Fig. 1: ADF image of the TiN/MgO interface along [1-10] zone axis. Ti, Mg and O atomic positions as marked in TiN and MgO filtered images in the insets. (angle > 90 mrad)

Fig. 2: ABF image of the TiN/MgO interface along [1-10] zone axis. Ti, N, Mg and O atomic positions as marked in TiN and MgO filtered images in the insets. (angle ~ 11-22 mrad)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3027 Structural transition by thermal annealing of ZnO:Al films sputtering-deposited on glass substrates

Chen Y. Y.1, Chen P. Y.1, Cheng S. L.2, Huang B. M.1, Yang J. R.1, Shiojiri M.3
1Department of Materials Science and Engineering, National Taiwan University, Taipei, Taiwan, 2Department of Chemical and Materials Science Engineering, National Central University, Taoyuan, Taiwan, 3Professor Emeritus of Kyoto Institute of Technology, Japan
d98527006@ntu.edu.tw

Transparent conducting oxide (TCO) films are widely applied in optical and electronic devices.1-3 Among these TCO materials, ZnO:Al has attracted much attention because it is an abundant, inexpensive, non-toxic and environmentally friendly raw material with high crystallinity and good conductivity that is easy to prepare. In this study, ZnO:Al (2 wt%) films with a thickness of 650 nm were deposited on Corning Eagle2000 glass substrates by RF magnetron sputtering. They consisted of columnar grains preferentially grown along the [0001] axis (Fig. 1). Then, the films were activation annealed at different temperatures for 1 h in vacuum. The optical and electrical properties of the ZnO:Al films were improved by the annealing; the optimum transmittance of light over a 400-800 nm wavelength and the electric resistivity of ZnO:Al film annealed at 400oC were 85.5% and 2.9×10-3 Ω-cm, respectively, while those of the as-deposited film were 81.2% and 3.53×10-2 Ω-cm. Fig. 2 shows the X-ray diffraction results of as-deposited and post annealed ZnO:Al films, it revealed that the lattice parameter c of the hexagonal ZnO:Al decreased with annealing temperature, from 0.524 nm for the as-deposited film to 0.5205 nm for the film annealed at 400oC, compared with 0.521 nm for pure ZnO. High-resolution electron microscopy lattice images (Fig. 3) suggested that the hexagonal unit cell of the ZnO:Al film is deformed to a triclinic structure, not only by shrinking along the c axis, but also by leaning of the c axis. The lean angle increased with annealing temperature and reached a maximum value of 4o in the film annealed at 400oC. The change in crystal structure during the activation annealing is attributed to the substitution of Zn2+ ions with Al3+ ions and the formation of oxygen vacancies, both of which also caused the observed change in the optical and electrical properties.

References
1 S. Fernández, O. de Abril, F.B. Naranjo, J.J. Gandía, Sol. Energy Mater. Sol. Cells 95, (2011) 2281.
2 J.K. Jeong, Semicond. Sci. Technol. 26, (2011) 034008.
3 T.W. Kuo, S.X. Lin, Y.Y. Hung, J.H. Horng, M.P. Houng, IEEE Photonics Technol. Lett. 23, (2011) 362.

This work was supported by the National Science Council (NSC), Taiwan, under Contract No. NSC-101-2221-E-002-088-MY3. The authors are graceful to Mr. Hsueh-Ren Chen for the high resolution TEM support.

Fig. 1: (a) TEM bright field image and (b) selected area electron diffraction pattern of an as-deposited 650 nm thick ZnO:Al film on a glass substrate.

Fig. 2: (a) X-ray diffraction patterns of the as-deposited ZnO:Al film and the films annealed at different temperatures. (b) The 0002 peaks in the X-ray diffraction patterns. (c) The lattice parameter c of the hexagonal unit cell evaluated from the peak positions in (b).

Fig. 3: HR-TEM lattice images and the corresponding FFT images of the as-deposited ZnO:Al film and the films annealed at 400oC. The angle between the [000] and [010] axes is indicated in each FFT image.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3100 Growth and Structure of Physical Vapour Deposition grown MAX phase on Graphene

Zan R.1, Vishnyakov V.2, Bangert U.3, 4, Guo Y.4, Halsall M.5, Proctor J.6, Colligon J.2
1Department of Physics, Faculty of Arts and Sciences, Niğde University, 51000, Niğde, Turkey, 2School of Engineering, Manchester Metropolitan University, Manchester, M1 5GD, UK, 3Department of Physics and Energy, University of Limerick, Limerick, Ireland, 4Materials and Surface Science Institute, University of Limerick, Limerick, Ireland, 5Photon Science Institute and School of Electrical and Electronic Engineering, The University of Manchester, Manchester M60 1QD, UK, 6Joule Physics Laboratory, University of Salford, Salford M5 4WT, UK
recepzan@gmail.com

Although graphene was the first proven two-dimensional (2-D) material, there are many other atomically layered materials which can be extracted/formed into 2-D structures [1]. As is the case of graphene these 2-D materials have very different physical and chemical properties from those of the bulk materials. This leads to the need to assess these materials properties by as many techniques as possible and answer questions such as: when going from 3-D to 2-D, do these materials retain their electronic character (i.e., insulator or semi-metal) and, if the materials have differing phases, which do this? The MAX-phases are an interesting group of such materials, their name derives from there structure being a combination of a transition metal M, an element from the A section of the periodic table and X which is either Carbon or Nitrogen. The phases represent but another class of natural nano-laminated materials encompassing characteristics of metals and ceramics with unique combination of chemical, physical and electrical properties [2]. Utilising a unique layer-by-layer magnetron deposition technique the synthesis temperature was recently significantly reduced to allow the thin film creation of MAX phases on common engineering substrates [3].
Here we present the evidence, for the first time, that one of the MAX-phases, Cr2AlC, can be successfully deposited onto suspended graphene (directly on the TEM grid) at 500 and 600 oC. The graphene membranes have survived deposition as evidenced by microscopic and spectroscopic measurements. The deposited phase formed a nano-crystalline thin layer with island-like morphology on the graphene surface as shown in Fig. 1. The structure, growth orientation, thickness and composition of those nano-structures were revealed via atomic resolution phase contrast (BF) and high angle annular dark field imaging (HAADF), and energy dispersive X-Ray spectroscopy (EDXS). We show that the nano-structures are observed in different epitaxialy arranged orientations leading to different structural appearances. In addition to that, based on spectroscopic measurements, the nano-structures with different orientations are rich either in Cr or Al (see Fig.2).

[1] X. Zhang et al., Angew. Chem. Int. Ed., 52 (2013) 4361-4365.
[2] M.W. Barsoum, M. Radovic, Annu. Rev. Mater. Res., 41 (2011) 195-227.
[3] V. Vishnyakov et al., Vacuum, 100 (2014) 61-65.

Fig. 1: BF images of the Cr2AlC nanostructures deposited on graphene at 600 oC, a) an overview, b) a closer look to (a), (c)&(d) show the thin film morphology and the two prevalent epitaxial orientation of the nano-crystallites.

Fig. 2: a) HAADF image of Cr2AlC nanostructures before taking EDX maps on the region highlighted by yellow rectangular, b) Cr map, c) Al map, d) combination of Cr, Al and C maps, e) combination of HAADF image and Cr, Al and C maps, f) EDXS results.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3153 TEM characterization of defect free heterostructures grown on a compact home-build molecular beam epitaxy system

Covre da Silva S. F.1, 2, Lanzoni E. M.1, Coelho Neto P. M.1, 3, Garcia Junior A. J.1, de Barroa A. T.4, Pimentel V.1, 5, Ospina C. A.1, Bettini J.1, Malachias A.3, Ferreira S. O.2, Deneke C. F.1
1Brazilian Nanotechnology National Laboratory, 13083-100, Campinas, SP, Brazil, 2Viçosa Federal University, 36570-000,Viçosa, MG, Brazil, 3Minas Gerais Federal University, 30123-970,Belo Horizonte, MG, Brazil, 4Synchrotron Light National Laboratory, 13083-100, Campinas, SP, Brazil, 5Current address: Center for Information Technology Renato Archer, 13069-901, Campinas, SP, Brazil
carlos.ospina@lnnano.cnpem.br

Basic Transmission Electron Microscopy (TEM) techniques were used to characterize four period 11 nm InGaAs/10 nm GaAs superlattices, grown on a compact home-build molecular beam epitaxy system. The MBE system, of low investment and low running cost [1], was design to obtain in a small reactor, defect free epitaxial heterostructures, on 10x10 mm substrates. Cross section TEM specimens were prepared along one of the <110> zone axis by mechanical and ion polishing. Compositional, stress sensitive and high-resolution images were obtained using a JEM-3010 microscope, operating at 300 kV, with point resolution of 0.17 nm. Compositional sensitivity dark field images, using the (004) reflection, showed the contrast between the Indium rich and Gallium rich constituents, along the four period superlattice, as seen in Figure 1. The crystallinity quality was observed by dark field, using the (111) reflection (Figure 2), and high-resolution images, which show high quality interface between layers (Figure 3). Dark field images, using the (022) reflection, were also obtained; no defects were observed, rather a strain contrast at the interface due to lattice mismatched between GaAs and InGaAs, as seen in Figure 4. All TEM analysis confirmed the results obtained also by x-ray diffraction (XRD) and atomic force microscopy (AFM), which denote coherent epitaxial growth in the different growth setups of GaAs and InGaAs. All the fundamental growth studies conducted here shown the home-build MBE system produce high quality defect-free InGaAs heterostructures, on GaAs (001) substrates, for optoelectronic applications.

Reference

[1] S. Filipe Covre da Silva, E. M. Lanzoni, P. M. Coelho Neto, A. J. Garcia Jr., A. T. de Barroa, V. Pimentel, C. A. Ospina, J. Bettini, A. Malachias, S. O. Ferreira and Ch. Deneke. Setup and characterization of a compact, home-build molecular beam epitaxy system for overgrowth of small sized substrates. Submitted.

Authors thank M. Cotta, R. M.-Paniago, H. Schuler, F. Huber, K. Eberl, R. Kubiak, FAPESP (2011/22945-1) and CNPq (482729/2013-9), LNLS and LNNano for beamline and TEM facilities.

Fig. 1: Dark Field TEM image, using (004) reflection, sensitive to element concentration, shows four period InGaAs/GaAs superlattice.

Fig. 2: HRTEM image shows the absence of structural defects by imaging lattice fringes of InGaAs/GaAs.

Fig. 3: Dark Field TEM image, using (111) reflection, shows high crystal quality (no defects).

Fig. 4: Dark Field TEM image, using (022) reflection, for strain contrast.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3179 Microstructural characterization of AlGaN/GaN heterostructures grown on low angle-off 4H-SiC substrates

Gkanatsiou A.1, Lioutas C. B.1, Frangis N.1, Prystawko P.2,3, Leszczynski M.3,2
1Department of Physics, Aristotle University of Thessaloniki 54124, Greece, 2Institute of High Pressure Physics “Unipress”, Polish Academy of Sciences, Sokolowska 29/37, 01-142 Warsaw, Poland, 3TopGaN Ltd, Sokolowska 29/37, 01-142 Warsaw, Poland
alexgkan@auth.gr

The present work concerns the microstructural characterization of a multi-component (based on GaN and related materials) and multi-layered (5 layers) film, grown on 4H-SiC substrate (with a misorientation of ±0.5-2 degrees off from (0001) plane), using High Resolution Transmission Electron Microscopy (HRTEM). A typical sequence of the epilayers from bottom to top is: AlN nucleation layer-GaN layer-AlN spacer layer-AlGaN layer-GaN cap layer (Fig. 1).
The layers are grown epitaxially, as it is confirmed from the corresponding electron diffraction patterns (with the [0001] SiC direction parallel to the [0001] GaN direction, as seen in Fig. 1 inset a). Conventional TEM images allow the measure of the layers’ thicknesses. Sharp interfaces are observed between the layers. However, the AlN spacer layer is not uniform in thickness and in several areas the GaN/AlN and AlN/AlGaN interfaces are not too clear. Moreover, in the AlN nucleation layer V-shaped formations are observed (Fig.1 inset b). It is remarkable that the surface of the ±0.5 deg. sample appears a characteristic roughness, which is not present in the other sample.
HRTEM micrographs clarify the quality of the interfaces and of the defects observed in the layers. In the case of the 2 deg. off sample, multiple steps are observed at the interface between the AlN nucleation layer and the 4H-SiC substrate (Fig.1 inset c). The height of the steps is one atomic layer (about 0.25 nm) and their period is about 5-6 nm. Moreover, characteristic contrast (in conventional TEM and HRTEM images) suggests the growth of threading dislocations in the AlN layer that begin from the SiC/AlN interface at the steps’ positions. On the other hand, as shown in fig. 2, no steps are observed in the case of the ±0.5 degrees off sample. Additionally, Fast Fourier Transforms (FFT), performed on HRTEM micrographs taken from the AlN layer, confirm the GaN diffusion from the epilayer forming the V-shapes.

This work was supported by the JU ENIAC Project LAST POWER Grant agreement no. 120218 and the Greek G.S.R.Τ., contract SAE 013/8 - 2009SE 01380012.

Fig. 1: A multilayer structure grown on 4H-SiC substrate. Five layers were grown epitaxially on the substrate. a) An electron diffraction pattern taken close to SiC/GaN interface, revealing the very good epitaxial growth of GaN on the 4H-SiC substrate, b) V-shaped formations in the AlN nucleation layer, c) Steps in the SiC/AlN interface.

Fig. 2: An HRTEM image of the ±0.5 deg. off sample revealing no steps at the SiC/AlN interface showing the very good growth of AlN on the SiC substrate.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3239 A direct bond analysis of SrTiO3/SrIrO3 (3 bilayers)/(111) SrTiO3 oxide heterostructures based on aberration-corrected HRTEM images

Xie L.1,2, Anderson T. J.3, Eom C. B.3, Pan X.1,2
1National Laboratory of Solid State Microstructures and College of Engineering and Applied Sciences, Nanjing University, Nanjing 210093, P. R. China., 2Department of Materials Science & Engineering, University of Michigan, Ann Arbor, MI 48109, USA, 3Department of Materials Science & Engineering, University of Wisconsin, Madison, WI 53706, USA
xielin@nju.edu.cn

Oxide heterostructures have been intensively studied in recent years due to their novel physical properties, e.g. metal-insulator transition, ferromagnetism and superconductivity. These phenomena are related to the interplay between the lattice, charge, orbital and spin degree of freedom at the interface. Hence, a direct measurement of strain, cation displacement and oxygen octahedral rotation-tilt is essential for understanding the structure-property relation of these materials. In this work, we demonstrate a direct measurement of the bond length and bond angle of each oxygen octahedron across the interface based on aberration-corrected HRTEM.

Figure 1 shows the TEM image of a SrTiO3/SrIrO3 (3 bilayers) film grown on (111) SrTiO3 oriented along [110] zone axis. Negative spherical aberration imaging condition is used in the experiment and thus the image reflects the projected atomic structure of the heterostructure. To understand the structure of the heterostructure, we carried out a direct measurement of the atom positions by fitting the intensity of individual atomic column to a two-dimensional Gaussian peak. Based on the fitting results, the projected bond length and bond angle of each oxygen octahedron (defined in the inset of Fig. 1) is analyzed and the results are shown in Figure 2. We first note that the Sr-Sr bond lengths of the SrIrO3 film (6th to 8th oxygen octahedron) are larger than that of the substrate. It is interesting to point out that the Sr-Sr bond length of the 7th oxygen octahedra is much shorter than its neighboring octahedron. This may be due to the variation of Sr-Sr bond length projected along [1 0] direction for orthorhombic SrIrO3, which are 2.88 Ǻ and 2.69 Ǻ, respectively. The projected Sr-Sr bond length along [001] direction is consistent with the bulk value ~3.94 Ǻ. We also note that the projected bond length and bond angle between B-site cation and oxygen changes abruptly at the SrIrO3/SrTiO3 interface and extends only several atomic layers. Furthermore, from Fig. 2b and 2c, it is clearly that the bond length and bond angle between B-site atoms and oxygen is maximum/minimum. These results are not compatible with the bulk SrIrO3 structure and indicate that the ultrathin 3 bilayers thick SrIrO3 film is possibly in a new displacive polar state.

In summary, we have shown that the bond length and bond angle of oxygen octahedron across the SrTiO3/SrIrO3 interface can be directly measured from an aberration-corrected HRTEM image. We found that the ultrathin SrIrO3 film has a new displacive polar state different from its bulk structure. Although the exact crystal structure of the film remains unclear, our work provides an insight into the atomic structure of these materials with peculiar properties at the interface.

Fig. 1: HRTEM image of [110] SrTiO3/SrIrO3/(111) SrTiO3. The projected bond length and bond angle of the oxygen octahedron is defined in the inset and the number in the image indicates the n-th oxygen octahedron used for the analysis.

Fig. 2: The projected bond length of (a) Sr-Sr atoms and (b) Sr-B and O-B atoms and the projected bond angle of (c) Sr-B-O atoms. It is clear that the bond lengths of Sr-Sr and O-B and bond angle between cations and oxygen of the film deviate from the substrate substantially.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3260 Investigation of initial stage of sputtered ZnO thin film by TEM

Medlín R.1, Novák P.1
1New Technologies Research Centre, University of West Bohemia, Pilsen, Czech Republic
medlin@ntc.zcu.cz

Zinc Oxide (ZnO) is a wide bandgap semiconductor material which can be successfully used for wide variety of potential applications such as biosensors and acoustic resonator devices. Recently was found that preferred orientation of sputtered ZnO films can be controlled by applied substrate bias and substrate temperature during deposition.
This work is focused on the initial growth stage of sputtered ZnO film. Films were deposited by BOC Edwards TF600 sputtering system to formvar coated copper TEM grid glued by carbon paste to silicon substrate. Thin films were deposited at floating potential as well as at bias -100V. Thickness of prepared films varied from 6 nm to 50 nm at both conditions. The films were studied immediately after deposition by HR-TEM JEOL 2200FS equipped with autoemission Schottky gun, in-column energy filter, Oxford EDS detector and Gatan CCD camera with 2048×2048 pixels handled by Digital Micrograph. SAED patterns were treated by Process Diffraction.
We found that the biased samples started crystallization process differently and exhibit more random oriented nanocrystals. Lately nanocrystals get predominant orientation of crystallites. Structure of the films prepared at floating have less nanocrystals with 002 reflections oriented parallel with the beam. Moreover, the initial growth stage affects significantly the resulting film structure and preferred orientation. Obtained results will help to understand evolution of sputtered ZnO film structure at different conditions.

The result was developed within the CENTEM project, reg. no. CZ.1.05/2.1.00/03.0088.

Fig. 1: HR-TEM study of 6 nm thickness ZnO films deposited on floating potential.

Fig. 2: HR-TEM study of 6 nm thickness ZnO films deposited with bias -100V.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3349 Structure of Ni doped ZnO thin films prepared by magnetron sputtering

Rajendran S.1, Savkova J.1, Sutta P.1, Medlin R.1, Novak P.1
1New Technologies Research Centre, University of West Bohemia, Pilsen, Czech Republic
savkova@ntc.zcu.cz

Zinc Oxide (ZnO) is of great interest for various photonic and electrical applications due to its unique physical and chemical properties. ZnO films doped with transition metals like Co, Mn, Ni, has been studied as a promising material for diluted magnetic semiconductors.

This work concentrate on structure of nickel doped ZnO films prepared by reactive magnetron sputtering. Thin films with nickel concentration 4.8, 5.3 and 8.4 wt% were prepared using BOC Edwards TF 600 coating system. Film thickness has been measured on cross-section and varies from 500 nm up to 670nm. X-ray diffraction (PANalytical) with Cu Kα radiation, scanning electron microscopy (JEOL JSM7600F), energy dispersive analysis using X-rays (Oxford Instruments) and high resolution transmission electron microscopy (JEOL JEM2200FS) has been used for thin film characterisation.

X-ray diffraction analysis reveals that the Ni was doped interstitially in Zn sites of ZnO without forming any detectable secondary phase. Preferred orientation is in [001] direction to the substrate surface for all three samples. The influence of Ni content on surface morphology of the films has been determined by field emission electron microscope. For lowest Ni content (4.8 wt%) grain size is not uniform and clusters of very fine grains are present with few large crystallites. Fig. 1 shows FE-SEM micrograph of the film with 5.3wt% of nickel with uniform grain size. Nickel content 8.4 wt% results in smaller grain size with homogenous distribution. Columnar structure with column width less than 100nm has been observed for all examined samples (Fig. 2). EDX map of nickel, zinc and oxide has been collected to analyse elemental distribution in thin films. EDX maps revealed homogenous distribution of nickel in all three samples. SAED profile of ZnO:Ni shows the structure [wurtzite] of pure ZnO monocrystals since the Ni doped interstitially in Zn sites. Cross-section on TEM shows ZnO monocrystals in columnar structure.

The result was developed within the CENTEM project, reg. no. CZ.1.05/2.1.00/03.0088 that is cofounded from the ERDF within the OP RDI programme of the Ministry of Education, Youth and Sports.

Fig. 1: As deposited surface, 5.3wt% Ni

Fig. 2: Cross-section, 5.3wt% Ni

Fig. 3: HRTEM study of cross-section ZnO:Ni film
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3385 Study of the Deposition of DLC in steels API 5L for corrosion protection

Bottega Peripolli S.1 2, Silva Gomes L.1, Trava-Airoldi V.3, Guimarães de Oliveira L.1, Lucchese Marcia M.4, Machado G.5
1Centro de Tecnologia SENAI Solda (Sistema FIRJAN), 2Universidade Estácio de Sá - UNESA, 3Instituto Nacional de Pesquisas Espaciais, INPE, 4Universidade Federal do Pampa, UNIPAMPA, 5Centro de Tecnologia Estratégico do Nordeste, CETENE
speripolli@gmail.com

The extreme low coefficient of friction, hardness and high thermal conductivity of CVD diamond like carbon coatings films, can play an important role in the oil, gas and petrochemical industries in different kind of components (for example valves and mating parts) for wide range of applications are susceptible to a build-up of frictional forces in very aggressive chemical conditions, including H2S hydrogen sulfide. In many cases, coating with protective films may prevent future problems and extend the devices lifetime [2]. The current work is an interface study for DLC/SiH/Steel to understand the DLC excellent adhesion proprieties on API 5L steel, endorses its use anti-corrosion and protection applications. In this work, the DLC coating were grown by Chemical Vapor Deposition (CVD) method using 1 kV deposition voltage and the films were studied by scanning electron microscopy (SEM) and Raman Spectroscopy to study the interface nature of CVD diamond films grown on API 5L steel substrate [1]. Cross-sectional and surface SEM images were to measure the thickness of DLC films and elemental composition of the DLC film was determined by energy dispersive X-Ray analysis (EDX) using Quanta 450 from FEI Company. Figure 1 shows the surface morphology of deposited DLC film was continuous, smooth and uniform. Typical cross-sectional interface SEM image of DLC film presents an uniform thickness about 4 µm and good adhesion including a silicon interlayer. The energy dispersive X-Ray analysis (EDX) mapping results (figure 2) confirm the Carbon, silicon and iron presents in the sample. The Raman spectra shows the typical peak from DLC films (sp2 and sp3) related to the microcrystalline size of the graphitic cluster to 1kV deposition voltage (Figure 3). It is clear that more investigations are necessary to better understand the good adhesion, like adhesion tests and transmission electron microscopy (TEM) samples will be prepared using focused ion beam (FIB) to explain what is the SiH interlayer role in good adhesion properties.

References

[1] S.B. Peripolli, et al., Microscopy and MicroAnalysis 15 (2009) 57-58.

[2] M. Frenklach et al., In Diamond and diamond-like films and coatings, Eds.; Plenun Press: New York, 1991, p 499.

Fig. 1: SEM images from surface and cross-section interface from DLC coating on API 5L Steel substrate.

Fig. 2: Energy dispersive X-Ray analysis mapping (EDS) from cross-section DLC film.

Fig. 3: DLC film Raman spectra to 1kV and 0.8 kV deposition voltages
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3394 Determining the Crystal Structure of Metastable cubic SiN at the TiN/SiN Interface

Fallqvist A.1, Hultman L. G.1, Persson P. O.1
1Linköping University, Sweden
perpe@ifm.liu.se

The TiN-SiNx system is subject to intense research, mainly as a model system for superhard nanocomposite (NC) materials.1 Although the elements are commonly deposited simultaneously as a thin film, the TiN-SiNx nanocomposite formation is a consequence of phase separation of the immiscible components, leaving sharp interfaces between the two phases. It has been reported that such nanocomposites exhibit high hardness, which is of use for wear-resistant coatings.1 As a consequence of these small dimensions, dislocation glide is prevented while also the thin matrix prevents grain boundary sliding due to its high cohesive strength.2 While the structure of the crystallites is well known, e.g. B1 (NaCl) TiN in the TiN-SiNx system, the structure of the TiN-SiNx interface and the thin intergranular SiNx matrix has been debated for some time. The spatially constrained dimensions makes it challenging to just “look at it and see”. To limit the complexity, but also to investigate the hardening mechanisms a number of studies have reported successful growth of transition metal nitride-SiNx (001)-oriented multilayers and that these ML also exhibited increased hardness for thin SiNx layers.3 Depending on thickness of the SiNx layer, the ML structure can be grown epitaxially, indicating a crystalline nature of the SiNx. Constituting an epitaxial nature, these multilayers are the key towards understanding the nanocomposite TiN to SiNx interface. With increasing SiNx thickness, the layer assumes an amorphous structure and the epitaxial nature of the ML is lost. Through the significance of the TiN-SiNx (001) interface, it’s structural nature has been subject to intense theoretical studies. In contrast, few results have been published by high resolution microscopy methods.

In this contribution, the structure of a SiNx layer, epitaxially stabilized on TiN(001), is determined by atomically resolved aberration corrected scanning transmission electron microscopy ((S)TEM), using parallel high angle annular dark field (HAADF-) and annular bright field (ABF)-(S)TEM in combination with STEM image simulations. Complementarily, spatially resolved electron energy loss spectroscopy (EELS) spectrum imaging (SI) of the nitrogen (N-K) near edge fine structure (ELNES) was applied to the SiNx and corroborated with full potential calculations of candidate structures. This work was carried out at the Linköping double corrected Titan3, equipped with a Gatan Tridiem ERS imaging filter.

The study localizes the N atomic position in the structure, identifying the SiN structure to exhibit a B1 (NaCl) - like structure.

L. Hultman, et.al., Phys. Rev. B 75, 155437 (2007).

J. Schiøtz, F.D. Di Tolla, K.W. Jacobsen, Nature, 391, 561 (1998).

H. Söderberg, et.al., J. Appl.Phys 97, 114327 (2005).

The authors wish to acknowledge funding from the Swedish Research Council, and from the Knut and Alice Wallenberg Foundation for the Electron Microscopy Laboratory in Linköping

Fig. 1: The figure shows a layer of cubic SiN (indicated by an arrow in the HAADF images) embedded between TiN layers. The same location was viewed from the two different orientations, and enable identification of the N position in the structure from the ABF images.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3411 Using STEM-EELS to Observe Effects of Metallic Adhesion Layers on Plasmon Resonance in Electron Beam Lithographically Patterned Gold Thin Films

Madsen S. J.1, Koh A. L.2, Kempen P. J.1, Sinclair R.1
1Stanford University, Department of Materials Science and Engineering, Stanford, CA 94305-4034 USA, 2Stanford Nanocharacterization Laboratory, Stanford University, Stanford, California 94305-4045, USA
smadsen1@stanford.edu

Noble metals are often used when producing lithographically patterned plasmonic resonators, biosensors and other optical devices. Cr and Ti are the standard adhesion layers for the deposition of these metals. However, recent evidence shows that even a thin adhesion layer of 1-2nm can negatively impact the optical properties of these structures, such as plasmon resonance [1] and Raman signal enhancement.[2] The present study uses monochromated electron energy-loss spectroscopy in a scanning transmission electron microscope (STEM-EELS) to study plasmon resonances in structures with either a Ti adhesion layer or an organic molecular alternative, 3-mercaptopropyltrimethoxysilane (MPTMS).


Polymethyl methacrylate (PMMA) resist was spin coated onto 35nm thick silicon nitride membranes. 140nm diameter cylindrical holes were made in the PMMA by electron beam lithography. An adhesion layer, either Ti (2nm e-beam evaporated) or MPTMS (vapor deposited) was applied next, followed by 30nm of Au. A cross sectional schematic of the final structure is shown in Fig. 1a, as well as plan view bright field STEM images in Fig. 1b-c.


Spectrum images (SI’s) with 2.5 nm pixel size were acquired by STEM-EELS. Energy windows were selected from the SI’s and the intensities were normalized, generating an intensity map of the probability that an electron will lose the specified energy. Bright features on these maps can be interpreted as regions where plasmons of the chosen energy are excited by the electron beam. A series of these images generated from the MPTMS/Au sample are shown in Fig. 3a-d. Four different plasmon modes are apparent – localized surface modes at points 1, 2, and 3 and a mode at the bulk plasmon energy at point 4. In the Ti/Au sample, Fig. 3e-h, there are no regions which appear to have strong coupling between the electrons in the beam and plasmons in the sample. This damping of optical frequency resonance is proposed to arise from the large imaginary component of the dielectric function of Ti.[1] The damping is also evident by viewing the EELS spectra. Spectra from a small region around each of the labeled points in Fig. 3 were summed to give the charts shown in Fig. 2. The Ti/Au sample has peaks with less intensity at every position sampled.


Thus it has been demonstrated that STEM-EELS is capable of clearly resolving changes in plasmon resonance due to the use of as little as 2nm of Ti. Additionally, this study has shown that MPTMS can be used to produce evaporated metal structures with improved plasmonic properties compared to the more widely used Ti. The influence of this effect on the intensity of Raman spectroscopy signals is described elsewhere.[2]

[1] T.G. Habteyes et al, ACS Nano, 6 (2012) pp 5702–5709.
[2] S. Madsen et al, M&M, 20 (2014). In press.

This research is supported by the Center for Cancer Nanotechnology Excellence and Translation (CCNE-T) grant funded by NCI-NIH to Stanford University U54CA151459.

Fig. 1: Cross-sectional schematic (A) of the structures produced. (B) and (C) show plan view bright field STEM images of the MPTMS/Au and Ti/Au samples respectively.

Fig. 2: Electron energy loss spectra from the areas marked in Fig. 3. (A) corresponds to regions 1 and 4, (B) to region 2, (C) to region 3 and (D) integrated over the whole spectrum image . Note that the plasmon peaks are much less prominent when 2nm Ti is used as the adhesion layer.

Fig. 3: Normalized energy slices from spectrum images showing locations of plasmon excitation. Slices are 0.1eV wide and centered around the energy shown. The MPTMS/Au sample (A-D) shows modes excited at several different energies and locations (points 1-4), while the Ti/Au sample (E-H) does not.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3428 Development of nanoporous substrate PS/Au for SERS.

Fragal V. H.1, Silva R.1, Muniz E. C.1, Madina Neto A.1, Rubira A. F.1
1Universidade Estadualde Maringá, Maringá-Paraná, Brazil
afrubira@gmail.com

PS nanoporous films on low density polyethylene - LDPE were evaluated as substrates for Surface-enhanced Raman spectroscopy (SERS). SERS is a powerful analytical method capable of providing information about the structure of a variety of analytes in a non-destructive way. The sensitivity, reproducibility and stability of the SERS signal depend on the selection of an appropriate substrate(1). Thus, this study demonstrated the formation of a polymeric substrate for SERS from PS nanoporous films covered with Au thin film (sputtered film). Nanoporous films were obtained from 150 µL of a 10% (w/v) PS/THF solution. The solution was placed on LDPE and then rotated at 3000 rpm in spin coating for 10 seconds to generate a PS nanoporous film. Different spin speed, 1000 and 9000 rpm, were also tested. The humidity during the polymer casting was kept constant at 81%. A similar procedure was carried out using chloroform as solvent, which provided a non-porous PS film. Gold thin layers were sputtered on PS film using a current of 6 mA at different times, 10, 5 and 1 min. The efficiency of the nanoporous PS substrate with gold layer deposited for 5 min for generating SERS signal was evaluated using 10 µM 4- mercaptopyridine aqueous solution. AFM images before and after Au deposition of were presented in the Figures 1, 2 and 3. The Au deposition changes the topology of the initial nanoporous film and the nanoporous are not completely filled. Since the conditions 1 and 5 minutes Au deposition of the average depth of the nanoporous remained almost constant compared with the films without the deposition of Au [Figures 1a, 2a and 3a] and [Figures 1, 2 and 3 (c) and (d)]. SERS effect of the films obtained in PEBD_PSnanoporoso revolutions of 1000, 3000 and 9000 rpm, that have average pore size of 303 ± 68, 123 ± 23 and 80 ± 24 nm, respectively, were tested. The result of Raman spectrum for SERS using the substrate obtained at 3000 rpm and 5 minute can be seen in Figure 4. A characteristic
4-mercaptopyridine Raman bands are verified, intense bands at 1492, 1276, 1100, and 1040 cm-1 (2,3). The great similarity between the spectra of solid 4-mercaptopyridine [Figure 4 (b)] and 4-mercaptopyridine in LDPE/PSnanoporous/ Au film [Figure 4 (c)] can also be observed. In conclusion, it was possible to synthesize rapid, simple and inexpensive one nanoporous polymeric substrate of PS and Au nanoparticles. The limit of sensitivity of this substrate is being evaluated

References

(1) Banholzer, M. J.; Millstone, J. E.; Qin, L.; Mirkin, C. A. Chem. Soc. Rev. 37, 885-897 (2008);

(2) Silva, R.; Biradar, A. V.; Fabris L.; Asefa, T.J. Phys. Chem. C, 115:22810-22817 (2011);

(3)Zou, X.; Silva, R.; Huang, X.; Al-Sharabc, J. F.; Asefa T. Chem. Commun.49, 382-384 (2013).

The authors would like to thank CAPES, CNPq and INCT-Inomat for the financial support.

Fig. 1: AFM images and the average Rz of PS nanoporous at 1000 rpm on LDPE (a) without deposition of Au and the following times after deposition of Au (b) 10 (c) and 5 (d) 1 minute.

Fig. 2: AFM images and the average Rz of PS nanoporous at 3000 rpm on LDPE (a) without deposition of Au and the following times after deposition of Au (b) 10 (c) and 5 (d) 1 minute.

Fig. 3: AFM images and the average Rz of PS nanoporous at 9000 rpm on LDPE (a) without deposition of Au and the following times after deposition of Au (b) 10 (c) and 5 (d) 1 minute.

Fig. 4: SERS spectrum of 4 - mercaptopyridine (10-5mol/L) (a) PEBD/PS non- porous/5minAu (c) PEBD/PSnanoporous/5minAu and (b) 4 - mercaptopyridine solid. The wavelength used was 785 nm
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3431 CHARACTERIZATION OF LOW ELECTROCHROMIC VANADIUM OXIDE THIN FILMS PRODUCED BY MAGNETRON SPUTTERING.

Acosta D.1, Pérez A.1, Magaña C.1, Hernández F.1, Arenas J.1
1Instituto de Física, Universidad Nacional Autónoma de México A. P. 20 364, 01000, México D.F., MEXICO
jarenas@fisica.unam.mx

Vanadium oxide is a material that shows a phase transition of semiconductor to metal when is heated around of certain critical temperature. For the V2O5 compound,,this phase transition occur at 257±5°C. The study of vanadium compounds in thin film configuration, has received special attention in recent times because of their interesting electrochromic and thermochromic properties and potential uses as thermal sensing, optical switches, optoelectronic devices and energy saving devices with emphasis in the development of smart windows 1.
In this work, vanadium pentoxide (V2O5) thin films were deposited by RF magnetron sputtering with different deposition conditions: with and without O2, using a V2O5 target 2. A power of 100 watts during 10 minutes was used to deposit vanadium oxide on corning glass pure and coated with a conductive layer of SnO2:F (FTO) with an average sheet resistance of 7Ω/sq . The films were deposited on substrates kept at room temperature and 400ºC respectively. The optical and electrical properties were characterized by optical spectroscopy in the visible and ultraviolet range and the Four Points Van der Pauw method, respectively. Likewise, changes in resistance as a function of temperature were performed. The surface composition and morphological properties were followed with X-ray photoelectron spectroscopy (XPS) measurements and electron microscopy techniques. Cyclic voltammetry experiments were performed in a potential range: E0= -2800mV to E= potential 2800 mV vs. a platinum reference electrode with a scanning rate of 1000 mV/s. The cyclic voltammogram exhibits the evolution of the formation of vanadium oxides until the electrochromic species be obtained. Cycling runs, were done for 1, 10 and 60 cycles respectively and the coloration and decoloration processes at different rates, were observed for all the cases. X-rays diffraction patterns reveals low crystallinity mainly in samples deposited at room temperature. For samples deposited at 400ºC , HREM micrographs confirm low crystallinity in our samples.From SEM micrographs obtained before and after voltammetry cycling it was observed that V2O5 films look regular and compact, with an uniform grain size distribution. From SEM micrographs of films deposited at room temperature and 400 ºC and after cyclic voltammetry experiments it were detected modifications in grains configuration and surface details that might be related with sample degradation and loss of electrochromic activity as a consequence of mass and charge transport during the experiment.

The finanncial support of DGAPA-UNAM Project IN 105514 is appreciated and recognized. Also we thanks to Roberto Hernandez for technical help the and the financial support of DGAPA–UNAM to the Posdoctoral Position of Dr. Francisco Hernández in our laboratory.

Fig. 1: Spectra XPS survey of V205/FTO and V205/FTO films deposited without O2(black) and with O2(red) , respectively. The inset in the figure is core level of O 1s, V 2p 1/2 and V 2p 3/2 of narrow scan.

Fig. 2: A typical HREM micrograph of the V2O5 films deposited onto FTO glass substrate at (a) room temperature . HREM details are not appreciated along the whole sample.

Fig. 3: SEM micrographs of the V2O5 films deposited onto FTO glass substrate at (a) room temperature and (b) 400°C . The right side images correspond to samples after 60 voltammetric cycles; the degradation of film surfaces as a consequence of charge and mass transport processes inside the electrochemical cell can be observed.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-5714 Atomic Force Microscope to study mechanical interactions between neurons and nanostructured materials

Daza J.1, Monsalve G.2, Sutachan J.1, Gonzalez E.3
1Faculty of Sciences Pontificia Universidad Javeriana, Bogotá, Colombia , 2Departament of Surgery, Fundación Santafé, Bogotá, Colombia, 3Faculty of Engineering, Pontificia Universidad Javeriana, Bogotá, Colombia
egonzale@javeriana.edu.co

The study of interactions between neural cells and nanostructured substrates -such as surfaces and nanoparticles- is currently attracting great interest due to their potential applications in the area of health care, especially in diagnostic and treatment of neurodegenerative diseases. It has been clearly demonstrated that mechanical interactions of neurons with their extracellular matrix play an important role in the processes related to growth and development. Mechanical parameters such as roughness of the surface, elasticity or adherence are critical to understanding and controlling neuronal growth. Within this context, atomic force microscope (AFM) provides an important alternative approach to systematically study the mechanical interactions that occur between the substrate and neuronal cells. In this work, nanostructured gold surfaces of type Au(111) functionalized with self-assembled monolayers (alkanethiols, molecules formed by a sulfur binding group, a spacer chain , and a functional head group) are used. These surfaces can be topographically programmed to control behaviors such as growth or adhesion capabilities. With the high resolution of atomic force microscope and its ability to measure adhesion forces and other mechanical properties, it is also possible to obtain and investigate correlations between the nanometric surface topography and neuronal behavior. This has allowed us to establish criteria for the use of nanostructured surfaces for sensing responses of neurons under external electrical stimulation.

We thank the Vicerrectoría de Investigaciones, Pontificia Universidad Javeriana, for supporting our research program.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-3540 Electron beam micro-, nanofabrication and TEM studies of fine crystalline spots in thin amorphous films

Kolosov V. Y.1, Veretennikov L. M.1, Schwamm C. L.1
1Ural Federal University, Ekaterinburg, Russia
Vladimir.Kolosov@usu.ru

Electron beam in TEM column can be used as convenient fine probe for local film heating initiating multiple amorphous-crystalline transitions, crystal growth, recrystallization, etc. The e-beam intensity can be varied in a wide range by changing condensers current and focusing. The transformations presented were traced mostly in situ and followed by TEM studies involving bend-contour method for lattice orientation analysis [1] (supported by HREM, correlative AFM-TEM in due cases). We report on complex zonal structure, lattice orientations, texture and film thickness influence for round-like crystallized spots (around µm scale) and its nuclei. The spots of explosive and radial/tangential spherulite crystallization, textures or/and transrotational crystals [2] were grown by e-beams in various TEMs (5-200Kv) in thin (10-100 nm) amorphous films of different chemical nature (VI group & Ge-based materials, oxides, several metals and alloys, some other substances) produced by diverse methods (thermal, e-beam and laser evaporation, solid state amorphization). Several types of spots with characteristic features and differences are shown at Fig. 1-8. Crystals with most sophisticated structure finally acquire azimuthal spherulite-like regular misorientations complicated by internal lattice bending (transrotation) round axes lying in the film plane (FP), Fig. 1-3: Fig.1a (hexagonal Se, growth rate ~1 µm/s) - corresponds to regular perturbation around 2 poles (where [001] ┴ FP) with nucleation center in between ([001] || FP); Fig.2a (α-Fe2O3) - alternating circular single-crystalline ([001] ┴ FP) odd zones and fine-grained ([001] || FP) even zones; Fig.3 (Te & Cu-Te) – 2-phases alternating zones: odd zones – Te (with strong orientation gradients ~ 200°/µm), even zones - Cu-Te phase (with low gradients ~ 10°/µm), Fig. 1b, 2b – colored schemes of lattice orientations (for [001] of hexagonal structure) in corresponding crystals (Fig. 1a, 2a). The alternating zones studied can differ in lattice orientation (Figs. 2a, 6), material phases (Fig. 3-4), lattice imperfection (Fig.2a, 6). Dynamical (Fig.7a-c) and non-circular (Fig.6, 8) features are described.

Most details are presented for transrotational crystals (with regular translation of the unit cell accompanied by its slight rotation) that have been eventually recognized/studied by other authors in some thin film systems, i.e. PCMs for optical memory [3, 4], STO [5], silicides.

[1] I. E. Bolotov and V. Yu. Kolosov, Phys. Stat. Sol. 69a (1982), 85.

[2] V.Yu. Kolosov and A.R. Tholen, Acta Mater. 48 (2000) 1829.

[3] B. J. Kooi and J. T. M. De Hosson, J. App. Phys. 95 (2004), 4714.

[4] E. Rimini et al, J. App. Phys. 105 (2009), 123502.

[5] V. Longo et.al., ECS J. Solid State Sci. & Tech., 2 (2013), N120.

Partial RFBR support (grant 12-03-01118) is acknowledged

Fig. 1: - 8: TEM of spots in amorphous films crystallized by e-beam (Fig.1b, 2b – schemes of [001] orientation changes shown by rainbow colors; Fig.2-4, 6 - circular zonal structures; Fig.7a-c - subsequent images of a crystal growing in Tl-Se with bending changes at the center; Fig.8 - Se bent crystal without transrtotation, “egg cells” bending).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-5702 Nanoanalytical investigations at the interface of 4H-SiC/SiO2 MOSFETs

Tan H.1, Beltran A. M.1, 2, March K.3, Mortet V.2, Bedel-Pereira E.2, Cristiano F.2, Strenger C.4, Bauer A. J.4, Schamm-Chardon S.1
1CEMES-CNRS and Université de Toulouse, nMat group, Toulouse, France, 2CNRS, LAAS, 7 avenue du colonel Roche, 31400 Toulouse, France, 3Univ Paris 11, CNRS, UMR 8502, Lab Phys Solides, F-91405 Orsay, France , 4Fraunhofer IISB, Schottkystrasse 10, 91058 Erlangen, Germany
sylvie.schammchardon@cemes.fr

Despite the continuous improvement in performance and stability achieved in the development of 4H-SiC MOSFETs, the 4H-SiC MOS system still suffers from heavy carrier trapping at the SiO2/SiC interface. It was proposed that interface states at the SiC/SiO2 interface are responsible for the electron trapping but also bulk traps in SiC1. Furthermore it was suggested that the density of these bulk traps is significantly increased by ion implantation followed by high-temperature anneals. At the same time several groups provided experimental evidence for a carbon rich transition region on the SiC side of the interface with a C/Si ratio higher than one, and even, the width of the carbon-rich transition region was found inversely related to the peak field effect mobility2. It was also proposed that C di-interstitial defects in the SiC side of the interface account for the increased bulk trap density in SiC3.

In this work, differently processed n-channel planar MOSFETs manufactured on p-implanted n-type 4H-SiC epitaxial layers are considered. In particular, the effect of different channel implantation concentrations is examined. We have investigated the structural and chemical state of these MOSFETs focusing on the structural state and C distribution at the interface using high resolution scanning transmission electron microscopy (HR-STEM) (0.1 nm) and spatially resolved electron energy loss spectroscopy (STEM-EELS). The Si-L edge (100 eV), C-K edge (284 eV) and O-K edge (532eV) were collected in the same spectrum. The n-channel MOSFETs were investigated after FIB sample preparation.

Based on the relative compositions extracted from our EELS data, no C excess is evidenced for the samples neither in the SiC substrate nor in the SiO2 gate oxide. However, modification of the Si-L ELNES was revealed and numerically exploited. In particular, fitting of the Si-L edge evolution across the interface with a linear combination of reference spectra (Si, SiC and SiO2) evidences the presence of a « suboxide » over a short distance (less than 2 nm) at the SiC/SiO2 interface. It implies a transition layer where the Si bonding is modified compared to what is observed either in SiC or in SiO2. These results will be commented with regard to electron mobility measurements4.

1. A Agarwal, S Haney, J. Elec. Mater 37, 646 (2008)

2. T.L.Biggerstaff et al., Appl. Phys. Lett. 95, 032108 (2009)

3. X. Shen et al., Appl. Phys. Lett. 98, 053507 (2011)

4. A. M. Beltran et al., Materials Science Forum 711, 134 (2012)

* A.M. Beltran now at CENEM, Universität Erlangen-Nürnberg, Erlangen, Germany

Work performed by the French-German Consortium MobiSiC, supported by the Programme Inter Carnot Fraunhofer from BMBF (G.A.312483). Thanks to the French METSA network for the access to the probe-corrected Ultra-STEM Nion microscope, Orsay, France.

Fig. 1: (a) Schematic cross-section of the studied MOSFETs; (b) STEM-EELS elemental maps across one 4H-SiC/SiO2 interface and the corresponding C/Si ratio map; (c) STEM Bright-field (left) and STEM-HAADF (right) images across one 4H-SiC/SiO2 interface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-5697 Sputtering Targets Produced from Magnetite Nanoparticles

Perez-Herrero G.1, Baggio-Saitovitch E.2, Solorzano I. G.3
1Instituto Nacional de Metrologia (Inmetro), 2Centro Brasileiro de Pesquisas Físicas (CBPF), 3Pontifícia Universidade Católica (PUC-Rio)
perezgeronimo@hotmail.com

The semi-metallic Fe3O4 films have attracted interest by the characteristic of combining a 100% spin polarization with a high Curie temperature [1] and have a relatively high conductivity [1]. These have been of great interest due to the properties of spin in an insulating material, therefore, are candidates for spintronic applications [2], such as magnetoresistive devices or magnetic tunneling junctions [1, 3]. It has also been of great interest to study the transition temperature Verwey and transport properties observed in thin films of magnetite [4].

Magnetite thin films were produced using the sputtering RF (radio frequency source) deposition system. The thin films were deposited on a silicon substrates. The formation of the magnetite after the deposition was confirmed by x-ray (XRD) diffraction and vibrating sample magnetometer (VSM). The magnetite films presented a magnetic saturation near 85 emu/cm3 at longitudinal direction (easy magnetization direction). The targets to sputtering were produced by compression of magnetite nanoparticles previously produced by chemical method of co-precipitation from mixing of iron salts and ammonium hydroxide. Fig. 1 shows TEM micrograph of magnetite nanoparticles: (a) bright field, (b) dark field, (c) its respective selected area electron diffraction pattern.

Periodic arrays of circles and squares were produced by electron beam lithography combined with sputtering deposition and lift-off process, a squares array of 1 μm and arrays of circles of 1 μm, 500 nm and 250 nm in diameter formed of a magnetite film 80 nm thick. The first step was the preparation of polymethylmethacrylate (PMMA) film of 250 nm thick by spin coat method on silicon substrate. At the second step, this substrate was written by electron beam and later, immersed into acetone solution for some seconds to produce the mask of the arrays. Then, the magnetite film was deposited onto the lithographed sample by RF sputtering. Finally the sample was immersed in acetone until all the PMMA film has been lifted-off. The film thickness, shape, size and separation between the figures which comprise standards lithographed can influence the ease with which the mask is withdrawn from PMMA.

Scanning electron microscopy (SEM) and atomic force microscopy (AFM) images provide additional topographical information. The AFM provides good topography and thickness information. Fig. 2 show the AFM topography images of the shapes corresponding to different arrays.

References:

[1] H. Takahashi, et al, J Appl Phys, 2003,93: 8029-8031.
[2] X. L. Tang, et al, Journal of Solid State Chemistry 179 (2006) 1618–1622.
[3] K. I. Aoshima and S. X. Wang, J. Appl. Phys. 93 (2003) 7954.
[4] L. Pan, et al, Thin Solid Films 473 (2005) 63– 67.

Fig. 1: TEM micrograph of magnetite nanoparticles: (a) bright field, (b) dark field, (c) selected area electron diffraction pattern.

Fig. 2: AFM images of the shapes corresponding to different arrays: (a) 250 nm circles, (b) 500 nm circles, (c) 1 μm circles, (d) 1 μm squares.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-5968 Significant increase of Cr-C-Cr bond length in the surface relaxation of Cr2AlC thin film

Chen Y. T.1,2,3, Music D.1, Shang L.1, Mayer J.2,3, Schneider J. M.1
1Materials Chemistry, RWTH-Aachen, D-52056 Aachen, Germany , 2Central Facility for Electron Microscopy, RWTH-Aachen, D-52056 Aachen, Germany , 3Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, PGI-5, Forschungszentrum Julich, 52425 Juelich, Germany
chen@mch.rwth-aachen.de

Cr2AlC thin film with MAX-phase structure was deposited by magnetron sputtering. Ab initio calculation of the structural was performed to obtain the relaxed lowest total energy and coordinates. Accordingly, a supercell was obtained and based on it, STEM-HAADF image was simulated with the consideration of temporal and spatial coherence of the electron beam. The result was matched with the experimental observation performed with atomic resolved STEM-HAADF images, as shown in Figure 1.

The lattice distortion in the vicinity of a growth-induced void was observed and analyzed. The distortions were observed in the vicinity of several voids, and can be as large as 23.5% of the surface layer, as shown in Fig. 2. 

In order to investigate the mechanism behind the distortion, ab initio calculation was performed for simulating the surface relaxation effect, as shown in Figure 3.

The ‘a’ lattice constant was controlled to shrink from 0% to 17.6%. The Cr atoms at the upper end shown in the figure are fixed and all the other atoms are allowed free to move during the relaxation calculation. As the result, the Cr-C-Cr bond was found to be expanded by as much as 49.0% in the basal plane direction while the perpendicular in-plane strain (17.6%) was applied on. Meanwhile, the Cr-Al-Cr lattices only increased by 7.2%. Although the total equilibrium energy increased from -242.3 eV to -199.5 eV by calculation, the structure remained stable therefore, the cause of the measured lattice distortion appears to be surface relaxation.

Fig. 1: Model construction, STEM-HAADF, and simulation[1] of the HAADF pattern has been performed. The result of experiments and calculation matched as shown.

Fig. 2: A void is investigated with atomic resolved STEM-HAADF. The lattice can be distorted to the vacuum as large as 23.5% in the direction of basal plane.

Fig. 3: Ab initio calculation shows the Cr-C-Cr bond is much larger affected (49.0% increase) by the applied lateral compressive strain, compared to the Cr-Al-Cr bond (7.2% increase).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-3-P-6008 Microstructure and phase modifications of CGDS and HVOF-sprayed CoNiCrAlY bond coats remelted by electron beam.

Gavendová P.1, Čupera J.1, Hanusová P.1, Čížek J.1, Dlouhý I.1
1Brno University of Technology, Faculty of Mechanical Engineering, Institute of Materials Science and Engineering, Brno, Czech Republic
gavendova@fme.vutbr.cz

The paper deals with CoNiCrAlY coatings manufactured by the high-velocity oxygen-fuel (HVOF) and cold gas dynamic spraying (CGDS) deposition techniques and modified by electron beam (EB) remelting. HVOF and CGDS spraying method was applied in order to obtain very dense and good adhesive CoNiCrAlY-coatings deposited onto nickel-based alloy. The bond coat having thickness of about 70 m. The electron beam remelting process is one of the most advanced convenient processes to reduce the main disadvantages of thermal spray coatings. The effect of high-energy electron beam surface remelting and microstructural modification in CoNiCrAlY bond coats have been investigated in this study. The electron beam remelting of both HVOF and CGDS – coatings has been proven to minimize the porosity and, in addition, modified the microstructure morphology and the phase composition of the CoNiCrAlY bond coats. Scanning electron microscopy, light microscopy and X-ray diffraction methods were performed to characterize the phase modifications and morphology before and after electron beam and thermal ageing treatment. The results obtained in this study could be summarised as follows: The pulsed electron beam surface remelting contributed to improvement of the bond coat to surface interface and, at the same time, to the refinement of this area. EB-treatment provided a smooth BC surface with low porosity level. The microstructure of the bond coat after this treatment has been is formed by Inconel fine grain layer being followed by the surface layer consisting of elongated dendritic microstructure. The longitudinal axis of dendrites has been oriented predominantly perpendicularly to the Inconel surface. Comparing to as-sprayed CGDS and HVOF coatings after standard vacuum heat treatment better operation properties are expected when using the EB remelting. The findings have also shown that bond coat achieved using low-temperature kinetic spraying appears to be perspective for practical applications.

The works have been partly supported by the financial support from the Operational Programme Education for Competitiveness no. CZ.1.07./2.3.00/30.0005 and within the project Netme plus centre (lo1202), project of ministry of education, youth and sports under the “national sustainability programme.

Fig. 1: CoNiCrAlY coatings before electron beam remelting.

Fig. 2: CoNiCrAlY coatings after electron beam remelting.
↑ Go to the Table of content ↑

MS-4. Metals, alloys and metal matrix composites

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-4-IN-1916 Crystal structure and deformation of long-period stacking-ordered intermetallic phases in the Mg-TM-RE systems

Inui H.1,2, Kishida K.1,2
1Department of Materials Science and Engineering, Kyoto University, 2Center for Elements Strategy Initiative for Structural Materials (ESISM), Kyoto University
inui.haruyuki.3z@kyoto-u.ac.jp

Mg alloys containing ternary Mg-TM(Transition-metal)-RE(Rare-earth) phases with long-period stacking-ordered (LPSO) structures have received a considerable amount of attention in recent years. Although reasons why these alloys can simultaneously exhibit high strength and high ductility have been remained largely unsolved, ternary LPSO phases have been believed to play important roles in endowing them with excellent mechanical properties. Mg-Zn-RE LPSO phases are reported to consist of structural blocks with five to eight close-packed atomic planes, forming various polytypes with different numbers of the close-packed atomic planes in the structural blocks and with different stacking of the structural blocks. In the absence of the in-plane long-range ordering of the constituent atoms (as usually assumed in most studies in Mg-TM-RE LPSO phases), polytypes expressed as 10H, 14H, 18R and 24R polytypes are reported to form, among which 14H and 18R polytypes are the most dominantly observed ones.　However, the details of the crystal structure are still controversial. We have very recently investigated the crystal structure of the 18R-type LPSO phase newly found in the Mg-Al-Gd system by scanning transmission electron microscopy (STEM) and transmission electron microscopy (TEM) and successfully determined the in-plane arrangement of the enriched layers [1,2]. The 18R-type Mg-Al-Gd LPSO phase is composed of 6-layer structural blocks with fully-ordered atomic arrangement. The enrichment of RE (and TM) atoms occurs in four consecutive close-packed atomic planes in each structural block and the long-range atomic ordering involving a periodic arrangement of Al6Gd8 clusters of the L12 type occurs in the four consecutive atomic planes (Figs. 1 and 2). However, it should be noteworthy that the stacking sequence of the 6-layer structural blocks does not exhibit any long-range order along the stacking direction (Fig. 3). Because of these characteristics, the LPSO phase in the Mg-Al-Gd system cannot be described as an ‘LPSO’ phase any longer in a strict sense but as an order-disorder (OD) intermetallic phase with a so-called OD structure [1-3]. In the presentation, we will present the details of the crystal structure of the OD/LPSO phases in Mg-TM-RE alloys on the basis of the OD theory. Deformation behavior of the OD/LPSO intermetallic phases will also be presented.

References

[1] M. Yokobayashi et al., Acta Mater., 59 (2011) 7287.

[2] K. Kishida et al., Intermetallics, 31 (2012) 55.

[3] K. Kishida et al., Philos. Mag., 93 (2013) 2826.

This work was supported by Grant-in-Aid for Scientific Research (Nos. 23360306 and 23109002) and the Elements Strategy Initiative for Structural Materials (ESISM) from MEXT, Japan.

Fig. 1: Atomic resolution HAADF-STEM images of the Mg-Al-Gd OD phase.

Fig. 2: Periodic arrangement of Al6Gd8 clusters with the L12-type atomic arrangement in the 6-layer structural block projected along [0001].

Fig. 3: Variation of the stacking sequence of the 6-layer structural blocks.
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-4-IN-2880 STEM studies of Ag-, Cu- and Zn-containing precipitates in Al-Mg-Si alloys

Saito T.1, Wenner S.1, Marioara C. D.2, Andersen S. J.2, Lefebvre W.3, Holmestad R.1
1Department of Physics, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway, 2SINTEF Materials and Technology, 7465 Trondheim, Norway, 3Université de Rouen, GPM, UMR CNRS 6634 BP 12, Avenue de I’Université, 76801 Saint Etienne du Rouvray, France
randi.holmestad@ntnu.no

The 6xxx series aluminium alloys, with magnesium and silicon as primary alloying elements, are widely used as structural materials, for example in the construction and automotive industry. The alloys are age-hardenable, as they acquire strength through the formation of nanoscale, needle-shaped, metastable precipitate phases during heat treatment. Our objective is to understand more of the fundamental physics going on at the atomic scale, which governs nucleation, phase stabilization and precipitation in these alloys. The morphology, structure and strengthening properties of age-hardening precipitates depend on the alloy composition and the thermo-mechanical history of the material. Being able to understand the atomic structure of the precipitates, how they affect each other and the material’s physical properties, composition and heat treatment can be optimized to tailor alloys with optimal properties. In recycled Al alloys, several heavier elements can be found, but they can also be added on purpose to obtain better properties. Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) is an excellent technique to study the distribution of elements such as Ag, Cu and Zn in the precipitates [1], also electron energy loss spectroscopy (EELS) has been used to study the composition of individual atomic columns in the precipitates[2].

The main hardening precipitates form and grow as needles along <100>Al, and are observed in cross-section in high resolution HAADF-STEM. When Cu, Zn or Ag is added, the precipitate structures often become disordered, with no unit cell. All precipitates still contain an ordered network of Si atomic columns. We see preferred local atomic configurations which do not exist in the more common β’’ phase. Cu, Zn and Ag atomic columns are observed to locate either in-between the Si-network columns or (fully or partly) substituting a Si-network column. In both cases, they form the center in a three-fold rotational symmetry on the Si-network. Another observation is that the disordered precipitates consist of fragments of known phases in the Al-Mg-Si alloy system, connected through the common Si-network. All elements reside also in Al fcc positions at the precipitate/matrix interface. STEM image simulations and quantitative analysis are used to estimate the occupancy of heavier elements in the different columns. The presentation will show an overview of recent work done in the group.

References:

[1] T Saito, CD Marioara, SJ Andersen, W Lefebvre and R Holmestad, Phil. Mag., (2014) 94, 520.

[2] S Wenner, CD Marioara, QM Ramasse, DM Kepaptsoglou, FS. Hage, R Holmestad Scripta Mat. (2014) 74, 92.

The authors want to thank the Norwegian Research Council and Hydro Aluminium for financial support, and prof. Kenji Matsuda, Toyama University for good collaboration.

Fig. 1: HAADF-STEM images of disordered precipitates viewed in cross-section along <001> Al (a) in Cu-containing, (b) Zn-containing and (c) Ag-containing precipitates in Al-Mg-Si alloys at peak hardness. The images are filtered to reduce noise using a circular low-pass mask that removes all period shorter than 0.15 nm.

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-4-IN-5743 The role of atom probe tomography in physical metallurgy

Blavette D.1, Cadel E.1, Chbihi A.2, Sauvage X.1
1Groupe de Physique des Matériaux, UMR CNRS 6634, Normandie University, France, 2CEA SRMA, Saclay, France
didier.blavette@univ-rouen.fr

Atom Probe Tomography (APT) is the only analytical microscope able to map out the distribution of chemical species in materials at the atomic-scale in the three dimensions. It has shown to be a quantitative instrument for the measurement of phase composition in solids including metals, oxides, and semiconductors. The volume that can be reconstructed is close to 50x50x100 nm3 (figure 1). The composition in a small selected volume (1 nm3) within this volume can be measured. The spatial resolution of the instrument is 0.1 nm in depth and a fraction of a nm at the specimen surface. The first French prototype (the tomographic atom probe-TAP) was designed and set up in the lab in the early nineties and subsequently marketed by CAMECA [1]. With the development of laser-enhanced instruments, the investigation of non-conducting materials (oxides, semiconductors) was made possible [2]. Together with the use of FIB techniques for the preparation of specimens, this innovation has opened a considerable development of APT in nanosciences (e.g. spintronic) including microelectronics [3].

Because of its ultimate spatial resolution and quantitativity in composition measurements, APT is a well suitable technique to investigate the early nucleation of a new phase in solids as well as the segregation of impurities to Cristal defects (grain boundaries, dislocations (figure 1), stacking faults…)[4]. APT has also been extensively employed to study precipitation kinetics (figure 2). One of the force of APT is that 3D images can be directly confronted to Kinetic Monte-carlo simulations (rigid lattice) [5]. The composition and structure of nuclei during the early stages of solid state phase separation in binary systems is a challenging problem both from a theoretical and an experimental point of view and is of utmost importance for applications. APT investigations showed that nuclei have sometimes a solute concentration smaller than the equilibrium phase [6]. In this presentation, the role of APT in material science will be illustrated through a few selected examples related to segregation to crystal defects and to the early stages of precipitation.

[1] D. Blavette, A. Bostel, J.M. Sarrau, B. Deconihout and A. Menand, 1993, Nature 363, 432

[2] B. Gault, F. Vurpillot, A. Vella, M. Gilbert, A. Menand, D. Blavette, B. Deconihout, Rev. Sci. Instr. 77, 043705 (2006)

[3] I. Mouton, R, Larde, E. Talbot, C. Pareige, D. Blavette, Journ. Appl. Phys. 115, 053515 (2014)

[4] D.Blavette, E. Cadel, A. Fraczkiewicz, A. Menand, SCIENCE Dec 17 (1999) 2317-2319

[5] C. Pareige, F. Soisson, G. Martin, And D. Blavette, Acta Met Mater. 47-6 (1999) 1889-99

[6] A. Chbihi, X. Sauvage, D. Blavette, Acta Materialia Volume 60, Issue 11, June 2012, 4575–4585

Fig. 1: 3D reconstruction of a Cottrell atmosphere in boron-doped FeAl intermetallics (iron is not represented for the sake of clarity). APT image reveals the segregation of boron atoms to the dislocation line. Boron concentration (2at.%) was found to be 400 times the nominal composition (400 at.ppm). Note the presence of an Al-depleted zone.

Fig. 2: 3D map (107 atoms - 40×40×150 nm3) of a model nickel base superalloy containing small Al-enriched precipitates (7 nm). Small Al-enriched precipitates (7 nm in diameter, 18at.%) embedded in a Cr-enriched parent phase are evidenced.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1455 Application of GPA and HRTEM for strain mapping of a graphite metal matrix composite

Hernandez-Rivera J. L.1, Aranda-Cstillo M. G.2, Cruz-Rivera J. J.2, Garibay-Febles V.3
1Centro de Investigación en Materiales Avanzados (CIMAV), Laboratorio Nacional de Nanotecnología, 2Facultad de Ingeniería-Instituto de Metalurgia, Universidad Autónoma de San Luis Potosí, 3Instituto Mexicano del Petróleo, Laboratorio de Microscopia Electrónica de Ultra Alta Resolución
jlhri10@yahoo.com.mx

Aluminium composite was processed by means of mechanical alloying with the aim to disperse graphite particles. Subsequent processing was cold compression, sintering and then hot extrusion. Samples for electron microscopy were obtained from extruded bars and were prepared using ultrasonic cutting, mechanical grinding, jet electropolishing and ion milling. It has been established previously that significant strain gradients are created in the metal matrix because of thermal mismatch between matrix and reinforcement particles. The presence of these strain gradients can produce significant increments in the flow stress and also an alteration in the kinetics of precipitation during the heat treatment.

We have applied the geometric phase analysis (GPA) technique to measure strain in the (001) plane of Al in the vicinity of the graphite particles. In order to avoid commonly observed artefacts in strain maps generated from HRTEM images we applied the GPA technique to the complex-valued exit face wave function reconstructed from a series of HRTEM micrographs recorded at different values of the defocus with the sample oriented in the [001] zone axis of the matrix. Strain maps were also obtained from individual defocus micrographs in order to compare it with reconstructions results. It was possible to establish that there were tension and compression elastic strain gradients that extend out to about 15 nm around the graphite particles. According to the results obtained the strain was heterogeneous and had an average value of 0.9 % in areas close to the particles

JLHR appreciates the endless and invaluable support from all members of the Stuttgart Electron Microscope group (StEM) at the Max Planck Institute for Intelligent Systems

Fig. 1: TEM micrograph that shows the graphite particle inside Al matrix.

Fig. 2: HRTEM micrograph that shows the interface between graphite and Al. Also it is indicated the direction on which strain was measured

Fig. 3: Strain map obtained in the ɛyy direction (transverse) on which it is demonstrated that strain was negligible

Fig. 4: Strain map obtained in the ɛxx direction (longitudinal) on which can be seen strain gradients
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1494 STEM Diffraction Imaging for Evaluating Ultra-fine MC Carbides Embedded in Steel

Nakamichi H.1, Yamada K.2, Sato K.3
1Steel Research Lab., JFE Steel Corp., 1-1, Minamiwatarida-cho, Kawasaki, 210-0855 Japan, 2Steel Research Lab., JFE Steel Corp., 1, Kokan-cho, Fukuyama, 721-8510 Japan, 3Steel Research Lab., JFE Steel Corp., 1, Kawasaki-cho, Chiba, 260-0835 Japan
h-nakamichi@jfe-steel.co.jp

[Introduction]
      Precipitation hardened high strength steel with an excellent formability has been developed [1] and a typical TEM micrograph is shown in Fig.1. MC type carbides (black contrast) formed as columnar in a row have a thin plate shape on three equivalent habit planes ({001}) with high coherency obeying Baker-Nutting relationships. For understanding the precipitation as well as hardening mechanism, precise and high throughput evaluation techniques are required. Because of their coherency and small volume fraction, it is difficult for imaging and analyzing those precipitates when the size of MC carbides is small. The objectives of present experiments are establishing high throughput and precise methods for evaluating distribution and morphologies of precipitates using STEM-ADF techniques.
[Experimental Procedure]
      800MPa tensile strength grade Ti-Mo bearing hot rolled steel is used the present experiment. STEM observations are carried out using a probe Cs-corrected STEM (FEI, TITAN80-300) operated at 300kV. STEM-ADF images are taken from an Fe [001] direction through an annular detector with inner scattered semi angle of from 18mrad to 230mrad. Elemental analysis is also conducted using EDS.
[Results and Discussion]
      Fig.2 shows STEM-ADF images, which are taken by changing the electron scattered angle between 18 mrad and 54mrad from Fe [001] direction. It is found that platelet precipitates on three equivalent habit plane, schematically shown in figure, are observed in Fig.2a) and b). Square shape precipitates indicated by arrows are not recognized under high scattered angle of larger than 40mrad. This result found that high angle scattered imaging (HAADF) is not useful in this situation. It is supposed that six g110 of diffraction spots from [001] pole figure are excited and those images are collected as an ADF image when the scatter angle is small. Based on this reason, it is difficult to recognize these three equivalent precipitates at one time through TEM.
      Fig.3 shows the interaction between dislocation and precipitates using this method. It is found that both needle shape and square shape precipitates are pinning the dislocation. Fig.4 is high resolution image of precipitate. EDS analysis is carried out with the same condition of imaging and it is found that MC has almost uniform composition of Ti/Mo=1 and no core-shell structure.
[Summary]
      From low to high magnification investigation with the same condition is possible using low angle scattered electron (around 20mrad) STEM imaging and this technique has huge advantage of high through put for evaluating precipitation distribution as well as their sub-nano structures.
[References]
      [1] Y. Funakawa et al. : ISIJ Int. 44 (2004) 1945

Fig. 1: TEM micrograph of precipitate hardened steel.

Fig. 2: STEM-ADF images of precipitates with various scattered angles at same area.

Fig. 3: STEM-ADF low magnification image of interaction between precipitate and dislocation.

Fig. 4: STEM high magnification image of precipitates.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1538 Grain microstructure and local texture in ball milled ODS steel particles revealed by the TEM ACOM method

Sallez N.1, Donnadieu P.1, Courtois-Manara E.2, Kübel C.2, Blat-Yrieix M.3, de Carlan Y.4
1laboratoire SIMaP Domaine Universitaire 38402 Saint Martin D’Hères, France, 2Karlsruhe Nano Micro Facility & Institute of Nanotechnology KIT 76344 Eggenstein-Leopoldshafen Germany, 3EDF Les Renardières 77818 Moret-sur-Loing, France, 4CEA DEN SRMA 91191 Gif-sur-Yvette, France
patricia.donnadieu@simap.grenoble-inp.fr

As expected from the extreme deformation involved in ball milling and confirmed by X ray diffraction (XRD) line profile analysis, powder particles prepared by this process are considered as formed by nanocrystallites. Indeed, in these systems, structural knowledge relies on global information given by XRD because the high strain contrast prevents from good TEM imaging condition. However the recent TEM ACOM method (1) should be able to overcome this difficulty. TEM-ACOM is based on the scanning by a narrow parallel beam combined to the acquisition of the local diffraction patterns. Indexation of the diffraction pattern data provides a map containing phase information and crystallographic orientation on each point of the scanned area. This method gives similar maps as SEM-EBSD with the specific advantage to be reliable even in presence of a high strain level. Therefore information like grain microstructure and local distortion can be reached.

In the present work, TEM-ACOM has been applied to ODS steel powders prepared by ball milling. According to XRD analysis, these materials developed for fuel cladding application have a fine scale microstructure (crystallite size ~ 30 nm).The TEM ACOM experiment was carried out with a parallel beam (probe size 1nm) scanned with a 2.5 nm step on FIB sections taken in ODS steel particles. The data indexation was refined in order to have an angular resolution of ~ 0.2°.

Figure 1 displays the orientation map obtained on an as milled ODS steel particle. The color coding of local orientation directly reveals that the microstructure is formed of long grains (width ~ 50-100 nm length up to several microns). At first sight, the microstructure does not seem made of 30 nm crystallites as indicated by XRD. But misorientation profiles taken across a grain (Fig. 2 and 3) reveal that within the grains, there are domains of about 30 nm separated by thick dislocation walls. These low misorientation domains correspond to the coherent domains measured by XRD line width. Indeed there are no contradictions between the XRD nanoscale domains and the TEM ACOM long grains; only XRD is blind to the local texture between domains.

TEM ACOM appears then as a very appropriate method to analyze complex micro /nanostructures and to provide relevant information. For instance in ODS steel powders, information on local texture as given by TEM ACOM are of significant importance for understanding the evolution under further processing like consolidation and extrusion.

(1) E.F. Rauch, M. Véron, Coupled microstructural observations and local texture measurements with an automated crystallographic orientation mapping tool attached to a TEM. Materialwissenschaft und Werkstofftechnik 36, 552 (2005)

We acknowledge for support to "CPR ODISSEE" program funded by AREVA, CEA, CNRS, EDF and Mécachrome (contract n°070551) and to the Karlsruhe Nano Micro Facility (www.kit.edu/knmf).

Fig. 1: TEM-ACOM orientation map of a ball milled ODS particle FIB section. Note that numerous grains exhibit a very high anisotropy (shape factor > 30). The gradual color change within the grains indicates local distortion. The orientation map is overlapped in each point with the indexation reliability value.

Fig. 2: High magnification view of TEM-ACOM map of the as-milled powder. Using ACOM analysis tool, a profile of misorientation showing the local and cumulated distorsion is taken in a elongated grain along the line marked in white. The cumulated misorientation corresponds to the global deformation

Fig. 3: Misorientation profile revealing domains without misorientation (i.e. coherent domains) or with high and uneven misorientation corresponding to dislocation walls or entangled dislocations
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1559 Structure-Property Relationships in Fe-Mn Austenitic TRIP/TWIP Steels Determined With Conventional and Aberration-Corrected Transmission Electron Microscopy

Wittig J. E.1, Pierce D. T.1, Bentley J.2, Beigmohamadi M.3, Mayer J.3
1Interdisciplinary Materials Science, Vanderbilt University, Nashville TN, 37235 USA, 2Microscopy and Microanalytical Sciences, PO Box 7103, Oak Ridge, TN 37831-7103, USA, 3Central Facility for Electron Microscopy, RWTH Aachen University, Aachen, Germany
j.wittig@vanderbilt.edu

A new class of austenitic steels stabilized with high Mn contents (instead of Ni) exhibits exceptional mechanical properties, such as large energy absorption and high work-hardening rate, owing to secondary deformation mechanisms such as mechanical twinning-induced plasticity (TWIP) and martensitic transformation-induced plasticity (TRIP) favored for low stacking-fault energy (SFE) [1]. The interaction of dislocations with twin boundaries and martensite interfaces during mechanical deformation enhances the work hardening, i.e., a dynamic Hall-Petch effect, with total elongations exceeding 70% and ultimate tensile strengths in the GPa regime.

In this investigation, the SFE and deformation mechanisms of Fe-(22,25,28)Mn-3Al-3Si (wt%) austenitic steels have been studied with a combination of conventional and advanced electron microscopy to make correlations with the work-hardening behavior and mechanical properties. The SFE measurements employed weak-beam dark-field (WBDF) imaging to measure the separation of partial dislocations. Figure 1 is a WBDF image from Fe-22Mn-3Al-3Si recorded with a Philips CM20. Using single-crystal elastic constants to determine the effective shear modulus on the (111) slip plane and effective Poisson’s ratio, the SFE energies for the 22, 25 and 28% Mn alloys are 15 ± 3, 21 ± 3 and 39 ± 5 mJ/m2, respectively [2]. Deformation mechanisms were characterized by bright-field (BF) imaging of interrupted tensile tests. Figure 2 shows epsilon-martensite lath formation in the 22% Mn alloy after 10% strain. As the SFE increases, the secondary deformation changes from martensite to mechanical twining as shown in figure 3 from the 28% Mn alloy with 10% strain.

In order to better understand the role of twin boundaries and martensite interfaces on work hardening, high-resolution imaging (HRTEM) using an image-corrected FEI-Titan provides both qualitative and quantitative information about the strain fields at these interfaces. Figure 4 is an HRTEM image from the 28% Mn alloy of a twin boundary in a sample with 20% strain. The twin plane exhibits a lack of mirror symmetry which could contribute to the strong work-hardening effect. Quantification of the strain fields at these interfaces is currently ongoing using real-space strain measurements. The relatively thick electropolished samples (t/λ maps indicate that t~ 20 nm) and 20% deformation limit image quality. Improved images may be obtained with planned aberration-corrected STEM imaging.

References

[1] O. Grassel, L. Kruger, G. Frommeyer, and L. W. Meyer, Int. J. Plasticity,16(2000) p.1391

[2] D.T. Pierce, et al., Acta Mater 68 (2014) 238-253

Financial support from the NSF DMR 0805295 and the SFB 761 “Steel –ab initio” and research at the Ernst Ruska Centre are gratefully acknowledged.

Fig. 1: Weak Beam Dark Field image of partial dislocations in Fe 22Mn-3Al-3Si for stacking fault energy (SFE) measurements (sg = 0.15 nm-1).

Fig. 2: Bright Field of the Fe 22Mn-3Al-3Si alloy after 10% deformation exhibiting two variants of epsilon martensite formation.

Fig. 3: Bright Field image of the Fe 28Mn-3Al-3Si alloy after 10% strain revealing multiple deformation twins.

Fig. 4: High Resolution TEM image of a twin boundary in the Fe 28Mn-3Al-3Si alloy after 20% deformation.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1581 Healing kinetics of voids in an Al-Mg-Er alloy investigated by in situ transmission electron microscopy and electron tomography

Song M.1, Du K.1, Wen S. P.2, Nie Z. R.2, Ye H. Q.1
1Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, 2School of Materials Science and Engineering, Beijing University of Technology
kuidu@imr.ac.cn

Extensive researches have revealed that stress concentration and crack nucleation usually take place around defects such as voids under external loadings, which would greatly shorten the incubation period of crack initiation. Voids also have adverse effects on the fatigue crack propagation and crack closure, while most of mechanical fractures in engineering are reportedly related to fatigue crack initiation and growth in metals. Therefore, investigations on the evolution of voids in materials have attracted great interests, and these investigations help to understand the recovery mechanism of materials properties, and consequently to improve the qualities of materials and to guide the development of processing techniques.

Using in situ transmission electron microscopy and electron tomography, we have studied the healing kinetics of voids with the dimension of submicron-scale embedded in a cold-rolled Al-Mg-Er alloy. The results show that voids are healed successfully within 50 minutes at a relative low temperature of 453 K. Quantitative analysis of the in situ micrographs reveals three stages for the void healing process: an initial fast-healing stage, then a constant healing stage, and finally a rapid-healing stage. The different healing rates are likely caused by varying surface curvatures due to the evolution of void morphology during the healing process. The entire evolution process of voids healing is actually completed together by surface diffusion, lattice diffusion and interface diffusion. However, as the voids are embedded inside Al alloy grains, lattice diffusion is considered to dominate the whole healing process. Mg enrichment was observed at the healed voids immediately after the healing. This indicates that the faster diffusion of magnesium atoms in aluminum matrix enhances the void healing in the Al-Mg-Er alloy, which is particularly essential for the void healing at low temperatures. The fatigue resistance and plasticity of the cold-rolled Al alloy are improved significantly after the annealing at 473 K.

References

[1] M. Weyland, P.A. Midgley, Nanocharacterisation (A.I. Kirkland, J.L. Hutchison, eds.), The Royal Society of Chemistry, 2007, p. 184.

[2] M. Song, K. Du et al, Acta Materialia, 2014, accepted.

The authors acknowledge financial support from the Special Funds for the Major State Basic Research Projects of China (Grant No. 2012CB619503) and the Natural Sciences Foundation of China (Grant Nos. 51171188, 51390473, 51221264, and 11332010).

Fig. 1: HAADF STEM image and reconstructed surface renders of a void between Al6Mn precipitates embedded in Al matrix. (a, b) HAADF images of the void with 0° and -50° tilting. (c-f) Surface renders of the void (gold) and the Al6Mn precipitates (purple) at two sides, obtained with electron tomography, while all of them are embedded in an Al matrix.

Fig. 2: The healing evolution process of voids embedded in the cold-rolled Al alloy during the in situ heating experiment.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1747 Characterization of multi-phase steel using collection-angle controlled BSE images

Sato K.1, Sueyoshi H.2, Yamada K.2
1JFE Steel Research Laboratory, Chiba, Japan, 2JFE Steel Research Laboratory, Fukuyama, Japan
ka-sato@jfe-steel.co.jp

Microstructural characterization is crucial for designing advanced steels. Cs-corrected STEM has been successfully applied to the studies of nanometer-sized precipitates and interfaces at sub-nanometer resolution. For the quantification of particle size and precipitation ratio of alloying elements, particle beams such as synchrotron radiation and neutron have been extensively used.
Compared to the abovementioned methods, SEM has been regarded as a supporting technique. However, improvements in both spatial resolution at low-voltages and multi imaging detector design are giving rich information on the “real” surfaces.
For optimizing the microstructure of steels, characterization using SEM is powerful because it allows both low and high magnification observation. SEM specimens are often etched in order to differentiate the different phases as topographic information in steels. This is an “indirect” method of characterization, which does not give precise structural information. Consequently, we have been searching for a more direct imaging technique. Aoyama et al have done a systematic measurement of oxide on steel by changing the acceptance angle of back-scattered electron (BSE) images1). Low angle (angle θ is measured from the surface) BSE images exhibit strong channeling contrast, whereas high angle BSE gives atomic number contrast. As shown in Fig.1a, sub-micron precipitates exhibit much higher visibility when low angle BSE is collected. The poor contrast in Fig.1b recorded at larger detection angles is not due to degraded probe size but due to the contribution of BSEs from larger volume. As can be seen in Fig.1d, the secondary electron image obtained at the same working distance as Fig 1b shows little image degradation.
We have found a new technique of selective imaging of martensite (M) phase in a ferrite (F)-M dual phase steel. BSE images at 10-15 kV were recorded by systematically changing θ. When θ was 30-45°, strong channeling contrast was observed. Under this θ, it is the low energy-loss electrons that mainly contribute to the contrast. As θ increases, M phase exhibits a high contrast. When θ exceeds 60°, a selective imaging of M phase was attained. This is not because martensite has a larger mean atomic number than ferrite, but is due to the fact that martensite has a high dislocation density. This is consistent with the fact that martensite always exhibits dark contrast in TEM bright field images regardless of the crystal orientation. Low angle BSE will allow high resolution characterization of lath structure and small precipitates, while high angle BSE gives quantitative measurement of the volume fraction and distribution of the second phase.
1) T. Aoyama et al,: ISIJ Int., 51, 1487 (2011).

Fig. 1: Nanometer-sized carbide in a high strength steel observed at 15 kV. BSE images a) and b) and SE images c) and d) were recorded at two working distances 2 mm (a and c) and 20 mm (b and d). A higher visibility of carbide was achieved for Fig. 1a where collection angles were between 31°-45° than 1b whose collection angles were 77°-81°.

Fig. 2: Selective imaging of martensite (M) in dual-phase steel. BSE images were recorded at 15 kV. Figs 2a and 2c were taken with the collection angles of θ=31°-45° whilst figs 2b and 2d were taken at θ=63°-70°. Lath structure is clearly seen at low θ, while selective imaging of martensite was attained at high θ.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1787 Re-examining the role of silver in aluminium alloys: interfacial segregation, growth kinetics and chemical order.

Rosalie J. M.1, Dwyer C.2,3, Bourgeois L.2
1National Institute for Materials Science, 2Monash University, 3Ernst Ruska-Centre and Peter Gruenberg Institute, Forschungszentrum Juelich
rosalie.julianmark@nims.go.jp

Aluminium alloys are an ideal example of materials where an understanding the microstructure at a nanometer scale is critical to effective materials design. A broad range of aluminium-based alloys develop high strength from the controlled precipitation of one of more intermetallic phases, with sizes in the nanometer regime. These alloys have formed the backbone of the aviation industry since its inception and continue to be widely used in transport applications ranging from military and civilian aircraft to automobiles and bicycles. Electron microscopy is a key technique used to study the nucleation and growth, morphology and orientation relationships and interactions with defects of these precipitates in order to understand these systems and thus optimise alloy design.

Although aluminium alloys are considered mature materials with a long history in industrial service, recent advances in microscopy have revealed a wealth of new information about alloys that were considered thoroughly-understood, including simple binary and ternary alloys used as models for the more complex industrial systems.

Recent studies have shown that interfacial Ag segregation is not limited to the well-known Ω-phase in Al-Cu-Mg-Ag alloys, but occurs in different, well-defined ways in other alloys. 0 In Al-Cu-Ag alloys this segregation takes the form of an Ag bilayer around  θ´ (AlCu2) precipitates [1]  and  affects the growth behaviour [2] but not the precipitate structure or orientation relationship to the matrix.n In contrast, Ag segregates as a monolayer to  γ´precipitates (AlAg2) in Al-Ag alloys. / The presence of  excess Ag solute around the precipitates suggests that growth of the  γ´ phase is initially controlled by the rate of migration of the interface, rather than the supply of solute [3].

These studies have also revealed surprising results about the  γ´ phase itself. E This hexagonal, close-packed phase was thought to be chemically ordered, with alternate Ag-rich and -poor layers and a high density of stacking faults. Recent investigations using aberration-corrected HAADF-STEM and CBED have not only found no evidence of long-range chemical order, but also determined that the phase is essentially free of  stacking-faults [3].

This study adds to a growing body of evidence calling for careful re-examination of these well-studied systems to better understand their precipitation behaviour and facilitate further improvements in the performance of industrial aluminium alloys.

[1] Rosalie and  Bourgeois, Acta Mater., 60:6033-6041, 2012.

[2] Rosalie and  Bourgeois, Light Metals, 365-371,   2013.

[3] Rosalie, Dwyer and Bourgeois,  Acta Mater., 69:224-235, 2014.

The authors  acknowledge the support of the Australian Research Council via the Centre of Excellence for Design in Light Metals. The authors are also grateful for the use of the facilities at the Monash Centre for Electron Microscopy and engineering support by Russell King.

Fig. 1: Ag segregation to a θ´ (AlCu2) precipitate.7 The micrograph shows Ag bilayers on the θ´-matrix interfaces in an Al-Cu-Ag alloy. The curve shows the HAADF-STEM intensity profile.

Fig. 2: Chemical order in γ´ precipitates. The HAADF-STEM micrograph (Exp.) is compared with simulations for ordered and disordered structures. The experimental image shows no evidence of chemical order.

Fig. 3: Ag segregation to a γ´ (AlAg2) precipitate in an Al-Ag alloy.b HAADF-STEM micrograph (Exp.) and simulations (for foil thickness=42.9 nm) with monolayer and bilayer Ag segregation. The boundary between the hcp γ´ phase and the fcc matrix is indicated. Monolayer segregation best reproduces the experimental image.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1796 Interfacial structure and mechanisms of precipitate growth in aluminium alloys

Bourgeois L.1,2, Medhekar N. V.2, Rosalie J. M.3, Smith A. E.4, Weyland M.1,2, Nie J. F.2, Dwyer C.1,2,5
1Monash Centre for Electron Microscopy, Monash University, Victoria, Australia, 2Department of Materials Engineering, Monash University, Victoria, Australia, 3National Institute for Materials Science, Tsukuba, Ibaraki, Japan, 4School of Physics, Monash University, Victoria, Australia, 5Present address: Ernst Ruska-Centre and Peter Grünberg Institute, Forschungszentrum Jülich, Germany
laure.bourgeois@monash.edu

The interfaces between solid-state precipitates and the crystalline matrix in which they are embedded are key to fundamental processes such as nucleation and growth. This is particularly true in high-strength aluminium alloys, which derive their mechanical properties from a fine and even distribution of high aspect ratio precipitates [1]. Understanding the development and preservation of precipitate shape requires knowledge of the precipitate-matrix interfaces at the atomic scale. Yet despite their critical importance, most such interfaces have not been characterised structurally or chemically. The chief reason for this deficiency has been the experimental difficulty in characterising embedded interfaces.

We used a dual-aberration-corrected FEI Titan3 80-300 scanning transmission electron microscope (STEM) at 300 kV, in high-angle annular dark field (HAADF) mode, to image the interfaces of the θ′ precipitate phase in an Al-1.7at.%Cu alloy. The Al-Cu alloy system is the textbook example of precipitation hardening [2]; it also forms the basis of a significant class of commercial alloys used in the aerospatial industry [1]. As shown in Fig. 1, we found that the two types of interfaces shared by θ′ precipitates with the Al matrix α, a coherent interface and a semi-coherent interface, exhibit structures that are not simple combinations of the structures of the bulk phases θ′ and α [3,4]. First-principles calculations using density functional theory (DFT) indicated that these unusual structures do not correspond to the lowest energy states [3,4], but to intermediate states with low activation energy barriers. The determined structures suggest atomic-scale mechanisms for the two growth modes: lengthening (Fig. 2) and thickening (Fig. 3).

Knowledge of the interfacial structures and growth mechanisms provides an explanation for the observed segregation behaviour of elements such Sn and Ag on the θ′ precipitate interfaces [5,6].

[1] B.C. Muddle, S.P. Ringer and I.J. Polmear, Trans. Mater. Res. Soc. Jpn 19B (1994) 999. [2] G. Kostorz (Ed.), Phase Transformations in Materials, Wiley-VCH, Weinheim, (2001). [3] L. Bourgeois, C. Dwyer, M. Weyland, J.F. Nie and B.C. Muddle, Acta Mater. 59 (2011) 7043. [4] L. Bourgeois, N.V. Medhekar, A.E. Smith, M. Weyland, J.F. Nie and C. Dwyer, Phys. Rev. Lett. 111 (2013) 46102. [5] L. Bourgeois, C. Dwyer, M. Weyland, J.F. Nie and B.C. Muddle, Acta Mater. 60 (2012) 633. [6] J.M. Rosalie and L. Bourgeois, Acta Mater. 60 (2012) 6033.

The authors acknowledge the staff and facilities of the Monash Centre for Electron Microscopy, and the Australian Research Council for funding of the FEI Titan microscope.

Fig. 1: HAADF-STEM images of θ′ precipitates showing (a) the microstructure and location of the coherent (C) and semi-coherent (SC) interfaces, displayed at high resolution in (b) and (c) along [100] and [110] respectively. Both interfaces exhibit structures that are not simple combinations of the structures of the bulk phases θ′ and α (Al matrix).

Fig. 2: Proposed atomistic mechanism for precipitate lengthening. (a) Starting interfacial structure deduced from Fig. 1(b)-(c); (b)-(d) atomic steps leading to motion of the SC interface. Cu atoms are shown in yellow/brown and Al atoms in blue.

Fig. 3: (a) Proposed atomistic mechanism for precipitate thickening from 2cθ’ to 2.5cθ’, based on the structure of the coherent interface; red/orange circles indicate Cu atoms and white circles vacancies; (b) energy per Cu atom, as calculated by DFT.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1867 Study of fine scale microtexture features associated with globularization in a near β titanium alloy using precession electron diffraction (PED) assisted orientation electron microscopy (OIM)

Balachandran S.1, Sharath K.2, Banerjee D.1,2
1Materials Engineering, Indian Institute of Science (IISc) Bangalore, India, 2Advanced facility for microscopy and microanalysis, Indian Institute of Science (IISc) Bangalore, India
shanoob.b@gmail.com

Commercial titanium alloys undergo a series of thermo mechanical processes and subsequent heat treatments at the high temperature β (BCC) phase and lower temperature α + β (HCP+ BCC) phase to achieve desired properties. The microstructure and global texture is heavily influenced by the processing parameters such as temperature, strain and strain rate. The widmansttaten α (HCP) laths form by slow cooling of β or isothermal aging at α + β phase maintaining a burgers orientation relationship (BOR) with β phase given by (1-10)β || (0001)α and <111>β || <11-20>α. Upon thermo mechanical processing and subsequent heat treatment, the lath structure transforms to equiaxed, a process known as globularization. The globularization does not lead to a completely random texture and many a times, we may retrieve the initial α orientation with certain spread even after heavy deformation. In addition, the microtexture associated with globularization is an interplay between the α and β phases and recrystallization in beta can happen in combination with α to form special angle, epitaxial grain boundaries in both α and β phases as suggested by some of previous studies in this direction [1][2].

In the present work, we incorporate transmission electron microscopy (TEM) based orientation electron microscopy (OIM) assisted by precession electron diffraction (PED) to investigate the triggering points of recrystallization events having special angle boundaries in both α and β phases, at resolutions beyond conventional scanning electron microscopy (SEM) based electron backscattered diffraction (EBSD). A 200 KV FEI T20 S-TWIN microscope coupled with Nanomegas-ASTAR precession and data collection system was used for this study. Events of epitaxial recrystallization of fine α associated with special angle boundaries in β around the alpha was frequently observed. Two interesting examples are shown here. In Figure.1, the recrystallized α maintains a common <10-11> pole with the other α with a special β grain boundary evolving from α / α interface. In Figure.2, a fine β layer is observed around the globularizing α laths maintaining BOR with α and in special angle boundary with parent β grain, suggesting altogether a new mechanism for α globularization.

Many more of the above discussed events were observed in our study. The resulting global texture is a sum of these discontinues recrystallization events and the deformation texture associated with parent β and α phase. We have observed consistently that the original BOR is restored by these events.

1. C. Cayron, Scripta Materialia. 59, 570 (2008).

2. E. Lee, R. Banerjee, S. Kar, D. Bhattacharyya, and H. L. Fraser, Philosophical Magazine. 87, 3615 (2007).

Laboratory for mechanical testing, Materials Engineering, IISc Bangalore

Fig. 1: Epitaxial recrystallization in α phase associated with special angle grain boundaries in β and the corresponding pole figures with 10-11 and 110 common poles.

Fig. 2: Globularization in α associated with formation of a fine β layer, which is in burgers orientation relationship (BOR) with α and maintains special angle relationship with parent β grain.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1871 High-resolution characterization of stress corrosion cracking in reactor grade stainless steels via analytical TEM

Meisnar M.1, Lozano-Perez S.1, Moody M.1
1University of Oxford, Department of Materials, Parks Road, OX1 3PH, Oxford, UK
martina.meisnar@materials.ox.ac.uk

Understanding crack propagation is fundamental for the investigation of the underlying mechanisms of stress corrosion cracking (SCC). While conventional surface techniques are well suited for an initial observation of the sample and selection of important areas, only high-resolution techniques such as Transmission Electron Microscopy (TEM) or Atom-Probe Tomography (APT) can deliver novel results with regard to the relationship between crack propagation and chemistry.

High-resolution analytical TEM has been used to study intergranular SCC in stainless steels (SS) that have been exposed to simulated Pressurized Water Reactor (PWR) primary water conditions for 702 h at 360°C under constant load. The main focus of this study is the oxide chemistry and the microstructure around the crack. As it will be shown, analytical TEM is capable of resolving and imaging nanoscale features in order to determine the exact location of the crack tip, the composition of the surrounding oxides and their interaction with the microstructure. Electron diffraction and tilt-series have been used to study the microstructure and oxide structure within and around the crack tip. Figure 1 illustrates a tilt series recorded of a crack tip in an SUS316 SS sample. It can be observed, that at different tilt angles, different crystallographic features come into view and its 3D morphology can be deduced (i.e. a Ni-rich region around an oxidized portion of the grain boundary in Figure 1a, deformation bands (DBs) in Figures 1c and 1d or the grain boundary ahead of the crack tip in Figure 1e). In Figure 1b grain 1 appears darker than grain 2, suggesting different crystallographic orientations which have been identified via electron diffraction patters (DP) as shown in Figures 1b-1 and 1b-2. Thus, it was verified that deformation bands appeared parallel to {111} planes as expected. Further analysis has been carried out via EDX, which allows the determination of the oxide types and compositions inside and around the crack. Figure 2 shows the HAADF image (Figure 2a) and the EDX elemental maps of O, Fe, Cr and Ni (Figure 1b-e) located at a crack tip in an SUS304 SS sample. The Z-contrast in the HAADF image (Figure 1a) allows the distinction between the matrix and the open crack which is filled with two different types of oxide. The O map (Figure 2b) indicates where oxides are present within the crack region. While the Fe map (Figure 2c) suggests the existence of an Fe-rich and an Fe-depleted oxide region, the Cr map (Figure 2d) shows the absence of Cr in the Fe-rich oxide. The Fe-depleted oxide seems to be Cr rich. Lastly, Ni enrichment can be observed ahead of the crack tip in the Ni map (Figure 2e) which could be an important factor in the study of crack propagation.

The authors are grateful to Areva (France) for sponsoring the project and INSS (Japan) for providing the samples.

Fig. 1: STEM (BF mode) tilt series of crack tip in an SUS316 stainless steel sample using a JEOL 2100; y tilt = 0° in all images a) x tilt = -24°; b) x tilt = -16°; b-1) DP in upper grain (grain 1); b-2) DP in lower grain (grain 2); c) x tilt = 4°; d) x tilt = 10°; e) x tilt = 25°

Fig. 2: EDX analysis of crack tip in an SUS304 stainless steel sample using a JEOL 2100 in STEM mode; a) HAADF image of crack tip region; b) Elemental map of Oxygen (O); c) Elemental map of Iron (Fe); d) Elemental map of Chromium (Cr); e) Elemental map of Nickel (Ni)
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-1948 Deformation-induced ultrahigh lattice rotation via phase transitions in body-centered cubic metals

Wang S. J.1, Wang H.1, Du K.1, Zhang W.1, Sui M. L.2
1Institute of Metal Research, CAS, Shenyang, China, 2Beijing University of Technology, Beijing, China
mlsui@bjut.edu.cn

When a material is loaded under stresses, the accumulating elastic energy eventually sets off dissipative processes such as plastic deformation. Such processes are mediated generally by dislocation slip and deformation twinning, or stress-assisted phase transformations. It is the nature of materials to take an easy way to deform, but sometimes the high-cost paths have to be taken when the easy deformation way is suppressed or bypassed. Among these deformation mechanisms, stress-assisted phase transformation is often an efficient outlet to accommodate the imposed straining, such as in transformation-induced plasticity steels and shape memory alloys. As far as is known, the occurrences of phase transformations under straining are mostly observed in the materials with polymorphs (such as iron with α-Fe, γ-Fe and ε-Fe). However, there are also materials, such as elemental molybdenum (Mo) with the body-centered-cubic (bcc) structure, for which no other polymorphs have ever been found before. Indeed, computer calculations have predicted that alternative crystalline lattices of Mo, for example a face-centered-cubic (fcc) phase, would have an energy much higher (0.2~0.4 eV/atom) than that of bcc Mo. As such, a fundamental question regarding such materials is whether their deformation can ever be coupled with structural transformation that leads to the emergence of new (metastable) crystalline lattices/forms.

In the present work, we report a direct observation of two sequential phase transitions bcc→fcc and fcc→bcc in Mo, a novel plastic deformation mechanism in bcc metals in response to the enhanced external loading, by carrying out in situ high-resolution transmission electron microscopy (HRTEM) investigation. Under tensile loading, the grain changes from the <001> (henceforth denoted as bcc1) to the <111> (denoted as bcc2) orientation via a bcc1→fcc→bcc2 phase-transition process, which corresponds to a grain rotation of 54.7°. Molecular dynamics simulations confirm the bcc1→fcc→bcc2 transition process under a shear stress. Here the refractory Mo is a powerful example to show that even for a highly stable bcc crystal, when it (or a local region) is subjected to very high shear stresses while other plastic deformation routes (such as twinning) are suppressed or bypassed, the crystalline structure can be forced to change to release the local stress concentration. Specifically, the energetically uphill bcc to fcc transition becomes viable in Mo, when driven by sufficiently high applied stresses.

Reference

1. S.J. Wang, et. al. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4433

This work is supported by the Major State Basic Research Projects of China (Grant No. 2012CB619503), the National Natural Science Foundation of China (Grants No. 51221264, 51171188, 51390473, 11374028 and U1330112), and the Cheung Kong Scholars Program of China. This work used resources provided by the Beijing National Center for Electron Microscopy.

Fig. 1: In situ HRTEM observations under tensile loading. a-d, Time-resolved HRTEM images of Mo (5nm scale-bar). A <111>-oriented bcc-2 grain, outlined by red dots, formed and grown inside the <100>-oriented bcc-1crystal. e-g, FFT patterns of bcc-1, bcc-2, and both together. h, A schematic illustration showing the distribution of bcc1 and bcc2 crystals.

Fig. 2: Three structural variationsin Mo. a, Atomic-resolution HAADF-STEMimage (0.5nm scale-bar) showing regions I (bcc-1,blue), II (fcc, red), and III (bcc-2, green). b,Distribution of the angle between two basic vectors (x and y) in the entire imaged area.c-g, The nanodiffractionpatterns from regions I,II, III, I+II and II+III, respectively.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-2400 Electron Microscopy of Oxide Layers and Secondary Phases in Ferritic High Chromium Cast Irons Containing 0-0.3wt%Si

Wiengmoon A.1, Pearce J. T.2, Chairuangsri T.3
1Department of Physics, Faculty of Science, Naresuan University, Phitsanulok, Thailand, 2National Metal and Materials Technology Center, Pathumthani, Thailand, 3Department of Industrial Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand
ampornw@nu.ac.th

Ferritic, high-chromium cast irons can be used as a heat-resistance material at up to 1050oC in applications such as furnace and sinter plant parts, burner nozzles and recuperator tubes [1]. The high level of chromium increases resistance to both oxidation and corrosion. Effect of Si addition on improving oxidation of these irons is of interest. In the present work, electron microscopy study on oxidation behavior and phase transition in 31wt%Cr-1wt%C irons containing up to 3wt%Si was therefore addressed. For oxidation tests, polished samples were exposed to air at 1000oC for 1-48 hours followed by weight-gain measurement at the different times. The oxidized surfaces and their cross-sections were characterized by light microscope (LM) and scanning electron microscopy (SEM). For phase stability test, the as-cast samples were held at 700-1000oC for 2-8 days. The nature of secondary phases was examined by SEM and transmission electron microscopy (TEM). It was found that the as-cast microstructure of these irons consisted of M7C3 eutectic carbide and ferrite matrix. The iron containing 1wt%Si possessed the highest oxidation resistance. SEM analysis revealed a formation of multi-oxide layers on the iron surface, including SiO2, Cr2O3, (Fe,Cr)2O3, (Fe, Cr)3O4 and Fe2O3. It is suggested that, at the optimum content of Si, the formation of continuous SiO2 layer reduce susceptibility for oxidation of these irons (Fig. 1). Excessive Si addition will leads to spalling and hence further oxidation, possibly because of reduced adhesion of the multi-oxide layers [2]. After stability test, a secondary phase was observed along M7C3 carbide/ferrite interfaces (Fig. 2). TEM analysis revealed that this phase is M23C6 carbide (Fig. 3 and 4). The volume fraction of M23C6 was increased with increasing Si content.

References :

[1] Boyes, J.W., (1966) “High-chromium cast irons for use at elevated temperature”, Iron and Steel, 39, 102-109.

[2] Bamba, G. et al, (2006) “Thermal oxidation kinetics and oxide scale adhesion of Fe-15Cr alloys as function of their silicon content”, Acta Materialia., 54, 3917-3922.

The authors gratefully thank the Thailand Research Fund: MRG5480285 and Naresuan University for funding support of this work.

Fig. 1: SEM-BEI of 31wt%Cr-2wt%Si cross section sample shows oxide layer (1) and SiO2 film (2) at the oxide-metal interface.

Fig. 2: SEM-BEI shows phase transformation along M7C3 carbide/ferrite (α) interfaces of 31wt%Cr-2wt%Si after holding at 800oC for 2 days.

Fig. 3: Bright-field TEM image shows the M23C6 secondary phase around M7C3 eutectic carbide.

Fig. 4: Selected area diffraction pattern from the M23C6 carbide.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-2559 Atomic resolution investigation of twin boundaries in Fe-Cr-Mn austenitic TWIP steels with aberration-corrected transmission electron microscopy and real-space strain field calculation

Beigmohamadi M.1, 2, Mayer J.1, 2, Mosecker L.3, Rezaei Mianroodi J.4, Svendsen B.4, Pierce D.5, Wittig J.5
1Central Facility for Electron Microscopy, RWTH Aachen University, Ahornstraße 55, 52074 Aachen, Germany, 2Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich, 52425 Jülich, Germany, 3Institut für Eisenhüttenkunde, RWTH Aachen University, Intzestraße 1, 52072 Aachen, Germany, 4Chair of Material Mechanics, RWTH Aachen University, Schinkelstr. 2, 52062 Aachen, Germany, 5Interdisciplinary Materials Science, Vanderbilt University, Nashville TN, 37235 USA
beig@gfe.rwth-aachen.de

The development of austenitic stainless Fe-Cr-Mn-steels alloyed with carbon and nitrogen was shown to result in an increased strength with good plasticity and toughness at the same time [1]. Adding Mn as an austenite stabilizer decreases the stacking-fault energy (SFE) in the steel, which favors mechanical twinning over dislocation glide. The strain-hardening of TWIP steels is commonly attributed to the formation of deformation twins, as twin interfaces act as strong obstacles to dislocation glide. This effect is consequently controlled by interaction of perfect and partial dislocations with twin boundaries and twinning kinetics [2]. In this investigation, an austenitic steel with the chemical composition of Fe–14 wt.% Cr –16% Mn–0.3% C–0.3% N was studied after tensile deformation to 1 and 20 % strain. Detailed microstructural properties were observed by high resolution TEM (HRTEM) and Scanning TEM (STEM) by utilizing image and probe-corrected FEI Titan microscopes.

Figure 1 exhibits the formation of two different deformation twins systems active in the same grain for 20% strain. The diffraction pattern (inset of Fig. 1) clarifies that the twin boundaries are parallel to the (111) plane. The same area investigated by HRTEM clearly shows the atomic arrangement in the twin boundary (see Fig. 2). In this image the grain was tilted to the [011] zone axis. In order to interpret the images obtained by HRTEM, they were used as a basis for the reliable measurement of atomic distances at the twin boundary or in the neighboring matrix area. The precise atomic column positions were calculated with the imTools software package. These coordinates were used as input for further real space structure analysis and calculation of the displacement field and the strain field in the twin boundaries. Figure 3 shows the real space analysis of a twin boundary which used to obtain precise atom coordinates in the twin boundary. Using the coordinates, the displacement vector and its magnitude were calculated. The strain field in the twin boundary was also calculated by different approaches and the implications for the materials properties will be discussed.

Refrences:

[1] H. berns, V.G. Gavriljuk, S. Riedner, and A. Tyshchenko: Steel Res Int 78, 9, 2007, P. 714

[2] T.-H. Lee, C.-S. Oh, S.-J. Kim, S. Takaki, Acta Materialia 55, 2007, P. 3649

Authors would like to acknowledge Dr. Lothar Huben for his technical advice on working with imTools and the SFB 761 “Steel –ab initio” for financial support of this work.

Fig. 1: Bright Field image of twins and stacking faults after 20% deformation exhibiting two sets of active twins.

Fig. 2: High Resolution TEM image of a ca. 10 nm wide twin after 20% deformation

Fig. 3: a) enlarged section of the HRTEM image of a twin boundary in Fig. 2, b) the real space analysis used to calculate atomic coordinates. From these coordinates, c) strain fields are calculated at the twin boundary and in its vicinity.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-2610 TEM characterization of oxidised Zr-1%Nb nuclear fuel cladding alloys

Hu J.1, Ni N.2, Lozano-Perez S.1, Frankel P.3, Allen V.3, Preuss M.3, Grovenor C.1
1Department of Materials, Oxford University, Parks Road, Oxford, UK, 2Department of Materials, Imperial College London , Royal School of Mines, London, UK, 3Materials Performance Centre, School of Materials, University of Manchester, Manchester, UK
jing.hu@materials.ox.ac.uk

Transmission Electron Microscopy has been used to study the microstructure of Zr-1.0Nb alloys, which show excellent corrosion resistance under autoclave conditions1. Samples oxidised for 225 days and 585 days have been chosen to be representative of the pre-transition and post-transition periods of the oxidation process respectively. Cracks and pores are found to be associated with small equiaxed grains near the sample surface,but at a lower density than commonly reported in oxides on other alloys. A periodic pattern of columnar-equiaxed-columnar grain structure is observed, where the width of the columnar grains is greater than normally reported in oxides on other alloys2. In the post-transition sample, the equiaxed grains are associated with cracks parallel to the metal-oxide interface. Two types of second phase particles (SPP) are observed, one containing only Nb, the other with both Nb and Fe. The second type of SPP is present at a lower number density and tends to be amorphous once incorporated into the oxide or near the metal-oxide interface. Near this interface, Fresnel imaging reveals the existence of both parallel and some vertically interconnected porosity along the columnar oxide grain boundaries towards metal-oxide interface, where disconnected porosity is seen. Electron Energy Loss Spectroscopy (EELS) analysis has also revealed a suboxide layer in the pre-transition sample with compositions in different regions of ZrO or Zr3O2, with significant local variations in thicknesses from 15nm to more than 260nm, much thicker than observed previously in other oxidised zirconium alloys3. High dislocation densities are found in the metal grains under the metal-oxide interface, some associated with second phase particles. These observations will be compared to previous reports of less corrosion-resistant alloys studied by the same techniques.

1. Wei, J. et al. Autoclave study of zirconium alloys with and without hydride rim. Corros. Eng. Sci. Technol.47, 516–528 (2012).
2. Yilmazbayhan, A. et al. Transmission electron microscopy examination of oxide layers formed on Zr alloys. J. Nucl. Mater.349, 265–281 (2006).
3. Ni, N. et al. How the crystallography and nanoscale chemistry of the metal/oxide interface develops during the aqueous oxidation of zirconium cladding alloys. Acta Mater.60, 7132–7149 (2012).

This research was funded by the MUZIC2 consortium and JH is supported by the China Scholarship Council.

Fig. 1: (a) A HAADF image of a region of the metal–oxide interface from the 225 day Zr-1.0Nb sample oxide. Three EELS oxygen line scans have been taken revealing suboxides of following thickness; (b) 150 nm with Zr:O=1:1, and (c) 15 nm with Zr:O=1:1 and 100nm with Zr:O=3:2.(d) 130 nm with Zr:O=1:1 and 100nm with Zr:O=3:2

Fig. 2: Through-focal imaging of fine pores (circled) on the 225 days Zr-1.0Nb sample. Three different types of porosity are revealed at different locations: 100nm from metal-oxide interface: (a) parallel interconnected pores; (b) vertically interconnected pores along the columnar oxide grain boundary; (c) near the metal-oxide interface: disconnected pores
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-2725 High-resolution Structural imaging and STEM-Spectroscopy Studies of Phase Transformation in V-Ti-Cr alloys

Ghosh C.1, Basu J.1, Divakar R.1, Mohandas E.1
1Materials Synthesis and Structural Characterisation Division, Physical Metallurgy Group, Metallurgy and Materials Group, Indira Gandhi Centre for Atomic Research, Kalpakkam-603102, Tamil Nadu, India
chanchal@igcar.gov.in

V-Ti-Cr alloys are potential candidate materials for many advanced engineering applications. Based on Miedema model three compositions of V-Ti-Cr alloys have been identified and prepared by vacuum arc melting. Phase and structural information have been studied through different transmission electron microscopy (TEM) techniques using FEI Tecnai G2 F30 U-TWIN TEM.
    Detailed TEM studies confirm the stability of a bcc solid solution phase at the V-rich corner of the V-Ti-Cr ternary system. For a ternary extension of the binary TiCr2 Laves phase, formed by addition of V to the extent of a 7 atom%, thermodynamic calculations predict the stabilization of an amorphous phase. TEM studies have been carried out to: (i) identify the atomic columns and (ii) determine whether the phase is binary or ternary. Zero loss phase contrast microscopy has been carried out to distinguish the atomic columns unambiguously. In case of zero loss imaging only the elastically scattered electrons contributes in the contrast generation and hence the system can be considered more closely to an ideal weak phase object. Results indicate that Ti and Cr atoms are not imaged simultaneously at all thickness – defocus combinations; this is further confirmed by phase contrast image simulation [Fig.1(a-c)]. Though the exit wave function contains information relating all atomic columns over the thickness range of 15nm to 70nm, variation in Ti and Cr exit wave profiles as a function of thickness convoluted with the instrument CTF results the disappearance specific atomic columns at specific thickness ranges. A comparison of the experimental and simulated electron intensity profiles confirm that V can substitute for both Ti and Cr lattice sites and V-doped TiCr2 exists as a pseudo-ternary Laves phase with a modified stoichiometry of (A,B´)(B,B´)2. 
For binary V-Ti alloys, thermodynamic calculations suggest the occurrence of a phase separation tendency which is reduced with the addition of Cr. The lamellar nanostructured domains with incoherent interfaces seen in the phase contrast mode are indicative of a phase separation event. STEM - HAADF studies indicate the compositional modulation perpendicular to the lamellar domains and further examined through STEM-XEDS and STEM-EELS confirming a Ti rich phase separates out from the matrix (Fig2). 
    Different TEM based techniques at near atomic resolution have been successfully applied to study phase transformations in V-Ti-Cr alloys. A combination of phase contrast and Z contrast microscopy coupled with spectroscopic imaging, phase contrast image simulation and atomic structure modelling have been successfully analyse the materials related issues of V-Ti-Cr alloys which is otherwise not possible through any of the conventional techniques.

The authors would like to acknowledge Dr. M. Vijayalakshmi, AD, PMG and Dr. T. Jayakumar, Director, MMG, IGCAR for their constant support and encouragement.  The authors would also like to acknowledge UGC-DAE-CSR for providing the experimental support.   

Fig. 1: (a) Zero-loss phase contrast image from a region of the V doped TiCr2 alloy. Annotations indicate planes indexed with cubic TiCr2 Laves phase cF24 structure. (b) & (c) show the phase contrast simulated image along with the atomic structure model confirming that only the Cr atomic columns of a specific location are imaged.

Fig. 2: (a) STEM-HAADF image from the V-Ti alloy exhibiting compositional fluctuations, (b) the corresponding XEDS profile for V and Ti along the marked line in (a).

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-2801 Electron-microscopical characterization of White Etching Areas in 100Cr6 bearings

Diederichs A. M.1, Schwedt A.1, Mayer J.1
1Central Facility of Electron Microscopy RWTH Aachen University, Aachen, Germany
diederichs@gfe.rwth-aachen.de

Early failures in 100Cr6 roller and ball bearings, which are caused by microstructural changes and material degradation leading to the formation of white etching areas (WEA) have become a serious problem in many technological applications, the most prominent example being wind turbines. [1] WEA appear in subsurface regions associated with the maximum of Hertzian stress and have been observed in numerous applications running under high cycle conditions and high loads. As a result of WEA formation, systems of cracks (WEC, cf. Fig.1a) are formed and extend to the surface resulting in catastrophic bearing failure as a consequence of massive pitting at the bearing raceway. Since the failure mechanism of WEA formation is still not fully understood, the lifetime of the components is unpredictable and WEA analysis is in focus of interest.

Cross-sections of bearings after failure were investigated in different etching conditions by a complementary use of Light Optical Microscopy, SEM with focus on the use of Electron Channeling Contrast Imaging (ECCI) and EBSD analysis, EPMA, FIB preparation and TEM.

ECCI was used for the first time to give insight in the grain structure within regions with altered microstructure considered to be regions of WEA formation in detail. It was observed that WEA are composed of areas with different grain sizes down to nanoscale. Electron Diffraction in TEM was performed to reveal that the very homogenous nanocrystalline parts of the WEA are consisting of a bcc structure with a slightly increased lattice parameter. Using EBSD analysis evidence was found that WEA formation and accompanying crack growth are without relation to prior austenite grain boundaries or other microstructural features (cf. Fig. 2). Furthermore, it was shown that the larger-sized grains inside the WEA usually are textured. The inhomogeneous chemical structure of WEA as a result of carbide dissolution was investigated using SEM (cf. Fig. 1b), EDX, EPMA and EFTEM. A detailed characterization of the role of iron-chromium carbide decay and chromium transport in WEA formation was obtained. Hence it was clearly shown that chromium and carbon are involved in the white etching area formation.

[1]H.Swahn and P.C.Becker. Martensite decay during rolling contact fatigue in ball

bearings. Chemical Analysis, 7, 1976,

M. H. Evans. White structure flaking (wsf) in wind turbine gearbox bearings: Effect

of ’butterflies’ and white etching cracks (wec). Material Science and Technology,

28(1):3–22, 2012,

O.H.E.West, A.M.Diederichs, K.V.Dahl. Application of Complementary Techniques for Advanced Characterization of White Etching Cracks. Practical Metallography, 50(6): 410-431, 2013.

Fig. 1: Figure 1a) LOM image showing WEA appearing in white and a typical example of a branching crack network as result of WEA formation in the subsurface of a bearing raceway (etched with 2% Nital), b) SEM image showing the dissolving iron-chromium carbide structures (highlighted by an arrow) within a WEA (etched with 2% Nital).

Fig. 2: Figure 2: EBSD IPF map of a branching crack faced with nanocrystalline WEA running through a prior austenite grain (marked with a white square), grains of different sizes embedded into the nanocrystalline parts of the WEA (too fine to be measured with EBSD, thus black in the image) are visible.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-2935 Structure and formation of novel LPSO structures in Mg-Co-Y alloy

Egami M.1, Abe E.1, Kimizuka H.2, Yamasaki M.3, Kawamura Y.3
1The Univeristy of Tokyo, Tokyo, Japan, 2Osaka University, Osaka, Japan, 3Kumamoto University, Kumamoto, Japan
egami@stem.t.u-tokyo.ac.jp

Recently, dilute Mg-Zn-Y alloys have attracted great attention because of their excellent mechanical properties, and their key microstructural feature strongly depends on a long period stacking/order (LPSO) phase. So far, four types of LPSO structures, 10H, 18R, 14H and 24R have been reported for Mg-Zn-Y alloy, and all of them are systematically described by two common structural units; AB stacking of hcp structure and AB’C’A stacking where B’ and C’ layer have local fcc environment [1]. The chemical order occurs to synchronize with stacking order. That is, Zn/Y atoms distribute at the particular four layers at AB’C’A.
The LPSO structures are also observed in several Mg-transition metal-rare earth systems. In Mg-Co-Y alloys, novel types of LPSO structures are observed [2] as well as those described previously (i.e., 10H, 18R, 14H and 24R). We investigate the details of these novel LPSO structures in Mg-Co-Y alloys, based on scanning electron microscopy observations and first principles calculations.
Figure 1 shows three novel LPSO structures, 15R, 12H and 21R. They are described by two structural units composed of AB stacking and AB’C stacking, and Co/Y atoms distribute at three particular layers AB’C. For the previous LPSO structures, the local AB’C’A stacking is attributed to intrinsic-2 (I2)-type stacking fault (SF) with respect to the original 2H stacking. However, the AB’C stacking represents intrinsic-1 (I1)-type SF; therefore, the present LPSO structures are systematically described as periodic introduction of I1-type SFs into 2H stacking and solute segregations at the SFs. Hereafter, we denote these LPSO structures as I2-LPSO and I1-LPSO structures.
Figure 2 shows the interfaces between the LPSO and 2H crystals. Generally, I2-SF is introduced into 2H by an <a> dislocation and I1-SF is introduced by an <a+c> dislocation. Fig. 2a shows the partial <a> dislocations at the end of LPSO, but in fig. 2b no dislocations with <c> component could be observed. At the interface, phase inversion between AB structural unit of LPSO and 2H periodically appear because AB’C stacking invert ABAB… into BABA..., forming I1-LPSO without <a+c> dislocation motion.

[1] E. Abe et al, Philos. Mag. Lett. 91 (2011)
[2] S. B. Mi and Q. Q. Jin, Scr. Mater. 68 (2013)

Fig. 1: Electron diffraction patterns and HAADF-STEM image of a: 15R-, b: 12H- and c: 21R-LPSO structure. d: structure model of three types of I1-LPSO structures.

Fig. 2: HAADF-STEM images of the interface between 2H-Mg and a: I2-LPSO, b: I1-LPSO and (c, d): structure models of them.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-2964 Kink-deformed microstructures in Mg-Zn-Y alloys with a unique long-period

Tanaka Y.1, Egusa D.1, Yamaguchi M.2, Abe E.1
1Department of Materials Science and Engineering, University of Tokyo, Japan, 2Japan Atomic Energy Agency, Japan
tanaka@stem.t.u-tokyo.ac.jp

Mg alloys containing a small amount of TM (transition metal) and RE (rare earth) form a unique long period stacking/ordered (LPSO) structures and show excellent mechanical properties. Kink-deformation is believed to play a key role for realization of the excellent properties of the LPSO-Mg alloys. Kink deformation is phenomenologically understood as a result of polygonizations of a large number of dislocations that have migrated on the (0001) basal plane, which is limited as single-variant for the anisotropic crystals such as a hcp structure.[1] In the present work, we investigate kink-deformed microstructures in the LPSO-Mg alloys, focusing on dislocation characters around the kink-band using advanced electron microscopy and first principles calculations. We selected two LPSO-Mg alloys; hot-extruded Mg97Zn1Y2 (at. %) alloy with an extrusion ratio of 10:1 and cold-rolled Mg85Ni6Y9 (at. %) alloy with 30% reduction. The microstructures were observed by a scanning transmission electron microscopy, and first principle calculation was performed with the Vienna Ab-initio Simulation Package (VASP).
TEM observations confirmed that kink-deformation occurred in these alloys (Fig. 1(a)). A number of dislocations are regularly arrayed along the c-axis to form a definite interface, across which the relevant crystal is sharply bended with an angle of 1~2 degree (Fig. 1(b) (c)). Atomic-scale STEM observations from [11-20]hcp direction were performed to determine the dislocation core structures. Fig. 2(a) shows that extra half planes are indeed inserted, and the corresponding burgers circuit is shown in Fig. 2(b). The results indicate that some dislocations on the kink-boundary are mixed dislocations; the dislocation line is inclined by ~60 degree with respect to the burgers vector (Fig. 2(c)); this feature is different from that expected for moving dislocations in hcp-metals. It is found that the slip plane in Fig. 2 (a) is located between the richest layer and the second richest layer (Fig. 3 (a), interlamellar2), the dislocation seems to be unfavorable for moving. We calculate γ-surface and theoretical shear strength (τmax) using first principles calculations, and derive a Peierls stress (σp) accroding to Joos&Duesbery equation[2]. As a result, the Peierls stress appear to be not much different from the lowest stress of interlamellar 1 (Fig. 3(b)).
References
[1] J. B. Hess, C. S. Barett, Metals Transactions, 1949
[2] B. Joos and M.S. Duesbery, Phys. Rev. Lett., 1997

A part of this research was supported by "Center for Fusion Research of Nano-Interface Devices, Tohoku University" of "Low-Carbon Research Network" funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Fig. 1: Fig. 1 Bright field TEM images obtained from (a),(b) Mg85Ni6Y9 (at %) alloy observed from [11-20]hcp direction and (c) Mg97Zn1Y2 (at %) alloy observed [1-100]hcp direction. White line shows the trace of the basal plane, orange dotted lines show the kink-boundaries, and red triangles show the end of the kink-boundaries.

Fig. 2: Fig. 2 (a), (b) HAADF-STEM images taken from [11-20]hcp direction. The red dotted line shows the slip plane. (c) Schematic model of the dislocation. The blue line shows the dislocation line and the red arrow shows the burgers vector.

Fig. 3: Fig. 3 (a) Ideal model of 10H-type LPSO structure. The rich layers (fcc-stacking layers) contains L12-type order structure. (c) Calculated theoretical shear strength (τmax) and Peierls stress (σp)
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-3040 (Y-Ti-O)-Cr core-shell structure and Ti oxidation states in ferritic ODS steels designed for sodium fast reactors by electron energy-loss spectroscopy

Badjeck V.1, Walls M.1, Chaffron L.2, Malaplate J.2, March K.1
1Laboratoire de physique des solides, Bât. 510, université Paris-Sud, 91405 Orsay Cedex, 2DEN/DMN/SRMA, CEA Saclay, 91191 Gif Sur Yvette cedex
vincent.badjeck1@u-psud.fr

Recent years have witnessed increasing research efforts concerning materials for the next-generation nuclear fission reactors (Gen IV – sodium fast reactor), which will need to work at higher temperature and radiation levels. For these applications, oxide dispersion strengthened (ODS) steels are the most promising candidates.

We present a study by scanning transmission electron microscopy coupled with electron energy-loss spectroscopy (STEM-EELS) of an ODS steel with the nominal composition Fe-14Cr-1W-0.3TiH2-0.3Y2O3 (wt.%). After denoising the spectrum-images via principal component analysis (PCA), elemental maps were generated (Y-M3, Ti-L2,3, O-K, Cr-L2,3 and Fe-L2,3), showing a (Y-Ti-O)-Cr core-shell structure (fig 1c) and Cr segregation at the matrix grain boundary. Y-M3 and Ti-L2,3 elemental maps (fig 1b) show alternately Ti-rich and Y-rich atomic planes with the interreticular distance of d~2.87Å, which correspond to the (222) family planes of cubic pyrochlore Y2Ti2O7 (d222=2.91Å). HAADF images showing alternately bright (Y) and dark (Ti) (222) planes (fig 1a) and oriented along [110] axis (fig 2) also confirm the Y2Ti2O7-pyrochlore structure. EELS quantification was performed on Ti2O3 powder as a reference sample and on these nanoparticles: the result O/Ti~3.25 is close to the value of 3.5 for stoichiometric Y2Ti2O7. The smaller O/Ti ratio and the non homogeneity of the interreticular distance d222 through the particle (fig 1a) was interpreted due to defects in the particles structure: Y-Ti-O nanoparticles in ODS can present numerous defects and are often non stoichiometric [Yamashita et al, J Nucl Mater 2004].

The Ti oxidation, bonding state and site symmetry was studied using the Ti-L2,3 and O-K fine structure (ELNES), which is sensitive to Ti local environement. Furthermore, novel multivariate statistical analysis such as independent component analysis (ICA) [De la Peña et al. Ultramicroscopy 2011] was used after PCA to separate the individual spectral reponses of the EELS signal. The Ti oxidation state is shown to vary from the center of the nanoparticles to their periphery from Ti4+ in distorted Oh symmetry to a valency often lower than 3+. After ICA, the obtained independent components allow us to generate bonding maps (fig 3, 4): the particle center presents Ti4+, O and Y signatures where its periphery presents a reduced-Ti signature without crystal field splitting (CFS), depleted in Y and O.

The sensitivity of the Ti “white lines” ELNES to local symmetry distortions is also shown to be useful when investigating the strain induced in the nanoparticles by the surrounding matrix as can be seen in fig. 4a where the asymmetric Ti-L3 eg peak reflects the tetragonal distortion of the octahedral symmetry.

We thank the French programs CPR ODISSEE (funded by AREVA, CEA, CNRS, EDF, Mécachrome - contract n°070551) and METSA as well as the European program ESTEEM2 for financial support.

Fig. 1: (a) HAADF image of a nanoparticle and its RGB elemental maps : (b) red=Y-M3, green=Ti-L2,3, blue=O-K and (c) red=Cr-L2,3, green=Ti-L2,3, blue=Fe-L2,3

Fig. 2: (b) HAADF image oriented along [110] axis with (222) family planes of Y2Ti2O7, (a) the FFT taken from the nanoparticle and (c) the HAADF intensity profile (orthogonal to (222) planes) of the nanoparticle

Fig. 3: results of the ICA with Y-M2,3, Ti-L2,3 and O-K edges; maps of the components corresponding to (a) (Ti4+, Y, O) and (b) reduced-Ti<3+, (c) Spectra of these two components

Fig. 4: results of the ICA with Ti-L2,3 and O-K edges; (b) RGB bonding map (red=native oxide layer on the sample surface, green=Ti4+, blue=reduced-Ti<3+), (c) spectra of these three components and (a) Ti-L2,3 of the component 0 (Ti4+)
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-3057 Imaging of Li and He distribution inside steel using low-loss EELS

Klimenkov M.1, Materna-Morris E.1, Möslang A.1
1Karlsruhe Institute of Technology (KIT), Institute for Applied Materials - Applied Materials Physics (IAM-AWP)
michael.klimenkov@kit.edu

Low-loss electron energy loss spectroscopy (EELS) was used to detect and study the spatial distribution of He and Li in boron-alloyed steel after neutron irradiation. Both products of the 10B(n, α)7Li nuclear reaction have a low solubility in the steel Fr/Cr matrix. As a consequence, they form nano-sized precipitates or bubbles, which influence the steel’s mechanical properties. Their detailed characterization is an important step towards understanding microstructural changes in steels caused by both products of the transmutation reaction.

Previous TEM studies of He detection and distribution revealed that the He 1s-2p line from the He filled bubble is clearly detectable on the plasmon matrix signal [1], whereas the detection of metallic Li precipitates in metals was not described in the literature. We present the new method for the direct detection of Li and He in the Fe/Cr matrix based on the analysis of EELS spectra in the low-loss range. Calculation of the Fe/Cr plasmon structure using the Gaussian function or, in some complex cases, linear extrapolation and separation of the Li plasmon (9eV) and He 1s-2p line (22.4eV) allow for the generation of elemental maps showing the spatial distribution of Li and He (Figs. 2-3).

Fig. 1a shows a plasmon spectrum of the Fe/Cr matrix. Fig. 1b presents the spectrum of a bubble containing both Li and He lines. The intense He line at 21.7eV and the same line after background subtraction (insert) are visible in Fig. 1b. Left from the He line, an intense Li plasmon peak at 12eV (9.5eV - 10.5eV after subtraction of the Fe plasmon) can be observed clearly.

In Fig. 2 a Li drop of 22 nm in size is located in a 50 nm large He-filled bubble. The Li fills approximately ¼ of the bubble volume. This is clearly visible in (b) the He map and (c) the Li map. The map obtained by the 2.5eV window positioned at 7.5 eV shows the increased plasmon intensity on the drop’s surface (part d), whereas the map obtained at 11eV (part e) reveals nearly the same spatial distribution as the entire Li plasmon shown in part (c).

Fig. 3 presents an area with numerous bubbles that appear dark in the HAADF image. Several bubbles do not contain He, because they are located on the specimen surface – for example, the large bubble at the bottom right of the scanned area. The observation of numerous bubbles with Li drops or Li/He-filled cavities shows that the width of the Li plasmon peak is variable.

The spatial distribution of Li inside the Fe matrix on the nano-scale level was detected and investigated by means of EELS plasmon spectra. Some bubbles are half filled with Li and half with He.

[1] S. Fréchard, et.al J. Nucl. Mater. 393 (2009) 102

[2] M. Klimenkov, et.al Micron 46 (2013) 51–56

Fig. 1: EELS spectra of the Fe/Cr matrix (a) and a spectrum of a bubble showing Li and He lines.

Fig. 2: HAADF image (a) as well as  He (b) and Li (c) elemental maps. Parts (d) and (e) show the spatial distribution of Li surface and bulk plasmons

Fig. 3: HAADF image and Li and He elemental maps
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-3093 Micro-structural evolution in age-hardening alloys revealed by atomic-scale in-situ heating electron microscopy

Liu C. H.1,2, Xu Q.1,3, Chen J. H.2, Malladi S. K.1, Erdamar A.1, Tichelaar F.1, Zandbergen H.1
1Quantum Nanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands., 2Center for High Resolution Electron Microscopy, College of Materials Science and Engineering, Hunan University, Changsha, China., 3DENSsolutions, Delft, The Netherlands
c.liu-1@tudelft.nl

Thermal treatment is of vital importance in producing metal alloys with the desired performance. Many industrial tempers are designated by the temperature and duration adopted to treat the materials. Though tremendous microstructure characterizations have been done to understand the transformation process induced by heating, ambiguities pertaining to the underlying mechanisms still persist even for the well-known phenomenon such as precipitation hardening. The reason is related mainly with the fact that the microstructure observation and heat treatment weren’t done simultaneously in most previous literatures. In the present study, by using MEMS based in situ heating holder (DENSsolutions) we successfully carried atomic-scale real-time scanning transmission electron microscopy (STEM) investigations on the micro-structural evolution in metal alloys (AlCu alloy, AlCuMg alloy, AMgSiCu alloy and NiAl superalloy). The same thermal history as that used for treating metal alloys in factory has been applied on the specimens inside TEM. Solution treatment, quenching and annealing can be precisely controlled regarding the temperature, time as well as heating/cooling rate. This detailed study provides valuable data for quantifying the thermodynamics and kinetics governing the micro-structural evolution including nucleation, growth and coarsening of individual strengthening precipitate (θ' (AlCu2) and γ' (Ni3Al)) (Fig. 1). The direct atomic-scale imaging of the thickening and lengthening of the plate-like θ' (AlCu2) phrase generates new mechanistic insights into the growth process (Fig. 2 and 3). It is noteworthy to mention that the tip of the θ' precipitate is slightly broader than the inner part (Fig. 2). Contrary to this, the tip of this precipitate in Fig. 3 is narrower than the inner part possibly because of the proximity to another precipitate. Such kinds of local changes provide new insights into the interaction of different growing precipitates due to the overlap of diffusion field. The alloying elements diffusion behaviours monitored during heating are critical for unravelling the factors affecting the formation of the effective strengthening particles especially those in the confusing multi-step ageing frequently used in processing high performance aluminium alloys. Our study also demonstrates that in-situ heating electron microscopy is a powerful tool to assess the effect of alloying elements and tempers on the microstructure and is fruitful for developing metal alloys with enhanced properties.

This work is financially supported by ERC NEMinTEM Project under contract no. 267922, and partly by the National Natural Science Foundation of China (No. 51171063, 51371081); Instrumental Innovation Foundation of Hunan Province (No. 2011TT1003). C.H. Liu gratefully thanks the financial support from China Scholarship Council and technical support by DENSsolutions B.V..

Fig. 1: Snapshots from a video showing the evolution of plate-like precipitates viewed along [001]Al direction in AlCu alloy aged at 180℃ from as-quenched to 10 h.

Fig. 2: Snapshots from a video showing the thickening of θ' precipitate viewed along [001]Al direction in AlCu alloy aged at 180℃. The arrow points to the changing position of the growth ledge.

Fig. 3: Snapshots from a video showing the lengthening of θ' precipitate viewed along [001]Al direction in AlCu alloy aged at 180℃. The arrow points to the changing position of interface. The red box represents the unit cell of θ' phrase.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-3123 Structural investigations on Cu-Ag alloys produced by high-pressure torsion

Kormout K. S.1, Yang B.1, Pippan R.1
1Erich Schmid Institute of Materials Science, Leoben, Austria
karoline.kormout@oeaw.ac.at

Severe plastic deformation (SPD) techniques are convenient methods to force a mixing of usually immiscible elements. Supersaturated solid solutions or extended solubilities were reported for several systems [1–3]. The microstructures of so-called “far-from-equilibrium” materials show specific characteristics compared to conventional coarse-grained materials from which unique material properties can arise [4, 5]. SPD deformation of two-phase alloys usually leads to highly defective microstructures with extremely small grain sizes and exceptional grain boundary morphology. The large volume fraction of grain boundary regions, the grain shape, which can vary from equi-axed to fiber-like, and supersaturation or amorphization processes strongly influence the mechanical properties. A correlation between microstructure and material properties is essential for tailoring the materials performance for specific applications. In order to study this relationship CuAg alloys were produced by powder consolidation and subsequent high-pressure torsion processing. Varying composition (Cu‑25/50/75wt% Ag) and process parameters led to a multitude of microstructures. The defect-rich microstructure of a Cu‑25wt%Ag is shown in Fig 1a with a grain boundary revealed by high-resolution TEM (HRTEM) in Fig 1b and c. Such non-uniform faceted grain boundaries are typical for SPD materials [6]. In Cu‑50wt%Ag alloys a very fine-grained, partially amorphous structure was observed (see Fig 1d and e). Disordering occurs mainly at former grain or phase boundaries, but also in the grain interior. Further investigations will include a detailed TEM analysis of the microstructure combined with mechanical testing and annealing experiments.

[1] H. Shen, Z. Li, B. Günther, A. V Korznikov, and R. Z. Valiev, Nanostructured Mater., vol. 6, pp. 385–388, 1995.
[2] A. Bachmaier, M. Kerber, D. Setman, and R. Pippan, Acta Mater., vol. 60, pp. 860–871, 2012.
[3] A. Bachmaier, J. Keckes, K. S. Kormout, and R. Pippan, Philos. Mag. Lett., vol. 94, no. 1, pp. 9–17, 2014.
[4] R. Z. Valiev, R. K. Islamgaliev, and I. V Alexandrov, Prog. Mater. Sci., vol. 45, pp. 103–189, 2000.
[5] A. Bachmaier and R. Pippan, Int. Mater. Rev., vol. 58, no. 1, pp. 41–62, 2013.
[6] X. Sauvage, G. Wilde, S. V Divinski, Z. Horita, and R. Z. Valiev, Mater. Sci. Eng. A, vol. 540, pp. 1–12, 2012.

We gratefully acknowledge the financial support of the Austrian Science Fund (FWF): P24429.

Fig. 1: Figure 1: (a) TEM bright-field micrograph of a Cu-25wt%Ag alloy with corresponding HRTEM images of a grain boundary in (b) and (c), and (d) TEM bright-field image of a Cu-50wt%Ag alloy with HRTEM image in (e).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-3131 Transmission Electron Microscopy analysis of sigma (σ)-phase formation mechanisms in high chromium content iron-chromium model alloys with small addition of molybdenum for high temperature application

Iskandar M. R.1, Niewolak L.2, Quadakkers W. J.2, Mayer J.1
1RWTH Aachen University, Central Facility for Electron Microscopy (GFE), Aachen, Germany, 2Forschungszentrum Jülich, Institute of Energy and Climate Research (IEK-2), Jülich, Germany
iskandar@gfe.rwth-aachen.de

One of the well-known intermetallic phases which form in the Fe-Cr system is the sigma (σ)-phase. It has a tetragonal unit cell (space group P42/MNM) with a = 8.80 Å and c = 4.54 Å [1]. In general, the σ-phase forms during long-time exposure in the temperature range of 565 to 980°C and it’s composition varies quite widely which makes it difficult to give an exact stoichiometry. The formation of the σ-phase is one of the main reasons for the deterioration of stainless steel properties. As an example, it has been reported that the precipitation of σ-phase on grain boundaries consumes chromium and as a result, there is a loss in corrosion resistance [2]. The present investigation was conducted to investigate the σ-phase formation caused by the addition of molybdenum as one of the ferrite stabilizing alloy elements in order to control the σ-phase nucleation mechanisms.
A high purity model alloy containing 68 wt% Fe, 30 wt% Cr and 2 wt% Mo, manufactured by ThyssenKruppVDM, was annealed in vacuum for 1000 h at 700°C. Later, the specimen was prepared using the focused ion beam (FIB) technique in a FIB Strata 205 from FEI. Microstructural analyses were carried out using the Libra 200 FE and Titan-T TEMs under an acceleration voltage of 200 KV and 300 KV respectively.
The formation of the σ-phase along the grain boundaries as well as within the grains are shown in Figure 1. Two types of precipitates with contrast difference are detected on σ-phases formed on grain boundaries (figure 1.b). Figure 2 shows the bright-field (BF) TEM images and SAED patterns of these two precipitates. The BF images, figure 2.a and 2.c, show that the bright precipitate (marked as L2 in fig 1.b) contains a higher density of planar faults than the dark one (marked as L1 on fig 1.b). Furthermore, the analyses of the SAED patterns also confirms that the dark and bright precipitates were identified as chromium-carbide and chi (χ)-phase, respectively (fig 2.c and 2.f). The BF images also revealed that part of the σ-phase close to the chromium-carbide precipitates has less planar faults than the one close to χ-phase precipitates.
The matrix/σ-phase interface from σ-phase formed on grain boundaries and inner grains (marked L1 and L3 in figure 1 respectively) were further investigated by means of high-resolution imaging technique. Figure 3 shows an example of the high-resolution (HR) image of σ-phase formed on grain boundaries which contains faults at the interface.

References
[1] B.G. Bergman and D.P. Shoemaker, The space group of the σ-FeCr crystal structure, J. Chem. Phys, 19, 515, 1951.
[2] N. Lopez, M. Cid, and M. Puiggali, Influence of σ-phase on mechanical properties and corrosion resistance of duplex stainless steels, Corr. Sci, vol. 41, no. 8, pp. 1615-1631, 1999.

The authors gratefully acknowledge the financial support granted by the Deutsche Forschungsgemeinschaft (DFG) within the research project DFG MA 1280/41-1

Fig. 1: (a) SEM-BSE micrographs of the investigated specimens showing σ-phase precipitated on grain boundaries and inside grains as well as selected areas marked by A1 (b) and A2 (c) where FIB lamella were prepared.

Fig. 2: Bright-field (BF) images and SAED patterns of two precipitates, Cr-carbides and χ-phase, which were found on σ-phase formed on grain boundaries (marked by L1 and L2 on fig 1.b). The BF and SAED pattern show that the σ-phase contains numerous planar faults.

Fig. 3: High-resolution (HR) image taken from matrix/σ-phase interface of σ-phase formed on grain boundary (marked L1 in figure 1.b) with the electron beam parallel to the [001] matrix zone axis.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-3282 Electron diffraction tomography study of MgZn precipatates in Mg matrix

Klementová M.1, Palatinus L.1, Němec M.1, Gärtnerová V.1
1Institute of Physics of the AS CR, v.v.i., Na Slovance 2, 182 21 Prague 8, Czech Republic
klemari@fzu.cz

Low density, high specific strength and the ease of recycling make magnesium and its alloys potentially good candidates for numerous structural applications [1]. One of the most common alloying elements in magnesium is Zn. Besides remarkable improvement of mechanical properties via solid solution and/or precipitation strengthening, Zn is together with Mg classified as a biocompatible element. Thus, Mg-Zn based systems can also be considered as an attractive material for implants.


Binary magnesium alloy with nominal composition Mg-12 wt.% Zn was prepared by die casting under Ar atmosphere and subsequently annealed at 320°C for 20 hours followed by warm water quenching. The goal of this work is to describe the crystal structure of Zn based particles present in the binary magnesium alloy.


Samples were studied by transmission electron microscopy performed on a Philips CM 120 (LaB6, 120kV) equipped with a NanoMEGAS precession unit DigiStar, an Olympus SIS CCD camera Veleta (2048x2048), and an EDAX windowless EDS detector Apollo XLTW. Precession-assisted electron diffraction tomography (EDT) in microdiffraction setup was used to acquire data for structure determination of MgZn precipitates and their orientation within the Mg matrix.


Precipitates of several micrometers in size (Fig. 1a) correspond to Mg21Zn25 phase with rhombohedral structure, space group R-3c, lattice parameters a~26 Å, c~8.9 Å (Fig. 1b). The structure was determined from 1711 independent reflections (averaged from 13026 measured intensities, Rint=24.09), and refined using kinematical approximation to R-value of 26.53 %. The structure model matches very well the previously reported structure of Mg21Zn25 [2]. The matrix is formed by hexagonal Mg, space group P63/mmc, lattice parameters a=3.2 Å, c=5.2 Å. Orientation relationship of MgZn precipitates in Mg matrix was observed as (10-1)Mg || (010)MgZn and [101]Mg close to [201]MgZn (Fig. 2a,b). However, this relationship might vary significantly as the precipitates are quite coarse and therefore loss of coherency is expected.

[1] Pollock T.M. Weight Loss with Magnesium Alloys. Science (2010) 328, 986-987.
[2] Cerny, R. and Renaudin, G. Acta Cryst. (2002) C58, 154-155.

Authros would like to acknowledge Grant Agency of the Czech Republic for support under project No. P108/12/G043.

Fig. 1: (a) Bright-field image of MgZn precipitates in Mg matrix, (b) results of structure solution from EDT data of a MgZn precipitate viewed down [001] (top - map of electrostatic potential, bottom - structural model).

Fig. 2: Oriented SAED patterns of a MgZn precipitate in Mg matrix. (a) viewed down Mg [101], (b) viewed down MgZn [201].
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-3460 Transmission electron microscopy characterization of copper-multiwall carbon nanotube interfaces in Cu-CNT nanocomposites.

Mendoza M. E.1, Campos A. P.1, Archanjo B. S.1, Machado I. F.2, Solorzano I. G.3
1Materials Metrology Division, National Institute of Metrology, Quality and Technology, Rio de janeiro, Brazil., 2Mechatronic Engineering and Mechanical Systems Department, University of São Paulo, São Paulo, Brazil., 3Materials Engineering Department, PUC-Rio, Rio de Janeiro, Brazil.
meoliveros-prometro@inmetro.gov.br

The increasing interest in nanostructured materials in recent years has provided incentive to develop new kind of composites containing carbon nanotubes (CNTs). In particular, Copper- multiwall carbon nanotube nanocomposites (Cu-MWCNT) have been considered as a promising material for applications in electronic materials, heat exchangers and structural elements [1,2].
It has been reported that the addition CNTs in a Cu matrix can increase or decrease the mechanical and transport properties of the matrix depending on the Cu-CNT interface, CNT integrity as well as on the CNT uniform distribution, which are directly correlated with the nanocomposite synthesis and processing(sintering).
The main objective of this work is to characterize structurally, morphologically and analytically a Copper –5 wt% MWCNT nanocomposite produced by chemical synthesis and thermo mechanical processing (Spark plasma sintering), by means of transmission electron microscopy (TEM).
Nanocomposite powders were produced by dissociation of a homogeneous suspension containing Cu(NO3)2.3H2O–MWCNT,previously functionalized in tetrahydrofuran solution, followed by H2 reduction of the obtained CuO-MWCNT precursor. Bulk nano-composite pellets were obtained using a Doctor Sinter Lab Machine (SPS 1050) applying 70 MPa pressure, at 600 °C for 5 minutes.
TEM samples were prepared from the powder synthesis material and from the obtained final pellets. The former sample was prepared using a dispersion of few milligrams of powder inisopropyl alcohol, followed by ultrasonic agitation. One drop was placed on a nickel TEM grid. The later was made using a FEI Nova FIB-SEM instrument. A TEM -FEI Titan operating at 300kV equipped with EDS, and EELS were used as main characterization tool.

After chemical synthesis Cu powder particles with spherical and faceted morphologies decorating the MWCNTs were observed. The particles were in the 5-100nm range (Fig.1a), showing good adherence at the interface (Fig.1b).
After consolidation into pellet and sintering, good consolidation and heterogeneous grain growth (50nm–2μm range) were observed in the Cu matrix. Remaining porosity and annealing twins are present at the lamella. TEM-BF images show regions with high dislocations density as well as the presence of CNTs at the Cu grain boundaries and its transformation into amorphous carbon, nanoribons and graphitization (Fig 2).
Elemental mapping using STEM-EELS allowed us to identify the presence of carbon, copper oxide and metallic copper at the interface Cu-MWCNT (Fig. 3). Notwithstanding, possible re-oxidation after sample preparation can be considered.

References
[1] K. T. Kim, et al. Materials Science and Engineering A 430 ( 2006) 27-33.
[2] K.Tae, et al. Materials Science and Engineering A 449-451 (2007)46-50.

The authors acknowledge to Air Force Office for scientific research (AFOSR,USA), CNPq- Brazil.

Fig. 1: Fig 1. MWCNTs decorated by copper nanoparticles and good adherence at the interface are observed in the TEM bright field images (a and b).

Fig. 2: Fig. 2. (a and b). TEM-BF images show regions with high dislocations density and typical strain fields as well as big damage of CNTs. HRTEM (Fig 2 c) shows a carbon nanostructure like ribbon as product of MWCNT transformation during the sintering processes.

Fig. 3: Fig. 3.Elemental mapping using STEM-EELS allowed us to identify the presence of carbon, copper oxide and copper at the interface Cu-MWCNT.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-4-O-3468 Multi-scale Observation of Microstructure Developments in Disorder-order Transformation in CoPt Alloy Heat-treated under a Magnetic Field

Akamine H.1, Farjami S.1, Itakura M.1, Nishida M.1, Fukuda T.2, Kakeshita T.2
1Kyushu University, Fukuoka, Japan, 2Osaka University, Osaka, Japan
3ES14001R@s.kyushu-u.ac.jp

CoPt alloy undergoes disorder-order transformation from a cubic disordered phase to a tetragonal ordered phase at 1045 K. Because of decrease in crystal symmetry, three ordered variants are formed. An ordering heat-treatment under a magnetic field was performed to control formation and selection of preferred variant with its tetragonal c-axis parallel to the applied magnetic field. However, the ordering process and mechanism of variant selection have not been clarified yet because of its multi-scale changes of microstructure over nm to µm scale. In the present work, multi-scale microstructure observation with the novel SEM was carried out using the channeling contrast imaging techniques to clarify the process of microstructure formation and variant selection in single crystalline CoPt alloy ordered by the two-step heat-treatment under a magnetic field of 10 T and without magnetic field. In order to understand the origin of various contrasts in the obtained SEM images, TEM and STEM observations were carried out and studied correspondence between SEM, TEM and STEM results.
In the early stage of growth process, many {101}L10 twins were successfully observed in SEM observations and the contrasts of channeling BSE images well corresponded to TEM observation results in nm scale (Fig. 1). In addition, the variant which has c-axis parallel to the applied magnetic field was developed preferentially compared to other two variants. After all of the heat-treatment process, those twins were vanished and single variant structure was obtained. On the other hand, without magnetic field, coarsening process of the micro-twins in the early stage of growth heat-treatment was strongly limited because of competition among three variants. In addition, it was clarified by wide-range observation with SEM that the three variants formed self-accommodation structure of {101}L10 twins (Fig. 2) which was similar to that in thermoelastic martensitic transformation. These results indicate, after the diffusional ordering process in the early stage, invariant deformation process becomes dominant and three variants accomodates martensite-like structure.
In this work, we successfully evaluated the microstructure development process of disorder-order transformation in single crystalline CoPt alloy in multi-scale by SEM observations with channeling contrast imaging technique. These results show promising performance of the novel SEM in multi-scale evaluation of microstructures.

The authors are grateful to Prof. S. Nishijima of Osaka University for providing the superconducting magnet through the accomplishment of this work.

Fig. 1: (a) Channeling BSE image and (b) TEM bright field image for the specimen heat-treated at 773 K for 30 min under magnetic field of 10 T and at 1023 K for 3 min in the first and second steps, respectively.

Fig. 2: Channeling BSE image for the specimen heat-treated at 773 K for 30 min under magnetic field of 10 T and at 1023 K for 180 min in the first and second steps, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1409 NANOPRECIPITATES OF PHOSPHORUS IN SUPERMARTENSITIC STAINLESS STEELS

Rodrigues C. D.1, Jorge A. M.2, Tremiliosi F. G.1
1Instituto de Química de São Carlos – Universidade de São Paulo, Brasil Av. Trabalhador Sãocarlense, 400, CEP: 13566-590, São Carlos (SP), Brasil, 2Universidade Federal de São Carlos, Departamento de Engenharia de Materiais Rodovia Washington Luiz, Km 235, cx 676, 13560-270 São Carlos, Brasil
cesaraug@sc.usp.br

Super martensitic stainless steels (SMSS) exhibit a good combination of strength, toughness, corrosion resistance and weldability properties. Due to these properties, they have been increasingly applied in the oil and gas industries to substitute the more expensive duplex stainless steels for onshore and offshore tubing applications [1]. The chemical compositions of SMSS are based on Fe–Cr–Ni–Mo system and lower contents of the C (≤ 0.02 wt %), N (≤ 0.002 wt %) and P, S (≤ 0.003 wt %). This steels present good strength, toughness and corrosion resistance mainly when microalloyed with Al, Nb, Ti and V [2]. In this work was used the SMSS with high phosphorus content (SMSS+P = 0.012 % C, 12.5 % Cr, 5.36 % Ni, 2.11 % Mo, 0.29 % Mn, 0.19 % Si, 0.0013 % S, ↑0.017 % P (≤ 0.003%), and 0.001 % N). The steel was produced with extra low residual impurity contents in a vacuum induction-melting furnace and hot rolled to 29 mm diameter round bars in Villares Metals Research Centre. The samples of SMSS+P steel were heat treated at: 1000 °C/45 min/oil + 610 °C/2 h/air. Chemical and microstructural characterizations were performed by transmission electron microscopy (TEM) in a TECNAI G2 F20 microscope (200KV), withh Energy Dispersive X-ray chemical microanalysis system. Fig.1 (a) presents a bright field STEM image showing ultra-fine martensite-lath morphologies with recrystallized grains inside and also the presence of subgrains. Fig. 1 (b) presents the corresponding selected area electron diffraction pattern (SAEDP) revealing the high deformation state of the sample and the presence of subgrains. The white circle in Fig. 1 (a) represents the region where it was zoomed in Fig.2 (a), which presents a bright field STEM image showing phosphorus-rich nanoprecipitates (point 1), and in in Fig.2 (b), which presents a dark field STEM image showing nanoprecipitates with sizes of about 8 nm, and that the precipitation occurs preferentially in dislocation lines. The SAEDP in the inset of Fig. 2 (b) shows a diffraction ring of such precipitation together with matrix spots. Indexation of SAEDP revealed the presence of CrP4 along [0,1,0] zone axis, which was confirmed by EDX analysis (Fig 2a, point 1 as indicated) revealing the higher content of phosphorous (0.07 weight%) when compared with the matrix (Fig 2a, point 2 as indicated) where phosphorous was not detected. The presence of such precipitates can justify the good obtained mechanical and corrosion resistance properties.
References: [1] X. P. Maa, et al. Mat. Sci. Eng. A-Struct., A539 (2012) 271.
                  [2] C. A .D. Rodrigues, et al., Mat. Sci. Eng. A-Struct., A460 (2007) 149.

This research was supported by CNPq, CAPES and FAPESP (Brasil).

Fig. 1: Fig.1. (a) Bright field STEM image showing the ultra-fine martensitic-lath morphologies with recrystallized grains inside and also the presence of subgrains, and the white circle corresponds to the region where this image was zoomed in Fig. 2. (b) the SAED pattern of the martensitic matrix.

Fig. 2: Fig.2. (a) Bright field STEM image showing phosphorus-rich nanoprecipitates (point 1) located into subgrains of the SMSS+P steel. (b) Dark field STEM image showing nanoprecipitates with size approximate of 8 nm, and top right hand side present the corresponding SAED pattern of the nanoprecipitates.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1414 Revealing the precipitation of Al alloys subjected to high pressure torsion

Li J. H.1, Renk O.2, Kutleša P.2, Zhang Z. L.2, Pippan R.2, Schumacher P.1,3
1Chair of Casting Research, Department of Metallurgy, the University of Leoben, A-8700, Leoben, Austria , 2Erich Schmid Institute of Materials Science, Austrian Academy of Sciences, Austria, 3Austrian Foundry Research Institute, Parkstrasse 21, Leoben, Styria, A-8700, Austria
jiehua.li@unileoben.ac.at

Al alloys have important applications in the automotive and aerospace industries because of their high specific strength for the weight reduction and better fuel economy. However, mechanical properties of conventional Al alloys are often not suitable for some extreme applications. High pressure torsion (HPT) has been found to be effective to refine the grain size, enhance the precipitation hardening, and finally improve the performance of Al alloys. The enhanced precipitation hardening can be directly correlated to the different type precipitates formed during HPT and subsequent aged treatments. Full characterisation and quantification analyses of the precipitates evolution is thus of great importance to optimize conventional Al alloys in service and develop new Al alloys.

In this contribution, advanced electron microscopy was employed to characterise the evolution and chemistry composition of precipitates formed along grain boundary and / or within the matrix in Al alloys subjected to HPT. Furthermore, in situ heating and cooling in high resolution TEM (STEM) was also employed to elucidate the evolution of precipitates, thereby optimize the heat treatment and finally improve the mechanical properties. It was demonstrated that advanced electron microscopy is of great necessity to reveal mysteries in conventional research fields, e.g. casting and / or solidification. However, more attentions have to be paid to the differences caused by the size effects. For example, the temperatures of precipitates observed by in situ heating of thin TEM foil is slightly higher than that measured by DSC heating of bulk samples. An combined application of advanced electron microscopy and other analysis techniques (i.e. DSC) can provide a comprehensive information of the precipitation of Al alloys.

The authors gratefully acknowledge the access to the TEM facility at the Erich Schmid Institute of Materials Science of the Austrian Academy of Sciences.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1422 Structural and Morphological study of nanocrystalline Fe-50%Al alloy during ball milling of elemental powders

Brajpuriya R. K.1
1Department of Physics, Amity University Haryana, Manesar, Gurgaon, India
ranjeetbjp@yahoo.co.in

The objective of the work is to synthesize and study the structural and morphological changes in nanocrystalline Fe-50%Al alloy prepared directly by mechanical alloying in a high energy rate ball mill. The phase transformations, structural and morphological changes occurring in the studied material during mechanical alloying (MA) were investigated by X-ray diffraction (XRD) and Scanning electron Microscopy. Fig. 1 shows the morphological evolution of Fe1-xAlx alloy samples as a function of milling time. As a result of intensive fracture and cold welding during the ball milling, the structure and shape of the particles have been changed drastically. The initial shape of crystallites disappeared completely, and their structure became an amalgam of small and large irregular and angular shaped particles with wide range of sizes. During ball milling, the grain size of constituents was decreased to the nanometer range and the constituents dissolved at the nanograin boundaries, which provided the strong conditions for the solid-state synthesis reaction. This phenomenon is a result of the existence of a balance between the fracture and re-welding processes. The formation of FeAl intermetallic alloy from elemental Fe and Al powders appears to be composed of two steps: progressive refinement of Fe and Al grains within the sandwich type microstructure, followed by FeAl formation, presumably at the interfaces between Fe and Al grains. From SEM images, it is clear that the iron and aluminium elemental distributions are closely correlated indicating that the two elements are completely alloyed and the FeAl solid solution is formed. These results are very consistent with the XRD analysis.

Fig. 1: SEM micrographs of Fe1-xAlx alloy as a function of milling time.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1433 Revealing the precipitation in Al-Cu based alloys with Sc addition

Li J. H.1, Albu M.2, Hofer F.2, Schumacher P.1,3
1Chair of Casting Research, Department of Metallurgy, the University of Leoben, A-8700, Leoben, Austria , 2Institute for Electron Microscopy and Nanoanalysis (FELMI), Graz University of Technology, Center for Electron Microscopy Graz, Steyrergasse 17, A-8100 Graz, Austria, 3Austrian Foundry Research Institute, Parkstrasse 21, Leoben, Styria, A-8700, Austria
jiehua.li@unileoben.ac.at

Al-Cu based alloys have important applications in the automotive and aerospace industries because of their high specific strength for the weight reduction and better fuel economy. However, their hot tearing tendency hampers their wider applications. Sc addition into Al-Cu based alloys has been found to be effective to refine the grain size, reduce the hot tearing tendency, enhance the precipitation hardening and finally improve the performance of Al-Cu based alloys. Grain refinement can be directly correlated to the enhanced heterogeneous nucleation of primary Al3Sc or Al3(Sc,Zr,Ti) phase for α-Al. While, enhanced precipitation hardening can be mainly attributed to different precipitates formed during heat treatments.

In this contribution, advanced electron microscopy was employed to characterize the precipitates formed along grain boundary and / or within the matrix in Al-4.5Cu-0.2Sc (wt.%) alloys after T6 heat treatment. It was found that (i) Sc partitions into the Al2Cu phase (Figure 1), which was believed to improve the thermal stability of Al2Cu phase, and (ii) Compared with Al-4.5Cu (wt.%) based alloys (not shown here), the size of the Al2Cu precipitates decreases, however, the number density of the precipitates increases (Figure 2). Furthermore, high resolution STEM was employed to characterize the Al2Cu precipitates and the interface between Al2Cu precipitates and α-Al matrix (Figures 3 and 4). The Al2Cu precipitates appear to be no-coherent with α-Al matrix when viewed from {011}α-Al (Figure 3c) and {001}α-Al (Figures 4c,d). The loss of coherency indicates that the precipitation process may be in the stage of peak ageing or over ageing. At this stage, the precipitation microstructure becomes more stable, and the mechanical properties is enhanced. It should be also noted that one lost atomic layer (step) was observed at the interface between Al2Cu precipitate and α-Al matrix, as shown in Figure 4c. Although this observation could be due to the beam damage, it also strongly demonstrates that advanced electron microscopy is of great necessity to reveal the mysteries (i.e. the precipitate interface structure and composition) in conventional research fields, e.g. solidification and / or precipitation. Full characterization on the precipitates from micro to atomic scale is of great importance to optimize conventional Al-Cu based alloys in service and develop new Al-Cu based alloys.

The author (J.H. Li) also gratefully acknowledge the access to the TEM facility at the Erich Schmid Institute of Materials Science of the Austrian Academy of Sciences.

Fig. 1: Figure 1: Sc partitions into the Al2Cu phase in Al-4.5Cu-0.2Sc (wt.%) alloys.

Fig. 2: Figure 2: The smaller precipitates with higher number densities in Al-4.5Cu-0.2Sc (wt.%) alloys.

Fig. 3: Figure 3: The interface between Al2Cu precipitates and α-Al matrix. The precipitates appear to be no-coherent when viewed from {011}α-Al.

Fig. 4: Figure 4: High resolution STEM images of the Al2Cu precipitates (a) and the interface between Al2Cu precipitate and α-Al matrix (b-c). The Al2Cu precipitate appears to be no-coherent when viewed from {001}α-Al.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1473 TEM Analysis of Carbides in As-cast High Chromium White Cast Iron Containing 0-10 wt.% Tungsten

Imurai S.1, Chomsaeng N.2, Thanachayanont C.3, Pearce J. T.3, Tsuda K.4, Chairuangsri T.1
1Department of Industrial Chemistry, Chiang Mai University, Chiang Mai, 50200, Thailand, 2Faculty of Gems, Burapha University, Chanthaburi, 22170, Thailand, 3MTEC, Pathumthani, 12120, Thailand, 4IMRAM, Tohoku University, Sendai, Japan
suttawani@gmail.com

To improve wear resistance, tungsten (W) has been added into high chromium white cast irons as a carbide-forming element. Recent work has reported that W at low content led to the formation of WC1-x, W6C2.54 and W3C [1]. However, at high tungsten content, Fe7W5C2 and possibly W2C or WC are formed [2]. In the present study, carbides in as-cast (27-28) wt.%Cr – (2.4-2.9) wt.%C alloys with 0-10 wt.% W addition were investigated by TEM. Effects of W addition on the microstructure of the alloys and on formation of different carbides are discussed and compared with the results based on SEM and XRD in previous work [1,2].
TEM thin foils were prepared by twin-jet electropolishing (Fishchion, Model 110) at 20 V and -10°C. The electrolyte was a solution containing 10 vol.% perchloric acid and 30 vol.% of 2-butoxyethanol in absolute ethanol. TEM investigation was performed at 200 kV using a TEM/STEM JEOL JEM2010 equipped with an EDS detector (Oxford, Inca).
SEM backscattered electron images of alloys studied are shown in Figure 1. The matrix is austenite partially transformed to martensite. TEM analysis of carbides is given in Figures 2 to 4. Only eutectic M7C3 was found in the reference alloy without W addition. Two forms of W-containing M7C3 were found in the alloys with 1 wt.% and 4 wt.% W : one as large blocky, primary M7C3 and another as eutectic M7C3. In the alloy with 10 wt.% W, only large blocky, primary M7C3 was found. Streaking in SAED patterns (shown in Figures 2(b) and 3(b)) and the low FeKα/CrKα (ca. 0.4) and WLα/CrKα (ca. 0.02) peak height ratios in EDS spectra (shown in Figures 2(c), 3(d) and 4(d)) are characteristics of M7C3. The W content in M7C3 increased as the overall W content of the alloys was increased. High W addition promotes formation of M23C6 and M6C. These carbides formed in the later stage of solidification from W-segregated liquid, resulting in carbides with higher M:C ratio than that of the M7C3. W-rich M23C6 and M6C are distinguishable by electron diffraction from certain zone axes and also by TEM-EDS. FeKα/CrKα and WLα/CrKα peak height ratios of ca. 0.6 and 0.1, respectively, are characteristics of M23C6, whereas those of ca. 1.7 and 0.9, respectively, are of M6C. Fish-bone M23C6 colonies encapsulating the M6C structure were found in the alloy with 10 wt.% W addition. In the present study, M3C, M2C or MC has not been found in any of the alloys. The reason for the absence of these carbides can be attributed to the higher M:C ratio in the alloys in the present study as compared to those in previous work [1,2].
References
[1] Lv Y., Sun Y., zhao J., Yu G., Shen J., Hu S., Mats Design 39 (2012) 303-308.
[2] Heydari D., Skandani A.A., Haik M.A., Mats Sci. Eng. A 542 (2012) 113-126.

The TGIST-NSTDA Scholarship as well as the Graduate School and Faculty of Science, Chiang Mai University, Thailand, are thanked for funding support.

Fig. 1: Backscattered electron images in SEM show the microstructure of the alloys : (a) the reference iron without tungsten, (b) 1 wt.%W addition, (c) 4 wt.%W addition, (d) 10 wt.%W addition.

Fig. 2: (a) Bright-field TEM micrograph shows austenite (γ) and M7C3 in the reference iron. (b) Corresponding SADP from the [100] M7C3 zone axis. (c) TEM-EDS spectra from austenite (γ) and M7C3, respectively.

Fig. 3: (a) Bright-field TEM micrograph shows M7C3 and M6C in the alloy with 4 wt.% W. (b) and (c) Corresponding SADPs from the [122] M7C3 and the [112] M6C zone axes. (d) TEM-EDS spectra from austenite (γ), M7C3 and M6C.

Fig. 4: (a) Bright-field TEM micrograph shows M23C6 and M6C in the alloys with 10 wt.% W. (b) and (c) Corresponding CBEDs from the [111] M23C6 and the [111] M6C zone axis. (d) TEM-EDS spectra from austenite (γ), M7C3, M23C6 and M6C.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1488 Advanced Electron Microscopy Characterization of spray formed and hot-extruded Al Alloy 7050 under different aging conditions

Afonso C. R.1, Mazzer E. M.1, Bolfarini C.1, Kiminami C. S.1
1Universidade Federal de São Carlos (UFSCar), Department of Materials Engineering (DEMa), São Carlos - SP, Brazil
conrado@ufscar.br

It is demonstrated here that the rapid cooling rate imposed by the spray forming equipment is convenient to reprocess the Al 7000 alloy machining chips from the manufacture of aeronautic components resulting in a good microstructure with refined hardening η’ precipitates within the matrix, refined grains and low segregation of the elements. The final microstructure after the hot-extrusion, solution and aging heat treatment is composed by GP zones, metastable phase η’, equilibrium η phase, Al3Zr nanometric precipitates and the coarse intermetallic Al7Cu2Fe phase. The microstructure was characterized using X-ray diffraction (XRD), differential scanning calorimeter (DSC) and scanning electron microscopy (SEM). Transmission electron microscopy (TEM) analysis together with associated techniques (HRTEM, STEM, spectral imaging and EFTEM) was realized in an FEI Tecnai G2 TEM/STEM 200 kV equipped with EDS (EDAX DX-4) and a Gatan Image Filter (GIF) Tridiem – Electron Energy Loss Spectroscopy (EELS) detector. After the homogenization heat treatment the only remaining phase was the intermetallic Al7Cu2Fe. After the extrusion, partial recrystallized regions were found in the microstructure. Precipitation of intermetallic phases occurred during processing, in this way, a solution heat treatment was needed. Then, the artificial aging provided a fine nanometric precipitates distribution of hardening (η’) precipitates and GP zones within the Al-fcc matrix and equilibrium precipitates (η) distributed along the grain boundaries. Advanced transmission electron microscopy techniques are fundamental to characterize nanometric precipitates and GP zones such as the ones formed during aging of 7050 Al alloy.

The authors would like to thank to CNPq and FINEP for financial support

Fig. 1: HRTEM image in the [100]Al-fcc zone axes showing the nanoscale precipitates and Fourier transfor (FT) for the whole image, equivalent to SAED pattern

Fig. 2: EFTEM (energy filtered TEM) images of 7050 Al alloy sample aged at 160oC for 16 h showing maps of Al, Mg, Zr and Zn edges.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1531 Particle reinforced Al-matrix composites

Lábár J. L.1, Balázsi K.1, Tóth A. L.1, Wéber F.1, Károly Z.2, Hargitai H.3, Dhar A.4
1Research Institute for Natural Sciences of the HAS, Institute for Technical Physics and Materials Science, Konkoly-Thege M. út 29-33, H-1121 Budapest, Hungary, 2Research Institute for Natural Sciences of the HAS, Institute of Materials and Environmental Chemistry, Magyar tudósok körútja 2, H-1117 Budapest, Hungary, 3Material Testing Laboratory, Széchenyi István University, Egyetem tér 1, 9026 Győr, Hungary, 4National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi, 110070 Delhi, India
labar.janos@ttk.mta.hu

Composites provide a possibility to tune materials properties by a combination of the properties of the matrix and the reinforcing material. An application specific good compromise can be reached in hardness, fracture toughness, tribological and other physical parameters. An improvement in properties is expected by reducing the grain size and particle size to the submicron level for the nanocomposites produced by powder metallurgy (PM).

Al-Al2O3 nanocomposites were produced by cryogenic milling and subsequent sintering. There are three problems to be solved in this process. First, production of nano-sized mixed powder required cryogenic milling. Second, spark plasma sintering (SPS) was used to prevent grain growth during sintering. Third, solution was needed to overcome of the problem of high melting point alumina (both native oxide on the surfaces of grains and the alumina nanoparticles in the mixture) surrounding the low melting point Al and so hindering the sintering process.

The sintered nanocomposites were characterized by densitometry, measurement of hardness, tribology, electron microscopy and related analytical techniques (SEM, TEM and EDS). Effect of additional elements (mixed to the Al-powder) was also studied. Optimization of the sintering parameters improved density and mechanical properties.

Both pure Al and alloyed Al were mixed with 30% Al2O3. Best densification (97%) and lowest wear rate (4.1*10-4 mm3/m) was achieved with pure Al, sintered at 600°C, while the hardest composite was obtained with an Al-27%Si-7%Ni alloy. The same hardness (2.75 GPa) was reached with two preparation conditions for the 70%( Al-27%Si-7%Ni alloy)+30%Al2O3 samples. First, 600°C sintering temperature was needed for powders milled in Ethanol at room temperature. Second, the same powder mixture milled in liquid nitrogen sintered to the same hardness at 560°C. Dry sliding properties were studied with pin-on-disc geometry at low speed (0.3 m/s) at low load (1 N). The wear mechanism involved is dominated by adhesion.

Support of the Hungarian-Indian co-operation R&D&I program TET_09_IN_DST (ALNANO09) and of the National Office for Research and Technology (REG-KM-09-1-2009-0005) is acknowledged.

Fig. 1: Figure 1. SEM secondary electron image of broken surface of a 70%Al+30%Al2O3 sample. Milled in Ethanol at room temperature for 1 h at 600 rpm with ZrO2 balls. Spark Plasma Sintered at 647°C for 5 minutes. Uniform distribution of the Al2O3 particles is seen.

Fig. 2: Figure 2. SEM secondary electron image from the surface of a 70%Al+30%Al2O3 sample after tribology measurement. Milled in Ethanol at room temperature for 1 h at 600 rpm with ZrO2 balls. Spark Plasma Sintered at 640°C for 5 minutes. Traces of adhesion are seen.

Fig. 3: Figure 3. TEM bright field image from a 70%(Al-27%Si-7%Ni alloy)+30%Al2O3 sample. Milled in Ethanol at room temperature for 1 h at 600 rpm with steel balls. Spark Plasma Sintered at 600°C for 5 minutes. Traces of porosity are seen next to the Al2O3 particles. EDS proved that Al and Si remained separated even after sintering.

Fig. 4: Figure 4. Apparent density (that includes open porosity) for samples prepared from pure and from alloyed Al. Mass fraction of Al2O3 is 30% in each case. Sintering (SPS) temperature is indicated as labels above the columns. Nominal density for pure Al is 2.70 g/cm3, while for the Al-27%Si-7%Ni alloy it is 2.72 g/cm3.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1556 Preparation of nitinol by reactive sintering

Novák P.1, Michalcová A.1, Čapek J.1, Pokorný P.1, Karlík M.2, Haušild P.2, Kopeček J.3
1Institute of Chemical Technology, Prague, Czech Republic, 2Czech Technical University in Prague, Czech Republic, 3Institute of Physics of the ASCR, Prague, Czech Republic
Miroslav.Karlik@fjfi.cvut.cz

Melting metallurgy processes of NiTi shape memory alloys can lead to undesirable contamination of the melt and forming of oxide inclusions. One possible solution of this problem is reactive sintering using pure elemental powders. Reactive sintering method enables to produce high-purity materials. In this process, compressed mixture of metallic powders is transformed to bulk intermetallic phases via thermally activated exothermic reactions. The evolved heat sustains and helps to propagate the reaction front through the body of the reactants. Therefore this process is called “Self-sustainable High-temperature Synthesis” (SHS). This work aims to optimize the parameters of the SHS process for the preparation of NiTi shape memory alloy to obtain a high-purity, low-porosity material.

Results revealed that heating rate strongly affects the structure of this alloy. Using slow heating (20 °C.min-1) leads to extremely heterogeneous structure composed of various Ni-Ti phases and high porosity (Fig. 1).The microstructure consisting of NiTi and Ti2Ni phases can be obtained in this material by rapid heating (approx. over 300 °C.min-1) (Fig. 2). Sufficient reactive sintering temperature to obtain NiTi phase is 900 °C. At 800 °C, the structure composed of Ni and Ti elemental powder particles was observed (Fig. 3). No Ni-Ti intermetallic phase was observed after sintering at 800°C. Further increase of the SHS initiation temperature to 1100 °C reduces porosity and the amount of the Ti2Ni phase. The lowest porosity and lowest amount of the undesirable Ti2Ni phase were achieved by the utilization of coarse titanium powder (200-600 µm) with fresh surface, produced by mechanical machining, or by the increase of nickel content in the alloy (Fig. 4). However, the change of the nickel content will affect the transformation temperatures and induce the formation of Ni4Ti3 phase. Reactive sintering process is completed during less than 20 min in the investigated material. It was proved that the formation of Ti2Ni phase always accompanies this process, even though its content can be minimized by the proper choice of the SHS parameters. To eliminate this phase, further thermal or thermo-mechanical treatment will be required.

This research was financially supported by the Czech Science Foundation, project 14-03044S.

Fig. 1: Microstructure of Ni-50at%Ti alloy prepared by SHS process at 1100°C for 20 min, heating rate of 20 °C.min-1, Ti particle size < 10 µm, light microscopy.

Fig. 2: Microstructure of Ni-50at%Ti alloy prepared by SHS process at 1100°C for 20 min, heating rate > 300 °C.min-1, Ti particle size < 10 µm, light microscopy.

Fig. 3: Microstructure of Ni-50at%Ti alloy prepared by SHS at 800°C for 20 min, heating rate > 300 °C.min-1, Ti particle size < 10 µm, light microscopy.

Fig. 4: Microstructure of Ni-50at%Ti alloy at 1100°C for 20 min, heating rate > 300 °C.min-1, Ti particle size 200 - 600 µm, light microscopy.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1570 The broad area detection of Mg micro-core in the graphite of the Spheroidal Graphite Cast Iron

Maeda H.1, Sugiyama A.2, Yasuda H.3
1Ryukoku University, Shiga, Japan, 2Osaka Sangyo University, Osaka, Japan , 3Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan
hidefumi@rins.ryukoku.ac.jp

The cast iron is accounting for the main part of industrial products, and improvement of the mechanical property is always an important issue. By performing the rounding of the graphite by additive elements (Mg) in the cast iron, various superiority of the mechanical property can be obtained. After this, many studies have been accomplished about the mechanism of rounding graphite. However, the role of the additive trace elements has not been reported yet.
In this study, the broad area detection by X-rays signal to the distribution of the Mg was performed. The broad area detection means to get to know the position of Mg in the entire organization of the Spheroidal Graphite Cast Iron. It is expected that especially the X-ray signal of Mg in graphite is extremely small, because its volume is very small and Mg is surrounded by graphite. Therefore, other than the example confirmed accidentally, there has been no example that showed Mg distribution as a core of spheroidal graphite. However, by estimating the minimum dose of electron beam that detect Mg in spheroidal graphite, high-speed detection was realized and enabled the broad area detection. The Spheroidal Graphite Cast Iron that added 0.05% Mg by weight was used, and the X-rays measurement performed by WDS-EPMA JEOL 8900R. By applying the trace element mapping method, the detection was performed in the broad area of approximately 1,500μm x 1,500μm. The spheroidal graphite having Mg micro-core is identified by putting Mg signals on the reflection electron (COMPO) image recorded at the same time.
A composition image by the Mg Kα line is shown in Fig.1 and COMPO image is shown in Fig.2. On each figure, the broad area detection (low electron dose condition) is compared with the usual trace element mapping (high electron dose condition). Although a noise seems to be large in the broad area detection, Mg micro-core can confirm to be in the inside of the spheroidal graphite. The distribution of graphite with Mg micro-core confirmed by broad area detection is shown in Fig.3. A red round mark shows the graphite position where Mg micro-core was detected, and a blue round mark is the graphite in which Mg micro-core was newly detected after polishing about 1~2μm from surfaces. Thus, the distribution of spheroidal graphite with Mg micro-cores is changed with polishing a slight micrometer from the surfaces. Only when the Mg micro-core in graphite is exposed to the observation surface, the X-rays signal can detect.
These data improve a possibility that Mg micro-cores exist also in the spheroidal graphite in which Mg was not detected, and these data also may become the proof that the micro-core of an additive element is deeply related with the rounding of graphite.

Fig. 1: Mg Kα images (the broad area detection (low electron dose :left) is in comparison with the usual trace element mapping (high electron dose :right)). The red dotted line is a shape of the spheroidal graphite by COMPO image in Fig.2.

Fig. 2: Reflection electron (COMPO) images (the broad area detection (left) is in comparison with the usual trace element mapping (right)).

Fig. 3: Distribution of spheroidal graphite with Mg micro-core in cast iron. Red round marks show the spheroidal graphite positions where Mg micro-core was detected, and a blue round marks show where Mg micro-core was newly detected after polishing the surfaces. The only red circle* remains Mg micro-core, after polishing.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1675 Mechanical alloying of Fe–X (X=Al, Mo, Ni) powders studied by analytical electron microscopy

Buršík J.1, Jirásková Y.1
1Institute of Physics of Materials, Academy of Sciences of the Czech Republic, Brno, Czech Republic
bursik@ipm.cz

It is well-known that materials of the same nominal composition processed in a different way yield different microstructure and phase constitution, which result in diverse physical properties. Mechanical alloying (MA) via ball milling represents a relatively simple way of preparing materials with a micro- to nanograin crystalline and/or amorphous metastable structure. MA is a high-energy process where the energy developed during high speed rotation of grinding vial and balls is transferred to the powder particles which are brought to contact and exposed to severe mechanical deformation. This leads finally to formation of an alloy of the required mostly thermodynamically unstable composition.
In this work we have studied three binary systems in various states of MA by means of analytical electron microscopy (SEM and TEM). The Fe–Al system is frequently exploited for its low cost, high temperature corrosion resistance and good mechanical properties. The studied composition Fe82Al18 is close to the phase boundary between bcc Fe–Al solid solution and Fe3Al phase. Poor mutual solubility of Fe and Mo restricts the fabrication of Fe–Mo alloys by conventional technologies. MA helps substantially as shown here for Fe80Mo20 composition. Fe20Ni80 is studied as a representative of Fe–Ni alloys with extraordinary magnetic, mechanical and electrical properties. For details of materials preparation see e.g. Jirásková et al, J. Alloys and Comp. 568 (2013) 106-111.
A TESCAN LYRA 3XMU FEG/SEM scanning electron microscope, a Philips CM12 STEM and a JEOL JEM-2010F transmission electron microscopes (all equipped with an XMax80 Oxford Instruments detector for energy dispersive X-ray (EDX) analyses) were used for microstructural studies. The comparison of evolution of the three systems during milling has shown how the rate of mixing visualized by powder  morphology and chemistry depends on the properties of constituents and on pertinent binary phase diagrams.
The alloying of the Fe-Al is observed already after 5 h of milling yielding bcc-Fe(Al) coexisting with α-Fe (Fig. 1). After 30 h EDX analyses have shown the dominant peak close to nominal composition. The alloying of Fe and Mo proceeds more slowly. Mo starts to dissolve in bcc-Fe and vice versa after 10 h of milling and bcc-FeMo and bcc-MoFe phases are formed (Fig. 2). Details observed in TEM after 250 h of milling (Fig. 2c) show the microstructure consisting of dense packed nanoparticle cores (< 10 nm). TEM yields a similar morphology also for Ni-Fe (Fig. 3) after 15 h of milling and the diffraction pattern confirms the formation of Ni3Fe phase. The SEM micrographs of all samples document similar final morphologies of the powders formed by small particles (< 500 nm) and the larger agglomerates up to tens of micrometers.

The work was supported by the Czech Science Foundation (project P108/11/1350).

Fig. 1: SEM micrographs of Fe-Al powders after ball milling for 5 h (a), 20 h (b) and 30 h (c).

Fig. 2: Electron micrographs of Fe-Mo powders after ball milling for 60 h (a, SEM image) and 250 h (b, SEM image and c, TEM image).

Fig. 3: Electron micrographs of Fe-Ni powders after ball milling for 2 h (a, SEM image), 20 h (b, SEM image) and 15 h (c, TEM image with selected area diffraction pattern).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1707 Al–Ni–Pt Alloy System: Isothermal Sections, Liquidus, Solidus and Reaction Scheme

Kapush D.1, Korniyenko K.1, Grushko B.2, Meshi L.3, Petyukh V. M.1, Shemet V.4, Velikanova T. Y.1
1Department of Physical Chemistry of Inorganic Materials, I.M. Frantsevich Institute for Problems of Materials Science of NASU, 03680 Kiev 142, Ukraine, 2PGI-5, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany, 3Department of Materials Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel, 4IEK-2, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
d.kapush@gmail.com

Pt-modified nickel aluminide coatings are used in order to improve the high-temperature oxidation resistance of the components of the gas turbines produced from Ni-based superalloys. This explains an interest in the low-Al part of the Al–Ni–Pt system. On the other hand, the interest in the Al-rich part is because of the formation of complex intermetallics attractive for both basic and applied research.

In the present work the phase equilibria in Al–Ni–Pt were studied in the whole compositional region. DTA, powder XRD, SEM/EDX and TEM were applied. The liquidus and solidus surfaces, isothermal section at 1100 °C and partial isothermal sections at 1000, 900 and 790 °C were constructed. The reaction scheme was proposed.

The total compositional region of the ternary phase diagram can coarsely be divided into three peculiar subregions (see Ref. [1] and references therein):

•Above ~70 at. % Al this alloy system is characterized by the formation of complex binary and ternary phases. The boundary Al–Pt phase diagram was completed with the recently revealed high-temperature ‘‘Al3Pt’’ ξ-phase (Bmmb, a = 1.9718, b = 1.6228, c = 1.4266 nm) [2], which was found to extend up to 1.6 at. % Ni. Two ternary structures were identified in this region: χ (P31c, a = 1.2095, c = 2.6932 nm), ε6 (Pnma, a = 2.3119, b = 1.6416, c = 1.2171 nm).

•Between ~40 and 70 at. % Al the structures based on the CsCl type configuration dominate. The ternary extension of the Al2Pt phase (β*) separates the compositional regions extending from Al3Ni2 and Al3Pt2. At 900–1100 °C no complete separation was revealed between the compositional fields of the β and β* phases. With decreasing temperature the total β + β* field shrinks around the compositional lines Al2Pt–Al2NiPt and AlNi–Al2NiPt.

•Below ~40 at. % Al the structures are based on the FCC (Ni, Pt) solid solution. Two ternary structures were identified: γ* (P4/mmm, a = 0.3872, c = 0.3548 nm) and γ♦ (Cmmm, a = 0.7902, b = 0.7258, c = 0.3932 nm). The low-Ni limit of the γ♦ phase region, probably extending at low temperatures from Al3Ni5, was found to be below 4 at. %.

[1] B. Grushko, D. Kapush, J. Alloys Compd. 594 (2014) 127.

[2] B. Grushko, D. Kapush, J. Su, W. Wan, S. Hovmöller, J. Alloys Comp. 580 (2013) 618.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1731 Scanning transmission electron microscopy study of Mg-RE solid solutions

Bugnet M.1, Kula A.1,2, Niewczas M.1, Botton G. A.1
1Materials Science and Engineering, McMaster University, Hamilton, ON, Canada, 2AGH-University of Science and Technology, Cracow, Poland
bugnetm@mcmaster.ca

Magnesium-Rare Earth (RE) alloys exhibit favorable combination of high specific strength and good ductility, making them suitable for various applications in aerospace, aircraft, and automotive industry. Understanding the effect of RE elements on alloys’ properties is considered a key aspect in alloy develepoment and processing. RE elements tend to segregate to the grain boudaries affecting kinetics of the grain growth, texture and mechanical properties of Mg-based alloys. The phenomenon generates much interest from the academic and industrial communities and recently has been a subject of intense studies [1].

In the present work, we investigate the distribution of solute elements in the structure of Mg-0.28at.%Gd and Mg-0.36at.%Sm binary alloys by high-angle annular dark field (HAADF) imaging in scanning transmission electron microscopy (STEM) and by electron energy loss spectroscopy (EELS) techniques [2]. The structure of Mg-0.28at.%Gd and Mg-0.36at.%Sm solid solutions is characterized by STEM in Z-contrast, down to the atomic scale. We show that Gd(Sm) are present in two different forms: (i) in solid solution as quasi-random atoms distributed in Mg matrix and (ii) as segregates at high angle grain boundaries forming 1-2 nm Gd(Sm)-rich clusters (Figure 1). The analysis of the structural models for atomically resolved images of the clusters at grain boundaries suggests the stabilization of the face-centered cubic Gd phase (Figure 2). The results validate the already reported predictions about segregation of RE elements at grain boundaries in Mg-RE alloys, and ultimately provide a direct visualization of the distribution of the solute atoms in the structure of Mg-Gd and Mg-Sm alloys. The segregation phenomenon of solute atoms at grain boundaries can be directly correlated to the decrease of the grain size in the Mg-Gd and Mg-Sm alloys as compared to pure Mg. The present study provides new insight towards understanding the effect of RE elements on the texture development during alloy processing and recrystallization, and thereby the mechanical behavior and properties of Mg-RE alloys [3,4].

References:

[1] J.D. Robson, Metall Mater Trans A, (2013). DOI: 101007/s11661-013-1950-1

[2] M. Bugnet, A. Kula, M. Niewczas, G.A. Botton, submitted.

[3] A. Kula, R.K. Mishra, M. Niewczas, in preparation.

The Authors are grateful to NSERC for financial support. The STEM work was carried out at the Canadian Centre for Electron Microscopy, a national facility supported by NSERC and McMaster University.

Fig. 1: HAADF-STEM image of a high angle grain boundary in a Mg-0.28at.%Gd alloy, and EELS line scans illustrating the substantial segregation of Gd.

Fig. 2: (a-c) High resolution HAADF-STEM image of Gd-rich clusters at high angle grain boundaries in a Mg-0.28at.%Gd alloy.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1896 TEM Study of Inclusions on the Nucleation of Acicular Ferrite in Weld Metal

Gallego J.1, de Araújo M. R.2, Ventrella V. A.1, Yamakami W. J.1, Tokimatsu R. C.1
1São Paulo State University - UNESP, Department of Mechanical Engineering, Av. Brasil Centro 56, CEP 15385-000, Ilha Solteira/SP, Brazil., 2Nove de Julho University - UNINOVE, Department of Mechanical Engineering, R. Vergueiro 1289, CEP 01504-001, São Paulo/SP, Brazil.
gallego@dem.feis.unesp.br

The welding of steel parts by Submerged Arc Welding (SAW) is one of most traditionally processes used in manufacturing of structural components. High levels of strength and toughness can be achieved if SAW weld metals are mainly composed by acicular ferrite (AF), a microconstituent whose its nucleation depends of the existence of a non-metallic inclusion distribution. However not all of these particles take effectively part in the formation of ferrite laths during the iron gamma-alpha phase transformation, being also autocatalytic nucleation a important mechanism for acicular ferrite formation. SAW was carried out on low-carbon steel substrate to produce a bead-on-plate weld metal with different heat input welding (HI: 1, 2 and 3 kJ.mm-1). Disks with 50-100 µm thick were carefully cut from cylinders with 3 mm diameter, which were machined parallel to the welding direction. Thin foil samples were obtained after double-jet electropolishing using Struers Tenupol-3. Transmission Electron Microscopy (TEM) observations were carried out in a Philips CM120, operated at 120 kV and equipped with EDAX DX-4 Materials Thin System for EDS microanalysis.

Figure 1 shows TEM bright field micrographs of the typical interlocking laths of AF, where each ferrite lath have usually measured between 1-10 µm length and 0.5-2 µm width. In these micrographs some of the inclusions appeared to be acted as substrate for acicular ferrite formation, i.e. some ferrite laths grow outwards from the particles while other ones seem like non-nucleant inclusions. That behaviour may be associated to size, shape and composition of the inclusions, become their effect on the formation of AF quite complex.

The role of the inclusions was investigated by TEM according to nucleant or non-nucleant behaviour for acicular ferrite. Figure 2 shows examples of both kinds of particles where clearly 1 and 2 no contribute to formation of ferrite laths, unlike of the particles 3 and 4. A nucleant particle may be able to form one or more AF laths on its surface. In general, larger and more intricate inclusions (lower roundness) are more efficient to nucleate AF laths than smaller and rounder ones. However, increasing of roundness was observed in the nucleant inclusions formed with higher HI, Figure 2(b), suggesting that effect may be related to composition of these particles. Figure 3 shows mean amounts (%wt) of elements such as Al, Si, S, Ti and Mn determined by EDS in the nucleant and non-nucleant particles. Non-metallic inclusions are essentially complex oxides but presence of higher concentration of titanium is expected for improving their nucleant AF behaviour in the SAW weld metal.

The authors gratefully acknowledge the Brazilian institutions FAPESP, CNPq and CAPES for their financial support provided for this research and to the Electron Microscopy Laboratory of the Federal University of São Carlos (Brazil) for allowing us to use their electron microscopy facilities.

Fig. 1: Typical thin foil TEM BF micrographs showing AF laths and rounded non-metallic inclusions in weld metal. SAW performed under different heat input (HI): 1 kJ.mm-1 in (a), 2 kJ.mm-1 in (b) and 3 kJ.mm-1 in (c).

Fig. 2: TEM BF micrographs showing examples of non-nucleant (1-2) and nucleant (3-4) particles. Effect of heat input on the size and morphology of nucleant particles for AF in (b).

Fig. 3: Variation of mean composition (%wt) of non-nucleant and nucleant particles for acicular ferrite laths with different SAW heat inputs.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1919 Structure and evolution mechanism of δ’-θ’-δ’ composite precipitates in an Al-Li-Cu Alloy

Duan S. Y.1, Chen J. H.1, Liu J. Z.1, Gao Z.1, Wu C. L.1
1Center for High-Resolution Electron Microscopy, College of Materials Science and Engineering, Hunan University, Changsha, Hunan 410082, China
huhuhu@hnu.edu.cn

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1949 TEM Observations of Nano-Scale MX Precipitates in Crept Super304H Austenitic Steel

Xing H.1, Ou P.1, Sun J.1
1Shanghai Key Laboratory of Advanced High-temperature Materials and Precision Forming, School of Materials Science and Engineering, Shanghai Jiaotong University, Shanghai, PR China
xinghui@sjtu.edu.cn

Super304H austenitic steels containing small amount of Cu and Nb elements are widely used as superheated tubes in 600°C USC power plants. The precipitation of Cu-rich phases in Super304H austenitic steels has been extensively investigated and it has been well recognized that nano-scale Cu-rich precipitates can improve creep strength of the Super304H steel. However, the precipitation of nano-scale MX phases received little attention until now. In this work, the precipitation of nano-scale MX Phases in the Super304H steel crept at 650°C/447 hours has been observed by TEM. Based on the TEM observations, the precipitation mechanism of nano-scale MX Phases was discussed additionally.

The chemical composition of the Super304H austenitic steel is 0.08C, 0.23Si, 0.80Mn, 0.027P, 0.001S, 9.5Ni, 18.5Cr, 2.81Cu, 0.51Nb, 0.11N, 0.0034B (in mass %) with the balance of Fe. The TEM samples were cut from the steel crept at 650°C/447 hours and were prepared by twin-jet electro-polishing in a 5 vol.% perchloric acid and 95 vol.% ethanol solution at about 243 K and at 60 V. TEM observations were conducted on JEOL 2100F machine operating at 200 kV. Fig. 1 is a TEM micrograph of crept Super304H steel, showing high density of nano-scale circular shaped Cu-rich precipitates with a weak contrast and small amount of cubical-shaped precipitates with nano-scale diameter and dislocations with a dark contrast in the austenitic matrix. Fig. 2(a) is a HRTEM micrograph along the [011] direction of the austenitic matrix, showing a character of Moire fringes of the cubical-shaped precipitate. The FFT diffractogram as shown in Fig. 2(b) and EDS result in Fig. 2(c) acquired from the precipitate indicate that the cubical-shaped precipitate is fcc-structured NbC with a lattice constant of about 0.4454 nm and a cubic/cubic crystallographic relationship with the austenitic matrix. The interface of nano-scale NbC with the austenitic matrix is the (111) plane. Fig. 3 are TEM micrographs, which showing the precipitation of nano-scale MX phases at different locations in the austenitic matrix. Fig. 3(a) shows that MX phases precipitate together with the Cu-rich phases owing to a relatively high concentration of Nb near the Cu-rich precipitates in the matrix. Moreover, MX phases often precipitate along dislocation line as shown in Fig. 3(b), because small carbon or nitrogen atoms are easily clustered at dislocation core. Fig. 3(c) shows MX phases precipitating along glide dislocations, which interacting with the Cu-rich precipitates. These TEM observations confirm an interaction of nano-scale MX and Cu-rich precipitates with glide dislocations in the austenitic matrix, which obviously enhances creep strength of the Super304H austenitic steel.

This research is financially supported by the NSFC under Contract no. 50931003 and by the STCSM under Contract no. 13dz2260300.

Fig. 1: TEM micrograph of Super304H austenitic steel crept at 650°C/447 hours.

Fig. 2: HRTEM micrograph (a), FFT diffractogram (b) and EDS result (c) of the MX precipitate in the austenitic matrix.

Fig. 3: TEM micrographs of MX phases precipitating together with the Cu-rich phase (a), MX phases precipitating along dislocations (b) and MX phases precipitating along glide dislocations interacting with Cu-rich phases (c) in the austenitic matrix.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-1984 The 3D imaging of 718Plus superalloy microstructure by FIB-SEM tomography

Kruk A.1, Cempura G.1, Czyrska-Filemonowicz A.1
1AGH University of Science and Technology, Al. A. Mickiewicza 30, 30-059 Kraków, Poland
kruczek@agh.edu.pl

Allvac 718Plus is a high strength, corrosion resistant nickel-chromium-iron based superalloy used for applications in power generation, aeronautics and aerospace [1]. Its typical chemical composition is: Ni-18Cr-10Fe-9Co-5.1(Nb+Ta)-1W-2.7Mo-0.7Ti-1.5Al-0.03C (wt%). The 718Plus microstructure consists of a γ matrix (Ni-based solid solution) with ordered face centred cubic γ' Ni3(Al,Ti)-type phase and some orthorhombic δ (Ni3Nb)-type particles precipitated at the grain boundaries. The primary strengthening mechanism for this alloy is a precipitation hardening, therefore properly sized and distributed precipitates are critical for good alloy performance. The aim of this study was to describe 718Plus microstructure and apply FIB-SEM tomography to 3D imaging and metrology of the precipitates.
Microstructural investigation and phase chemical compositions were performed using SEM-NEON CrossBeam 40EsB (Zeiss) and TEM – a probe Cs corrected Titan3 G2 60-300 (FEI) equipped with ChemiSTEM, a Super-X EDS detector with 4 windowless diodes and an X-FEG high brightness electron gun. The TEM study was conducted on the lamella prepared by FIB. FIB-SEM tomography is based on a serial slicing technique employing a FIB-SEM dual beam workstation [2]. The NEON 40EsB CrossBeam with Ga-ion beam was used to perform a precise in-situ milling. Consequently, the acquired stack of images was transformed directly into a 3D data volume with a voxel resolution of 12×12×12 nm. Repeated removal of layers as thin as a 12 nm allowed exploring a total volume of 10.7 x 7.2 x 7.7 μm.
Fig. 1 shows 718Plus microstructure observed using SEM-EsB detector (Z-contrast). Needle shaped particles, identified as δ-phase by EDS analysis, were observed at grain boundaries and occasionally at twin boundaries. Distribution of chemical elements between γ, γ' and δ phases was determined using STEM-EDS (ChemiSTEM) (Fig.1c). It can be seen that precipitates with round-to-blocky morphology are randomly dispersed within the matrix. Some primary Nb-rich MC and Ti-rich M(C,N) particles were also observed. Fig. 2 shows 3D visualization of the shape and distribution of δ phase at γ grain boundary in 718Plus. The results achieved confirm the ability of FIB-SEM tomography to reconstruct 3D structures with dimensions in the range of 100 nm or even smaller. Such 3D reconstructions can serve as a basis for quantitative analysis of complex structures in a nanoscale. Further investigation of 718Plus superalloy by analytical TEM is in progress.
References
[1] R. M. Kearsey, J. Tsang, S. Oppenheimer, E. Mcdevitt; JOM, 64(2)(2012)241.
[2] A. Kruk, A. Czyrska-Filemonowicz; Archives of Metallurgy and Materials, 58(2013)387

The authors acknowledge the financial support from EU 7FP under Grant Agreement 312483 - ESTEEM2 and the AGH-UST statutory project 2014.

Fig. 1: Microstructure and chemical composition of 718Plus superalloy: a, b) microstructure of non-etched sample observed in Z-contrast (SEM – EsB detector), c) chemical element distribution maps obtained by STEM-EDS.

Fig. 2: Three-dimensional visualization of tomographic reconstructed volume of 718Plus superalloy by FIB-SEM tomography: a) reconstructed volume 10.7 x 7.2 x 7.7 μm at different angle of view, b) shape and distribution of δ phase precipitated at the γ grain boundary.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2008 The hardening precipitates in AlCuMg alloys

Chen J. H.1
1Hunan University, Changsha, Hunan, China
jhchen123@hnu.edu.cn

Developments of high-strength aluminum alloys have always faced a difficult problem: owing to their small size, the early-stage strengthening precipitates are difficult to characterize in terms of composition, structure and evolution. Even for the widely used AlCuMg alloys, in which the phenomenon of precipitation hardening in metals was first discovered by Wilm more than a century ago, the essential questions remain: how many different precursors exist for the most effective strengthening precipitates (referred to S-phase), and how do they transform to the S-phase? Here we employ atomic-resolution electron microscopy imaging and first-principles energy calculations to address these problems. Our study demonstrates that the early-stage S-phase precipitates are highly dynamic in both composition and structure. Having their own genetic double Cu–Mg atomic walls to guide their evolution, these dynamic precipitates initiate, mature and grow with thermal aging following three evolution paths, leading to the S-phase precipitates formed, without exception, with even numbers of Cu–Mg atomic layers. By employing atomic-resolution imaging to follow the growing S-phase precipitates in the Al-matrix, it is demonstrated that the growth of a S-phase crystal is rather anisotropic and temperature-dependent, and is furthermore accompanied by low-dimensional phase transformations. There are actually two types of well-defined characteristic Guinier-Preston (GP) zones that determine the growth mechanism of a S-phase crystal at an elevated temperature.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2026 Development and growth of Omega-phase in a TiAl-Nb-Mo alloy and its effect on hardness

Rashkova B.1, Schloffer M.1, Schöberl T.2, Zhang Z.2, Mayer S.1, Clemens H.1
1Department of Physical Metallurgy and Materials Testing, Montanuniversität Leoben, 8700 Leoben, Austria, 2. Erich Schmid Institute of Materials Science, Austrian Academy of Sciences, 8700 Leoben, Austria
boryana.rashkova@unileoben.ac.at

Advanced intermetallic γ-TiAl based alloys are considered for high temperature application in the aerospace and automotive industry as lightweight materials which can withstand temperatures up to 750°C, while maintaining attractive thermal and mechanical properties [1] [2]. TNM alloys, exhibiting a nominal base composition of Ti-43.5Al-4Nb-1Mo-0.1B (in at.%), are multi-phase γ-TiAl-based alloys, where the microstructure is determined by the manufacturing process as well as subsequent heat treatments. It has been established that the formation of ωo (B82)-phase takes place in βo (B2)-phase during thermal processing [1-3]. In this work we studied the development and growth mechanism of ωo-phase which forms in the βo-phase during heat treatments (HT) and creep tests conducted at 750°C, 780°C and 800°C. In situ high energy X-ray diffraction experiments were conducted to investigate the decomposition behaviour of the ωo-phase as well as to determine their dissolution temperature. High resolution transmission electron microscopy (HRTEM) was used to study the coarsening of wo-grains during creep. Figs. 1 and 2 show a Cs-corrected HRTEM image of the fine wo precipitates within the βo-matrix after HT (i.e. before the creep test) and one, dislocation free, segment of the atomically abrupt and coherent βo/ωo interface after the creep test, respectively. The analysis of the Fourier transformed image shows that the {11-20} and {-1010} lattice planes from the ωo-phase are parallel to the {1-10} and {11-2} planes from the βo-matrix. The chemical composition of bo and wo was determined by means of energy dispersive X-ray microanalysis (EDX). In particular, the impact of the Mo content on the growth of the ωo-grains within the βo-matrix was investigated. Fig. 3 presents EDX line scans of a region with ωo-grains in the βo-matrix. The intensity of Mo reveals an inverse trend at the transition to the βo/ωo-interface. In the ωo-grains the Mo content decreases whereas in the βo-phase it increases (Fig. 3b). Additionally, nano-hardness measurements in γ,α2, βo, and (βo+ωo) grains were performed by cube corner indentation and the trend how the hardness develops was established. The results show that pure βo is not the hardest phase in the TiAl-Nb-Mo alloy system. More details about the experimental conditions and the obtained results are given in the original full-length paper in [4].

[1] F. Appel, J. Paul, M. Oehring Gamma Titanium Aluminide Alloys Science and Technology. WILEY-VCH; 2011.

[2] H.Clemens, S. Mayer, Adv. Eng. Mat.,15 (2013) p.191.

[3] W. Wallgram, T. Schmolzer, L. Cha, G. Das, V. Güther, H. Clemens, Int. J. Mat. Res., 100 (2009), p.1021

[4] M. Schloffer,B. Rashkova, T. Schöberl, E. Schwaighofer, Z. Zhang, H. Clemens, S. Mayer, Acta Mat., 64 (2014) p. 241.

Fig. 1: Microstructure and distribution of the constituent phases after HT. a) TEM BF image of βo-phase containing ωo precipitates, surrounded by γg-grains and (α2/γ)-colonies. The substructure inside the βo-phase arose from the presence of fine ωo-domains interrupted by γp-platelets b) HRTEM image showing the ωo-precipitates within the βo-phase.

Fig. 2: Microstructure after the creep test. a) TEM BF image shows ωo-grains with a globular shape uniformly distributed in the βo-phase, surrounded by γp- and γg-grains. b) a Cs-corrected HRTEM image of one, dislocations free, segment of the coherent βo/ωo interface along the <111>βo and [0001]ωo zone axes.

Fig. 3:  a) TEM BF image of a βo-grain with ωo-particles and γp-grains. The inset shows the position of the EDX-line scan. b) EDX line scans of region with alternating ωo-grains in the βo-matrix. The intensity of the Mo content increases at the transition to βo-phase and shows an inverse trend to that in the ωo-phase.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2044 Structural evolution of T91-ODS steel during high temperature deformation

Litvinov D.1, Strassberger L.1, Aktaa J.1
1Institute for Applied Materials, Karlsruhe Institute of Technology (KIT), D-76344 Eggenstein-Leopoldshafen, Germany
litvinov@kit.edu

Scanning and transmission electron microscopy (TEM) was applied to study the structure of oxide dispersion strengthened (ODS) ferritic–martensitic steel T91 (9 wt.% Cr, 1 wt.% Mo) before and after mechanical deformation at elevated temperatures.

The microstructure of investigated samples consists of two kinds of ferrite grains, with nearly no defects, and with a high defect density due to formerly martensite. In the as received samples, the grain boundaries are decorated by relatively large, non-regular shaped M23C6 carbides where M is metal. Additionally, there are finely distributed Y2O3 round ODS nanoparticles.

Figure 1a shows high angle annular dark-field (HAADF) scanning TEM (STEM) image of as received sample. The small precipitates with a dark contrast are Y2O3 particles showing energy-dispersive X-ray (EDX) linescan through the precipitate as illustrated in Figure 1b. The high resolution TEM (HRTEM) images (here not shown) yield the Y2O3 particle with bcc structure and a lattice parameter of 1.06 nm. Note that Y2O3 particles are arranged close to dislocations D inside the ferrite grain as is shown in enlarged region in Figure 1a.

In Figure 2a, conventional bright-field TEM image of as received sample is presented. Analysis of corresponding diffraction pattern in Figure 2b shows the coherence relationship between the ODS particles and the matrix: <100> Y2O3 || <110> Fe.

HRTEM analysis of M23C6 in as received samples (here not shown) reveals that carbide has fcc structure with a lattice parameter of ~ 1.07 nm. The relation between the carbide and ferrite is: <112> M23C6 || <110> Fe. The detail EDX analysis of the carbide regions shows the existence of Cr21Mo2C6. Many {111} stacking faults in the large carbide inclusions are observed.

The EDX study combined with the diffraction analysis of other inclusions in the investigated steel shows the presents of MX phases with fcc (rock salt) structure and lattice parameter of 0.44 nm, where M is metal and X is C,O or N.

The first investigations of the samples deformed at elevated temperatures from 500 to 700 °C show, that mean particle diameter of ODS particles in these samples increases and their size distribution broadens. The number of the grains without defects extremely decreases. Additionally, some extremely large non-regular shaped M23C6 carbides have been found that is caused by Ostwald ripening during the deformation at higher temperatures.

The present work was composed at the Institute for Applied Materials at the Karlsruhe Institute of Technology with support of the Helmholtz Association (Germany).

Fig. 1: a) HAADF STEM image of as received sample. In insert enlarged region by D marked dislocations. b) EDX linescan taken along dash line of a).

Fig. 2: a) Bright-field TEM image of as received sample close to [001] Fe-zone axis with b) corresponding diffraction pattern.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2089 The Study of Nanoparticles of Au2O3 using Tannic Acid and Gallic Acid

San German-Perez S.1, Zorrilla-Cangas C.2, Herrera-Becerra R.2
11 Posgrado en Ciencia e Ingeniería de Materiales, Instituto de Física, UNAM. Circuito Exterior, Ciudad Universitaria, Coyoacán, 04510, México., 22 Departamento de Materia Condensada / Instituto de Física UNAM. Circuito de la Investigación Científica Ciudad Universitaria CP 04510 México.
rherrera@fisica.unam.mx

It is attempted to determine the differences in shape, size, and structure between Au2O3 particles synthesized using the Bioreduction [1] method, using tannic acid or galic acid as a reducing agent, controlling the value of pH with the addition of NaOH, and working at room temperature. Oxidized gold nanoparticles have been subject of several studies since they are a matter of interest due to the different applications they have [2], however it has been observed that when they are synthesized in aqueous solutions they might get oxidized becoming at once an interesting subject of study. The solutions are prepared in 5 ml distilled water with a concentration of 0.3 mM tannins (tannic acid or gallic acid), which has been subjected to an ultrasonic treatment for 1 minute. After fixing the pH, the solutions are taken to the ultrasonic cleaner again for 15 minutes and then centrifuged at 5000 rpm for 15 minutes. Afterwards, HAuCl4 deluted in 5 ml distilled water is added and for it to mix homogeneously, the ultrasonic cleaner is used again for 20 minutes before introducing it to the centrifuge at 5000 rpm for 20 more minutes. The whole process is performed at room temperature. Electron microscopy characterization included transmission electron microscopy (TEM), high resolution transmission electron microscopy (HRTEM). The samples were prepared by adding a drop of the solution on carbon-coated copper grids and then they were left to dry. Electron microscopy was performed in a JEOL JEM-2010F FasTem, equipped with analytical devices. High resolution images were obtained under several different conditions and the images were analyzed by obtaining digital spectra by FFT (Fast Fourier Transforms), to achieve more precise measurement. The FFT was obtained from the images with the Digital Micrograph software and indexation was performed with the program DPIP developed within our group [3]. The analysis performed was developed in three stages in order to observe size, structure and shape of the particles. Using the same molar proportions and fixing the pH value, we find that the number of particles in the case of the gallic acid is relatively smaller than the one obtained with tannic acid. The sizes and structures found in both cases are relatively similar. The study is also performed for different pH values. [1] R. Herrera-Becerra, J. L. Rius, y C. Zorrilla, Tannin biosynthesis of iron oxide nanoparticles, Applied Physics A. (2010) 453-459. [2] Luis K. Ono and Beatriz Roldan Cuenya, Formation and thermal stability of Au2O3 on gold nanoparticles: size and support effects, J. Phys. Chem., C 112, (2008) 4676-4686. [3] Galicia, R., Herrera, R., Rius, J L & Zorrilla, C. & Gómez, A., Revista Mexicana de Física 59, (2013)102–106.

Our gratitude to Roberto Hernández Reyes for his aid with the Electronic Microscope at IFUNAM and the financial support from DGAPA with grant PAPIIT IN105112.

Fig. 1: HRTEM image of an Au2O3 particle and its FFT where tannic acid was used.

Fig. 2: HRTEM image of an Au2O3 particle and its FFT where gallic acid was used.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2091 Development of ultra-fine grained Mg-Fe alloys for hydrogen storage

Neves A. M.1, Coimbrão D. D.1, Gallego J.2, Ishikawa T. T.1, Bolfarini C.1, Kiminami C. S.1, Leiva D. R.1, Botta W. J.1
1Federal University of São Carlos, São Carlos, SP, Brazil, 2São Paulo State University, Ilha Solteira, SP, Brazil
wjbotta@ufscar.br

The excellent hydrogen storage properties of Mg-based hydrides are dependent of a refined microstructure to provide the required interfaces to enhance the kinetics of hydrogen sorption. The usual way to obtain such characteristic was the processing by high-energy ball milling (HEBM), reactive or not, which result in nano-grained MgH2 powders containing, most often, additions of transition metals, their oxides or their fluorides [1]. More recently, bulk Mg and Mg-based alloys have been produced by different severe plastic deformation techniques [2,3]. In this work we present the production of ultra-fine grained Mg-Fe alloys using a combination of rapid solidification followed by extensive cold rolling. A fine mixture of Mg and Fe (8 wt%) was obtained by high-energy ball milling (HEBM) after 2h processing in a planetary mill; the mixture was then cold compacted before further processing by melt spinning. Melt spinning was carried out under argon atmosphere, using a graphite crucible in a single roller melt-spinning wheel at a tangential wheel speed of 42 m/s. Microstructural characterization in ion beam milled foils was carried out by transmission electron microscopy (TEM), in a Philips CM120 and in a FEI TECNAI G2 F20 200 kV microscopes. Figure 1 shows a bright field TEM micrograph of the as-spun Mg-Fe ribbons; the rapid solidification processing resulted in grain sizes in the range of few microns. The dark particle is Fe, which was distributed in the microstructure in a large size range. The smaller size Fe particles can be observed in Figure 2, which shows a dark field STEM micrograph of the as-spun ribbon. Fe particles are distributed in a rather uniform way inside and in the grain boundaries. Figure 3 shows a STEM dark field image of the Mg-Fe ribbons after being further processed by cold work. An important grain refinement was observed after this step, with grain sizes in the range of 100 to 200 nm. Figure 4 shows a HAADF image of the same cold rolled ribbon. Very small Fe particles are distributed in the ultra-fine grained Mg both inside and in the grain boundaries and such refined structure are expected to contribute to good hydrogen sorption properties.

References :

[1] A. R. Yavari, A. LeMoulec, F. R. de Castro, S. Deledda, O. Friedrichs, W. J. Botta, G.Vaughan, T. Klassen, A. Fernandez and Ǻ. Kvick. Scripta Mater. 52 (2005) pp. 719-724.

[2] D.R. Leiva, D. Fruchart, M. Bacia, G. Girard, N. Skryabina, A.C.S. Villela, S. Miraglia, D.S. Santos and W.J. Botta. Int.J.Mat.Res., 100 (2009) 12, pp. 1739-1747.

[3] W.J. Botta, A.M. Jorge Jr, M.Veron, E.F. Rauch, E. Ferrie, A.R. Yavari, J. Huot, D.R. Leiva. J.AlloysComp 580 (2013) S187–S191.

The authors gratefully acknowledge the financial support of the Brazilian institutions FAPESP, CNPq, CAPES and FINEP

Fig. 1: Bright field TEM micrograph of the as-spun Mg-Fe ribbons with grain sizes in the range of few microns.

Fig. 2: Dark field STEM micrograph of the as-spun ribbon with Fe particles distributed in uniform way inside and in the grain boundaries.

Fig. 3: STEM dark field image of the Mg-Fe ribbons processed by cold work, with grain sizes in the range of 100 to 200 nm.

Fig. 4: HAADF image of the Mg-Fe ribbons processed by cold work with very small Fe particles distributed in the ultra-fine grained Mg both inside and in the grain boundaries
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2108 TEM observation of microstructure in the early stage of Mg-Gd-Y alloy aged at 473K

Matsuoka Y.1, Matsuda K.2, Watanabe K.1, Nakamura J.3, Lefebvre W.4, Saikawa S.2, Ikeno S.5
1Graduate School of Science and Engineering for Education, University of Toyama,Toyama, Japan, 2Graduate School of Science and Engineering for Research, University of Toyama, Toyama, Japan, 3Tohoku University, Sendai, Japan, 4Universite de Rouen, France, 5Hokuriku Polytechnic College, Toyama, Japan
ikenolab@eng.u-toyama.ac.jp

Magnesium alloys containing rare earth elements are known to show good heat resistance.[1] The Mg-Gd alloy shows good age-hardenability, and the Mg-Gd-Y alloys have been developed for practical Mg alloys by Kamado et. al. to reduce the density of alloy and these alloys have a good creep resistance, even 523 - 573 K.[1] In our previous study, Mg-Gd-Y alloys show the mono-layer structure has been discovered before β” phase with DO19 structure in the aged sample at 473 K, and the β” and β’ phases with bco structure co-existed at the peak aged condition.[2] Recently, Nishijima et al. detailed examinations on the precipitation behaviours of Mg-Gd alloy and Mg-Y alloy by high angle annular dark field - scanning transmission electron microscopy (HAADF-STEM) technique.[3] They concluded that the arraignment of bright dots indicates the short range ordered state and the β’ phase nuclide in the short range ordered structure. And they have a doubt for the existence of the β” phase with DO19 structure. Moreover, they presented a new structure model for the β’ phase, an Mg7RE-type structure different from the previously proposed Mg15RE-type. In this study, the early stage of aging in Mg-2.9at.%Gd-0.8at.%Y alloy has been observed by high resolution transmission electron microscopy (HRTEM), HAADF-STEM and calculations of images and electron density and bond overlap population (BOP) by first principal to understand the origin of precipitation in this alloy.

Fig.1 shows HRTEM image in as-quenched specimen. In HRTEM image, some lines which has brighter ( or darker ) dots having spacing of 0.64 nm on the mono-layer of {1-100}Mg plane in the as-quenched sample.

Fig.2 shows HRTEM image obtained for the alloy aged at 473K for 7.2ks. The β’ phase has four atomic layers periodicity in the [1-100]Mg, and the arrangement of the bright dots with space of 0.64nm is observed.

At the under-aged condition, precipitates observed by HRTEM were classified as follows; mono-layer, a part of β”, β’. By HAADF-STEM observation, zig-zag structure, small hexagonal network, and β’ can be recognized. The small hexagon of 0.37 nm is the first precipitate in this alloy, and this is the evidence of short range ordering close to DO19 structure. This is referred as the pre β”-phase. Finally, we concluded that the proposed precipitation sequence is as follows; S.S.S.S. → pre β” phase having DO19 SRO → β” → β’.

[1] S. Kamado, Y. Kojima, S. Taniike, I. Seki and S. Hama : Proc. of the International Conf. on Magnesium Alloys and Their Applications, (1998), 169-174 .

[2] T. Kawabata, D. Nakagawa, S. Saikawa, J. Nakamura, S. Ikeno and K.Matsuda, Materials Transactions, Vol. 54, (2013), 225-230.

[3] M. Nishijima and K. Hiraga, Materials Transactions, Vol. 48, (2007), 10-15.

Fig. 1: HRTEM image obtained for as-quenched sample.

Fig. 2: HRTEM image obtained for aged sample at 473K for 7.2ks.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2119 Transmission Electron Microscopy of 935 Silver Alloys Containing Sn and Be

Sakultanchareonchai S.1, Nisaratanaporn E.1, Chairuangsri T.2
1Innovative Metals Research Unit, Department of Metallurgical Engineering, Chulalongkorn University, Bangkok10330, Thailand , 2Department of Industrial Chemistry, Chiang Mai University, Chiang Mai, 50200, Thailand
siriwanschai@gmail.com

Silver alloys with high spring property have been developed for using in jewelry spring articles. Usually copper is a main alloying element for improving the formability and strength [1-4]. To improve spring property and maintain anti-tarnish ability, a combination of Cu, Sn and Be additions was introduced. In the present work, effects of heat treatments on the microstructure of 935 silver alloy with 5.9 wt.%Cu, 0.35 wt.%Sn and 0.25 wt.%Be has been investigated by transmission electron microscope(TEM).
TEM samples were prepared by cutting the cast bars as thin slices with 200 µm in thickness using a high precision cutting machine (Struers, Accutom 50). The thin slices were ground down to 30-50 µm, then punched out as a discs of 3 mm, and subsequently electropolished at voltage of 7-9 V in potassium cyanide solution 10%(wt/vol) using a twin-jet electropolishing machine (Fishchion, Model 110) at room temperature. A JEOL2010 TEM/STEM was utilized and operated at 200 kV with attached Oxford Instrument, Inca, energy dispersive X-ray spectroscopy (EDS) detector.
TEM analysis of the silver alloy after-solution heat treatment at 750°C for 1 hr and subsequently aging at 350°C for 1 hr revealed a dispersion of precipitates with Moire’ fringes and size of 3-10 nm in diameter as shown in Figure 1. Strain fields around precipitate at P1 were found by coherency effect, SADP and TEM-EDS analyses suggested that the precipitate is fcc α-(Cu,Sn), a = 3.655 A. Comparable precipitation was also observed in the silver alloy after aging at 350°C for 30 minute without solution heat treatment. Effects of this precipitation behavior on improvement of spring property of the silver alloy will be discussed.

References
[1] Gardam G.E., Metallurgia, 1953; 47 (279):29–33.
[2] Nisaratanaporn S. and NisaratanapornE., J Met. Mat. Min., 2003; 12(2): 13-18.
[3] Nisaratanaporn E., Wongsriruksa S.,Pongsukitwat S. and Lothongkum G., Mat Sci. Eng. A-Struct, 2007; (445-446): 663-668.
[4] Sakultanchareonchai S. and Nisaratanaporn E., Proceedings of the 26th Annual Conference of The Microscopy Society of Thailand held at the Empress Hotel, Chiang Mai, 28-30 January 2009: 27-28.

The authors wish to acknowledge the financial and facility support by the Oldmoon Co., Ltd. And the Thailand Research Fund (TRF).

Fig. 1: a) Bright-field TEM micrograph the silver alloy after solution heat treatment at 750°C for 1 hr and subsequently aging at 350°C for 1 hr, b) Corresponding SADPs form the [001]α-Ag, c) corresponding SADP analysis (• is fcc α-Ag , ♦is fcc α-(Cu,Sn), d is double diffraction) and d) TEM-EDS spectra from precipitates and the silver matrix.

Fig. 2: a) Bright-field TEM micrograph of the silver alloy BF-TEM after aging at 350°C for 30 minute (b) corresponding SADP from [011 ̅]α-Ag
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2150 Focused Electron Beam Induced Deposited multi-metal nanoalloys

Shawrav M. M.1, Wanzenboeck H. D.1, Gavagnin M.1, Belić D.1, Wachter S.1, Schinnerl M.1, Bertagnolli E.1
1Institute of Solid State Electronics, Vienna University of Technology, Vienna, Austria
mostafa.shawrav@tuwien.ac.at

Focused Electron Beam Induced deposition (FEBID) is a mask-less, resist-less technique that has gained popularity due to its superior precision. In FEBID, precursor molecules are introduced inside a scanning electron microscope (SEM) using gas injection system. High energy electron beam of the SEM decomposes the precursor molecules adsorbed on the surface. This technique has already proven it’s potential for fabricating magnetic materials for nanomagnet logic devices, MOS capacitors and platinum based humidity sensors. FEBID has not been used so far to produce i) Fe-Au multilayer structure or ii) Fe-Au nanoalloys. This work will present an approach to produce multilayer and multi-material nanoalloys deposited by FEBID.

Multi-layer nanostructure of multi-material is required for various other applications such as CMOS transistors, photonic crystals etc. In addition, multi-metal structures such as nanoalloys have made substantial advances due to their promising magnetic, optical properties.

To investigate the possibility of a FEBID multi-layer structure and Fe surface oxidation issue, a 1×1 μm2 of Au was deposited on top of 2×2 μm2 Fe using 3kV acceleration voltage and 1nA beam current. Experimental results points that the deposited Au structure reduced the oxidation of underlying Fe deposit in a large degree. The resulting structure is presented in Figure 3. The FIB cross section showed no compositional changes to the initial Fe layer due to FEBID of the top Au structure. Both surfaces seen homogenous with no gaps in between the Fe and Au section, as expected.

Among different multi-metal nanoalloys, Au-Fe nanoalloys are considered as prospective materials for data storage application. Due to the possibility to directly write nanostructures at nanometer resolution, FEBID is also an appropriate candidate to realize such Fe-Au nanoalloys.

In order to fabricate nanoalloys, Fe and Au metal precursors were injected simultaneously to deposit Fe and Au within the same structure. Au precursor flux was kept constant, while Fe precursor flux was varied. These structures showed a uniform mixture ratio of iron and gold. The chemical composition confirmed the formation of Fe-Au nanoalloys. The resulting nanoalloys is show in Figure 4. The effect of Fe precursor flux on the deposition rate and elemental composition of the alloy structures will be addressed.

In summary, this work will show that FEBID is a suitable technique for fabricating multilayer nanodevices. FEBID is also a promising technique to deposit multi-metal nanostructures for potential data storage applications in nanoelectronics.

Shawrav, M. M., Belić, D., Gavagnin, M., Wachter, S., Schinnerl, M., Wanzenboeck, H. D. and Bertagnolli, E. (2014), Electron Beam-Induced CVD of Nanoalloys for Nanoelectronics. Chem. Vap. Deposition, 20: 251–257. doi: 10.1002/cvde.201407119

The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement number ENHANCE-238409 and the Austrian Science Fund Project P24093.

Fig. 1: Schematics of Focused Electron Beam Induced Deposition process.

Fig. 2: SEM image of a complex structure deposited by FEBID

Fig. 3: SEM image of a multilayer Au/Fe structure

Fig. 4: SEM image of Fe-Au nanoalloys
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2197 EBSD analyses of the dynamic recrystallisation in high-Mn steels

Wentz S. C.1, Krämer A.2, Schwedt A.1, Bambach M.2
1Central Facility for Electron Microscopy (GFE), RWTH Aachen University, Ahornstr. 55, 52074 Aachen, Germany, 2Institute of Metal Forming (IBF), RWTH Aachen University, Intzestr. 10, 52056 Aachen, Germany
schwedt@gfe.rwth-aachen.de

With the increasing demand for steel properties tailored to specific applications, understanding the microstructure development in all stages of the manufacturing chain becomes more and more important.

In order to get a deeper insight into the dynamic recrystallisation processes during hot rolling, a series of 12 differently deformed samples from Rastagaev compression tests have been examined by performing EBSD and SEM analyses after the test.

As material, a fully austenitic high-Mn-steel of the composition X30MnAl23-1 was chosen. Cylindrical Rastagaev samples of a diameter of 10mm and a height of 15mm were compressed in a servo-hydraulic testing machine by Schenck at a temperature of 1050°C and a strain rate of 0.1/s. The analysed target strains ranged from 0.05 to 0.5 (cf. Fig. 1). In order to ‘freeze’ the state of microstructure evolution after the test, the samples were quenched in water directly after they had been deformed to their final height.

The SEM / EBSD analyses were performed with a Hikari camera by EDAX-TSL attached to a JSM-7000F by JEOL.

The results (cf. Fig. 1) of the EBSD analyses clearly show the different degrees of microstructure evolution from the starting growth of subgrain boundaries at the existing high-angle grain boundaries to the growth of recrystallized grains.

These observed microstructure changes are compared to existing models for the recrystallisation process (e.g. Ponge & Gottstein 1998).


Reference:

D. Ponge and G. Gottstein, 1998, Acta materialia 46(1), 69-80.

The authors gratefully acknowledge the support of the Deutsche Forschungsgemeinschaft (DFG) within SFB761 - Stahl ab initio: Quantenmechanisch geführtes Design neuer Eisenbasis-Werkstoffe.

Fig. 1: Stress over strain hardening rate- and stress over strain curve during the compression test and corresponding Kernel Average Misorientation plots, KAM(450nm,5°). (a) and (b) show subgrain boundaries growing from the large-angle grain boundaries. (c) and (d) show the presence and growth of recrystallised grains (marked in (c)).

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2201 Formation of different carbide phases in the Nb-1%Zr-0.1%C alloy

Bathula V.1, Tewari R.1, Dey G. K.1
1Materials Science Division, Bhabha Atomic Research Centre, Mumbai, India
visubathula@gmail.com

In the Nb-Zr-C based ternary alloy system, Nb-1%Zr-0.1%C shows optimum high temperature properties. Adding 1wt.% of zirconium to niobium greatly improves its resistance to oxygen absorption and also its strength by solid solution strengthening. Carbon, the other alloying element in the alloy, increases the strength of the material by forming various carbide precipitates. Solubility of carbon in niobium is limited (~0.1 at.% at 1200 oC) at low temperatures. Therefore, excess carbon atoms precipitate out to form carbon-rich Nb-C phases during cooling from high temperatures. In the present study, the Nb-1%Zr-0.1%C alloy was prepared by electron beam melting technique. Subsequently, the as-solidified Nb alloy was extruded at different temperatures. These deformed samples were recrystallized by annealing at 1300 OC for 3 hrs. These three samples (as-solidified, deformed and annealed) were characterized using optical microscopy, X-ray diffraction and electron microscopy techniques. Microstructural characterization of all the samples revealed that the formation of type of carbide phase depends on the processing condition of the sample. The as-solidified sample had needle morphology of γ-Nb2C type of carbide phase (Fig.1), deformed samples had needle morphology of α-(Nb,Zr)2C as well as cuboidal morphology of (Nb,Zr)3C2 carbide phases (Fig.2) and the recrystallized samples had spherical morphology of (Nb,Zr)C type of carbide phase (Fig.3). This study showed that among all the carbides the (Nb,Zr)C carbide is the stable carbide phase in the Nb alloy. Based on the crystallographic analysis, the formation mechanism for all the carbide phases has been inferred. It showed that transformation of Nb to Nb2C take place by the occupation of carbon atoms at octahedral sites in bcc Nb lattice. This transformation does not involve a large movement of Nb atoms. The detailed crystallographic analysis of this structure based on orientation relationship showed that (Nb,Zr)C has formed by diffusional phase transformation and to form (Nb,Zr)C carbide phase, large movement of Nb and C atoms is required. Detailed carbide phase formation study has shown that phase formation sequence for the formation of stable (Nb,Zr)C carbide phase as: γ-Nb2C → α-(Nb,Zr)2C + (Nb,Zr)3C2 → (Nb,Zr)C.

Fig. 1: A typical bright field TEM micrograph of needle morphology precipitates present in the as-solidified Nb alloy. The inset figure shows the presence of heavy faults within the precipitate.

Fig. 2: TEM micrographs of the deformed Nb alloy sample showing the presence of (a) needle and (b) cuboidal morphology precipitates.

Fig. 3: Bright field TEM image of the annealed sample showing the presence of fine spherical morphology precipitates.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2306 Microstructural evaluation of PM S390 MC high-speed steel using Transmission Electron Microscopy

Jenko D.1, Šolić S.2, Dražić G.3, Leskovšek V.1, Jenko M.1
1Institute of Metals and Technology, Ljubljana, Slovenia, 2Faculty of Mechanical Engineering and Naval Architecture, Zagreb, Croatia, 3National Institute of Chemistry, Ljubljana, Slovenia
darja.jenko@imt.si

The research deals with the study of the impact of microstructure on the tribological properties of PM S390 MC high-speed steel (Böhler), prepared by powder metallurgy with chemical composition in weight % of 1.64 C, 0.60 Si, 0.30 Mn, 4.80 Cr, 2.00 Mn, 4.80 V, 10.40 W, 8.00 Co and the rest was Fe. Different microstructure conditions of high-speed steel were obtained by heat treatment in two ways: conventional (vacuum hardening, three high-temperature temperings) and deep cooling (vacuum hardening, deep cryogenic treatment, high-temperature tempering). In some experiments, nitriding in plasma of ionising gases was also carried out.

Thin foil specimens for transmission electron microscopy (TEM) were prepared using Jeol EM-09100IS Ion Slicer and further analyzed by TEM (Jeol JEM-2100 and Jeol ARM 200F) using conventional TEM (CTEM) and energy dispersive X-ray spectroscopy (EDS, Jeol JED-2300 Series) at 200 kV electron accelerating voltage. Detailed characterization of the microstructure was performed on thin foil specimens, and electron diffraction method was used as well to carry out the microstructure–crystallographic analysis of the phases. In the martensite microstructure some nanometre sized areas of possible other phase were observed. This study tries to explain whether these areas are nanometre sized carbide precipitates.

The research showed that the microstructure obtained by deep cooling and nitriding compared to conventional heat treated resulted in an increase of wear resistance of PM S390 MC high-speed steel.

This work has been supported by the research program ''Surface physics and chemistry of metallic materials'' P2-0132.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2310 Understanding the failure of oxidized grain boundaries

Dohr J.1, Armstrong D.1, Tarleton E.1, Couvant T.2, Lozano-Perez S.1
1Department of Materials, University of Oxford, Oxford, United Kingdom, 2Électricité de France R&D, Écuelles, France
judith.dohr@materials.ox.ac.uk

Stress Corrosion Cracking (SCC) of Alloy 600 (Ni-base alloy) in Pressurized Water Reactors (PWRs) is known to be one of the most expensive and challenging phenomena in the nuclear industry. SCC is difficult to observe, investigate and predict and often it occurs although no obvious signs of corrosion are present. Over the last decades great research efforts have been made to understand SCC of Alloy 600 under PWR conditions and many mechanisms based on different theories have been proposed to explain crack initiation and propagation. One thing they all have in common is the lack of definite experimental proof in favour of one of them. Although Alloy 600 suffers from intergranular failure under PWR primary water conditions, experimental evidence capable of explaining the failure mechanism and its link to microstructure is still insufficient. A combination of detailed microscopy investigations and micromechanical testing of individual grain boundaries (GBs), oxidized in simulated PWR primary water, now provides a novel tool for studying the specific fracture behaviour (e.g. brittle failure, plastic deformation etc.) of oxidized GBs. Utilizing a recently developed novel approach to fabricate and micromechanically test micron-sized cantilevers (Figure 1), we are now able to obtain information about the elastic moduli, yield stress and fracture toughness of tested GBs. The same GBs are also characterized by 3D FIB Slicing and (S)TEM, thus enabling the correlation of their measured mechanical response to the specific 3D microstructure and degree of oxidation (Figure 2). This includes the extraction of the grain orientations via Selected Area Diffraction (SAD) as well as analytical mapping of the grain boundary region after testing (Figure 2), providing further insights on how the crack propagates. Employing the crystal plasticity Finite Element Method (CPFEM) the experimental data can then be used to gain quantitative information about the SCC initiated failure of GBs by building realistic computer models of the fracture experiments. These models enable us to not only simulate the basic fracture experiments but also to consider the realistic plastic response of the tested microcantilevers. The presented approach consequently allows for the quantification of the stress necessary to fracture individual oxidized GBs and adds valuable qualitative and quantitative data to the study of stress corrosion cracking as well as the role of (intergranular) oxidation with unprecedented detail.

The authors want to thank INSS (Japan) and EDF (France) for the provision and autoclave testing of the samples. EDF is further acknowledged for the financial support of this work.

Fig. 1: SEM images of a FIB-machined microcantilever before (top) and after (bottom) the micromechanical bending test. The bottom image shows how the oxidized portion of the grain boundary failed intergranularily after testing.

Fig. 2: HAADF image of the crack region along an oxidized GB after the test (left). EDX elemental maps revealing the chemistry of the GB region (right). The results show that (a) the oxide grows along the GB and around a carbide, (b) the crack proceeds along the metal/oxide interface and (c) that the crack stops exactly where the oxide vanishes.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2454 Effects of arcing on the Microstructure, Morphology and Photoelectric Work Function of Ag-ZnO Electrical Contacts.

AKBI M.1, BOUCHOU A.2
1Department of Physics, University M'hammed Bouguerra of Boumerdes, Independence Avenue, Boumerdes (35000), Algeria, 2Faculty of Physics, University of Algiers (USTHB), 16000, Algiers, Algeria.
akbim656@gmail.com

Contact materials used for electrical breakers are often made with silver alloys. Mechanical and thermodynamical properties as well as electron emission of such complicated alloys present a lack of reliable and accurate experimental data. At present, new types of contactors with longer duration are marketed, but manufacturers do not understand well why this improvement.
The influence of industrial conditioning (polishing, mechanical shocks and electrical arcs in air) on the microstructure, the composition, and the morphology of internally oxidized Ag-ZnO contacts was investigated. The microstructure, physical properties and electronic emission behavior of Ag-ZnO materials were studied.
Contacts were mounted in a contactor working repeatedly on air (laboratory atmosphere). When submitted to 500 opening electric arcs, the electron work function of an electromechanically conditioned contact Ag-ZnO (92/8), measured photoelectrically by using Fowler’s method of isothermal curves, is F = (4.82 ± 003) eV.
The melting temperature of the zinc oxide is greater than that of silver. Thus, zinc oxide melts after evaporation of silver base material. However, the zinc oxide increases the viscosity of the molten composite and reduced mass losses by radial ejection of the liquid metal. Microanalyses EDS indicate the presence of Ag, Zn, O, C, Al, Si, Cl, ... etc. The additives migrate to the periphery of the contact, as usual. Examination by SEM and and microanalyses by EDS of the conditioned cathode AgZnO have been performed to produce proofs of these phenomena.

 

Fig. 1: Micrographs for (a) a virgin and unpolished contact, Ag-ZnO (92/8) SEM magnification x 2000, for (b) a virgin and unpolished contact, Ag-ZnO (92/8) SEM magnification x 8000, and for (c) a virgin and polished contact, Ag-ZnO (92/8) SEM magnification x 8000

Fig. 2: EDS line scans of two representative points (one bright and one dark) for the central contact surface of the conditioned cathode (500 arcs), Ag-ZnO (92/8). SEM magnification x 2000.

Fig. 3: EDS line scans of two representative points (one bright and one dark) for the peripheral contact surface of the conditioned cathode (500 arcs), Ag-ZnO (92/8). SEM magnification x 2000.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2473 Interface characterization of a W-coated diamond/Al composite for thermal management applications: towards interface design and engineering

JI G.1,2, Tan Z. Q.3, Lu Y. G.2, Schryvers D.2, Li Z. Q.3, Addad A.1, Zhang D.3
1Unité Matériaux et Transformations, UMR CNRS 8207, Bâtiment C6, Université Lille 1, 59655 Villeneuve d'Ascq, France, 2Electron Microscopy for Materials Science (EMAT), University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 3State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, China
gang.ji@univ-lille1.fr

Over the last decade, a diamond particle reinforced Al matrix, namely diamond/Al, composite has been developed for thermal management applications, mainly in the microelectronic industry. This composite has demonstrated an excellent combination of a Thermal Conductivity (TC) as high as 600 W/m k and a Coefficient of Thermal Expansion (CTE) lower than 10 ppm/K, being compatible with that of electronic components. However, considering the TC (~ 1800 W/m K) of the diamond particles incorporated, it is clear that the overall TC enhancement has not been completely exploited in the composite. The key is then pointed to the diamond/Al interface, which should provide good adhesion as well as maximal Interfacial Thermal Conductance (ITC) in order to facilitate thermal exchange across the interface.

Our analytical modeling has recently predicted that introduction of a W interface nanolayer is one of the most efficient ways to achieve high ITC, which provides a practical guide for interface engineering [1]. Accordingly, a cost-effective sol-gel process has been tentatively used to deposit a W coating for diamond surface metallization. Compared with the diamond/Al counterpart without a W nanolayer, TC of the composite with a W nanolayer has been improved more than 20 % [2]. In this work, Scanning Transmission Electron Microscopy (STEM)/Energy-Dispersive X-ray spectroscopy (EDX) has been performed in order to investigate interface configurations of a W-coated diamond/Al composite. The aim is studying the effect of interface formation, reaction and diffusion on the ITC.

The deposited W coating is discontinuous, which is made of nanoparticles with a size in the range 30-400 nm and homogenously covering the surface of the diamond particle (Figs. 1a and 1b). The average coating thickness is estimated to be around 200 nm (Fig. 1c). The STEM/ADF image in Fig. 1d shows that the formed diamond/Al interface has a heterogeneous configuration at the nanoscale where Al grain contrasts and a particle with high Z contrast are revealed. STEM/EDX mapping in Fig. 2 displays a W-rich (or W-Al rich) interfacial particle. Visible O traces can be related to fine microstructural features. Alternatively, a 'clean' diamond/Al interface is tightly-adhered and is not rich in O (Fig. 3). Different chemical nature of the bonds at the interface can have a pronounced effect on the local ITC.

[1] Tan Z Q, Li Z Q, Xiong D B, Fan G L, Ji G, Zhang D, Materials and Design 55 (2014) 257–262.

[2] Tan Z Q, Li Z Q, Fan G L, Guo Q, Kai X Z, Ji G, Zhang L T, Zhang D, Materials and Design 47 (2013) 160–166.

We thank the financial support of the FWO project of Belgium (G.0576.09N), the National Natural Science Foundation (No. 51131004) and the National Basic Research Program (973 Program) (No. 2012CB619600) of China.

Fig. 1: Scanning electron microscopy images showing the morphologies of (a) a W-coated diamond particle, (b) deposited nanoparticles, (c) deposition thickness measured by scanning probe microscopy and (d) STEM/Annular Dark Field (ADF) image of a diamond/Al interfacial area prepared by focus ion beam.

Fig. 2: STEM/EDX analysis of the diamond/Al interfacial area containing a deposited particle: (a) ADF image, (b) W, (c) O and (d) mixed Al and C elemental maps.

Fig. 3: STEM/EDX analysis of the 'clean' diamond/Al interface: (a) ADF image, (b) Al, (c) C and (d) O elemental maps.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2491 Effect of heat treatment on the creep behavior and microstructural evolution of P92 steel

Shin K.1, He Y.1, Kim J.1, Chang J.2
1Changwon National University, Changwon, Korea, 2Korea Electric Power Research Institute, Daejeon, Korea
keesam@changwon.ac.kr

The 9-12% Cr creep-resistance steels are mainly used as components of high temperature boilers, steam turbines and heat exchangers in power plants. The wonderful high temperature properties of these steels are based on the complex microstructure of stable precipitates and dislocations. It’s known that the steels are strengthened by: i) solid solution with a high concentration of Cr, Mo, ii) refined grains by the formation of tempered martensitic lath structure, iii) uniformly dispersed M23C6 (M: Fe, Cr, Mo) and MX carbonitrides (M: Nb, V, Ti, X: C, N), and iv) high dislocation density in the lath matrix. The inhibition of dislocation migration, annihilation and/or recovery by precipitates, and grain boundaries during service are considered to be the predominant mechanism of the strengthening. The creep properties of the steels, in turn, depend on the stability of microstructure. In the past decades, microstructural study of the P92 steel upon aging or creep rupture test has been extensively carried out.
In this work, to investigate 1) the effect of heat treatment on the creep rupture behavior, 2) microstructural evolution after heat treatment and creep rupture, 3) the role of the Laves phase on the creep properties, as-received (AR) P92 steels were heat treated at 632°C for 500 and 1,000 hrs before creep rupture test. Both the AR and heated treated specimens were creep tested at 650°C with constant applied stress of 120 and 100MPa. The electron backscattered diffraction (EBSD) and backscattered electron (BSE) imaging was used to study the tempered lath and Laves phase. Both the 3mm disk thin foils and carbon extraction replicas of precipitates specimens were analyzed by using transmission electron microscopy (TEM).
Up to 500hrs of heat treatment, the creep life did not change (Fig. 1a). However, the life has reduced sharply after aging of 1000hrs or longer, which showed a direct relation to the growth of Laves phase from 245nm to 354nm. Microstructural observation shows that the formation and growth of the Laves phase with heat treatment time (Fig.1b, c). Slight coarsening of M23C6 was observed at the heat treated and crept ruptured specimens (Fig.2). In addition, upon heat treatment and/or creep test, the elongated lath grains evolved to equiaxial grains with the decrease of low angle grain boundary, suggesting occurrence of grain recovery (Fig.3). The investigation suggests that Laves phase also played a role on the strengthening mechanism of the heat resistant steel when it was in fine size and homogenously distributed in the matrix. The effect of creep parameters on the evolution of M23C6 and Laves phase will be also discussed.

Acknowledgements: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No.2012-0009454), and partially supported by the Korea Electric Power Research Institute.

Fig. 1: (a) Heat treatment and creep test parameters, (b) and (c) BSE images showing the formation of Laves phase in the 500 and 1,000hrs heat treated specimens, respectively.

Fig. 2: TEM images of the test P92 steels: (a) AR, (b) 1,000hrs heated treated and (c) creep ruptured at 650°C/120MPa/408hrs.

Fig. 3: EBSD inverse pole figure images of the test P92 steels: (a) AR, (b) 1,000hrs heated treated, and (c) creep ruptured at 650°C/120MPa/408hrs.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2525 Multi-Scale-Analysis of Modern Aluminium-Alloys

Schroettner H.1, 2, Panzirsch B.3, Albu M.2, Mitsche S.1, 2, Mertschnigg S.1, 2, Gspan C.2, Rattenberger J.2, Wagner J.2, Hofer F.1, 2
1Institute for Electron Microscopy and Nanoanalysis (FELMI), Steyrergasse 17, 8010 Graz, Austria, Graz University of Technology (TU Graz), 2Graz Centre for Electron Microscopy (ZFE), Steyrergasse 17, 8010 Graz, Austria, 3Austrian Foundry Research Institute (OGI), Parkstraße 21, 8700 Leoben, Austria
hartmuth.schroettner@felmi-zfe.at

During the last years a lot of new light metal alloys for automotive and aerospace applications have been developed. Most of them have been generated based on practical experience. So there is a demand for a deeper insight of the inner structure and the resulting mechanical properties of these materials. In this project four Austrian institutes (OFI, OGI, SZA, ZFE) – all members of the Austrian Cooperative Research (ACR) – are focusing their knowledge and efforts to receive a holistic view of these materials.

Heat resistant aluminium-alloys possess good creep strength and high thermal stability and can be used for permanent casting. Their strength properties are based on specific precipitations in the

structure of the materials.

The main goal of this project is to characterise the fundamental mechanism, which affect the high temperature strength and the creep strength, the connectivity and the corrosion resistance. Based on these findings an optimization of these alloys should be achieved.

The focus of this work is the multi-scale-analysis of the structure of modern aluminium-cast-alloys. Classical metallography is combined with micro- and nano-characterisation methods using scanning electron microscopy (SEM) (Fig. 1) and transmission electron microscopy (TEM) as well as 3D-methods as computer-tomography (CT) and in-situ-ultramicrotomy (3viewTM) [1] (Fig. 2) in an environmental scanning electron microscope (ESEM) or focused ion beam (FIB) techniques.

Special attention is turned on the micro- and nano-analysis of phases and precipitations and the influence of dopant elements on the structure and behavior of these aluminium-cast-alloys.

For the micro-characterisation the SEM is used in combination with energy dispersive x-ray spectroscopy (EDXS), wavelength x-ray spectroscopy (WDXS) and electron backscatter diffraction (EBSD). Out from regions of interest TEM-lamellas from regions of interest are prepared with the FIB and transfered to the TEM for the nano-characterisation of the samples, using EDXS and electron energy loss spectrometry (EELS/EFTEM) methods and electron diffraction techniques [2]. High resolution scanning transmission electron microscopy analysis on an atomic scale level is focused on the dopant elements (Fig. 3) and is compared with HREM simulations (Fig. 4). This versatile combination of methods leads to a multi-scale-analysis of the investigated materials and helps materials scientists to deepen their know-how.

Ref.:

[1] Zankel, A.; Reingruber, H.; Schröttner, H.: 3D Elemental Mapping in the ESEM - Imaging & microscopy 2 (2011) 35–37

[2] Albu, M.; Li, J.; Kothleitner, G.; Schumacher, P.; Hofer, F.: Atomic resolution STEM analysis of Sr and Yb addition in Al-Si alloys - Mg; Al; Ti Science and Technology (2013) 162 – 162

The authors want to thank the Austrian Research Promotion Agency (FFG) for financial support (PN 839958).

Fig. 1: SEM Backscatter electron image of an AlSi7 alloy.

Fig. 2: 3D reconstruction of the intermetallic phases of an Al-Si-Cu-Mg alloy based on in-situ-ultramicrotomy in an ESEM [1].

Fig. 3: Scanning Transmission Electron Microscopy High Angle Annular Dark Field (STEM-HAADF) image of the eutectic silicium phase with strontium atoms in the interstitial position and EDXS spectra of the marked area [2].

Fig. 4: HAADF simulation of the eutectic silicium phase with strontium atoms in the interstitial position.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2526 Influences of Dy content and heat treatments on the formation of hydrides in Mg-Dy alloys

Huang Y.1, Yang L.1, Wang Z.1, Kainer K.1, Hort N.1
1MagIC-Magnesium Innovation Centre, Helmholtz-Zentrum Geesthacht Max-Planck-Str. 1, 21502 Geesthacht, Germany
yuanding.huang@hzg.de

Owing to their suitable mechanical properties and acceptable corrosion resistance, Mg-RE (rare earths) alloys were recently regarded as one of the most potential degradable biomaterials [1]. From their preparations to their final applications these magnesium alloys are subjected to the environments containing hydrogen (H).  Yang et al. reported that NdH2 was formed in Mg-2wt.% Nd alloy with T4 treatment [2]. The formation of NdH2 was attributed to the reaction of Nd with the previously dissolved hydrogen during casting. In the recent, another different explanation was suggested by Peng et al. [3]. They suggested that the formation of hydride in Mg-Gd alloys can also proceed during sample preparations or mechanical deformation if subjected to H-containing environment.

The present work investigates the influences of alloying element Dy and heat treatments on the formation of hydrides in Mg-Dy alloy. The alloys with different contents of Dy were prepared by permanent mould direct chill casting. The heat treatments such as T4 or T6 were performed. The samples for the observations of optical microscopy and scanning electron microscopy (SEM) were machined and polished with water or without water. The X-ray diffraction (XRD) was used to identify the phases .

Figure 1 shows SEM micrographs of Mg-20Dy alloy with T4 treatment. Lots of hydride particles are observed on its surface when the sample was wet machined and mechanical polished with water. In contrast, the hydride particles are hardly found on the surface of the sample with dry machined and electropolished. The formed hydrides have a cuboid morphology with a size of 2 to 5 micrometers. The content of Dy influences the formation of hydrides (Figure 2(a)). With the increment of Dy content, the amount of hydrides increases. In addition, the amount of hydrides is also affected by heat treatment (Figure 2(b)). On the surfaces of the samples with as-cast or T6 treatment, XRD test cannot identify the existence of hydrides, indicating that the amount of hydrides is very less.

In conclusion, the formation of hydrides in Mg-Dy alloys is affected by Dy content and heat treatment. Their formation mechanism is attributed to the surface reaction of Mg-Dy alloys with water.

References

[1]. Hort N, Huang Y, Fechner D, Störmer M, Blawert C, Witte F, Vogt C, Drücker H, Willumeit R, Kainer KU and Feyerabend F (2010). Acta Biomaterialia, 6, 1714-1725.

[2]. Yang Y, Peng L, Fu P, Hu B and Ding W (2009). Journal of Alloys and Compounds, 485, 245-248.

[3]. Peng Q, Huang Y, Meng J, Li Y and Kainer KU (2011). Intermetallics, 19, 382-389.

Fig. 1: SEM micrographs of Mg-20Dy alloy with T4 treatment, (a) machined and polished with water, and (b) dry machined and electropolished.

Fig. 2: X-ray diffraction patterns showing the phases, (a) effects of Dy content and (b) effects of heat treatments.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2645 Microstructure of Duplex-Heat-Treated Ti-6Al-4V Alloy

Khalil A. S.1, Abdel Rehim R. A.2
1Tabbin Institute for Metallurgical Studies (TIMS), Cairo, Egypt, 2Central Metallurgical Research and Development Institute (CMRDI), Cairo, Egypt
askhalil2004@yahoo.com

In pure titanium, solidification occurs at 1668 OC with the formation of body centred cubic (BCC) crystal structure known as β titanium. An allotropic transformation occurs at 888 OC where the BCC transforms to hexagonal close-packed (HCP) structure known as α titanium. The temperature above which the microstructure is 100% β titanium is referred to as the β transus. Certain elements will raise the β transus and are known as α-stabilizers such as aluminium while others like vanadium will lower the β transus and are known as β-stabilizers [1]. In Ti–6wt%Al–4wt%V, the two phases coexist together. The alloy has excellent combinations of desired properties making it a preferential alloy in many modern applications [1].

In this work, scanning electron microscopy (SEM) and optical microscopy were used to study the effects of duplex heat treatment on the microstructure of a commercial cast Ti-6Al-4V. The duplex heat treatment (two stage treatment; solution treatment then aging, each followed by water quenching) the solution treatment was at 975 OC for 15 minutes followed by water quenching and then aging conducted at two different temperatures of 490 OC and 595 OC for 6 hour duration (each aging followed by water quenching) as depicted schematically in figure 1.
In figure 2(a), the microstructure of the as-received cast sample shows a fully lamellar dendrite structure consisting of alternating laths of α and β phases intertwined in a “basket-weave” pattern (Widmanstätten pattern).
In figure 2(b) for the quenched sample from 975 OC which is below the β transus temperature, and is within the α+β phases field in the phase diagram [1], the primary α is interlaced with β phase in which a fraction had transformed into acicular α\ martensite phase (supersaturated α HCP) [2].
The aging leads to the decomposition of the nonequlibrium α\ into fine β and α and delineating grain boundaries as shown in figure 2(c) and 2(d) for 480 OC and 595 OC respectively. No martensitic structure was observed or expected by quenching from these temperatures [3]. However, aging at 595 OC leads to a finer observed structure. Optical microscopy complimented the SEM observations as shown in figure 2(e) for the sample aged at 480 OC . These observed microstructures strongly affect the hardness, wear and corrosion rates relative to the as-received alloy as wil be presented.

References

[1] G. Welsch in “Materials Properties Handbook: Titanium Alloys”, ed. G. Welsch, (ASM-Ohio) (1994), p. 1501.
[2] E. Morita et al, Materials Transactions 46 (2005), p. 1681.
[3] P. Pinke et al, Proceedings of 12th CO-MAT-TECH Conference 2004, Bratislava, Slovakia, p.1042.

Fig. 1: Figure 1: A schematic of the heat treatment regime followed in this study.

Fig. 2: Figure 2: SEM micrographs (x1000) of as-received in (a) and quenched from 975 OC in (b) and aged for 6 hours at 480 OC in (c) and at 595 OC in (d), the scale bar = 100 μm. And an optical micrograph (x200) in (e) for the sample aged at 480 OC, the scale bar = 50 μm.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2649 TEM Investigation on the Surface Oxide Layer of an Annealed AISI-316L Stainless Steel Microfiber

Ramachandran D.1, 2, Egoavil R.1, Verbeeck J.1, Abakumov A.1, Schryvers D.1
1Electron Microscopy for Materials Science (EMAT), University of Antwerp, Antwerp, Belgium, 2SIM vzw, Technologiepark 935, BE-9052 Zwijnaarde, Belgium
DhanyaR.Puthenmadom@uantwerpen.be

Steel fibre reinforced polymers are gaining considerable attention because of their remarkable flexural strength, fatigue resistance and the ability to tailor directional mechanical properties. Here, we investigate an annealed AISI-316L stainless steel microfiber sample using advanced electron microscopy and spectroscopy to understand the chemical composition and morphology of surface oxide layers on these fibres. When a steel fibre surface is thermally treated or even simply exposed to natural atmospheric conditions, a thin layer of oxide is instantaneously formed at the surface of the steel. This oxide layer not only plays an important role in protecting the steel from further corrosion, [1] but also acts as the interface with the matrix when using these fibres in composite materials. There are several literature reports which explain the effect of annealing on the thickness of the oxide layer [2]. The objective of the present study was to characterize this surface layer using HRTEM in combination with energy-dispersive X-ray (EDX) analysis and electron energy loss spectroscopy (EELS). SEM images from the surface of the steel microfibers showed the presence of small chromium rich particles with spinel structure and spherical morphologies. TEM specimens for the studies were prepared using Focussed Ion Beam (FIB) technique enabling the preparation of cross-section as well as on-axis samples. HRTEM images showed the presence of a very narrow gradient surface oxide layer with approximately 5.0 nm thickness at the surface of the steel fibre. EDX analysis revealed that this narrow gradient consists of three sublayers – an external layer rich in iron, an intermediate layer rich in chromium and an inner layer rich in nickel. ELNES studies reveal that the fine structure of the Fe-L2, 3, Cr-L2, 3 and Ni-L2, 3 edges obtained from the surface are different from those obtained from the bulk of the alloy, confirming the oxidised character of the surface layer. The insight gained from these studies is valuable considering the fact that these types of metal fibre alloys are widely used commercially.

References:

[1.] G. Renaud, Oxide surfaces and metal/oxide interfaces studied by grazing incidence X-ray scattering, Surface Science Reports, 32, 1-90 (1998).

[2.] H. Singh, D. Puri, S. Prakash, Rabindranath Maiti, Characterization of oxide scales to evaluate high temperature oxidation behavior of Ni–20Cr coated superalloys, Materials Science and Engineering A, 464, 110–116 (2007).

The authors would like to thank the Strategic Innovative Materials (SIM) project for the support through research projects.

Fig. 1: SEM image showing particles on the surface of AISI-316L stainless steel microfibre.

Fig. 2: SEM image of a cross-section of AISI-316L stainless steel microfiber.

Fig. 3: HRTEM image of an oxide layer formed on the surface of AISI-316L stainless steel microfibre.

Fig. 4: EDX map of the phases present within the surface oxide layer formed on AISI-316L stainless steel microfibre.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2657 TEM study of low carbon steel subjected to severe plastic deformation by multistage compression

Vilotic M.1,2, Sidjanin L.1, Jeng Y.2, Alexandrov S.2,3, Kakas D.1
1University of Novi Sad, Faculty of Technical Science, Serbia, 2National Chung Cheng University, Advanced Institute of Manufacturing with High-tech Innovation (AIM-HI), Taiwan (ROC), 3Ishlinski Institute for Problem in Mechanics, Russian Academy of Sciences, Russia
markovil@uns.ac.rs

Steels are the most used engineering material in many industrial fields, due to their relative low cost, availability and excellent properties. Development of new metal forming processes such as severe plastic deformation (SPD) makes it possible to extend, the range of steels application. However, the SPD can be an effective method of producing ultra fine-grained with submicron and nanocrystalline structure, even in steel bulk semiproducts, as well as increasing in mechanical properties [1]. Thus, some products of high-alloy steels might be replaced by carbon steel or low alloy ones. For that reason, at the Department of Production Engineering, an attempt was made to develop one discontinuous SPD method for upsetting square shaped billet by V-shape dies, Fig.1(a). The V-shape die compression is multistage process in which, after single compression stage, sample is removed from the dies and rotated for 90º in anti-clockwise direction and returned into the dies.
Experimental application of the compression by the V-shape dies was conducted using a normalized carbon rod steel CK15 with 0,14%C and ferrite-pearlite microstructure, Fig.1(b). The samples of 14x14x70 mm were compressed in eighteen turns, without any lubrication on hydraulic press. The influence of the processing parameters was evaluated by TEM microstructure analysis using FEI Tecnai F20. For TEM sample preparation FIB (Quanta 3D FEG) TEM sections of the carbon steel upsetting specimens were prepared with in-situ, lift-out technique, Fig.1(c).
TEM micrographs and corresponding diffraction patterns of the ferrite region in the as-compressed samples are present in Fig.2(a-d). After the second turn, Fig.2(a), the ferrite microstructure mainly consisted parallel bands of elongated grains having width 0,2-0,3 μm. The band boundaries are predominantly in low-angle missorientations. It is also apparent that inside the band, the interior dislocation cell boundaries are also present. This kind of boundary microstructure is typical in heavily deformed metals.
The TEM microstructures and corresponding ring patterns, after eight, twelve and eighteen turns are shown in Fig.3(b-d) respectively. Equiaxed grains with an average size of 0,15-0,3 μm were formed in all three samples. The presence of equiaxed grains, i.e. the grains with high angle boundaries confirmed by ring patterns with large number of reflections and a TEM contrast at the grain boundaries.
Moreover, the grain refinement during the eighteenth turn is not significant compared to twelfth. In addition, from the results presented it can be concluded that multistage compression by V-shape dies might be used as a severe plastic deformation method for the steels.

[1] R.Z. Valiev, R.K. Islamgaliev, I.V. Alexandrov, Progr. Mat. Sci. 45 (2000), 103-189.

The research described was supported by grants RFBR-13-08-00969 (Russia), TR035020 (Serbia) and NSC 100-2923-E-194-001-MY3 (Taiwan R.O.C.).

Fig. 1: a) V-shape die b) as-received microstructure c) FIB sample preparation

Fig. 2: TEM micrographs of as-compressed low-carbon steel after: a) second b) eight c) twelve d) eighteen turns
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2744 Characterization of high strength Aluminium alloys with coherent precipitates

Makineni S.1, Meher S.2, Banerjee R.2, kumar S.1, Chattopadhyay K.1
1Department of Materials Engineering, Indian Institute of Science, Bangalore, India, 2Center for Advanced Research and Technology and Department of Materials Science and Engineering, University of North Texas, Denton, TX, USA
surendramaterials@gmail.com

The present paper deals with a characterization of a class of aluminium alloys that are developed through a unique processing route that contains two different types of strengthening coherent precipitates in the aluminium matrix. The two types of precipitates are Al2Cu (θ’, tetragonal structure) and Al3Zr (L12 structure). The alloy belongs to Al-Cu binary system with minor additions of Zr, Nb and Zr, Hf. A unique three stage sequence of heat treatment was given to a chill cast alloy yielding final microstructure containing coherent finely dispersed L12 Al3Zr precipitates that formed at high temperature and coherent disk shaped θ’ precipitates heterogeneously nucleated on the prior Al3Zr particles. Therefore, tuning the heat treatment time and temperature for Al3Zr precipitation, it is possible to achieve a finely spaced precipitate distribution of θ’ disks that are resistant to coarsening at high temperature. We show that this leads to remarkable high temperature strength with 0.2% proof stress of about 250 MPa at 250ºC, thus opening a new window for high temperature aluminium alloy development. The first stage of the heat treatment was done at three different temperatures that are 375ºC, 400ºC and 450ºC till the time the peak hardness was achieved. The precipitation and coarsening was monitored through hardness measurements (Vickers hardness, Hv) and STEM HAADF contrast images were used to calculate the change in length of θ’ disks, number of disks and their spacings for different temperatures and time. The final microstructures were also characterized by High Resolution Transmission Electron Microscopy (HRTEM) and atom probe tomography (APT).

The authors would like to acknoledge the microscopy facility available at materials department (JEOL 2000FX), at AFMM centre (FEI F30) and for Atom Probe Tomography (APT) facility at CART, deparment of materials engineering, University of North Texas, Denton.

Fig. 1: TEM darkfield image taken from a superlattice spot in [110] Al direction
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2976 Microstructure of a CoCrFeMnNi high-entropy alloy investigated by advanced TEM techniques

Dlouhý A.1, Otto F.2, 3, Somsen C.4, Bei H.2, Eggeler G.4, George E. P.2, 3
1Institute of Physics of Materials, Academy of Sciences of the Czech Republic, 616 62 Brno, Czech Republic, 2Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA, 3Materials Science and Engineering Department, University of Tennessee, Knoxville, TN 37996, USA, 4Institut für Werkstoffe, Ruhr-Universität Bochum, 44780 Bochum, Germany
dlouhy@ipm.cz

An equiatomic CoCrFeMnNi high-entropy alloy, which crystallizes in the face-centered cubic (fcc) crystal structure, was produced by arc melting and drop casting. The drop-cast ingots were homogenized, cold rolled and recrystallized to obtain single-phase microstructures with three different grain sizes in the range 4–160 μm. Quasi-static tensile tests at an engineering strain rate of 10-3 s-1 were then performed at temperatures between 77 and 1073 K [1]. Microstructures after heat-treatment and tensile deformation were investigated using advanced high angular annular dark field (HAADF) and high resolution transmission electron microscopy (HRTEM) techniques. The recent HAADF stereo-imaging method [2] helped to characterize planar slip of {111} a/2<110>-type dislocations and their dissociation into {111} a/6<112>-type Shockley partials. HRTEM experiments yielded full local information on the type and geometry of nanoscale deformation twins that were observed after interrupted tensile tests at 77 K. We have shown that the relative orientations of the parent crystal and the twins can be described by the twinning elements K1 = (-11-1), η1 = [-112], K2 = (-111), η2 = [-11-2] and a shear s = 1/√2, analogous to a compound fcc twin commonly reported in fcc crystals. Results of the two TEM techniques provided insight into the relation between microstructure and the rather unique mechanical properties of this compositionally complex alloy.

[1] F. Otto et al.: Acta Mater. 61 (2013) 5743 - 5755.

[2] L. Agudo Jácome, G. Eggeler, A. Dlouhý: Ultramicroscopy 122 (2012) 48 - 59.

Research sponsored by the U.S. Department of Energy, Basic Energy Sciences, Materials Sciences and Engineering Division; AvH Foundation (F.O.) and CSF project no. 14-22834S (A.D.).

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2800 Microstructural evolution in Ti-Fe-O-N alloys during heating at intermediate temperature

Mitsuhara M.1, Nagase T.2, Masuda T.2, Nishida M.1, Kunieda T.3, Fujii H.3
1Department of Engineering Sciences for Electronics and Materials, Kyushu University, Fukuoka, Japan, 2Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, Fukuoka, Japan, 3Steel Research Laboratories, Nippon Steel & Sumitomo Metal Co, Chiba, Japan
mitsuhara@asem.kyushu-u.ac.jp

In dual phase (α + β) Ti alloys, the phase ratio of α-Ti (hcp) and β-Ti (bcc) can be controlled by adjusting chemical compositions and heat treatments. This feature realizes the wide range of mechanical properties such as strength, ductility and so on. Note, however, that most of the alloying elements are expensive, by which α-Ti and β-Ti phases are stabilized. Then, production cost of the dual phase Ti alloys should be reduced in order to widely apply them to various fields. Ti-Fe-O-N alloy is one of the commercially available dual phase Ti alloys consisting of low-cost alloying elements. The maximum tensile strength of Ti-1.5Fe-0.5O-0.05N is increased more than 1 GPa by exposing at 573 K and 623 K for 400 hr and more [1]. So, Ti-1.5Fe-0.5O-0.05N alloy has been recognized to be a suitable alternative material to replace Ti-6Al-4V, since mechanical properties in both alloys are equivalent. In this study, we observe the microstructural evolution during heat-treating at 623 K and unveil the strengthening mechanism in Ti-1.5Fe-0.5O-0.05N. In addition, we report a novel secondary phase precipitated in α-Ti phase with prolonged heating. Figure 1 is a SEM-BSE image in solution treated Ti-1.5Fe-0.5O-0.05N alloy. The dark and light gray areas correspond to α-Ti and β-Ti phases, respectively. The area fraction of α-Ti is approximately fifth times as large as that of β-Ti. We confirm that the phase ratio is almost the same after the heat-treatment for more than 1000 hr at 623 K. Figure 2 is hardness of α-Ti and β-Ti as a function of heating time at 623 K. The hardness of β-Ti increases with heating time. In contrast, that of α-Ti is hardly changed by long-term heating. It means that the strengthening of alloy is caused by microstructural evolution in β-Ti. Figure 3 is taken in the alloy with heating for 720 hr. (a) is a DF-TEM image of ω particles in β-Ti taken by selecting 1-101ω reflection. (b) is a BF-TEM image in α-Ti under a two beam condition of g = 11-20α. In Fig. 3 (a), there are many cuboidal ω particles in the size of range of 20 to 40 nm, which is well known to be harder than β-Ti. From other TEM observations in various heat-treated alloys, the size of ω particles is confirmed to be gradually increased with heating time. This precipitation and growth of ω phase lead to the strengthening of β-Ti as shown in Fig. 1. In Fig. 3 (b), there are many coherent precipitates with “coffee-bean” strain contrast in α-Ti. This precipitate is firstly observed in α-Ti of the Ti-Fe system with long-term heating, although the precipitate hardly contributes to the strengthening.
[1] H. Fujii, K. Fujisawa, M. Ishii, Y. Yamashita, Nippon Steel Technical Report, 85 (2003), 107-112.

Fig. 1: SEM-BSE image in solution treated Ti-Fe-O-N alloy.

Fig. 2: Hardness of α-Ti phase and β-Ti phase in Ti-Fe-O-N alloy as a function of heating time at 623 K.

Fig. 3: TEM observation in Ti-Fe-O-N alloy with heating at 623 K for 720 hr. (a) is a DF-TEM image in β-Ti phase taken by selecting 1-101ω reflection. (b) is a BF-TEM image in α-Ti phase showing strain contrast due to novel secondary phase.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3007 TEM observation of HPT-processed Cu-added excess Mg-type Al-Mg2Si alloys

Maruno S.1, Watanabe K.1, Matsuda K.2, Saikawa S.2, Hirosawa S.3, Horita Z.4, Lee S.4, Terada D.5
1Graduate School of Science and Engineering for Education, University of Toyama, 2Graduate School of Science and Engineering for Research, University of Toyama, 3Department of Materials Science and Engineering, Faculty of Engineering, Yokohama national University, 4Department of Materials Science and Engineering, Faculty of Engineering, Kyushu University, 5Department of Materials Science and Engineering, Faculty of Engineering, Kyoto University
m1371529@ems.u-toyama.ac.jp

Al-Mg-Si alloys are known as the age-hardening alloy. Addition of transition elements and thermo-mechanical treatments have been reported as methods to improve the mechanical properties of these alloys[1]. Ultrafine-grained (UFG) materials produced by SPD have attracted considerable attention over the last decade because of their superior mechanical and physical properties. HPT is an attractive processing route because there is good evidence that it leads to a greater refinement of the microstructure and to a higher incidence of high-angle boundaries. The HPT samples, in the form of disks with diameters in the range of ~10 mm, are held between anvils and subjected concurrently to a high pressure and torsional straining [2]. The aim of this study, to study the effect of HPT on aging behavior and microstructure in Cu-added excess Mg-type Al-Mg-Si alloys by means of hardness tests and TEM.

The Al-1.1%Mg2Si-0.4%Mg alloys (at.%) including Cu were obtained by the laboratory casting. The specimens were solution heat treated at 848K for 3.6ks in an air furnace, quenched in chilled water and subsequently HPT processed or rolled, followed by an aging treatment at 343 and 373K for different periods. The specimens were processed by HPT using imposed pressures of 6.0GPa for 5 rotations at a rotation speed of 1 rpm. The Micro vickers hardness was measured using Mitutoyo HM-101 hardness tester. After the aging treatment, samples were polished by using electrolytic solution (perchloric acid: ethanol=1:9) for making thin films for TEM observation. The microstructure was observed using TEM(TOPCON EM-002B) operated at 120kV.

Fig. 1 shows the age-hardening ability (ΔHV) of HPT processed ex.Mg-type alloys aged at 373K. ΔHV is the difference between each point of the value of hardness and the hardness of as HPT.ΔHV increases in aging treatment at 373K in Fig. 1. ΔHV is high in order of ex.Mg, ex.Mg-0.2%Cu and ex.Mg-0.7%Cu alloy. This is regarded as influence of amounts of the additional element. These results were same as the result on aging treatment at 343K

Fig.2 shows the bright field images of HPT-processed ex.Mg-0.7%Cu alloy aged at 373K for 6000 ks. Some fine-grains were observed, and a few dislocations in the crystal grain were observed. The typical needle-shaped precipitates of Al-Mg-Si alloys were not observed in the matrix. But precipitates were observed on grain boundary. β, S and Q phase were observed from the analysis of SAED ring pattern.

References

[1] Kenji Matsuda, Kosuke Kido, Tokimawa Kawabata, Yasuo Uetani, Susumu Ikeno: Journal of Japan Institute of Light Metals, Vol.53 (2003) 528-533.

[2] Zenji Horita : Journal of Japan Institute of Light Metals, Vol.60 (2010) 134-141.

Fig. 1: Age-hardening ability of HPT-processed ex. Mg-type alloys aged at 373K.

Fig. 2: The bright field image of HPT-processed ex. Mg-0.7%Cu alloy aged at 373K for 6000 ks.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2829 Carbides Transformation Phenomena in AISI M42 Steel

Godec M.1, Večko Pirtovšek T.2, Šetina Batič B.1
1Institute of Metals and Technology, Lepi pot 11, 1000 Ljubljana, Slovenia, 2Metal Ravne d.o.o., Koroška cesta 14, 2390 Ravne na Koroškem, Slovenia
matjaz.godec@imt.si

In convention AISI M42 tool steel production route the steel is cast and then forged in temperature window between 1100 °C to 1150 °C. At those temperatures metastable M2C carbide phase transforms in stable carbides as M6C and MC following the phase transformation M2C + matrix → M6C + MC. This is well known and accepted equation. In the present study SEM based EBSD and EDS analysis was applied to characterize the transformation of eutectic carbides during annealing of AISI M42 steel at the typical forging temperature of 1100 °C. Specific large eutectic carbide grains were characterized before and after annealing for different times. It was found that MC carbides appeared to form independently from the transformation of M2C to M6C. During M2C transformation some vanadium diffuses out of the newly formed M6C and enriches the surrounding matrix. Due to a higher concentration of vanadium in matrix, formation of vanadium rich MC carbides is favourable (Fig. 1). Results have also shown an interesting difference in carbide transformation reactions on the surface versus the bulk of the alloy, presumably due to the operation of different diffusion processes. Observing the transformation in the bulk shows that the transformation of M2C to M6C started on the M2C/matrix boundary and grew into inner region, while observing the carbide transformation phenomena on surface shows that the metastable carbides as well as the matrix is covered by a thin layer of M6C. It has been proven that in-situ analysis might not give the same results as you get it when the sample is annealed and cut in order to observe inner region.

Fig. 1: Transformation of carbides during annealing at 1100 ºC for 30 minutes. The arrows show the EBSD spot analysis performed at certain carbides.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2890 Understanding the role of silver in the NiTiAg4 SMA functional biomaterial: a microstructural and chemical insight

Álvares da Silva G. H.1, Otubo J.1
1ITASMART, Instituto Tecnológico de Aeronáutica, São José dos Campos, SP, Brazil
alvares@ita.br

The NiTi Shape Memory Alloys are known by two unique behaviors, both crystallographic dependent. Thermally or mechanically activated they are: the shape memory effect, and the pseudoelastic effect; the former, ensuring the recovery of the original shape even after large deformations and, the latter, the maintenance of a constant applied force in correspondence of significant displacements. Biomedical applications are an outstanding one of its alloys, so, a NiTiAg is believed to be a functional/antibacterial biomaterial. Samples of an Ni53,1Ti42,9Ag4%wt alloy was carefully sectioned and prepared metallographically by an specific procedure. Analysis of the microstructure alloy were performed in a TESCAN Vega3 SEM equipped with x-act SDD EDS detector and AZtec EDS analysis software – Oxford Instruments – installed in Scanning Electron Microscopy Laboratory of Instituto Tecnológico de Aeronáutica – ITA. In large columnar grains, Ag particles do not present any pattern distribution (Fig. 1a-b). As-cast microstructure show preferred precipitation at the interdendritic regions (Fig. 2a), in addition to a large range of particles sizes (Fig. 2b). Regarding the Ag content higher than the solubility limit and the process of solidification – e.g., upward in Figure 1a – is acceptable that interdendritic precipitation occurs. After solution treatment at 900°C for 1 hour, Ag particles re-arrange in a cellular-like structure, suggesting a high-temperature-austenite grain boundary segregation (Fig.3a). Etching to martensitic microstructure (Fig. 3b), one can see that there are left few austenitic phase, with no Ag surrounding it, as well as the same Ag particles arrangement. Accounting the alloy composition – equiatomic between Ni and Ti – a martensitic microstructure should be expected at the room temperature, however few austenite phase areas are identified after etching; the Ag content reduces the martensitic transformation temperature, yielding a martensitic/austenitic microstructure at room temperature. Inciting the formation of a surface oxide, EDS X-ray maps show the constitution of the scale (Fig.4). Ti, thermodynamically stronger to oxide formation, diffuses from inner regions to the surface of the sample, leaving back a Ni-rich layer, besides no Ag oxidation was observed; the scale formation in NiTiAg alloys behaves like any other NiTi alloy. Microstructural and chemical concluding remarks are: 1) the Ag precipitates has spherical shapes ranging from nano to micrometer; 2) beyond the influence of Ni and Ti content on the final microstructure of the alloy, Ag effectively lower the martensitic transformation temperature, then changing the microstructure of the alloy; 3) no Ag are lost in scale formation during heat treatment.

Grant 2012/15302-0, São Paulo Research Foundation (FAPESP)

Fig. 1: Fig. 1:Cross section of Ni53,1Ti42,9Ag4%wt. (a) Large columnar grains (stereoscope), and (b) SEM micrograph of the surface (a).

Fig. 2: Fig. 2: As-cast polished surface. (a) Interdendritic preferred segregation (BSE image), and (b) nano to micrometer spherical Ag precipitates.

Fig. 3: Fig. 3: Solution-treated sample (900°C, 1h). (a) Cellular-like structure, and (b) martensite + austenite microstructure and the cellular arrangement of Ag particles; etched.

Fig. 4: Fig. 4: Qualitative chemical data of the Ni53,1Ti42,9Ag4%wt and the scale formed during heat treatment. (a) Oxide and carbide layer (scale) –from middle to the left – and NiTi matrix containing Ag precipitates. (b) Ni, (c) Ti and (d) Ag X-ray maps.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2968 Effect of Cu/ Ag addition on the Two-Step Aged Al-Mg-Si Alloys

Matsuda K.1, Oe Y.2, Ikeno S.3, Nakamura J.4
1Graduate School of Science and Engineering for Research, University of Toyama, 3190 Gofuku, Toyama, 930-8555 Japan, 2Graduate school of Science and Engineering for Education, University of Toyama, 3190 Gofuku, Toyama, 930-8555 Japan, 3Hokuriku Polytechnic College, 1289-1 Kawaberi, Uozu, Toyama, 937-0856 Japan, 4Graduate school of Environmental Studies, Tohoku University, Japan
matsuda@eng.u-toyama.ac.jp

It has been known that Cu-addition or Ag-addition to Al-1.0mass%Mg2Si alloy (Al-Mg-Si-Cu alloy and Al-Mg-Si-Ag alloy) has higher hardness and elongation than those of Al-1.0mass%Mg2Si alloy [1]. It has been reported that the “negative effect” is observed for Al-Mg-Si alloy with two-step aging while the “positive effect” is observed for Al-Zn-Mg alloy with two-step aging [2]. Cu-addition or Ag-addition to Al-1.0mass%Mg2Si alloy shows higher hardness and elongation than that without Cu or Ag. In this study, the 2- step aging behavior in Al-Mg-Si alloy with Cu or Ag has been investigated by hardness test and TEM observation to understand the effect of Cu and Ag addition on precipitation.

The chemical composition of the alloys are Al- 0.7%Mg – 0.35%Si(base), Al -0.68% Mg -0.37%Si -0.35%Cu (0.35Cu), and Al -0.67% Mg -0.35%Si -0.33%Ag (0.35Ag) alloys (at%). Sheets of these alloys with 1 mm thickness for micro-Vickers hardness and 0.2mm for TEM observation were made by hot extrusion and cold-rolling. The specimens were solution heat treated at 878K for 3.6ks in an air furnace and quenched in chilled water. Two step aging was for The pre-aging treatment was performed at 343K for 600ks and followed by aging at 473K in oil bath. Thin specimens for TEM observation were prepared by electrolytic polishing method and microstructures were observed by Topcon EM-002B operation at 120kV.

The age hardening curves obtained for two-step aged these alloys showed typical age-hardening, and there was no remarkable difference for the peak-hardness of those alloys between with and without pre-aging. On the other hand, the hardness decreased once for the 0.35Ag and 0.35Cu alloys aged at 473K just after pre-aged at 343K for 600ks. This means that the 0.35Ag and the 0.35Cu alloys show the reversion. Fig. 1 shows TEM bright-field images obtained from base and 0.35Ag and 0.35Cu alloys peak aged at 473K without pre-aging. There are only needle-shape precipitates parallel to <100>Al direction. The number density of the precipitate in the 0.35Ag and 0.35Cu alloys were higher than that of the base alloy. Fig. 2 shows TEM bright-field images of three alloys peak aged at 473 K after pre-aged at 343K for 600ks. There are also only needle-shape precipitates parallel to <100>Al direction. The number density of the precipitate was higher in the base alloy peak-aged at 473K after pre-aged at 343K than the same alloy just aged at 473K. The classification of those precipitates using by HRTEM and its result will be reported in the presentation.

[1]K.Yokota, T.Komatubara, T.Sato, A. Kamio, J. Japan. Inst. Light Metals, vol.42 (1992)149-154.

[2]D.W.Pashley, M.H.Jacobs, J.T.Vietz：Philo.Mag, 16（1967）51．

This research was supported by JSPS, Grants-in-Aid for Scientific Research, (Scientific Research C, #23560893). A part of this research was also supported by The Light MetalEducational Foundation.

Fig. 1: TEM bright field images of (a)base, (b)0.35Ag and (c)0.35Cu alloys peak-aged at 473K.

Fig. 2: TEM bright field images of the (a)base, (b)0.35Ag and (c) 0.35Cu alloys peak aged at 473K after pre-aged at 343K for 600ks.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2974 Z-phase in Cr-Nb-N system studied by analytical electron microscopy

Buršík J.1, Kroupa A.1
1Institute of Physics of Materials, Academy of Sciences of the Czech Republic, Brno, Czech Republic
bursik@ipm.cz

Z-phase is a complex nitride with CrNbN stoichiometry and tetragonal crystal lattice [1]. It was first found in 1950s in austenitic steels and its precipitates have been attributed beneficial strengthening effects. A renewed interest in Z-phase launched in 1990s when it was detected in 9-12% Cr creep resistant ferritic steels for power plant applications [2-3]. Slowly precipitating Z-phase in ferritic steels consumes fine dispersion of (Nb,V)(C,N) particles and severely deteriorates long term creep properties. A precise knowledge of thermodynamic parameters of Z-phase is crucial for complete assessment of multicomponent systems and for reliable prediction of creep life.
In this work we study the Z-phase in model Cr-Nb-N alloys by means of analytical electron microscopy (SEM and TEM). Cr-Nb alloys with 1-14 at.% Nb were arc melted under the Ar+N2 atmosphere. The resulting content of N was 15-20 at.%. Samples were annealed for a long time at 1100 and 1300 °C to reach states close to thermodynamic equilibrium. Here we present the results obtained on 70Cr-14Nb-16N (at.%) sample annealed at 1100 °C for 48 days, i.e. the one after the longest annealing time where the Z-phase was found. A TESCAN LYRA 3XMU FEG/SEM×FIB scanning electron microscope and a Philips CM12 STEM transmission electron microscope (both equipped with an XMax 80 Oxford Instruments detector for energy dispersive X-ray (EDX) analyses) were used for microstructural studies.
Figure 1a shows microstructure with a distribution of bright coarse needles/plates and with a mixture of two phases between needles. A closer look (Fig. 1b) reveals fine two-phase microstructure also inside needles. This indicates the dissolution of (Cr,Nb)2N phase originally present in as cast alloy and its substitution by Z-phase and Cr. The stable equilibrium of the reported sample at 1100 °C is presumably Cr solid solution + Z-phase. X-ray analysis confirmed the Z-phase together with a bcc Cr phase and traces of (Cr,Nb)2N. EDX analyses in SEM support the conclusion that there are only two equilibrium phases; one of them (the dark regions in SEM micrographs) around 98Cr-1Nb-1N (at.%) and the second one close to 1:1:1 stoichiometry. N content in both phases was confirmed also by wave dispersion X-ray analyses. Elemental distribution is clearly shown on EDX maps in Figure 2. Identification of phases was accomplished by TEM inspection of thin lamella prepared by focused ion beam (FIB) in SEM (Figures 1c and 3).
[1] Jack DH, Jack KH: J. Iron Steel Inst. 209 (1972) 790-792.
[2] Buršík J, Merk N: In: Mechanical Behaviour of Materials at High Temperature, NATO ASI series, Vol. 15. Eds. Branco CM et al. Kluwer Academic Publishers (1996) 299-307.
[3] Strang A, Vodárek V: Mater. Sci. Tech. 12 (1996) 552-556.

The work was supported by the Czech Science Foundation (Project 14-15576S).

Fig. 1: SEM micrographs of Cr-Nb-N sample, signal of backscattered electrons. Low magnification overview (a), a detail of needle microstructure (b) and a scene from TEM lamella preparation (c).

Fig. 2: A detail of microstructure (SEM, signal of backscattered electrons) with elemental distribution maps measured by EDX.

Fig. 3: TEM micrographs of lamella prepared by FIB. The whole lamella at low magnification (a), a detail of two-phase microstructure (b) and SAD patterns of the two phases – Cr solid solution and Z-phase (c).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-2991 Influence of Sb on spheroidal graphite in ductile cast iron

Kuroki K.1, Matsuda K.2, Hara T.1,3, Ikeno S.4, Saikawa S.2
1Graduate School of Science and Engineering for Education, University of Toyama, 2Graduate School of Science and Engineering for Research, University of Toyama, 3Komatsu Castex ltd., 4Hokuriku Polytechnic College
m1371511@ems.u-toyama.ac.jp

Ductile cast iron that is developed in spheroidizing flake graphite by added Mg alloy is broadly used for autoparts and mechanical structure parts by it has sterling mechanical property. And, RE is added as spheroidizing agent. But, RE makes for formation of chunky graphite on heavy sectioned castings. Chunky graphite decrease mechanical property. Sb is added in order to avoid formation of chunky graphite. It has been reported that Sb brings forward to pearlite on matrix. But, it is not clear what the effect of Sb on shapes of graphite in ductile cast iron is. After development of ductile cast iron, there are many reports about the formation and growth mechanism of spheroidal graphite. In addition, it is not clear what the effect of Sb on shapes of graphite in ductile cast iron. Therefore, the aim of this study is the observation of spheroidal graphite in ductile cast iron to clarify the effect of Sb addition for formation of spheroidal graphite.　For scanning electron microscopy of graphite structure, the metallic matrix was dissolved away by deep etching in a 10% nitric acid solution. The cross-sectional TEM specimens of the spheroidal graphite were prepared by Focused Ion Beam System (FIB). The microstructures of spheroidal graphites were observed by SEM with S-3500H (Hitachi, Co., Ltd.) and TEM with Topcon EM-002B.Fig.1 shows the optical microscope image of base alloy and 0.097Sb. As a result of observation, chemical compound and porosity existed mostly in region of less than 5µm. Therefore, the image analysis performed in region of more than 5µm. As a result of image analysis, almost all the graphite particles present in the heats have circular in shape. Compared with the base alloy, graphite like a perfect circle increased in 0.097Sb. Fig.2 shows the particle size distribution of base alloy and 0.097Sb obtained by image analysis. Spheroidal graphite in 0.097Sb increased below 15µm in particle size distribution compared with base alloy and the average size of spheroidal graphite decreases. In SEM observation, the surfaces on spheroidal graphite were asperity in base alloy, while it was smooth in 0.097Sb. In TEM observation, cross-section of the central part in spheroidal graphite was observed the shape of a fan that it was framed blocks, while the internal structure was observed smoother with the spheroidal graphite in 0.097Sb. The domain below 5µm in nearly center of the spheroidal graphite was analyzed in the incidence orientation [0001] of graphite structure. The incidence orientation was analyzed that is [0001] Tilted 90 degrees in circumference of the spheroidal graphite.

Fig. 1: The optical microscope images of base alloy and 0.097Sb alloy.

Fig. 2: Particle size distribution of basic alloy and 0.097Sb.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3014 The microstructure characterization of unalloyed austempered ductile iron

Rajnovic D.1, Eric Cekic O.2, Labus D.1, Dramicanin M.1, Balos S.1, Sidjanin L.1
1Faculty of Technical Sciences, University of Novi Sad, Novi Sad, Serbia, 2Innovation Centre, Faculty of Mechanical Engineering, University of Belgrade, Belgrade, Serbia
draganr@uns.ac.rs

The Austempered Ductile Iron (ADI) is advanced material used increasingly for many tough engineering components in automotive, trucks, construction, agricultural and railway industry. The ADI is produced by austempering of ductile iron, where a unique microstructure - ausferrite (mixture of carbon enriched, stable, retained austenite and ausferritic ferrite) is obtained. By varying austempering parameters the different morphologies of ausferrite and amounts of retained austenite could be achieved. For that reason, characterization of microstructure and its influence on mechanical properties is of great importance.
In this paper, the ADI materials were produced by austenitisation at 900°C/2h and austempering at 300°C/1h, 400°C/1h and 400°C/3h. The microstructure was examined by “Leitz-Orthoplan” light microscope, while fracture mode by SEM JEOL JSM 6460LV, at 20kV. To identify microstructures a standard etching by 3% nital and heat tinting etching (heating in air, at 260°C/5h) was carried out.
The microstructure of ductile iron was mostly ferritic, with spheroidisation >90%, graphite amount 10.9%, nodule size 25÷30 μm and nodule count 150÷200 per mm2; while the mechanical properties were: Rm=473 MPa, Rp0.2=326 MPa, A=22.2%, KO=119 J. The ADI austempered at 300°C/1h posses high strength and low ductility (Rm=1513 MPa, Rp0.2=1395 MPa, A=3.8%, KO=68 J), while ADI austempered at 400°C/1h had low strength and high ductility (Rm=1042 MPa, Rp0.2=757 MPa, A=14.2%, KO=140 J). At 400°C/3h, strength of ADI remained at same level, but ductility decreases (Rm=1060 MPa, Rp0.2=780 MPa, A=9.8%, KO=95 J).
The difference in mechanical properties is due to different microstructures, Fig. 1-2. After 1 hour austempering, microstructure is fully ausferritic. When temperature increases from 300 to 400°C the ausferritic morphology is changing, from needle-like (Fig. 1a) to more plate-like (Fig. 1b), while amount of retained austenite increases from 16 to 31.4%. The 3h austempering time results in decrease of retained austenite to 24.1%, due to decomposition to bainite (mixture of ferrite and carbides), Fig. 1c, 2c. The occurrence of carbides (white in Fig. 2c) makes the material brittle and thus, it should be avoided. The effect of microstructure on fracture mode is presented in Fig. 3, where the fracture is changing from mix mode to fully ductile with increases of retained austenite amount, Fig 3a and 3b. On the contrary, the carbides presence, formed during prolonged austempering time, has opposite effect on fracture mode, i.e. fracture becomes fully brittle produced by quasi-cleavage mechanism, Fig. 3c.
Finally, based on presented results it could be summarized that the optimum mechanical properties of an ADI can be achieved upon achieving appropriate microstructure.

The authors gratefully acknowledge research funding from the Ministry of Education, Science and Technological Development of the Republic of Serbia under grant number TR34015.

Fig. 1: Microstructure of ADI: a) 300°C/1h, b) 400°C/1h, c) 400°C/3h; etched by 3% nital

Fig. 2: Microstructure of ADI: a) 300°C/1h, b) 400°C/1h, c) 400°C/3h; heat tinting (purple - reacted, carbon enriched retained austenite - the higher the carbon, the darker the purple color; light blue - unreacted, low carbon retained austenite; beige - ausferritic ferrite; white or cream - carbides, and dark blue - martensite)

Fig. 3: Fracture mode of ADI: a) 300°C/1h, b) 400°C/1h, c) 400°C/3h
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3017 Characterization of microstructure evolution in 9-12% Cr steels using SEM, HR-TEM, and EDS

Kafexhiu F.1, Žužek B.1, Vodopivec F.1, Godec M.1, Jenko M.1, Tuma J. V.1
1Institute of metals and technology, Ljubljana, Slovenia
fevzi.kafexhiu@imt.si

Microstructure evolution in terms of coarsening and redistribution of carbide and nitride precipitates in two grades of 9-12% Cr steels, X20CrMoV121 and X10CrMoVNb91 after long-term tempering was investigated. Samples for SEM imaging were prepared from three characteristic microstructures, namely base metal (α) and two heat affected zone (HAZ) regions, i.e., inter-critical (α+γ) and coarse-grained (γ) microstructures. All three microstructures were tempered for 6 months at 750 °C and 2 years at 650 °C. Applying an accelerating voltage of 15 kV, SEM images were taken at magnifications of 5000× and 10000×, which were found to be optimal for analysis of size and distribution of precipitates. Additional image enhancements were necessary for more reliable results before automatic image analysis. Besides this statistical SEM characterization, a more detailed microstructure examination was performed using HR-TEM and the EDS analysis. The main reason of using these techniques was the observation and characterization of very small carbide particles, which were impossible to characterize using the SEM at the magnifications applied. The EDS point analysis and mapping of carbide and nitride precipitates were performed, focusing on carbide and nitride forming elements such as Cr, V and Nb. The results of size, distribution and inter-particle spacing of carbide and nitride precipitates were then compared to the measured stationary creep, where important correlations were found and as such they can be used as a means of predicting the long-term behavior and the lifetime of studied materials.

Institute of metals and technology

Fig. 1: Steel X20CrMoV121 at the initial state (tempered martensite).

Fig. 2: Steel X20CrMoV121 after 1 year of tempering at 750 °C.

Fig. 3: Steel X10CrMoVNb91 at the initial state (tempered martensite).

Fig. 4: Steel X10CrMoVNb91 after 1 year of tempering at 750 °C.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3136 Hf-containing precipitates in the Al-Si-Mg-Hf aluminum alloy

Jia Z. H.1, Wang X. L.1, Huang H. L.1, Xing Y.1, Liu Q.1
1College of Materials Science and Engineering, Chongqing University, Chongqing, China
zhihongjia@cqu.edu.cn

Hf element has the same group as Zr and Sc elements in the elemental table. For the latter two, there are many research reports because of their efficient effect on the alloy properties by adding a small amount of them. Particularly, co-addition of Zr and Sc could form core-shell structure which is more stable and homogeneous distributed comparing to either individual addition. However, fewer studies on Hf addition into aluminum alloys can be found. A new precipitate were reported by one of the present authors, which shows nanobelt-like morphology with Si2Hf structure [1].

In this work, different Hf-containing precipitates in Al-Si-Mg-Hf alloys were found. The precipitates show various kinds of morphologies, such as nanobelt-like, rectangle, triangle, trapezoid etc.(Figure 1) The chemical compositions and crystal structures of these precipitates were investigated by STEM-EDS and HRTEM with diffraction techniques. It was shown that the formation of the Al-Hf or the Si-Hf precipitate is closely related to the heat treatment conditions. The precipitates formed at high temperature of 560oC were investigated in more detail. The distribution and the orientation relationship of the nanobelt-like precipitate were studied by FIB 3D-imaging reconstruction combined with EBSD and HRTEM as well. In addition, the growth mechanism of the nanobelt-like precipitate preferentially along one-dimension will be discussed.

Keywords: Aluminum alloy, Precipitation, TEM, FIB

Reference:

[1] Z.H. Jia, L. Arnberg, Nanobelts in multicomponent aluminum alloys, Appl. Phys. Lett., 2008, 093115.

Financial support from the National Natural Science Foundation of China with project No.51271209 is gratefully acknowledged.

Fig. 1: Figure 1 TEM investigations of the Hf-containing precipitates in the Al-Si-Mg-Hf alloy. (a) nanobelt-like precipate; (b-c) triangle and trapezoid shaped precipitates; (d) rectangle shaped precipitate.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3056 TEM investigation of nanosized interphase-precipitated carbides and fibrous carbides in a Fe-V-C steel

Yang J. R.1, Chen M. Y.1
1National Taiwan University, Taipei, Taiwan
jryang@ntu.edu.tw

In this work, a vanadium-containing medium-carbon steel was studied. Through the isothermal treatments, the nanometer-sized fibrous carbides adjacent to the nanometer-sized interphase-precipitated carbides were obtained. Thereby, TEM could provide direct orientation information about the transition of different precipitation modes [1-3]. The montage of TEM micrographs in Figure 1 was taken from the region covering the interphase-precipitated carbides, the fibrous carbides and pearlite in a ferrite matrix in the treated specimen. It is revealed that the interphase-precipitated carbides are intimately connected with the fibrous carbides in the ferrite grain. The direct crystallographic information for the transition from interphase-precipitated carbides to fibrous carbides within a ferrite grain is presented in Figure 2, which provides TEM bright-field and dark-field images with corresponding diffraction patterns. As analyzed in Figures 2d, e and f, both interphase-precipitated carbides and fibrous carbides are identified as VC carbide with NaCl-type crystal structure; both carbides are also within the same ferrite grain. Interphase-precipitated carbides adopt only one variant of Baker-Nutting orientation relationship (B-N OR) with respect to the ferrite: (0 0 1)VC || (0 0 1)ferrite and [-1 1 0] VC || [0 1 0]ferrite, as identified in Figure 2e. Fibrous carbides also exhibit only one variant of B-N OR with respect to the same ferrite: (0 0 1)VC || (1 0 0)ferrite and [ -1 1 0]VC || [0 1 0]ferrite , as identified in Figure 2f. The TEM results in Figures 2e and f clearly indicate that the transition from the mode of interphase precipitated carbides to that of fibrous carbides leads to a new selected variant via a 90° rotation of VC carbide crystal around [-1 1 0]VC. The results provide strong evidence to suggest that during the transition the carbide broad plane (0 0 1)VC|| (0 0 1)ferrite shifts from the position most closely aligned to the α/γ interface to that almost perpendicular to the α/γ interface.

References

1. D.V. Edmonds, J. Iron and Steel Inst., 210 (1972) 363.

2. A.D. Batte, R.W.K. Honeycombe, J Iron Steel Inst., 211 (1973) 284.

3. R. Okamoto, A. Borgenstam, J. Agren, Acta Mater., 58 (2010) 4783.

This work was carried out with financial support from National Science Council (Taiwan) under Contract NSC 102-2622-E006-032.

Fig. 1: A montage of TEM bright-field image taken from the region covering interphase-precipitated carbides, fibrous carbides and pearlite in ferrite matrix.

Fig. 2: (a) TEM BF image; (b) DF image (002)VC reflection of interphase-precipitated carbides; (c) DF image (002)VC reflection of fibrous carbides; (d) SADP revealing two variants of Baker-Nutting B-N OR; (e) interphase-precipitated VC carbides adopt 1st variant of B-N OR; (f) fibrous VC carbides adopt 2nd variant of B-N OR with the same ferrite.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3083 Three-dimensional characterization of Nanobelt-like Precipitates in Al-Si-Mg-Hf Aluminum Alloy

Wang X. L.1, Jia Z. H.1, Xing Y.1, Huang H. L.1, Liu Q.1
1College of Materials Science and Engineering, Chongqing University,Chongqing,China
20120901015@cqu.edu.cn

Nanobelt-like precipitates with high density were found in the Al-Si-Mg-Hf-Y aluminium alloy after high temperature heat treatment, and identified as Si2Hf structure by combining HRTEM, EDX techniques[1]. The alloy with these precipitates was found to evident improvement on creep property which is beneficial for application of automobile cylinder head.

In this study, we used FIB-SEM technique to investigate Si2Hf nanobelt-like precipitation to understand how the precipitations distributed in three-dimensional space. From Fig.1a, we could clearly observe the nanobelt-like precipitates growing along some certain directions. The close-up of the precipitates in red marked area from Fig.1a was analysed, and ten different growing orientation naonbelt-like precipitations were found as shown in Fig.1 b-d. In addition, many other precipitations with rectangle shape were also observed, and their chemical composition and crystal structure are under investigation.

Fig.2 shows the EBSD inverse pole figure (IPF) map of the same sample as described above, from which one can obtain the crystallographic orientation of the Al matrix in the studied region. By combining FIB 3D-reconstructed result with the EBSD result we could calculate the orientation index of these nanobelt-like precipitations in the Al matrix. The work is still going on.

Keywords: FIB, EBSD, precipitate, Al alloy

Reference:

[1] Z.H. Jia, L. Arnberg, Nanobelts in multicomponent aluminum alloys, Appl. Phys. Lett., 2008, 093115.

Financial support from the National Natural Science Foundation of China with project No.51271209 is gratefully acknowledged.

Fig. 1: Fig. 1 3D reconstruction of the Al-Si-Mg-Hf-Y aluminium alloy: (a) 3D visualization of the nanobelt-like precipitates, (b)-(d) close-up view of marked area from (a). The precipitates with different distributed directions were indicated by arrows with corresponding colours and numbers.

Fig. 2: Fig. 2 An inverse pole figure map of the Al-Si-Mg-Hf-Y alloy
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3084 Electron and Scanning Probe Microscopy Characterization of Shape Memory Alloy Micro-Pillars Array Along Nano-Compression Test

Gómez-Cortés J. F.1, San Juan J.1, López G. A.2, Jiao C.3, Nó M. L.2
1Dpto. Física de la Materia Condensada/Facultad de Ciencia y Tecnología/Universidad del País Vasco, Bilbao, Spain, 2Dpto. Física Aplicada II/ Facultad de Ciencia y Tecnología/Universidad del País Vasco, Bilbao,Spain, 3FEI, Achtseweg Noord 5, 5651 GG Eindhoven, The Netherlands
josefernando.gomez@ehu.es

Shape memory alloys (SMA) are materials that exhibit a martensitic phase transformation, leading two interesting thermo-mechanical properties, the shape memory and the superelasticity effects. The martensitic phase transformation is a first order and diffusionless crystal phase transition between two crystallographic phases, which can be induced thermally (shape memory effect) or by the application of stress (superelasticity effect). These thermo-mechanical properties make SMA very useful in a wide variety of applications like actuators and sensors [1].

The current device miniaturization tendency has led to growing interest in developing SMA for microelectromechanical systems (MEMS) motivated by its high work output per volume unit. This approach has been applied mainly using NiTi based alloys, with which some MEMS have already been fabricated [2]. Nevertheless, in recent years it was demonstrated that Cu-Al-Ni SMA have also notable properties at micro and nano-scale [3-5], opening a new promising possibility in this area.

In this work we present the microscopic characterization of the top shape of Cu-Al-Ni micro-pillars during a long superelastic cycling in nano-compression tests. The array of 4x4 micro-pillars studied were milled by Focused Ion Beam technique using a 3D-nanoprototyping program on a (100) Cu-Al-Ni single crystal. All pillars were tested in an instrumented nanoindenter with a sphero-conical diamond indenter. Electron and scanning probe microscopy images were taken along nano-mechanical tests (Figure 1). A residual plastic deformation was observed on the top of all tested pillars and its evolution and stabilization during de tests is explained and discussed. Fully recoverable and reproducible superelastic behaviour has been obtained during long term cycling tests above thousand cycles (Figure 2). These promising results open the door for designing potential applications doing use of 3D devices of SMA, which could be integrated in MEMS technology.

[1] K. Otsuka and C. M. Wayman in “Shape memory alloys”, ed. Cambridge University press, (1998).

[2] S. Miyazaki et al in “ Thin film shape memory alloys” ed. Cambridge University press, (2009).

[3] J. San Juan, M. L. Nó, and C. A. Schuh, Advanced Materials 20 (2008), p. 272.

[4] J. San Juan, M. L. Nó, and C. A. Schuh, Nature Nanotechnology 4 (2009), p. 415.

[5] J. San Juan, J. F. Gómez-Cortés, G. A. López, C. Jiao, and M. L. Nó, Appl. Phys. Lett. 104 (2014), p.011901

The authors thanks to the Spanish Ministry of Economy and Competitivity MINECO, project MAT2012-36421 and CONSOLIDER-INGENIO CSD2009-00013, the Consolidated Research Group IT-10-310 and the ACTIMAT-2013 from ETORTEK from the Basque Government. J. San Juan and M.L. Nó also acknowledge the support from EOARD through the Grant FA8655-10-1-3074. J.F. Gómez-Cortés thanks the Ph.D. Grant from the MINECO

Fig. 1: CuAlNi 4x4 micro-pillars array. a) SPM image of array before nano-mechanical tests, b.) SPM image of single pillar before nano-mechanical tests, c.) and d.) SEM and SPM of array and single pillar respectively after nano-mechanical tests

Fig. 2: Superelastic nano-compression tests performed on single pillar, after 1005 previous cycles, in load control mode at a loading rate of 250 mN s-1. The loading-unloading cycles for cycles 1006 to 1010 have been superimposed to show the reproducibility of the behavior
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3127 The Effect of Cu, Ag and Ge on Strength and Precipitation in a Lean 6xxx Alloy

Mørtsell E. A.1, Marioara C. D.2, Røyset J.3, Holmestad R.1
1Department of Physics, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway, 2SINTEF Materials and Chemistry, N-7465 Trondheim, Norway, 3Hydro Aluminium R&D Sunndal, N-6600 Sunndalsøra, Norway
eva.mortsell@ntnu.no

The main alloying elements in 6xxx aluminium alloys are Silicon and Magnesium. Lean alloys where the content of alloying elements is typically less than 1 atomic percent (at %), are often used in rolling and extrusion, yielding the material medium strength and more ductility than for higher alloying contents. The goal in this work has been to replace a small amount in at % of Si and Mg with an even lower amount of other elements such as Cu, Ag and Ge and keep the peak hardness at the same level or higher. Achieving this goal would open the possibility of producing alloys with improved extrudability. It is known from previous studies that additions of Cu, Ag and Ge improve material strength, and these elements enter the crystal structure of hardening precipitates [1-3].
Three alloys were investigated, defined as a lean reference, a denser reference and a Cu, Ag and Ge-added lean reference, with the total solute content higher than the lean reference, but lower than the dense reference. A final heat treatment of 4 hours of artificial ageing at 195 ⁰C produced conditions close to peak hardness for all alloys. It was found that adding Cu, Ag and Ge leads to an increased hardness compared to the lean reference, but the hardness was still lower than that of the denser reference. Precipitate type, size and volume fraction were determined for the three alloys using conventional transmission electron microscopy (TEM) and are presented in Table 1. Further characterization of precipitate types was performed using aberration corrected high angle annular dark field scanning transmission electron microscopy (HAADF-STEM). The distribution of the heavy Cu, Ag and Ge elements in the precipitate structure was determined.The denser reference alloy and the alloy with Cu, Ag and Ge additions are illustrated in Fig. 1 (a) and (b) respectively. The images are taken from similar thicknesses in the material and are therefore directly comparable. High number densities of relatively small needles (Fig. 1 (a)) produce a higher hardness than the lower number densities of larger needles (Fig. 1 (b)).

[1] C. D. Marioara, S. J. Andersen, T. N. Stene, H. Hasting, J. Walmsley, A. T. J. Van Helvoort and R. Holmestad, The effect of Cu on precipitation in Al-Mg-Si alloys, Philos. Mag., ISSN 1478-6435, vol. 87, 2007, 3385-3413.
[2] S. Wenner, C. D. Marioara, Q. M. Ramasse, D. M. Kepaptsoglou, F. S. Hage and R. Holmestad, Atomic-resolution electron energy loss studies of precipitates in an Al–Mg–Si–Cu–Ag alloy, Scripta Mater., ISSN: 1359-6462, Vol. 74, 92-95, 2014.
[3] R. Bjørge, S. J. Andersen et al., Scanning transmission electron microscopy investigation of an Al-Mg-Si-Ge-Cu alloy, Philos. Mag., ISSN: 1478-6443, DOI:10.1080/14786435.2012.700129, vol. 92(32), 3983-3993, 2012.

Hydro Aluminium and the Research Council of Norway are appreciatively recognised for their financial support through the BIA project no 219371. The authors also thank Birgitte Karlsen for performing the heat treatments and hardness measurements in this work.

Fig. 1: TEM micrographs of the microstructure after 4 hours of artificial ageing at 195 ⁰C in a) the denser reference alloy and b) the alloy containing Cu, Ag and Ge.

Fig. 2: TEM micrograph of a β" - like precipitate cross section in the alloy containing Cu, Ag and Ge, after 4 hours of artificial ageing at 195 ⁰C.

Fig. 3: Average cross sections, needle lengths and number densities for the alloys. The numbers are based on measurements of at least 100 cross sections and 1000 needle lengths from one grain in each condition.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3143 The study of Sn effect on recrystallization of hot rolled Ti-Nb-Ta-Sn biomedical alloy

Málek J.1,2, Hnilica F.1, Veselý J.1
1UJP PRAHA, Prague, Czech Republic, 2CTU in Prague, Faculty od Mechanical Engineernig, Department of Materials Science, Prague, Czech Republic
jardamalek@seznam.cz

Beta-titanium alloys are used as materials for bio-application. Their properties suitable for this purpose are low Young’s modulus, high strength, good corrosion resistance etc. These properties are attached with microstructure (phase composition, grain size etc.). These alloy exhibit very coarse grains in as-cast state. Therefore hot rolling (or hot forging) is desirable process step in order to obtain fine grained microstructure with desired mechanical properties. Fine grained microstructure is also suitable for subsequent cold deformation that may result in further improvement of mechanical properties. In this work hot rolling of-as-cast beta titanium alloys (type TiNbTaSn) was performed at temperatures between 900 and 1080°C in several steps. The total section reduction was about 50%. Subsequently rolled rods were solution treated at 850°C/0.5h/water quenched. The effect of Sn addition (6, 8 and 10 wt.% - marked 6Sn, 8Sn and 10Sn) has been studied by using light microscopy (LM) and electron microscopy (electron back scatter diffraction – EBSD).
The microstructure of hot rolled specimens consists mainly of very coarse and elongated grains of β-Ti (bcc) phase. These grains originate from as-cast grains. Inside these grains significant amount of low angle grain boundaries has been detected by EBSD analysis for all studied materials. This signs, that these coarse grains are deformed. Along their grain boundaries relatively fine grains occured in all studied specimens (e.g. Fig.1). This means that dynamic recrystallization took place during hot rolling on grain boundaries (places with higher accumulated deformation). Recrystallization was observed in solution treated 6Sn and 8Sn specimens, where fine equiaxed β-grains has been observed (Fig.2). In 6Sn specimen areas consisting of α”-martensitic phase are present (Fig3). These areas were not indexed during EBSD analysis (dark areas in Fig.2) This signs that MS (martensite start) temperature is lowered below room temperature by Sn addition, because no martensite has been observed in specimens 8Sn or 10Sn addition. In 10 Sn specimen both recrystallized and deformed grains has been observed (Fig.4). The recrystallization took place in this specimen only in highly deformed regions (original grain boundaries) during solution treatment, so the Sn hinders recrystallization in this type of alloys.

This work was supported by Technology Agency of the Czech Republic (project No. TE01020390).

Fig. 1: Inverse pole figures of hot rolled 6Sn specimen (RL)

Fig. 2: Inverse pole figures of solution treated 6Sn specimen (RL)

Fig. 3: Light micrograph of and solution treated 6 Sn specimen (RL)

Fig. 4: Inverse pole figures of solution treated 10Sn specimen (RL)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3237 HRTEM observation for precipitates structure of Al-Mg-Ge alloys aged at 473K

Kawai A.1, Matsuura K.1, Watanabe K.1, Matsuda K.2, Ikeno S.3
1Graduate school of Science and Engineering for Education, University of Toyama, 3190 Gofuku, Toyama, 930-8555, 2Graduate school of Science and Engineering for Research, University of Toyama, 3190 Gofuku, Toyama, 930-8555, 3Hokuriku Polytechnic College, 1289-1 Kawabuchi, Uozu, Toyama, 930-0856
m1371528@ems.u-toyama.ac.jp

The Al-Mg-Ge alloy is one of the age-hardening aluminum alloy after solution heat treatment.

It has been proposed that the age-precipitation behavior of Al-Mg-Ge alloy is different from that of Al-Mg-Si alloy according to our previous works about the microstructure on Al-Mg-Ge alloy over-aged at 523K1). There a few reports about microstructure on Al-Mg-Ge alloys observed by TEM for different aging temperature. But the age-precipitation structure of Al-Mg-Ge alloy has not been became clear. In this work, Al-Mg-Ge alloys observed age-precipitates were analyzed about their types of crystal lattice by HRTEM to understand the age-precipitation.

The alloy of Al-1.0mass%Mg2Ge was obtained by laboratory casting. This alloy sheet with 1.5mm thickness and 15mm width was made by hot extrusion. The specimens were solution heat treated at 873K for 3.6ks in an air furnace, quenched in chilled water. Aging treatment was done in oil bath at 473K. After the aging, specimens are polished by using two type electrolyte, perchloric acid: ethanol=1:9, nitric acid: methanol=1:3 and make specimens for TEM. The microstructure was observed using TOPCON EM-002B operated at 120kV.

Figure 1 shows HRTEM images obtained for Al-1.0mass%Mg2Ge alloy aged at 473K for 600ks.

The hexagonal network of the bright dots in this cross section of precipitate was observed with the spacing about 0.72nm.It is recognized as the b’-phase in Al-Mg-Ge alloy.

Figure 2 shows HRTEM images obtained for Al-1.0mass%Mg2Ge alloy aged at 473K for 600ks. The large precipitate is identified as the type-A precipitate in this alloy. It shows a rectangle network of 0.36nm and 0.69nm. This large precipitate are similar to the A-type precipitate in the Al-Mg-Si alloy with excess Si. It is found that the age-precipitates are b’phase and type-A precipitate in Al-1.0mass%Mg2Ge alloy over aged at 473K for 600ks.

References

1) K. Matsuda , T. Munekata , T. Kawabata , Y. Uetani and S. Ikeno: J. Inst. Light. Metals,56,11

Fig. 1: HRTEM images of obtained for samples aged for 600ks: b’ phase.

Fig. 2: HRTEM images of obtained for samples aged for 600ks: Type-A precipitate.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3267 Microstructural analysis of cold-forged duplex stainless steel using EBSD and TEM techniques

Fabrizi A.1, Bassan F.1, Bonollo F.1
1University of Padua - Department of Management and Engineering, Stradella S. Nicola, 3 - 36100 Vicenza, Italy
fabrizi@gest.unipd.it

Duplex stainless steels (DSSs) have been increasingly used for a variety of applications in marine construction, chemical industries and power plants, due to their excellent combination of mechanical property and corrosion resistance. It is well known that such good properties depend on the two-phase microstructure of a mixture of approximately equal amounts of austenite (γ) and ferrite (α). Generally, the steel cold forging process is a very cost effective metal forming method for mass produced parts and it includes several benefits with respect to other forging methods such as a reduction in waste material, energy saving and production of a net shape or near-net part with little or no machining requirements. In most production processes, in order to obtain high product rate, the initial billets usually are hot processed to forging parts by rough forge at the beginning and subsequently processed by finish hot-forge. Even so, the processing of DSS steels still requires special care due to the poor hot workability, which may lead in many cases to the presence of cracks if hot working parameters are not in the optimum conditions. However, there is still a lack of knowledge concerning the study on cold working characteristics of duplex stainless steels and it is possible to obtain a more fundamental understanding of microstructural behavior of duplex stainless steels under these conditions.

The aim of the present work was to undertake a detailed investigation of the microstructural evolution of a dual-phase α-ferrite/γ-austenite 2205 duplex stainless steel deformed by cold forging process. The cold forged parts were produced using a multi-station cold forming press and deformed through an uniaxial compression by three deformation passes with the same loading direction. Microstructural analyses were carried out by means of Electron Back-scattered Diffraction (EBSD) and Transmission Electron Microscopy (TEM). The results indicate that the applied deformation causes significant microstructural modifications in terms of grain size, misorientation distribution, grain subdivision and texture. The EBSD orientation maps qualitatively show the formation of deformation bands within the α and γ grains as the deformation is applied. Furthermore, from the grain boundary analysis, an increased density of Low Angle Grain Boundaries (LAGBs) appears, mainly in austenite grains, and the beginning of subgrains' formation is suggested. In addition, phases orientation relationships evolution between neighbouring grains of α and γ phases were analyzed as a function of the deformation. Moreover, in this paper, a direct comparison of images obtained by using EBSD and TEM techniques for the deformed microstructure is presented by inspecting exactly the same zone of the sample.

The authors are grateful to Zoppelletto Spa ( Torri di Quartesolo, Vicenza, Italy) for the material supplying and cold forging tests.

Fig. 1: a) EBSD grain map and inverse pole figures of b) ferrite and c) austenite phase of the as-received sample.

Fig. 2: a) EBSD grain map and inverse pole figures of b) ferrite and c) austenite phase of cold-forged sample. Compression axis is orthogonal to the investigated area.

Fig. 3: a) EBSD orientation map and b) phase map of cold-forged sample. c) TEM micrograph of the corresponding EBSD area; fine deformation twins are marked by a white arrows whereas red lines indicate the grain boundaries.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3269 Shell-wise growth of (Nb,Ti)(C,N) precipitates during cyclic cooling of microalloyed steel

Wojcik T.1, Kozeschnik E.1
1Institute of Materials Science and Technology, Vienna University of Technology, Vienna, Austria
tomasz.wojcik@tuwien.ac.at

The nucleation and growth characteristics of NbC- and TiN-precipitates, as well as complex Ti-Nb-carbonitrides in microalloyed steel, have been investigated numerous times. Due to different solubility products, TiN nucleates at higher temperatures than NbC in austenite. Both phases have the same fcc crystal structure with similar lattice constants and are able to form complex Nb-Ti-carbonitrides. In the present work, Nb-Ti-microalloyed steel samples are exposed to a cyclic cooling profile with nine quenching and reheating cycles between 1200°C and 800°C. The temperature oscillations have amplitudes of over 100K. The actual composition of the precipitates depends on the temperature and chemistry of the matrix. A TEM-investigation of samples treated in this way shows various particle populations. The largest ones, which nucleated at the first stages of the heat treatment, are platelet shaped with diameters of over 200 nm. The EDX and EFTEM characterisation shows an alternating structure of shells with local Nb- and Ti-enrichments. In HRTEM micrographs of these particles, local variations in the lattice parameter are measured. The number of shells corresponds to the number of applied thermal cycles. The smallest precipitates are spheroidal NbC nanoparticles with diameters up to 10 nm. The experimental setup is modelled with the thermo-kinetic software MatCalc, which allows for the simulation of the precipitate evolution in the course of this cyclic treatment. The calculated results are in good agreement with the experimental data.

The research leading to these results has received funding from the Research Programme of the Research Fund for Coal and Steel by European Commission. Grant Agreement number: RFSR‐CT‐2011-00008

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3305 simultaneous determination of carbon concentration and misorientation angle in bainitic ferrite laths via convergent beam electron diffraction

Tsai Y. T.1, Tsai S. P.1, Huang C. Y.2, Yang J. R.1
1National Taiwan University, Taipei, Taiwan, 2China Steel Corporation, Kaohsiung, Taiwan
f99527004@ntu.edu.tw

In this reserach, the carbon content of multiple bainitic laths are determined via accurate convergent beam electron diffraction method (CBED). The misorientation angles betweeen bainitc ferrite laths can also be determined simultaneously. A 0.1C-3Mn-2Si steel was used in this research, and carbide-free bainite was formed, which is currently widely studied due to potential mechanical behaviour as a result of TRansformation Induced Plasticity effect (TRIP). For the first time, misorientation angles between and carbon content in bainitic laths are gathered at the same time, allowing more clear understanding of the characteristics of bainite transformation. Misorientation angles are identified using Kikuchi lines and simulating software, while the carbon concentration was deduced CBED high order Laue zone (HOLZ) lines shift due to lattice parameter expansion.  It was found out that the misorientation angles between the adjacent bainitic ferrite laths generally has two type, either KS-variants related or in very small angle. The prior one is due to variant selection during austenite to bainite transformation, while the latter one is possibly due to plastic accomodation of austenite after the formation of previous bainitic ferrite. The carbon concentration of each lath was also individually determined, and results are in accord with previous researches using atom probe tomography (ATP), suggesting the reliability of CBED method. Simultaneous morphological, crystallographical and chemical informations can be obtained using CBED method.   

This work was carried out in the Advanced Steel Microstructural Center - Engineering Research Center (ASMC-ERC) in National Taiwan University, founded by China Steel Corporation and Companhia Brasileira de Metalurgia e Mineração. The authors also thank Dr. Hung-Wei Yen, Dr. Bian and Professor Hardy for invaluable advices and discussions.

Fig. 1: STEM image of multiple bainitic ferrite laths. The laths are seperated by M/A phase. Misorientation angle can be determined by Kikuchi lines fitting, while carbon concentration can be deduced by HOLS lines shift.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3338 TEM identification of precipitates in the as-welded P91 steel performed by the flux cored arc welding (FCAW) process

Marzocca A. L.1, Luppo M. I.2, Zalazar M.3
1Instituto Sabato, National University of San Martín – Argentina Atomic Energy Commission, Buenos Aires, Argentina, 2Materials Department, Argentina Atomic Energy Commission, Buenos Aires, Argentina., 3School of Engineering, National University of Comahue, Neuquén, Argentina.
almarzocca@gmail.com

The superior properties of the 9Cr1MoNbV steel (P91) at high temperatures depend on the maintenance of its microstructure throughout its service life: a high density of dislocation with M23C6 and MX type I (NbCN), type II (VN) or type III (“wings”) precipitates.
As precipitation strengthening is one of the most effective mechanism active in the P91 steel, the precipitates present in the fusion zone (FZ), coarse-grained (CGHAZ), fine-grained (FGHAZ) and intercritical (ICHAZ) heat affected zones generated by a FCWA process, with a rutilic slag wire as filler material, were identified by means of TEM on carbon extraction replicas, before to doing the PWHT.
Precipitates found in the base material (BM) were shown in a previous work. The M23C6 carbides (M = 55.4Cr-32.2Fe-11.5Mo-0.9V) were the major observed precipitates They were followed by isolated or clusters of VN precipitates with a chemical composition of 54.9V-22.3Cr-22.8Nb and “wings”.
The FZ exhibited inclusions (18.8Cr-2.9V-41Mn-34.2Ti-3.1Nb) and a fine distribution of elongated M3C (M = 16.6Cr-83.4Fe) formed as a result of autotempering during cooling (Fig. 1).
The CGHAZ subzone (Fig. 2) showed a reduction in the quantity and size of M23C6 carbides compared to the base material. Almost all had a spherical shape and the EDS analysis of these carbides showed that the most of them had M = 51.2Cr-36.6Fe-8Mo-4V, but few carbides with M = 56.2Cr-22.2Fe-21.6Mo were also detected. Equiaxed particles, approximately 90 to 290 nm in diameter, were identified as primary NbCN (M = 89.9nb-5.7V-3.2Cr-1.1Ti). The size and morphology suggest they are residual precipitates undissolved during the thermal cycle experienced by CGHAZ subzone. Minority, small spherical NbCN (M = 83.4Nb-6.9V-9.6Cr) and VN (M = 24.4V-49Nb-26.6Cr) precipitates were also observed.
The FGHAZ subzone M23C6 (M = 50.4Cr-34.5Fe-10.9Mo-3.5V) isolated or clusters as shown in Fig. 3. Then, the NbCN precipitates (M = 87.5Nb-6.4V-6.1Cr) were identified and a few “wings” with very little VN particles were observed.
In the ICHAZ subzone, M23C6 carbides with M = 57.1Cr-31.6Fe-9.3Mo-2V and M = 42.3Cr-38.1Fe-19.6Mo were observed. Then, all types of MX were identified: NbCN (M = 82.8Nb-9.7V-7.5Cr), VN (M = 61.8V-21.3Nb-16.8Cr) and elongated wings. (Fig. 4).
The M23C6 carbide was the major observed precipitate in all zones. According with thermodinamic calculations its dissolution is completed at ~900 ºC but it was observed even in the CGHAZ. Only traces of the VN were detected in the CGHAZ and in the few “wings” observed in the FGHAZ. A change in a chemical identity of the MX precipitates, from V-rich to Nb-rich was observed in the FGHAZ. The ICHAZ showed large M23C6 particles and all types of MX precipitates, included the NbCN which was not observed in the BM.

Fig. 1: TEM micrograph of a carbon replica extracted from the FZ.

Fig. 2: TEM micrograph of a carbon replica extracted from the CGHAZ.

Fig. 3: TEM micrograph of a carbon replica extracted from the FGHAZ.

Fig. 4: TEM micrograph of a carbon replica extracted from the ICHAZ.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3413 Comparison of the 3D Calcium and Aluminium distribution in standard and ECO-Mg alloys

Wagner J.1, Schroettner H.2, Rattenberger J.1, Mitsche S.2, Panzirsch B.3
1Graz Centre for Electron Microscopy, Graz, Austria, 2Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Graz, Austria, 3Austrian Foundry Research Institute, Leoben, Austria
julian.wagner@felmi-zfe.at

Due to their corrosion resistance, excellent mechanical properties, low density (one-quarter that of steel) and formability, magnesium-aluminium alloys are widely used. Especially automotive industry applications make many efforts to increase the fuel efficiency, but the amounts of magnesium per vehicle are very small in comparison to other materials such as steel, aluminium and plastics and therefore as one alternative step magnesium developed as a serious candidate for light weighting. And even for artificial replacements and biomedical implants magnesium alloys are considered to be the new basic material because of its good biocompatibility and biodegradation.

Continually improved and pushed by an ecological friendly awareness the early magnesium-aluminium alloys turned into the Environment COnscious magnesium alloys (ECO Mg). Some examples of alloys in use are AM50, ECO AM50, AZ91 and ECO AZ91. However, the properties of all are attributed to the combined effects of chemistry, heat treatment and microstructure. Especially the diffusion behaviour of aluminium in magnesium and in case of an ECO alloy the presence of calcium is important (Figure 1 and 2). But also knowledge of temperature caused the θ´´ to θ´ phase transition is essential. Optimizing the functionality of materials often depends on a precise control of the size, shape, crystal structure and composition of the material being synthesized. Many analysing methods were established in order to characterise solids in an appropriate way. Among several investigative tools and techniques like electron back scatter diffraction (EBSD) and transmission electron microscopy (TEM) the 3D micro-structural characterization of four different alloys (AM50, ECO AM50, AZ91 and ECO AZ91) were carried out on the FEI Nanolab Nova200 dual beam focused ion beam (DB-FIB) equipped with an energy dispersive X-ray detector (10 mm²) Quantax 400 system from Bruker using the Esprit software version 1.8.5. The serial sectioning thickness was selected to be 100 nm. Final data visualization was performed using the Avizo Fire software. The results give a three dimensional comparison of the differences in the elemental distribution, the chemical composition of the precipitates and the cell volumes. As an example Figure 3 and 4 shows a quantitative line scan of ECO AZ91.

The authors gratefully acknowledge the Austrian Research Promotion Agency (FFG) for the financial support (PN 839958).

Fig. 1: Aluminium distribution in ECO AZ91

Fig. 2: Calcium distribution in ECO AZ91

Fig. 3: Scan direction

Fig. 4: Quantitative elemental distribution
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3421 Transmission electron microscopy and X-ray diffraction studies of Ni-W/ZrO2 metal-matrix composites

Indyka P.1,2, Beltowska-Lehman E.2, Kania B.2, Jany B. R.3
1Jagiellonian University, Faculty of Chemistry, Krakow, Poland , 2Polish Academy of Sciences, Institute of Metallurgy and Materials Science, Krakow, Poland , 3Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland
paulina.indyka@uj.edu.pl

          Studies on preparation and microstructure-property relationship of particle reinforced metal-matrix composite (MMC) coatings are of fundamental importance mainly due to the potential breakdown of classical scaling laws and the accompanying demands for advanced materials of physical properties in the nanostructured limit. With the emergence of nanostructured materials, electrodeposition techniques have been widely applied to obtain a variety of new nanomaterials, including nanocomposites of enhanced mechanical properties [1]. Such MMC coatings could be relevant for many technological applications like thermo-resistant, hard-wearing materials [2] and as important alternatives to hard chromium coatings. Electrodepositited composites containing hard ceramic particles incorporated into a Ni-W alloy matrix has been the subject of few papers [3,4], but none was related to the Ni-W/ZrO2 system.

          The sample structural features were investigated using X-Ray diffraction and high-resolution transmission electron microscopy (HRTEM), high-angle annular dark-field scanning transmission electron microscopy (HAADF STEM), in combination with electron energy loss spectroscopy (EELS) and energy dispersive X-ray spectroscopy (EDX) at nanometer scale.

          A detailed characterization on the structural and chemical composition of ZrO2 nanoparticles embedded in a Ni-W supersaturated alloy matrix of about 10 nm grain size is presented. As seen in Fig. 1. deposited composite material was crack free, compact, and well adherent to the steel substrate. We report structural and compositional inhomogenities found in the Ni-W matrix, where the local changes in Ni-W alloy composition were found to reach 10 wt%. EDX results exemplified in Fig. 1a show tungsten segregation/enrichement in between the ceramic nanoparticles regions, being consistent with complementary EELS data. In addition, changes in the O K edge was observed at the surface of the ZrO2 particles.

          These observations, complemented by microhardness, wear and corrosion resistance tests, allow for detailed understanding of tungsten-based alloy and composite systems functional properties.

[1] Z. Zhang, D.L. Chen, Scripta Materialia 54 (2006) 1321–1326.
[2] C. Kerr, D. Barker, F. Walsh, Transaction of the Institute of Metal Finishing 78 (2000) 171–178.
[3] B. Han, X. Lu, Surface & Coatings Technology 203 (2009) 3656–3660.
[4] E. Beltowska-Lehman, P. Indyka, A. Bigos, M. Kot, L. Tarkowski, Surface & Coatings Technology 211 (2012) 62–66.

Funding from the Polish National Science Centre under grant number NCN 2011/01/B/ST8/03974, 2011 – 2014 is acknowledged.

Fig. 1: In the background SEM BSE overview image of the Ni-W/ZrO2 MMC deposited on a steel substrate,(a) STEM HAADF overview image and EDX color maps of the system, showing the Zr, O, Ni and W distribution, notice different Ni/W repartition, (b) EELS signal of Ni (Ni-L3,2 edge 855 eV), W (W-M5,4 edge 1809 eV) and Zr (Zr-L3,2 edge 2222 eV).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3457 Explanation of observed unusual X-ray Kossel reflection doubling at the ferromagnetic shape memory alloy Co-Ni-Al

Langer E.1, 2, Däbritz S.1, Potapov L.1, Krátká K.1, Kopeček J.3
1Technische Universität Dresden, Institut für Festkörperphysik, 01062 Dresden, Germany, 2Technische Universität Dresden, Institut für Halbleiter- und Mikrosystemtechnik, 01062 Dresden, Germany, 3Academy of Sciences of the Czech Republic, Institute of Physics, Na Slovance 2, 18221 Prague, Czech Republic
langer@physik.tu-dresden.de

Among the ferromagnetic shape memory systems the metallic compound Co-Ni-Al shows particularly advantageous properties such as good oxidation resistance, low density and appreciable ductility at room temperature, therefore current research activities are focussing on a better understanding of the structure and the behaviour of this alloy.

In the present work austenitic single crystals with nominal composition Co38Ni33Al29 were studied by means of X-ray Kossel diffraction within a scanning electron microscope. The samples were grown in [100] direction by the Bridgman method and wet-polished using conventional metallographic techniques. Fig. 1 shows a SEM micrograph with two crystal phases present in the sample, the matrix B2-β-phase with filigrane precipitates of A1-γ-phase.  In the X-ray diffraction studies, a further unusual doubling of X-ray Kossel reflections was observed close to the phase boundary besides the Co-Kα and Ni-Kα (111) reflections in each case as can be seen in Fig. 2. This doubling could be explained by an abnormal overlapping of Kossel reflections of the two different crystal phases whilst allowing to determine precisely the orientation relationship as Kurdjumow-Sachs:

                                                         (111)A1 || (110)B2, [110]A1 || [111]B2.

      

Moreover, remarkable dark regions (lower backscatter coefficient η due to a channeling effect) between the B2 matrix and the γ-phase were seen using backscattered electrons (see Fig. 1). On the basis of Kossel investigations it may be concluded that this structure along the boundary is connected to the measured exact plane orientation relationship between the phase and the matrix: (111)A1 || (110)B2 (misorientation within a few tenths of a degree) and therefore reveals areas of excellent crystal quality with very low dislocation density.

The authors are grateful to M. Melo for his experimental help.

Fig. 1: SEM micrograph showing the different crystal phases using backscattered electrons.

Fig. 2: X-ray Kossel diffraction pattern of the ferromagnetic shape memory alloy Co-Ni-Al at the matrix close to the phase boundary. Overlaps of two Kossel reflection systems between the A1 and the B2 crystal phases can be seen.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3474 The influence of the number of constrained groove pressing cycles on the microstructure of twin-roll cast aluminum alloy.

Cieslar M.1, Bajer J.1, Poková M.1, Zimina M.1, Zrník J.2
1Charles University in Prague, Faculty of Mathematics and Physics, Prague, Czech Republic, 2COMTES FHT a.s., Dobřany, Czech Republic
cieslar@met.mff.cuni.cz

Mechanical properties of metallic materials are very sensitive to the grain size. A reduction of the mean grain size increases the yield stress and the tensile strengths according to the Hall-Petch relationship. Severe plastic deformation (SPD) is frequently used for the grain refinement of metals and alloys [1]. The basic principle of the SPD process consists in inducing the extremely high plastic strain into the material resulting in a substantial grain refinement and improved strengths. Among them three methods are the most suitable for processing of continuous UFG strips. They are the accumulative roll-bonding [2], continuous confined strip shearing [3] and constrained groove rolling or its discontinuous version - constrained groove pressing (CGP) [4]. In our study the mechanical properties and stability of the microstructure of aluminum alloy after 1 – 3 CGP steps were studied using the microhardnes measurements and light and electron microscopy characterization. One of the main benefit of the CGP technique is the improvement of the mechanical properties withoutanydimensional changes of the material. The homogeneity and final thermal stability of the grain structure in aluminum alloys depends on the processing temperature, number of CGP cycles but also on the grain size of the initial material and the size and distribution of coarse primary particles which are generally present in the ingot cast and cold-rolled sheets. Therefore the thermal stability and homogeneity is improved in materials with fine particles and small grain size which is typical for continuously twin-roll cast (TRC) aluminum alloys. Microhardness mappings were done on the material with ultra-fine grained structure prepared by CGP of twin-roll cast AA3003-based aluminum strips. A commercial TRC alloy modified by a small addition of Zr was used in the study. Light optical microscopy and electron microscopy were used for the microstructure investigations.

[1] R. Z. Valiev, A. V. Korznikov, and R. R. Mulyukov, Mater. Sci. Eng. A168 (1993), p.141.

[2] Y. Saito, H. Utsunomiya, N. Tsuji, T. Sakai. Novel ultra-high straining process for bulk materials – development of the accumulative roll-bonding (ARB) process. Acta Mater. 47 (1999) 579-583.

[3] J.-Ch. Lee, H.-K. Seok, J.-H. Han, Y.-H. Chung: Controlling the textures of the metal strips via the continuous confined strip shearing (C2S2) process. Materials Research Bulletin 36 (2001) 997-1004.

[4] J.W. Lee, J.J. Park: Numerical and experimental investigations of constrained groove pressing and rolling for grain refinement. J. Mater. Process. Technol. 130-131 (2002) 208-213.

Acknowledgment: The authors are grateful to the financial support from the Czech Science Foundation under the project P107-12-0921 and to the grant SVV-2014-269303.

Fig. 1: Light optical micrograph of the twin-roll cast AA3003 aluminum alloy after 3 cycles of constrained groove pressing.

Fig. 2: Distribution of microhardness HV 0.1 (in MPa) in the specimen from Figure 1.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-3537 Effects of arcing on the Microstructure, Morphology and Photoelectric Work Function of Ag-Ni Electrical Contacts.

Bouchou 1 Akbi 2
Faculty of Physics, University of Algiers (USTHB), Algiers, Algeria 1 Faculty of Sciences, University M'hammed Bougherra of Boumerdes, Boumerdes, Algeria 2
aissabouchou@yahoo.fr

Materials used to provide both electrical current flow and creation of electric arc in low voltage switchgear devices such as contactors, relays, circuit breakers and switches, are made of silver based alloys, (Ag-CdO (88/12), Ag-ZnO (92/8), Ag-SnO2 (88/12), Ag-Ni (70/30), Ag-Ni (60/40) and Ag-W (50/50)). Properties both in terms of mechanics and thermodynamics, and from point of view of electronic emissivity remain very poorly known for these complex aggregates where oxides are more or less dispersed in the silver matrix. Currently new contact types with increased longevity were marketed but manufacturers do not have enough knowledge about this improvement. Unfortunately, one must note the lack of rational scientific explanations on improving the performance of new materials; new switches currently on the market perform thousands of operations under load without failure. The aim of this work is to define, measure and explain from both theoretical and experimental point of view, the phenomena of electron emission for the following material (Ag-Ni (60/40)), pure silver is the material reference. To better define this work, one can say that we will be able to identify at the end of this study the bases for a better understanding of the electron emission at the surface of the alloy formed from the silver matrix; thus, the role played by the metal alloy nanoparticles (Ni, W) will be more clearly identified.

The observations made in the present investigation reveal the multiple layer structure of the silver-nickel alloys. Indeed, segregation of nickel interfaces of a solid solution Ni-Ag is important. A multilayer model taking into account the strong intergranular and volume segregation gives a good interpretation of the results. The observations with the scanning electron microscope and analyses by energy dispersive X-ray spectroscopy (EDS) have shown the evolution of the surface composition for contact material after heating treatment.

Fig. 1: EDS line scans of two representative points (one bright and one dark) for the central contact surface of the conditioned cathode (500 arcs), Ag-Ni (60/40). SEM magnification x 2000.

Fig. 2: EDS line scans of two representative points (one bright and one dark) for the peripheral contact surface of the conditioned cathode (500 arcs), Ag-Ni (60/40). SEM magnification x 2000.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5706 Analysis of nanocrystals embedded in Co3Ti made amorphous by severe plastic deformation

Noisternig S.1, Ebner C.1, Gammer C.2, Rentenberger C.1, Gspan C.3, Hofer F.3, Karnthaler H. P.1
1University of Vienna, Physics of Nanostructured Materials, 1090 Vienna, Austria, 2National Center for Electron Microscopy, LBNL, Berkeley, California, USA , 3TU Graz, Institute of Electron Microscopy and Nanoanalysis, 8010 Graz, Austria
stefan.noisternig@univie.ac.at

Pure components were used to make the L12 long range ordered intermetallic Co3Ti alloy that was homogenised for 100 hours at 950 °C to dissolve dendritic structures. To accomplish an amorphous phase the alloy was severely plastically deformed (SPD) by high pressure torsion at 4 GPa with 80 rotations. Methods of transmission electron microscopy (using a Titan operatingat 300 kV) were applied to analyse the specimens. After SPD the specimens contain both, crystalline and amorphous regions. In the amorphous phase nanocrystals are encountered. Fig.1 shows an annular dark field (ADF)image with nanocrystals having dark or bright contrast corresponding to the given diffraction conditions. It is the aim of this study to find out whether the nanocrystals contain retained crystalline structures or if they were formed during the SPD process.

Local chemical variations in the specimens are studied by high angle annular dark field (HAADF) images and electron energy loss spectroscopy (EELS). Fig.2 shows a HAADF image of the amorphous region with embedded nanocrystals (cf. Fig.1). The contrast variations in the HAADF image indicate chemical variations in the specimen. To analyse the chemical composition of the nanocrystals EELS line scans were acquired for the nanocrystals and the surrounding matrix. The elemental concentrations of Ti, Co and O are deduced from the integrated EELS intensities between different positions; their concentration profiles are shown in Fig.3. The evaluation leads to the result that the nanocrystals exhibit a higher ratio of Ti / Co than the surrounding amorphous phase. The concentration of Ti at the positions of the nanocrystals is about 15% higher than the one in the surrounding amorphous phase. This is an indication that the embedded nanocrystals contain Laves phases (Co2.1Ti0.9 or Co2Ti) and are not retained crystalline material. Therefore, we conclude that the nanocrystals are formed in the amorphous phase during SPD by a dynamic non-polymorphic crystallisation process.

In addition, a few nanoparticles revealing pronounced dark contrast in the HAADF images were encountered. As shown in Fig.4 the concentration profiles indicate that they contain preferentially Ti and O. Their Ti concentration is about 50% higher than the one of the surrounding amorphous phase. These nanoparticles are therefore interpreted as titanium oxide particles (cf. [1]).

[1] ''Fluctuation electron microscopy of an amorphous-crystalline composite material'', Ebner C., Gammer C., Karnthaler H.P., Rentenberger C., this conference

We acknowledge support by the Austrian Science Fund (FWF):[I1309, P22440, J3397] and C.G. by the National Center for Electron Microscopy, Lawrence Berkeley Lab, supported by the U.S. Dept. of Energy under Contract # DE-AC02-05CH11231.

Fig. 1: Annular dark field image of a HPT deformed Co3Ti specimen showing nanocrystals embedded in an amorphous phase.

Fig. 2: HAADF image of the specimen area shown in Fig.1. The outlined regions correspond to segmented nanocrystals in the ADF image.

Fig. 3: Concentration profiles deduced from an EELS line scan across a nanocrystal showing a higher amount of titanium and a lower one of cobalt at the nanocrystal.

Fig. 4:   Concentration profiles deduced from an EELS line scan across a nanoparticle showing both, a high titanium and oxygen concentration.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5730 Microstructure of M152 rotor steel

Ramar A.1, Oruganti R.1, Nalawade S.1, Swaminathan S.1, Vishwanath T.1, Sivakumar V.1, Mastromatteo F.2, Giovannetti I.2
1GE Global research , GE Technology center, Bangalore, India, 2GE O&G, Nuovo Pignone, via F. Matteucci 2, Florence 50127, Italy
amuthan.ramar@ge.com

M152 steel belongs to the class of Fe-12Cr (12 wt.% Cr) martensitic stainless steels. It has a good combination of properties which include good ductility, high strength, uniform properties through thick sections and favorable strength at temperatures up to about 480°C [1]. Martensitic stainless steels possess a body-centred tetragonal (bct) crystal structure after quenching from an austenite phase (face-centred cubic structure) at high temperature. The chromium content is generally in the range of 10.5 to 18.0 wt.%, and carbon content may exceed 1.2 wt.%. The actual chemical composition of the investigated alloy is presented in Figure.3. The M152 alloys finds application in steam turbine and gas turbine parts where their outstanding fracture toughness and good oxidation resistance up to approximately 425°C [1,2] come into play.

This paper describes the microstructure in an M152 rotor that had seen significant service in a power generation turbine. Detailed microstructural analyses were performed using the JEOL 2011 high resolution TEM equipped with a LaB6 gun and a high resolution pole piece. The TEM samples were prepared using standard electro-polishing technique. The steel showed microstructure typical of tempered martensitic steel with body centered cubic structure as shown in Figure.1. The precipitates present in this steel and their sizes are shown in Figure.2 and 4. Ni was observed to form a thin austenitic film in between two neighboring laths which has been proposed as a mechanism to increase the fracture toughness of the alloy [2]. Cr rich precipitates are homogenously distributed in the matrix with sizes ranging from 200 – 600nm. In addition a high density of MC (M=W, Mo, V) and M2C (M=Cr) precipitates were also observed. Both the MX and M2X are oriented either parallel or perpendicular to g (200) in the {001} zone axis of the matrix. They are found pinned to dislocations indicating therefore that they are strong contributors to creep strengthening.

References:

1 Davies JR (Ed). Stainless steels, ASM Specialty Handbook, ASM International, Ohio, 1994.

2. J.W. Schinkel, P.L.F. Rademakers, B.R. Drenth, and C.P. Scheepens: Ferritic Steels for High-Temperature Applications, ASM, Metals Park,OH, 1982, pp. 131-49.

Fig. 1:  Structure typical of martensitic steel with laths boundaries

Fig. 2: Types of precipitate observed (a) MX; (b) M2X; (c) Ni rich; (d) Cr-O;(e) SiO2; (f) Ni rich austenite film (g) Cr rich precipitates. 

Fig. 3: Alloy composition of M152 steel

Fig. 4: Precipitate size distribution
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5737 Study of fine scale microtexture features associated with globularization in a near β titanium alloy using precession electron diffraction (PED) assisted orientation electron microscopy (OIM)

Balachandran S.1, Sharath K.2, Banerjee D.1,2
1Materials Engineering, Indian Institute of Science (IISc) Bangalore, India, 2Advanced facility for microscopy and microanalysis, Indian Institute of Science (IISc) Bangalore, India
shanoob.b@gmail.com

Commercial titanium alloys undergo a series of thermo mechanical processes and subsequent heat treatments at the high temperature β (BCC) phase and lower temperature α + β (HCP+ BCC) phase to achieve desired properties. The microstructure and global texture is heavily influenced by the processing parameters such as temperature, strain and strain rate. The widmansttaten α (HCP) laths form by slow cooling of β or isothermal aging at α + β phase maintaining a burgers orientation relationship (BOR) with β phase given by (1-10)β || (0001)α and <111>β || <11-20>α. Upon thermo mechanical processing and subsequent heat treatment, the lath structure transforms to equiaxed, a process known as globularization. The globularization does not lead to a completely random texture and many a times, we may retrieve the initial α orientation with certain spread even after heavy deformation. In addition, the microtexture associated with globularization is an interplay between the α and β phases and recrystallization in beta can happen in combination with α to form special angle, epitaxial grain boundaries in both α and β phases as suggested by some of previous studies in this direction [1][2].
In the present work, we incorporate transmission electron microscopy (TEM) based orientation electron microscopy (OIM) assisted by precession electron diffraction (PED) to investigate the triggering points of recrystallization events having special angle boundaries in both α and β phases, at resolutions beyond conventional scanning electron microscopy (SEM) based electron backscattered diffraction (EBSD). A 200 KV FEI T20 S-TWIN microscope coupled with Nanomegas-ASTAR precession and data collection system was used for this study. Events of epitaxial recrystallization of fine α associated with special angle boundaries in β around the alpha was frequently observed. Two interesting examples are shown here. In Figure.1, the recrystallized α maintains a common <10-11> pole with the other α with a special β grain boundary evolving from α / α interface. In Figure.2, a fine β layer is observed around the globularizing α laths maintaining BOR with α and in special angle boundary with parent β grain, suggesting altogether a new mechanism for α globularization.
Many more of the above discussed events were observed in our study. The resulting global texture is a sum of these discontinues recrystallization events and the deformation texture associated with parent β and α phase. We have observed consistently that the original BOR is restored by these events.

1. C. Cayron, Scripta Materialia. 59, 570 (2008).

2. E. Lee, R. Banerjee, S. Kar, D. Bhattacharyya, and H. L. Fraser, Philosophical Magazine. 87, 3615 (2007).

Laboratory for mechanical testing, Materials Engineering, IISc Bangalore

Fig. 1: Epitaxial recrystallization in α phase associated with special angle grain boundaries in β and related 10-11 and 110 pole figures with common poles.

Fig. 2: Globularization in α associated with formation of fine β layer, which is in BOR with α and maintains special angle relationship with parent β grains
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5773 Application of Electron Backerscattering Diffraction: Qualification of granular bainite in Nb-Mo containing low carbon bainitic hot rolled strips

Huang B. M.1, Chang Y. L.1, Chien Y. C.1, Yang J. R.1, Yen H. W.1, Huang C. Y.2
1Department of Materials Science and Engineering, National Taiwan University, Taipei, Taiwan, ROC, 2Steel & Aluminum Research & Development Department, China Steel Corporation, Kaohsiung, Taiwan, ROC
r95527042@ntu.edu.tw

In this work, three experimental steel strips involved the investigation; they had the same base composition of 0.05C-1.7Mn-0.08Nb (wt%), one without Mo addition and the other two with 0.1 and 0.3 Mo (wt%), respectively. The steel strips were fabricated by a combined process of controlled-rolling and accelerated-cooling. Through microstructural characterization of optical microscopy, scanning electraon microscopy, and transmission electron microscopy, it was found that the microstructure consisted of allotriomorphic ferrite and granular bainite in all hot rolled strips. The morphology of granualr bainite is considered as similar as that of allotriomorphic ferrite in observation of SEM images. Therefore, the effect of Mo on the suppression of allotriomorphic ferrite in Nb containing hot rolled strips will not be resolved unless the character of granular bainite can be clearly identified and then quantitated in SEM images. The subunits within bainite sheaves were studied and appeared the high gradient of misoreintation, 3°/10μm with "point to origin" linescan in electron backscattering diffraction (EBSD) orientation mapping.1 The gradient of misoreintation is definely different from that of allotriomorphic ferrite.2 Thus, present study aims to utilized the difference to recognize the granular bainite in sequence in EBSD orientation mapping then complete the qualification of phase balance in all hot rolled strips. It is surprised to find that only high addition of Mo, 0.3wt% can effectively raise granular bainite from 64 Vol% to 80 Vol% in Nb-Mo containing hot rolled strips.

[1]. E. Keehan, L. Karlsson, H.K.D.H. Bhadeshia and M. Thuvander. Electron backerscattering diffraction study of coalesced bainite in high strength steel weld metals. Mater. Sci. Technol. 2008; 24: 1183-1189.
[2]. M. Calcagnotto, D. Ponge, E. Demir, and D. Raabe. Orientation gradients and geometrically necessary dislocations in ultrafine grained dual-phase steels studied by 2D and 3D EBSD. Mater. Sci. Eng. A. 2010; A527: 2738-2746.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5779 Microstructural Investigation of the Nickel-Based Superalloy ATI 718Plus

Whitmore L. C.1
1National Institute of Research & Development for Technical Physics, 47 Mangeron Boulevard, Iasi, ROMANIA
lw@rubyway.org

The microstructure of thermally-aged nickel-based 718Plus superalloy is investigated using transmission electron microscopy (TEM). Samples are annealed at 980 °C for 30 minutes, quenched to room temperature, and then isothermally aged at 788 °C for a range of aging times 1, 5, 10, 25 and 50 hours. The size, number density and phase fraction of gamma-prime precipitates are measured using dark-field TEM. The coherent gamma-prime phase gives rise to super-lattice reflex spots additional to the fcc diffraction pattern of the nickel-base matrix; and using these supplementary spots it is possible to uniquely image the population of gamma-prime precipitates. Using energy-dispersive x-ray spectroscopy (EDX), elemental maps have been obtained which reveal the distribution of alloying elements within the matrix and the gamma-prime precipitates, as well as the larger incoherent delta phase precipitates. It is observed that Al, Mo, Nb, Ni, Ti and W are all increased within the gamma-prime phase, while Fe, Co and Cr are excluded. In the case of the delta phase, the distribution is the same as that for the gamma-prime phase with the exception of Co, which is found to collect in the precipitates. In order to relate the microstructure to macroscopic properties of the alloy, yield strength tests have been made at each of the aging times. This data is then compared with simulated data, based on traditional theoretical models of precipitate strengthening, which take into account the shearing of preciptates by dislocations based on the effects of coherency, modulus and the anti-phase boundary which forms in the gamma-prime phase.

Material was supplied by Dr. Martin Stockinger, Böhler Schmiedetechnik GmbH, Kapfenberg, Austria.

Fig. 1: STEM image of 718Plus superalloy showing a dense population of gamma-prime precipitates and three larger delta precipitates

Fig. 2: STEM image showing details of individual gamma-prime precipitates
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5797 TEM microstructure characterizations of Al-Mg-Si aluminum alloy processed by severe plastic deformation

Yu Y. D.1, Liu M. P.2, Hjelen J.1, Roven H. J.1
1Department of Materials Science and Engineering, Norwegian University of Science and Technology, N-7491 Trondheim, Norway, 2School of Materials Science & Engineering, Jiangsu University, 212013 Zhenjiang, P. R. China
yingda.yu@ntnu.no

Nanostructured materials (NSM) processed by severe plastic deformation (SPD) have provided new opportunities for nanostructure refinements in metals and alloys with unusual properties which are very attractive for various structural and functional applications [1]. In the present work, a commercial 6061 Al-Mg-Si alloy (Al-1.0Mg-0.6Si in wt.%) was processed by equal channel angular pressing (ECAP) at 110 °C. Post ECAP microstructure characterization was carried out by using a JEM2010 (HRTEM).

All SPD procedures rely upon imposing a very heavy strain to the material so that a very high dislocation density is formed. By using the weak-beam dark-field (WBDF) method under special diffraction conditions, dislocations can be imaged as narrow lines. Figure 1(a) and (b) show WBDF TEM micrographs of dislocations in an ECAPed alloy by using the diffraction vectors, g =11-1 and g = 200 respectively. Under the present ECAP condition, the average projected dislocation density expected to be in the order of 1.4x10E17m-2 [1]. However the observed dislocation density in Fig.1 is much lower than this, suggesting that the majority of perfect (unit) dislocations had already passed through the Al alloy during the ECAP process. This can be further confirmed in the HRTEM image of Fig.2, where typical dislocation structures involve two partial dislocations connected by a stacking fault. The detailed configurations of partial dislocations in the present face-centered-cubic Al system has earlier been reported by the present authors [2], and an example is shown in Fig. 2(b) where a unit screw dislocation dissociated into two 30 ° partial dislocations connected by an intrinsic stacking fault is seen. The precipitation sequence in this alloys is generally accepted to be supersaturated solid solution → GP-I (II) zones → β’’ → β’. All these hardening phases are formed by nucleation and growth along Al {100} planes. Figure 3(a) shows a low magnification micrograph under multi-beam bright-field TEM mode from one [001] orientated Al grain. The homogenous disk-like precipitates can be found in the figure. Further HRTEM results of these disks-like precipitates microstructure are shown in Fig. 3(b), where it can be seen precipitates without any obvious lattice structure. These precipitates could be identified at very initial stage of dynamic precipitation.

TEM microstructure characterizations revealed most possibly in supersaturated solid solution is identified from the HRTEM investigations. Dislocation distribution investigations by using WBDF TEM reveal that most unit dislocations possibly already passed through this Al alloy during ECAP, which could also serve as driving force for promoting present dynamic precipitation.

MP Liu gratefully acknowledges funding from the National Natural Science Foundation of China (NSFC) under grant number 50971087.

[1] AP Zhilyaev and TG.Langdon, Progress in Materials Science 53 (2008), p. 893.
[2] MP Liu, HJ Roven and YD Yu, Zeitschrift für Metallkunde, 3 (2007), p. 184.

Fig. 1: Weak-beam dark-field TEM micrographs show dislocation microstructures by using diffraction vectors of (a) g = 11-1 from Al [011] orientation and (b) g = 200 from Al [001] orientation.

Fig. 2: (a) Typical dislocation structure in Al [011]. (b) A unit screw dislocation dissociated into two 30 ° partial dislocations connected by an intrinsic stacking fault.

Fig. 3: (a) Bright-field TEM micrograph shows homogenous precipitates, and non-sharp precipices on the lower part image are caused by specimen bending and locally missed accurate Al [001] orientation. (b) The corresponding HRTEM image shows precipitate lattice structure.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5826 Microstructural characterization of detonation sprayed MMC coatings

1 Dina V. Dudina, 2 Igor S. Batraev, 2 Vladimir Yu. Ulianitsky, 1 Michail A. Korchagin, 1 Oleg I. Lomovsky, 3 Ivan A. Bataev
1 Institute of Solid State Chemistry and Mechanochemistry SB RAS, Kutateladze str., 18, Novosibirsk, 630128, Russia 2 Lavrentiev Institute of Hydrodynamics SB RAS, Lavrentiev Ave., 15, Novosibirsk, 630090, Russia 3 Novosibirsk State Technical University, K. Marx Ave, 20, Novosibirsk, 630073, Russia
dina1807@gmail.com

In thermal spraying of powders, deposition in the solid state is accompanied by intensive plastic deformation; deposition of fully or partially molten particles proceeds through the splat formation and particle dispersion upon impact. Thermal action on the powder particles can cause chemical interactions of the powders with the gaseous environment and between the phases of the composite materials. Due to involvement of several physical and chemical phenomena, the microstructure development in thermally sprayed coatings is a complex process while the coatings are challenging objects for Electron Microscopy. In our investigations, we use Scanning and Transmission Electron Microscopy to study the microstructure of detonation sprayed metal matrix composite (MMC) coatings. Their phase composition is either inherited from the powders or evolves during spraying. In the TiB2-Cu system, we found that the size of the titanium diboride particles depends on the O2/C2H2 ratio increasing with increasing O2 content in the mixture. The ceramic reinforcement in TiN-Ti, TiCxNy-Ti and titanium oxides-Ti MMC coatings formed in situ as a result of chemical reactions of titanium with the gaseous phase during spraying, the coatings possessing a layered hierarchical structure composed of the matrix metal and the reaction products (Fig.1). The Ti3SiC2-Cu system is of great interest, as it can be sprayed in both solid and molten states. In the latter, reaction Ti3SiC2 + Cu → TiCx + Cu(Si) occurs. We have shown that it is possible to preserve the Ti3SiC2-Cu phase composition in the coatings only in cold conditions of detonation spraying ― at an explosive charge of 30% of the barrel volume at O2/C2H2=1.1. Calculations show that the temperature of copper particles 40 μm in size sprayed under these conditions does not reach the copper melting point (Fig.2). Chemical etching of the polished surface of the cross-sections of the coatings helped us better reveal the microstructural features of the coatings. In the coating deposited at an explosive charge of 30%, the lines reflecting the microstructural features of the Ti3SiC2-Cu powder agglomerates produced by mechanical milling show random orientation (Fig.3). This indicates solid-state deposition of the Ti3SiC2-Cu composite particles. In the coating deposited at an explosive charge of 40%, these lines have preferential orientation parallel to the coating/substrate interface (Fig.4) indicating the presence of a characteristic layered structure formed by partially molten composite particles.

This study was partially supported by RFBR, research project No. 13-03-00263a.

Fig. 1: Cross-section of the titanium oxides-Ti coating produced by detonation spraying of titanium at O2/C2H2=2.5 and air as a carrier gas, BSE image.

Fig. 2: Calculated temperatures of detonation sprayed copper particles 40 μm in size upon leaving the gun barrel at different explosive charges (O2/C2H2=1.1; carrier gas – air).

Fig. 3: Cross-section of the coatings produced by detonation spraying of the Ti3SiC2-Cu composite powder (etched in FeCl3 solution; the coating/substrate boundary is horizontal): O2/C2H2=1.07, explosive charge 30%, BSE image.

Fig. 4: Cross-section of the coatings produced by detonation spraying of the Ti3SiC2-Cu composite powder (etched in FeCl3 solution; the coating/substrate boundary is horizontal): O2/C2H2=1.07, explosive charge 40%, BSE image.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5850 Experimental and simulated crystal structure of phases in Mg-12wt.%Zn alloy

Němec M.1, Gärtnerová V.1, Drahokoupil J.1, Klementová M.2
1Institute of Physics, Academy of sciences of The Czech Republic, Na Slovance 2, 182 21 Praha 8, 2Institute of Physics, Academy of sciences of The Czech Republic, Cukrovarnická 10, 162 00 Praha 6
nemecm@fzu.cz

Magnesium and its alloys are promising lightweight materials with great potential in industrial fields such as automotive, aerospace or biomaterials engineering. Zinc is one of the most common alloying elements in magnesium alloys. Although Mg-Zn system has been thoroughly studied for many years, there are many unsolved issues so far.
For example, the crystal structure of the phases, which are in equilibrium with α-Mg at lower Zn concentrations, is still uncertain. These phases emerging as precipitates are, however, often responsible for significant strengthening effect of Zn in Mg solid solution. The goal of this work is to describe the microstructure of binary magnesium alloy with nominal composition Mg-12wt.%Zn and to clarify phase distribution and crystal structure of Zn based particles in Mg matrix.
Binary Mg-12wt.%Zn alloy was prepared by die casting in Ar atmosphere and subsequently annealed at 320°C for 20 hours followed by quenching in warm water. The main experimental technique used in this work is the transmission electron microscopy (TEM), particularly the selected area electron diffraction (SAED) and the three-dimensional precession electron diffraction (3D PED). On the base of experimental results crystal structure of the phases was obtained. Simulation of the zone axes diffraction patterns using JEMS software was compared with experimental data. Instead of commonly reported Mg12Zn13, the most common phase at this temperature was found to be Mg21Zn25 with complex structure. Some other Mg-Zn phases were also detected. The results are supported by X-ray diffraction data, which confirmed the majority of the Mg21Zn25 phase in the Mg matrix.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5862 A Novel Method for Microstructural Characterization of Cast Iron

J P Arul Mozhi Varman1
1Indian Institute of Technology - Madras, Indian
aruliitm2010@gmail.com

A typical compressor crankcase made up of grey cast iron is developed for truck applications by optimizing the shell casting process with superior mechanical & metallurgical properties. This crankcase requires high wear resistance and increased tensile strength combined with good machinability properties. When the product was developed it did not meet the required material specification. Even though many trials were being carried out, some of the properties were adhered and some of them weren’t particularly metallurgical properties like ferrite content and desired graphite flake size. Also the method to differentiate between steadite & ferrite is not so precise. This deteriorates product performance and dissatisfies the customer. This practical problem is addressed here and the bottleneck for the problem is identified and completely eliminated after developing the desirable method of casting; and the etchant solution is developed for differentiating and quantifying the steadite and ferrite micro constituents under an ordinary metallurgical microscope.

The difficulty in the metallography of gray cast iron lies in the differentiation of ferrite and iron phosphide eutectic (steadite) microconstituents by using normal etchant like nital, picral and both these phases appears as bright under normal light microscope. Hence, it is difficult to find out the relative amounts of phases, either photo micrographically or using sophisticated image analysis software. In general industry practice, the samples are etched with 2% to 3% nital for quantification of ferrite, steadite and cementite plus carbide particles under optical (light) microscope. This method of inspection requires high skill to differentiate the ferrite and iron-phosphide eutectic (steadite) microconstituents and also sometimes it leads to misinterpretation. This led to the development of a novel etching method called Selenic etchant, in which the steadite and ferrite constituents are differentiated at 100 % confidence level and the precise quantification of phases were done.

I thank the management and staff of WABCO India who supported me for completing this project succesfully during my internship

Fig. 1: The microstructire (x100) of gray cast iron etched with 3% nital reveals presence of ferrite and steadite microconstituents as same colour under normal microscope.

Fig. 2: The microstructire (x1000) of gray cast iron etched with Selenic composition reveals presence of ferrite as white or yellow colour and steadite as green colour
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5864 Anisotropic creep properties in single crystal superalloys at low temperature and high stress

Long H. B.1, Wei H.2, Mao S. C.1, Li Q.1, Xiang S. S.1, Zhang Z.1, 3, Han X. D.1
1Beijing University of Technology, Beijing, China, 2Institute of Metal Research, CAS, Shenyang, China, 3Zhejiang University, Hangzhou, China
scmao@bjut.edu.cn

The nickel-based single crystal (SC) superalloys have been widely used to fabricate turbine blades and vanes due to their excellent high temperature mechanical properties, oxidation and hot corrosion resistance [1, 2]. Creep deformation was usually used to evaluate the higher temperature mechanical properties of superalloys. There are two types of creep test, one is the high temperature and low stress while the other is the low temperature and high stress [3-5]. The superalloys are strengthened by ordered g′ phase embedded in a continuous g matrix. The orientation of superalloys has a strong influence on the creep properties of superalloys [6-8]. It is important to study the deformation behaviors in superalloys with different orientation.

In this study, the creep deformation behaviors in [001] and [011] orientated SC superalloys under low temperature and high stress were studied (750 ºC/750 MPa). The results show that the [001] superalloys has a much longer creep life (~1500 h, Figure 1a) than those of [011] superalloys (~70 h, Figure 1b). Analysis on fracture surfaces (Figures 1c and d) show that the [011] superalloys, having a regular cleavage plane, was fractured in a brittle way, while the [001] superalloys, having both cleavage plane and holes, fractured in a ductile way. TEM studies show that fracture of the superalloys was mainly caused by stacking fault cutting into the g′ phase (Figures 1e and f). <span>As can be seen from Figure 1e, the stacking faults in [001] superalloys has a orientation angle, indicating more than one type slipping systems are activated during creep. On the other hand, only one type of stacking fault can be find in [011] superalloys (Figure 1f), indicating only one slipping system is activated. 

<span>
[1] M. Gell, Superalloys 1980.Warrendale, PA: TMS, (1980) 205-214.
[2] R. Hashizume, TMS (The Minerals, Metals & Materials Society), (2004) 53-62.
[3] S. Tian, Y. Su, B. Qian, X. Yu, F. Liang, A. Li, Materials &amp; Design, 37 (2012) 236-242.
[4] M. Sakaguchi, M. Ike, M. Okazaki, Materials Science and Engineering: A, 534 (2012) 253-259.
[5] X.P. Tan, J.L. Liu, T. Jin, Z.Q. Hu, H.U. Hong, B.G. Choi, I.S. Kim, C.Y. Jo, Materials Science and Engineering: A, 528 (2011) 8381-8388.
[6] S. Tian, S. Zhang, C. Li, H. Yu, Y. Su, X. Yu, L. Yu, Metallurgical and Materials Transactions A, (2012).
[7] V. Sass, Acta mater, 44 (1996) 1967-1977.
[8] G. Xie, L. Wang, J. Zhang, L.H. Lou, Scripta Materialia, 66 (2012) 378-381.

This work was supported by Beijing Municipal Education Commission research project in grant KM201410005033, Beijing Nova program (Z141103001814108), NSFC (11327901), Beijing university of technology in 2014 young teachers ability of internationalization development plans.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5920 Possibility of two kinds of nucleation-sites of α-phase precipitates in a β-type Ti-15V-3Cr-3Sn-3Al alloy aged due to a two-step aging at high and low temperatures

Sukedai E.1
1Okayama University of Science, Okayama, JAPAN
sukedai@mech.ous.ac.jp

Introduction

Bi-modal structure formed by a two-step aging (high to low temperature) of Ti-15-3 alloy improves mechanical properties1). To clarify the origin, optical microscopy and TEM observation have been performed. The results suggest a possibility of independent nucleation-sites of α-phase precipitates depending on aging temperatures.

Experimental procedure

A commercial Ti-15-3 alloy was used. Solution treatment (ST) was done at 1123 K for 3.6 ks. A two-step aging method was adopted; 873 K for 11 ks as the 1st-step aging and 673 K for 90 ks as the 2nd-step aging. For TEM observations, a JEM 4000 EX electron microscope operated at 400 kV was used.

Results and discussion

1) Hardness testing

The  values of ST-specimen, 1st-step aged specimen and 2nd-step aged specimen were 265, 276 and 358, respectively. The hardness after 2nd-step aging indicated the highest value. The low temperature aging following the high temperature aging improves a mechanical property.

2) Optical microscopy observation

Figure 1(a) and (b) show optical micrographs of 1st-step and 2nd-step aged specimens. Fig. 1(a) shows precipitates formed along one side of a grain-boundary. Precipitates are hardly observed inside of grains. The feature of precipitates in Fig. 1(b) is more complicate; fine precipitates appeared along grain-boundaries and inside of grains. They also appeared around large precipitates due to 1st-step aging. These results suggest independent nucleation-sites of precipitations due to 1st-step aging and 2nd-step aging.

3) TEM observation

Figure 2(a) shows a dark field image of 1st-step aged specimen. Fig. 2(b) indicates a diffraction pattern from the central part of Fig. 2(a) and its key-diagram. Since Fig. 2(a) was taken using -103α spot, bright and parallel line-like precipitates from a grain-boundary are α-phase precipitates.

Figure 3 shows a dark field image of a 2nd-step aged specimen. Fig. 4(a) and (b) indicate diffraction patterns of the central part and a large bright precipitate in Fig. 3. Fig. 4(a) indicates overlap of diffraction patterns of 110 reciprocal plane of the matrix and 001 reciprocal plane of α-phase. Burger’s relationship is satisfied. Since Fig. 3 was taken using an α-phase spots indicated by an arrow in Fig. 4(a), all precipitates are α-phase. Large precipitates formed in 1st-step aging and fine precipitates formed in 2nd-step aging. This image also suggests fine precipitates formed independently from large ones.

Summary

The obtained results make us understand that fine precipitates improve mechanical properties, and nucleation-sites of α-phase precipitates due to high temperature and low temperature aging are independent each other.

Reference: 1) N. Niwa: Tetsu to Hagane, 78 (1992), 493.

Author would like to sincerely appreciate collaboration of Messrs. T. Hashiguchi and K. Naruse, undergraduate students of Okayama University of Science.

Fig. 1: Figure 1 Optical micrographs of 1st-step aged specimen (a) and 2nd-step aged specimen (b). (a) shows precipitates formed mainly along grain-boundaries. (b) shows fine precipitates formed along grain-boundaries, inside of grains and around precipitates due to 1st-step aging.

Fig. 2: Figure 2 Dark field image of 1st-step aged specimen (a) and a diffraction pattern from the central part in (a) with its key-diagram (b). The image was taken using α-phase spot -103 in (b). Bright and parallel line-like precipitates formed from a grain-boundary are visible.

Fig. 3: Figure 3 Dark field image of 2nd-step aged specimen, taken using α-phase spot indicated by an arrow in Fig. 4(a). Large and fine precipitates formed during 1st-step and 2nd-step aging, respectively. Their distribution is good correspondent with the distribution in the optical micrograph of Fig. 1(b).

Fig. 4: Figure 4 Diffraction patterns from the central part of Fig. 3 (a) and a large precipitate (b). (b) indicates only α-phase 001 pattern. (a) denotes overlap of 110 pattern of the matrix and 001 pattern of α-phase.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5924 Investigation of Nanocrystalline Nickel and Severely Plastically Deformed Ag-Cu Nano-Eutectic using TEM Automated Crystal Orientation Mapping

Cizek P.1, van Driel R. R.1, Wen M.1, Barnett M. R.1, Hodgson P. D.1
1Institute for Frontier Materials, Deakin University, Waurn Ponds, Australia
rosey.vandriel@deakin.edu.au

Detailed orientation and phase studies of nanocrystalline and severely plastically deformed submicron-sized materials on a large scale present a challenging experimental task. A novel automated crystal orientation and phase mapping technique in a transmission electron microscope (TEM) has recently been introduced that benefits from the high spatial resolution of TEM. This technique has been implemented onto a single instrument called ASTAR, which enables incident electron beam precession and simultaneous scanning over up to 10 micron sized sample area. The crystal orientation determination is performed through template matching of experimental electron diffraction spot patterns to their pre-calculated theoretical counterparts.
In the present work, the ASTAR technique has been applied to the study of electrodeposited nanocrystalline Ni and a severely plastically deformed Ag-28.1 wt.% Cu nano-eutectic alloy. The nanocrystalline Ni was studied in both the as-deposited state and after in-situ annealing up to a temperature of 400 oC. The Ag-Cu alloy was subjected to surface mechanical attrition treatment (SMAT) performed at room temperature for 60 min. The investigation was performed using a Digistar device, manufactured by NanoMEGAS, attached to a JEM 2100F TEM operated at 200 kV in a nanobeam mode. Spot/step size was ranging from 2 to 10 nanometers and the beam precession angle used was 0.7o. The orientation data obtained were exported to the electron back-scattered diffraction (EBSD) Channel 5 software for post-processing and visualization.
The as-deposited nanocrystalline Ni contained, apart from nanograins, also coarse (sub)grain clusters having large internal misorientation gradients (Fig. 1). Contrary to some previous suggestions, during annealing these clusters neither served as nuclei for the observed abnormal grain growth nor displayed a tendency for (sub)grain coalescence. The abnormal grain growth appeared to originate from randomly distributed nanograin nuclei and involved the profuse formation of annealing twins. During the SMAT processing, the original nanostructured eutectic Ag–Cu alloy with alternate Ag and Cu lamellae (Fig. 2a) became progressively transformed, from the top surface towards the specimen core, into a composite with the isolated Cu regions dispersed in the Ag matrix (Fig. 2b). The interphase boundaries simultaneously changed from those having cube-on-cube and hetero-twin orientation relationships (Fig. 2c) into general large-angle boundaries (Fig. 2d). In summary, the obtained results have demonstrated that the ASTAR technique is well suited for the orientation and phase study of nanocrystalline and heavily strained materials with a reasonable angular resolution of around 1o.

The financial support provided by the Australian Research Council is gratefully acknowledged.

Fig. 1: (a) ASTAR orientation map of the as-deposited nanocrystalline Ni having <001> fibre texture (see the inset). Nanograins and (sub)grain clusters are coloured in blue and green to yellow to red, respectively; (b) Enlarged rectangular area highlighted in (a) containing a cluster; (c) Misorientation linescans along the arrow shown in (b).

Fig. 2: ASTAR orientation maps (a,b) and interphase misorientation spectra (c,d) for the nano-eutectic Ag-Cu specimen core and SMAT treated surface, respectively. Ag and Cu are grey and white, respectively. The black, red, blue and green lines are grain, twin, cube-on-cube interphase and non-cube-on-cube interphase boundaries, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5943 Transmission Electron Microscopy Study of the Strain-Induced Precipitation in a Model Austenitic Fe-30Ni-Nb Steel

Poddar D.1, Cizek P.1, Beladi H.1, Hodgson P. D.1
1Institute for Frontier Materials, Deakin University, Geelong, Australia
dpoddar@deakin.edu.au

The present work has investigated the evolution of strain-induced NbC precipitates in a model austenitic Fe-30Ni-Nb steel deformed at 925 °C to a strain of 0.2 during post-deformation holding between 3 and 1000 s and their effect on the reloading flow stress. The microstructural examination was performed using a JEM 2100F transmission electron microscope operated at 200 kV. A range of imaging and diffraction techniques was used to determine the crystal structure, coherency state, size, shape, number density and volume fraction of the precipitates in conjunction with the dislocation substructure. Extensive use of moiré-fringe technique allowed to distinguish down to 3 nm scale precipitates clearly from the dislocation contrast background. Foil thickness measurements, required for the precipitate volume fraction estimation, were performed using the convergent beam electron diffraction. The precipitate particles preferentially nucleated on the nodes of the periodic dislocation networks constituting microband walls (Fig. 1a). Holding for 10 s resulted in the formation of fine, coherent NbC particles with a mean diameter of about 5 nm (Figs. 1b and 1e) that displayed the cube-on-cube orientation relationship with austenite (Figs. 1c and 1d) and caused the maximum increase in the reloading steady-state flow stress. A further increase in the holding time from 30 to 1000 s led to the formation of semi-coherent (Fig. 1f), gradually coarser and more widely spaced particles with a mean diameter of 8 nm and above, which led to a gradual decrease in the reloading steady-state flow stress. The holding time increase resulted in progressive disintegration of the dislocation substructure and dislocation annihilation through static recovery processes, which was also reflected by the measured softening fractions. The precipitate particle shape changed during post-deformation annealing from elliptical to faceted octahedral and subsequently to tetra-kai-decahedral.

The financial support provided by the Australian Research council is gratefully acknowledged.

Fig. 1: TEM observations of the NbC particles at a strain of 0.2 and holding for 10 s (a-e) and 1000 s (f). (a) Bright-field micrograph of the particles (circled) on the dislocations network constituting a microband wall; (b) Enlarged particle; (c) Nanobeam diffraction pattern for (b); (d) Pattern indexing; (e,f) NbC/austenite interphase lattice images.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5948 Nano-scale analysis on the cementite dissolution behavior of high carbon tire-cords by using atom probe tomography

Bang C. W.1, Yang Y. S.3, Park C. G.1, 2
1Department of Materials Sci. & Eng. Pohang Uni. of Sci. & Eng. (POSTECH), Pohang, Korea, 2National institute for Nanomaterials Tech. (NINT), Pohang, Korea, 3POSCO Technical Research Lab., Pohang, Korea
specialone@postech.ac.kr

Pearlitic steel filaments are usually twisted together to form the steel tire cords used for truck/bus radial tires, due to their outstanding strength as well as acceptable ductility. In response to the market trend toward lighter and higher performance tires, the strength required for the steel filaments has been increased. In order to enhance the strength, it is generally tried to increase the carbon content and to increase the drawing strain. [1] When the drawing strain is progressively increased, cementite dissolution, in which carbon atoms in the cementite lamellae diffuse into ferrite phase, occurs upon high drawing strain [2, 3]. It is known that cementite dissolution affecting the fatigue resistance is related to the carbon composition of specimen and the final drawn strain.[4] However, the effects of morphology and behavior during drawing have been not understood yet. In this study, the cementite dissolution behavior depending on drawing strain and cementite morphology have been investigated by transmission electron microscopy (TEM) and laser-assisted atom probe tomography (APT).
Chemical composition of specimens used in the present study was as follows: C 0.8%, Si 0.3% and Mn 0.5%. The specimen were fabricated with various drawing strain(0, 0.63, 1.26, 1.82, 2.38, 2.96, 3.48). Fig. 1 shows lamella spacing and cementite thickness depending on drawing strain. It was found that the thickness of cementite and lamellae spacing were dramatically decreased at the early stage of the drawing, and then, slowly decreased. The cementite dissolution could be observed right after the early stage. APT results suggest that cementite dissolution occur only at above critical thickness of cementite and/or lamellae spacing. With a drawing strain more than 2.38, the cementite dissolution could be suppressed, due to limited carbon solubility of ferrite. Fig. 2 shows Microstructural changes during wire drawing by APT. The result shows the existence of broken cementite clearly. It revealed that cementite which had perpendicular direction to drawing axis was broken during drawing. The amount in dissolution of the large angle cementite to drawing axis was higher than that of low angle cementite (Fig.3). APT results also exhibited an important clue that the carbon in the cementite lamellae actively diffused into ferrite region in case of cementite lamellae that perpendicular to drawing direction.

Reference

1. G. Langford : Metall. Trans., 1A 465, 1970
2. V. N. Grindev, V. G. Gavrilyuk, I. V. A. Dekthyar, Y. Y. Meshkov, P. S. Nizin and V. G. Gavrilyuk : Phys. Stat. Sol. A, 14 : 689, 1972
3. V. N. Grindev and V. G. Gavrilyuk : Phys. Metals, 4 : 531, 198
4. Y.S. Yang and J.G. Bae and C.G. Park : Materials Science and Engineering A, 508, 148, 2009

 

 

Fig. 1: Lamella spacing and cementite thickness measured by TEM. Thickness of cementite and lamellae spacing were dramatically decreased at the early stage of the drawing, and then, slowly decreased

Fig. 2: APT result showing the microstructural changes occurred during the wire drawing total (ε=2.96). cementite which had perpendicular direction to drawing axis was broken during drawing. The circled line presents deformation behavior of cementite lamellae.

Fig. 3: Atom map and quantitative analysis of two colony (ε=2.38). (a) Many broken cementite lamellae in the colony 1 and cementite plate in the colony 2. (b) Projected image from the direction of dotted line (c) Amount of carbon in the broken cementite in colony1. (d) Amount of carbon in the cementite plate in colony 2.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5949 In-situ TEM annealing study of void shrinkage in aluminium

Zhang Z.1, Nakashima P. N.1, Smith A. E.2, Medhekar N. V.1, Bourgeois L.1,3
1Department of Materials Engineering, Monash University, Victoria, Australia, 2School of Physics, Monash University, Victoria, Australia, 3Monash Centre for Electron Microscopy, Monash University, Victoria, Australia
laure.bourgeois@monash.edu

A void is a three-dimensional cluster of vacancies embedded inside of a matrix. The presence of voids can significantly impact on the performance of engineering alloys. To eliminate voids or suppress void formation, it is important to understand the stability of voids and their evolution under different condition such as temperature. Aluminium, as the most used structural light alloy, is well-known to possess voids with truncated octahedron shape.[1] The kinetics of void evolution as a function of temperature was also studied many years ago.[2,3] However, no in-situ annealing or high-resolution imaging studies exist to date. Such studies are important in order to develop an understanding of the atomic-scale mechanisms of void evolution.

To fill this gap, we utilised a transmission electron microscope (TEM) JEOL 2100F at 160 kV and 200 kV and a Gatan 652 heating holder to observe the shrinkage of voids in aluminium under in-situ annealing. The samples were 99.9999 wt% pure aluminium heat treated at 550°C for 30 mins, and quenched to room temperature. Isothermal annealing experiments were conducted at various temperatures on void-containing aluminium tilted along the [110] direction, using bright-field TEM mode.

Figure 1 shows a typical TEM image of a void in its early stage of evolution at 100°C.. The void has clear {111} and {002} facets, as expected.[1] In our in-situ experiments, we recorded movies of the void evolution at various temperatures and characterised the void shape and size by measuring the distances between the main facets.

Void shrinkage was found to be a two-stage as shown in Figure 2: for aspect ratios larger than a specific value corresponding to the equilibrium state, the void shrinks along <001> only, while retaining the distance between the {111} facets constant. Once the aspect ratio reaches equilibrium, the void keeps the ratio constant and shrinks equally in all directions. The vacancy emission rates are distinctly different for the two stages, as only the {002} facets are activated for shrinkage during the first stage. We explained this phenomenon through a surface energy analysis: the void shrinks so to maximise the reduction in total surface energy per vacancy emitted.

We observed that void shrinkage takes place layer-by-layer as shown in Figure 3. Initiation of a new atomic layer starts from the kink at the facets' intersections and progressively glides along the surface. It indicates surface diffusion is an important process in void shrinkage; however bulk diffusion remains the rate-limiting process.

[1] M. Kiritani and S. Yoshida, J. Phys. Soc. Japan, 18 (1963) 915.

[2] T.E. Volin and R.W. Balluffi, Phys. Status Solidi, 25 (1968) 163.

[3] K.H. Westmacott, R.E. Smallman and P.S. Dobson, Met. Sci., 2 (1968) 177.

The authors acknowledge the financial support of the Victorian State Government and Monash University for instrumentation, and use of the facilities within the Monash Centre for Electron Microscopy.

Fig. 1: High-resolution TEM image of a typical void. The arrows indicate the distances between facets. These distances are used to characterize the shape and size of the void: the aspect ratio is defined as D(111)/D(002).

Fig. 2: High-resolution TEM images of the void evolution process under in-situ annealing at 100°C showing the two-stage process.

Fig. 3: (a) Layer-by-layer shrinkage initiated from a kink at the intersection of {002} and {111} facets. (b) Spreading of atoms along a {002) facet; the size of the new layer is indicated by the yellow dashed line. (c) Almost complete new layer. (d) Resulting movement of the {002} facet by one atomic layer.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5953 NI-AFM measurements of phases in 2nd generation PWA1484 and 4th generation PWA1497 single crystal nickel-base superalloys

Ziętara M.1, Czyrska-Filemonowicz A.1
1International Centre of Electron Microscopy for Materials Science and Faculty of Metals Engineering and Computer Industrial Science, AGH University of Science and Technology (AGH-UST), Al. A. Mickiewicza 30, PL-30 059 Kraków, Poland
zietara@agh.edu.pl

In the recent years, the nanoindentation in AFM (NI-AFM) technique has become a valuable tool for materials science. The NI-AFM technique was used for quantitative measurements of nano-hardness of the γ and γ’ phase. Investigation was performed on the 2nd generation (PWA 1484) and 4th (PWA 1497) single crystal nickel base superalloys for the samples of baseline material (after heat treatment) and after creep tests at 980 °C.
Figures 1 show PWA 1484 surface topography revealed by AFM, before and after nano-hardness measurements and Figs 2 those of PWA 1497 superalloy, after creep test. Ten indents for each phase (γ and γ’) were performed for each sample. Scan size for each AFM micrograph is 10 μm x 10 μm. Brighter color indicates γ matrix phase and γ’ phase precipitates are represented by darker shade, the contrast is obtained due to differences in height on the surface of the samples which are caused by difference in nano-hardness of the phases.
The comparison of the mean nano-hardness of both phases precipitated in both superalloys show that the γ’ phase is always harder than the γ matrix phase. There is no tendency or clear difference in the nano-hardness of γ and γ’ phases in the samples after different creep deformation of both superalloys. This means that the nano-hardness does not change with increasing creep strain or that the hardness changes are very small, therefore beyond a resolution of applied measurement technique.
The nano-hardness of the γ’ precipitates in the PWA 1484 superalloy is slightly higher than the nano-hardness of the γ’ phase of PWA 1497. The reason is most probably due to a difference in alloy chemical composition; the PWA 1497 contains less Ta which act as a strengthening element for γ’ phase. The γ matrix of the PWA 1497 superalloy is on average 0.8 GPa harder than that of the PWA 1484, that might be explained by higher content of Re and Ru, since they partition predominantly to the matrix phase.
The higher stability of 4th generation superalloy PWA 1497 is related to significantly stronger solid solution strengthening, what results in higher hardness of γ matrix in comparison with that of second generation PWA 1484 superalloy.

This work was partially supported by AGH statutory project (11.11.110.290) The authors would like to acknowledge also Pratt & Whitney for providing the materials used in this investigation. We acknowledge gratefully the help of S. Neumeier and F. Pyczak from University of Erlangen-Nürnberg.

Fig. 1: PWA 1484 superalloy, baseline material surface topography before (a) and after (b) NI-AFM tests (indents in γ and γ’ phases are visible) revealed by AFM

Fig. 2: PWA 1497 superalloy, after creep test, surface topography before (a) and after (b)NI-AFM tests (indents in γ and γ’ phases are visible) revealed by AFM
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5984 TEM STUDY OF THE INFLUENCE OF PRE-AGING AT 100ºC ON PRECIPITATION DURING ARTIFICIAL AGING IN Al-Mg-Si.

Castro Riglos M. V.1, Noseda Grau V.2, Cuniberti A.3, Stipcich M.3, Tolley A.1
1Centro Atómico Bariloche, Comisión Nacional de Energía Atómica and CONICET, Av. Bustillo 9500, Bariloche, Argentina., 2Instituto de Física de Materiales (IFIMAT), Universidad Nacional del Centro de la Provincia de Buenos Aires (UNICEN) and CICPBA, Tandil, Argentina., 3IFIMAT-UNICEN and CONICET, Pinto 399, Tandil, Argentina.
victoria.castroriglos@cab.cnea.gov.ar

Age hardenable Al-Mg-Si alloys are widely used as structural materials in automotive and architectural industries due to their low density, high formability and corrosion resistance (1). In these alloys, strengthening is due to the formation of needle-shaped β” precipitates, that is controlled by the time and temperature of artificial aging. However, it has been reported that room temperature (RT) pre-aging delays the formation of the strengthening precipitates (2), whereas by pre-aging at temperatures of 70ºC or 100ºC, this delay is reduced (3). The detailed process by which these effects take place is not yet fully understood. In this work, the influence of pre-aging at RT and at 100ºC on the subsequent precipitation process during artificial aging at 180ºC in an Al-Mg-Si alloy is addressed.
A commercial AW-6082 alloy with composition Al-0.64Mg–0.50Si–0.60Mn–0.05Cu–0.05Fe (wt%), determined by optical emission spectrometry, and a mean grain size was 0.3 mm was used. Specimens were solution-treated for 1 h at 530 ºC and water quenched. Before artificial aging at 180ºC, one batch was aged at room temperature (RT) and another at 100ºC. The resulting microstructure was characterized using a Tecnai F20 transmission electron microscope.
Figure 1 shows two-beam bright field images with g = 200 obtained near the [011] zone axis of specimens pre-aged for 3 hours at RT (a) or at 100ºC (b), and subsequently aged at 180ºC for 30 minutes. The observed contrast corresponds to needle-shaped β” precipitates whose projections are parallel to the [0 1-1] direction. The density of precipitates is higher and the average size is smaller in the specimen pre-aged at 100ºC than those in the RT pre-aged specimen.
Figure 2 shows STEM annular bright field (ABF) images along the [001] zone axis of specimens with 3 hours pre-aging at RT (a) or 100ºC (b), subsequently aged for 2 hours at 180ºC. Three needle-shaped precipitate variants, oriented along the <100> matrix directions, are indicated. Comparison of the precipitate lengths was carried out using such images, while that of the precipitate diameters was done using High Resolution images (Figure 3). The results are shown in Table I. Pre-aging at RT leads to fewer precipitates that grow to larger sizes, indicating that pre-aging at 100ºC favours the nucleation of β” precipitates.

Table I: Comparison of precipitate sizes after pre-aging treatments of 3h at RT or 3h at 100ºC, subsequently aged for 2 h at 180ºC (fig. 2).
Pre-aging | Mean length (nm) | Mean diameter (nm) |
RT | 20 ± 1 | 2.2 ± 0.2 |
100ºC | 14 ± 1 | 2.3 ± 0.2 |

References.
(1) I. Polmear, Light Alloys, 4th Edition, Butterworth-Heinemann (2005).
(2) C. Marioara et al., Acta Materialia 51 (2003), 789-796.
(3) T. Abid et al., J. of Alloys and Compounds 490 (2010), 166-169.

Acknowledgements: Financial support from the ANPCyT is acknowledged through grant PICT 0643.

Fig. 1: Bright field 2 beam images near the [011] zone axis in specimens 3h room temperature pre-aged (a) and 3h 100ºC pre-aged (b), subsequently annealed for 30 minutes at 180ºC.

Fig. 2: Annular Bright Field STEM images along the [001] zone axis in specimens pre-aged for 3h at RT (a) or at 100ºC (b), and subsequently annealed for 2 hours at 180ºC.

Fig. 3: High Resolution images along the [001] zone axis in specimens pre-aged for 3h at RT (a) or at 100ºC (b), and subsequently annealed for 2 hours at 180ºC.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5987 TEM Characterization of oxide dispersions in Al compacts from powder metallurgy.

Castro Riglos M. V.1, Moreno M.1, Balog M.2, Krizik P.2, Tolley A.1
1CONICET. División Física de Metales – Centro Atómico Bariloche – CNEA. Bariloche. Argentina, 2IMMS. Bratislava. Slovakia
victoria.castroriglos@cab.cnea.gov.ar

Due to their high specific strength and their relatively low cost, Al alloys are of great interest in applications related to the automotive and aerospace industries. Because pure aluminium is not hard enough for many of its target applications, different methods have been applied to enhance its mechanical properties [1,2]. Among such approaches, powder sintering has gained attention as an alternative method to obtain Al alloys that can provide the benefits of conventional Al alloys but offering superior mechanical properties even at elevated temperature exposures. Over the past years, increasing reliability has made it possible for Al powder metallurgy to expand its implementation scope [3].
In this kind of alloys the mechanical properties are related to the distribution of the in situ introduced Al2O3 dispersoids from the Al powder native oxide skin. The properties of the resulting powder compact can vary significantly depending on the process by which the compact is prepared and the original powder quality. Thus, microstructure must be studied in order to understand the properties variations.
The aim of the present research was to study how oxide dispersoids distribute into an Al alloy obtained by forging from ultra fine powders, and the oxide crystalline structure. In addition, the evolution of the microstructure after annealing over 24 h at 520ºC was addressed. Microstructural characterization was carried out with Transmission Electron Microscopy (TEM) in a Philips CM200 microscope.
Figures 1 and 2 illustrate the microstructure of the “as forged” alloy. It can be observed that the oxide distributes over the grain boundaries in the form of an oxide “skeleton” (Fig.1). High resolution images of the oxide indicate that its structure is amorphous (Fig.2). Figures 3 and 4 display the microstructure after annealing. Whereas the mean grain size was not appreciably altered, the structure and morphology of the oxide was significantly modified. Distinct oxide particles are observed mainly along grain boundaries, but also inside grains (Fig.3), and their structure is shown to be crystalline. Figure 4 shows a high resolution image and its corresponding diffractogram. Analysis of such images indicates that the reflections are compatible with the structure of γ-Al2O3.
Annealing was found to be detrimental to the strength of the alloy. Such changes are related to the transformation of the oxides from amorphous to crystalline and the modification of their distribution, leading to a loss in efficiency of grain boundary strengthening.

[1] I.Polmear. “Light Alloys: Metallurgy of the Light Alloys.” Butterworth-Heinemann (1995).
[2] J.Gilbert Kaufman. “Introduction to Aluminum Alloys and Tempers.” ASM International, 2000.
[3] M.Balog et al. Mat.Sci.Eng.A 504(2009)1-7.

The financial support from the following projects is acknowledged:
- CONICET-SAS RD Nº 182/13
- APVV-0556-12 projects
- VEGA 2/0025/14 and VEGA 2/0158/13 projects

Fig. 1: Overview of the “as forged” Al alloy. The oxide distributes as an almost continuous “skeleton” over the grain boundaries.

Fig. 2: The oxide structure in the “as forged specimen showed to be amorphous.

Fig. 3: Microstructure of the annealed alloy. Oxide particles are distributed preferentially over grain boundaries but also within grains.

Fig. 4: HRTEM image of an oxide particle showing crystalline structure.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-5991 Identification of phases in titanium alloy and correlation between EBSD, High Voltage, Medium Voltage and Low Voltage BSE imaging

Čupera J.1, Jan V.1
1Institute of Materials Engineering, NETME centre, Faculty of Mechanical Engineering, Brno University of Technology, Technická 2896/2, 616 69 Brno, Czech Republic
cupera@fme.vutbr.cz

The imaging of composite materials is steadily growing in importance and since the contribution of the multi-phase composites combining metallic and non-metallic is rising therefore imaging of these materials becoming more significant. When ceramic (or generally non-conductive) matrix contains metallic phase components problems with phase analysis due to charging of the sample is evident. Coating the sample with conductive layer is the standard solution which may obscure fine surface details. This problem can be reduced using electron microscopy at low landing energy while the information quality of signals usually connected with high landing energies is preserved.
The aim of this investigation is to evaluate the possibility to distinguish different phases in intermetallic alloy Ti - 46Al - 7Nb - 0.7Cr - 0.2Ni - 0.1Si using energy filtered Low Voltage Backscattered electron images (impact energy less than 5 kV). Microstructure of the specimens was examined using an Electron Backscatter Diffraction (EBSD) and results were correlated with High Voltage BSE (HVBSE – impact energy 20 kV), Medium Voltage BSE (MVBSE –impact energy 12 kV) and Low Voltage BSE (LVBSE – impact energy 2 kV) images.
The intermetallic alloy samples were analysed by electron diffraction technique and four main phases were identified: α – Phase (hexagonal; SG 194), β – Phase (BCC; SG 229), γ – Phase (tetragonal; SG 123) and Ti3Al – Phase (hexagonal; SG 194) (Fig. 1). These phases exhibit different chemical composition, therefore at 20 keV in HVBSE image (Fig. 2) an obvious difference in contrast between phases is generated. A decrease in impact energy to 12 keV of the primary electrons causes an increase in image contrast (Fig. 3). For this case, the increase in contrast is caused by combination of composition contrast and channelling contrast. When the impact energy is lowered to 2 keV, detector positioned in the electron optics (in-lens) for BSE has to be used. For low impact energies the contrast corresponding to phase differences is still evident (Fig. 4) although it is accompanied by surface features (scratches, surface deformation). The contrast mechanism at low impact energies does not depend on atomic number or material density, but only on the bonding structure of the outer shell electrons. There we get ionization losses, resonances (phonons, plasmons) or band gap losses [1]. Therefore contrast in LVBSE images is still visible and can even bring additional information in some cases.
Low Voltage BSE imaging allows phase contrast imaging while decreasing charging of the non-conductive components in composite materials.

[1] JAKSCH, H. CONTRAST MECHANISMS LOW-LOSS BSE IN A FIELD EMISSION SEM. In: MODERN DEVELOPMENTS AND APPLICATIONS IN MICROBEAM ANALYSIS, 2012, p. 255-269.

NETME CENTRE project CZ. 1.05/2.1.00/01.0002 ED0002/01/01, Czech Grant Agency under the project GACR 13-35890S and OPVK project n.: CZ.1.07./2.3.00/20.0197 are acknowledged.

Fig. 1: Reconstructed EBSD phase map of Ti - 46Al - 7Nb - 0.7Cr - 0.2Ni - 0.1Si alloy.

Fig. 2: High Voltage BSE image at 20 kV of Ti - 46Al - 7Nb - 0.7Cr - 0.2Ni - 0.1Si alloy.

Fig. 3: Medium Voltage BSE image at 12 kV of Ti - 46Al - 7Nb - 0.7Cr - 0.2Ni - 0.1Si alloy.

Fig. 4: Low Voltage BSE image at 2 kV of Ti - 46Al - 7Nb - 0.7Cr - 0.2Ni - 0.1Si alloy.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-6003 Electron microscopy and spectroscopy for the study of helium cavities and radiation damage in oxide-dispersion-strengthened steels

Walls M.1, Badjeck V.1, Meslin E.2, Bhattacharya A.2, March K.1
1Laboratoire de physique des solides, Bât. 510, université Paris-Sud, 91405 Orsay Cedex , 2CSNSM/IN2P3/CNRS, Université Paris-Sud, 91405 Orsay Cedex
michael.walls@u-psud.fr

The understanding and the assessment of neutron irradiation in nuclear materials is critical in the design of the next-generation nuclear fission reactors (Gen IV – sodium fast reactor) which will need to work at higher temperature and higher radiation levels. Recent years have witnessed increasing research efforts in this area. For these applications, oxide dispersion strengthened (ODS) steels are amongst the interesting candidates. Here, one of the most promising structural and fuel cladding materials, an oxide-dispersion-strengthened (ODS) steel was implanted with He and Fe ions in order to simulate the transmutant He and the damage (He/dpa) caused by neutron irradiation. The fine distribution of Y-Ti-O nanoparticles (NPs, 1-20 nm) in the Fe-Cr ferritic matrix is expected to improve thermal and mechanical properties [1].

Scanning transmission electron microscopy coupled with electron energy-loss spectroscopy (Nion USTEM 200) was used to investigate at the nano- and atomic scale the structure and chemistry of these NPs and the He bubbles generated. The ODS material (Fe-14Cr-1W-0.3TiH2- 0.3Y2O3, wt.%) was prepared by mechanical alloying of Fe-Cr-W-TiH2 and Y2O3 powders, followed by hot extrusion. Ion irradiationwas carried out at 500°C, producing 5 dpa damage (Fe) with 1000 appm/dpa He implantation. Core loss spectrum-images were denoised using principal component analysis [2]. Implanted He is shown to be trapped in some Ti-O NPs (figs 1d-f) although bubbles also exist outside the NPs. The He-K line (21.2 eV for free atoms) shifts to higher energy in the bubbles (fig. 2c, E=1.75 to 4.6 eV); this has been shown to be correlated to the bubble He density and these shifts imply fairly high densities of up to ~150 at.nm-3[3]. The ion irradiation has also changed the Cr distribution, removing the Cr-shell observed around the NPs in non-irradiated ODS samples, as seen in Fig.3 [4,5]. A detailed study of the helium densities and pressures in the bubbles associated with the nanoparticles and in the steel matrix will be presented.[Yamashita S et al, J Nucl Mater 2004].

[1] T. Yamamoto et al. J. of Nucl Mater 367–370 (2007) 399–410
[2] www.hyperspy.org
[3] S. Fréchard et al. Journal of Nuclear Materials 393 (2009) 102–107
[4] A. Hirata et al. Nature Materials 2011;10:922.

[5] V. Badjeck et al submitted to Phys. Rev.

We thank the French “Contrat de Programme de Recherche: ODISSEE” funded by AREVA, CEA, CNRS, EDF and Mécachrome under contract n°070551,the METSA network and the European 7th framework program “ESTEEM2” for financial support.

Fig. 1: (b) HAADF image of nanoparticles and He bubbles, (a) spectra of the bubble n°4 (He-K) and of the Fe-Cr matrix around this bubble with the iron plasmon. (c) He-K normalized spectra of 4 He bubbles numbered in the HAADF image, after subtraction of the iron plasmon signal

Fig. 2: (a) HAADF image and (b) Fe-L2,3, (c) Cr-L2,3, (d) O-K, (e) Ti-L2,3 and (f)He-K elemental maps with a the left and right arrows showing He bubbles without and with Ti presence respectively

Fig. 3: Elemental maps of Ti,O Cr and Fe in a sample of the same steel before irradiation, showing the chromium shell present around unirradiated particles
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-6009 TEM Investigation of the Effect of NbC Precipitates on Microband Alignment during Multipass Hot Deformation of Model Fe-30Ni-Nb Microalloyed Steel

Poddar D.1, Cizek P.1, Beladi H.1, Hodgson P. D.1
1Institute for Frontier Materials, Deakin University, Geelong, Australia
dpoddar@deakin.edu.au

Strain-induced NbC precipitation in multipass hot rolling of microalloyed steels is of a high practical importance, however, its direct study is precluded by the phase transformation on cooling from the hot rolling temperatures in these steels. The current investigation was performed using Fe-30Ni-Nb model austenitic steel, that preserves the hot deformed microstructure on cooling to ambient temperature. The hot deformation was carried out in two passes, separated by isothermal holding (10, 100 and 300 s), in uniaxial compression at 925 °C at a strain rate of 1 s-1 using the first pass strain of 0.2 and the second pass strain of 0.2 to 0.6. High resolution EBSD and a wide range of TEM imaging and diffraction techniques were employed to characterise the dislocation substructure and its interaction with strain-induced precipitates. EBSD investigation after the first pass revealed that the microstructure in the <110> fibre grains consists of the crystallographic microbands (MBs) aligned parallel to highly stressed {111} slip planes. This is consistent with reported findings that, during straining to medium levels, the MBs represent “transient” microstructure features that maintain their crystallographic character, through continuously rearranging themselves, and do not follow the rigid body rotation imposed by the plastic deformation. During inter-pass holding after the first pass, the strain-induced NbC particles were observed to nucleate preferentially on the nodes of periodic dislocation networks constituting MB walls. Over the increase of holding time from 10 to 300 s, the precipitates grew in size from 5 to 31 nm and the MB walls became increasingly disintegrated. Interestingly, after second pass deformation the MBs maintained their crystallographic character irrespective of holding time (Fig, 1). For shorter holding time, NbC particles still occupied the MB nodes (Fig, 1a), which indicates that during reloading these particles remained strongly pinned and became dragged by the rearranging MB walls. During reloading after increased holding time the particles tended to become increasingly detached from the MB walls and to follow the rigid body rotation (Fig. 1b). Ultimately, precipitate-free MBs were created for the prolonged inter-pass holding (Fig. 1c). This might be attributed to both the progressively reduced pinning force exerted by coarsening precipitates and the reduced dislocation density in MB walls obtained with an increase in the inter-pass holding time, which would allow the precipitates to move out of these walls during reloading. The present observations made on partially pinned MB walls (see Fig, 1b) revealed that their rearrangement on reloading occurred through cooperative migration of the corresponding dislocation networks.

The financial support provided by the Australian Research council is gratefully acknowledged.

Fig. 1: TEM dark-field micrograph of the microband wall occupied by NbC particles, obtained after double-pass deformation with a strain of 0.2 at each pass and inter-pass holding of 10 s. The compression direction, diffraction vector g and (111) trace are marked on the micrograph.

Fig. 2: TEM bright-field micrograph of the microband wall partly detached from NbC particles, obtained after double-pass deformation with a strain of 0.2 at each pass and inter-pass holding of 100 s. The compression direction, diffraction vector g and (111) trace are marked on the micrograph.

Fig. 3: TEM bright-field micrograph of the microband wall fully detached from NbC particles (arrowed), obtained after double-pass deformation with a strain of 0.2 at each pass and inter-pass holding of 300 s. The compression direction, diffraction vector g and (111) trace are marked on the micrograph.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-6024 The microstructure of Al-Ni-Fe-La polycrystalline alloys.

Kolobylina N. N.1, Lopatin S.2, Presniakov M. Y.1, Vasiliev A. L.1, 3, Bakhteeva N. D.4, Todorova E. V.4, Ivanova A. G.3
1National Research Center “Kurchatov Institute”, Moscow, Russia, 2Electron Optics, Achtseweg Noord 5, GG Endhoven, the Netherlands, 3Shubnikov Institute of Crystallography of the Russian Academy of Sciences, Moscow, Russia, 4Institute of Metallurgy and Material Science , Moscow, Russia
kolobylina@gmail.com

One possible way to obtain nanocrystalline multiphase composites is the intensive plastic deformation (IPD) of alloys under a high pressure. This method was applied to Al-RE-TM alloys, where RE- rare earth (La) metal and TR - transition metal (Ni, Fe). Two as received alloys, namely Al85Ni9Fe2La4 and Al85Ni7Fe4La were studied by scanning/transmission electron microscopy (STEM), energy dispersive X-ray (EDX) microanalysis and X-ray diffraction (XRD). The microstructural analyses were performed in an aberration corrected TITAN 80-300 TEM/STEM (FEI, USA) and TITAN 60-200 S/TEM attached with a high sensitivity Super-X EDX detector system and X-FEG source (ChemiSTEM technology). The specimens for transmission electron microscopy (TEM) were prepared by an electrochemical or ion etching.
It was found that as received alloys exhibits similar mixed polycrystalline microstructure containing four phases: face-centered cubic c-Al, binary phases Al3(Ni,Fe) and Al11La3 and a quaternary phase, contained Fe (Fig.2). The structure of Al3(Ni,Fe) particles were orthorhombic (Fe3C-type crystal structure, Pnma, a=0.661 nm, b=0.736 nm, c=0.481 nm) and Al11La3 also orthorhombic (Immm, a=0.44 nm, b=1.31 nm c=1.01 nm). The electron diffraction together with HAADF STEM analysis (Fig.2) and XRD of the quaternary phase indicated that it was Al8Fe2-xNixLa adopted Al8Fe2Eu type structure (S.G. Pbam) and unit cell parameters a=1.2530(6) nm, b=1.4503(4) nm, c=0.4036(1) nm. The Ni and Fe were homogeneously distributed in the compound (see Fig.3) The planar defects with Al3.2Fe type structure (monoclinic, S.G. С2/m) was observed inside the Al8Fe2-xNixLa particles (Fig.2). The atomic resolution EDX (Fig.3) unambiguously demonstrated that these insertions were Al3.2FexNi1-x. Again, the distribution of Ni and Fe in these defected areas was homogeneous.The interfaces between Al8Fe2-xNixLa and Al3.2FexNi1-x were defect free because the atomic structure of the correspondent (001) Al8Fe2-хNiхLa and (010) Al3.2FexNi1-x crystal planes match perfectly to each other.

This work was supported by RFBR grant 13-02-12190 OFI-m.

This work was supported by RFBR grant 13-02-12190 OFI-m.

Fig. 1: Optical image of Al85Ni7Fe4La alloy: 1– c-Al, 2 –Al3Ni, 3 –Al11La3, 4 – Al8FeNiLa.

Fig. 2: The HAADF HR STEM image of Al8Fe2-xNixLa particle in B=[001] zone axis. The crystal structure of Al8Fe2-xNixLa is in the inserts. The Al3.2FexNi1-x planar defects adopted Al3.2Fe structure type are shown by arrows.

Fig. 3: The STEM image of Al8Fe2-xNixLa particle in B=[101] zone axis. The atomic resolution Ni and Fe maps are in the inserts.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-6043 Effect of microstructure development on mechanical properties of ECAP-processed AE42 and LAE442 magnesium alloys.

Minárik P.1, Král R.1
1Charles Univeristy, Department of Physics of Materials, Prague, Czech Republic
peter.minarik@mff.cuni.cz

Extruded magnesium alloys AE42 and LAE442 were processed by Equal channel angular pressing, what led to significant grain refinement and specific texture formation. The resulting average grain size was ~1 µm in both alloys. Investigation by EBSD showed, that evolution of the texture was defined by channel parameters and slip system activity during the processing. In AE42 alloy was during the processing activated preferentially basal slip system. Fiber texture formed by extrusion was replaced with specific texture previously observed in magnesium alloys processed similarly: basal planes preferentially oriented 45 ° from the processing direction. However, lithium addition in LAE442 alloy resulted in decrease of c/a ratio, what facilitated prismatic and pyramidal slip during the thermomechanical processing. Compared to AE42 was the texture after final stage of the thermomechanical processing formed furthermore by grains with basal planes preferentially oriented parallel to the processing direction. Therefore final texture was not so strong, when comapred to AE42 alloy. Both grian refinement and texture formation had substantial effect on the evolution of yield tesile strength in both alloys. Pronounced decrease of yield strength due to texture evolution was measured in AE42 alloy despite significant grain refinement. Texture formed after final stage of the processing was not so strong, moreover, the element representing basal planes rotated 45 ° from the processing direction was weak. Therefore decrease of yield strength was not measured.

The present work is a part of the Czech Science Foundation project 14-36566G.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-6045 Combine EBSD stage and beam long scan on two Titanium metal sheets assembled by friction stir welding

Brisset F.1, Baudin T.1, Helbert A. L.1
1ICMMO, CNRS, Université Paris-Sud, Orsay, France
francois.brisset@u-psud.fr

Two metal sheets of Titanium 21S have been stir welded at a rotation speed of 200 tr/min. and a linear speed of 50 mm/min. The microstructure was studied by EBSD. The particularity of this study is the very long duration of the EBSD acquisition experiment that was run over 9 days in continuous. To set this experiment a combine stage/beam acquisition was started up after having selected different areas including some large joined zones located at different positions over the sample. This type of experiment is only possible if the SEM can be stable enough over such a long period, which means that only a few schottky type FEG-SEM could be used. Figure 1 shows some typical areas (in blue) that were recorded. It shows also an orientation map acquired on an area of 15 mm x 4 mm at a step size of 2 microns although the blues areas (and others) were acquired at a step size of 0.2 micron (figure 2). The first one allowed a general overview of the microstructure evolution and the others the detail observations on specific parts. Doing some large areas combining stage en beam moves allow to be sure to get all the needed data in one go all over some interfacial zones (like area 4 and similar, by example).

At the initial state, the material is formed of an equiaxe grain size of about 35 microns. The microstructure is becoming very fine at the centre of the welding and evolve trough the thickness of the sheets. Orientation maps with the smallest step confirm the mean grain size in the area number 1 of about 1 micron and in zones 2 and 3 of about 2 microns. This important reduction is characteristic of a severe plastic deformation process. These small grains show a very small intragranular misorientation not more than 1°. In the contrary, in area number 4 grains show large intragranular misorientations (figure 3).

The analysis of the texture clearly indicates that the tool generates, during its rotation, a very strong shearing of the material.

Authors thank Mrs Millet and Goussain respectively from Timet and Institut de Soudure for providing the material and the welding.

Fig. 1: Weld section of the whole sample with typical small step acquisition areas, in blue. Left side: the retreating side, right side: the advancing side.

Fig. 2: Grain sizes in zones 1, 2 and 3

Fig. 3: Variation of intragranular misorientation along an interface containing small and large grains.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-6051 Various modes of α phase precipitation in metastable β titanium alloys

Šmilauerová J.1, Harcuba P.1, Holý V.1, Stráský J.1
1Faculty of Mathematics and Physics, Charles University in Prague, Czech Republic
smilauerova@karlov.mff.cuni.cz

Metastable β titanium alloys are widely used as construction materials in automotive and aerospace industry. These applications demand materials with superior properties, such as high specific strength, good ductility and excellent fatigue and corrosion resistance. The improvement of strength can be achieved through ageing treatment which results in the formation of small precipitates of thermodynamically stable α phase in the metastable β matrix [1]. When the α precipitates are very fine and homogeneously distributed, the strength of the alloy increases without a significant deterioration of the ductility [2]. This fine microstructure can be achieved by employing such heat treatment in which the α phase precipitation is preceded by ω phase formation. ω phase is a metastable phase occurring as nanometric-sized, homogeneously dispersed particles. ω-assisted α phase nucleation results in very fine α phase microstructure [3].
In this study, very small precipitates of the α phase were studied by scanning electron microscopy (SEM). This observation allowed us to investigate the dependence of the resulting α + β microstructure on various ageing conditions (time, temperature, cooling regime). The SEM study was supplemented by the measurement of small-angle x-ray scattering (SAXS) which provides precise information on crystallographic orientation between the β and α lattices and on the shape and size of α precipitates. In order to assess the relationship of microstructure and mechanical properties of the material, Vickers microhardness was measured.

References:
[1] Banerjee D., Williams J. C. Perspectives on titanium science and technology, Acta Materialia 2013; 61:844.
[2] Lütjering G., Williams J. C. Titanium. Berlin; Springer-Verlag; 2007.
[3] Blackburn M. J. , Williams J. C. Transactions of the Metallurgical Society of AIME 1968; 242:2461.

The work was supported by the Grant Agency of the Czech Republic under the grant No. 14-36566G.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-4-P-6062 Effects of the Localized Overheat On the Microstructure of Centrifugally Cast HP-Steels Used In Steam Reformer Furnaces

Mendes M. C.1, de Almeida L. H.1, Dille J. A.2
1Federal University of Rio de Janeiro, 2Free University of Brussels
lha@metalmat.ufrj.br

High strength at elevated temperatures is an important characteristic of heat-resistant cast stainless steels, an example of these steels is ASTM A297, grade HP, which is used as radiant tubes in pyrolysis and reformer furnaces. In operational conditions the wall temperature in these tubes can reach a maximum of 950°C. During that operational condition, the gradual deterioration of the catalyst in the radiant tubes can result in increased temperature over 1000°C in localized regions[1]. The radiant tubes usually take 100000 h as a reference lifespan but, nowadays requirements for higher productivity have raised the demand for improved performance. In this context Nb and Ti were added in the chemical composition of the normal grade, resulting in the modified HP steels. Despite the improvement in the structural stability by the additions of Nb and Ti, during service at high temperatures the microstructure changes due to aging time [2]. Modified HP steels have a fully austenitic matrix with primary Cr and Nb carbides along the interdendritic region. During aging time, besides the coalescence of the primary interdendritic carbides, a fine secondary precipitation in the matrix and a partial transformation of the primary NbC to a G-Phase (Ni16Nb6Si7) occurs [3]. Despite this, some uncertainties remain concerning the integrity of tubes that don’t collapse during an overheating events and the possibility of reusing them. In this context, the aim of this work is evaluate the effects of the localized overheating on these modified HP steels aged in different conditions during long term exposure in service by SEM and TEM. The microstructural analysis was performed using samples in aged, over-aged and localized overheated conditions, obtained from radiant tubes reached 70.000 h of service. The SEM samples were prepared by conventional grinding and polishing techniques and TEM samples were prepared by electro-polishing techniques. Despite the fragmentation of primary carbides, including the G-Phase, the SEM analysis of the samples in the same region of different tubes showed mainly a massive secondary precipitation in the aged condition and an apparently complete dissolution of these precipitates in the overheated tube, Fig. 1. But in fact, the TEM analysis, Fig. 3, showed a partial dissolution of the secondary precipitates. This fact broad the discussions about the possibility of reuse these radiant tubes, who suffered the temperature burst and showed a microstructure near than tubes in the aged condition.

[1] F.C. Nunes et al., Mater Charact. 58 (2007) 132. [2] G.D.A. Soares et al., Mater Charact. 29 (1992) 387. [3] R.A.P. Ibañez et al., Mater Charact. 30 (1993) 243.

The authors would like to acknowledge the support of TSEC, CNPq, CAPES and ULB for the samples preparation, financial support, and use of the microscopes.

Fig. 1: SEM image of the microstructure an overheated condition.

Fig. 2: TEM image of secondary precipitation.

Fig. 3: TEM image of secondary precipitation, aged condition.

Fig. 4: TEM image of secondary precipitation, overheating condition.
↑ Go to the Table of content ↑

MS-5. Ceramics and inorganic materials

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-5-IN-1583 Structural and Chemical Adsorption Transitions at Ceramic Interfaces

Kaplan W. D.1
1Department of Materials Science and Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel
kaplan@tx.technion.ac.il

It is recognized that grain boundaries (GBs) can adopt a diffuse structural nature, and be described using diffuse interface theory, where the structure and chemistry of GBs, interfaces and surfaces can go through 2-D transitions between thermodynamic states (termed complexions) in order to minimize the interface energy [1-2]. As such, complexions for interfaces are analogous to phases in the bulk.

To date, almost all of these studies have been conducted at GBs in single phase polycrystalline systems, which by definition are not at equilibrium, and in some cases it is not even clear if the identified complexions are at steady-state [3-4]. Similar questions have been raised regarding interfaces in thin film studies, where the deposition process may be very far from equilibrium.

This presentation will focus on an experimental approach to address the structure, chemistry, and energy of complexions at metal-ceramic interfaces which are fully equilibrated, from which it can be demonstrated that a change in complexion reduces interface energy [5-6]. This will be compared with complexions at solid-liquid interfaces, where a region of ordered liquid exists adjacent to the interface at equilibrium [7-10], and the details of a reconstructed solid-solid interface where the reconstructed interface structure accommodates lattice mismatch for a nominally incoherent interface [11]. These three systems will be compared to known reconstructed solid surfaces, which can also be described as complexions, within a more generalized Gibbs adsorption isotherm.

References

1. D.R. Clarke, J. Am. Ceram. Soc. 70:15-22 (1987).

2. M. Tang, W.C. Carter, R.M. Cannon, J. Mat. Sci., 41:7691-7695 (2006).

3. R.H. French, H. Mullejans, D.J. Jones, G. Duscher, R.M. Cannon, M. Ruhle, Acta Mat., 46:2271-2287 (1998).

4. S.J. Dillon, M.P. Harmer, J. Am. Ceram. Soc., 91:2304-2313 (2008)

5. M. Baram, D. Chatain, W.D. Kaplan, Science, 332:206-209 (2011).

6. H. Sadan, W.D. Kaplan, J. Mat. Sci., 41:5099-5107 (2006).

7. S.H. Oh, Y. Kauffmann, C. Scheu, W.D. Kaplan, M. Ruhle, Science, 310:661-663 (2005).

8. S.H. Oh, M.F. Chisholm, Y. Kauffmann, W.D. Kaplan, W. Luo, M. Rühle, C. Scheu, Science, 330: 489-493 (2010).

9. Y. Kauffmann, S.H. Oh, C. T. Koch, A. Hashibon, C. Scheu, M. Ruhle, W.D. Kaplan, Acta Mat., 59:4378-4386 (2011).

10. M. Gandman, Y. Kauffmann, C. T. Koch, W.D. Kaplan, Phy. Rev. Let., 110:086106 (2013).

11. H. Meltzman, D. Mordehai, W.D. Kaplan, Acta Mat., 60:4359-4369 (2012).

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-5-IN-2401 Grain Boundary Atomic Structures, Vacancies and Dopant Segregation in Oxide Ceramics

Ikuhara Y.1,2,4, Shibata N.1,3
1Institute of Engineering Innovation, The University of Tokyo, Tokyo, Japan, 2Nanostructures Res. Lab., Japan Fine Ceramics Center, Nagoya, Japan, 3PRESTO, Japan Science and Technology Agency, Saitama, Japan, 4WPI-AIMR Research Center, Tohoku University, Sendai,Japan
ikuhara@sigma.t.u-tokyo.ac.jp

Properties of ceramics are strongly dependent on the grain boundaries (GBs) which has different atomic structures due to the disorder in periodicity. The grain boundary structures are also influenced by dopants and vacancies segregated at GBs, providing various functional properties, which cannot be observed in a perfect crystal. In order to control GB structures to improve the properties, we need to understand the relationships between GB characters, atomic structures and chemistry. It is considered that the formation of vacancies reconstructs the GB atomic structures, depending on the GB characters such as misorientation angle and GB plane. In addition, the segregated dopants also should play an crucial role to change the GB atomic structures, which is related to the GB structural transition.

In this study, well-defined GBs structures of CeO2 and co-doped MgO and Al2O3, which are fabricated by the bicrystal techniques, are used as the model samples, and the behavior of the GB structure reconstruction due to vacancies and co-dopants are systematically investigated by combining aberration-corrected STEM, EELS and theoretical calculations. STEM observations were performed using JEM-2100F and ARM-200F (JEOL) equipped with CEOS Cs-corrector. EELS spectra were acquired in STEM mode by an Enfina spectrometer (Gatan Inc). For theoretical approach, static lattice and density functional theory (DFT) calculations were used complementary.

CeO2 has attracted much attention as electrolyte materials for solid oxide fuel cells. It has been reported that GBs play an important role in the oxygen transport properties in CeO2, which must be influenced by the vacancies introduced in GBs. Various types of GBs of CeO2 were characterized by STEM, in which the periodic structural units are formed. In the case of Σ5 GB, nonstoichiometric GB core structure with oxygen vacancies is considered to be the most suitable model for the experimentally observed Σ5 GB. On the other hand, the Σ3 GB has the stoichiometric GB core structure. According to the DFT calculations, the structural distortions at the Σ3 GB are not as clear as those at the Σ5 stoichiometric GB. These results suggest that the oxygen stoichiometry at the GBs does not only depend on the atmosphere but also on the GB atomic structure, which is closely related to the GB energies, dangling bonds and strain. Co-dopant systems with aliovalent dopants were also investigated by the same method. For the model GB with the co-dopants, Ca and Si doped Al2O3 and Ca and Ti doped MgO were are selected for the present investigation. It was found that the two different dopants form the periodic structures along the respective GBs to compensate the charge neutrality at the GBs to relax their atomic structures.

This work was partly supported by the Grant-in-Aid for Scientific Research on Innovative Areas "Nano Informatics" (Grant No. 25106003) from JSPS and "Nanotechnology Platform" (Project No. 12024046) of MEXT, Japan.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-1585 Direct imaging of surface non-stoichiometry in nanoscale magnesium aluminum oxide spinel

Young N. P.1, Sawada H.2, Takayanagi K.3, Kirkland A. I.1
1Department of Materials, University of Oxford, Parks Road, Oxford, U.K., 2JEOL Ltd 1-2 Musashino 3-Chome, Akishima, Tokyo 196 Japan, 3Department of Physics, Tokyo Institute of Technology, 2-12-1-H-51 Oh-okayama, Meguro-ku, Tokyo 152-8551, (Japan) and CREST Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 (Japan)
neil.young@materials.ox.ac.uk

Spinels form a class of mixed-metal oxides with a general composition XY2O4, where X and Y are divalent and trivalent cations respectively. The spinel structure is robust against irradiation via fast neutron or ion beams, with minimal formation of defects or dislocations. Such radiation tolerance in spinel structures has yielded applications in the nuclear industry, including use as an inert matrix for fuel containment. Cation mobility and an ability to easily invert antisite defects in the cation sublattice are thought to contribute to the radiation tolerance of spinel and associated retention of an ordered anion sublattice. Direct imaging of the constituent ionic sublattices under neutron or ion-beam irradiation is crucial in understanding the nanoscale response of spinel to irradiation. Here we report on in-situ electron irradiation of spinel crystals and direct visualisation of the atomic structure via aberration-corrected high-resolution electron microscopy. Specimens of spinel prepared by ultramicrotomy were imaged using a JEOL R005 aberration-corrected transmission electron microscope, operating at a beam energy of 300keV and with Cs compensated to -4mm. The surfaces of spinel crystals were studied as a function of dose, with figure 1(a) showing the initial surface structure and figure 1(b) the same following a beam exposure of approximately ten minutes. Initially the crystal surface profile is stepped, with small terrace lengths and short {111} facets showing enhanced surface contrast. Following exposure to the electron beam elongated {111} facets are formed. Figure 2 shows a more detailed view of the structure of the {111} facets in a crystal that has been exposed to the electron beam. The terminating atomic layer deviates from the bulk atomic arrangement and shows enhanced contrast on the {111} facets. The indicated region in figure 2(a and b) has been compared to simulated exit-wavefunctions calculated for a range of crystal thicknesses. Figure 2 (c) shows a simulated exit-wavefunction for crystal thicknesses of 4.6nm and yields a qualitative match of atomic column positions with the experimental data. The location of the more intense of the two surface features is consistent with the positions expected for Mg sites in a {111} surface terminated with Mg. The second less intense column is consistent with either an aluminium or oxygen site. MgO layers have been reported to form when spinel is subject to electric fields under high temperature. Here we confirm via direct imaging, that a non-stoichiometric surface layer exists in nanoscale spinel, with electron irradiation enhancing the formation of elongated {111} surfaces and the Mg ion diffusion that contributes to the surface MgO layer.

Fig. 1: Phase of the reconstructed exit-wavefunction of spinel specimens. (a) Initial specimen. (b) The same area following approximately 10 minutes of exposure to a 300kV electron beam.

Fig. 2: (a) Enlarged area of figure 1(b). Strong surface contrast is evident as marked with an arrow. (b) False-colour image of the highlighted region in 2(a) with locations of the Mg sites indicated with arrows. (c) Simulated exit-wavefunction phase for a 4.6nm thick section of spinel crystal.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-1765 Angle-resolved STEM and monochromated EELS characterization of electrode and electrolyte materials for all-solid-state Li-ion battery

Gao X.1, Ikuhara Y. H.1, Kimura T.1, Fisher C. A.1, Kuwabara A.1, Moriwake H.1, Oki H.2, Kohama K.2, Ikuhara Y.3
1Japan Fine Ceramics Center, Nagoya, Japan, 2Toyota Motor Corporation, Shizuoka, Japan, 3The University of Tokyo, Tokyo, Japan
g.xiang@outlook.com

The development of next-generation secondary batteries continues to promote integration and miniaturization. Al-solid-state Li-ion batteries using nonflammable solid electrolyte and thin film electrode materials not only offer significant advantages in terms of improved safety and chemical and thermal stability, increased power and energy densities, and large potential windows, but also can be produced to much smaller dimensions than conventional Li-ion batteries containing liquid electrolytes. As with conventional batteries, the performance of thin-film batteries is influenced strongly by the nature of the embedded interfaces, such as electrode/electrolyte and electrode/current-collector interfaces, as well as the grain and domain boundaries within the electrode and/or electrolyte materials. Detailed knowledge of the interface structures, which provides insights into formation mechanisms of the interfaces and the effects of microstructure on electrochemical properties, is essential for efficient materials and device design. Here based on a systematic study using state-of-the-art scanning transmission electron microscopy (STEM), we report the epitaxial growth mechanism of a typical cathodic LiMn2O4 thin epifilm by exploring the detailed structural and compositional variations in the vicinity of a film/substrate interface, while reveal the structures and composition of the domain boundaries (DBs) and consider their effect on Li-ion mobility and ionic conductivity of La2/3-xLi3xTiO3 (LLTO) electrolytes.
Direct observation of atom columns shows the LiMn2O4 epifilm forms an atomically flat and coherent heterointerface with the Au(111) substrate, but that the crystal lattice is tetragonally distorted with a measurable compositional gradient from the interface to the crystal bulk. The growth mechanism is interpreted in terms of a combination of chemical and physicomechanical effects, namely a complex interplay between the internal Jahn-Teller distortions induced by oxygen non-stoichiometry and the lattice misfit strain.
DBs in LLTO are shown to consist essentially of two types: frequently occurring 90° rotation DBs and a much less common antiphase-type boundary. It is found that the 90° DBs are coherent interfaces consisting of interconnected steps that share La sites, with occupancies of La sites higher than in the domain interiors. The DBs suffer different degrees of lattice mismatch strain depending on Li content. The lattice strain and resultant Li and O vacancies and the high La occupancy at DBs are expected to result in lower interdomain Li-ion mobility, which will have a deleterious effect on the overall Li-ion conductivity.1,2
[1] X. Gao, et. al., Chem. Mater. 2013, 25, 1607.
[2] X. Gao, et. al., J. Mater. Chem. A 2014, 2, 843.

A part of this research was supported by the Japan Society for the Promotion of Science (JSPS) through its “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)”.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-1828 Microstructure Characterization of VO2 Thin Films and its Effect on Metal-Insulator Transition

Li X.1,2, Gloter A.2, Zobelli A.2, Gu H.1, Luo J.3, Colliex C.2
1State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, China, 2Laboratoire de Physique des Solides, Univ. Paris Sud 11, CNRS UMR 8502, Orsay, France, 3Department of NanoEngineering, Program of Materials Science and Engineering, University of California – San Diego, La Jolla, U. S. A.
leery@student.sic.ac.cn

VO2 undergoes a reversible metal-to-insulator transition (MIT) at 68 oC from a high temperature rutile tetragonal structure (R) to a low temperature monoclinic structure (M) accompanied with a great change in both resistivity and IR transmittance, which are promising for broaden applications such as smart window, switching and storage media. Since the microstructures of a thin film, including interface [1], grain boundary, strain distribution, and doping effect, etc. play a crucial role in its performance, the understanding of their relationship is essential for an effective manipulation of MIT and application design.

In our work, the structures and chemistries of VO2/Al2O3 thin films are systematically investigated by a Cs-corrected STEM. The twinning epitaxial structures, as well as the highly orientated twin boundaries (TB) are reported for the pure M-VO2 thin film. As revealed by the strain-sensitive low angle annular dark field (LAADF) imaging where a “colossal” bright contrast is raised only for some TB, they can be classified into two families by their different strain behaviors (Fig.1). Direct observations and measurements of the atomic displacement in the vicinity of TB quantitatively demonstrate a local structure distortion from monoclinic toward tetragonal accordant with their strain (Fig.2). Thus, these structural heterogeneities will cause unsynchronized dynamic MIT process and broaden the transition [2].

Moreover, for most VO2 applications, a transition temperature around RT is generally required, thus W doping, as established to be the most efficient method, is also studied. Distributions of the W atoms in R-VO2 thin film are analyzed by EELS mapping and HAADF imaging, showing an ordered W substitutional structure with the atomic clustering, and in particular along <010>R orientations (Fig.3). These clustering are further confirmed by DFT simulations, which find the most energetically favored W-W distances to be 4.23 Å along <010>R. The simulations further suggest that the clustering may be an effective mechanism to localize and reduce the W-induced structural distortions in the V-V chains, therefore, it may produce an efficient balance between the electron doping and the weak structural distortion and contribute to reduce the MIT temperature [3].

[1] Li et al. Scripta Mater. (2014) http://dx.doi.org/10.1016/j.scriptamat.2014.01.029

[2] Li et al. Acta Mater. 61 (2013) 6443–6452

[3] Li et al. submitted

This study was supported by Shanghai Key Basic Research Project (No. 09DJ1400200), National Natural Science Foundation of China (No. 51228202), NanOxyDesign ANR-10-BLAN-0814 program and European Union 7th Framework Programme (No. FP7/2007- 2013) under No. n312483 (ESTEEM2).

Fig. 1: Fig. 1 (a) STEM-BF and (b) LAADF images of VO2 thin film. Six orientations for twin boundaries are found and classified into two sets of families.

Fig. 2: Fig.2 (a) STEM-HAADF image, (b) zoomed HAADF images boxed in (a) and the scheme descriptions of the off-position measurement of the vanadium atom position. (c) Profiles of the atomic displacements.

Fig. 3: Fig. 3 (a) STEM-HAADF image of the W 0.02V0.98O2 thin film at zone axis [100]R, (b) Enlarged HAADF images of the boxed grain. (c)the intensity profile of (b) along the bR axis.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-1946 Spatially-Resolved Quantitative Analysis on Composition and Bonding of Grain and Phase Boundaries in Ceramics

Gu H.1
1Shanghai Institute of Ceramics (CAS)
gu@mail.sic.ac.cn

Structure, composition and bonding at grain boundary and interfaces affect the sintering process, the microstructure control and the thermo-mechanical properties of advanced ceramics. TEM has always played the leading role in pursuit of grain boundary nature: in the pre-Cs time, equilibrium amorphous film was found at grain boundaries in Si3N4; in the beginning of Cs era, rare-earth cations were directly imaged at specific sites of intergranular films, which opened the door of detailed study of chemical bonding. However, between structure and bonding, the composition of grain boundaries was not sufficiently studied as compared either on scale or quality. Here I would like to present a comprehensive approach to probe chemical composition and bonding based on a combined EELS methodology [1]. The resultant quantitative knowledge on grain boundary chemistry may help us to gain deeper insights into the nature of ceramic materials.

Difference in ELNES characters between the probed grain boundary film and the adjacent grains allow the overlapping spectrum to be separated in a systematic procedure, which is based on the “spatial difference” method combined with the principle of “Multiple-variant Statistic Analysis”. Indeed the ~1nm thick amorphous film is made of silicate, hence the SiO4 bonding must be quite different from the Si-N4 bonding. As illustrated in Fig. 1, this approach separates not only the specific ELNES for grain boundary film, but also the associated volume, which results in corresponding chemical width and composition for the grain boundary film [1]. This is effectively an orthogonization process to find two independent vectors in the vector-space of EELS spectra. This indirect approach could even obtain atomic level EELS spectrum in the pre-Cs era in a suitable case [2].

The exclusive EELS spectrum reveals that the amorphous film is made of silicon oxynitride instead of silicate. Ratio of N:O remains close to 1:2 in several doping systems, but it could vary when the sintering could not fully densify [3]. More detailed study reveals further that dopant cations segregated to the grain boundary film in a trend different from the film composition, and the grain surfaces play also a role in the structure and chemistry of amorphous films [4].The ELNES separation could also be applied to phase boundary to reveal the exclusive spectrum corresponding to the boundary width [1]. It leads to finding amorphous double-layer film at the boundary between a grain and a devitrified triple pocket phase in a doped Si3N4 (Fig. 2). More details and implication of such a phenomenon will come soon.

[1] Ultramicroscopy 76 (1999), 159; 173.

[2] Ultramicroscopy 78 (1999), 22.

[3] Mater. Trans. 45 (2004), 2091.

[4] Int. J. Mater. Res. 101 (2010), 66.

 

Fig. 1: ELNES separation results in the specific spectrum of grain boundary film that contains no overlapping signal from the adjacent grains, thanks to their difference in bonding nature [1].

Fig. 2: EELS line-scan across triple pocket in a La2O3-doped Si3N4 to reveal the double-layer for amorphous film at the two-phase boundary. The EELS spectra were processed according to their ELNES differences between both phases as described in [1].
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-2723 Probing the modulated structure and Li ordering in Li0.5-3x Nd0.5+x TiO3 using atomic-resolution STEM and EELS

Zhu Y.1, Withers R. L.2, Bourgeois L.1, Dwyer C.3, Etheridge J.1
1Monash Centre for Electron Microscopy (MCEM) and Department of Materials Engineering, Monash University, VIC 3800, Australia, 2Research School of Chemistry, College of Physical and Mathematical Sciences, The Australian National University, Canberra, ACT, Australia, 3Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, and Peter Grunberg Institute, Forschungszentrum Julich, D-52425 Julich, Germany
ye.zhu@monash.edu

Perovskite-related Li0.5-3xLn0.5+xTiO3 (Ln = lanthanides) are among the best Li ion conductors, with potential for use as Li-battery solid electrolytes. In particular, Li0.5-3xNd0.5+xTiO3 (LNT) exhibits spontaneous self-assembled nano-domains with well-defined 2-D periodicity, which appears to be tuneable by Li concentration. Its potential application as a template for the assembly of nanostructures has attracted significant interest in the nature of the 2-D modulated nanostructure. However, due to the complexity of the structure together with difficulties in detecting light and highly-mobile Li-ions and Nd-vacancies, the nano-domain structure of LNT remains a subject of controversy.

In this work, we reveal further the intriguing nanoscale structure of LNT using scanning transmission electron microscopy (STEM) and electron energy loss spectroscopy (EELS). We used an aberration-corrected Titan 80-300 S/TEM, which can give a sub-Ångström STEM probe with sufficient intensity for both atomic-resolution imaging and EELS mapping. The optimized 300kV STEM-EELS setup on our instrument is sensitive enough to measure the Li distribution in [100]-oriented LNT, when care is taken to minimise beam damage. Using Ti-L and Nd-M EELS signals (Fig. 1(c)), we reveal the atomic-scale elemental distribution in LNT along both [100] and [001] zone-axes (Fig. 1(d-e)). The [100]-oriented Nd-map in Fig. 1(d) shows that along the [001] direction (vertical direction) only every two perovskite unitcells contain one Nd atomic-layer, indicating that the other A-cation sites are occupied by Li ions. This observation provides the direct evidence of the Nd/Li cation-ordering in alternating (001) planes of LNT. Similar atomic-resolution EELS mapping on defects, such as anti-phase boundaries and 90° boundaries shown in Fig. 2, uncovers that the Ti-sublattice remains unchanged around the defects, while Nd-ions are restructured to form different types of boundaries.

Based on atomic-resolution STEM imaging on [001]-oriented LNT, a strain modulation associated with the twinning of the Ti-O octahedral tilt system is identified, which is at least partly responsible for the formation of the 2-D modulated structure. In addition, we image directly the tilted octahedra in LNT using annular bright-field (ABF) imaging in STEM and measure the tilt angle to be 15°. Moreover, we show that the nano-domain contrast diminishes when STEM collection angle increases - an indication that strain contrast is dominant, rather than chemical contrast. These two observations suggest that strain plays a significant role in the nano-domain formation.

This work was supported by the Australian Research Council (ARC) grant DP110104734. The FEI Titan at Monash Centre for Electron Microscopy was funded by the ARC Grant LE0454166.

Fig. 1: Atomic structure of LNT in (a) [110] and (b) [001] zone axes. (c) EEL spectrum taken from LNT showing Ti-L, O-K, and Nd-M edges. (d) Atomic-resolution STEM-EELS maps of [100] LNT. (e) Atomic-resolution STEM-EELS maps of [001] LNT.

Fig. 2: Atomic-resolution STEM-HAADF image and EELS maps showing defects in LNT: (left) 90° grain boundary and (right) anti-phase boundary.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-2345 High-resolution STEM-EELS characterization of co-doped lanthanum niobate proton conductors

Palisaitis J.1, Ivanova M. E.2, Meulenberg W. A.2, Mayer J.1
1Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons (ER-C), Forschungszentrum Jülich GmbH, Jülich and Central Facility for Electron Microscopy (GFE), RWTH Aachen University, Aachen, Germany, 2Institute of Energy and Climate Research (IEK-1), Forschungszentrum Jülich GmbH, Jülich, Germany
j.palisaitis@fz-juelich.de

Lanthanum niobate (LaNbO4) is a promising material system in application as a novel proton conductor due to its combination of properties allowing to withstand high levels of humidity and CO2 containing atmospheres [1]. The materials conductivity can be further enhanced by suitable choice of the type and amount of dopants and in particular a co-doping strategy is highly attractive as a route towards improving functional properties of the LaNbO4 [2].
We present a detailed study of co-doping influence on the microstructure and compositional homogeneity of LaNbO4 proton conductor using combination of high-resolution STEM-HAADF imaging and spectroscopy techniques.
Series of co-doped LaNbO4 alloys were synthesized, where Ca, Ba or Sr acted as substitutes on La-sites, and Ge, Ti or Al on Nb-sites. As an example, 1%-Ca and 1%-Ti co-doped LaNbO4 proton conductor with nominal formula La0.99Ca0.01Nb0.99Ti0.01O4-δ (LCNT) is discussed in more detail in the following. A low-magnification STEM-HAADF image acquired from an as-sintered sample is shown in Fig. 1. LCNT is predominantly composed of low temperature monoclinic, randomly oriented and well packed large grains showing hexagonal shapes and stress-induced stripy patterns. Strong elemental contrast STEM-HAADF images provided indication for curved shape secondary phase grains present in the host matrix, like grains denoted by letter ‘S’ in Fig. 1. Core-loss EELS spectra revealed the compositional nature and established the presence of a dopant-rich phase in LCNT (see Fig. 2). Furthermore, valence EELS spectroscopy confirmed the same chemical nature for all studied secondary phase grains (not shown). Fig. 3 shows a high-resolution STEM-HAADF image together with the corresponding electron diffraction pattern acquired from one of the ‘S’ grain oriented along a low index zone axis. The crystal structure of the secondary phase grain is highly ordered, layered and defect-free in the observed projection. The layering was characterized by a representative periodicity of ~12.92 Å perpendicular to the layers. High spatial resolution core-loss EELS line profiles of the Ca-L23, Ti-L23, and La-M45 absorption edge intensities are shown in Fig. 4. The ‘S’ grains contain La-rich layers (also accommodating some amount of Ca) which are separated by Ti-Ca rich layers accommodating some amount of La. By correlating experimental data with crystal structure models, we identified that the secondary phase grains possess the lanthanum titanate (La2Ti2O7) crystal structure with substantial amounts of Ca incorporated into them, where Ca partly substitutes La.

References:
[1] R. Haugsrud, T. Norby, Nature Materials 5 (2006) 193.
[2] M. Ivanova, S. Ricote, W. Meulenberg, R. Haugsrud, M. Ziegner, Solid State Ionics 213 (2012) 45.

Financial support by the Helmholtz-Society in the framework of the Portfolio Project MEMBRAIN is gratefully acknowledged.

Fig. 1: Overview STEM-HAADF image acquired from La0.99Ca0.01Nb0.99Ti0.01O4-δ sample showing the presence of secondary phase grains denoted by ‘S’.

Fig. 2: Core-loss EELS spectra recorded from the host matrix and the ‘S’ grain.

Fig. 3: High-resolution STEM-HAADF image acquired from the ‘S’ grain together with corresponding electron diffraction pattern in the inset.

Fig. 4: High spatial resolution core-loss EELS elemental line profiles plotted as a function of probe scanning position perpendicular to the layers in the ‘S’ grain.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-2601 TEM and STEM Investigations of SrO-doped Sr(Ti,Nb)O3-δ Thermoelectrics

Čeh M.1,2, Jerič M.1, Ow-Yang C.3, Gülgün M. A.3
1Department for Nanostructured Materials, Jožef Stefan Institute, Ljubljana, Slovenia, 2Center for Electron Microscopy and Microanalysis, Jožef Stefan Institute, Ljubljana, Slovenia, 3Materials Science & Engineering, Sabanci University, Tuzla, Istanbul, Turkey
miran.ceh@ijs.si

Sr(Ti1-xNbx)O3-δ solid solutions are promising materials for n-type high-temperature thermoelectrics1. In our study 10 mol% of SrO excess was added to stoichiometric composition with x=0.2 in order to introduce Ruddlesden-Popper (RP) type-planar faults2,3 into the material, thus minimizing thermal conductivity. TEM and STEM were used to study possible ordering and/or distribution of Nb on Ti sites in the perovskite structure. All results were obtained in a Jeol ARM-200F with a CFEG and Cs probe corrector. HAADF imaging was performed at angles from 70 to 175 mrad, while ABF imaging from 11 to 23 mrad. EDXS spectra were acquired using JEOL Centurio Dry SD100GV SDD Detector.
RP planar faults, as viewed along [001] zone axis, are shown in HRTEM micrograph in figure 1. The commonly observed number of perovskite unit cells between the planar faults is >2, which corresponds to various homologous compounds with the formula Srn+1(Ti,Nb)nO3n+1. However, solid solution Sr(Ti,Nb)O3-type grains with no RP faults can also be observed (bottom inset in Fig. 1). A HR HAADF STEM image of ordered RP faults (Fig. 2) shows that while the measured intensities of individual Sr atomic columns along a single fault do not scatter significantly, the (Ti,Nb)O atom columns exhibit quite large differences in measured intensities, thus indicating significant variation in Nb and Ti content within a single atom column. Quantitative analysis of measured intensities is in progress. The comparison between simultaneously acquired HAADF and ABF images of a single RP fault is shown in figure 3. While pure oxygen atomic columns cannot be resolved in the HAADF image, they can be readily observed using ABF imaging. The positions of oxygen atom columns along the planar faults are in full agreement with the structural model of a RP planar fault. Additional information on Nb distribution within perovskite matrix/RP faults was obtained by EDXS. While low magnification EDXS mappings show enrichment of Sr at RP faults accompanied by a corresponding decrease in Ti and Nb content, atom-resolved EDXS mappings confirm that individual mixed (Ti,Nb)O atom columns contain different Nb content (annotated atom column). Additionally, the spot EDXS line analysis (net counts) again shows much larger scatter in accumulated net counts for Ti as compared with Sr. The results being presented clearly show that no Nb is incorporated into the SrO RP faults and that the Nb is inhomogeneously incorporated within (Ti,Nb)O atom columns.

References
1. S. Ohta et al., App.Phy.Lett., 2005, vol. 87, p. 092108.
2. S.N. Ruddlesden, P. Popper, Acta cryst., 1958, vol. 11, p. 54 -55.
3. S. Sturm et al, J. Mater. Res., 2009, Vol. 24, No.8, p. 2596-2604.

The authors acknowledge financial support from the Scientific and Technological Research Council of Turkey (TÜBITAK) under Fellows Program and from EU under Seventh Framework Programme under grant agreement n°312483 (ESTEEM2).

Fig. 1: HRTEM micrograph of RP faults as seen in the [001] zone axis with the corresponding diffraction pattern. The lower inset shows coexistence of Sr(Ti,Nb)O3 grains with grains containing RP faults.

Fig. 2: HR HAADF STEM micrograph of ordered RP faults. Sr and mixed (Ti,Nb)O atomic columns are clearly distinguished due to different intensities. Large scatter in measured intensities of (Ti,Nb)O atom columns is observed.

Fig. 3: HR HAADF and ABF STEM micrographs of a single RP fault. The positions of oxygen atomic columns in APB micrograph are clearly resolved at the faults as well as within the perovskite matrix viewed along the [001] zone axis.

Fig. 4: Low magnification and atom resolved EDXS mappings of RP fault(s). The latter clearly shows atom columns with different Ti and Nb content (marked). Zig-zag pattern of Sr atom columns at the fault is also resolved. EDXS line analysis is showing large scatter in Ti Kα net counts across perovskite matrix.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-2632 Microstructures of Nanoscale Mixed Phase BiFeO3 Thin Film with Electrically Controllable Spontaneous Magnetism

Huang R.1,3, Duan C.1, Chu Y.2, Ikuhara Y.3,4
1East China Normal University, Shanghai, China, 2National Chiao Tung University, Hsinchu, Taiwan, 3Japan Fine Ceramics Center, Nagoya, Japan, 4The University of Tokyo, Tokyo, Japan
rhuang@ee.ecnu.edu.cn

BiFeO3 (BFO) is a promising multiferroic for technological applications because its electric polarization is coupled to antiferromagnetic ordering at room temperature. Recently, a strain-driven rhombohedral (R phase) and super-tetragonal (T phase) mixed phase in BFO thin films grown on LaAlO3(LAO) substrates has also been reported to exhibit electrically controllable spontaneous magnetism[1]. To reveal the stability of both T-BFO and R-BFO, the detailed local structures of the BFO/LAO interfaces and the T/R morphotroic phase boundary were investigated by means of state-of-the-art Cs-corrected scanning transmission electron microscopy (STEM) techniques.
Figure 1(a) shows the detailed atomic structure of the R-BFO/LAO interface observed by the high angle annular dark-field (HAADF) STEM. A continuous expansion of the BFO crystal lattice, and displacement of Fe ions occur at coherent super-tetragonal BFO/LAO and rhombohedral BFO/LAO heterointerfaces, measured as c/a ratios given in Fig 1(b). A similar pinned interface transition layer about two unit cells thick was directly observed at both interfaces. The continuous ferroelectric polarization relaxations observed at both T-BFO/LAO and R-BFO/LAO interfaces show different features, which are the result of competition between the tiliting of Fe-O6 octahedra and the displacement of Fe ions, depending on the strain state of BFO thin film [2]. However, the phase boundary between T-BFO and R-BFO is very sharp. The lattice relaxed within 1-2 unit cells and no defects can be observed, as shown in Fig. 2. Such information is important for rationaliz ing the varied and complex phenomena of BFO, and adds to our rapidly growing understanding of the ferroelectric behavior of complex oxide heterointerfaces.

References:
[1] Q. He et al., Nat. Commun., 2(2011) 255.
[2] R. Huang et al., Adv. Func. Mater., 24 (2014) 793.

This work was supported by the National Key Project for Basic Research of China (Grants No. 2013CB922301), NSFC under Grants No. 61125403, PCSIRT, NCET, Shanghai Pujiang talents plan (Grant No. 11PJ1402900), Program of Shanghai Subject Chief Scientist and Fundamental Research Funds for the central universities (ECNU).

Fig. 1: (a) HAADF image of R-BiFeO3/LaAlO3 interface along the [010]c zone axis. (b) c/a ratios of T-BFO and R-BFO as a function of distance from the interface.

Fig. 2: Atomic structure of T/ R phase boundary.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-2730 Initial Growth Behavior of ZnO Nanomaterials on Various 2-D Layered Materials

Jo J.2, Oh H.1, Kim M.2, Yi G. C.1
1Department of Physics and Astronomy, Seoul National University, Seoul 151-747, Korea., 2Department of Materials Science and Engineering, Seoul National University, Seoul 151-744, Korea.
jjhjjh0218@snu.ac.kr

Atomically thin layered crystals isolated by mechanical exfoliation method have exhibited new physical properties and provided novel applications. Moreover, hybrid structures of these 2-dimensional (2-D) layered materials with semiconductor thin films and nanostructures offer additional functionalities, such as flexibility and transferability, thereby greatly extending the applicability to the fabrication of electronic and optoelectronic devices [1,2]. Accordingly, many efforts have focused on nanomaterials growth using 2-D materials as substrates. In order to fabricate such nanomaterials with desired shapes and physical properties, however, the study on the initial growth mechanisms, such as nucleation, nuclei growth, and orientational relationship with substrate, should be accompanied in detail.
Here, we report on the initial growth behavior of nanomaterials on various 2-D layered materials, including graphene, h-BN, and MoS2. “Direct growth and imaging” method was used to image nanomaterials at the early growth stages to avoid unintentional damages arising from conventional transmission electron microscopy (TEM) sample preparation processes, as described in Fig. 1 [3]. In this method, electron-beam transparent 2-D layered materials were exploited as supporting layers for TEM measurements as well as substrates for nanomaterial growth. After 2-D layered materials were transferred onto a TEM grid by mechanical exfoliation, ZnO nanomaterials were grown directly on these 2-D materials by metal-organic chemical vapor deposition and TEM measurements were conducted.
Using this method, we could clearly observe the growth behavior of ZnO nanomaterials on 2-D layered materials. At the initial stage of growth, ZnO clusters, a few tens of nanometer in size, were sparsely observed, and diffraction peaks from ZnO were rarely obtained (Fig. 2). However, they showed different growth behavior as the growth proceeded. Whereas rectangular-shaped and randomly grown nanomaterials were grown on h-BN and MoS2, respectively, only hexagonal-shaped nanomaterials grown along typical [0001] direction were observed on graphene (Fig. 3). Disordered diffraction patterns of ZnO in the insets of Figs. 3a and c suggest that each nanomaterial grew with different growth directions and in-plane orientations on h-BN and MoS2. In addition, dispersive patterns from ZnO in the inset of Fig. 3b indicate no epitaxial relationship with graphene. We believe that this study will lay down the building block for fundamental understanding of the effect of substrates on material growth by bottom-up approach.

[1] K. Chung et al. Science, 330, 655 (2010)
[2] D. I. Son et al. Nat. Nanotechnol. 7, 465 (2012)
[3] J. Jo et al. Advan. Mater. (2014) (online published)

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF 2013034238), and the Ministry of Science, ICT & Future Planning (2013K1A3A1A32035597).

Fig. 1: Schematic diagram of the experimental technique for growing ZnO nanomaterials on 2-D layered materials and performing TEM measurements. 2-D materials were transferred onto a TEM grid by mechanical exfoliation. Afterward, ZnO nanomaterials were grown directly on these 2-D materials by metal-organic chemical vapor deposition and observed by TEM.

Fig. 2: Initial stage of ZnO nanomaterials growth on various 2-D layered materials. Bright-field images were obtained for ZnO grown on (a) h-BN, (b) graphene, and (c) MoS2 for 2 min, and the insets represent their corresponding SAED patterns. ZnO clusters with a size of a few tens of nanometer were sparsely observed. A 4.15-μm-diameter aperture was used.

Fig. 3: Morphology of ZnO nanomaterials grown on various 2-D layered materials. Bright-field images were obtained for ZnO grown on (a) h-BN, (b) graphene, and (c) MoS2 for 15 min, and the insets represent their corresponding SAED patterns. These images show different growth behavior of ZnO on different 2-D materials. A 4.15-μm-diameter aperture was used.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-2795 Atomic resolution studies of grain boundaries in ionic conducting materials by aberration corrected STEM-EELS and DFT calculations

Sanchez-Santolino G.1, 2, Salafranca J.1, 3, 2, Frechero M. A.1, Rocci M.1, Schmidt R.1, Rivera-Calzada A.1, Mishra R.5, 3, Pantelides S. T.3, 5, Varela M.3, 1, 2, Pennycook S. J.4, Santamaria J.1, Leon C.1
1GFMC, Dept. de Fisica Aplicada III, Universidad Complutense de Madrid, 28040 Madrid, Spain, 2Instituto Pluridisciplinar, Universidad Complutense de Madrid, 28040 Madrid, Spain, 3Materials Science and Technology Div., Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA, 4Dept. of Materials Science and Engineering, University of Tennessee, Knoxville, TN 37996, USA, 5Dept. of Physics and Astronomy, Vanderbilt University, Nashville, TN 37235, USA
gabriel.sanchez.sant@ucm.es

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-3068 DETERMINATION OF STRUCTURE AND CHEMISTRY OF LONG-PERSISTENCE STRONTIUM ALUMINATE PHOSPHOR COMPOUNDS IN ABERRATION-CORRECTED TEM/STEM

Inan G.1, Ceh M.2,1, Sturm S.2,1, Ow-Yang C. W.1
1Sabanci University, Istanbul, Turkey, 2Jozef Stefan Institute, Ljubljana, Slovenia
gulizinan@sabanciuniv.edu

Representing a source of short-term stored energy, strontium aluminate phosphor compounds of nominal stoichiometry (SrO)•(Al2O3)2 co-doped with 1 mol% Eu2+ and 1 mol% Dy3+ (SA2ED) exhibit long persistence that is even further extended by the incorporation of boron1. To elucidate the effect of boron on afterglow persistence, we synthesized the phosphor powders using a sol-gel (i.e., modified Pechini) method2 and investigated the chemistry and structure by applying high-resolution STEM imaging, energy dispersive X-ray (EDX) spectroscopy, and electron energy-loss spectroscopy (EELS). Large single-crystal grains were analyzed from as-reduced powders suspended on carbon-coated lacey formvar on copper support grids. Individual crystalline particles were tilted onto a low-index [0001] zone axis and imaged in both high resolution TEM and STEM, using a JEOL JEM-ARM 200CF, equipped with a cold field emission tip and a probe-side Cs aberration corrector. High-angle annular dark-field (HAADF) images were formed using an annular detector with an inner diameter of 70 mrad and an outer diameter of 175 mrad, while annular bright-field (ABF) images were obtained from an annular detector of 11-mrad inner diameter and 23-mrad outer diameter. EDX spectra were collected using a JEOL Centurio Dry SD100GV SDD detector. EELS analysis was enabled by a Gatan GIF Quantum ER spectrometer.
Rietveld refinement of XRD spectra obtained from the powders revealed a mixture of (SrO)4•(Al2O3)7, (SrO)•(Al2O3)2 , and (SrO)•(Al2O3)6 phases. Single crystal particles of the (SrO)•(Al2O3)6 phase were the most stable and allowed for tilting onto the [0001] zone axis for qualitative identification of the atomic columns in HAADF and ABF micrographs. Quantitative image simulations of the measured intensities are in progress. Local variations were observed in the energy loss near-edge fine structure of the B-K, O-K, Al-L2,3 edges.

References

1. A.V. Uluc, Sabanci University, M.Sc. Thesis, 2008.

2. M.G. Eskin, Sabanci University, M.Sc. Thesis, 2011.

The authors acknowledge financial support from the Scientific and Technological Research Council of Turkey (TÜBITAK) from project #110M426, #112M360, and project #212T177.

Fig. 1: High-resolution HAADF STEM micrographs of a SrO•(Al2O3)6 crystal tilted onto the [0001] zone axis. (a) Experimental image, (b) noise filtered image. Inset: HRTEM.

Fig. 2: High-resolution ABF STEM micrographs of a SrO•(Al2O3)6 crystal tilted onto the [0001] zone axis. (a) Experimental image, (b) noise filtered image.

Fig. 3: The positions of the oxygen atomic columns in the ABF micrograph are clearly resolved in the SrO•(Al2O3)6 crystal matrix, as viewed along the [0001] zone axis.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-5-O-3359 Local tetragonality of epitaxial BaTiO3 thin films on Si for ferroelectric applications

Schamm-Chardon S.1, Denneulin T.1, Hÿtch M.1, Mazet L.2, Bachelet R.2, Saint-Girons G.2, Dubourdieu C.2
1CEMES-CNRS and Université de Toulouse, nMat group, Toulouse, France, 2Institut des Nanotechnologies de Lyon, CNRS, Ecole Centrale de Lyon, Ecully France
sylvie.schammchardon@cemes.fr

Ferroelectric oxides integrated on a semiconductor substrate are of particular interest for various applications such as memory or logic devices1,2. Among the ferroelectric materials, BaTiO3 is an attractive candidate for large-scale applications compared to Pb- or Bi-based compounds. It is a well-known perovskite largely studied for its dielectric, piezoelectric and ferroelectric properties. However, the direct epitaxy of BaTiO3 on silicon is challenging due to the oxidation of the silicon surface and due to the large lattice mismatch (4%) and thermal expansion mismatch between the oxide and the semiconductor. Moreover, the control of the ferroelectric polarization is a crucial topic for the targeted applications. The polarization must point perpendicular to the Si channel.

In this study, a quantitative analysis of high-resolution transmission electron microscopy (HRTEM) images using the geometric phase analysis (GPA)3 is proposed in order to support the growth strategy of epitaxial BaTiO3 films with the desired orientation, i.e. with the c-axis of the tetragonal structure perpendicular to the Si substrate. With GPA, maps of the strain in the BaTiO3 films with respect to the Si substrate are determined with a high precision (0.1%) at the nanometric scale (1-2nm). Strain maps with improved precision (0.05%), 5 nm spatial resolution and with a large field of view (1 μm) are also proposed for selected samples, using dark field electron holography4. From these maps, the local lattice parameters and thus the tetragonality (c/a ratio) of the BaTiO3 films can be evidenced5. The HRTEM images and holograms were acquired on a Hitachi HF3300S microscope (I2TEM-Toulouse) fitted with the new aplanatic spherical aberration corrector B-COR from CEOS and a 4096x4096 camera.

Growth of the epitaxial BaTiO3 films was performed by molecular beam epitaxy (MBE) using an SrTiO3 epitaxial buffer layer to reduce both thermal and lattice mismatches on Si (001). Different process parameters like the growth temperature and cooling conditions were explored to optimize the quality of 20 nm thick BaTiO3 films and to minimize the SiO2 interfacial layer regrowth between Si and the SrTiO3 buffer. The influence of the thickness of the SrTiO3 buffer layer on the growth mode of 10 nm thick films was examined. Finally, in order to investigate the first steps of the BaTiO3 formation, the growth behavior of thin films with thicknesses ranging from 1.2 to 4 nm (3 to 10 monolayers) was considered.

1. J. Scott, Ferroelectric memories (Berlin: Springer), chapter 2 and 12 (2000)

2. S. Salahuddin et al., Nano Lett. 8(2), 405 (2008)

3. M.J. Hytch et al., Ultramicroscopy 74, 131 (1998)

4. MJ Hÿtch et al., Nature 453, 1086 (2008)

5. C. Dubourdieu et al., Nature Nanotechnology 8, 748 (2013)

This work has been supported by the French National Research Agency under the reference No. ANR-10-EQPX-38-01. The authors acknowledge the "Conseil Regional Midi-Pyrénées" and the European FEDER for financial support within the CPER program.

Fig. 1: HRTEM image of a MBE grown BaTiO3 film on a Si substrate using an SrTiO3 epitaxial buffer layer ; Strain maps in the BaTiO3 film along two perpendicular directions ; Corresponding ratio profile of the retrieved BaTiO3 lattice parameters revealing the tetragonality of the film with the largest parameter (c axis) being perpendicular to the substrate.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-1520 TEM investigation of wound highly porous oxide (WHIPOX) ceramic matrix composites (CMC)

Müller M. G.1, Mayer J.1, Eils N.2
1Central Facility for Electron Microscopy, RWTH Aachen University, Aachen, Germany, 2German Aerospace Center, Cologne, Germany
mueller@gfe.rwth-aachen.de

Oxide ceramics are widely used as structural materials for long-term high-temperature applications in oxidizing atmospheres. Monolithic ceramics are not suitable for many applications due to their brittle fracture behavior. This brittleness can be overcome by fiber reinforcement of ceramics. The so called ceramic matrix composites (CMCs) show pseudoplastic behavior, if the fibre-matrix bonding is relatively weak [1]. Weak fibre-matrix bonds can be achieved e.g. by porous or low toughness fibre-coatings. A less expansively alternative is according to Lange et al., the use of a highly porous Si3N4 or mullite matrix instead of a dense matrix and a weak-fibre-matrix interphase [2]. In this work a CMC with Al2O3-fibres and a porous Al2O3-matrix, produced at the German Aerospace Center Cologne, the so called WHIPOX (wound highly porous oxide) CMC, which is a promising material for the application in gas turbines, was characterized by Transmission Electron Microscopic (TEM) methods. The WHIPOX-material was manufactured by filament winding as described in [3]. Thin cross sections from the samples embedded in an epoxy-resin were prepared by the Focused Ion Beam (FIB) technique. The lamellae were investigated using a FEI Tecnai F20 (TEM) operated at 200 kV. For TEM investigation of the sinter degree within the Al2O3-matrix and of the matrix-fibre interface TEM bright field images, selected area diffraction patterns as well as STEM HAADF images and EDX spectra have been acquired experimentally. In addition STEM Tomography has been performed. Figure 1 shows the overview STEM BF image of a matrix area (sintering temperature 1200 °C) enclosed by two fibers. The Al2O3-grain size within the fibers is homogeneous with a maximum diameter of about 200 nm, whereas the Al2O3-grains within the porous matrix show a maximum diameter of about 700 nm. The STEM HAADF image in Figure 2 shows the existence of small, below 100 nm in size, bright ZrO2-particles with homogeneous distribution within the matrix, which can be found also between matrix-grains and between matrix-fibre-grains. The ZrO2-particles got into the matrix during preparation. The comparison with a sample which was manufactured without ZrO2, leads us to the assumption that the ZrO2-particles decrease the sintering activity and hence have a positive effect on the matrix-structure. Figure 3 shows a tomogram, which was reconstructed from a STEM ADF tilting series (-60° till +60°). Besides the influence of the ZrO2-particles also the influence of the sintering temperature onto the matrix-structure was investigated.

References
[1] K. K. Chawla, Springer, New York, 2012.
[2] F. F. Lange, W. Tu, C. A. G. Evans, Mat. Sci. Eng. A 195 (1995) 145-150.
[3] M. Schmücker, P. Mechnich, W. Krenkel (Ed.), Weinheim, 2008.

The authors kindly acknowledge the financial support through funding of the EU and North Rhine-Westphalia within the Ceramic Materials for Energy Research (CeraMER) project.

Fig. 1: STEM BF image of the matrix area between two fibers

Fig. 2: STEM HAADF image of the matrix area between two fibers

Fig. 3: Reconstructed tomogram: Al2O3-matrix grains and Al2O3-fibre (blue) und ZrO2 particle (red)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-1528 TEM/STEM investigation of Ni/YSZ boundary in solid oxide fuel cell

Liu S. S.1, 2, Matsumura S.1, 2, 3, Koyama M.1, 2, 4
1INAMORI Frontier Research Center, Kyushu University, Fukuoka, Japan, 2CREST, Japan Science and Technology Agency, Tokyo, Japan, 3Research Laboratory for High Voltage Electron Microscopy, Kyushu University, Fukuoka, Japan, 4International Institute for Carbon-Neutral Energy Research, Kyushu University, Fukuoka, Japan
ssliu@ifrc.kyushu-u.ac.jp

Solid oxide fuel cell produces electricity directly from chemical reactions between a fuel and an oxidant with high efficiency, fuel flexibility and low emissions. Ni/yttria stabilized zirconia (Ni/YSZ) cermet is now the main anode material for commercial products. Ni/YSZ interface and Ni/YSZ/pore triple-phase boundary (TPB) play important roles in SOFC. For specially designed boundaries, the crystallographic orientation relationship between Ni and YSZ has been investigated by transmission electron microscopy (TEM), and scanning TEM (STEM). The orientation relationships are found to be easy and contact surfaces are low index, such as Ni[1-10]//YSZ[1-10], Ni(111)//YSZ(111) and Ni[1-10]//YSZ[001], Ni(111)//YSZ(100). For conventional cells, little is known on the orientation relationship, which is quite important for the performance. In this study, we used a conventional Ni/YSZ anode of a button cell and focused on the TEM and STEM investigations at the boundaries.

Ni/YSZ cell was prepared by conventional screen-printing/sintering/reduction procedures. The TEM specimen was lifted out in a focused ion beam & scanning electron microscopy (FIB-SEM, FEI Quanta 200i 3D). An advanced mill machine (Fischione NanoMill 1040) was used for the post-FIB processing to remove damage layers. Microstructure observations were done using two machines: JEOL JEM3200FSK and ARM200F. Elementary mappings were used to identify the phases. High resolution TEM (HRTEM) and atomic resolution STEM images were taken at different Ni/YSZ interfaces or TPBs.

As an example, Fig. 1 shows the diffraction patterns of YSZ(a), Ni(b) and interface(c), respectively. Fig. 2a is the bright-field image of region of interest while Fig. 2b shows the elemental mapping of Ni. These two images agree well with each other and the boundary of Ni and YSZ is at the center line. The STEM image in Fig. 2c shows the boundary clearly. By evaluating Fig. 1 and Fig. 2c, the facets like (111) and (113) of YSZ could be indexed easily. Meanwhile, Ni is very close to [-112] direction. Its lattices could also be indexed with the help of IFFT (Inverse Fast Fourier Transform). The orientation relationship is Ni[-112]~//YSZ[1-10], Ni(220)~//YSZ(113), Ni(311)//YSZ(111). Step contact surfaces were expected. The misfit between three layers of Ni(1-11) and two layers of YSZ(-1-11) is only 2.8% while it is 3.2% between three layers of Ni(311) and one layer of YSZ(111). So Ni and YSZ match well.

Other cases will be presented. Compared with specially designed boundaries, the orientation relationship between Ni and YSZ in a conventional cell was found to be much more complicated. Misorientation could be frequently found.

The authors greatly acknowledge the support from members (especially Dr. Y. Shimada and Dr. T. Yamamoto) in HVEM lab of Kyushu University. Activities of INAMORI Frontier Research Center are supported by KYOCERA Corporation.

Fig. 1: Diffraction patterns of YSZ (a), Ni (b) and interface(c) along YSZ zone axis.

Fig. 2: Ni/YSZ boundary: left is Ni and right is YSZ. (a) bright-field (BF) image; (b) elemental mapping of Ni; (c) atomic resolution STEM image, inset is the IFFT image of Ni.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-2408 Microstructure evolution of NiO-YSZ cermet sintering process by HRSEM

Liu S. S.1, 2, Matsumura S.1, 2, 3, Koyama M.1, 2, 4
1INAMORI Frontier Research Center, Kyushu University, Fukuoka, Japan, 2CREST, Japan Science and Technology Agency, Tokyo, Japan, 3HVEM lab, Kyushu University, Fukuoka, Japan, 4I2CNER, Kyushu University, Fukuoka, Japan
ssliu@ifrc.kyushu-u.ac.jp

Solid oxide fuel cell (SOFC) is considered as a highly efficient device to convert chemical fuels directly into electrical power through the electrochemical oxidation of fuels in the anodes, typically NiO-YSZ (yttrium-stabilized zirconia). The synthesis procedure will influence the microstructure and further the performance. Sintering is one of the most important procedures. Although quite a lot of work has been done on the shrinkage of NiO or YSZ as well as the microstructure of cells sintered at high temperature (>1200 °C), little is known on the microstructure evolution of electrodes during the sintering process. Herein, we use high-resolution scanning electron microscopy (HRSEM) to observe the microstructure of NiO-YSZ cells after sintering to different temperatures.
Four NiO-YSZ half-cells were prepared by using ethyl cellulose as a binder or pore former through the screen-printing technique (C1). C1 was sintered in air to 700 °C (C2), 1100 °C (C3), 1250 °C (C4) and 1400 °C (C5) respectively without holding, as well as 1400 °C with holding for 3 h (C6). The heating/cooling rates were 2 °C/min. The surfaces and interfaces morphology was observed in SEM (Zeiss ULTRA55) with four detectors: lateral secondary electron (SE), InLens SE, energy selective backscatter (EsB) and angle selective backscatter (AsB).
In Fig. 1(C1 & C2), the particles with sharp edges in SE images correspond to the gray colour in the ESB images and they are NiO. NiO particles have relatively larger sizes than YSZ particles, which are apparently round shape. ESB signal gives planar image and it is difficult to see the boundary between two particles of one phase if they are closely connected. The connection of particles becomes closer in C2 than that in C1 since ethyl cellulose is already burned away at 700 °C and the space is easily occupied by fine YSZ or NiO. The morphology of C3 is obviously changed. Different particles start to sinter together and form short skeletons. The porosity increases significantly compared with C2. In Fig. 1(C4), the ESB and ASB images are compared. The ASB signal contains not only the contrast information as ESB, but also the morphological information. So it is favourable to separate connected particles of one phase in ASB image. Sintering becomes significant and porous skeleton is formed with pore size ranging from ~100 nm to ~2 μm. For C5 and C6, only the ASB images are shown. The sintering continues and the skeleton becomes coarser and stronger from C4 to C6. The porosity is decreasing due to the coarsening and shrinkage of particles. Also, it is noticeable more and more YSZ particles migrate onto the top surface to form larger networks.

The particle size distributions as well as the activition energy were evaluated based on these images.

The authors greatly acknowledge the support and guidance by Prof. Hiroshige Matsumoto, Dr. Yuji Okuyama and Dr. Takaaki Sakai for cell preparation. The authors also acknowledge the support from KYOCERA Corporation.

Fig. 1: SEM images of the surfaces of C1 ~ C6. Images were taken by different detectors at the same position. YSZ: bright; NiO: gray.
Fig. 2:
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-1576 Element/Site-Selective Local Ligand Analysis using High-Angular Resolution Electron Channeled Fluorescent Spectroscopy

MUTO S.1, Ohtska M.2, ICHIKAWA T.2, TATSUMI K.1, BOSMAN M.3, YAMANE H.4
1EcoTopia Science Institute, Nagoya University, Nagoya 464-8603, Japan, 2Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan, 3Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602, 4Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan
s-mutoh@nucl.nagoya-u.ac.jp

The current state-of-the-art scanning transmission electron microscope (STEM) equipped with aberration correctors allows it handy for atomic scale imaging and elemental/electronic structural analysis, due to its high-brightness electron source and highly focused subatomic probe size available. The technique inevitably leads to a drawback associated with a high density of focused electron probe and its small illuminating area (i.e., a small number of sampling points), so that the probe can drill the sample or otherwise the obtained data may contain high level noise accordingly. We have hence been engaged in an alternative microanalysis method, instead of exploiting the atomic resolution in real space, using inelastic scattering by channeled electrons in a crystal [1]. We have developed an ‘integrated electron spectroscopic STEM’, where electron energy-loss spectroscopy (EELS), energy/wavelength dispersive x-ray spectroscopy (E/WDXS) and cathode-luminescence (CL) are implemented in a single STEM.

The incident electron beam is rocked about a pivot point on a sample, acquiring the spectroscopic intensity data as a function of the incident beam direction (and the momentum transfer vector in EELS) (Ionization channeling pattern: ICP). The sample orientation and beam direction is monitored by the rocking image recorded by the ADF detector. The present method exploits site selective information of the material associated with different electron densities propagating along the specific atomic planes/columns by varying Bloch wave symmetries excited in the crystalline sample.

We have examined Eu/Y doped Ca2SnO4 with red emissions associated with f-f transitions: Eu(Y) occupies the Ca and Sn sites by the ratio of 7:3 (3:7) determined by EDXS (cf., Fig. 1), both taking the tri-valent state by EELS [2] and the Ca site with the asymmetric ligand as the active light emitting site by CL (Fig. 2). It is noted that the electric dipole transition (615 nm: 5D0-7F2) changed in its intensity with the diffraction condition, while the magnetic dipole transitions (5D0-7F1, 5D1-7F2) were independent of the diffraction condition. The present results directly support the conventional idea [3] that Eu3+ occupying the asymmetric Ca site is a primary light emitting specie induced by the enhanced electric dipole moment.

[1] K. Tatsumi, S. Muto and J. Rusz, Microsc. Microanal. 19 (2013) 1586; K Tatsumi and S. Muto, J. Phys.: Condens. Matter 21 (2008) 104213.

[2] Y. Fijimichi et al, J. Solid State Chem. 183 (2010) 2127; S. Muto et al, Opt. Mater. 33 (2011) 1018.

[3] H. Yamane et al, J. Solid State Chem. 181 (2008) 2559.

A part of this work was supported by a Grant-in-Aid on Innovative Areas "Nano Informatics" (Grant number 25106004) from the Japan Society of the Promotion of Science.

Fig. 1: Electron beam rocking pattern (a) and ICPs of Ca-K(b), Sn-L(c), Eu-L(d) and Y-K(e) characteristic x-ray emissions from Eu/Y doped Ca2SnO4 near [100]. It is seen that Eu(Y) mainly occupies the Ca(Sn) site. The crystal structure of Ca2SnO4 is inset lower left corner.

Fig. 2: CL spectra from Ca2SnO4 with the excitation error S of 100 reflection positive (red) and negative (blue).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-1599 Preparation and Characterization of Mixed CeO₂-Nb₂O₅-Bi₂O₃ Nanoparticles

Moore K. L.1, Jefferson D. A.1
1University of Cambridge
klm70@cam.ac.uk

Mixed CeO2-Nb2O5-Bi2O3 nanoparticles were prepared using a resin-gel synthesis [1] and subsequently analysed using transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDS), powder X-ray diffraction (PXRD), and X-ray photoelectron spectroscopy (XPS). These oxides are of importance because of their interesting crystal structures and industrial applications [2-4]. CeO2 and δ-Bi2O3 are both excellent ionic conductors, making them suitable as the electrolyte component in solid oxide fuel cells (SOFCs). Nb2O5 has been used mainly as a solid support to take part in redox reactions and as a solid acid catalyst. As each of the parent metal oxides possesses useful properties, if all three metal ions can be contained in one particle, they could show novel structures and characteristics.


The primary purpose was to form nanoparticles containing appreciable proportions all three metal atoms, as at present there have been no investigations into the quaternary system CeO2-Nb2O5-Bi2O3. The nanoparticles were then probed to see what crystal structures and atomic arrangements were exhibited, as it was assumed that, due to the more relaxed crystal structure prevailing at the nanoscale as compared to the bulk material, alternative cations would be more easily accommodated, hence facilitating the formation of a solid solution.


The mixed metal oxide nanoparticles were synthesized via a resin-gel method using polyethylene glycol (PEG mw=20,000) as the binding agent. The method used low temperatures (350 °C) to calcine the samples and prevent sintering of the nanoparticles. The synthesis was successful in producing mixed metal oxide nanoparticles with Scherrer analysis indicating crystallite sizes of 5-8 nm. EDS analysis showed many of the crystalline nanoparticles contained all three metals, however, elemental compositions calculated from EDS data varied significantly with elemental compositions calculated from XPS data. This evidence suggests that the distribution of elements within the particles is not homogenous, with bismuth showing a strong preference for surface or near-surface sites. Using FFT data from the electron microscope, d-spacings for the crystal lattices could be calculated. These values corresponded to the fluorite, pyrochlore and perovskite phases. This agrees with the PXRD data, which shows peaks indicative of these three phases.

[1] X.Li, H. Zang, F. Chi, S. Li, B. Xu and M.Zhao, Mat Sci Eng B., 10, 209-213, (1993).
[2] E.A Mamedov, Catal Rev., 36, 1-23, (1994).
[3] V.V.Kharton, F. M. Figueiredo, L. Navarro , E. N. Naumovich, A. V. Kovalevsky, A. A. Yaremchenko, A. P. Viskup, A. Carneiro, F. M. B. Marques, J. R. Frade, J Mater Sci, 36, 1105– 1117, (2001).
[4] I.Nowak and M. Ziolek, Chem. Rev., 99, 3603−3624, (1999).

The authors would like to thank the EPSRC for financial support.

Fig. 1: (a) HRTEM micrograph with showing a highly crystalline region of the CeNbBi oxide with d-spacings calculated from the FFT giving values of 6.16 Å, 3.21 Å, and 2.83 Å indicating the pyrochlore structure has been formed. The d-spacings correspond approximately to the {111}, {311} and {400} lattice planes respectively.

Fig. 2: (b) FFT of the region of the CeNbBi oxide shown in (a).

Fig. 3: (c) EDS spectrum of the area imaged in (a).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-1617 Structural examination of bioactive, low cost and environmentaly friendly biogenic hydroxyapatite

Balazsi K.1, Tapaszto O.1, Lee S. W.2, Kim S. G.2, Balazsi C.1,3
1Institute for Technical Physics and Materials Science, Research centre for Natural Sciences, Budapest, Hungary, 21Department of Oral and Maxillofacial Surgery, Collage of Dentistry, Gangneung-Wonju National University, Gangneung, Republic of Korea, 3Institute for Materials Science and Technology, Bay Zoltan Nonprofit Ltd. for Applied Research, Budapest Hungary
balazsi.katalin@ttk.mta.hu

The incidence of bone fractures worldwide is constantly increasing, due to increasing traffic accidents and natural disasters. Filling of bone defect is a significant question in every day clinical work. Hydroxyapatite (HAp) is a one of most used biomaterial. The natural-biological origin HAp have several important advantages: worldwide availability in almost unlimited supply, very low cost of raw materials, utilization of very simple and inexpensive apparatuses, rapid and very efficient transformation from raw materials into HAp.

We prepared the nanostructured HAp from eggshells or seashell in different forms as powder or polymer/HAp composite fibers. High efficient attritor milling was used for HAp production at 5 or 10 h. Structural characterization, mainly morphology, grain size, structure and elemental composition of different types of HAp powders were studied. Structural observation confirmed the average grain size about few 100 nm.  Elemental composition of HAp prepared from eggshells showed  higher magnesium (Mg) content. On the other hand, HAp prepared from seashell showed the higher sodium (Na) and strontium (Sr) contents.

In co-operation with Gangneung-Wonju National University, Korea we compared the biological behavior of different HAp; prepared from eggshell, seashell or synthetic commercial HAp. In vivo and in vitro studies showed that the recylcled HAp form eggshell and seashell showed more regenerated bone volume than that the synthetic HAp. This biogenic HAp can be considered a possible useful bone graft materials.

This work was supported by the Hungarian Scientific Res. Fund, OTKA 105355, PD 101453, the János Bolyai Res. Scholarship of the HAS. The research leading to this result has received funding for European Community Seventh Framework Programme FP7/2007-2013 under grant agreement Nr. 602398 (HypOrth) and the Next-Generation BioGreen21 Program ( no. PJ009013), Rural Devel. Admin., Republic of Korea.

Fig. 1: SEM images of nanosized HAp prepared from eggshells

Fig. 2: SEM images of nanosized HAp prepared from seashells
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-1638 TEM on LiNi0.5Mn1.5O4 Thin-Film Cathodes on Gold-Coated Stainless Steel Substrates

Gries K. I.1, Gellert M.2, Ott A.1, Zakel J.2, Spannenberger S.2, Yada C.3, Rosciano F.3, Roling B.2, Volz K.1
1Philipps-Universität Marburg, Faculty of Physics and Materials Science Center, Marburg, Germany, 2Philipps-Universität Marburg, Faculty of Chemistry, Marburg, Germany, 3Toyota Motor Europe, Advanced Technology 1 Division, Zaventem, Belgium
katharina.gries@physik.uni-marburg.de

Nowadays, lithium-ion batteries are an integral part of our mobile life. The cathode materials used in such batteries should exhibit a high capacity and a high redox potential vs. Li+/Li. A promising material is LiNi0.5Mn1.5O4 (LNMO), which crystallizes in a spinel structure allowing for three-dimensional Li+ ion transport [1]. While the mass production of composite battery electrodes with carbon additives and polymeric binders is well established, thin-film electrodes are mostly prepared via costly sputtering techniques. A cheaper alternative is sol-gel chemistry combined with spin or dip coating [2]. Here, liquid organic precursors are coated onto a substrate and are converted to oxides via a heating process. High temperatures of about 700 °C are needed to form crystalline oxides. However, many metallic substrates used as current collectors cannot withstand such high temperatures in air. In basic research, pure gold current collectors were used successfully [3-5]. As a cheaper alternative, we have deposited Au layers onto stainless steel substrates.
To get information on the structure, the formation and the chemical composition of interlayers between the LNMO films and the gold-coated substrate, we prepared a cross sectional lamella using focused ion beam (FIB). This sample was than analyzed with scanning transmission electron microscopy (STEM) in combination with energy dispersive X-ray spectroscopy (EDX).
These investigations reveal the existence of a mixed oxide interlayer at the LNMO/gold interface containing oxygen, iron, nickel and chromium as can be seen in Fig. 1. The formation of this layer is caused by the diffusion of iron and chromium from the stainless steel substrate through the gold layer. Since the knowledge on the structural and chemical composition is essential for understanding the influence of interlayers on the electrochemical performance of thin-film batteries, STEM/EDX turns out to be a very useful method for this kind of materials.

[1] K. M. Shaju et al., Dalton Trans. 40 (2008) 5471.
[2] H. Liu et al., Journal of Solid State Electrochemistry 8 (2004) 466.
[3] Y. H. Rho, et al., Solid State Ionics 151 (2002) 151.
[4] K. Hoshina, et al., Solid State Ionics 209-210 (2012) 30.
[5] J. C. Arrebola, et al., Journal of Power Sources 162 (2006) 606.

Fig. 1: STEM image and EDX maps of a FIB prepared lamella. Between the LNMO film and the gold layer a mixed oxide interlayer consisting of oxygen, iron, nickel and chromium has developed.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-1662 A Rhombic Dodecahedral Honeycomb Structure with Cation Vacancy Ordering in a γ-Ga2O3 Crystal

Mitome M.1, Kohiki S.2, Nagai T.1, Kurashima K.1, Kimoto K.1, Bando Y.1
1National Institute for Materials Science, 2Kyusyu Institute of Technology
MITOME.Masanori@nims.go.jp

γ-Ga2O3 is a metastable form and is transformed to a stable β form at high temperature. However, it might be a more attractive because it has been reported that the metastable γ form shows some unexpected properties; a blue light emission, room temperature ferromagnetism on Mn doping, and selective catalytic reduction of NO in γ-Ga2O3-Al2O3 system. We found a strange structure in γ-Ga2O3 layer grown on an MgO substrate. Many forbidden reflections including both systematic absences and lattice absences were excited in electron diffraction patterns and phase boundaries were observed in atomic column images using high angle annular dark field images.

We proposed a structure model to explain the experimental results. First, cation vacancy ordering is supposed to distort the γ-Ga2O3 crystal lattice. From an ab initio calculation, it is found that the crystal lattice expands along one axis and matches a substrate lattice. Some grains are formed and alter the directions to reduce the distortion for the other axis. Next, it is supposed that the grains are truncated by {110} lattice planes and form rhombic dodecahedrons. The grains are stacked to form honeycomb with {110} phase boundaries. A TEM image and a diffraction pattern simulated by the structure model reproduce the experimental results consistently. The systematic absences are excited by cation vacancy ordering and the lattice absences are excited by double reflections between grains over the phase boundaries.

The rhombic dodecahedral honeycomb structure with cation vacancy ordering is stabilized by the lattice mismatch between the γ-Ga2O3 crystal and the MgO substrate, and it disappears at a depth of 170 nm from the interface.

Fig. 1: A TEM image of γ-Ga2O3 layer grown on an MgO substrate (left) and electron diffraction patterns taken from circled areas (right). Many forbidden reflections are seen in a middle panel of the diffraction patterns.

Fig. 2: An experimental diffraction pattern (left) and a simulated diffraction pattern based on a structure model proposed in this paper. All forbidden reflections are reproduced in the simulated pattern.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-1753 High-precision chemical analysis and structural determination of functional oxides by STEM-EELS

Kepaptsoglou D. M.1, Azough F.2, Jackson S.2, Freer R.3, Ramasse Q. M.1
1SuperSTEM Laboratory, STFC Daresbury Campus, Daresbury WA4 4AD, U.K., 2School of Materials, University of Manchester, Manchester M13 9PL, U.K.
dmkepap@superstem.org

The physical properties of complex oxides depend crucially on small changes in their chemical composition, as they can dramatically alter the local atomic configuration. These effects often occur at the sub-angstrom scale, due to the presence of point or extended defects for instance, and are therefore too local to be fully understood through bulk characterization methods. Scanning transmission electron microscopy (STEM) is thus an essential tool for their study, thanks to HAADF imaging and EELS chemical mapping. When these techniques are combined with advanced statistical image analysis [1,2] it is possible to determine statistically the chemical distribution of the different sites in these structures across a range of compositions and to relate those to accurately measured small local atomic displacements generated by these compositional changes [3]. Here, we present examples of combined STEM/EELS and statistical image analysis in functional complex oxide systems where the cation contents are varied to alter the materials' properties. For instance, the magneto-electric oxide Ga1-xFexO3 shows an inherent spontaneous polarization which is dependent on the distortions caused by a structural asymmetry of the cation sites [4]. Analysis of the HAADF image intensities as well as detailed 2D EELS mapping is used to demonstrate that in the intermediate compositions, cation intermixing occurs mainly in the Fe2 and Ga2 sites. Template matching analysis of the HAADF STEM images [1] is used to determine the respective displacement of the Fe2 and Ga2 sites in the order of a few pm between different compositions (Fig. 1). Furthermore, the thermoelectric properties of the perovskite system (1-x)SrTiO3-xLa2/3TiO3 can be fine-tuned by adjusting the La/Sr ratio, which in turn affects the distribution of deficient sites. HAADF imaging revealed that in the La-rich side of the compound series there are two distinct A lattice sites with strikingly different column intensities (fig. 2). In combination with 2D EELS mapping it is revealed that one of the two A sites (site A1) is fully occupied by La, while the other (A2) exhibits either a shared occupancy of Sr and La, or is fully La-deficient. Further analysis of the EELS data as well as statistical analysis of the image intensities [2,3] is used to determine the cation concentration at La-deficient sites (Fig. 2). The structural variations derived from image and chemical map analysis are in turn related to fine structure changes in the EELS maps, notably for the Ti L2,3 edge.

[1] P. Galindoet al , Ultramicroscopy 107 (2007), p.1186
[2] M.C. Sarahan et al., Ultramicroscopy 111 (2011), p.251
[3] S. I. Molina, et al., Ultramicroscopy 109 (2009), p.172
[4] A. Roy et al, J. Phys.: Condens. Matter 23 (2011) p. 325902

We gratefully acknowledge the support of EPSRC through award EP/H043462. SuperSTEM is the UK National Facility for Aberration corrected STEM, funded by the EPSRC

Fig. 1: Ball-and-stick model and HAADF images (averaged from a stack of consecutive frames) of two Ga1-xFexO3 compositions, showing the atomic site displacements, template selection and selected and averaged motif, for the HAADF images.

Fig. 2: a) Stack-averaged HAADF STEM image showing two distinct A sites in La0.6Sr0.1TiO3 and intensity profile of the A2 site, (b) histograms of the intensity distributions of the A2 sites from HAADF image and atomically resolved EELS maps.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-1788 Measurement of Piezoelectric Properties of BaTiO3 Nanoparticle

Kim S.1, Kim G.1, 2, Rhyim Y.1, Choi S.1
1Korea Institute of Materials Science, 2Pusan National University
sdkim@kims.re.kr

Recently, various kinds of piezoelectric nanocomposite materials have been developed for self-powered devices, high power capacitors and large-scale flexible sensors [1-2]. The nanocomposite materials are usually fabricated by dispersing piezoelectric nanoparticles on the polymer matrix [3]. Therefore, control of uniformity and density of nanoparticles is a critical issue to fabricate the high quality nanocomposite materials. Besides, the piezoelectric properties of the nanoparticle itself determine the characteristics of the nanocomposite materials. For all that, there are little discussions about piezoelectric properties of a single crystalline nanoparticle. In this study, we investigated the piezoelectric properties of a single BaTiO3 nanoparticle using the indentation technique. In other words, we measured the electrical response when a single crystalline nanoparticle was mechanically compressed by a conductive indenter. To make an accurate indentation of a single nanoparticle whose size is below a few hundred nm, we used a Hysitron PI95 TEM pico-indentation system. Also, we measured the mechanically induced currents on the BaTiO3 nanoparticles with Keithley 6485 picoammeter. Figure 1 is a typical TEM image which shows a cross-sectional view of the indentation process of a BaTiO3 nanoparticle. Controlling the displacements of the conductive diamond indenter, we could induce compressive stress to the single nanoparticle placed on the Au film coated Si wedge fixture and also measure the mechanical (load) induced on the particle (figure2) and electrical charge from the piezoelectric nanoparticle at the same time. From the TEM-indentation results, we calculated the piezoelectric coefficient value (d33) of a single BaTiO3 particle. The d33 value of a 100nm size particle is about 800pC/N which is about 6 times larger than that of polycrystalline BaTiO3 ceramics [4]. The present study reveals that size effect of nanoparticles on the enhancement of the piezoelectric property.

References

[1] P. Kim et al., Adv. Mater., 7 (2007), 1001-1005.

[2] Z. L. Wang and J. Song, Science, 14 (2006), 242-246.

[3] K. Park et al., Adv. Mater., 24 (2012), 2999-3004.

[4] S. Sridhar et al., J. Appl. Phys., 85 (1999), 380-387.

This work was supported by the Global Frontier R&D Program(2013-M3A6B1078872) on Center for Hybrid Interface Materials(HIM) funded by the Ministry of Science, ICT and Future Planning”.(GFHIM- 2013M3A6B1078872)

Fig. 1: A cross-sectional view of the indentation process of aBaTiO3 nanoparticle.

Fig. 2: Compressive load induced on a BaTiO3nanoparticle, (b) a cross-sectional view of the indentation process of aBaTiO3 nanoparticle. The piezoelectric charge generated at surfaces(red dotted line) was measured.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-2035 Electron microscopy study of vitrified products containing chromium-rich tannery ash

Varitis S.1, Kavouras P.1, Kaimakamis G.1, Dimitrakopulos G. P.1, Pavlidou E.1, Vourlias G.1, Komninou P.1, Karakostas T.1
1Department of Physics, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
karakost@auth.gr

Chromium-rich ash produced from incineration of tannery sludge was vitrified using SiO2, CaO and Na2O vitrifying agents in various relative proportions. The aim of this work was to monitor the extent of chromium incorporation inside a vitreous silicate matrix, considering its low solubility in silicate melts. The experimental methods used include X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDS), Conventional and High Resolution Transmission Electron Microscopy (TEM and HRTEM). These methods provide morphological and structural information in a multiscale range, defining the sub-microscopic properties that govern macroscopic performance. The motivation behind this work was to investigate the ability to produce stabilized/solidified glass and glass-ceramic products, either for safe disposal or for construction and/or decorative applications.
Processing of tannery sludge was carried out via incineration at 500οC for 1.5 h in anoxic conditions, in order (a) to remove its organic content and (b) to suppress Cr(III) oxidation to Cr(VI). Three different glass batch compositions (GL1, GL2 and GL3) were prepared using various proportions of vitrifying agents and the Cr-rich tannery ash (20-15-10 wt% respectively). The batch mixtures were heated for 2h at 1400οC and casted rapidly on a refractory steel plate. In GL1 and GL2 products the most of the chromium is separated from the matrix and formed Cr2O3 crystals [Figure 1]. Such phase separation was not detected for GL3 product, which mesoscopic appears to be amorphous and chromium appears to be incorporated in the glass matrix.
The devitrification of the as-casted products was carried out at temperatures determined after DTA study of the as-casted samples. The as-casted products were thermally treated for 30 minutes. GL1 and GL2 devitrified products displayed bulk crystallization of the devitrite (Na2Ca3Si6O16) crystal phase. On the other hand GL3 devitrified products displayed both bulk and surface crystallization of the combeite (Na4Ca4(Si6O18)) crystal phase [Figure 2]. The nanoscale structure of the samples was investigated using TEM. Fig 3 illustrates a HRTEM image of GL3 showing dispersed crystalline nanoparticles in an amorphous matrix.

This research has been co-financed by the European Union (European Social Fund) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework-Research Funding Program: THALES: Reinforcement of the interdisciplinary and/or inter-institutional research and innovation. G.P.D acknowledges support by the Reseach Committee-AUTH

Fig. 1: SEM micrograph from as-casted GL1 which contains Cr2O3 crystallites

Fig. 2: Devitrified GL3 at 880οC, which contains needle like combeite (Na4Ca4(Si6O18)) crystallites

Fig. 3: HRTEM image of GL3 showing dispersed crystalline nanoparticles in the amorphous matrix. The corresponding selected area electron diffraction and an magnified nanocrystal are given as insets
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-2087 Grain boundary domain dynamics investigated by Transmission Electron Microscopy in modified BaTiO3 displaying PTCR effect

Holsgrove K. M.1, Douglas A. M.1, Einsle J.1, Gregg J. M.1, Arredondo M.1
1Centre for Nanostructured Media, Queen’s University, Belfast, U.K
kholsgrove04@qub.ac.uk

Positive temperature coefficient of resistivity (PTCR) materials are long-established in the electronics industry and used for many applications such as temperature control, current stabilisers and industrial sensing. Interestingly the PTCR effect can be observed in donor-doped barium titanate near the ferroelectric transition temperature TC. The most accepted model to explain the PTCR behaviour in modified BaTiO3 materials is the Heywang-Jonker model, whereby the PTCR behaviour is said to arise from the presence of a potential barrier at the grain boundaries; the barrier arising from the trapping of electrons by acceptor species at the grain boundary1. Much work has been done studying the structural aspects and grain boundary potential2, but still many important factors of the PTCR phenomena are yet to be explored in depth. Transmission electron microscopy (TEM) has been used here to investigate the relationship between domains and grain structure in BaTiO3 based ceramics. As shown in Figure 1 and 2, domains can be continuous across a grain boundary (allowing the material to behave more as a single crystal) and conversely domains can abruptly change close to the grain boundary. Also, regions of different domain variant can occur within grains where areas of fine domain patterns and changes in domain orientation can be observed (Figs. 1 and 3). Internal stresses and defects are factors that add to the complexity of this material, the thick domain walls displayed in Figure 1 reflect internal strain generated due to the paraelectric-ferroelectric phase transition. The structural intricacy of crystallographic axes dependence on domain dynamics will be discussed in further detail. In addition, conductive-atomic force microscopy (c-AFM) studies will be presented to relate the macroscopic PTCR behaviour in specific grains with domain dynamic behaviour. In-situ TEM experiments such as heating and electrical bias, along with high resolution imaging, are being undertaken in an attempt to bring further understanding in a complex phenomena; by exploring the strong and delicate interplay between macroscopic polarization, switching mechanism and crystal structure, both within grains and across grain boundaries.

1 W. Heywang, Solid State Electron., 3, 51, 1961
2 R.D. Roseman and N. Mukherjee. J. of Electroceramics, 10, 117-135, 2003

We would like to acknowledge the department for employment and learning.

Fig. 1: STEM image of domains continuing across a grain boundary. Fine-scale domains can also be seen in some regions where the domain configuration changes orientation.

Fig. 2: STEM image of domains abruptly changing at the grain boundary.

Fig. 3: STEM image of complex regions of different domain variant within a grain.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-2110 STRUCTURE AND MICROSTRUCTURE STUDY OF HYDROXYAPATITE SYNTHESIZED BY CO-PRECIPITATION METHOD ASSISTED BY THREE DIFFERENT STIRRING MECHANISMS

De León E C.1,2, Vargas B N.1, Téllez J L.2, Reyes G J.3, Álvarez P M. A.1
1Laboratorio de Bioingeniería de Tejidos; División de Estudios de Posgrado e Investigación de la Facultad de Odontología, UNAM, D.F., México., 2Departamento de Ingeniería Metalúrgica y de Materiales E.S.I.Q.I.E-I.P.N, D.F., México. , 3Instituto de Física, UNAM, D.F., México.
ailicec28@yahoo.com

The effect of stirring mechanism on the microstructure and morphology synthesis of hydroxyapatite (Ca10(PO4)6(OH)2, HA) was investigated in this work. The HA samples were prepared by wet method (co-precipitation), employing calcium hydroxide and phosphoric acid as precursors [1-4]. A solution with 8 mL of H3PO4 and 235 mL deionizated H2O was prepared and slowly dropped into a solution of Ca(OH)2, prepared with 14.75g Ca(OH)2 and 200 mL deionizated H2O. Meanwhile the samples were stirring assisted by three different mechanisms: ultrasonic bath (sample HA-b), magnetic-plate (sample HA-p), and ultrasonic tip/ultrasonic bath (sample HA-s). All samples were carried out at a pH in the range of 8.5-9.0. Afterwards powders were filtered, washed with distiller H2O, and dried at 100°C for 24h. The structure of the samples was characterized by scanning electron microscopy (SEM), X-ray powder diffraction (XRD). Lattice parameters and grain sizes were obtained by Rietveld refinement. Figure 1 shows the SEM images of the three HA samples. Samples HA-p and HA-s show elongated grains in the range of 100 nm, being more elongated in sample HA-s. Sample HA-b shows rounded grains smaller than 100 nm. The SEM images suggest an important influence of the stirring mechanism: it enhances or reduces the grain growth along a-axis and/or c-axis producing elongated and/or rounded grains [2-3]. The hexagonal unit cell of the samples was supported by XRD and Rietveld analysis. As shown in table 1, and in using the unit cell parameters reported in reference [5], a decrement of the a-axis and an increment of the c-axis were registered in the all samples. Sample HA-b presented the biggest increment (0.17%) in the c-axis while sample HA-s was the lowest (0.11%). In the case of the a-axis, sample HA-p presented the biggest decrement (0.17%) while the sample HA-b presented the lowest (0.02%). Therefore, sample HA-b was the most influenced in the c-axis while samples HA-p and HA-s present the same behavior practically: the percentage of decrement in the a-axis was the percentage of increment in the c-axis. Therefore, the stirring mechanism plays an important role on the replacement of atoms inside the unit cell of HA, producing type A and/or type B materials [4].

[1] L. B. Kong, J. Ma, F. Boey, J. Mater. Scien. 37 (2002) 1131. [2] X. Guo, P. Xiao, J. Europ. Ceram. Soc. 26 (2006) 3383. [3] W. Kim, F. Saito, Ultrason. Sonochem. 8 (2001) 85. [4] R. Roy, D. K. Agrawal, V. Srikanth, J. Mater. Res. 6 (1991), 11. [5] M. J. Hughes, M. Cameron, D. K. Crowley, Am. Mineral. 74 (1989) 870.

Authors thank to CONACYT for supporting scholarship to CLE and to UNAM-DGAPA postdoctoral scholarship to NVB during the course of this study. This research were financially supported by funds from the UNAM-DGAPA: PAPIIT project IN213912 and Graduate Program of High Quality UNAM-CONACYT No. I010/480/2013 C-736/213.

Fig. 1: Table1 Lattice parameters, average crystal size and deviation percentage from the reported HA data: a=0.94166 nm, c=0.68745 nm [5].

Fig. 2: Figure1 SEM images of samples A)HA-b, B)HA-p, C)HA-s
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-2515 Transmission electron microscopy investigation of transparent α-alumina

Nabiei F.1, Tewari A.2, Cantoni M.1, Bowen P.2, Hebert C.1
1Interdisciplinary Centre for Electron Microscopy, EPFL, Lausanne, Switzerland, 2Powder Technology Laboratory, EPFL, Lausanne, Switzerland
farhang.nabiei@epfl.ch

Transparent polycrystalline alumina is being promoted as a replacement for sapphire because of its excellent mechanical and high temperature properties as well as the possibility to form different shapes. Transparency in this material is only achievable by controlling porosity and grain growth which is usually done by doping. Computational studies have been done on these materials to simulate dopant segregation at the grain boundaries (e.g. Tewari et al. 2012, Galmarini et al. 2011); however, there is lack of experimental analysis to quantify the dopant concentration in the grain boundaries and validate the modelling which could then be used as a predictive tool.

In this study α-alumina doped by 445 ppm of La and Y and sintered by spark plasma sintering (Stuer et al. 2009) is used to investigate dopant segregation at grain boundaries with different orientations. Thin sections about 300nm thick were made by focused ion beam (FIB) for investigation by transmission electron microscopy (TEM).

Energy dispersive X-ray (EDX) spectroscopy shows strong cosegregation of La and Y atoms to all grain boundaries observed. This is in agreement with atomistic simulation where no energetic gain or loss for cosegregation over single dopant segregation was predicted. EDX quantification on individual grain boundaries showed the average of 2.79 atoms/nm2 and 2.29 atoms/nm2 for Y and La respectively which is in a good agreement with the optimum total dopant concentrations found in simulations 4-6 atoms/nm2 (Tewari et al. 2012). Also, we detected Cl segregation in grain boundaries and triple points.

Finally, large angle convergent beam electron diffraction (LACBED) is performed to characterize the grain boundaries studied by EDX. By using LACBED to identify the planes parallel to the grain boundary in each adjacent grain, the angle between these planes can be calculated. It was found that the grain boundaries studied here were all high angle ones (e.g. (-7, -2, 4) & (1, 5, 5)). This data was used to simulate the same grain boundary for direct comparison between simulations and experimental results for dopant segregation. It was found that in contrast to the twin grain boundaries, which favor lower order GB complexions, general grain boundaries favor higher order grain boundary complexions. It would also lead to higher grain growth in general grain boundaries in comparison to twin grain boundaries.

Tewari, A., Galmarini, S., Stuer, M., Bowen, P., 2012. . Journal of the European Ceramic Society 32, 2935–2948.

Galmarini, S., Aschauer, U., Tewari, A., Aman, Y., Van Gestel, C., Bowen, P., 2011. . Journal of the European Ceramic Society 31, 2839–2852.

Stuer, M., Zhao, Z., Aschauer, U., Bowen, P., 2010. . Journal of the European Ceramic Society 30, 1335–1343.

I would like to express my appreciation to Prof. Pierre Stadelmann for his great advices on LACBED method and indexing by JEMS software.

Fig. 1: From left to right: bright field image of La ,Y codoped alumina, La intensity map and Y intensity map.

Fig. 2: From left to right: La intensity map, Y intensity map and normal line scan on one grain boundary in La, Y codoped alumina.

Fig. 3: LACBED pattern from a grain boundary (left) and one of its grains (right). Grain boundary direction (known from the left pattern) is shown with red line in the right pattern.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-2639 The modulated structure of Fe2O3(ZnO)7 studied by HAADF-STEM

Eichhorn S.1, Schmid H. K.1,2, Mader W.1
1Inst. Inorganic Chemistry, University Bonn, Bonn, Germany, 2now with JEOL (Germany) GmbH, Eching, Germany
mader@uni-bonn.de

The existence of phases ABO3(ZnO)m in the system ZnO-Fe2O3 is known up to a composition of Fe2O3(ZnO)12 at 1350 °C [1]. These phases share the structural characteristics of the better known compounds InFeO3(ZnO)m [2] which exist in the range m ≥ 1. ABO3(ZnO)m compounds consist of wurtzite slabs separated by layers of AO6 octahedrons fully occupied by A3+ cations such as In3+. Within the wurtzite layers B3+ ions occupy tetrahedral positions. The crystal structures of compounds with low ZnO content were verified by single crystal X-ray diffraction [3], however Fe2O3(ZnO)m compounds show an obvious super-structure in diffraction.
We obtained Fe2O3(ZnO)7 by solid state reaction of ZnO and ZnFe2O4 powders at 1600 °C. The reaction product was quenched in water to retain the metastable high temperature phase. XRD proves the layer structure of Fe2O3(ZnO)7, however, EDS measurements show a metal content of 30% Fe and 70% Zn vs. nominal composition of 22% Fe and 78% Zn as derived from the formula. Hence, the correct formula should be written as Fe2O3(Zn1-xFexO)7 with x ≈ 0.1. The Compound crystallizes in the monoclinic system with lattice constants a = 5.566(8) Å, b = 3.234(5) Å, c = 24.00(3) Å and β = 95.35° with possible spacegroups C2, Cm or C2/m. EDS and electron diffraction was conducted on a Philips CM30 with a Noran System 7 EDS system and a twin lens providing large tilt angles of up to 45°. HAADF was conducted on a probe Cs-corrected JEOL ARM200F equipped with a cold FEG.
Figure 1 shows an SAED pattern from Fe2O3(ZnO)7 in [100] orientation where additional superstructure reflections 0klx are clearly visible as satellites to the main reflections. The modulation vector q is about 34 Å measured from electron diffraction patterns. The periodicity of the (00l) reflections corresponds to a 24 Å lattice plane spacing.
A HAADF micrograph in the same orientation is shown in figure 2. Multiple layers of Zn2+ cations show a wave like modulation along [010] direction. Also Fe3+ octahedral layers show considerably less detail in atomic resolution which is attributed to significant displacements of Fe3+ cations in FeO6-octahedrons. The misfit of the rather small Fe3+ cation in octahedral coordination is considered to be the origin of the structure modulation.
These materials offer challenging crystallographic and analytical questions, such as Fe2+ distribution in the wurtzite layers, which can be tackled by advanced electron microscopy methods, i.e. quantitative EDS and EELS analyses with high spatial resolution in TEM/STEM [4].

[1] N. Kimizuka et al., J. Solid State Chem. 103 (1993) p. 394
[2] N. Kimizuka et al., J. Solid State Chem. 74 (1988) p. 98
[3] I. Keller et al., Z. Anorg. Allg. Chem. 635 (2009) p. 2065
[4] H. Schmid et al., Ultramicroscopy. 127 (2013) 76-84

Fig. 1: SAED pattern of Fe2O3(Zn1-xFexO)7 in [100]. Periodicity of <00l>reflections is 24 Å, of <0k0> reflections is 34 Å.

Fig. 2: HAADF STEM micrograph of Fe2O3(Zn1-xFexO)7 in [100] orientation. Layers of FeO6-octahedrons are marked by arrows.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-2683 Structure determination of an iron perovskite lanthanum material using precession and convergent beam electron diffraction

Martinez-Amesti A.1, Ecija A.1, Jacob D.3, Vidal K.1, Nó M. L.2, Arriortua M. I.1
1Departamento de Mineralogía y Petrología, Universidad del País Vasco, Facultad de Ciencia y Tecnología, Apto. 644, 48080-Bilbao, Spain, 2Departamento de Departamento Física Aplicada II, Universidad del País Vasco, Facultad de Ciencia y Tecnología, Apto. 644, 48080-Bilbao, Spain, 3Laboratoire de Structure et Propriétés de l’Etat Solide-UMR CNRS 8008, Université des Sciences et Technologies de Lille-Bât. C6, Villeneuve d’Ascq Cedex, France.
ana.martinez@ehu.es

Perovskite-type oxides with formula ABO3 have received much attention due to their high oxygen mobility and extensive applications in oxygen separation membranes, fuel cells, sensors and catalysts [1-3]. In ABO3 perovskite structure, A denotes the rare earth element and B denotes the transition metal. These oxides can tolerate significant partial substitution and non-stoichiometry, while still maintaining the perovskite structure [4]. In this way, divalent cation-doped lanthanum ferrite materials (La3+1-xM2+xFeO3-δ) (M= Ba2+, Sr2+ or Ca2+) exhibit a variety of useful properties to be used as IT-SOFC cathodes due to their mixed ionic-electronic conducting properties. Consequently, it is important to understand structural and magnetic phase transformations that may occur at elevated temperatures to optimize the performance.
A perovskite sample of composition La0.5Ba0.5FeO3 has been prepared by two different synthetic routes, solid-state reaction (LBFss) and glycine-nitrate route (LBFgn), in order to study the sample preparation influence in the structure. The structure of these compounds cannot be determined by X-ray or neutron diffraction unambiguously. Precession electron diffraction (PED) (Figure 1) and convergent beam electron diffraction (Figure 2) techniques were used to solve the structure of the compounds
TEM examinations were carried out using a TECNAI G2 20 TWIN microscope operating at 200 kV and equipped with a “SpinningStar” electron precession unit from Nanomegas. PED patterns were obtained with a semi-angle of 2º.
The obtained results show that at room temperature the space group of samples synthesized by ceramic (LBFss) and glycine-nitrate (LBFgn) synthetic routes is the same, Pm-3m, with similar lattice parameters as determined by neutrons diffraction a= 0.39292nm and 0.39381nm and with small differences in the oxygen content, La0.5Ba0.5FeO2.91 and La0.5Ba0.5FeO2.98, respectively.

[1] Chroneos, A, Vovk, R. V., Goulatis, I. L. and Goulatis, L. I., J. Alloy Compd., 494(2010)190-195.
[2] Dailly, J., Fourcade, S., Largeteau, A., Mauvy, F., Grenier, J. C. and Marrony, M., Electrochim. Acta, 55(2010)5847-5453.
[3] Blasin-Aube, V., Belkouch, J. and Monceaux, L., Appl. Catal. B: Environ., 43(2003)175-186.
[4] Pena, M. A. and Fierro, J. L. G., Chem. Rev., 101(2001)1981-2017.

 

This work has been financially supported by the Ministerio de Ciencia e Innovación (MAT2010-15375 and CONSOLIDER-INGENIO 2010 CSD2009-00013) and by Dto. Educación, Política Lingüística y Cultura (IT-630-13) of the Basque Government. The authors wish to thank SGIker technical support (UPV/EHU, MEC, GV/EJ and European Social Fund). A. Martínez-Amesti whises to thank UPV/EHU for funding.

Fig. 1: Experimental precession electron diffraction pattern of the ZOLZ/FOLZ of [001] zone axe.

Fig. 2: Experimental CBED for ZOLZ of [-1-11] zone axe.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-2811 Accelerated decomposition of Y2O3-doped ZrO2 by dissolved Ni — Unveiling the atomistic processes by in situ TEM

Butz B.1, Stark M.1, Michel S.2, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany, 2Chair of Metals Science and Technology (WTM), Friedrich-Alexander University Erlangen-Nürnberg (FAU), Erlangen, Germany
benjamin.butz@ww.uni-erlangen.de

Yttria-stabilized Zirconia (YSZ) has been widely used as structural and functional ceramic in harsh chemical environments or at high operating temperatures. For most applications its long-term stability is of importance. As the system Y2O3-ZrO2 exhibits a miscibility gap, spinodal decomposition occurs depending on the Y2O3 content and the applied conditions. This also holds for 8.5 mol% YSZ [1] one of the most common electrolytes for solid oxide fuel cells (SOFC). During operation at 950 °C the spinodal decomposition of 8YSZ, which is characterized by the microstructural coarsening of metastable t’’-YSZ precipitates (Fig. 1a) and the accompanied evolution of chemical variations (Fig. 1b), leads to a significant degradation of the oxygen-ion conductivity (40% within 5000 h). Besides the fact that the decomposition rate depends on the temperature, it can also strongly be enhanced by pO2-sensitive trace elements possessing strongly different solubility for 8YSZ in oxidizing or reducing atmosphere. Here, the accelerated decomposition of Ni-containing 8YSZ in reducing atmosphere, which proceeds more than 50 times faster than for pure 8YSZ (Fig. 1c), is investigated [2]. To understand the underlying mechanisms, the fundamental processes like Ni indiffusion (oxidizing atmosphere) and Ni exsolution/precipitation (reducing atmosphere) are investigated. The study comprises the local analysis of the oxidation state of the dissolved Ni by EELS. Therefore, an optimized procedure has been established to quasi-in situ prepare ideal metallic reference samples (Fig. 2a). It comprises the preparation of purely metallic Ni nanoparticles on Lacey carbon using a H2-reactor (thermal treatment: 1 mbar Ar/H2, 650 °C). Such samples are directly transferred into the microscope utilizing an inert-gas glove box plus a transfer holder. The high quality of such metallic Ni particles is shown in Fig. 2b. It has to be mentioned here, that organic residuals are pyrolyzed upon treatment in Ar/H2 (Fig. 2b, graphitic microstructure around the particles) solving the common contamination problem. Figure 2c shows the obtained Ni‑L2,3 reference spectra for metallic Ni and NiO that were already used to determine the composition of Ni precipitates (distribution of Ni2+, Ni0) that are typically found at 8YSZ grain boundaries after the annealing in reducing atmosphere. The introduced procedure is applicable to preserve the state of almost any TEM sample and will prospectively be extended even to prepare the principal diffusion couples.

[1] B Butz et al, Acta Mater. 57 (2009) 5480-5490

[2] B Butz et al, Solid State Ionics 214 (2012) 37–44

We gratefully acknowledge provision of materials by the IWE (KIT, Germany) and the IEK-1 (FZ Jülich, Germany). Furthermore we thank the group of Prof. J. Janek (Physikalisch-Chemisches Institut, Uni Gießen, Germany) for the PLD-deposition of NiO (Mr. Kleine-Boymann). The research was supported by the DFG (Cluster of Excellence EXC 315, GRK1896).

Fig. 1: a) Spinodal decomposition of 8YSZ at 950 °C: a) coarsening of t’’-YSZ precipitates within single 8YSZ grains and b) chemical variations across a Y-depleted region. c) Accelerated degradation of Ni-containing 8YSZ in reducing atmosphere (red data points) vs. oxidizing atmosphere (black data points).

Fig. 2: a) Ideal metallic reference particles for oxidation-state analysis: reduction in H2 and subsequent inert-gas transfer (reactor → glove box → transfer holder → microscope). b) Microstructure and structure of metallic Ni particles. c) Obtained Ni-L2,3 reference spectra. d) Oxidation-state analysis of Ni precipitates at 8YSZ grain boundary.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-2837 Real structure of (Sb1/3Zn2/3)GaO3(ZnO)3 revealed by ED and HAADF-STEM

Garling J.1, Hühne S. M.1, Schmid H.1 2, Assenmacher W.1, Mader W.1
1Institut für Anorganische Chemie, Universität Bonn, Römerstr. 164, 53117 Bonn, Germany, 2Now with JEOL (Germany) GmbH, Oskar-von-Miller-Straße 1, 85386 Eching, Germany
assenmacher@uni-bonn.de

A high temperature (1350 °C) route in sealed Pt tubes was used for the synthesis of pure powders of (Sb1/3Zn2/3)GaO3(ZnO)3. Single-crystal X-Ray diffraction of crystals grown from K2MoO4 flux revealed the space group R-3m (a = 3.2366(3) Å, c = 41.793(8) Å) and the structural characteristics as known from other members with general formula ABO3(ZnO)m [1]. (Sb1/3Zn2/3)GaO3(ZnO)3 consists of an alternate stacking of [(Sb1/3Zn2/3)O2]- and [(GaZn3)O4]+ units corresponding to CdI2 and wurtzite structure type motifs, respectively. Inversions of the ZnO4 tetrahedra occur at the octahedral layers and halfway in the wurtzite type. There is no indication for a cation-ordering on octahedral sites from X-ray data, but the Sb1/3Zn2/3 occupation of the octahedral is confirmed by refinement of the occupation factor.
Surprisingly, electron diffraction and HRTEM show an ordering of the cations within the [(Sb1/3Zn2/3)O2]- octahedral layer by presence of superstructure reflexions (Fig 2) and contrast modulations of the cation columns [2]. This cation ordering can be described by a model deduced earlier for single defect layers in ZnO doped with Sb [3]. The description of the ordered structure succeeds in space group P3112 (a = 5.60 Å, c = 42.02 Å). The observed streaks parallel to 000l (Fig 2c) can be described by a statistic displacement of well ordered octahedral layers among themselves.
To confirm the deduced structure model HAADF-STEM imaging was carried out on a JEOL JEM-ARM 200CF . The present setup provides sub-Å resolution capability in HAADF STEM imaging, whereas BF and particularly ABF with increased sensitivity for light elements enable the elucidation of true atomic structures [4]. The QSTEM software [5] was used for simulation of HAADF images (Fig 3).
HAADF imaging reveals the periodic order of Sb and Zn in the octahedral layers. The intensity ratio of the Zn and Sb columns can be measured to 0.42. This corresponds to an exponent of 1.7 (Int(Zn)/Int(Sb)=301.7/511.7= 0.41), which is in agreement with theoretical predictions [6,7]. In some areas the stacking of the octahedral layers corresponds well with the structure model in P3112. However, the stacking of larger regions does not correlate to the ABC stacking, i.e. stacking disorder occurs. This leads to streaks in SAED patterns (Fig 2).

References
[1] N. Kimizuka, T. Mohri, M. Nakamura, J. Solid State Chem. 81 (1989), p. 70-77.
[2] J. Garling, Diploma Thesis, Uni Bonn (2012).
[3] A. Recnik, N. Daneu, T. Walther, W. Mader, J. Am. Ceram. Soc. 84 (2001), p. 2657-68.
[4] H. Schmid, E. Okunishi, W. Mader, Ultramicroscopy 127 (2013), p. 76-84.
[5] C. Koch, PhD Thesis, Arizona State University (2002).
[6] R.F. Klie, Y. Zhu, Micron 36 (2005), p. 219.
[7] P.D. Nellist, S. J. Pennycook, Ultramicroscopy 78 (1999), p. 111.

We thank E. Arzt for continuous support through INM and G. Schnakenburg for single crystal X-Ray data collection.

Fig. 1: Crystal structure of (Sb1/3Zn2/3)GaO3(ZnO)3 in P3112, drawn as ball and stick model and as polyhedron representation, respectively.

Fig. 2: Electron diffraction patterns of (Sb1/3Zn2/3)GaO3(ZnO)3 in principal orientations and calculated patterns using the structure model in P3112. Diffuse scattering in (c) is caused by stacking disorder of the octahedral planes.

Fig. 3: HAADF image (a), simulated HAADF image (b) and structure model (c) of (Sb1/3Zn2/3)GaO3(ZnO)3 in a-axis orientation.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-2839 Synthesis of rare earths oxides from waste minerals

MADIGOU V.1,2, PEREIRA DE SOUZA C.3, ARAB M.1,2, GAVARRI J. R.1,2, LEROUX C.1,2
1IM2NP-CNRS, UMR 7334, 2Université de Toulon, Campus de La Garde Bât.R, BP 20132, F- 83957, 3Universidade Federal do Rio Grande do Norte, DEQ/PPGEQ-LTRC
madigou@univ-tln.fr

Rare earth elements like cerium and lanthanum are currently involved in a wide range of advanced technologies and in industrial chemistry. The initial rare earth (RE) mineral is extracted, mechanically and physically concentrated, giving a main concentrate and mineral wastes. These waste materials can be considered as “zero cost” initial sources of rare earth oxides. Thus, the development of a simple chemical process allowing synthesis of rare earth oxide phases from these waste minerals is part of sustainable and economic development. The waste is rich in monazite and allanite which contain different proportions of rare earth elements.

Different chemical processes were explored, in order to extract the interesting oxide phases from these complex mixed materials. Transmission Electron Microscopy coupled with Energy Dispersive Spectroscopy was performed at each step of the chemical process: it was the most efficient way to check the efficiency of the removal of chemical species, and the crystallisation and composition of the products. The first way explored was a solubilization of the minerals through an acid attack followed by a selective precipitation of Re (OH)x [1]. The precipitation of RE oxalates and their final annealing at 900 °C led to a new pyrochlore phase RE2Ce2O7 [2]. Another process started with an alkaline fusion, followed by a solubilization in nitric acid led to two distinct crystallographic phases, related to the initial compounds: monazite and allanite. Fig 1 shows two distinct morphologies: one can found spherical grains (Fig1: A grains) and platelets (Fig.1: B grain). The spherical grains crystallize in the cubic CeO2 structure (Fig.2). EDS analyses lead to the chemical formula of this oxide: Ce0.65La0.25Nd0.1O2. The diffraction pattern of the cubic shaped grain can be indexed in a double cell of a tetragonal structure along the [001] axis or in an ordered cubic double perovskite along the [001] axis (Fig.3). The EDS analyses show that this oxide phase contains mainly lanthanum and iron, with a La/Fe ratio around 2.

References

[1] J. C. Gomes, C. P. de Souza, U. U. Gomes, J. R. Gavarri, J. P. Dallas, Ch. Leroux, Materials Research Forum, advanced Mat. Forum III, vols. 514-516, (2006) 1653.

[2] F. W. Bezerra Lopes, C. Pereira de Souza, A. M. Vieira de Morais, J.-P. Dallas and J.-R. Gavarri, Hydrometallurgy, 97, (2009) 167.

This research was supported by CAPES –COFECUB, and by the PACA region (ARCUS Brazil)

Fig. 1: Grains obtained after alkaline fusion. The grains labelled A are spherical and contain Ce,La,Nd,Th. The grain labelled B contains mainly La and Fe.

Fig. 2: diffraction pattern of a grain labelled A on figure 1. It is indexed in the CeO2 fluorite structure along the [001] direction.

Fig. 3: diffraction pattern of a grain named B on figure 1, showing a doubling of the perovskite cell.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3060 Characterization by aberration corrected STEM-EELS of YSZ epitaxial thin films

Cabero M.1, Rivera Calzada A.1, Pennycook T. J.2,3, Varela M.4,1, Pennycook S. J.5, Leon C.1, Santamaría J.1
1GFMC, Universidad Complutense de Madrid. Madrid 28040, Spain, 2Department of Materials, University of Oxford, Parks Road, Oxford, OX1 3PH, UK, 3SuperSTEM Laboratory, Daresbury, WA4 4AD, UK, 4Oak Ridge National Laboratory, Oak Ridge TN 37831, 5The University of Tennessee, Department of Materials Science and Engineering, Knoxville TN 37996-2200, USA
marionacabero@ucm.es

The search of novel materials with enhanced ionic conductivity for their application in energy devices is an exciting direction in the expanding field of oxide interfaces [1]. In particular, exploiting size effects in ionic conducting materials by the controlled reduction of sample dimensions down to the nanoscale holds the promise to yield large improvements in the performance of oxide based devices for energy generation and storage [1-3]. Epitaxial strain is a key parameter to tune the enhancement of ion mobility at interfaces. A good example can be found in the colossal increase of ionic conductivity observed in SrTiO3/Y2O3:ZrO2 (STO/YSZ) superlattices, which has been the subject of intense debate [4-9]. It has been suggested that disorder in the O sublattice at the interface, transfer of vacancies or interface reconstructions may be responsible for the enhancement in ionic conductivity. Aberration-corrected scanning transmission electron microscopy combined with electron energy loss spectroscopy (STEM-EELS) is a powerful technique capable of providing simultaneous information about structure, chemistry and electronic properties of materials. In this work we apply these techniques to the study of STO/YSZ interfaces and we will discuss the structure, chemistry and physical properties of fluorite/perovskite interfaces (as shown in Figure 1) of (111) oriented ZrO2:Y2O3 (YSZ) films epitaxially grown on top of (1-10) YAlO3 (YAP) substrates (with small epitaxial mismatch), where an enhancement of 5 orders of magnitude in ionic conductivity has been measured near room temperature. Research at ORNL supported by U.S. Department of Energy (DOE), Basic Energy Sciences (BES), Materials Sciences and Engineering Division. Research at UCM supported by the ERC starting Investigator Award, grant #239739 STEMOX. SuperSTEM is the EPSRC UK National Facility for Aberration-Corrected STEM.

[1] B. C. H. Steele and A. Heinzel, Nature (2001) 414, 345.

[2] N. Sata, K. Eberman, K. Eberl and J. Maier, Nature (2000), 408, 946.

[3] J. Maier, Nature Materials (2005) 4, 805-815.

[4] J. García-Barriocanal et al., Science (2008) 321, 676-680.

[5] X. Guo et al., Science (2009) 324, 465a.

[6] A. Cavallaro et al., Solid State Ionics (2010) 181, 592-601.

[7] T. J. Pennycook et al., Physical Review Letters (2010) 104, 115901.

[8] A. Rivera-Calzada et al., Advanced Materials (2011) 23, 5268.

[9] T. J. Pennycook et at., The European Physical Journal Applied Physics (2011) 54, 33507.

We also acknowledge support from Spanish MICINN through grants MAT2011-27470-C02 and Consolider Ingenio 2010-CSD2009-00013 (Imagine), from CAM through grant S2009/MAT-1756 (Phama).

Fig. 1: Z-contrast STEM image of a 70 nm YSZ thin film with an amorphous YAP capping. The yellow arrows show the orientation of the substrate. a. Low magnification image of the thin film. b.High resolution image of the interface. Data acquired in a Nion UltraSTEM200, operated at 200 kV.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3155 Characterisation of thermally degraded polycrystalline diamond

Westraadt J. E.1, Neethling J. H.1, Sigalas I.2
1Nelson Mandela Metropolitan University, Port Elizabeth, South Africa, 2University of the Witwatersrand, Johannesburg, South Africa
johan.westraadt@nmmu.ac.za

Polycrystalline diamond (PCD) materials can be found in a wide range of applications, from machining of abrasive alloys, wood and plastics, to wire drawing and rock drilling. PCD compacts consist of diamond powder that has been sintered at high pressure (5.5 GPa) and temperature (>1400 °C) onto a WC/Co substrate. During this process the cobalt from the substrate melts, infiltrates and causes the diamond grains to bond to each other, thus creating a strong network of diamond grains. The cobalt metal is left behind as small pockets within the composite material. An unfortunate consequence of the residual cobalt, is that it leads to a degree of thermal instability when the material is exposed to temperatures exceeding 700 ºC [1]. The cobalt metal acts as a carbon solvent and will graphitise diamond at atmospheric pressure and elevated temperatures. In this study, PCD material was heated in an inert atmosphere and the various microstructural changes were investigated, in order to gain an understanding of this thermal degradation process.

Cobalt based PCD samples were heated in an argon atmosphere for various times and temperatures. Temperatures of 700 ºC, 750 ºC, 800 ºC and 850 ºC were used at time intervals of 30 min, 2 hours, 4 hours and 6 hours. X-ray diffraction (XRD) was performed to determine the resulting phases and lattice parameters. In-situ XRD was performed in vacuum at 800 ºC in order to track the changes in the material as a function of time. Transmission electron microscopy (TEM) using the techniques of High Angle Annular Dark Field (HAADF) Scanning-TEM (STEM) and Electron Energy Loss Spectroscopy (EELS) were used in order to investigate the chemical changes of this material during the heat treatment.

The cobalt lattice parameter decreased as a function of heating temperature (Figure 1) and time. The formation of graphite was preceded by η-phase formation at the cobalt/diamond interface (Figure 2). At heating temperatures of 800 ºC and above, graphite formed in the cobalt pools (Figure 3). The results of this study were used to propose a possible mechanism, whereby the dissolved tungsten in the cobalt pool will combine with dissolved carbon and cobalt to form η-phase instead of graphitic carbon. The dissolved tungsten will then delay the process of graphite formation in the cobalt pool. If the tungsten levels are depleted or if the rate of carbon influx is too high, then graphitic pools will form in the cobalt pool. In this model, dissolved tungsten in the binder is thought to be beneficial to the thermal resistance of PCD.

References
1. Bex, P.A. and Shafto, G.R. (1984) Ind. Diam. Rev. 3 128.

The authors wish to acknowledge the financial support from the National Research Foundation and the Department of Science and Technology in South Africa. 

Fig. 1: X-ray diffraction pattern showing a shift in cobalt lattice parameter as a function of temperature for 2 hour exposure.

Fig. 2: Bright-field TEM image showing a degraded cobalt pool after heating at 700 ˚C for 2 hours. Dark particles identified as the η-phase carbide with the aid of EDS analysis and electron diffraction.

Fig. 3: HAADF-STEM image with EELS spot analysis of a degraded cobalt pool after heating at 800 ˚C for 2 hours. The EELS analysis show the presence of graphite inside the cobalt pool.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3174 HRTEM studies of crystalline growth to unlock the formation mechanisms of titania nanodandelions

Narrandes A.1, Franklyn P. J.1
1Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, Johannesburg, South Africa
ashvir.narrandes@students.wits.ac.za

Titania in the rutile phase is able to form self-assembled, mesocrystalline, three dimensional superstructures [1,2]. These dandelions are formed by hierarchical arrangements of nanorods aligning radially on a common axis and have been documented during different synthetic routes [1,2]. The cause of rutile dandelion formation and the crystallinity of the structure at the common point have yet to be described.

In this study a form of rutile dandelions have been prepared using resin-gel synthesis. This technique involves addition of a long chain coordinating polymer to a stable solution of metal ions in the presence of a solvent [3]. The classic theory of resin-gel is that metal ions are held apart by the polymer until ignition of the resin causes polymer degradation and forces the metal ions to agglomerate and form a metal oxide. The strength of this technique lies in its ability to form homogeneous non-stoichiometric mixed metal oxides nanoparticles. Evidence supporting the formation of a polymer reaction chamber has previously been reported [4].

The primary aim of this study was to use HRTEM coupled with cryo ultra-microtome to obtain crystalline information about the dandelion superstructure and polymer chambers as they formed during the ignition stage of the resin-gel synthesis.

TiCl4 was added to distilled water containing HNO3. The solution was added to a stoichiometric quantity of polyethylene glycol with a molecular weight of either 3000 or 10000 g/mol. After complete solvent evaporation, the resins were heated until auto-ignition. Formvar coated TEM grids were used to sample the molten intermediates at different points during the ignition in an attempt to view the forming reaction chamber. Selected samples were cured in a resin before being sliced using cryo ultra-microtome with the intention of obtaining superstructure slices.

The results showed carbon lattice fringes surrounding growing nanoparticles supporting the reaction chamber hypothesis. Nanorod tips were either rounded or tapered to a point with various faces presenting at the tips. Imaging the common point of the nanorods showed several ‘seed points’ that gave rise to fragments of the superstructure. These fragments then formed the dandelion. Using the generated information, understanding of the polymer reaction chambers within the gel is obtained allowing for the formation of either anatase, rutile nanorods or mesoporous dandelions.

1. Zhang. D, Li. G, Wanga. F, Yu. C, CrystEngComm 12 (2010), 1759
2. Hu. W, Li. L, Tong. W, Li. G, Yan. T, J. Mater. Chem 20 (2010), 8659
3) Lin, J. et al., J. Phys Chem. C. 111 (2007), 5835
4) Franklyn, P. J, Narrandes. A, Proc. Nap 1 01 (2012), 40

The authors wish to thank Dr. James Wesley-Smith from the Council for Scientific and Industrial Research (CSIR) for the use of the Jeol JEM 2100 HRTEM.

Fig. 1: Polymer surrounding growing nanoparticles during the early stages of heating.

Fig. 2: Polymer sheets begin to wrap around growing particles after heating for a while.

Fig. 3: Formation of a crystalline carbon reaction chamber.

Fig. 4: Ruitle nanodandelion formation with several seed crystals at the common point.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3185 The effect of precursor variation on the formation of TiO2 synthesized by the resin-gel method

Narrandes A.1, Franklyn P. J.1
1Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, Johannesburg, South Africa
ashvir.narrandes@students.wits.ac.za

The resin-gel synthesis method is a modification of the Pechini method for the fabrication of mixed-metal oxide nanoparticles and involves the addition of a long chain coordinating polymer to a stable solution of metal ions in the presence of a solvent [1]. Literature describes the metal ions fixed in positions apart from each other when coordinated to the polymer. During the ignition stage of synthesis, the metal ions are forced to agglomerate and a mixed metal oxide forms. This technique has the ability to form nonstoichiometric multi mixed metal oxide nanoparticles provided that the mechanism of particle formation is fully understood.

Previous parameter variation work on TiO2 synthesis showed that using TiCl4 as the source of Ti ions: the solvent, polymer chain length, heating rate and organic acids affect both the amount of anatase or rutile formed and the size of the respective crystals produced. The incorporation of citric acid inhibited the formation of rutile and hence no rutile mesoporous heirarchichal superstructures were formed. The effects of citric acid were due to protection of the growing anatase particles and caused the inhibition of its transformation to rutile.

Therefore, the aim of this study was to vary the Ti ion precursor to try to obtain similar results as was achieved with citric acid. This would strengthen the proposed resin-gel particle formation hypothesis. HRTEM would aid in individual crystal polymorph determination.

TiOSO4 was added into excess distilled water. HNO3 was added to aid complete dissolution. The solution was then introduced into a stoichiometric amount of polyethylene glycol of various molecular weights from 200 – 20000 g/mol. Following complete solvent evaporation, the resins were heated in a sand bath to their auto-ignition point and ignited with a flame source. The resulting powders were calcined at 773 K for 1 hour. All samples were analysed using HRTEM.

The results obtained showed an interesting trend: An increase in PEG molecular weight caused an increase in the agglomeration of the formed anatase particles. Comparing the obtained and simulated FFT of selected crystals showed that the particles were composed of anatase with no evidence of rutile formation. This result is in accordance with that obtained when citric acid was added to the reaction mixture. Due to the bulky structure of TiOSO4 it is possible that the SO4 group had coordinated with developing anatase nanoparticles, protecting the high energy surface of the particles and inhibiting their transformation into rutile. This experiment is currently being repeated with a variety of different bulky Ti precursors. Similar results will strengthen the proposed mechanism of particle formation.

1) Lin, J. et al., J. Phys Chem. C. 111, (2007) 5835

The authors wish to thank Dr. James Wesley-Smith from the Council for Scientific and Industrial Research (CSIR) for the use of the Jeol JEM 2100 HRTEM.

Fig. 1: TiOSO4 forming anatase particles exclusively. The insert compares the calculated and detemined FFT illustrating that the outlined crystal is in the 001 orientation.

Fig. 2: Anatase particles agglomerating into a cluster.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3207 Nanocharacterization and Electrical Properties of Grain Boundaries in Gd/Pr Doubly-Doped CeO2

Bowman J. W.1, Zhu J.1, Crozier A. P.1
1Arizona State University
wjbowman@asu.edu

Grain boundaries (GBs) in O2- conducting ceramics like doped ceria significantly degrade ionic conductivity especially at intermediate temperatures (300 – 550 °C) [1]. High GB resistivity has been attributed in part to a space charge double layer which creates a vacancy depletion region emanating from grain interfaces. Other factors such as dopant segregation (and segregated dopant species) may also influence GB electrical properties. Recent high resolution elemental analysis in the TEM of 20 at% Gd-doped ceria by our group and others shows significant Gd segregation to GBs yielding enrichment zones of approximately 60 at% Gd, far exceeding the optimal Gd concentration (10 – 20 at%) for maximum ionic conductivity [1].

We have synthesized Ce0.8Gd0.2O2-δ (GDC), and Ce0.85Gd0.11Pr0.04O2-δ (GPDC) powders and fabricated bulk samples for characterization of electrical properties via AC impedance spectroscopy, and GB structure and composition via TEM. Electron energy-loss spectroscopy (EELS) in a JEOL ARM200F probe corrected scanning TEM (STEM) has been performed to map the distribution of dopant cations in the vicinity of GBs.

Fig. 1b shows an annular dark field (ADF) STEM image of a GB in GPDC with inset 2D EELS spectrum image color map indicating the segregation of dopant cations to the GB. The color map is created from integration of the background-subtracted Ce, Pr and Gd M45 white lines (fig. 1a). Fig. 1c illustrates the compositional variation near the GB estimated by k-factor analysis of background-subtracted white line integrated intensities. A distinct Ce M4:M5 white line ratio decrease characteristic of the reduction of Ce4+ to Ce3+ was also observed, possibly indicating an increased oxygen vacancy concentration associated with the structural disorder of the GB core. The cation segregation zone was measured at full width half maxima (FWHM) to be 1.6 and 2.0 nm in the GDC and GPDC, respectively. The average GB core composition in the GDC was approximately 61%, and the preliminary results presented here indicate GPDC GB core compositions of [Pr] ≈ 16% and [Gd] ≈ 29%. The relative dopant concentration (i.e. [Gd]/[Pr]) also appears to vary with position near the GB.

We present characterization of electrical conductivity, microstructure, and nano-scale grain boundary structure and chemistry of nominally Gd-doped and Gd/Pr doubly-doped ceria fabricated using mixed oxide nanopowders synthesized by spray drying. We discuss correlations between Ce4+ oxidation state variations, dopant segregation and resultant electrical properties in these materials. In an attempt to elucidate the O2- vacancy environment near GBs, the GPDC O K edge fine structure will also be discussed.

[1] Avila-Paredes, H.J. et al, Solid State Ionics 177 (2006) 3075-3080

We thank NSF GRFP-1311230 & DMR-1308085, and the J.M. Cowley HREM Center at ASU

Fig. 1: (a) GPDC EELS with M45 white lines and background windows BPr and BGd (BCe omitted for clarity). (b) ADF STEM of a GPDC GB with inset EELS spectrum image colored using the integrated intensities of Ce, Pr and Gd M45. (c) Cation concentration profiles and Ce M4:M5 white line ratio across the grain boundary in (a).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3261 Fiber Optic Dielectric Nanoparticle Characterization by Atom Probe Tomography

Francois-Saint-Cyr H.1, Ulfig R. M.1, Martin I. Y.1, Blanc W.2, LeCoustumer P.3, Hombourger C.1, Neuville D.4, Larson D. J.1, Guillermier C.5
1CAMECA Instruments Inc., 5500 Nobel Drive, Madison, WI, 53711, USA, 2Université Nice Sophia Antipolis, CNRS, LPMC, UMR7336, 06100 Nice, France, 3Université Bordeaux 3, Géo-ressources et Environnement, EA4592, 33607 Pessac, France, 4Institut de Physique du Globe de Paris, 1 rue Jussieu, 75005 Paris, France, 5National Resource for Imaging Mass Spectroscopy, Cambridge, MA 02139, USA
rulfig@hotmail.com

The engineered processing of luminescent ions-doped dielectric nanoparticles (DNPs) embedded in silica-based optical fibers aims at providing an enhanced spectroscopic behavior compared to pure silica. These DNPs should positively impact applications in high power fiber lasers, light sources with new wavelengths and telecommunications.

The prevalence of large phase immiscibility domains in silicate systems containing divalent metal oxides (Mg for instance) promotes the formation of DNPs through phase separation since heat treatments take place during the MCVD process.

Even after 60 years of glass-ceramics research, lack of experimental data concerning early nucleation stages imposes variations in composition and heat treatments as processing steps [1]. Although classical nucleation theory was the first model proposed to explain those phenomena, growth rate mismatches remain wide. According to this capillary assumption-based model, nuclei and bulk share similar structure-composition relationship. Recent articles disprove assumption of structure, pointing toward DNPs structural changes [2] and transition from amorphous nuclei to crystalline DNPs [3]. Compositional changes for small particle sizes (~1-10 nm) have been measured in alloys with Anomalous Small Angle X-Ray Scattering (ASAXS) [4]. Recent advances in APT techniques and the development of the Local Electrode Atom Probe™ have allowed the extension of high accuracy, sub nanometer compositional measurements to glass-ceramics [5], and in the current work, we report APT data disproving the second capillary assumption at the early stage of nucleation-growth process.

The APT generated atomic distribution map of Mg DNPs in silica-based glass doped with Mg, P, Ge and Er is reported in Figure 1. In addition, quantitative assessment of Mg, P and Er content levels in DNPs smaller than 10nm in diameter (Figure 2) could refine the theories behind nucleation and growth mechanisms.

References
[1] N. Karpukhina et al., Chemical Society Review (2014), DOI 10.1039/C3CS60305A.
[2] S.Y. Chung et al, Nature Physics 5 (2009), p. 68.
[3] P. Tan et al, Nature Physics 10 (2014), p. 73
[4] D. Tatchev et al, Journal of Applied Crystallography 38 (2005), p. 787.
[5] D.J. Larson et al., “Local Electrode Atom Probe Tomography” (Springer, New York 2014).


The authors would like to thank the CAMECA applications team at Madison Wisconsin for access to the laboratory and fruitful discussions to enable this work.

Fig. 1: Mg-based dielectric nano-particles (pink) surrounded by silica matrix (blue).

Fig. 2: Proximity histogram displaying the evolution of Mg, Er, P, and Ge concentrations from the silica matrix toward the center of the Mg-based dielectric nanoparticles.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3290 Monitoring of Ti Atoms Pathway at Nano-Scale in Joined Ceramics by Electron Microscopy

Tunckan O.1, Yurdakul H.2, Turan S.3
1Faculty of Aeronautics and Astronautics, Eskisehir, Turkey Republic, 2Institute of Materials Science and Engineering, Kutahya, Turkey Republic, 3Institute of Materials Science and Engineering, Eskisehir, Turkey Republic
orkun.tunckan@gmail.com

Joining is great importance in materials science to make complex shape products. Many different methods serve for this purpose. Among them, capacitor discharge joining comes to the fore with its fascinating properties, e.g. superfast and explosion occurrence. However, the scientific mysteries behind the reactions during capacitor discharge process have not so far unraveled. Here, we report a nano-scaled study of Ti atom behavior across the grain boundaries, interfaces and lattices in Si3N4-SiAlON/Ti capacitor discharged joint ceramics as a function of temperature through electron microscopy. Firstly, to make a bulk joint material with ceramic and metal foil, the experimental setup and design were organized. Later, samples were heat-treated in elevated temperatures between 900 and 1200°C under atmospheric conditions. Afterwards, electron transparent specimens from the special regions including Si3N4-SiAlON grains and Ti foil were prepared by using focused ion beam (FIB) lift-out method. Finally, the investigations of resulting samples were carried out by the use of different scanning and transmission electron microscopes (SEM and TEM). Based on the electron energy loss (EEL)-spectrum imaging (SI) results that acquired in energy filtering transmission electron microscopy (EFTEM) and scanning transmission electron microscopy (STEM) modes, the intense movement of Ti atoms toward to Si3N4-SiAlON grains was observed. This gives rise to a new type of nano-scaled phase formations in metal-ceramic interface and within foil by the combination of Si and N atoms diffusion arising from the Si3N4-SiAlON grains. The chemical compositions of these phases are well convenient with Ti3N2, Ti3N, Ti2N and Ti5Si3Nx. More interestingly, depending on the heat-treatment temperature, the morphology of the phases varies from the dendritic- to flower-type. Furthermore, Ti-rich cathodoluminescence (CL), energy dispersive X-ray (EDX), electron backscatter diffraction (EBSD), EFTEM-3 window and EELS elemental maps at grain boundaries and Si3N4-SiAlON grains revealed the nano-scale pathway of Ti atoms. Thus, we now thermodynamically make a clarification on the reactions that occur during the capacitor discharge process. These sequential ones will be also presented.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3380 Effect of Film Thickness and Strain on Octahedral Tilt in Epitaxial LaCoO3 Thin Films

Jang J.1, Mishra R.2, He Q.1, Qiao L.1, Biegalski M. D.1, Lupini A. R.1, Pantelides S. T.2, Borisevich A. Y.1
1Oak Ridge National Laboratory, Oak Ridge, United State, 2Vanderbilt University, Nashville, United State
jangj@ornl.gov

The corner sharing network of transition metal - oxygen octahedra (BO6) in the ABO3 perovskite can exist in a a variety of octahedral tilt patterns which affect materials properties.[1] Octahedral tilt behavior at the homo- and hetero-interfaces has recently attracted considerable attention as an important contributing factor for interface properties. Quantitative annular bright field (ABF) images combined with annular dark field (ADF) constitute a powerful tool that enables unit-cell-by-unit-cell analysis of octahedral tilt patterns.[2] In this study, we investigate octahedral tilt behavior of epitaxial LaCoO3 (LCO) films (a-a-a- tilt pattern in the bulk) for different film thicknesses and different epitaxial strain on SrTiO3 and LSAT substrates.
LaCoO3 is a particularly interesting object of study since thin films show ferromagnetic ordering under low temperatures, while the bulk compound is antiferromagnetic. For this study, LCO thin films were deposited by pulsed laser deposition. X-ray photoelectron spectroscopy studies of the films have demonstrated that Co 3p edges shift up to 2 eV for 15 u.c. and 20 nm films, indicating possible presence of 2D electron gas at the interface. Differences in properties could be due to differences in the structure or the differences in interface chemistry, which we investigated with STEM and EELS, respectively.
Octahedral tilts were examined for films of different thicknesses using ABF imaging. We found that from 15 u.c.thin film had fully developed tilted structure, with indications of tilt starting from the last 2 unit cells of STO and gradually increasing into LCO. However, similar behavior was not observed in the 5 u.c. film, which is apparently not tilted. (Fig.1) EELS mapping at the interface reveals some Ti/Co intermixing, which was however identical in the two films (Fig.2), suggesting that the tilts are responsible for the difference in properties.
Finally, to study substrate strain effect we examined LCO films grown on LSAT. We found that, compared to films on STO, octahedral tilts are substantially suppressed on LSAT substrate (Fig.3). STO and LSAT subject LCO to different degrees of tensile strain. However, first principles calculations suggest that the differences in tilt behavior cannot be explained by changing strain alone. One possible factor is very rigid lattice of the LSAT due to ordering of Al and Ta atoms on the B-site, suggesting even more complex picture of coupling of different structural order parameters inside perovskites.
[1] J. M. Rondinelli, S. J. May and J. W. Freeland, MRS Bulletin 37 261 (2012).
[2].Y.-M. Kim et al. Advan. Mater. 25 2497 (2013).

The MSE Division, US DOE; through a user project in ORNL’s CNMS, sponsored by the SUF Division, Office of BES, US DOE; The CSGB Division, Office of BES, US DOE

Fig. 1: (A) HAADF images of 15 u.c. / 5 u.c. LCO on STO (B) enlarged BF/ABF images of 15 u.c. / 5 u.c. LCO on STO at the interface. (C) Octahedral rotation angles show gradual increase and saturation of 15 u.c. LCO but it is suppressed in 5u.c. LCO.

Fig. 2: EELS spectra image and the corresponding intensity profiles of La M, Co L, and Ti L edges in (A) 5 u.c and (B) 15 u.c. LCO/STO thin film and. Evidence for Ti interdiffusion is highlighted with a blue arrow.

Fig. 3: HAADF/ABF images of LCO on LSAT (B) Octahedral rotation angles of LCO on LSAT are suppressed (note that the scale of Fig. 3(b) is the same as for Fig. 1(b)).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3418 Comparative microstructural evaluation of red ceramics incorporated with inorganic secondary materials

Spiliotis X.1, Karayannis V.2, Koukouzas N.3, Ntampegliotis K.1, Papapolymerou G.1
1Technological Education Institution of Thessaly, Larissa, Greece, 2Technological Education Institution of Western Macedonia, Kozani, Greece, 3Centre for Research and Technology Hellas, Athens, Greece
spil@teilar.gr

Various inorganic industrial by-products are currently under investigation as substitute secondary materials in standard ceramics manufacturing, because they contain several valuable oxides, and, consequently, their addition into clays used in the fabrication of ceramics appears attractive both from the environmental and economical point of view.
In the present research, microscopic techniques (Scanning Electron Microscopy, JEOL-JSM 6510 coupled with Energy Dispersive X-Ray Spectroscopy) were used for the comparative microstructural examination of red ceramics derived from fired clayey raw materials incorporated with different inorganic solid residues.
Fly ash, the by-product of solid fuel conventional burning for power generation, as well as fly ash originated from circulating fluidized bed combustion (a rapidly growing technology), and also steel making by-products, were considered as useful secondary resources. Specimens were shaped from clay/inorganic material mixtures by extrusion, followed by sintering at different peak temperatures, and their microstructure was studied as a function of the admixture used and the sintering temperature applied. Moreover, the relationship between structure and properties of the ceramic microstructures obtained and characterized was also evaluated. In conclusion, microstructural examination is important to assess and optimize the quality of red ceramics containing secondary inorganic residues, towards beneficial utilization of material and energy resources.

This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: ARCHIMEDES III. Investing in knowledge society through the European Social Fund.

Fig. 1: SEM micrographs of red ceramics incorporated with fly ash, sintered at 850 (a), 950 (b), 1050 (c), 1150 oC (d).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3426 TEM studies on in-situ formation of SiC in AlN-Si-Al composites

KAYA P.1, YURDAKUL H.1,3, TURAN S.1, KALEMTAS A.1,2, ARSLAN G.1, KARA F.1
1Anadolu University, Department of Materials Science and Engineering, Iki Eylul Campus, 26555 Eskisehir, Turkey, 2Mugla University, Engineering Faculty, Department of Metallurgical and Materials Engineering, 48000, Kotekli, Mugla,Turkey, 3Dumlupinar University, Evliya Celebi Campus, Faculty of Engineering, Department of Materials Science and Engineering, TR-43100, Kutahya, Turkey
pkaya1@anadolu.edu.tr

Monolithic ceramics such as Si3N4, AlN and SiC possess a good combination of properties including high hardness, high elastic modulus and high strength due to their strong covalent bonding but are inherently brittle. The use of ceramic components for structural, electrical and electronic applications is rapidly increasing, but difficulties in machining, low reliability and expensive production equipments have hindered the in cost-effective use of these materials [1]. Pressureless melt infiltration is generally considered to be a more attractive technique to produce ceramic-metal composites due to its cost effectiveness and easiness when compared with more conventional methods such casting and powder metallurgy [2]. The aim of the current study was to reduce metal content in final composite through incorporating active agents to the starting Si3N4 powder. A further objective was to fully characterize the microstructure of the composites, especially the distribution, content and morphology of the in-situ formed nano-sized SiC particles. For this purpose relatively dense AlN-SiC-Si-Al composites were produced by pressureless reactive infiltration of aluminum into porous Si3N4 preforms. The composite samples were characterized by employing XRD, SEM and TEM techniques. XRD patterns of the samples were recorded using a (Rigaku Rint 2200, Tokyo, Japan) was performed using monochromatic CuKα radiation (λ = 1.5406). Scanning electron microscope (SEM) investigations were carried out using a ZEISS SUPRA 50 VP microscope. For TEM investigations 200 kV field emission TEM (JEOL™ JEM-2100F) equipped with STEM high angle annular dark field (STEM-HAADF) detector (Model 3000, Fischione), parallel electron energy loss spectrometer (PEELS) and energy filter (Gatan™ GIF Tridiem), and energy dispersive spectrometer (EDS) (JEOL™ JED-2300T) were used. Phase analysis of the produced composites revealed that Si3N4 was consumed completely during the infiltration process via reacting with the Al metal and leading to formation of in-situ SiC, AlN and Si (Figure 1). Detailed TEM studies such as EFTEM results confirmed the XRD results (Figure 2).

References
[1] Janssen, R., Scheppokat, S., and Claussen, N., J. Eur. Cer. Soc., (2008), 28, 1369–1379.
[2] Development of Si3N4/A1 composite by pressureless melt infiltration, Akhtar Farid, GUO Shi-ju, Trans. Nonferrous Met. SOC. China 16, (2006), 629-632

Fig. 1: XRD patterns of (a) 100SN, (b) 92SN8C and (c) 96SN4C ceramic-metal composites.

Fig. 2: EFTEM 3 window elemental mapping showing general distribution of the elements in the 92SN4C composite.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-3495 Influence of the drying on the characteristics of ZnO particles

Mohallem N. S.1, Silva J. B.2, Resende C. P.1, Santos W. M.1
1Universidade Federal de Minas Gerais, 2CDTN/CNEN
silvajb@cdtn.br

ZnO is a simple compound, which shows different textural and morphological characteristics according its synthesis route. These changes lead to the expansion of its applications in electronic and photonic industry due to the wide range of technological applications in devices including sensors, light-emitting diodes, laser diodes, solar energy conversion, catalysts, solar cells, varistor, etc. Some studies are related with applications in nuclear medicine area. When ZnO sintered pellets are irradiated in a cyclotron, it is possible produced the radionuclides 66Ga, 67Ga and 68Ga, which are widely used for diagnosis of diseases. In this work, ZnO nanoparticulate was obtained by reaction between aqueous solution of Zn(CH3COO)2 with NH4OH, whose precipitate obtained was washed with distilled water to remove impurities, and centrifuged at 3000 rpm for 20 minutes. After that, the material was dried using various drying routes: controlled drying in oven, freezing drying and spray drying, and calcined at 800 °C for 2 h. The powders were pressed into pellet form and sintered at 1200 °C for 2 hours. With the use of different drying methods, the formation of the ZnO occurs at different stages and it was observed different morphologies and specific surface areas for the samples. The figures show the morphology of the samples heated at 800oC. The sintering mechanism was different for each sample.Some of the tablets were successfully used in the production of Ga radioisotopes by irradiation.

CNPq, FAPEMIG, CAPES and Centre of Microscopy of UFMG

Fig. 1: SEM image of the ZnO powders dried  by controlled temperature and heated at 800°C.

Fig. 2: SEM image of the ZnO powders dried by freezing drying temperature and heated at 800°C.

Fig. 3: SEM image of the ZnO powders dried by spray drying and heated at 800°C.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-5819 Mechanical behaviour of potassium niobate under compression between room temperature and 900°C

Mark A. F.1, Castillo-Rodriguez M.2, Sigle W.1, van Aken P. A.1
1Stuttgart Center for Electron Microscopy, Max Planck Institute for Intelligent Systems, Stuttgart, Germany , 2Instituto de Ciencia de Materiales, CSIC-Universidad de Sevilla, Sevilla, Spain
alison.mark@is.mpg.de

A detailed study was carried out of the mechanical properties under compression of potassium niobate, a ferroelectric perovskite that has the potential to replace lead-based materials in electromechanical (piezoelectric) applications. Potassium niobate (KNbO3) has recognized excellent optical properties and is widely used in optical mechanisms, e.g. for laser frequency doubling. However, its mechanical response to conditions such as it might see in electromechanical applications has not been widely studied. These conditions include both mechanical and thermal loading, in an uncontrolled atmosphere. A series of compression tests over a range of temperatures between room temperature and 900°C was performed on single crystals of potassium niobate and the specimens were subsequently examined at multiple length scales.

The results reveal the interesting and very complex behaviour of the potassium niobate material. The stress-strain response was unexpectedly consistent with temperature (Fig. 1). Macroscopic characterization of the compressed crystals revealed cracking in all the specimens tested above 200°C. The cracking coexisted with plasticity; dislocations were observed in all tested specimens (Fig. 2). At all temperatures the dominant slip system was {110}<1-10>. Finally, domain walls were observed in large numbers only in the specimens tested between 300°C and 500°C, indicating that unlike the cracking and plasticity responses, the strain accommodation through domain change was not consistent with temperature. The tests performed in this study have turned out to be an excellent first step in understanding the mechanical behaviour of this material. They have provided valuable data on the material’s response to compressive loading and have established the important areas for further study and the critical conditions for useful further modelling work.

This work was supported by DFG Project MR 22/4-2 and the EU Seventh Framework Programme [FP7/2007-2013] - grant 312483 (ESTEEM2). The authors gratefully acknowledge U. Salzberger for TEM specimen preparation and S. Kühnemann for SEM images. MCR acknowledges a JAE/DOC contract from the Spanish Consejo Superior de Investigaciones Cientificas and the European Social Fund.

Fig. 1: Critical flow stress of potassium niobate single crystals strained in compression at constant cross-head speed, at various temperatures.

Fig. 2: TEM BF image of potassium niobate deformed at 400°C showing dislocations and domain walls. Arrow indicates direction of g = [001].

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-5729 TEM-investigation of sulphur contamination on the SOFC material La0.6Sr0.4CoO3 

Gspan C.1, Bucher E.2, Sitte W.2, Hofer F.1
1Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Austria, 2Chair of Physical Chemistry, Montanuniversität Leoben, Austria
christian.gspan@felmi-zfe.at

For the intermediate temperature solid oxide fuel cell (IT-SOFC) the mixed ionic–electronic conducting perovskite oxide La0.6Sr0.4CoO3-δ is a promising material for cathodes due to its high activity for oxygen reduction even at relatively low temperatures of 600–800 °C. Recent investigations have shown that during long-term applications in this temperature range the reaction of the cathode with impurities from the gas phase may cause a significant degradation of the oxygen exchange kinetics. In particular under real conditions the cathodes underlay a time-dependent degradation. In addition to Cr and Si especially the influence of sulphur dioxide is critical for the cathode degradation. In the present work the sulphur poisoning was studied by scanning electron microscopy (SEM) and analytical transmission electron microscopy (TEM). Furthermore chemical depth profiles were performed by X-ray photoelectron spectroscopy (XPS). After 1000 hours of exposition to a test gas of O2-Ar with 50 ppm SO2 TEM images of the cross section show a strongly degraded sample (Fig. 1). The reason for degradation is a strong enrichment of S, Sr and La at the surface (0 – 500 nm) [1] which could be identified by energy-dispersive X-ray spectroscopy (EDXS). Quantification of EDX spectra reveal two different sulphate phases from this region (Fig. 2). Phase 1 of layer I could be identified as La2O2SO4 and Phase 2 as SrSO4. Beneath this layer I is a nano-crystalline region (layer II) within a range of approximately 500 to 1400 nm (Fig. 3). This second layer is rich in cobalt and features a reduced content of strontium. With EDXS also a small amount of sulphur can be found in this deeper region. Below a depth of 1400 nm no significant S-peak can be observed in the EDXS analysis of the bulk phase La0.6Sr0.4CoO3-δ.

References

[1] E. Bucher et al. Solid State Ionics 238 (2013) 15-23.

This research has received funding from the European Union within the 7th Framework Programme [FP7/2007-2013] under Grant Agreement no. 312483 (ESTEEM2).

Fig. 1: Cross-sectional STEM-HAADF image showing three different layers.

Fig. 2: EDXS of layer I with the two coexisting phases La2O2SO4 (1) and SrSO4 (2).

Fig. 3: EDXS (3) from the nano-crystalline layer with increased Co content and traces of S.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-5861 Microstructure of Si3N4 with graphene multipatelets sintered with different sintering additives

Kašiarová M.1, Michalková M.2, Dusza J.1, Šajgalík P.2
1Institute of Materials Research, Slovak Academy of Sciences, Košice, Slovak Republic, 2Institute of Inorganic Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic
mkasiarova@imr.saske.sk

Work shows the relation between the sintering additives, evolution of microstructure and some of the properties of Si3N4–GNPs composites. Si3N4 with graphene multiplatelets (GNPs) as the reinforcement were sintered with five different sintering additives. Initial ceramic powders containing 1 wt.% GNPs were milled in planetary ball mill in isopropanol for 2 hours and hot pressed at 1600°C or 1550°C for 60 min with an external load of 30 MPa and 40 mbar nitrogen overpressure. The relative densities of all Si3N4 composites with 1 wt. % of GNPs were above 98.5% of the theoretical density. The grain size distribution, the shape of silicon nitride grains and the location of graphene multiplatelets in the silicon nitride matrix has been studied using TEM and SEM. Observed changes in the microstructure depend on the used combination of sintering additives especially their viscosity at sintering temperature. Evolution of the grains and the phase transformation from α to β Si3N4 was suppressed by the presence of the graphene multiplatelets. The incorporation of such  carbon-nanostructures into a ceramic matrix inhibits the sintering driving force leading to a lower grain size in at higher temperatures and to a suppression of α to β transformation of silicon nitride at lower temperatures. Due to the relatively mild conditions of the sintering (1600°C/ 1 hour), no coarsening of the silicon nitride matrix or any significant change of this phase was observed. Young’s modulus measurement was performed in order to investigate the orientation of GNPs in the composites. Young’s moduli measured in both perpendicular as well as parallel directions to the sintering plane shows only slight difference which is caused by the arrangement of the silicon nitride grains due to the uniaxial stress applied during hot press sintering not due to the arrangement of graphene platelets. The difference of the grain evolution influences the brittleness, hardness and strength of composites. The presence of the elongated β Si3N4 grains increased the value of fracture toughness due to so called toughening mechanisms – crack bridging, deflection, mechanical friction.

The authors gratefully acknowledge the financial support from APVV 0161-11. Part of the work was realized within the frame of the projects NanoCEXmat II: ITMS No: 26220120035, NanoCEXmat I: ITMS No: 26220120019 and Centre of Excellence SAS CLTP-MREC.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-5947 Influence of use of plastic as fuel in cement clinker production on phase distribution: SEM as a QC-tool

Semsari Parapari S.1, Mokhtari P.1, Papila M.1, Gülgün M. A.1, Uysal Tüten P.2
1Sabancı University, FENS and SUNUM, No:27, 34956, Orhanlı Tuzla, Istanbul, Turkey, 2AkçanSA, No:38, 34662, Altunizade, Üsküdar, Istanbul, Turkey
sorour@sabanciuniv.edu

Use of plastic wastes as alternative fuels in cement producing kilns is currently performed at industrial scale in many countries. Beside of the many advantageous points of disposing residual plastics with this method, there are also a few drawbacks. For example, incorporation of minor elements coming from the fuel during the firing may affect the clinker phase distribution. It has been suggested that these alterations in clinker phases have influences in the subsequent hydration products and thus, could change the strength development of the cement [1-4]. Therefore, investigating the effects of alternative fuels in microstructural evolution and chemical composition of cement clinker is of utmost importance.
In this study, a scanning electron microscope (JEOL JSM-6010LV) was used to investigate the morphology of the cement clinker phases with and without the usage of plastic wastes as the fuel in production process. Backscattered electron images (Fig. 1 and Fig. 2) showed the phase distribution in each of the two clinker specimens. Alite (3 CaO • SiO2: C3S) and Belite (2 CaO • SiO2: C2S) phases are recognizable because of their atomic weight differences. The average atomic weight of Alite is 228.31 g/mol and for Belite is 188.23 g/mol. A slight increase in Belite phase formation can be seen in the clinker produced with plastics as fuel. Also, it is obvious that the distribution of phases all over the clinker granules is different for two samples. These alterations in microstructure are probably due to the influence of impurity elements like Sulfur (S) and Chlorine (Cl) which are the result of combustion of plastic wastes.
Energy dispersive x-ray spectroscopy was utilized to perform the chemical analysis of the clinker phases. Fig. 3 illustrates EDS maps of Cl and S for the same region from the sample produced with plastic waste as fuel. Sulphur appeared to have concentrated preferentially to Belite phase whereas chlorine was concentrated in pore volume.
ImageJ software was used to measure the variation of Alite/Belite phase ratio in two samples. Fig. 4 shows that this ratio decreased from 5.15 to 3.13 for the samples without and with plastic waste fuel, respectively.
[1] Husillos Rodríguez, et al., (2013). The effect of using thermally dried sewage sludge as an alternative fuel on Portland cement clinker production. Journal of Cleaner Production, 52(0), 94-102.
[2]Shirasaka, T., Hanehara, S., & Uchikawa, H., (1996). Influence of six minor and trace elements in raw material on the composition and structure of clinker. World Cement, 27(3).
[3] Woo-Teck KWON et al., (2005). Effect of P2O5 and Chloride on clinkering reaction. Online Journal of Materials, 1.
[4] Lin, K.-L., Lin, D. F., & Luo, H. L. (2009). Journal of Hazardous Materials, 168(2–3), 1105-1110.

Fig. 1: Backscattered electron image of cement clinker prepared without plastic fuels.

Fig. 2: Backscattered electron image of cement clinker prepared with plastic fuels.

Fig. 3: Energy dispersive spectroscopy maps of Cl and S for the same region in cement clinker prepared with plastic fuel.

Fig. 4: Alite/Belite ratio calculated with ImageJ software for two clinker samples.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-5-P-6058 Characterization of niobium carbides obtained via temperature programmed synthesis assisted with metallic magnesium.

Morgado-Vargas M.1, González G.2, Urbina de Navarro C.1, Betancourt P.3
1Centro de Microscopía Electrónica "Dr. Mitsuo Ogura”. Facultad de Ciencias. Universidad Central de Venezuela, Caracas, Venezuela, 2Departamento de Ingeniería y Ciencias de los Materiales. Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela, 3Laboratorio de Tratamiento Catalítico de Efluentes, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela
gemagonz@gmail.com

Transition metal carbides are ceramic refractory with unique properties such as a high melting, extreme hardness, excellent electrical and thermal conductivity, and high resistance to oxidation and corrosion. Therefore, the carbides of transition metals have a broad application in the area of metallurgy. However, the chemical stability of these compounds and their similarity in the catalytic properties of the noble metals of group 8 to 10, make of the transition metal carbides of great interest in the area of catalyst for a variety of reactions including hydrotreatment reactions. These materials are generally obtained by the synthesis method programmed temperature (TPS) of the metal oxide using a gas mixture to high temperatures, greater than 1500ºC. In this work, niobium carbides were synthesized using an alternative methodology via a decomposition-reduction route by the reaction of metallic magnesium powder with niobium oxide at 650ºC. With this method can prepare niobium carbide powder at a lower temperature in comparison with conventional methods. Gas mixture used for reduction and carburetion of metal was hydrogen (H2) and liquefied petroleum gas (LPG) in 9:1 ratio. The products were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and specific surface area by Brunauer-Emmett-Teller (BET) methods. The solids obtained show a specific surface area of 4m2/g. Mixture of niobium carbide (NbC) and niobium oxide (Nb2O5) was observable by XRD patterns, because the niobium carbide was passive with a mixture of O2 /N2 at 1%. SEM images showed particles like corals and wide size range, due to sintering of the particles (Figure 1). The TEM image of the NbC is shown in Figure 2, which indicates the synthesized NbC consists of particles 0.2 μm in diameter. This alternative route to the synthesis method allows obtaining niobium carbides at lower temperatures.

[1] Oyama, S. Catalysis Today 15 (1992) 179-200
[2] Furimsky, E. Applied catalysis A: general, 2003. 240(1-2) 1-28.
[3] Hwu H. and Chen J. Chem. Rev. 105 (2005) 185-212
[4] Jianhua Ma, et al. Journal of Alloys and Compounds 475 (2009) 415-417.
[5] S.V. Aydinyan. Materials Science and Engineering B 172 (2010) 267–271.

Fig. 1: SEM image niobium carbide

Fig. 2: TEM image niobium carbide
↑ Go to the Table of content ↑

MS-6. Polymers and organic materials

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-6-IN-3180 Control and fabrication of polymer nano-structures studied by electron microscopy

Jinnai H.1
1Institute of Materials Chemistry and Engineering, Kyushu University, Fukuoka, Japan
hjinnai@cstf.kyushu-u.ac.jp

In order to fabricate nano-scale structures for variety of applications, e.g., nanotechnology, photovoltaic devices, drug delivery etc., choice of three-deimsnional (3D) structures is a key issue. Block copolymers, consisting of multiple polymer chains (blocks) connected with covalent bond, self-assemble to form various kinds of morphologies due to immiscibility between the dissimilar blocks. We found an ABC-type tribock terpolymers self-assemble 3D helical morphology using electron tomography (ET) [1-3]. The ET observations revealed that the double-helical structure was composed of B helical microdomains around hexagonal-packed A cylinder cores in C matrix, even though none of the blocks is chiral. Under some condition, helical handedness of B domain was found to be uniform, the reason of which will be discussed with the help of computer simulation. This kind of interesting morphologies could be used as templates for materials with attractive properties.

Another way to assemble nano-structures may be to directly polymerize monomers in a controlled manner. We found that (conductive) polymers can be polymerized when electrons are injected into their monomers (Electro-polymerization) in liquid state. Namely, atmospheric Scanning Electron Microscope (ASEM) [4] was used to generate array of nano-scale pillars by irradiating focused electron beam in the monomer solution. The well-controlled pillar-morphology may be essential in some energy and optical properties.

References:


1) H. Jinnai, T. Kaneko, K. Matsunaga, C. Abetz, V. Abetz,
Soft Matter, 5, 2042-2046 (2009).


2) T. Higuchi, H. Sugimori, X. Jiang, S. Hong, K. Matsunaga, T. Kaneko, V. Abetz, A. Takahara, H. Jinnai, Macromolecules, 46(17), 6991–6997 (2013).


3) S. Hong, T. Higuchi, H. Sugimori, T. Kaneko, V. Abetz, A. Takahara, H. Jinnai, Polymer J., 44, 567-572 (2012).


4) M. Suga et al., Ultramicroscopy, 111, 1650-1658 (2011).

We would like to thank Dr. T. Higuchi, Dr. D. Murakami (JST ERATO Takahara Soft Interfaces Project), H. Nishiyama, M. Suga (JEOL Ltd.) and Prof. A. Takahara (IMCE, Kyushu University) for their help and support in ET and ASEM experiments. The authors are grateful to Dr. H. Morita of National Institute of Advanced Industrial Science and Technology for his valuable discussions and comments.

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-6-IN-5772 Insight into structure, morphology and device architectures in plastic electronics using transmission electron microscopy

Brinkmann M.1, Biniek L.1, Kayunkid N.1, Crespo-Monteiro N.1, Girleanu M.2, Ersen O.2
1Institut Charles Sadron, CNRS-Université de Strasbourg, 23 rue du loess , 67034 Strasbourg, France, 2Institut de Physique et Chimie des Matériaux de Strasbourg, Université de Strasbourg, 23 rue du loess, 67034, Strasbourg, France.
martin.brinkmann@ics-cnrs.unistra.fr

Transmission electron microscopy (TEM) is a unique tool to address fundamental issues regarding the structure and the morphology of electro-active materials used in plastic electronics. Various examples are provided to illustrate recent advances in the understanding of crystallization and structure of conjugated polymers and co-oligomers used as active layers in field effect transistors or organic solar cells. It is demonstrated that low dose TEM operated in bright field, electron diffraction, dark field and high resolution modes provides a new and unique insight into the structure and nano-morphology of key materials used in plastic electronics for the elaboration of electronic devices such as organic solar cells and organic field effect ransistors. In particular, we demonstrate the importance of growth control using either epitaxy, high temperature rubbing or other crystallization methods to address properly the structure of conjugated polymers by TEM. Examples concern the polymorphism and nanomorphology of both p- and n-type semiconducting polymers e.g. regioregular poly(3-hexylthiophene) and p(NDI2OD-T2) as well as donor-acceptor co-oligomers used in organic photovoltaic cells. Finally, we show first results on the importance of TEM investigations on the device structure of non volatile memories. More specifically, TEM tomographic investigations and STEM-HAADF investigations on cross sections provide important informations on metal diffusion and nanoparticle distribution in the bulk of polymer layers.

Financial support by the European Community via the Interreg IV-A program (C25, Rhin Solar) and the FP7 Project HYMEC (Grant
No. 263073 is gratefully acknowledged.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-6-O-1635 TEM Characterization of Organic Semiconductor Thin films

Felix R.1, Gries K. I.1, Haas B.1, Breuer T.1, Witte G.1, Volz K.1
1Philipps-Universität Marburg, Faculty of Physics and Materials Science Center, Marburg, Germany
rocio@staff.uni-marburg.de

Organic semiconductors have gained a great interest in the last years, due to their potential applications in electronic devices such as organic field-effect transistors (OFETs) and light-emitting diodes (OLEDs). Among these organic molecules, pentacene (PEN, C22H14), perfluoropentancene (PFP, C22F14), and above all, mixtures between both attract a special attention, because on the one hand they form donor/acceptor systems, and on the other hand they are expected to be structurally compatible due to their similar molecular geometry. Thus, they have been widely studied as a semiconductor p-type, n-type or p-n-junction, respectively, on various substrates such as halides for PFP [1] and PEN [2], SiO2/Si for PFP [3] or polymer gate dielectrics for PEN [4], and with optimal imaging conditions which minimize the radiation damage that destroys the organic materials [3].

Molecular orientation and ordering of different organic semiconductors depends on substrate interaction and substrate roughness. In this study we use Conventional TEM (CTEM) Bright Field (BF) and Dark Field (DF) as well as Electron Diffraction (ED) to show the difference between PEN:PFP (1:1) grown on crystalline substrates such as KCl (100) or NaCl (100) and amorphous substrates such as SiO2. In case of PEN:PFP grown on KCl well ordered films consisting of domains of elongated PEN fibers are formed directly over the substrate. Above these PEN films, bigger individual fibers consisting of PEN:PFP are distributed.The fibers of PEN films are oriented parallel to the KCl <100> directions. ED Patterns of these samples reveal a PEN [001] zone axis orientation and a fourfold ordering (figure 1). In contrast to PEN:PFP grown on SiO2, where no global ordering of the PEN molecules appears. In this case ED Pattern (figure 2) shows mainly a polycrystalline arrangement of the PEN molecules within the sample.   

In this way, substrates can also determine the orientation and arrangement of crystalline molecular films in organic semiconductors, playing an important role in electronic and optical properties of such materials. TEM characterization is an useful tool to understand local and extended crystal orientation by means of a combination of imaging and diffraction techniques.

[1] T. Breuer et al. Phys. Rev. B 83, 155428 (2011)
[2] T. Kiyomura et al. Thin Solid Films 515, 810-813 (2006)
[3] B. Haas et al. J. Appl. Phys. 110, 073514 (2011)
[4] H. Klauk et al. J. Appl. Phys. 92, 5259 (2002)

The authors gratefully acknowledge funding from the SFB 1083

Fig. 1: ED Pattern of PEN:PFP grown on KCl and simulated pattern of PEN in [001] zone axis orientation. The reflections match PEN in the [001] zone axis orientation with fourfold ordering.

Fig. 2: ED Pattern of PEN:PFP grown on SiO2. The Diffraction Pattern reveals the polycrystalline structure of the PEN molecules.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-6-O-1661 Micro-domain imaging in a copolymer by LAADF-STEM

Isoda S.1, Tsujimoto M.1, Aso R.2, Kurata H.2
1Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan , 2Institute for Chemical Research, Kyoto University, Kyoto, Japan
seiji.isoda@gmail.com

It is often difficult to observe nano-scale structures of polymeric materials using conventional TEM because of its weak scattering contrast. As one of methods to produce image-contrast without any artificial staining, LAADF-STEM was applied in this study, and examined on its quantitative feature for observation of fine structures in a block copolymer. Since LAADF-STEM provides a kind of dark-field image like HAADF-STEM, the contrast can be improved drastically from conventional bright-field imaging. Moreover, LAADF-STEM is powerful not only to produce a contrast but also to utilize more scattered electrons than the case of HAADF-STEM, indicating that the LAADF-STEM could be more practically usable for radiation-sensitive materials. Actually, it is expected to visualize micro-phase-separated morphology of block copolymers with sufficient contrasts by LAADF-STEM, corresponding quantitatively to the mass-thickness difference among micro-separated phases. LAADF-STEM was applied for observation of a diblock copolymer of poly(vinyl phenol)-block-polystyrene (PVPh-b-PS), in which a low detection angle (β=19–50 mrad in JEM-2200FS) was adopted for creating a contrast without artificial staining. FIG. 1 shows well-ordered lamellae morphology observed in cross-sections of the copolymer prepared by sequential living anion polymerization and subsequent hydrolytic deprotection. The bright domain corresponds to high density layer of PVPh, and the dark to low density layer of PS. Firstly, the density difference between PVPh (ρPVPh) and PS (ρPS) domains was evaluated from an intensity profile in FIG.1, resulting into ρPVPh/ρPS = 1.09±0.03. This ratio is reasonable although it is slightly smaller than 1.12 estimated from the reported densities of both homopolymers. On the base of the quantitative contrast, it is possible to examine quantitatively the morphology of phase separation, the local distribution of micro-domains and the interfaces in separation of phase domains. As shown in FIG. 2, for instance, one may see at glance that the mechanical cleaving is happened only at the high density domain of PVPh with bright contrast, presumably owing to its brittleness. As a further application, the temperature dependence of image contrast showed a kink at 90 ˚C, relating obviously to a glass-transition of PS (FIG. 3), which allows us to estimate the Tg and difference in thermal expansion coefficients of rubbery and glassy states of PS. These observations demonstrate that LAADF-STEM is an effective tool to image quantitatively nano-scale domains of polymers.

We thank Profs. S.W. Kuo and F.C. Chang for providing the copolymer samples. This work was partly supported by Grants-in-Aid for Scientific Research, Grants No. 20550188 and 23310075 from MEXT, Japan.

Fig. 1: LAADF-STEM image of PVPh-b-PS with lamellae structure of bright PVPh and dark PS layers with a regular spacing of 40 nm. Intensity profile was measured along the blue box in the figure to estimate the density ratio of both layers.

Fig. 2: LAADF-STEM image of cleaved part in PVPh-b-PS thin section. The outer layer after cleaving is always the bright PVPh layer, presumably owing to its high brittleness.

Fig. 3: LAADF-STEM intensity change against temperature around Tg of PS. A kink is observed around 90 ˚C, indicating the change in thermal expansion coefficient of PS layer against that of the glassy PVPh at the glass transition.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-6-O-2023 Annular Dark-Field Transmission Electron Microscopy for Low Contrast Materials

Bladt E.1, Leroux F.1, Timmermans J.2, Van Tendeloo G.1, Bals S.1
1EMAT, University of Antwerp, Belgium, 2Laboratory of Cell Biology & Histology, University of Antwerp, Belgium
eva.bladt@uantwerpen.be

Imaging soft matter by transmission electron microscopy (TEM) is anything but straightforward. Bright-field TEM (BF-TEM) applied to soft materials, and samples that consist of light elements in general has always been hampered by the lack of object contrast, resulting from the fact that light elements will not substantially alter the amplitude and/or the phase of the incident electron wave, particularly not for thin samples. Recently, interest has grown in developing alternative imaging modes that generate contrast without additional staining. Here, we present a TEM technique based on the use of an annular objective aperture: annular dark-field transmission electron microscopy (ADF-TEM) [1]. The objective aperture acts as a central beam stop in the back focal plane of the objective lens. In this manner, the central beam and all electrons scattered up to a certain semiangle are excluded from imaging. ADF-TEM has already been used successfully in materials science where it is applied to avoid diffraction contrast when recording a tilt series for tomographic reconstruction [2]. Here, we show that the technique also has advantages for soft materials, where diffraction contrast is not dominant.

Using ADF-TEM, our experiments demonstrate an increase in both contrast and signal-to-noise ratio in comparison to conventional BF-TEM. Annular dark-field imaging is also advantageous if hybrid structures with high and low mass thicknesses are to be imaged simultaneously. Especially to study the interaction between the soft and hard compound, it is of importance to visualize both compounds at the same time (figure 2). In addition, ADF-TEM is compared to annular dark-field scanning transmission electron microscopy (ADF-STEM). Although ADF-TEM and ADF-STEM are comparable to a certain extent, their electron dose dynamics is completely different. This has important consequences toward radiation damage when imaging low-Z materials. It can be expected that the decreased dose rate of ADF-TEM in comparison to the higher dose rates in STEM mode will be favored in specific studies.

 

[1] F. Leroux, E. Bladt, J.-P. Timmermans, G. Van Tendeloo, and S. Bals, “Annular dark-field transmission electron microscopy for low contrast materials.,” Microsc. Microanal., vol. 19, no. 3, pp. 629–634, Jun. 2013.

[2] S. Bals, G. Van Tendeloo, and C. Kisielowski, “A new approach for electron tomography: Annular dark-field transmission electron microscopy,” Adv. Mater., vol. 18, p. 892–895, 2006.

The authors acknowledge financial support from European Research Council (ERC Advanced Grant # 24691-COUNTATOMS, ERC Starting Grant #335078-COLOURATOMS) and FWO.

Fig. 1: (A) Secondary electron image of an annular aperture during fabrication using focused-ion-beam system. (B) Schematic overview of the column of an electron microscope with an annular aperture inserted in the back focal plane of the objective lens.

Fig. 2: Electron micrographs of silver-coated amyloid fibrils obtained using (A) BF-TEM with an objective aperture of 20 μm and (B) ADF-TEM.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-6-O-2276 In situ characterization of polymers in the ESEM

Poelt P.1, Nachtnebel M.1, Zankel A.1
1Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Graz, Austria
peter.poelt@felmi-zfe.at

The fracture behaviour of polymers is, amongst others, strongly dependent on their microstructure and especially also the type, size and distribution of modifier particles. But the conventional stress-strain diagrams resulting from tensile tests are global quantities, integrated over all microscopic processes occurring during the test in the fracture zone. Therefore a direct observation of these processes during the tensile test is impossible.
Yet tensile tests performed in an environmental scanning electron microscope (ESEM) enable the simultaneous recording of both the stress-strain diagram and the crack propagation at the crack tip [1, 2]. Whereas the former is a macroscopic measurement, the microstructures developing at the crack tip and their variation during the tensile test provide direct insight into the impact of the microstructure of the polymer on the fracture behaviour. Because strong stress concentration takes place at the crack tip, structures forming there and processes going on there are decisive for the fracture behaviour of the material.
In case of inorganic filler particles also the local strain distribution at the surface of the specimen and its change during the tensile test can be tracked (Figures 1 and 2). For this purpose generally the skin of the polymer has to be removed. Care has to be taken that no pre-cracks are formed during the polishing. Every pair of particles can be regarded as a micro-extensiometer. As a consequence the resolution of measured strain fields depends on the distances between the filler particles.
But what is happening at the crack tip does not provide full information about the fracture behaviour of the polymer. Especially the distribution of the cracks and their correlation to the distribution of the filler particles is very interesting. To extract this information the full 3D reconstruction of at least part of the sample is necessary. For this aim the tensile test has to be stopped at a predefined force or elongation. Subsequently automated serial sectioning and imaging by use of a microtome mounted in the ESEM can be used [3]. Finally from the resulting stack of images the 3D reconstruction is possible (Figure 3).
Thus a great wealth of information both on the micro- and macroscale can be gained by performing tests in the ESEM. Correlation of all these results should provide greater insight into the fracture behaviour of polymers.

References:
[1] P. Poelt, A. Zankel, M. Gahleitner, H. Herbst, E. Ingolic, C. Grein, Proc. PPS 24, Salerno, Italy (2008).
[2] P. Poelt, A. Zankel, M. Gahleitner, E. Ingolic, C. Grein, Polymer 51, 3203 (2010).
[3] W. Denk, H. Horstmann, PLoS Biol. 2(11), e329 (2004).

The authors want to thank the company BOREALIS for providing the specimens and for support in the discussion of the results.

Fig. 1: ESEM image (low vacuum mode) recorded during a tensile test (v = 0.2 mm/min) of polypropylene modified with glass spheres (image width: 679 µm). The three bright lines mark the distances between particles used to track the change in the local elongation during the test.

Fig. 2: The local elongation (as determined by the particles marked in Figure 1 during a tensile test) as a function of the overall elongation (specimen length: 42 mm).

Fig. 3: 3D representation of part of a crack in EPR (ethylene propylene rubber) modified polypropylene after a tensile test (v = 1 mm/min) stopped at 25% yield (axis labels: µm). The sample got stained with RuO4. Close to the centre and at the top edges EPR particles can be seen.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-1521 Synthesis and In Situ-TEM Investigation of (Thermo-)Responsive Microgels Stained With Gold

Caumanns T.1, Gelissen A.2, Mayer J.1, Richtering W.2
1Central Facility for Electron Microscopy (GfE), RWTH Aachen, Ahornstrasse 55, 52074 Aachen, Germany, 2Institute for Physical Chemistry (IPC), RWTH Aachen, Landoltweg 2, 52074 Aachen, Germany
caumanns@gfe.rwth-aachen.de

More than ever polymer science focus on complex molecular structures and supramolecular assemblies. Beyond this, responsive polymer materials are structures, which can be manipulated in e.g. charge or size change by external parameters like pH or temperature variation. This leads to Microgels. Microgels are soft particulate polymer networks that can be dispersed in a aqueos medium. They reveal unique features providing new opportunities to develop smart bio-inspired materials. In contrast to rigid colloidal particles, which lack the possibility to adapt their size and shape to enviromental requirements, microgels have switchable properties of form and function that makes them very useful in a wide range of e.g. biological sciences and medical applications. They combine properties of dissolved macromolecules with those of colloidal particles.

In the present work, thermoresponsive microgels were studied in their ambient enviroment by in situ-experiments in TEM/STEM. The microgels are made from N-isopropylacrylamide (NiPAAm) as is described in [1].

In order to stain the microgel complexes with gold nanoparticles, they were redispersed in water (1mg per 1ml water). HCl (aq) is used to set a defined pH. By adding chloroauric acid (HAuCl4*3H2O), centrifuging, redispersing in HCl (aq) and reducing with NaBH4 (aq) the stained microgels in aqueos solution are obtained.

In our studies, these microgels were observed by in situ-TEM in liquid environment. In the experiments, a thin layer of liquid was embedded between two hermetically sealed, electron transparent Si3N4-windows. The used holder is an in situ-liquid cell holder manufactured by Hummingbird Company and the microscope is a Zeiss Libra 200FE with an acceleration voltage of 200 kV. The resolution is mainly limited by the thickness of the liquid. To increase the contrast, an energy filter window of about 100 eV is inserted at the most probable energy loss, which reduces the background scattering of the solvent. A big challenge is to focus and get sufficient resolution because of the high mobility of the almost freely moving particles through the liquid.

Figure 1 shows an energy-filtered TEM-image of single microgel particle moving through the liquid, Figure 2 a energy-filtered TEM-image of a cluster of agglomerated microgel particles and Figure 3 a STEM image of a particle that adhere on the surface of the Si3N4-membrane with an additional EDX-spectra that shows the presence of gold, a EELS-Spectra to prove the presence of water and an intensity profile through the particle.

References:

[1] S. Hiltl et. Al., Soft Matter, 7, 8231-8238, 2011

The authors kindly acknowledge the financial support through the SFB 985 through the DFG.

Fig. 1: Single microgel particle observed by a TEM-magnification of 20.000x. The right corner shows a part of a second particle moving through the liquid.

Fig. 2: Aggregation of several microgel particles observed by a TEM-magnification of 8000x.

Fig. 3: STEM image of a microgel particle adhereing on the surface (top left). The EDX-Spectra (top right) shows the presence of gold and the EELS-spectra (bottom right) the presence of water. An intensity profile (bottom left) shows the structure of the microgel.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-1639 Electron Energy Loss Spectroscopy Of Codeposited Pentacene:Perfluoropentacene

Gries K. I.1, Felix R.1, Haas B.1, Breuer T.1, Witte G.1, Volz K.1
1Philipps-Universität Marburg, Faculty of Physics and Materials Science Center, Marburg, Germany
katharina.gries@physik.uni-marburg.de

Over the last several years organic semiconductor materials gain more and more importance. Two examples for such materials are pentacene (PEN) and perfluoropentacene (PFP).
PEN is a polycyclic aromatic hydrocarbon (C22H14) with a HOMO-LUMO gap of approximately 2.1eV. Because of its high hole mobility it acts as an p-type semiconductor. The unit cell of PEN crystals contains two nonequivalent PEN molecules and crystallization takes always place in a triclinic crystal structure. In case of PFP (C22F14) the hydrocarbon atoms are replaced by fluorine atoms. The strong electronegativity of fluorine results in quite different properties of PFP from PEN. PFP acts as an n-type semiconductor and crystallizes in a monoclinic crystal structure.
Transmission electron microscopy (TEM) is a useful method to investigate such structures at a high resolution level und thus to analyse the quality of PEN:PFP composite materials. Besides electron diffraction, dark field imaging, high resolution TEM and energy dispersive X-ray spectroscopy also electron energy loss spectroscopy (EELS) is a suitable method to learn more about the sample structure and composition. In case of organic materials the Plasmon peak located at energy losses in the range of 23 and 27 eV provides a possibility to obtain this information.
In our work we investigated codeposited PEN:PFP samples that have been grown on potassium chloride (KCl) via organic molecular beam deposition (OMBD). These samples are composed of a thin PEN film with fibers consisting of a mixture of PEN:PFP on top of it. Scanning transmission electron microscopy (STEM) enables the detection of EEL spectra from separate regions like the PEN:PFP fibers and the PEN film. For comparison also pure PFP samples have been investigated. The energetic position of the Plasmon peak for the three materials PEN, PFP and PEN:PFP is different. This provides information on the composition and thus on the structure of the sample.
In our presentation we will summarize the influence of different material compositions on the Plasmon peak position in EEL spectroscopy and show how EELS mapping in STEM can be used to characterize mixed organic films.

The authors gratefully acknowledge funding from the SFB 1083.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-1711 DualBeam (FIB-SEM) methodology enables linking 3D spatial orientation & bulk structure in polymeric composite materials to performance

Phifer D. W.1, Rosas-Aburto A.2, Baken E.1, Lei M.3, Pérez-Salinas P.2
1FEI Company, Eindhoven, The Netherlands, 2Universidad Nacional Autónoma de México, Mexico City, Mexico, 3FEI Company, Houston, Texas, USA
daniel.phifer@fei.com

The spatial relationship & composition of functional components within polymer materials is theorized to control bulk properties such as conductivity [1,2,3], however, characterizing 3D structure within a bulk material is not always easy due to the “soft” nature of the materials and similarities in filler components and bulk composition. Similar material investigation of components within bulk material are achieved by traditional FIB-SEM, slice and view techniques where the focused ion beam is used to cut through a bulk material and an electron beam of the SEM is used to characterize the composition and structure within successive slices of a 3D volume. After the slice images are obtained a 3D model can be generated and analyzing the elucidated structural relationship is possible.

Typically FIB-SEM 3D characterization is done with hard materials where the technique is capable of ~4nm resolution in x/y/z directions creating a symmetrical voxel for 3D reconstruction. “Soft” polymeric materials, however, exhibit various milling and imaging artifacts when traditional methodologies are used requiring the development of new low dose imaging and milling procedures to be used for 3D volume and (S)TEM sample preparation. In Fig. 1, images showing the effects of standard (33.6 nC/µm2) and low dose (7.57 nC/µm2) techniques on shrinkage, melting and general structural damage is compared as applied to extraction of a (S)TEM lamella from a bulk polymer material. This illustrates the operational conditions will not add artifacts for the 3D data set acquisition which was subsequently performed. A conductive rubber developed in Mexico (patent application MX/a/2013/014435) was tested by this technique to see if it is possible to image the component materials in the bulk to understand the inter particle spacing and composition.

Investigation of the conductive rubber was done with an automated slice and view procedure on the FEI Versa 3D DualBeam and slice-image data was processed with FEI Aviso Fire software to yield particle orientation and spacing. This data appears to align with the basic models presented in the text book by Milton [1]. This new low dose technique appears suitable for analyzing composite soft materials and should be applied to additional samples.

References:

1. G.W. Milton, The Theory of Composites, Cambridge University Press, 2004.
2. G.R. Ruschau, S. Yoshikawa, R.E. Newnham, Journal of Applied Physics. 72 (1992) 953-959.
3. M.M. Tomadakis, S.V. Sotirchos, Journal Of Chemical Physics. 98 (1993 ) 616-626

Fig. 1: Damage occurs in polymeric materials when traditional FIB milling conditions are used that result in a high ion dose (top image). Reducing the focused ion beam dose (by over 75%) eliminates damage to the sensitive polymeric materials (lower image).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-1757 Impact of ultrasonication on the morphology and structure of cellulose microfibrils and nanocrystals

Barbat-Rogeon A.1, Pétrier C.2, Molina-Boisseau S.1, Putaux J. L.1, Heux L.1, Nishiyama Y.1
1Centre de Recherches sur les Macromolécules Végétales, UPR CNRS 5301, BP 53, F-38041 Grenoble Cedex 9, France, 2Laboratoire Rhéologie et Procédés, UMR 5520, BP 53, F-38041 Grenoble Cedex 9, France
putaux@cermav.cnrs.fr

Cellulose microfibrils are extracted from biomass using chemical treatments and mechanical defibrillation. Our study aimed at characterizing the effect of ultrasonication on the morphology and crystal structure of model cellulose microfibrils and nanocrystals dispersed in water. Algal cellulose microfibrils were extracted from Glaucocystis and Valonia cell walls. Shorter nanocrystals were obtained by sulfuric acid hydrolysis of these microfibrils. Nanocrystals from tunicin, the cellulose found in Halocynthia, a marine animal, were prepared as well. 0.1 wt% aqueous suspensions of microfibrils or nanocrystals were submitted to low and high frequency ultrasounds (20 and 600 kHz, resp.). The suspensions were sonicated for 3 h, their temperature being thermoregulated at 25-30°C. Negatively stained preparations were observed before and after sonication by transmission electron microscopy (TEM). Thin films were prepared by air-drying concentrated suspensions and X-ray diffraction (XRD) patterns were recorded. Solid-state 13C NMR spectra were recorded from dry powders using magic angle spinning and cross-polarization (CP/MAS) techniques. Glaucocystis (Fig. 1a) and Valonia cellulose microfibrils were initially rectilinear and nearly defect-free. The damage resulting from sonication was extensive for low-frequency treatments and due to the many defects (kinks, subfibrillation), the general impression was that the microfibrils had lost their rigidity (Fig. 1b). 600 kHz sonication seemed to induce a smaller number of defects which remained separated by linear segments (Fig. 1c). Native cellulose is a mixture of two allomorphs: Iα (triclinic) and Iβ (monoclinic, thermodynamically more stable). Tunicin is Iβ-rich (90%) while Glaucocystis and Valonia celluloses are Iα-rich (90 and 65%, resp.). XRD profiles revealed that the structure of the latter two specimens changed during sonication (Fig. 1d), the effect being stronger at low frequency. The decreasing distance between 100 and 010 peaks indicated a transition to the Iβ structure while the shift of the 110 peak to lower angles would be due to the high number of defects and lower crystallinity. The quantitative analysis of the CP/MAS NMR spectra allowed to evaluate the Iα/Iβ ratio in the samples and showed that the transition was incomplete. The origin of the structural transition has not been fully identified yet and several hypotheses exist [Briois et al., Cellulose 20 (2013), 597-603]. Damage would be caused by the shocks and shears generated by acoustic cavitation that may also induce longitudinal translations along some crystal planes, resulting in the structural change. In addition, so-called "hot spots" are known to occur near cavitation bubbles which may promote a thermal transition in cellulose.

We gratefully acknowledge funding from Institut Carnot PolyNat and contributions from B. Briois, M.-F. Métral (Glaucocystis culture), B. Jean (tunicin nanocrystals) and H. Chanzy.

Fig. 1: Negatively stained Glaucocystis cellulose microfibrils (GCMs): initial (a), sonicated for 3 h at 20 and 600 kHz (b,c); d) XRD profiles of GCMs and tunicin nanocrystals (TNs): initial GCMs (1); GCMs sonicated for 3 h at 20 and 600 kHz (2,3); initial TNs (4); TNs sonicated for 3 h at 20 kHz (5). t and m refer to triclinic and monoclinic indexations.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-1758 Grain boundaries in nanoparticles made of self-assembled amphiphilic β-cyclodextrins

Putaux J. L.1, Lancelon-Pin C.1, Choisnard L.2, Gèze A.2, Wouessidjewe D.2
1CERMAV, UPR CNRS 5301, ICMG FR 2607, BP 53, F-38041 Grenoble Cedex 9, France, 2Département de Pharmacochimie Moléculaire, UMR CNRS 5063, ICMG IFR 2607, UFR de Pharmacie, Université de Grenoble 1, BP 53, F-38041 Grenoble Cedex 9, France
putaux@cermav.cnrs.fr

One major challenge of nanomedicine is to design nanocarriers that deliver active compounds to a target site, at a sufficient concentration and without premature degradation, in order to maximize the efficiency of the substance while limiting secondary effects. In this context, we have developed colloidal nanovectors based on cyclodextrin (CD) amphiphilic derivatives obtained by an enzymatically-catalyzed transesterification by thermolysin. We have shown that after dissolution in acetone, depending on the length of the grafted alkyl chain, the derivative had the ability to self-organize in water, forming nanoparticles with various shapes and ultrastructure [Gèze et al., Mater. Sci. Eng. C29 (2009), 458; see also the communication by Putaux et al. in this conference]. The knowledge of the morphology and ultrastructure of these nanovectors is crucial in order to optimize their formulation and lyoavailability. The present communication focuses on the βCD-C14 derivative, i.e. βCDs (made of 7 glucosyl units) acylated on their secondary face with C14 chains. The resulting nanoparticle suspensions were quench-frozen and observed by cryo-transmission electron microscopy (cryo-TEM). The βCD-C14 particles exhibited tortuous multidomain shapes (Fig. 1a) and the corresponding small-angle X-ray scattering (SAXS) pattern collected from a concentrated suspension contained peaks whose distribution was consistent with a columnar hexagonal structure (Fig. 1c). Depending on the orientation of the particles in the embedding film of vitreous ice, the cryo-TEM images showed that some particles consisted of misoriented domains separated by sharp interfaces (Fig. 1b). A direct view of the hexagonal organization was obtained when the incident beam was parallel to the columns and grain boundaries with various tilt angles were observed. The structure of the grain boundaries was analyzed using the concepts of coincidence site lattice (CSL) and structural units (SUs) frequently used to describe the atomic structure of interfaces in metallic alloy and semiconductor polycrystals [Thibault et al., Mat. Sci. Eng. A 164 (1993), 93-100]. An example of stepped tilt boundary is shown in Fig. 1d. Assuming that the repeating motif in each grain correspond to the projection of hollow columns made of βCD-C14 molecules (Fig. 1e), the boundary was described with a series of SUs differing by the number of neighboring columns (5, 6 or 7), each of them exhibiting a distinct contrast (Fig. 1f) To our knowledge, it is the first time that such grain boundaries are observed in nanoparticles of self-organized amphiphilic molecules and described at the nanometric scale.

We thank Agence Nationale de la Recherche, ESRF and Institut de Chimie Moléculaire de Grenoble for financial support, and D. Levilly for the synthesis of the βCD-C14 derivative.

Fig. 1: Cryo-TEM images of βCD-C14 particles (a,b,d) and corresponding SAXS pattern (c). The particle in (d) is made of 2 grains separated by a stepped tilt boundary whose structure is outlined in (f): the black dots and larger circles correspond to columns with 5 and 7 neighbors, resp. (e) Model of the columnar hexagonal organization of βCD-C14 molecules.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-1820 Polyhedral iron oxide core-shell nanoparticles in a biodegradable polymeric matrix: Preparation, characterization and application in magnetic particle hyperthermia and drug delivery.

Filippousi M.1, Angelakeris M.2, Pavlidou E.2, Bikiaris D.3, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium, 2Solid State Physics Section, Physics Department, AUTH, GR-54124 Thessaloniki, Greece, 3Laboratory of Polymer Chemistry and Technology, AUTH, GR-54124 Thessaloniki, Greece
maria.filippousi@uantwerpen.be

Nanotechnology is at the leading edge of rapidly developing new therapeutic and diagnostic schemes in diverse areas of biomedicine. Different materials from natural to synthetic polymers as well as inorganic materials with variable structural and physical properties are used as building blocks of biomaterials. Recently, a new term ‘theranostics’ is used in order to encompass two distinct definitions which is the combination of therapeutic and diagnostic agents on a single platform. The development of theranostic nanoparticles is emerging as a new form of “smart” nano-materials that may simultaneously monitor and treat diseases. [1]
The aim of the present study is to characterize the polyhedral iron oxide nanoparticles (IOs) and their magnetic properties that can then be used for the encapsulation of the Paclitaxel drug using two different polymer matrices such as PPSu and its block copolymer mPEG-PPSu-mPEG. [2] Both have been chosen because of their excellent biocompatibility and biodegradability and also because they have melting point temperatures close to the body temperature (Tm=42°C and Tm=44°C). This is very essential in case these IOs will be used for combinatory cancer treatment with hyperthermia and drug release and therefore the drug release was studied at 37°C and at 42°C (Figure 1). The encapsulation of iron oxide nanoparticles into a polymer matrix is confirmed by transmission electron microscopy and further corroborated by high angle annular dark field scanning transmission electron microscopy (HAADF-STEM) (Figures 2a, b). Energy dispersive X-ray spectroscopy mapping allowed us to determine the presence of the different material ingredients in a quantitative way (Figure 2c). HAADF-STEM tomography proved that the iron oxide nanocrystals consist of well-defined polyhedral structures with multiple facets (Figure 3). The magnetic features were found in good agreement with their structural and morphological features. The high heat capacity, which can be maintained in the nanovehicles of IOs encapsulated in the polymeric matrix, is sufficient to provoke damage of the cancer cells. Therefore, this nanosystem, in which polyhedral magnetic nanoparticles are incorporated in a biocompatible and biodegradable polymeric matrix, can be used as a multifunctional magnetic particle hyperthermia agent together with heat-assisted drug-delivery addressing directly the current theranostic trends.

1.Filippousi et al. International Journal of Pharmaceutics 2013, 448, 221.

2.Filippousi et al. RCS Advances 2013, 3, 24367.

GVT and MF acknowledge funding from the ERC grant N°246791 under the 7th Framework Program (FP7),COUNTATOMS. This work is also performed within the framework of the IAP-PAI.

Fig. 1: Drug release profile pattern of Taxol from the prepared PPSu-IOs and mPEG-PPSu-mPEG-IOs nanoparticles.

Fig. 2: (a) Bright field TEM image of mPEG PPSu-mPEG -IOs (b) HAADF- STEM image of the particles of Figure (a) and (c)HAADF-STEM EDX mapping (C- blue, Fe - green, O-red) of mPEG- PPSu- mPEG –IOs. The scale bar stands for all images.

Fig. 3: (a) and (b) 3D representation of the reconstructed volume of a single iron oxide nanoparticle along different views. The occurrence of different facets is obvious and the shape of iron oxide nanoparticle seems to be that of a rhombicuboctahedron. (c) An orthoslice through the volume. (d) Theoretical model of a rhombicuboctahedron.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-1885 Phase structure development and properties of HDPE/COC immiscible polymer blends

Ostafińska A.1, Vacková T.1, Šlouf M.1, Nevoralová M.1, Fortelný I.1
1Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
ostafinska@imc.cas.cz

Melt-mixing of immiscible polymer blends can form a broad range of heterogeneous structures [1]. Polymer blend morphology depends on various processing factors and affects their resulting physical properties [2]. The aim of this study was: 1) to describe development of the phase structure in high-density polyethylene/cycloolefin copolymer (HDPE/COC) blend, 2) to investigate the impact of phase structure on selected mechanical properties and compare them with predictive theories [2,3].

Phase structure and its development. The specimens of HDPE/COC systems (composition: 100/0, 90/10, 80/20…0/100 wt.%) were obtained by melt mixing followed by extrusion, compression or injection molding. Scanning electron microscope (SEM) Vega Plus TS 5135 (Tescan, Czech Republic) was used for observing the phase morphology. The samples were cut from the center of test specimens, smoothed under liquid nitrogen [3], etched with toluene (5 min, room temperature) and observed in SEM (30 kV, SE imaging). The SEM micrographs were processed with image analysis software (NIS-Elements) to calculate the average size of particles (Fig. 1; morphological descriptor EquivalentDiameter [4]) and the average length of segments of the two phases (Fig. 2; descriptor MeanChord [4], appropriate also for co-continuous morphologies). Tensile tests were carried out by using an Instron tester 5800R (Instron, United States) (dumb-bell-shaped specimens, room temperature, 50.0 mm/min).

Phase structure and mechanical properties. The impact of phase structure on selected mechanical properties (such as yield strength; Fig. 3) was experimentally determined and theoretically predicted using equivalent box model (EBM) and linear rule of mixtures (RoM). HDPE/COC morphology analysis showed the fibrous structure at compositions 70/30 and 60/40 The fibrous morphology which is a rare type of structure in the polymer blends occurred in this case due to careful selection of initial polymers and processing conditions. Moreover the resulting structure had a positive impact on mechanical properties: they proved positive deviations from EBM predictions, as indicated in our previous study [2].

References

1. Z. Horák, I. Fortelný, J. Kolařík, D. Hlavatá, A. Sikora, In: J. Kroschwitz,ed. Encyclopedia of Polymer Science and Technology. Indianapolis: John Wiley & Sons, Inc. 2005, 1–59.

2. T. Vacková, M. Šlouf, M. Nevoralová, L. Kaprálková, Europ. Polym. J. 2012, 48, 203–2039.

3. M. Šlouf, J. Kolařík, J. Kotek. Polym. Eng. Sci. 2007, 47, 582–92.

4. Laboratory Imaging s.r.o., NIS-Elements AR User's Guide, 2010, 114, 118.

GACR P106/11/1069 and TACR TE01020118.

Fig. 1: SEM micrograph of smoothed and etched surface    of injection molded HDPE/COC=70/30 blend with fibrous morphology perpendicular to the injection direction.

Fig. 2: SEM micrograph of smoothed and etched surface    of injection molded HDPE/COC=70/30 blend with fibrous morphology parallel to the injection direction.

Fig. 3: Yield stress of HDPE/COC blends as a function of COC content and their theoretical predictions: rule of mixtures (RoM), equivalent box model (EBM).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-1931 Preparation and morphology characterization of tunable conducting polyaniline nanoparticle materials

Huang W. Q.1, Miao X. P.1
1SINOPEC Beijing Research Institute of Chemical Industry，Beijing 100013, China
huangwq.bjhy@sinopec.com

Among the conducting polymers, polyaniline (PANI) is found to be one of the most promising materials because of ease of synthesis, high electrical conductivity, nontoxic property, good environmental and chemical stability, and low cost. Abundant research findings show that PANI nanomaterials exhibit photoelectric properties and chemical properties different from non-nano PANI when the particle size of PANI is in the nanometer scale, owing to the quantum tunneling effect, small size effect, surface effect and so on. At present, the synthesis of conductive PANI nanomaterials with controlled structure, size, morphology and property is the key to realize their applications in technology. Dispersion polymerization is one of the most commonly used method for preparing PANI nanoparticles (NPs). The general system of dispersion polymerization consists of monomer, dispersant, stabilizer and initiator, which forms an isotropic system. In the dispersion polymerization of PANI, water is generally used as the dispersion medium, and macromolecular polymer soluble in water, surfactant or inorganic nanoparticle is chosen as a stabilizer. Our previous research found that the size and shape of PANI particles in dispersion polymerization depends on the balance between the polymerization rate of the monomer and the adsorption rate of formed particles to stabilizers. It is easy to form spherical PANI NPs when the adsorption rate is greater than the rate of polymerization. Therefore, how to effectively speed up adsorption rate of particle to the stabilizer is essential to prepare PANI NPs.
In this work, we have synthesized PANI nanoparticle materials with different morphologies through decreasing the concentration of stabilizer from a in Fig.1 to d  in Fig.4. In the polymerization system, stabilizer incorporation into nanoparticle is as the role of adhesive. It is more difficult to disperse NPs due to the larger cohesive action of more stabilizer. In Fig.1, the whole dispersity deteriorates when the content of stabilizer is high. With the decrease of the stabilizer content, particles are easily disintegrated to NPs under otherwise identical experimental conditions. It can form rambutan like PANI NPs dispersing well with an average size of 140nm at a appropriate content of stabilizer, as shown in Fig.3. In Fig.4, if we continue to reduce the concentration of stabilizer, PANI NPs tend to aggregate into sub micro-scale particles with a size distribution of 300-400nm in diameter which limit its application areas. Consequently, the concentration of stabilizer(i.e.the solubility of stabilizer in water) has an important influence on the formation of PANI NPs. The worse solubility of stabilizer, the more unfavorable the formation of PANI NPs.

This research was supported by the Analytical Research Division of SINOPEC Beijing Research Institute of Chemical Industry.

Fig. 1: Scanning electron microscope image of polyaniline nanoparticles with the stabilizer concentration of a.

Fig. 2: Scanning electron microscope image of polyaniline nanoparticles with the stabilizer concentration of b.

Fig. 3: Scanning electron microscope image of polyaniline nanoparticles with the stabilizer concentration of c.

Fig. 4: Scanning electron microscope image of polyaniline nanoparticles with the stabilizer concentration of d.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2319 Comparative measurement of microplasticity by means of 3D-microscopy methods

Kokoskova M.1, 2, Slouf M.1, Nevoralova M.1, Vackova T.1, Kopecek J.3
1Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic, 2Dept. of Physical and Macromolecular Chemistry, Charles University in Prague, Hlavova 8, 128 40 Prague 2, Czech Republic, 3Institute of Physics, Academy of Sciences of the Czech Republic, Na Slovance 1999/2, 182 21 Prague 8, Czech Republic
marketa.kokoskova@natur.cuni.cz

Microplasticity (MP) is one of the parameters characterizing viscoelastic behavior of synthetic polymers [1,2]. MP is defined as the normalized ratio between the width and depth of the indent from microhardness tester after elastic recovery (Figure 1). In the past decades, MP was scarcely measured due to experimental difficulties [3]. Nevertheless, modern microscopic techniques made MP measurement feasible. Our preliminary results have showed that microscopically measured MP values are in a good agreement with material properties [3]. In this contribution we focus on four microscopic methods which can measure MP, i.e. which yield 3D surface maps with submicrometer accuracy, especially in Z-axis direction.

We tested the precision of the following four microscopic techniques: (i) wide-field light microscope with motorized Z-stage and software for combining Z-stack images into 3D-surface maps (DM6000 M, Leica), (ii) digital microscope, which provides 3D-surfaces with higher precision (VHX-1000, Keyence), (iii) standard SEM microscope (Vega Plus TS 5135, Tescan), in which the depth information is calculated from precisely tilted micrograph as described by Lawn et al [2], and (iv) advanced SEM microscope (Quanta 200 FEG, FEI) with eucentric stage and Stereo software (Scandium software with Stereo module, Olympus), which enables the user to create stereoscopic images and to compute the height of image points from two images tilted to small angle (typically +/- 3 degrees). The testing samples were two polymers (two types of ultrahigh molecular weight polyethylene – UHMWPE – with different thermal treatment and plasticity) and one metal (B2-ordered intermetallic alloy Fe - 41 at.% Al). MP of each sample was determined from at least five indents, which were left to equilibrate for 2 days and subsequently investigated by all above described methods.

Typical outputs from 3D microscopic methods are shown in Figure 2. With exception of standard LM microscope, all methods were in agreement with theoretical prediction that metal sample is >75 % plastic and polymer materials exhibit MP values within 20–30 % [3]. Final comparison of all results (Figure 3) suggested the following precision of the methods: standard LM microscope << digital microscope = SEM microscope (calculation according to [2]) = SEM microscope (calculation from stereo images using commercial software).

References: [1] Balta-Calleja F. Microhardness of polymers. Cambridge: Cambridge university press 2000, [2] Lawn BR. J. Mater. Sci. 1981, 16, 2745., [3] Slouf, M. et al Microhardness, microcreep and microplasticity of virgin, crosslinked and/or aged ultrahigh molecular weight polyethylenes, Conference Proceedings and Abstracts. 2013, p. 12-13 International UHMWPE Meeting /6./.

TACR TE01020118, IGA MZ CR NT12229-4/2011 and GAUK 558213.

Fig. 1: A schematic illustration of the principles of MP determination by means of Vickers pyramid indenter and the MP calculation formula [1].

Fig. 2: A - SEM micrograph of 50° tilted indent, B – micrograph of the indent obtained by means of digital microscope, C – typical depth profile of an indent used for MP determination.

Fig. 3: The overall results obtained by all 3D-microscopic methods. Note: remelting = thermal treatment above melting temperature, annealing = thermal treatment below melting temperature.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2125 Microstructure and micromechanical properties of PP/HDPE/EPDM blends

Situm A.1, Nevoralova M.1, Slouf M.1, Vranjes N.2, Rek V.2
1Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic, 2University of Zagreb, Faculty of Chemical Engineering and Technology, Marulic Sq. 19, 10 000 Zagreb, Croatia
Slouf@imc.cas.cz

Introduction. Like most polymers, isotactic polypropylene (PP) and high-density polyethylene (HDPE) are immiscible. Consequently, PP/HDPE blends exhibit phase separation, coarse phase morphology, poor interphase adhesion and bad mechanical properties [1]. Nevertheless, morphology and properties of immiscible polymer blends can be improved by compatibiliziation, i.e. by the addition of a copolymer called compatibilizer that improves interphase adhesion [1]. This contribution is focused on morphology and micromechanical properties of PP/HDPE blends, both non-compatibilized and compatibilized with ethylene, propylene, and diene-component terpolymer (EPDM).

Materials and methods. PP (HC206TF, Borealis), HDPE (Lupolen 5031L, Basel) and EPDM (Nordel IPNDR 4520, Dupont Dow Elastomers) are commercial products. PP/HDPE/EPDM blends with various compositions were prepared by melt-mixing (extrusion and injection-moulding) [2]. Morphology was visualized by SEM microscopy of fracture surfaces and smoothed, permanganic mixture-etched surfaces [3]. Microhardness (MC) and microcreep (MC) were measured by Vickers microhardness tester and compared with theoretical predictions as described elsewhere [4, 5].

Results and conclusions. SEM micrographs of fracture surfaces exposed the two-phase morphology of non-compatibilized blends (Fig. 1a). Voids could be observed, indicating a low interphase adhesion between the components. Finer morphology, with particles of minority phase better incorporated in the matrix, was obtained by addition of the EPDM compatibilizer (Fig. 1b). Smoothed surfaces revealed the inhomogeneity of the systems (Fig. 2a) and the lamellar structure of HDPE (Fig. 2b). The PP/HDPE interphase reduces MH values below the prediction based on the Additivity law [4]. The systems have also shown some trend in the measured values of MC (Fig. 3b), which slightly increased with the concentration of PP. We conclude that agreement among composition, micromechanical properties and theoretical predictions [4, 5] was found (Fig. 3). Nevertheless, the inhomogeneous morphology (Fig. 2a) explained why both MH and MC values were rather scattered (Fig. 3a). This re-confirms close relationship between morphology and micromechanical properties and points out the importance of microscopic investigation for correct interpretation of MH and MC results.

References

[1] Horak Z et al.: Polymer Blends. Encyclopedia Of Polymer Science and Technology (2005)

[2] Vranjes N et al.: Macromol Symp, 258 (2007) 90-100

[3] Slouf M et al.: Polym Eng Sci, 47 (2007) 582-592

[4] Calleja F et al: Microhardness of polymers. Cambridge university press (2000)

[5] Vackova T et al.: Eur Polym J, 48 (2012) 2031-39

TACR TE01020118 and MZOS (Croatia) 125-1252971-2578.

Fig. 1: SEM micrographs showing phase morphology of (a) non-compatibilized blend PP/HDPE (80/20), and (b) compatibilized blend PP/HDPE/EPDM (20/80/7).

Fig. 2: SEM micrograph showing (a) non-homogeneity and phase inversion of PP/HDPE (80/20) system and (b) crystalline lamellae in PE particles within PP/HDPE/EPDM (60/40/5) blend.

Fig. 3: Micromechanical properties of polymer blends: (a) microhardness and its comparison with linear model and equivalent-box model, (b) microcreep measurements.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2135 Morphology of PCL/TiX composites: comparison of standard and advanced microscopy methods

Slouf M.1, Situm A.1, Pavlova E.1, Nevoralova M.1, Hromadkova J.1, Vystavel T.2, Navratilova L.2, Govorcin Bajsic E.3, Ocelic Bulatovic V.3
1Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic, 2FEI, Podnikatelská 6, 612 00, Brno, Czech Republic, 3Faculty of Chemical Engineering and Technology, University of Zagreb, Marulic Sq. 19, 10 000 Zagreb, Croatia
Slouf@imc.cas.cz

Introduction. Biodegradable polymer composites attract increasing attention in recent years [1]. Polycaprolactone (PCL) is a polyester, frequently used in bio-based polymer systems. We prepared composites of PCL and three different types of TiO2-based nanoparticles (TiX; Figure 1). This contribution is aimed at detailed characterization of morphology of PCL/TiX systems.

Experimental. PCL polymer (6-Caprolactone polymer, M = 80,000 g/mol) and TiO2 nanoparticles with average size 200 nm (TiNP2; Fig. 1a) and 100 nm (TiNP1; Fig. 1b) were bought from Sigma Aldrich. Titanate nanotubes (TiNT; Figure 1c) were hydrothermally synthesized in our laboratory [2]. PCL/TiX composites (2 wt.% of TiX) were prepared by melt mixing (Brabender, 60 rpm, T=120C), followed by compression molding (T=140C). Morphology of PCL/TiX composites was characterized by light microscopy (LM; 10um sections, transmitted light) and scanning electron microscopy (SEM; cut surfaces, BSE imaging). The SEM/BSE microscopy was performed with various SEM/FEGSEM microscopes in high vacuum (HV), variable pressure (VP) and multi-energy deconvolution (MED) modes.

Results and conclusions. Three types of TiO2-based nanoparticles used for preparation of PCL/TiX composites are shown in Fig. 1. Overall morphology of PCL/TiX was visualized by LM (Fig. 2). The best dispersion of nanoparticles was found in the composite with biggest nanoparticles (PCL/TiNP2), which could be attributed to higher shear forces during melt mixing [2]. As the homogeneous dispersion of nanoparticles is crucial for good mechanical properties, the following SEM studies focused on PCL/TiNP2 (Fig. 3). 2D SEM/BSE microscopy yielded higher resolution, but suffered from electron-beam-induced sample damage and showed lower amount of nanoparticles due to lower penetration depth of electrons. In order to obtain 3D distribution of TiNP2 particles, the MED-SEM technique [4] was used. Schematic example of the technique is given in Fig. 3b showing BSE images, where one sample area is observed with energies 4.6 keV, 7.6 keV and 9 keV. Using deconvolution from sequence of images taken at distinct landing energies a 3D model up to depth of several hundred nm with voxel size of 4 nm can be achieved. If we combine MED-SEM with physical slicing [5], even larger 3D volumes can be reconstructed. We conclude that detailed morphology study of electron-beam sensitive polymer composites is a challenging task even for modern SEM methods.

References: [1] Mohanty et al.: Macromol Mater Eng 276 (2000) 1-24. [2] Kralova et al.: Mater Chem Phys 124 (2010) 652–657. [3] Tadmor et al.: Principles of polymer processing. J. Wiley (1979). [4] Boughorbel et al.: Microsc Microanal 18 (Suppl 2) (2012) 560. [5] Hovorka et al.: this proceedings.

GACR P108/14-17921S, TACR TE01020118 and MZOS Croatia, project no. 110001.

Fig. 1: TEM micrographs of TiX nanoparticles: TiO2 nanoparticles with average size 200nm (a; TiNP2), 100 nm (b; TiNP1), and titanate nanotubes (c; TiNT).

Fig. 2: LM micrographs (10 mm thin sections, transmitted light) showing the overall morphology of the PCL/TiX composites with 2 wt.% of (a) TiNP2, (b) TiNP1 and (c) TiNT nanoparticles.

Fig. 3: SEM/BSE micrographs of PCL/TiNP2 (cut surfaces): (a) high vacuum SEM and FEGSEM, (b) variable-pressure SEM images of one area taken at 4.6, 7.6, and 9 keV, demonstrating possibility to reconstruct 3D-volumes.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2374 Folding Structures of Polypeptide Chains in Silk

Schaper A. K.1, Ogawa T.2, Yoshioka T.3, Kameda T.4
1Philipps University of Marburg, Marburg, Germany , 2Institute for Chemical Research, Kyoto University, Uji, Japan, 3Toyota Technological Institute, Nagoya, Japan , 4National Institute of Agrobiological Sciences,Tsukuba, Japan
schaper@staff.uni-marburg.de

Fibrous structures represent a basic construction principle in nature and are of interest in research aimed at the development of advanced materials for biomimetic and other applications. The amino acid sequences and the environment determine the ability of a peptide chain to fold in a particular manner and assemble into a complex protein. This talk presents an overview of the structural organization in fibers and films of natural and regenerated silk as revealed by 200-400 kV electron microscopy and diffraction under cryo-protection.
The high mechanical stability of silk is provided by stiff nano-sized crystallites acting as reinforcing elements within an oriented fiber network [1], helical chain conformations, e.g., 31 helices, are responsible for the elastic and contractional behavior. The β-structure of spidroin from spiders is formed by peptide chains with dominating alanine groups in a pleated-sheet arrangement. Silkworm fibroin differs in its contributing amino acids through the replacement of parts of alanine repeats by glycine resulting in additional small and poor crystals and an increased number of random molecules. The silk-I phase and metastable hydrogen-bonded linker regions control the amazing reversible contraction behavior. Increasing the degree of silk-II β-crystallinity induces dramatic changes which cause irreversible supercontraction due to lamellar overgrowth of pre-existing β-crystals.
Silk from ants, wasps, bees and hornets exemplify a different fiber type that is dominated by α-helices in a coiled coil superstructure (Fig. 1) [2]. Characteristic diffraction features are the sharp reflection arc on the meridian, belonging to the 5.1 Å spacing of one turn of the α-helix, and the broad equatorial maximum related to a spacing of ~9 Å (Fig. 2a). The pitch of the supercoil of 172 Å gathered from the off-equatorial diffraction intensity distribution as well as from the orders of meridional scattering fits well into the range predicted for four-stranded coiled coils. Only a minor component forms pleated β-sheet structures (Fig. 2b).
[1] Schaper, A.K., Yoshioka, T., Kawahara, Y.: Fascinating silk - electrospinning, contraction and diffraction experiments. Imaging & Microscopy 14, 25-27 (2012).
[2] Kameda, T., Nemoto, T., Ogawa, T., Tosaka, M., Kurata, H., Schaper, A.K.: Evidence of α-helical coiled coils and β-sheets in hornet silk. J. Struct. Biol. 185, 303-308 (2014).

T.K. acknowledges support by JSPS grant no. 24580086, A.K.S. is grateful for JSPS/DAAD fellowships.

Fig. 1: Cocoon silk wowen by larvae of the giant hornet Vespa mandarinia japonica and model of the four-stranded coiled-coil structure.

Fig. 2: ED pattern of V. mandarinia cocoon silk showing typical features of an α-helical coiled-coil structure.

Fig. 3: ED pattern of the crystalline β-sheet component within V. mandarinia cocoon silk. 

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2272 Analytical TEM: a powerful technique in the field of organic electronics research

Kraxner J.1, Schmied R.1, Rothländer T.2, Stadlober B.2, Grogger W.1
1Institute for Electron Microscopy and Nanoanalysis of the TU Graz (FELMI), Graz Centre for Electron Microscopy (ZFE Graz), 2MATERIALS, JOANNEUM RESEARCH, Weiz, Austria
johanna.kraxner@felmi-zfe.at

Organic electronics is a field of research with growing interest in the last years. Many applications are already entering industrial commercialization, but the search for new materials and the improvement of the used processes are still in progress.
Considering the recent ongoing development, to smaller structures, cheaper manufacturing and the transition to printable and flexible materials, the cross section investigation via TEM is becoming more and more important but also more and more challenging.[1]
mainly prepare our TEM specimens by the usage of the focused ion beam instrument, because of the undoubted advantage of a target preparation and less problems compared to preparation by ultramicrotomy (delamination, artefacts). We use the established technique of milling out a window of interest and thereby providing mechanical stability of our lamellas and also the newly developed strategy of interlacing [2], to reduce the chemical damage and morphological instabilities on our materials due to heating effects. This provides us with lamellas for analytic characterization, which becomes increasingly important due to the lack of differences (e.g. contrast) between the materials.
The lamellas are analysed on a FEI Tecnai F20 and a FEI Titan 60-300 (ASTEM) by scanning TEM (STEM), energy filtered TEM (EFTEM) as well as Electron Energy Loss Spectroscopy (EELS) and Energy Dispersive X-ray Spectroscopy (EDXS). We will present results of organic thin film transistors (OTFTs) [3] made of partly and all printable materials as well as different sample layer stacks which illustrate the suitability of the TEM for the investigation of such material stacks. Due to the optimized sample preparation we are able to clearly visualize all materials individually. This is possible either by the usage of the heteroatomic differences (EDXS, EELS) in the materials or by careful analysis of the features in the EEL spectrum: On the one hand the fine structure of the C K edge was used for calculating ratios of the σ* and π* peaks. On the other hand we found that the accurate determination of the plasmon peak energies was very useful to assist the distinction between the different organic materials. In Figure 1 an EFTEM image (5eV-7eV) of an OTFT is shown. The dielectric layer can easily be separated from the semiconductive layer. The inset in Figure 1 shows a typical scheme of an OTFT. Figure 2 shows a polymer layer sample stack (made by spin coating, inkjet printing). The layers are very well separated which can be seen very well by the extracted elemental edges from an EEL spectra.
[1] S. Fladischer et al. Ultramicroscopy, 136 ( 2014), 26
[2] R. Schmied et al. RSC Adv., 2 (2012), 6932
[3] U. Palfinger et al. Adv. Mater. 22 (2010), 5115

The authors want to thank the Nanoinitiative Austria (FFG) for financial support. This work was done in the NILaustria cluster, in the project NILechoII 830269. Furthermore this research has received funding from the European Union within the 7th Framework Programme; ESTEEM2.

Fig. 1: EFTEM image (5.0eV-7.0eV) of a cross section of an OTFT. The scheme above shows the layer set up (Foil: modified PC, G (gate): aluminium, IL (dielectric layer): PMMA, S/D (source and drain electrode): gold, SC (semiconductive layer): pentacene). Due to energy filtering the semiconductive layer can be distinguished from the dielectric layer.

Fig. 2: EELS SI of an all polymer layer stack (Kapton, PVCi (poly(vinyl cinnamate), BCB (benzocyclobuten), PVCi, PEDOT:PSS, PVCi). Extracted different elemental edges as well as the superimposed elemental composition (C…red, O…white, N…yellow, S...blue and Si…green).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-5817 A New Perspective for Morphologies Observation in Ziegler-Natta Based Polymerization Processes

R. Selleri1, M. Casinelli1
1. Basell Polyolefine Italia SrL, a LyondellBasell Company, G.Natta Research Centre, 44122 Ferrara (Italy)
roberta.selleri@lyondellbasell.com

     In the field of heterogeneous Ziegler-Natta polymerization, morphological studies applied to industrial processes have allowed, in the last years, a good control of the forming polymer particles inside the reactors [1,2 ].

In the present study, using ESEM-LV and AFM microscopy techniques, applied to the samples chain formed by support → catalyst → polymer, a comprehensive evaluation of the replica phenomena of the internal morphology has been obtained. A proper sample preparation procedure for microscopy observation, consolidated in a previous work [3], has allowed to preserve the true internal morphology of each sample step, connecting the obtained final polymer porosity to the original support and evidencing differences presence due to the typical micro globules network formed during the reaction process [4,5].

The obtained images could be interpolated by image analysis software, connecting results with  compositional information of the studied system in order to create semi-quantitative models for morphological behavior of different support and catalyst types during the polymerization process.

Some examples of the typical internal morphologies are reported in Figures 1-3.

 

 

 

 

1.       P. Blais, R. St. J. Manley, Journal of Polymer Science. Part A-1(1968),6  p.291-334

2.       A. Munoz-Escalona, C. Villamizar and P. Frias , Polymer Science and Technology (1982), p. 95-113

3.       R. Selleri and M. Casinelli, MC2013 Regensburg,MS.1.P010

4.       G. Cecchin, E. Marchetti and G.Baruzzi, Macromol.Chem.Phys.(2001),202 p.1987-1994

5.       J. Kosek, B. Horackova Basell Symposium, (2006), p.65-81

Fig. 1: Support 1

Fig. 2: Catalyst 1

Fig. 3: Polymer 1
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2411 Influence of selected substrates and nanoparticles on crystallization of isotactic polypropylene

Pavlova E.1, Strachota B.1, Slouf M.1
1Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic
ewapavlova@seznam.cz

Nucleated polymer crystallization is used to fine-tune the morphology and properties of semicrystalline polymers, such as polyethylene and polypropylene. The polymer crystallization rate is increased by numerous substances (nucleating agents), but also the substrate influences the crystallization process. The nucleation facilitates the formation of crystal nuclei, which results in higher concentration of nucleation centers and smaller average size of spherulitic crystals [1].

We investigated crystallization of isotactic polypropylene (PP; Mosten GB005) on three different surfaces: microscopic cover glass, atomically flat mica, and highly oriented pyrolitic graphite (HOPG). Moreover, mica and HOPG surfaces were covered with: (i) vacuum-sputtered gold nanoparticles, whose morphology and size were controlled by a combination of sputtering time and thermal treatment [2], (ii) commercial TiO­2 nanoparticles (size ~ 200nm, Sigma-Aldrich), titanate nanotubes (TiNT) [3], and commercial alpha-nucleating agent (Hyperform HPN-68).

PP film (350 um) was placed on one of the above-described substrates, molten at 200 C and isothermally crystallized at 120 C. The concentration of nucleation centers was determined by microscopic methods: The PP films were removed from the substrates, etched with permanganic mixture [4], sputtered with platinum and observed by FEGSEM.The micro- and nanostructure of PP contact surface after its detachment from the support was studied also by light microscopy (LM) and atomic force microscopy (AFM).

FEGSEM micrographs (Fig. 1) of PP surfaces crystalized on clean substrates showed that PP crystallization is affected only by HOPG, while glass and mica did not influence the size of spherulites. The impact of Au nanoparticles (Fig. 2a–d) deposited on mica on PP crystallization was very weak (Fig. 2e–h), which was in agreement with our previous work [1]. Small Au nanoparticles deposited on HOPG also did not have any nucleating effect on PP crystallization. In contrary, they acted as obstacles for crystallization of PP on the nucleating surface of HOPG.

References:

[1] Pavlova E et al.: J. Polym. Sci., Part B: Polym. Phys. 49 (2010) 2504.

[2] Tracz et al.: Eur. Polym. J. 41 (2005) 501.

[3] Kralova et al.: Mater. Chem. Phys. 124 (2010) 652.

[4] Slouf et al.: J. Biomed. Mater. Res. B Appl. Biomater. 85 (2008) 240.

Acknowledgement: P205/10/0348, TACR TE01020118.

Fig. 1: Fig. 1: SEM and LM micrographs of the PP contact surface crystallized on: (A and D) cover glass; (B and E) mica; (C and F) HOPG.

Fig. 2: Fig. 2: Morphology of Au nanoparticles with different size, deposited on mica: (A) Au~5nm; (B) Au~100nm, (C) Au~300nm, and (D) Au islands. Lower row (E-H) shows the FEGSEM micrographs of the PP crystallized on the substrates above.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2429 HRTEM study of the ZnO/sexiphenyl inorganic/organic hybrid interface

Kirmse H.1, Sparenberg M.2, Polzer F.1, Sadofev S.2, Blumstengel S.2, Henneberger F.2
1Humboldt University of Berlin, Institute of Physics, TEM group, 2Humboldt University of Berlin, Institute of Physics, Photonics group
holm.kirmse@physik.hu-berlin.de

The structure of interfaces between inorganic and organic materials (here ZnO and sexiphenyl - 6P) strongly influences the resulting, e.g. optoelectronic properties of the hybrid inorganic/organic system (HIOS). In order to understand the interfacial structure, high-resolution transmission electron microscopy (HRTEM) and corresponding image contrast simulations have to be performed. From this, parameters like interface separation and lateral arrangement can be derived.

The growth of hybrid system was performed at extreme low temperature of 100 °C according to [1]. Sample preparation for TEM was done by ultramicrotomy in order to preserve the crystalline structure of 6P [2]. HRTEM imaging was performed at a JEOL2200FS (200 kV) equipped with in-column filter. For image contrast enhancement the energy slit was set to select the zero loss intensity within an energy window of 10 eV.

Fig. 1 shows a cross-sectional HRTEM image of an about 10 nm thick 6P layer grown on (0001)ZnO. Atop 6P a 70 nm thick ZnO layer was deposited which is textured with preferred orientation of the [00.1] axis along the growth direction. For sufficient HRTEM image contrast of 6P a defocus of -1000 nm was applied. The Fresnel fringe near the ZnO/6P interface hampers the interpretation of the interfacial structure. Therefore numbering of the (100) lattice planes of 6P (d100 = 2.6 nm) starts with n-th lattice plane (Fig. 1b). In the intensity profile four dark fringes can be detected corresponding to the intended thickness of the 6P layer of about 10 nm.

For the interpretation of the lattice fringe contrast, HRTEM simulations were performed using the JEMS software package of P. Stadelmann (Fig. 2). Since the azimuthal orientation of 6P on ZnO is arbitrary, the 6P structure model was arbitrarily rotated by about 10° around the (100) lattice plane normal. The calculated fringe contrast visible for 25 nm specimen thickness and a defocus of -1000 nm is in good agreement to the lattice fringe contrast observed in the experimental image of Fig. 1.

In order to predict the HRTEM image contrast of an interface with known azimuthal alignment a supercell comprising (10-10)ZnO substrate and (100)6P layer was set up. The resulting thickness/defocus map is given in Fig. 3. Near Scherzer defocus and for a specimen thickness of 25 nm the interfacial structure can be resolved. However, at larger underfocus applied so far experimentally the simulated image contrast is dominated by edge effects of the supercell.

References:

[1] S. Blumstengel et al., New Journal of Physics 10 (2008) 065010

[2] H. Kirmse et al., J. of Phys.: Conf. Series 471 (2013) 012034

Acknowledgement:

Research was financially supported by the DFG in the framework of CRC951 HIOS. The authors are grateful to E. Oehlschlegel for ultramicrotomy preparation of TEM samples.

Fig. 1: Fig. 1: Phase contrast imaging of ZnO/6P/ZnO hybrid system: a) zero-loss filtered HRTEM image defocused by -1000 nm for contrast enhancement of the organic component, b) intensity profile across the hybrid system for lattice plane analysis along path A-B marked in Fig. a).

Fig. 2: Fig. 2: HRTEM image contrast simulation of 3 molecular layers of 6P. The thickness-defocus map was calculated for a 6P crystal rotated by about 10° around the (100) lattice plane normal (supercell on the right). Best agreement with Fig. 1a (defocus -1000 nm) is found for a thickness of 25 nm.

Fig. 3: Fig. 3: HRTEM image contrast simulation of a ZnO/6P(2MLs) HIOS interface. The orientation relationship is [00.1]ZnO aligned parallel to [010]6P. Strong edge effects of the supercell modify the image contrast of 6P compared to Fig. 2.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2640 MORPHOLOGICAL CHARACTERIZATION OF HIGHLY POROUS BIODEGRADABLE POLYMER SCAFFOLDS FOR TISSUE ENGINEERING

Ninago M. D.1, Yañez M. J.2, Ciolino A. E.1, Villar M. A.1
1Planta Piloto de Ingeniería Química, 2Centro Científico Tecnológico Bahía Blanca (CCT BB)
mninago@plapiqui.edu.ar

Poly (ε-caprolactone) (PCL) is a hydrophobic, semi-crystalline polymer used in biomedical applications, such as controlled release systems, implantable biomaterials, surgical sutures and dental implants, among other devices that help internal fixation of bone fractures. In this work, different methods of PCL modification in order to improve hydrophilicity by introduction of functional groups on the surface were evaluated. Maleic anhydride (MA) was employed as a chain-end modifier agent. Modified samples were subjected to alkaline hydrolysis in order to increase their hydrophilicity. Polymeric scaffolds were obtained by employing a solvent casting/particle leaching technique, and scaffolds with porosity as high as 85 % were obtained, having both open and interconnected pores. Bioactivities tests for PCL after 7 days of immersion in SFB showed that apatite crystals effectively adhere to their surface. Two methods were employed to modify the hidrophilicity of the synthesized polymers. In the first method, polymer samples were immersed in a solution of MA, in THF, in the presence of pyridine as catalyst for 24 hours [1]. The second method was a simple alkaline hydrolysis of the samples by immersion in a NaOH solution during 8 or 20 h at room temperature. Interconnected porous membranes were prepared by solvent casting and particulate leaching by dissolving in chloroform (20 wt %) using NaCl as porogen [2]. Bioactivity of the scaffolds was studied in a phosphate buffered saline (PBS) at pH 7.4 to simulate in vivo conditions [3]. Figure 1(a) shows a cross-section of the scaffolds obtained where a highly and interconnected porous membrane can be observed. Internal photographies of the cross-section after immersion in SFB are shown in figures (b and c). In Fig. 1(b) apatite crystals can be found on the scaffold´s surface, whereas in Fig. 1(c) no apatite crystals appeared on the neat PCL scaffold. Chemical modification of PCL with MA yielded PCL samples bearing COOH groups at the chain-end. Porous scaffolds were prepared with open and interconnected pores ranging in size from 50 to 150 µm. Bioactivities tests after 7 days immersion in SFB showed apatite crystals growing on the surface of the scaffolds prepared using modified PCL. These results suggest that chemical treatment provides a polymer surface with nuclear precursors for apatite deposition.

References.

[1] M. Avella, M.E. Errico, P. Laurienzo, E. Martuscelli, M. Raimo, R. Rimedio. Polymer 41 (2000) 3875-3881.

[2] J. Wei, X. Wu, C. Liu, J. Jia, S. Heo, S. Kim, Y. Hyun, J.-W. Shin. Journal of the American Ceramic Society 92 (2009) 1017-1023.

[3] T. Kokubo, H. Kushitani, S. Sakka. Journal of Biomedical Material Reserch, 24, (1990) 721-734.

We express our gratitude to the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina (CONICET, Argentina) for the financial support

Fig. 1: (a) SEM photograph of the cross-section of a PCL scaffold. Internal surface of the PCL scaffold after 7 days of immersion in SBF: (b) PCL modified with MA, and (c) neat PCL.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2648 Study of Surface Morphology and Shell Thickness of Melamine-Formaldehyde Microcapsules using FEG-SEM

Ramachandran D.1, 3, Teixeira R.2, 3, Rivero G.2, 3, Leroux F.1, Du Prez F.2, Abakumov A.1, Schryvers D.1
1Electron Microscopy for Materials Science (EMAT), University of Antwerp, Antwerp, Belgium, 2Department of Organic Chemistry, Ghent University, Ghent, Belgium, 3SIM vzw, Technologiepark 935, BE-9052 Zwijnaarde, Belgium
DhanyaR.Puthenmadom@uantwerpen.be

Microcapsules (MC) are used in many research areas such as the development of self-healing materials [1], drug delivery [2] and other areas where controlled release of an active material, protected from its environment by encapsulation, is desired. In this work, we compared the surface morphology and shell thickness of melamine-formaldehyde microcapsules using a scanning electron microscope (SEM) equipped with a field emission gun (FEG). Cyclohexane was encapsulated by a melamine-formaldehyde wall via polycondensation in an oil-in-water emulsion. Capsules with different amounts of core content were prepared. A later removal of the core by soxhlet extraction provided the hollow microcapsules used for characterization. The capsules were embedded in an epoxy matrix and samples were prepared using microtomy to study the shell thickness. These capsules, designed to encapsulate self-healing agents, are spherical in shape with an approximate diameter between 5 and 30 µm. SEM images show that the shell thickness varies from 650 to 700 nm for MC-VI (F/M ratio = 3.25, pH = 5.0, core content ~ 85%) whereas in the case of MC-VII (F/M ratio = 3.25, pH = 5.0, core content ~ 60%), the variation is from 1.10 to 1.25 µm. In order to measure the shell thickness without the epoxy matrix, a thin strip was removed from the surface of an individual microcapsule using Focussed Ion Beam (FIB). In this case, the measured thickness values are nearly half of the values when the capsule is embedded in an epoxy matrix, which may be due to the diffusion of the epoxy into the microcapsule during the embedding and curing stages of the latter preparation. Although the surface of the capsule appears smooth, higher magnification shows a granular type surface composed of grains having sizes in the range 100-160 nm, which indeed allows for some porosity. The difference in shell thickness between the two types of capsules as observed in the SEM images can further imply that these microcapsules are having different mechanical strengths.


References:

[1.] H. Jin, C. L. Mangun, D. S. Stradley, J. S. Moore, N. R. Sottos, S. R. White, Self-healing thermoset using encapsulated epoxy-amine healing chemistry, Polymer, 53, 581-587 (2012).

[2.] K. Wang, Q. He, X. Yan, Y. Cui, W. Qi, L. Duan and J. Li, Encapsulated photosensitive drugs by biodegradable microcapsules to incapacitate cancer cells, J. Mater. Chem., 17, 4018-4021 (2007).

The authors would like to thank the Strategic Innovative Materials (SIM) project for the support through research projects.

Fig. 1: SEM image of a melamine-formaldehyde microcapsule MC-VII.

Fig. 2: SEM image of a single MC-VII microcapsule (inset) and a magnified image prepared for thickness measurement using FIB.

Fig. 3: SEM of a cross-section of MC-VII microcapsules in an epoxy matrix.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2660 Application of Atomic Force Microscopy in Morphology Characterization of Industrial Polymers

Yang L.1, Guise O.1
1SABIC Innovative Plastics, Department Technology and Innovation (T&I)
lanti.yang@sabic-ip.com

Atomic force microscopy (AFM) has emerged as a key microscopy tool for the characterization of the morphology of multiphase polymer systems with the development of AFM tapping mode height and phase imaging. In this abstract, we highlight the development and the benefits of morphology studies by AFM on variety of key industrial polymer multiphase systems containing hard and soft components.

First we demonstrate that AFM offers significant safety (no staining requirement) and productivity (no need for thin-sections) benefits over TEM while achieving similar resolution for the complete morphology characterization of polycarbonate (PC) / Styrene-acrylonitrile (SAN) / Acrylonitrile-butadiene-styrene (ABS) rubber materials (Figure 1). After this initial application of AFM in PC/SAN/ABS, new methods based on using a combination of AFM and image analysis are developed for the quantitative morphology characterization of PC-siloxane copolymer materials to further understand the influence of morphology on the materials aesthetic properties. As shown in Figure 2, conventional TEM approach can provide morphology images of PC-siloxane copolymer materials. However, performing accurate quantitative image analysis on these TEM images proves very challenging due to the lack of a strongly defined contrast between the siloxane domains and the PC phase. Using AFM in tapping mode, an enhanced contrast between siloxane and the PC phase (Figure 2) is generated vs. TEM. This high contrast morphology imaging enables accurate quantitative morphology studies on both the siloxane domain size distribution and the siloxane domain dispersion in a variety of PC-siloxane copolymers. The results of these quantitative morphology studies provide insight into the relationship between the material structure and its properties and significantly support further improvements in materials properties.

In the case of PC / Polybutylene Terephthalate (PBT) polymer blends containing saturated impact mordifiers (IMs), AFM in combination with TEM provides a complete morphology evaluation of the material. TEM imaging provides a high contrast between the PC and PBT phases via differential staining. However, as saturated IMs cannot be stained with the common staining techniques, they typically appear as white particles in the PBT phase in TEM images. AFM phase imaging provides a significant improvement in the contrast between the IM and the PC/PBT matrix. The high contrast of IMs in the AFM images allows us to perform quantitative analysis of IMs size distribution to further understand the influence of processing conditions on the material morphology and properties (Figure 3) and to further fine-tune the properties of these materials by dialing into specific processing conditions.

Fig. 1: Figure 1. (a) TEM and AFM sample preparation procedures for general impact modifier contained polymer system. (b) AFM phase images and (c) TEM image of polymer blends (PC/SAN/ABS HRG). Different components as indicated in the images: PC is the continuous phase, SAN is in the PC matrix, and polybutadiene rubber is dispersed in SAN.

Fig. 2: Figure 2. Chainarchitecture of opaque PC-siloxane copolymer (BPA-PDMS-1) (a) and transparent PC-siloxanecopolymer (BPA-PDMS-2) (b). Morphology of “BPA-PDMS-1” by TEM (c) and AFMtapping mode imaging (e) and siloxane size distribution (g). Morphology of “BPA-PDMS-2” by TEM (d) and AFMtapping mode imaging (f) and siloxane size distribution (h).

Fig. 3: Figure3. TEM (a & c) and AFM (b & d) phaseimages of PC/PBT blends containing saturated IM. TEM images show thePC/PBT matrix but the saturated IM Lotader† shows unclear contrast. AFM phase images show the morphology ofLotader† IM as darker phase. (e) and (f) Lotader† IM domain sizedistribution and median particle size of sample a and b.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2669 Morphological characterization of surface modified cellulose nanocrystals for application in polymeric nanocomposites

Taipina M. O.1, Ferrarezzi M. F.1, Battirola L. C.1, Gonçalves M. C.1
1Instituto de Química, Universidade Estadual de Campinas - P.O. Box 6154, 13083-970, Campinas, Brazil
liliane.battirola@iqm.unicamp.br

Cellulose nanocrystals (CNC) can be obtained by submitting native fibers to acid hydrolysis. These nanostructures can be used to enhance polymer nanocomposite properties due to their high crystallinity and aspect ratio (1-4). However, as a result of the hydrophilic character of CNC, several studies have been performed to promote the CNC surface modification aiming at the improvement of compatibility with hydrophobic polymers. For this purpose, silane coupling agents are widely used (5). Thus, the aim of this work was to characterize the CNC surface modification, which was carried out by using a silane which has isocyanate groups. CNC were obtained by cotton acid hydrolysis, which was carried out by treatment for 2h in a 10 wt% alkali solution, followed by hydrolyzation in a 4M hydrochloric acid at 80 °C for 3 h and 45 min under stirring. The excess acid was removed by repeated cycles of centrifugation. Afterwards, the CNC suspension was dialyzed, lyophilized, dispersed in DMF and kept under an inert atmosphere. Silane was added to the nanocrystal suspension and the reaction was maintained under stirring for 8 h. Catalyst and water were added and the mixture was kept under stirring for 30 min. Lastly, the product was dried and nanocrystal powder was obtained. The morphologies of the samples were investigated in a Carl Zeiss CEM 902 transmission electron microscope (80 kV) equipped with a Castaing-Henry energy filter spectrometer within the column and a Proscan Slow Scan CCD camera. To examine the CNC, a droplet of the diluted suspension was deposited on carbon coated parlodion film supported on a copper grid. Images were acquired using electrons with zero-loss energy and processed using the AnalySis 3.0 software. Fig.1a,b confirm that the hydrolysis conditions gave rise to needle-like structures with a 10 nm approximate diameter and a 166 nm average length. The silylation step did not cause any change in the CNC morphology. Fig. 2 presents ESI-TEM images of the modified nanocrystals and the silicon maps, before and after solvent extraction. Fig.2a,c show regions with high CNC concentration, while Fig.2b,d show the silicon mapping images in the same areas, where the bright regions correspond to silicon-rich domains. These images clearly show that there is a higher silicon concentration around the vicinity of the nanocrystals. Furthermore, a strong contrast can be observed between adjacent nanocrystals, suggesting the formation of a rather uniform polysilsesquioxane layer wrapping of the nanocrystals.
Ref.
[1] Alloin et al. Eletrochimica Acta 55:5186–5194 (2010);
[2] Habibi et al. ChemRev 110:3479–3500 (2010);
[3] Siqueira et al. Biomacromolecules 10:425–432 (2009);
[4] Taipina et al. Cellulose 20:207-226 (2013)
[5] Xie et al. Comp Part A 41:806–819 (2010).

The authors would like to thank CAPES, CNPq and FAPESP for the financial support and Dr. Carlos Alberto Paula Leite for his cooperation in the ESI-TEM analysis.

Fig. 1: TEM micrographs of cotton nanocrystals: (a) before and (b) after silylation reaction.

Fig. 2: ESI-TEM images of the modified nanocrystals: (a) bright field and (b) silicon map, obtained before solvent extraction, (c) bright field and (d) silicon map, obtained after solvent extraction.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2714 Analytical TEM cross section investigation of blue OLED display pixels

Graff A.1, Hübner S.1, Altmann F.1, Freitag B.2
1Fraunhofer Institute for Mechanincs of Materials, Halle, Germany, 2FEI Company, Eindhoven, The Netherlands
andreas.graff@iwmh.fraunhofer.de

Organic light emitting diodes (OLEDs) are getting more and more important for smartphones and television displays. OLED structures consist of thin organic layers between two electrodes. Dedicated organic layers near the electrodes transport charge carriers into the emission layer, where the radiative recombination of electrons and holes takes place. Information on thickness, composition and structure of the different thin organic layers is important for technology development and process qualification since it influences color, efficiency and lifetime of OLED devices.
In this paper we demonstrate the capability of advanced TEM investigations on blue OLED pixels of a commercial smartphone display. The challenge for TEM investigations is to prepare high quality electron transparent cross sections of certain small sized OLED pixels, obtain sufficient contrast to distinguish the organic layers and avoid beam induced damages during FIB preparation and TEM investigation. The present paper describes the complete workflow including FIB preparation of a TEM cross section out of a fully functional mobile phone display and EFTEM/STEM/EDX investigations of the OLED stack.
We applied FEI Versa 3D focused ion beam (FIB) system for the preparation of site specific electron transparent samples of a single blue OLED pixel of a Samsung Galaxy S3 AMOLED display using the in situ lift out technique (Figure 1). TEM investigations were performed in an image corrected microscope (Titan G2 60-300, FEI) equipped with an image filter, a high brightness field emission gun (X-FEG) and a dedicated EDS detector (SuperX) with high sensitivity.
The lamellas, fixed in a special TEM grid, are thinned by FIB from both sides finishing with low voltage FIB polish to reduce sidewall artefacts. TEM and STEM images at 80 kV show that the uppermost organic layers could be distinguished due to their different density (Figure 2). Even more information can be gained by using XEDS mapping. The highly sensitive EDS detector allows the collection of a reasonable amount of element specific X-rays at low beam current. Element distributions can be acquired before beam damage changes the organic layer stack. The organic layers differ in Carbon, Nitrogen and Oxygen content and can clearly be distinguished in the XEDS maps and related intensity profiles (Figure 3). Impurities like Fluorine can be found near the Silver anode but also between the electron transport layer (ETL) and the emission layer (EML) influencing their functionality.
We prove that low voltage TEM imaging combined with XEDS mapping of FIB prepared cross sections can provide important structural and chemical information of nm sized functional organic layer stacks valuable for further developments of electronic devices.

Fig. 1: Left: Micrograph of an OLED display. Right: Site specific TEM cross section before in situ transfer to the TEM grid.

Fig. 2: Left: TEM bright field image of the organic layer stack. Right: STEM HAADF image of the organic layers. Density variations between the organic layers are visible.

Fig. 3: Left: Color coded results of the XEDS mapping of the OLED layer stack. Right: Line profile out of the EDXS mapping across the layer stack.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-2746 Analytical Transmission Electron Microscopy (TEM) of Crystalline Organic Materials

Brydson R.1, Cattle J. E.1, Hondow N.1, Brown A. P.1
1Institute for Materials Research, SPEME, University of Leeds, Leeds, LS2 9JT, United Kingdom
mtlrmdb@leeds.ac.uk

Owing to the non-specific nature of intermolecular bonds, molecular organic materials can often crystallise in a number of different polymorphic structures with different packing arrangements. This is important in several industrial applications as many of the solid-state properties of a compound are dependent on the exact crystal form. In the pharmaceutical industry it is a requirement that the polymorphic behaviour of a drug compound is thoroughly investigated and understood as different crystal phases dissolve at different rates affecting the adsorption of the active compound in vivo, making it is essential to control which crystal form is dosed to patients. Here TEM is an important tool since it allows structural and chemical analysis to be done at a single particle level and complements X-ray based methods which average of an ensemble of particles [1]. Furthermore it allows investigation of apparently “X-ray amorphous materials” which may in fact be poorly crystalline or of ultrafine crystallite size. However a major drawback of using TEM is the electron beam sensitivity of organic crystals.
This paper focuses on the construction of a methodology for the study of organic crystalline materials using measurements on model materials such as theophylline, para-terphenyl and para-aminobenzoic acid. Initially we study the radiation damage of a particular compound by monitoring structural order (via measurement of the relative intensities of diffraction spots) as a function of electron fluence which allows us to derive a critical fluence [2] (see figure 1). We undertake these measurements at different microscope accelerating voltages, fluence rates, as function of sample cooling, as function of carbon coating of the sample and as a function of different TEM support films. Such measurements then allow us to define a set of experimental parameters for subsequent imaging, diffraction and also energy dispersive X-ray (EDX) or electron energy loss (EEL) spectroscopy measurements [3] specific to this particular material.
We demonstrate the use of particle morphology to identify different polymorphic forms of organic crystals which can be confirmed with selected area electron diffraction at the single particle level. We also discuss the potential use of bright field imaging to highlight growth or processing defects within the particles and present the use of low loss and core loss EELS to identify polymorphs and to determine light element chemical composition (figure 2). Finally we speculate on the use of scanning TEM for such measurements.

References
[1] Eddleston MD et al. 2010 Journal of Pharmaceutical Sciences 99, 4072
[2] Glaeser RM 1971 J. Ultrastructure Research 38, 466
[3] Eddleston et al. 2012 Proceedings European Microscopy Congress 2012, abstract 529

We are grateful to very useful discussions and provision of samples from Prof Bill Jones and Dr Mark Eddlestone from the Department of Chemistry at the University of Cambridge.

Fig. 1: Critical dose plots for different diffraction spots (1-3) in the selected area diffraction pattern of theophylline viewed down [100].

Fig. 2: Core loss EEL spectrum (total acquisition time 20 seconds at 4 x10-5 A/cm2) from a Theophylline crystal showing the nitrogen K-edge (ca. 400 eV) and the oxygen K-edge (ca. 530 eV) superimposed on the tail of the background subtracted carbon K-edge at ca. 285 eV. Note the poor signal to noise ratio.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-3217 HRTEM Studies of Micro and Nanocapsules of Self-Healing Polymeric Materials

Rossinyol E.1, Cardoso F.1, Garcia S.2, Cano-Sarabia M.2, Maspoch D.2
1Microscopy Service. Science Faculty. Autonomous University of Barcelona. Bellaterra 08193, Barcelona, Spain., 2ICN2, Institute Catalan of Nanoscience and Nanotechnolgoy. Bellaterra 08193,Barcelona, Spain.
rossinyol@gmail.com

Microencapsulation is a widely used technique to prepare micro and nanocapsules containing active materials. They can be used for many applications such as drug deliver, functional textiles or self-healing materials. All these applications are based in the same principle: the capsule retains the active agent until some stimuli triggers the release of the stored material.

In order to obtain a full control of the releasing process, it is important to fabricate capsules with optimal wall thickness and mechanical properties. The wall thickness determines the easiness of rupture, and therefore, the efficiency in the active agent release. The mechanical properties and the relation between the capsules and its surroundings affect the propagation of the product liberation.

In this work, we have focus on the structural characterization of different micro and nanocapsules with self-healing applications. All the analyzed capsules are based on polymeric materials with different encapsulated agents.

Characterization has been carried out in a HRSEM Merlin from Zeiss equipped with an INCA EDS detector from Oxford Instruments. The samples have been embedded in an EPON matrix and sectioned with a Leica EM UC7 ultramicrotome using a diamond knife. Section thickness has kept below 150 nm to reduce the charging during the SEM observations.

Combining the images acquired with the in-lens secondary electron detector and the energy selective backscattered detector (ESB), we can easily visualize the wall thickness of the polymeric microcapsules. With low voltage EDS studies we can also detect the differences in the carbon and nitrogen amounts, related with the compositional change between the polymeric capsule and the resin matrix.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-3252 Revealing the distribution of low molecular weight materials in the SEM using cathodoluminescence spectroscopy

Stowe D. J.1, Pakzad A.1, Mantei J. R.2, Green J. B.2
1Gatan Inc, R&D Headquarters, Pleasanton, CA, USA, 2Baxter Helathcare, Technology Resources/Particles and Interfaces, Round Lake, IL, USA
dstowe@gatan.com

Composite materials are commonly used because, compared to their individual components, they have improved qualities such as higher strength, lighter weight, and lower production price. Two of the most important factors affecting the final properties of such materials are the composition and size/spatial distributions of their individual components. These properties have been studied using various microscopy techniques such as atomic force microscopy (AFM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM).

When an SEM is available, some of the most common techniques are: Backscatter Electron Imaging (based on atomic number contrast), X-ray Computed Tomography (based on phase and absorption contrast) and Energy Dispersive X-ray Spectroscopy (based on elemental characteristic X-rays). One common limiting factor of all of these techniques is that in the case of organic materials (mostly made of light elements such as carbon, hydrogen and oxygen), acquisition of images with sufficient contrast and signal/noise ratio can be challenging. In such cases, an alternative technique that can differentiate materials independent of their elemental composition is necessary.

Cathodoluminescence (CL) analysis inside an SEM is a well-established analytical technique used in many industrial and academical laboratories to study minerals, glasses, ceramics, gemstones, semiconductors, rare earths and optoelectronic materials. In such experiments, the primary electron beam excites electrons in a crystal or a molecule ground state. When these excited electrons go back to their original state (through electron-hole pair recombination), photons with energy corresponding to this transition energy (0.3 to 6 eV) are emitted. These photons are then used for imaging and spectroscopy to gather information on the crystallographic and chemical properties of these materials at the microscopic level.

In the current study, we used a Gatan MonoCL4 installed on a Zeiss ∑igma VP SEM to study the change in CL emission of various types of polymers. Experiments were performed at room temperature, using a 12 kV electron beam, at variable pressure mode (20 Pa, to reduce charging artifacts).  Luminescence is exhibited by many organic compounds which possess aromatic molecules or molecules having conjugated double bonds. We observed significant spectral differences between the studied polymers and demonstrated that these differences can be used as a means to distinguish them in a compound system. Such experiments can be applied in a large scale to assess the homogeneity of polymer compounds and to improve and optimize their manufacturing processes.

Fig. 1: Figure 1. Example of CL spectra acquired from three polymers. The vertical and horizontal axes in the spectra show the CL counts and wavelength (nm), respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-3310 Electron tomography of PTB7:PC70BM

AlAfeef A.1, Alekseev A.2,3, Hedley G. J.2, Samuel I. D.2, Cockshott W. P.1, MacLaren I.4, McVitie S.4
1School of Computing Science, University of Glasgow, Glasgow, G12 8QQ, UK, 2SUPA, School of Physics and Astronomy, University of St. Andrews, KY16 9SS, UK, 3NURIS, Nazarbayev University, Astana 010000, Kazakhstan , 4School of Physics and Astronomy, University of Glasgow, Glasgow G12 8QQ, UK
a.al-afeef.1@research.gla.ac.uk

Electron tomography [1] plays an essential role in the study of 3 dimensional (3D) nanostructures. It involves reconstructing 3D objects from a series of 2D images by sequential tilting of the sample about a single axis. This technique, although originally designed for use in the life sciences, has also been applied to the study of polymer blends using bright-Field TEM (BFTEM) as in [2, 3]. Recently, the single layer polymer solar cell (PSC) based on bulk heterojunction (BHJ) blend of two materials: fullerene derivative [6,6]-phenyl-C71-butyric acid methyl ester (PC71BM) and a polymer with alternating units of thieno[3,4–b] thiophene and benzodithiophene (PTB7) has received significant attention [4], since high power conversion efficiency (PCE) of 9.2% has been reported for devices based on this mixture [5]. Unfortunately, little is known about the nanoscale organisation of PTB7:PC71BM blends beyond some 2D imaging. In this work, we use electron tomography (ET) [1] to investigate the 3D organisation of such blends. Different electron microscopy and atomic force microscopy (AFM) methods to determine bulk structure of PTB7:PC71BM film. In particular, energy filtered electron tomography [6] was performed with TEM Tecnai T20 (FEI) operated at 200 kV using thin cross-sections through the blend cut using a FIB (Nova Nanolab, FEI). Three-dimensional Reconstruction was performed using the simultaneous iterative reconstructive technique (SIRT) using IMOD. The volume rendering visualisations of the reconstructions are shown in Figure (2). Orthoslices through the reconstructions are shown in Figure (3).
EFTEM results clearly show that domains in the blend are PC71BM-rich (i.e. contain more carbon). The EFTEM tomography clearly reveals that these ellipsoids and are not spherical. TEM and SEM measurements of device cross-section show existence of thin skin layer covering domains. The fine structure inside domains and matrix was observed by TEM and AFM (Solver P47H and Next, NT-MDT). Evolution of (photo) current distribution measured by AFM equipped with conductive probe on surface of PTB7:PC71BM blend was studied. We also used plasma etching of blend to study internal structure of this film by AFM.
References
[1] Weyland M, et al. 2004 Materials Today 7 32–40.
[2] van Bavel S S, et al. 2009 Nano letters 9 507.
[3] Oosterhout S D, et al 2009 Nature materials 8 818–824.
[4] Liang, Y. et al. Adv. Mater. 22, E135–E138 (2010).
[5] He, Z. et al. Nat. Photon. 2012 6, 591–595.
[6] Weyland M, et al. 2003 Microsc. Microanal 9 542–555 .

The work of A.AlAfeef, P.C, I.M, and S.M was supported by Kelvin Smith scholarship -University of Glasgow. Work of A.A., G.J.H. and I.D.W.S. was supported by the EPSRC (grant number EP/I013288/1).

Fig. 1: background estimation image produced from two pre-edge EFTEM images, This background estimation is subtracted from the B) Post-edge image to obtain an C) Elemental map.

Fig. 2: Orthoslice through SIRT 3D reconstruction, the y-direction labelled is parallel to the tilt axis, x-direction is perpendicular and z-direction is parallel to the optic axis. E) Volume rendering of reconstruction, F) A domain is selected G) to be visualized by rotating to H) 45 I) 90 Degrees around the tilting axes.

Fig. 3: Cross-sections (Orthoslices) through 3D reconstruction of PTB7:PC70BM .
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-3470 POLYMERIZATION BY ATOMIZATION - AN ALTERNATIVE TECHNIQUE FOR PRODUCING POLYMERIC PARTICLES

Fernandes L. S.2, Souza E. B.2, Oliveira J. A.2, Cellet T. S.1, Rubira A. F.1
1Universidade Estadual de Maringá-UEM, Maringá-Paraná, Brazil, 2Universidade Federal do Rio Grande do Norte – UFRN, Natal, Brazil
afrubira@gmail.com

Nanoparticles are of great scientific interest due to a wide variety of potential applications in biomedical, optical and electronic fields. Many studies have been carried out in order to produce polymeric particles in nanometric scale. The main reason for this is the higher ratio of surface area per volume, which results in specific characteristics. Such considerations have driven researchers to develop techniques to obtain polymeric nanoparticles with properties that allow application in different areas. In the present work is presented a polymerization process by atomization applied to miniemulsion and suspension systems for formation of submicron particles of poly(methyl methacrylate) (PMMA) and polystyrene (PS), in the form of homopolymer and copolymer. In this technique, a simple atomizer device is used as an alternative method to break the monomer droplets before feeding and dispersing them in the reaction medium. The monomer droplets formed with the atomizer are directed to the liquid reaction medium and suspension or miniemulsion polymerizations are performed. Reactions using the proposed technique were carried out and the particles of PMMA and PS obtained by suspension or miniemulsion polymerizations were analyzed by dynamic light scattering (DLS) analysis, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). It was observed through DLS, polymeric particles with size between 40 and 300 nm and with spherical morphological characteristics, according to the results of SEM and TEM. The results demonstrated that the proposed technique is promising in obtaining polymeric nanoparticles. Even in suspension polymerization, it was possible to obtain particle in submicron scale. So, the atomization method proposed in this study seems to be very useful, since it could be applied in larger scale processes, different of the conventional methods, as for example mechanical stirrers at high rotation or ultrasonic waves systems that present some limitations for scale up. Additionally, tests are being performed to explore more extensively this process concerning formation of carrier nanoparticles. Therefore, the technique of polymerization by atomization can be an alternative way to produce carrier particles too

The authors thank CAPES for financial support to the project NANOBIOTEC, as well as the scholarships granted.

Fig. 1: TEM of PMMA obtained by suspension polymerization

Fig. 2: TEM of PMMA obtained by minemulsion polymerization

Fig. 3: TEM of PS obtained by suspension polymerization

Fig. 4: TEM of PS obtained by miniemulsion polymerization
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-5859 Designing of polymeric nanoparticles for drug delivery system based on FE-SEM analysis

Takahashi C.1, Ogawa N.1, Kawashima Y.1, Yamamoto H.1
1Pharmaceutical Engineering, School of Pharmacy, Aichi Gakuin University
chisato@dpc.agu.ac.jp

Introduction: A biofilm is an assemblage of surface-associated microbial cells that is enclosed in an extracellular polymeric substance (EPS) matrix. Biofilm protect microbial cells from treatment with antibacterial drug. In order to overcome this problem, polymeric nanoparticle for biofilm infection disease has been developed in our laboratory. In the present study, we revealed the fine morphology of the biofilm with moist condition using field emission electron microscope (FE-SEM) in order to optimal design of polymeric nanoparticles which can work as high anti-bacterial nanocarrier.

Materials and methods: S. epidermidis was used as a model biofilm forming bacterial strain. Ionic liquid (IL): 1-butyl-3-methylimidazolium tetrafluoroborate was used for FE-SEM observation. Structure of the biofilm, and, adherence and intrusion behaviors of nanoparticles into biofilm were observed using FE-SEM. Moreover, quantitative characterization and confocal laser microscopic observation of adherence of nanoparticles loaded fluorescence dye were determined. Polymeric nanoparticles were prepared with poly (DL-lactide-co-glycolide) (PLGA) and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (SoluplusR).

Results and discussion: We successfully characterized the fine morphological structure of the biofilm and its formation mechanism with the aid of IL by FE-SEM observations. In addition, the observation method using an IL allowed the exact morphology to be observed of water-containing biological sample with nano-particles. The adherence and permeation behaviors of nanoparticles with different polymer were also revealed. This adherence and intrusion abilities were enhanced by modification on nanoparticulate surface with chitosan. On the other hand, SoluplusR nanoparticles showed strongly adherence and intrusion abilities regardless of the chitosan modification. Between two particles, particle size was the most different physicochemical property, i.e. particle size of chitosan modified nano-particle and SoluplusR nano-particle was 300 nm and 100 nm, respectively. Therefore, particle size might be related to the adherence and permeation abilities.

Fig. 1: FE-SEM images of (a) cells of S. epidermidis and (b) biofilm treated with polymeric nanoparticles.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-5887 Controlling the parameters of self assembly to functionalise peptide hydrogels

Li R.1, Rodriguez A. L.2, Bruggeman K. F.2, van Driel R. R.3, Barrow C. J.1, Nisbet D. R.2, Williams R. J.1
1Centre for Biotechnology, Chemistry and Systems Biology, School of Life and Environmental Sciences, Deakin University, Geelong, AustraliaSciences, 2Research School of Engineering, College of Engineering & Computer Science, The Australian National University, Canberra, Australia, 3Institute for Frontier Materials, Geelong Technology Precinct, Deakin University, Geelong, Australia
rosey.vandriel@deakin.edu.au

Molecular self-assembly is a promising route for the formation of nanostructured materials through bioinspired processes for technological and biomedical applications (1). Here, We will demonstrate that by manipulating the supramolecular ordering of the final materials – especially important for biomaterial design - the final properties can be tuned in an application driven, context defined manner.

Self-assembling peptides (SAPs) are short, easily synthesised molecules that belie their simplicity by containing a host of structural and biochemical information. Recently, we have demonstrated that the SAP assembly process can be rationally designed to present specific amino acid sequences within a nanoscale fibril under physiological conditions; thereby allowing the high-density presentation of biochemical signals within a hydrogel (2).

To achieve this, we have employed well-characterised Fmoc-SAPs; these form hydrogel scaffolds through a combination of ionic and hydrophobic interactions to allow a β-sheet and π-stacking assembly mechanism known as Π-β assembly that is thermodynamically driven by the peptide sequence and the conditions of assembly [3]. Building upon this work, we report on mechanisms to determine the morphological and mechanical properties of hydrogel formed by the SAP fibrils by controlling the conditions of assembly, and introducing naturally occurring macromolecules. We will discuss how these, individually and in tandem, can be manipulated to determine the rate of formation, introduce templating, and alter the final order of the supramolecular arrangement. These processes are observed to have significant effects on the final material properties by manipulating the fibrillar interactions during the self-assembly process.

Such insights into the role of the fabrication process to the biochemical and biomechanical properties of these materials are relevant to controlling the biocompatibility, functionality and suitability of these materials for a range of biomedical applications.

References
[1] Nisbet, D. R., & Williams, R. J. (2012). Self-Assembled Peptides: Characterisation and In Vivo Response. Biointerphases , 7 (2).
[2] Rodriguez, A. L., Parish, C. L., Nisbet, D. R., & Williams, R. J. (2013). Tuning the Amino Acid Sequence of Minimalist Peptides to Present Biological Signals via Charge Neutralised Self Assembly. Soft Matter , 9, 3915-3919.
[3] Williams, R.J., Smith, A.M., Collins, R., Hodson, N., Das, A.K., & Ulijn, R.V. (2009) Enzyme-assisted self-assembly under thermodynamic control. Nature Nanotech. 4, 19 - 24

This work was supported by ARC Discovery Grant DP 13011301

Fig. 1: Illustration (A), self-assembly (B), and hydrogel (C) of Fmoc-FRGDF, and the application of cell line (D).

Fig. 2: TEM images (A and C) and AFM images (B and D) of Fmoc-FRGDF (A and B) and Fmoc-FDGRF (C and D) hydrogel.

Fig. 3: FTIR (A), CD (B) and fluorescence spectrum (C) of both peptides. Tunable mechanical properties of Fmoc-FRGDF (D).

Fig. 4: Human mammary fibroblast cells show high levels of viability in Fmoc-FRGDF system (A) and reduced viability in Fmoc-FDGRF (B). Cell morphology of cells in RGD system (top) and migrating through DRG system (bottom) (C).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-6-P-5979 AM-FM and Loss Tangent Imaging – New Tools for Quantitative Nanomechanical Properties

Johann F.1, Proksch R.1, Revenko I.1, Hohlbach S.1, Cleveland C.1, Geisse N.1, Moshar A.1, Bemis J.1, Callahan C.1
1Asylum Research, an Oxford Instruments Company
florian.johann@oxinst.com

Nanoscale information on mechanical properties is critical for many advanced materials and nanotechnology applications. Atomic Force Microscopy (AFM) techniques for probing mechanical properties of samples in the nanometer range have emerged over the past decades. Here we present two resonance techniques which allow mapping the elastic modulus and viscoelastic damping with high spatial resolution and sensitivity.

Amplitude-modulated AFM (AM-AFM), also known as tapping mode, is a proven, reliable and gentle imaging method with widespread applications. Previously, the contrast in AM-AFM has been difficult to quantify. Here, we introduce AM‐FM imaging, which combines the features and benefits of normal tapping mode with quantitative and high sensitivity of frequency modulated (FM) mode. Briefly, the topographic feedback operates in AM mode while the second resonant mode drive frequency is adjusted on resonance. With this approach, frequency feedback and topographic feedback are decoupled, allowing much more stable, robust operation. The FM image returns a quantitative value of the frequency shift that depends on the sample stiffness and can be applied to a variety of physical models.

In the example shown in Figure 1, a micro‐cryotomed, cross-sectioned area of a coffee bag packaging material has been imaged. The AM‐FM image clearly shows the soft “tie” layers (dark purple) connecting the stiff metal layer (bright yellow) with two vapor-barrier polymer layers (orange).

In addition, loss tangent imaging is a recently introduced quantitative technique that recasts the interpretation of phase imaging in AM‐AFM into one term that includes both the dissipated and stored energy of the tip sample interaction. Quantifying the loss tangent depends solely on the measurement of cantilever parameters at a reference position.

Fig. 1: A micro‐cryotomed, cross-sectioned area of a coffee bag packaging. The AM‐FM image clearly shows the soft “tie” layers (dark purple) connecting the stiff metal layer (bright yellow) with two vapor-barrier polymer layers (orange).
↑ Go to the Table of content ↑

MS-7. Composite materials and hybrids

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-7-IN-1636 High temperature in-situ ceramic eutectic composite and functional glass matrix composite with a novel microstructure

Waku Y.1
1Department of Material Science, Graduate School of Engineering, Tohoku University, Aoba-yama, Sendai 980-8579, Japan
mgcwaku@material.tohoku.ac.jp

   We have developed unique ceramic eutectic composites, which are named Melt Growth Composites (MGC). The MGCs have novel microstructures (Fig. 1 for Al2O3/Y3Al5O12 (YAG) binary MGC and Fig. 2 for Al2O3/GdAlO3 (GAP) binary MGC), in which continuous networks of single-crystal Al2O3 phases and single-crystal complex oxide compound (YAG or GAP) interpenetrate without grain boundaries. The reconstructed three-dimensional images showed that binary MGCs have a chain structure. The existence of amorphous phases at interfaces generally leads to a reduction in the strength of the material at high temperature. Fig. 3 shows typical HREM images of the interface between Al2O3 and YAG phases in unidirectionally solidified Al2O3/YAG binary MGC. No amorphous phases are observed at the interfaces between the Al2O3 and YAG phases and relatively compatible interfaces are formed. The MGCs, therefore, have excellent high-temperature characteristics such as high temperature strength, thermal stability of microstructure and oxidation resistance in an air atmosphere at very high temperatures. We have also developed a new compositional binary MGC comprised of Al2O3 and SmAlO3 phases. The Al2O3/SmAlO3 binary MGC displays the highest high temperature flexural strength at 1773 K in conventional MGC binary systems.
   On the other hand, we have recently developed functional glass matrix composites reinforced with thin discal metallic particles (cross-sectional microstructures to perpendicular to pressed plane showed in Fig. 4). The volume resistivity of the glass matrix composites can be controlled by the volume fraction and the aspect ratio of thin discal Ni-Cr alloy particles. In addition, it is achieved by simultaneous improvement of the strength and fracture toughness by microdispersion of thin discal Ni-Cr alloy particles. Therefore, several useful applications can be considered, for example, household electrical instruments as a board-shaped heater such as hot plates, etc.
   In this paper, microstructural and high temperature characteristics of the binary MGCs and new functional glass matrix composites with superior characteristics of electric resistance will be briefly introduced.

We thank prof. H. Yasuda at Department of Material Science and Engineering, Graduate School of Engineering, Kyoto University for the 3 D observation of eutectic structure in the ceramic eutectic composite.

Fig. 1: SEM image showing the microstructure of a cross-section perpendicular to the solidification direction of the unidirectionally solidified Al2O3/YAG binary MGC.

Fig. 2: SEM image showing the microstructure of a cross-section perpendicular to the solidification direction of the unidirectionally solidified Al2O3/GAP binary MGC.

Fig. 3: HREM image of the interface between Al2O3 and YAG phases of the unidirectionally solidified Al2O3/YAG binary MGC.

Fig. 4: Optical micrographs showing the microstructure of a cross-section perpendicular to the SPS pressed planes of the soda-lime glass matrix composite reinforced with 30 vol % Ni-Cr alloy particles with shapes of the thin disc.
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-7-IN-2844 Characterization of Hybrid Gradients between Bulk Metallic Glasses and High Entropy Alloys

Fraser H.1, Welk B.1, Huber D.1, Gibson M.2
1Center for the Accelerated Maturation of Materials, Department of Materials Science and Engineering, The Ohio State University, Columbus, OH, USA, 2CSIRO, Clayton, VIC, Australia
fraser.3@osu.edu

There has been much current interest in bulk metallic glasses (BMG), high entropy alloys (HEA), and combinations of both of these phases as potential composites. The chemical compositions of both of these types of materials usually involve combinations of a number of elemental species, present in reasonably significant concentrations. To develop a detailed understanding of these two different types of material, it is important to compare and contrast the evolution of microstructures as the compositions are varied from that of a BMG to that of an HEA. This has been effected by making use of a combinatorial technique involving laser deposition of metallic powders to produce hybrid samples, of approximately 3cm in length, starting with a composition corresponding to a BMG and ending with a composition of an HEA. Alloy systems currently under investigation include a graded samples varying in composition from (Ti25 Zr25 Cu25 Nb25) to (Ti17.5 Zr17.5 Cu17.5 Nb17.5 Ni30), and from (Cu47 Zr45 Al8) to (Cu Ni Al). Of particular interest are the structure of the constituent phases and the nature of the interfaces between them in the graded (hybrid) samples. The various samples have been characterized using aberration-corrected (S)TEM, and the tomographical atom probe, in addition to the more conventional techniques (e.g., SEM). Currently, the deformation behavior of these materials is being studied, and the defects in deformed samples are being characterized using both diffraction contrast in the TEM and high resolution (S)TEM. Of particular interest is the deformation behavior of composites of the two types of material, especially ones with a matrix of the HEA phase with a distribution of particles of BMGs. The results of these various characterizations will be compared and contrasted.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-7-O-1542 Revealing the Morphology of Mesoporous Perovskite Solar Cells Using Analytical Transmission Electron Microscopy

Kast A. K.1,2, Nanova D.2,3, Veith L.5, Wacker I.5, Agari M.6, Hermes W.6, Erk P.6, Kowalsky W.2,3, Lovrincic R.2,3, Schröder R. R.1,2,5
1CryoEM, CellNetworks, Universitätsklinikum Heidelberg, Germany, 2InnovationLab GmbH, Heidelberg, Germany, 3IHF, TU Braunschweig, Germany, 4EMAT, Antwerp, Belgium, 5Center for Advanced Materials, Universität Heidelberg, Germany, 6BASF SE, Ludwigshafen, Germany
anne.kast@bioquant.uni-heidelberg.de

Organometal trihalide perovskites, like CH3NH3PbI3, have recently become of great interest in the search for cost-efficient solar cell materials due to their remarkable power conversion efficiencies of up to 15% [1] and the possibility of solution based device fabrication. These devices are based on the principle of dye-sensitized solar cells, using a mesoporous TiO2 scaffold as a transparent electron contact and spiro-MeOTAD as holetransport layer (HTL) [2]. The question of the device nature (DSSC-like or p-i-n), morphology, infiltration of absorber and HTL into the scaffold, and its correlation to device performance arises.
The morphology of solution processed mesostructured perovskite solar cells was studied using Analytical Transmission Electron Microscopy (ATEM). After determining the characteristic excitation energies of TiO2 and Pb using Electron Energy-Loss Spectroscopy (EELS) and confirming this with known data from literature, material contrast was achieved using Electron Spectroscopic Imaging (ESI). A series of monochromatic images was acquired from 2 to 100 eV in 1 eV steps and analyzed using Multivariate Statistical Analysis (MSA) [3]. Classification of the TiO2- and perovskite-rich regions was possible with knowledge of the material specific excitations at different energies.
Since investigations were done on full devices, cross-section samples were prepared using Focused Ion Beam (FIB). Electron diffraction confirms the perovskite structure as known from literature [4] and excludes alterations induced by the preparation method.
Fig. 1a shows the zero-loss filtered TEM image of a part of the perovskite filled TiO2 scaffold. Fig. 1b and c are energy filtered images of the same sample area acquired at 15 eV and 48 eV, respectively. Examples of contrast inversion due to excitations of different materials at these energies are marked by circles. EEL spectra showed a higher excitation around 48 eV for TiO2 due to the Ti M2,3 edge. Knowledge of spectral differences aided in classifying perovskite- and TiO2-rich areas after MSA (fig. 1d, perovskite: green, TiO2: red). Segmentation and extraction of spectra from ESI data lets us ascertain the infiltration of perovskite and hole-transport layer into the mesoporous TiO2.
We demonstrate that our methods can be applied to give insight into the morphology of perovskite-based solar cells. Correlation of the morphology with the electronic properties and preparation methods of different devices can bring us one step further in understanding and improving these solar cells.
[1] M Liu, MB Johnston, HJ Snaith, Nature 501 (2013) 395–398
[2] MM Lee, et al. Science 338 (2012) 643–647
[3] M Pfannmöller, et al. Nano Lett. 11 (2011) 3099–3107
[4] A Poglitsch, DJ Weber, Chem. Phys. 87 (1987) 6373–6378

Financial support by the BMBF (FKZ 03EK3505K, FKZ 13N10794) is gratefully acknowledged.

Fig. 1: Zero-loss image of part of the perovskite-filled TiO2 scaffold (a), no material contrast. The same sample area imaged at 15 eV (b) and 48 eV (c) electron energy-loss, examples of contrast inversion marked by circles. Segmentation of the ESI data for two materials (d). TiO2-rich areas shown in red, perovskite in green, scale bar: 50nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-7-O-1575 Emission color mapping of white-luminescent mesoporous carbon-silica nanocomposite

MUTO S.1, SATO K.2, ISHIKAWA Y.2,3, BOSMAN M.4, ISHII Y.5, KAWASAKI S.5
1EcoTopia Science Institute, Nagoya University, Nagoya 464-8603, Japan, 2Japan Fine Ceramics Center, Nagoya 456-8587, Japan, 3Department of Frontier Materials, Nagoya Institute of Technology, Nagoya 466-8555, Japan, 4Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602, 5Department of Materials Science and Engineering, Nagoya Institute of Technology, Nagoya 466-8555, Japan
s-mutoh@nucl.nagoya-u.ac.jp

Luminescent materials widely used for optical devices, such as light-emitting-diode, plasma display and liquid crystal display rely on the inner-shell transition of rare earth (RE) elements. Unstable RE supply, however, accelerates the importance for developing RE-free luminescent materials. Silicon-oxycarbide (SiOC) has been a promising candidate for white luminescent materials [1], and recently, the present research group found that the mesoporous carbon silica (MPCS) nanocomposite with large surface area emits strong white PL, depending on the degrees of hydrolysis and polycondensation reactions in preparing MPCS [2]. The MPCS nanocomposite consists of a periodic honeycomb silica framework with pores of ~8 nm in diameter.

The synthesis procedures of the MPCS nanocomposite was described in detail elsewhere [3]. The sample was ground by an agate mortar and pestle into fine powder, which was sprinkled on a carbon coated micro-grid. The sample thus prepared was examined using a JEOL JEM-ARM200F STEM (double Cs-corrected) operated at 80 kV and a Gatan Image Filter Quantum ER and an FEI Titan equipped with a Gatan Vulcan TEM-CL system, operated at 80 kV at LN2 temperature for elemental/emission mapping.

Figs. 1(a) and (b) show the side- and top-view of the spatial distributions of silicon/carbon, reconstructed from the STEM-EELS spectrum imaging data sets. As expected, the framework of honeycomb is made of silica, the inner walls of which are covered by amorphous carbon layer. The CL spectrum consists of several emission components (Fig. 2). CL-spectrum imaging scanned along the line shown in Fig. 1(b) revealed that the primary two emission bands (#1 and #2 in Fig. 2) seemed to be derived from the silica frame and inner surface carbon layers, respectively, as shown in Fig. 3. The origin of #2 component is particularly discussed.

References

[1] S. Hayashi, et al, Jpn. J. Appl. Phys. 32 (1993) L274; A. V. Vasin, et al, Jpn. J. Appl. Phys. 46 (2007) L465; S. Y. Seo, K. S. Cho, and J. H. Shin: Appl. Phys. Lett. 84 (2004) 717.

[2] K. Sato, et al, IOP Conf. Ser.: Mater. Sci. Eng. 18 (2011) 102022.

[3] K. Sato, et al, Jpn. J. Appl. Phys. 51 (2012) 082402.

A part of this work was supported by a Grant-in-Aid on Innovative Areas "Nano Informatics" (Grant number 25106004) from the Japan Society of the Promotion of Science.

Fig. 1: HAADF-STEM (Left) and energy filtered image (Right) of MPCS nanocomposite reconstructed by STEM-EELS spectrum image using p-p* plasmon of sp2 carbon (green) and SiO2 volume plasmon (red) respectively for top- (a) and side-view (b). Step width for STEM-EELS ~ 0.5 nm.

Fig. 2: CL spectrum from MPCS nanocomposite flake, which was fit by 5 gaussians.

Fig. 3:   BF image contrast, #1 and 2 primary emission intensities (cf. Fig. 1) obtained by line scan along the broken line in Fig. 1(b). The red emission (red line) is well correlated with the projected amount of silica, while green emission (green line) with the inner surface of the silica frame.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-7-O-2384 Respective influence of the organic and inorganic components on the optical properties of a hybrid layered molybdate: an EELS and DFT study

Lajaunie L.1, Boucher F.1, Dessapt R.1, Moreau P.1
1Institut des Matériaux Jean Rouxel, (IMN) – Université de Nantes, CNRS, 2 rue de la Houssinère - BP 32229, 44322 Nantes Cedex 3, France
luc.lajaunie@cnrs-imn.fr

Introduction Investigation of photochromic materials has been extensive over the past decade due of their potential technological and marketable applications for high-density optical data storage and optical switching. Among them, polyoxometalate-based hybrid organic-inorganic materials exhibit remarkable solid-state photochromism at room temperature with reversible and multicolor possibilities, visible-light coloration and fast photoresponse compared to binary oxides. However the question concerning the respective influence of the organic and inorganic components on the optical properties is not clearly answered yet. On this aspect, valuable insights might be gained from a combination of Valence Electron Energy-Loss Spectroscopy and DFT calculations to properly determine and interpret dielectric properties.1

Materials & Methods In this work, we report for the very first time VEELS experiment before photo-irradiation on (N,N’-H2DMED)[Mo5O16] (N,N’-H2DMED2+=N2C4H142+), here after labeled Mo5O16. This photochromic hybrid crystallized material is based on ²/∞[Mo5O16]2- layers linked together via organoammonium chains (Fig. 1.a).2 EELS experiments were performed at liquid-nitrogen temperature using a Hitachi HF2000 TEM (100 kV, cold-FEG). In addition, electronic band structures and optical properties were calculated at the DFT level with the WIEN2k and VASP codes. In particular, the macroscopic dynamic dielectric functions including local-field effects (LFE) were obtained from the inversion of the full microscopic dielectric matrices. To highlight the influence of the inorganic chains on the material properties, the results are compared to those obtained on α-MoO3, which presents strong structural similarities with the inorganic framework of Mo5O16 (Fig. 1.b). 1,2

Results The experimental and calculated EELS spectra of Mo5O16 and MoO3 are compared in Fig. 2. An excellent overall agreement is observed and our calculations reproduce correctly the presence of the peak situated around 13 eV (labeled B) in the spectrum of MoO3 and its absence in the spectrum of Mo5O16. This excellent agreement confirms the validity of our calculations and allows us to use them for further investigations. Total and partial DOS for MoO3 and Mo5O16 are shown in Fig. 3. This figure highlights the lack of significant contributions directly linked to organic species in the band gap vicinity. This suggests that the low energy part of the dielectric function is mainly dominated by optical transitions involving the inorganic framework. Such influences on the optical properties will be discussed in details.

1. L. Lajaunie, F. Boucher, R. Dessapt and P. Moreau, Phys. Rev. B 81 115141 (2013)

2. R. Dessapt, D. Kervern, M. Bujoli-Doeuff et al., Inorg. Chem., 49, 11309 (2010)

Fig. 1: Figure 1. Representations of the Mo5O16 (a) and α-MoO3 (b) structures. The [MoO6] polyhedra are drawn in blue and the oxygen atoms of the ²/∞[Mo5O16]2- layers implied in the hydrogen-bonding networks (dotted lines) are drawn in red.

Fig. 2: Figure 2. Comparison of the experimental VEELS spectra (thick black) of Mo5O16 and MoO3 with the calculated ones including local-field effects (thin red).

Fig. 3: Figure 3. Electronic densities of states for Mo5O16 and MoO3. The circles highlight the main contribution of the organic part of the hybrid compound.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-7-O-2931 Spatial distribution of different diamond phases in compressed graphite studied by STEM-EELS

Sato Y.1, Bugnet M.2, Terauchi M.1, Botton G.2, Yoshiasa A.3
1Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2Department of Materials Science and Engineering, McMaster University, 3Graduate School of Science and Technology, Kumamoto University
y-sato@tagen.tohoku.ac.jp

Electronic structure of hexagonal diamond (Lonsdaleite, h-DIA) is different from that of cubic diamond (c-DIA) [1]. h-DIA specimen was synthesized by direct transfer from graphite under high pressure and high temperature, where c-DIA and graphite phases always coexist. It has been an interesting subject to clarify how graphite phase (sp2) transfers to h-DIA or c-DIA phases (sp3) in the compression process. X-ray diffraction analyses have reported the h-DIA, c-DIA and graphite phases in the specimen exist as particle with a few nanometer in diameter [2]. However, direct observation of spatial distribution of the h-DIA and other allotropes has not been reported.
  As the electronic structure of those carbon materials are different, K-shell excitation spectra, which reflect the density of states of conduction bands, should be different and effective to distinguish each crystal phases. In this study, spectroscopic imaging method by using STEM-EELS technique was applied. By fitting the spectra with linear combination of the three kinds of reference spectra of h-DIA, c-DIA, and graphite crystal phases (MLLS fitting) [3], spatial distribution of three kinds of the carbon crystal phases in the compressed graphite specimen was investigated.
  Carbon K-shell excitation spectra was obtained by using FEI Titan 80-300, equipped with monochromator and a high-resolution spectrometer operated at 80 kV. K-shell excitation spectrum of h-DIA shows a different intensity distribution from that of c-DIA (Fig.1) [1]. There is no π* peak intensity (indicated by an arrow) means that present spectral profile of h-DIA specimen produced consist of only σ* component formed by sp3 orbital. Intensity distribution of K-shell excitation spectrum at each measured points was fitted with a linear combination of the three reference spectra (Figure 2b). Figure 2c shows a spectroscopic imaging of the compressed graphite specimen, where blue, green, and red colors indicate h-DIA, c-DIA, and graphite, respectively. The image shows the individual crystal phases are a few tens nanometer in diameter, which is about ten times larger than the estimated value by X-ray diffraction. This indicates that actual crystal phases exist as larger sized masses, which should include many crystal defects. The spectroscopic imaging is effective to distinguish different carbon crystal phases in real space and it makes easy to understand how three kinds of carbon phases exist in this compressed graphite.

References
[1] Y. Sato et al., Diamond & Related Materials 25, 40-44 (2012).
[2] A. Yoshiasa et al., Jpn. J. Appl. Phys., 42, 1694-1704 (2003).
[3] R. D. Leapman and C.R. Swyt, Ultramicroscopy, 26, 393-404 (1988).

The experimental work was carried out at the Canadian Centre for Electron Microscopy (CCEM), a national facility supported by NSERC and McMaster University.

Fig. 1: Carbon K-shell excitation spectra of h-DIA and c-DIA.

Fig. 2: (a) ADF image of compressed graphite specimen. (b) Spectrum fitting of carbon K-shell excitation spectra with reference spectra. Dots and black line are the experimental and the fitting spectra. (c) Spectroscopic image of the specimen. Blue, green, and red colors indicate h-DIA, c-DIA, and graphite phases, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-1732 Morphology analysis of sponge-like Si-SiO2 nanocomposites using energy-filtered electron tomography and electron holographic tomography

Hübner R.1, Wolf D.2, Friedrich D.1, Liedke B.1, Schmidt B.1, Heinig K. H.1
1Institute of Ion Beam Physics and Materials Research, Helmholtz-Zentrum Dresden-Rossendorf, 01314 Dresden, Germany, 2Triebenberg Laboratory, Institute of Structure Physics, Technische Universität Dresden, 01062 Dresden, Germany
r.huebner@hzdr.de

Due to the possibility of band-gap engineering by quantum confinement, Si nanosponge structures embedded in SiO2 formed by spinodal decomposition of metastable silicon-rich silicon oxide are promising absorbers for 3rd generation solar cells. According to SiOx → ½x SiO2 + (1 - ½x) Si, annealing of thermodynamically metastable, silicon-rich oxide SiOx with x < 2 leads to phase separation of elemental Si from stoichiometric SiO2. While this phase separation results in disconnected Si nanoclusters for 1.2 ≤ x < 2, percolated Si nanostructures with a sponge-like morphology are observed for x < 1.2 [1].


To visualize the sponge-like morphology in SiOx films for x ≈ 1 after thermal treatment, energy-filtered transmission electron microscopy (EFTEM) imaging, EFTEM tomography, and electron holographic tomography (EHT) [2] were carried out. To this end, 200 nm thick SiOx layers were prepared on p-type (100) Si wafers by magnetron sputtering in Ar plasma from two simultaneously operating Si and SiO2 targets. During subsequent annealing, samples were heated up to 1150 °C. Sponge-like nanostructures were investigated by EFTEM imaging using an image-corrected FEI Titan 80-300 microscope equipped with a GIF 863. For EFTEM tomography, a tilt series between ±70° was acquired in a Philips CM200 FEG microscope with a GIF 678, and for EHT, a tilt series from -74° to +79° was recorded in an image-corrected FEI Tecnai TF20 microscope. Tomographic reconstruction of the Si 3D morphology was performed with the Weighted Simultaneous Iterative Reconstruction Technique [3].


Valence-band plasmon energy-loss imaging is an appropriate approach to visualize the Si morphology in phase-separated Si-SiO2 nanocomposites [4]. As an example, Figure 1 shows the Si plasmon EFTEM images (Eloss = 17 eV) of a SiOx≈1 layer decomposed into Si and SiO2 after thermal treatment at 1100 °C for 3 min (left) and 3 h (right). As indicated by the selected area electron diffraction patterns, coarsening of the Si nanostructure is accompanied by Si crystallite growth. Although Si plasmon EFTEM imaging can show the Si phase distribution in a planar projection, it does not provide 3D information. Therefore, EFTEM tomography was applied, revealing that a spinodal sponge-like morphology of Si is only partially visible in a volume of ca. (30 nm)³ (Figure 2). However, in a larger volume of ca. (140 nm)³ - as demonstrated by applying EHT on a needle-shaped specimen prepared by FIB - both isolated nanoparticles and percolated Si nanostructures with a sponge-like morphology are observed (Figure 3).


[1] T. Müller et al., Appl Phys Lett 85 (2004) 12.
[2] D. Wolf et al., Curr Opin Solid St M 17 (2013) 126.
[3] D. Wolf et al., Ultramicroscopy 136 (2014) 15.
[4] D. Friedrich et al., Appl Phys Lett 103 (2013) 131911.

The authors kindly acknowledge TEM sample preparation by Annette Kunz and Martina Missbach.

Fig. 1: Cross-sectional Si plasmon EFTEM images (Eloss = 17 eV) of a SiOx≈1 layer decomposed into Si (bright) and SiO2 (dark) during annealing at 1100 °C for 3 min (left) and 3 h (right) together with corresponding selected area electron diffraction patterns (insets).

Fig. 2: Si plasmon EFTEM tomogram (Eloss = 17 eV) of phase-separated SiO0.9. Si (red) and SiO2 (transparent) are distinguished by applying an intensity threshold resulting in approximately 30 vol.-% Si within a (28 x 31 x 24) nm³ volume.

Fig. 3: 3D potential reconstructed by EHT of phase-separated SiO0.9. Si (red) and SiO2 (transparent) are distinguished by selecting an iso-potential surface of 11 V, i.e. the mean value between the mean inner potentials of Si and SiO2.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-1942 THE STUDY OF THE DIFFERENT PERCENTAGE PERFORMANCE OF NANOPARTICLES ON THE PROPERTIES OF EPOXY RESIN

Hudec J.1,2, Neděla V.1, Polsterová H.2
1Institute of Scientific Instruments of the ASCR, v. v. i.in Brno, 2Brno University of Technology Faculty of Electrical Engineering and Communication
hudec@isibrno.cz

This paper deals with the study of impact of different percentage filling of nanoparticles on the electrical properties of epoxy resin, which has very good mechanical and electrical properties. The sample is the blended mixture which is evacuated, subjected to ultrasound and then cured. It is expected that the formation of lumps should be minimised due to the influence of microwaves. Nanoparticles should be equally distributed in epoxide volume for this case. Unfortunately, this assumption was not proven. The mixture contains an epoxy resin CY228, hardener HY918, softener DY045 and accelerator DY062. Nanoparticles of alumina (Al2O3), sulfur dioxide (SiO2), titanium dioxide (TiO2) and tungsten oxide (WO3) from Sigma Aldrich Company were used as a filler. There were made samples for each filler with 0.25, 0.5, 1, 2 weight percent for our experiment and were determined values of the dissipation factor tgδ, permittivity εr and resistivity ρv by measuring.

We are able to prepare samples with better electrical properties. Unfortunately, despite the advanced procedure of samples production, our main problem is the inhomogeneity of distribution of nanoparticles in the sample manifested by the formation of lumps, documented by figures 3 and 4. It can be assumed that the optimization of the manufacturing process will be achieved to increase the quality of the samples and particularly their final properties of measured electrical parameters.

Figures 3 and 4 show randomly chosen samples observed using a detector of secondary electrons in a scanning electron microscope REM Jeol JSM 6700F at a magnification of 10.000x and 50.000x.

The lowest permittivity was encountered in the samples with the 2% filling of Al2O3 and SiO2, in the case of TiO2 it was 1% (see Fig. 1).

In the samples containing Al2O3 a SiO2 the impact of the nanoparticles on the intrinsic resistivity is evident in the full temperature range (Fig. 1). The highest intrinsic resistivity is in the sample with the 0,5% content of SiO2, apart from the sample with 0,5% of Al2O3which has a lower resistivity than pure epoxide.

The most pronounced improvement in electrical properties of the resulting nanocomposite was achieved by adding Al2O3 and SiO2. The influence of TiO2 was less obvious, and adding the nanoparticles of WO3 caused no change in any of the measured parameters.

This work was supported by the Grant Agency of the Czech Republic: grant No. GA 14-22777S.

Fig. 1: The final samples of epoxy resin with nanofiller

Fig. 2: Comparison of temperature dependences of dissipation factor and permittivity of pure epoxy and specimens with 2% filler content

Fig. 3: Microstructure of the surface of the epoxy resin with 2 weight percent of filler Al2O3 nanoparticles, REM Jeol JSM 6700F

Fig. 4: Microstructure of the surface of the epoxy resin with 2 weight percent of filler Al2O3 nanoparticles, REM Jeol JSM 6700F
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-2024 Optimization of electron tomography for the three dimensional study of hybrid nanoparticle assemblies

Bladt E.1, Heidari H.1, Leroux F.1, Van Tendeloo G.1, Bals S.1
1EMAT, University of Antwerp, Belgium
eva.bladt@uantwerpen.be

Recently, hybrid materials which contain both organic and inorganic components have gained interest due to a broad range of potential applications such as catalysis [1]. The nano-architecture of such self-assembled structures plays a crucial role in the development of tunable systems. In order to understand the structure-property relation, a thorough characterization of the 3D nanoparticle organization is crucial. Conventional transmission electron microscopy only provides 2D projections of a 3D structure which may prohibit an appropriate interpretation of the morphology. This limitation can be overcome by using electron tomography. However, the technique has been used in only a few analyses of hybrid systems [2], [3]. This is related to the fact that both compounds of the hybrid structure require completely different imaging conditions. Electron tomography for inorganic-organic hybrid systems can therefore be considered as challenging. Here, we discuss a dedicated route towards 3D characterization of hybrid structures consisting of Au nanoparticles suspended in a flexible polymeric matrix.

The preparation method of the TEM sample for electron tomography studies of nanoassemblies is of crucial importance when investigating the 3D arrangement of nanoparticles in a nano-assembly. The native structure of the nanoassembly, as present in the solution, should be maintained. The conventional approach of drying the sample on a carbon coated TEM grid will alter the morphology of the structures. Plunge-freezing results in instantanteously embedding the nanostructures in a thin layer of vitreous ice. The structure is maintained, but the sensitivity of the specimen to the electron beam presents the limiting factor in cryotomography, therefore the images are acquired at a low dose mode [4]. As a result, the reconstructions suffer from several artifacts caused by low-SNR of the projection images as well as the presence of diffraction contrast in the projection images. Therefore, we propose to freeze-dry the samples after vitrification, where low pressure ensures sublimation of the ice into the gaseous phase. The native 3D structure is preserved and by using HAADF-STEM tomography diffraction contrast is avoided. In this manner an accurate 3D reconstruction of the hybrid nanoassemblies is obtained.

 

[1] C. J. Newcomb, et al.,” Curr. Opin. Colloid Interface Sci., vol. 17, no. 6, pp. 350–359, 2012.

[2] T. Altantzis,et al., Part. Part. Syst. Charact., vol. 30, no. 1, pp. 84–88, 2013.

[3] M. Grzelczak, et al., Nano Lett., vol. 12, no. 8, pp. 4380–4384, 2012.

[4] A. Koster and M. Bárcena, Electron Tomography SE - 5, J. Frank, Ed. Springer New York, 2006, pp. 113–161.

The authors gratefully acknowledge financial support from the Fund for Scientific Research-Flanders. We also thank Prof. Luis M. Marzán for provision of the samples.

Fig. 1: (a) A droplet of suspension is deposted on a TEM support grid, (b) as a consequence of the behaviour on a hydrophilic surface; the solution tries to extend upon the surface, (c) the soft polymer is absorbed towards the grid upon surface tension effects.

Fig. 2: A schematic overview of the optimized route for the 3D characterization of nanodumbbells in an assembly.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-2112 Characterization of Self-Hardening CrAlN/BN Nanocomposite Coatings

Sugita H.1, Nose M.2, Chiou W. A.3, Hanyu H.1, Matsuda K.4
1R&D Center, OSG Corporation, 1-15 Hon-nogahara, Toyokawa 442-8544, Japan, 2Faculty of Art and Design, University of Toyama, 180 Futagami-machi, Takaoka 933-8588, Japan, 3NISP Laboratory, NanoCenter, University of Maryland, College Park, MD 20740-2831, USA, 4School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan
nose@tad.u-toyama.ac.jp

The “self-hardening phenomena” of TiBN and TiCrBN coatings after annealing have been observed in vacuum or inert gas atmospheres. An increase of the indentation hardness of CrAlN/BN nanocomposite coatings after annealing may also be performed in air in the range of 700 to 800 oC. This peculiar phenomenon of the CrAlN/BN coatings has been studied by examining the microstructure and microchemistry of the coatings.
Cr50Al50 alloy (99.8%) and h-BN (99.5%) targets were sputtered in a mixture of highly purified argon and nitrogen gases (6N). Total thickness of the film was in the range of 1.8 ~ 2.2 μm. Coating morphology, microstructure and microchemistry were examined with a FEG SEM and a FEG TEM (JEM-2100F and ARM-200F) that were equipped with Oxford and JEOL EDS system, respectively.
Figure 1 illustrates the change of indexed indentation hardness, HIT (%), of the CrAlN, CrAlN/8vol% BN and CrAlN/18 vol.% BN coatings at different annealing temperatures in ambient air. The hardness of these coatings as-deposited was 42, 43 and 39 GPa, respectively. Self-hardening of the CrAlN/BN coating was evidenced by the increases of indentation hardness that occurred after annealing to 800 oC in air. Changes in surface morphology of CrAlN and CrAlN/8 vol.% BN coatings after annealing were observed in SEM. After oxidation at 800 oC, scale-like precipitates were strewed over a large area surface of CrAlN coatings (Fig. 1b). The surface morphology of CrAlN/BN coating is similar to that of the as-deposited coating that consisted of granulated particles (Figs.1c/1d). TEM cross-sectional images of CrAlN/18vol%BN coating, both as-deposited and annealed samples, appeared rather similar to each other with columnar structure (Figs. 2a/2b). However, a disruption in the columnar structure by a very thin layer (20 ~ 40 nm) of film was observed in the annealed sample (Figs.2c/2d) but not in the as-deposited sample. HRTEM image revealed that the top most layer is characterized by amorphous materials with embedded nanocrystalline particles (white circles in Fig. 2d). EDS line profiles of cross-sectional samples showed a high concentration of O in the uppermost layer of the annealed sample (Fig. 3a). The O content remained constant at a lower level throughout the film surface, up to a depth of ~100 nm in the as-deposited sample (Fig. 3b). This indicates that the oxidized layer formed near the top surface is likely to be one of the factors responsible for the self-hardening phenomena in CrAlN/BN coatings. Our recent investigation using ARM confirmed that the as deposited CrAlN/BN coatings have nanocomposite structure consisting of CrAlN nanocrystalline grains embedded in amorphous BN phase. This structure probably causes the self-hardening effect.

TEM work performed at NISP Lab was partially supported by NSF-MRSEC (DMR 05-20471)and　UMD. ARM TEM work was provided by Mr. Y. Sasaki and Mr. T. Suzuki of JEOL.

Fig. 1: Diagram shows variation of indexed indentation hardness, HIT(%), of CrAlN, CrAlN/8vol%BN and CrAlN/18vol%BNcoatings at different annealing temperature in air for 1 h. SEM micrographs of CrAlN (a/b) and CrAlN-8%BN(c/d) coatings show morphological change after annealing.(a) and (c): As-deposited; (b) and (d): annealed at 800 oC in air for 1h.

Fig. 2: Cross-sectional TEM images and SAD patterns of as-deposited CrAlN/18vol%BN coating (a and b) and after annealing at 800 oC for 1 h. in air (c and d) reveal columnar structure. An oxide layer formed on the surface of CrAlN/18vol%BN thin film (c), and the HRTEM image of the oxide layer depicts nanocrystallites embedded in the amorphous layer.

Fig. 3: EDS line profiles show elemental concentration across the film from the top surface of the annealed sample (a) and as-deposited sample (b). Note the change of O concentration in both samples.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-2118 Fibrous scaffolds of PLA and PLA/HA for bone tissue

Vargas-Becerril N.1, Granados-Hernández M. V.1, Téllez-Jurado L.2, Hipólito-García M.3, Álvarez-Gregoso O.3, Álvarez-Pérez M. A.1
1Laboratorio de Bioingeniería de Tejidos; División de Estudios de Posgrado e Investigación de la Facultad de Odontología, UNAM. Circuito Exterior s/n. Cd. Universitaria, 04510 Coyoacán México D. F., México., 22Departamento de Ingeniería Metalúrgica y de Materiales E.S.I.Q.I.E-I.P.N, Unidad Professional Adolfo López Mateos 7, Lindavista. 07738 Gustavo A. Madero, México D. F., México. , 3Instituto de Investigaciones en Materiales, Circuito Exterior s/n. Cd. Universitaria, 04510 Coyoacán México D. F., México.
nancyvb09@gmail.com

Scalffolds have been became an important materials for the tissue engineering, which provide temporal mechanical and structural support to cells [1]. The scalffolding materials for bone tissue should be osteoconductive such that osteoprogenitor cells can adhere and migrate on the scalffolds, differentiate, and finally form new bone. Some inorganic materials have been incorporated to polymeric scalffolds improving, the properties like bioactivity and biological functions [2].
In the present work we present poly (lactic acid) (PLA) and poly (lactic acid)/hydroxyapatite (PLA/HA) hybrid scalffolds. They were prepared by jet spinning, using five concentrations of PLA: 2.25, 6.25, 7.25 and 10 %, and 1g of HA was added to form the hybrid scalffolds. Morphology and surface were characterized by scanning electron microscopy (SEM), Atomic force microscopy (AFM). Thermal properties were carried out by Thermo gravimetric analysis (TGA) and infrared spectroscopy (FT-IR) was employed to analyze the atomic structure.
SEM images (figure1), show the effect of HA on the structure of PLA scalffolds. The width of PLA/HA scalffolds increase. The morphology changes from scalffolds like fibers to fibers with a form like staggered flakes. AFM images displayed higher roughness on the surface of hybrid scalffolds. FTIR of the hybrid scalffolds display the bands attributed to PO4+3 groups, suggesting the incorporation of HA into the organic PLA matrix. Thermal properties change with the incorporation of HA into the matrix of PLA.

References

[1] Nora Nseir, Omri Regev et al, Tissue Engineering: Part C, Volume 19, 4, (2013).

[2] Abdalla Abdal-hay et al, Colloids and Surfaces B:Biointerfases 102 (2013).

Authors want to thank to UNAM-DGAPA for postdoctoral scholarship supporting to NVB during the course of this study. This research were financially supported by funds from the UNAM-DGAPA: PAPIIT project IN213912 and Graduate Program of High Quality UNAM-CONACYT No. I010/480/2013 C-736/213.

Fig. 1: SEM images show the morphological changes when the HA is added on the PLA and hybrid schalffolds are formed by jet spinning; (a) PLA scalffolds and (b) PLA/HA hybrid scalffolds.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-2360 EELS study of the carbon speciation of the pyrocarbon interphase in SiC/SiC composites before and after neutron irradiation

Fave L. G.1,2, Hébert C.2, Pouchon M. A.1
1Paul Scherrer Institut, Villigen, Switzerland, 2École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
loic.fave@psi.ch

Silicon carbide ceramic matrix composites (SiC/SiC), made of SiC fibers embedded in a SiC matrix, are considered as a potential material for advanced fission as well as for fusion structural applications. A thin layer of pyrolytic carbon (PyC) is an integral part of the composite material since it transfers the mechanical loads from the matrix to the fibers. These 20 to 50 nm coatings deposited on the SiC fibers also play an important role in the effective thermal conductivity of the composite material. The effects of irradiation on this graphite-like layer and the link to subsequent changes in the thermal conductivity of the composite requires further research.
To accomplish this, an electron energy loss spectroscopy (EELS) study is carried out on the PyC layer of nuclear grade SiC/SiC samples which were exposed in pile in the High Flux Reactor (HFR) at Petten [1]. TEM lamellas are prepared from small capsules which were exposed as well as from unexposed material from the same batch. The amorphization of the PyC layer due to neutron irradiation might result in a change in the carbon speciation, from sp2 to sp3. To verify this, the core-loss part of the EELS spectra of the irradiated and pristine materials are compared to identify a possible amorphization. This method has been successfully reported by Yan et al. in [2], whereby the mechanical properties of unirradiated SiC/SiC samples were studied. Last, the effect of this microstructural change on the mechanical and thermal properties of the SiC/SiC composite are discussed. Indeed, a highly orientated PyC layer is desirable as far as mechanical properties are concerned [3,4], whereas the thermal conductivity of amorphous carbon is higher than that of pyrolytic carbon along its c-axis.
Bibliography
[1] The High Flux Reactor (HFR) at Petten. Retrieved from European Material Testing Reactors: http://www.emtr.eu/hfr.html, CEA. (2014, March 4)
[2] J.Y. Yan, C. C. (2004). The investigation of crack mechanism for Tyranno-SA SiC/SiC composites with ESI method. Journal of Nuclear Materials, 513-517.
[3] E. Buet, C. S.-N.-G. (2014). Influence of surface fiber properties and textural organization of a pyrocarbon interphase on the interfacial shear stress of SiC/SiC minicomposites reinforced with Hi-Nicalon S and Tyranno SA3 fibres. Journal of the European Ceramic Society, 179-188.
[4] T. Hinoki, W. Y. (2001). Improvement of mechanical properties of SiC/SiC composites by various surface treatments of fibers. Journal of Nuclear Materials, 23-29.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-2416 Application of scanning and transmission electron microscopies for the hybrid silica and silicone structures characterization

Staszewska M.1, Lewandowska-Łańcucka J.2, Wierzbińska M.1, Nowakowska M.2
1Department of Materials Science, Rzeszow University of Technology, Pola 2, 35-959 Rzeszów, Poland, 2Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland
staszews@prz.edu.pl


Nowadays the expectations concerning the new materials are very high so to meet them the scientists have created so called hybrid materials. They are composed of inorganic, organic or both types of these components with the fragmentation less than 1 μm. The properties and potential applications of the material depend strongly on the morphology thus the precise characterization is necessary. In this field scanning and transmission electron microscopy seems to be the most suitable. These techniques ensure not only imagining with high magnification, but also other analyses (e. g. energy dispersive X-ray spectroscopy, diffraction).
In this work we have presented the preparation method and characterization of the new hybrid materials, in which silica or polysiloxanes were used as a matrix. These substances were chosen due to their unique properties: silicon dioxide is chemically, mechanically and thermally resistant, and exhibits the UV-Vis permeability. Occurring on the surface Si-OH groups provide with further modification. While silicones are composed of polysiloxanes – high-molecular compounds whose main chain is built by alternating connected silicon and oxygen atoms. To silicon atoms might be attached hydrogen atoms or organic groups. In comparison with the typical organic polymers silicones possess exceptional properties such as: high chemical, thermal resistance, permeability for gases and vapors and in most cases biocompatibility.
They were combined with 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin. Porphyrines are group of compounds, which have strong absorption and emission properties, very interesting for industry and medicine. Unfortunately, their applications are limited by low mechanical and thermal stability. In order to solve this problem, we have proposed the synthesis of the new hybrid material with silica and silicone as a matrix [1].
The second group of the investigated materials were core-shell type particles - superparamagnetic iron oxide nanoparticles modified with cationic chitosan (SPION-cchit) and cover with silica. SPIONs are applied in many fields of science and medicine. However, the pristine SPIONs are not very stable and exhibit the tendency for aggregation which considerably limits their applications. In order to improve the stability and make possible their further modification we prepared the silica shell on the surface of the SPION-cchit and thus obtained the hybrid core-shell type particles [2].

References
[1] M Staszewska, M Dzieciuch, J Lewandowska, M Kępczyński, S Zapotoczny, M Oszajca, A Łatkiewicz, M Nowakowska, J Sol-Gel Sci Techn Vol 59 (2011), p 276
[2] J Lewandowska-Łańcucka, M Staszewska, M Szuwarzyński, M Kępczyński, M Romek, W Tokarz, A Szpak, G Kania, M Nowakowska, J Alloy Compd Vol 586 (2014), p 45

This work was supported by the European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, No POIG.01.01.02-00-069/09).

Fig. 1: SEM and TEM micrographs of hybrid materials: a, b silica-porphyrin, c, d polysiloxanes-porphyrin, e, f SPION-cchit-SiO2.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-2653 TEM investigation of Multilayer-Heusler Systems for ferroic cooling applications

Hetaba W.1, 2, Teichert N.2, Helmich L.2, Hütten A.2, Schattschneider P.1, 3
1University Service Centre for Transmission Electron Microscopy, Vienna University of Technology, Austria, 2Thin Films and Physics of Nanostructures, Department of Physics, Bielefeld University, Germany, 3Institute of Solid State Physics, Vienna University of Technology, Austria
walid.hetaba@tuwien.ac.at

Materials showing a magneto-caloric effect (MCE) are promising systems for diffusion-less cooling applications. In Heusler-alloys, the effect is related to a structural martensitic phase transformation [1]. Current research projects investigate this transition in multilayered systems incorporating different Heusler-alloys. In this work, we examine a system of alternating Ni2MnGa (direct MCE) and Ni2MnSn (indirect MCE) layers epitaxially grown on a MgO substrate. Transmission electron microscopy (TEM), scanning TEM as well as high resolution (HR) TEM investigations were used to study the structure of the samples. The elemental composition was determined with energy-dispersive X-ray (EDX) line scans while the magnetic properties of the individual Heusler-layers were investigated with energy-loss magnetic chiral dichroism (EMCD) [2].

The investigations were performed using a FEI TECNAI G2 F20 microscope operated at 200 kV. Figure 1 shows an image of the examined multilayer Heusler-alloy where the three layers (Ni2MnSn-Ni2MnGa-Ni2MnSn), each with a thickness of about 30 nm, can be seen. In Figure 2, a HRTEM micrograph of the Ni2MnSn layer is depicted.

Figure 3 shows the results of an EDX line-scan across the three Heusler-layers. It is obvious that Sn and Ga are diffusing between the layers during the growth process. A diffusion zone of about 10 nm can be identified. Furthermore, it can be seen that the stoichiometry of the two Ni2MnSn layers is slightly different.

Figure 4 shows an example of EMCD spectra clearly demonstrating that magnetic moments are present on the Mn sites. Multiple EMCD spectra at different positions were acquired to study the effect of the compositional changes on the magnetic properties of the sample. As EMCD is highly sensitive to e.g. sample thickness and changes in the crystallinity, simulations were performed in order to elucidate the underlying physics. The measurements are in good agreement with the model used for the calculations.

This analysis of the structural and magnetic properties of different Heusler alloys is of paramount importance to understand and develop functional materials which can be used as ferroic-cooling devices.

[1] A.Planes et al., J. Phys.: Condens. Matter 21, 233201 (2009)
[2] Schattschneider et al., Nature 441, 486 (2006)

WH thanks Stefan Löffler and Michael Stöger-Pollach for fruitful discussions and the Austrian Science Fund (FWF) for financial support, grant number I543-N20.

Fig. 1: TEM micrograph of a multilayer Heusler-alloy. From top to bottom: MgO substrate, Ni2MnSn-layer (red), Ni2MnGa-layer (green) and Ni2MnSn-layer (red). The three layers are about 30 nm thick.

Fig. 2: HRTEM image of the MgO-substrate (top), Ni2MnSn-layer (center) and Ni2MnGa-layer (bottom).

Fig. 3: EDX-line scan across the three Heusler-layers. The diffusion zones of about 10 nm are marked in grey.

Fig. 4: Electron energy-loss spectra of the Mn-L2,3 edge exhibiting an EMCD effect. This measurement was acquired in the centre of the Ni2MnSn-layer.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-2770 Imaging the dispersion of 2D materials in nanocomposites

Balades N.1, Sales D. L.1, Raya A. M.1, Galindo P. L.2, Molina S. I.1
1Dpto. de Ciencia de los Materiales e I. M. y Q .I., Facultad de Ciencias, Univ. de Cádiz, Campus Río San Pedro, 11510 Puerto Real, Cádiz, Spain., 2Dpto. de Ingeniería Informática, CASEM, Universidad de Cádiz, Campus Río San Pedro. 11510 Puerto Real, Cádiz, Spain.
nuria.baladesruiz@alum.uca.es

2D materials such as MoS2 and graphene are ideal candidates for developing novel nanocomposites with improved properties for the transport industry. A challenge to overcome for polymer matrix composites manufacture is to obtain high homogeneity filler dispersion within the matrix. Imaging simulation techniques can help us to explore the limits of characterizing bidimensional structures with only a few atoms at atom-scale and facilitate a correct interpretation of molecular studies performed by novel electron microscopes. However, when the composition of the filler and polymer matrices are similar, in terms of average atomic number, as it is the case of graphene, the structural characterization of these composites using Scanning Transmission Electron Microscopy (STEM) imaging techniques is hard. We show in this communication, based on STEM image simulations, that this technique can be helpful to know the dispersion of fillers in amorphous matrices.

In relation to filled-graphene nanocomposites, our study based on simulation techniques and structure-modeling images shows the possibility of functionalizing graphene layers with gold atoms to enable their location in a carbon matrix by Z-contrast STEM. Firstly several atomic models representing graphene composites marked with gold atoms and surrounded by amorphous carbon were built (fig.1A-B). Secondly, high-angle annular dark-field (HAADF) STEM images of these specimens were simulated by applying the multislice algorithm using SICSTEM [Pizarro et Al. Appl. Phys. Lett. 93, 153107 (2008)] and analyzed. All simulations were performed using CAI supercomputer [http//supercomputacion.uca.es].

Results ensure that unmarked graphene simulated images present a homogeneous contrast, making impossible to distinguish where the graphene sheets are placed in an amorphous material representing the matrix of a polymer-based nanocomposite. However, when the graphene sheet orientation coincides with the beam orientation of the microscope, the position of unmarked graphene can be clearly detected inside the amorphous matrix because of the channeling effect. Furthermore, the results demonstrate that marking graphene sheet with individual gold atoms allows identifying and locating graphene in reinforced amorphous areas, regardless of their spatial orientation (fig.1C). Finally, it has been observed that the focal series simulations enable us to know the optimum focus.

Regarding MoS2, due to the high atomic number of molybdenum, it is not necessary to mark the layers with heavier atoms to localize them within the matrix. Nevertheless, it is worth to explore the limits of detectability of HAADF-STEM, so we are currently simulating different configurations and orientations of these layers within an amorphous carbon matrix.

This work has been supported by the Spanish MINECO (Projects NANOTICS, TEC2011-29120-C05-03 and IMAGINE CONSOLIDER), and J.A. (groups TEP-946 and TIC-145).

Fig. 1: A)Isolated graphene sheet viewed from [100] direction marked with gold nanoparticles. B)Complete model dimensions: 8x4x8nm of amorphous carbon (blue) encircling a parallel graphene sheet (green), which is marked with tree gold nanoparticles (red). C) HAADF image. D) Intensity values at optimum focus represented on a surface with model dimension.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-2805 Analytical(S)TEM Characterization of the Morphology and Composition of Organic HybridQuantum Dot LEDs

Kübel C.1, Maier-Flaig F.2,3, Höfle S.2, Rinck J.4, Scherer T.1, Colsmann A.2, Powell A.1,4, Ozin G.5, Lemmer U.2
1Institute of Nanotechnology and Karlsruhe NanoMicro Facility, KIT, Karlsruhe, Germany, 2Light Technology Institute and Center for Functional Nanostructures, KIT, Karlsruhe, Germany, 3Performance Materials Division, Merck KGaA, Darmstadt / Germany, 4Institute of Inorganic Chemistry, KIT, Karlsruhe, Germany, 5Department of Chemistry, University of Toronto, Toronto, Canada
christian.kuebel@kit.edu

Organic light emitting diodes (OLEDs) and hybrid quantum dot organic LEDs (QD-OLED) have received much scientific interest as fully solution-processable, tunable solutions for lighting applications with high efficiency. However, accurate structural characterization of the devices and correlation with their degradation behavior is often limited due to the beam sensitivity of the organic layers. Here, however, we are able to describe results with such systems on a complete morphological and compositional study of stacked OLEDs and Si Quantum Dot LEDs (SiLED) [1]. These high quality contributions to nanoscience were achieved using an image corrected FEI Titan 80-300 operated at 300kV. Initial BF-TEM and HAADF-STEM imaging was performed under strict low-dose conditions (dose <100 e/nm²), but as the morphology of the organic multilayers remains stable even at significantly higher doses after pre-illumination at low current. A more detailed compositional analysis using EFTEM and STEM-EDX mapping was performed to image the composition of the different 2.5-35 nm thick organic and hybrid layers. The resulting elemental distribution is in very good agreement with the nominal composition of the different layers, e.g. shown in Fig. 1 and 2 for the C, N, O, S, Si and F distribution in a SiLED device.

A comparison of the as-fabricated and electrically driven SiLEDs as well as SiLEDs prepared using monodisperse and polydisperse SiQDs [2] was carried out to correlate the morphological and compositional features with the degradation behavior and was combined with electroluminescence and photoluminescence life-time studies. This analysis showed that the morphology and composition of the SiLED is preserved during normal operation of the devices even though the electroluminescence is reduced to 20% during this operation, which is attributed to atomic scale processes within the SiQDs themselves. In contrast, at high voltage/current, significant electromigration of SiQDs into the hole blocking layer TPBi is observed, whereas no change for the other organic layers can be detected. For non-size separated SiQDs, device life-times are significantly reduced compared to SiLEDs built from monodisperse SiQDs. This seems to be related to both percolating path of larger nanoparticles inside the SiQD layer as well as diffusion/electromigration of extremely small nanoparticles into the hole-blocking layer. We expect that these results are not only valid for SiLEDs but also transferable to other QD-based LEDs.

References:

[1] F. Maier-Flaig, et al., Nano Letters, 2013, 13, 475-480.

[2] F. Maier-Flaig, et al., Nano Letters, 2013, 13(8), 3539–3545.

This work was in part support by the Karlsruhe Nano Micro Facility (www.kit.edu/knmf).

Fig. 1: Schematic representation of theSiLED stack and HAADF-STEM cross-section image with corresponding EDX maps(scale bars 20 nm).

Fig. 2: Zero-loss filtered BF-TEM imageof a SiLED cross-section together with low-loss EFTEM images and elemental mapsfor silicon, carbon, oxygen and nitrogen (scale bars 50 nm).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-3160 STEM and EDS studies of Li4Ti5O12 modified with Ag nanoparticles

Andrzejczuk M.1, 5, Roguska A.2, Michalska M.3, Czerwiński A.4, Hebert C.5, Lewandowska M.1
1Warsaw University of Technology, Faculty of Materials Science and Engineering, Warsaw, Poland, 2Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland, 3Institute of Electronic Materials Technology, Warsaw, Poland, 4University of Warsaw, Faculty of Chemistry, Warsaw, Poland, 5Ecole Polytechnique Federale de Lausanne, CIME, Lausanne, Switzerland
mandrzejczuk@inmat.pw.edu.pl

Scanning transmission electron microscopy (STEM) and X-ray energy dispersive spectroscopy (EDS) was applied to characterize a Li4Ti5O12 (LTO) powder modified by Ag nanoparticles. Lithium-titanium oxide is one of the most promising materials to replace graphitic anodes in lithium-ion batteries but its surface modification is required to improve the rate capability. However, a comprehensive microstructure characterization using advanced electron microscope is necessary for further development of these materials. Nanocomposites with different Ag content were fabricated in chemical process from suspensions, with Ag to LTO weight ratio of 0.01, 0.04 and 0.10. The presence of pure silver on ceramic surface powder was confirmed by XRD and XPS analysis. The microstructure and chemical composition of the nanocomposites were analyzed using EDS maps performed on a FEI Tecnai Osiris microscope. This microscope is optimized for high speed and high sensitivity EDS measurements. Calculation of silver content on ceramic and statistical analysis of the nanoparticles size distribution was performed based on EDS maps after filtration and segmentation of signal. The results have shown relatively homogenous distribution of silver nanoparticles on LTO powder surface. Measured mean diameter of Ag particles on the LTO powder was about 4 nm and only slightly increased with higher metal concentrations. Higher Ag concentration, very often resulted in the creation of individual, few tens of nanometers in diameter, silver particles, located between LTO particles. The crystallinity of the Ag nanoparticles deposited (also the smallest ones) was confirmed by high resolution STEM observations.

This work was supported by The National Centre for Research and Development through the research grant PBS1 (contract no. PBS1/A1/4/2012). The work of Monika Michalska was supported by The National Science Centre through the research grant DEC-2011/03/N/ST5/04389.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-3162 Spatial-Resolved EELS Analyses of Antibody Distribution on Bio-functionalized Magnetic Nanoparticles

Arenal R.1, 2, De Matteis L.1, Custardoy L.1, Mayoral A.1, Grazu V.1, de la Fuente J. M.1, 2, Marquina C.3, 4, Ibarra R.1, 3, 4
1Instituto de Nanociencia de Aragon (INA), U. Zaragoza, Spain, 2Fundacion ARAID, Zaragoza, Spain, 3Dpto Física Materia Condensada, U. Zaragoza, Spain, 4Inst. Ciencia Materiales Aragon, U. Zaragoza-CSIC, Spain
arenal@unizar.es

The bio-functionalization of nanoparticles has a huge interest due to their use in biotechnology and bio-nanomedicine [1, 2]. However, in order to achieve the functionalization in a more efficient way, a deep knowledge of the bio-functionalizing moieties and their spatial distribution on the nanoparticle surface is required. Thus, we have showed that cryo-spatial-resolved EELS (SR-EELS) is a very appropriate and powerful technique for providing very rich information at the sub-nanometer scale on complex hybrid nanomaterials [3, 4]. The nanostructures on which we focus in these works are magnetic nanoparticles (NP) functionalized with a Protein-G/antibody (PG-Ab) system.

SR-EEL spectra were recorded using a VG-HB501 dedicated STEM, operated at 100 kV. Furthermore, in order to avoid electron beam damage, these measurements have been performed using a liquid-nitrogen-cooled cryo-stage [3], and relatively low electron doses. EELS-STEM studies have been also carried out using a FEI Titan Low-Base microscope, working at 80 kV, which is equipped with a Cs probe corrector. Fig. 1 displays an EEL spectrum-image (SPIM) recorded on PG/Ab-functionalized nanoparticles, where Fig. 1 (a) and (b) correspond to the bright-field and HAADF images of the nanoparticles, respectively. Carbon and nitrogen maps, extracted, after background subtraction, from the EEL spectra displayed in Fig. 2 (b), are shown in Fig. 1 (c) and (d), respectively. From these maps, we can observe that there is a clear correspondence between the spatial distributions of these elements which are localized at the surface of the NP. This finding indicates that they correspond to the organic constituents (PG/Ab) bio-functionalizing the magnetic nanoparticles, see Fig. 2 (a). In this contribution we will also show the analysis of the ELNES which provides rich information about those materials.

In summary, we have showed that the bio-functional moieties are only anchored in specific areas of the surface of the NP. This result showing that the biological entities are discontinuously distributed over the NP shell is very relevant because validates our selective functionalization protocol [4]. This will have a significant impact on biotechnological applications, as for instance biosensors, where an adequate NP functionalization approach for antibody immobilization is critical to improve the test sensitivity.

[1] L. R. Khot, S. Sankaran, J. M. Maja, R. Ehsani, E. W. Schuster, Crop Protec. 35 (2012).
[2] Ferrari, M. Nat. Rev. Cancer 5, 161 (2005).
[3] M. van Schooneveld, Gloter A., Stephan O., et al., Nature Nanotech. 5, 538 (2010).
[4] R. Arenal, L. De Matteis, L. Custardoy, A. Mayoral, V. Grazu, J.M. de la Fuente, C. Marquina, M.R. Ibarra, ACS Nano 7, 4006 (2013).

We acknowledge the LPS-STEM group for their support. The research leading to these results has received funding from the EU 7th Framework Program under Grant Agreement 312483-ESTEEM2.

Fig. 1: Fig. 1. (a) BF image of an agglomerate of bio-functionalized core-shell-shell NP. (b) HAADF image of this agglomerate where a 300x300 EELS-SPIM has been recorded at 150 K. (c) and (d) C and N chemical maps extracted, from the EELS-SPIM. For the sake of clarity these elemental maps have been colored with a temperature color scale.

Fig. 2: Fig. 2. (a) Model/scheme of the hybrid-nanostructures: anti-HRP Ab bound onto the NP shown in panel a, through the PG, which electrostatically interacts with the nanoparticle surface. (b) The individual EELS spectra, after the background removal, corresponding to the sum of the spectra collected in the positions marked in Fig. 1 (c).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-5747 Porous glass - epoxy resin systems investigated with AFM and SEM

Ostrowski A.1, Filimon M.1, Baller J.1, Sanctuary R.1
1Laboratory for the Physics of Advanced Materials, University of Luxembourg, Luxembourg
aleksander.ostrowski@uni.lu

Porous glasses are particularly interesting materials as they can represent inverse nanocomposites, where the interconnected pores with dimensions of nanometer scale are filled with a reactive polymer. Furthermore, confined reactive polymers are able to react within the pores. This is of particular interest in the polymer research as the reaction kinetics may be strongly driven by the confined environment.
In our studies, a reactive mixture of bisphenol A diglycidyl ether (DGEBA) and diethylenetriamine (DETA) was introduced to porous glass system. In the scope of interest was the influence of pore size, temperature and DGEBA/DETA ratio on the formation of the interphase. Process of preparation of the interphases for the measurements was a particular challenge as the samples based on porous glass were extremely fragile. Successful establishment of the polishing procedure allowed to produce very smooth epoxy - porous glass cross - sections, which were investigated by means of atomic force microscopy (AFM) (Figure 1 and 2) and scanning electron microscopy (SEM) (Figure 3). The studies revealed that depending on the conditions, at which the epoxy - porous glass interphase was formed, the thickness of the interphase and the degree of filling of the pores varied. Moreover, penetration depth of epoxy into porous glass could be influenced by various contributing factors: kinetics of the curing and viscosity. It was found that one of this factor can have a dominant role in porous glass penetration depending on the specific DGEBA/DETA ratio.

The present project is supported by the National Research Fund, Luxembourg

Fig. 1: AFM height map of epoxy resin - porous glass system. Left side shows porous glass filled with epoxy (interphase), while right side presents the unfilled porous glass.

Fig. 2: AFM phase map of epoxy resin - porous glass system. Left side shows porous glass filled with epoxy (interphase), while right side presents the unfilled porous glass.

Fig. 3: SEM image of epoxy resin - porous glass system. Upper region presents unfilled porous glass, and lower part: porous glass - epoxy resin interphase.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-5994 QUANTIFICATION OF THE DEGREE OF DISPERSION OF TUBULAR NANOFILLERS WITHIN A POLYMER MATRIX

Dehonor M.1, Flores-Santos L.2, González-Montiel A.3, Terrones M.3
1Centro de Tecnología Avanzada CIATEQ A.C., Lerma, Mexico., 2Rheomod de México, S.A. de C.V., Mexico, Mexico., 3Penn State University, Pennsylvania, USA.
mariamne.dehonor@ciateq.mx

Properties enhancement in polymer composite materials, as controlled release, thermal, electric and magnetic behavior, using natural nanofillers with tubular shapes strongly depends on the nanotubes individual separation as well as their homogeneous distribution in the polymer matrix. The nanofillers degree of dispersion in a polymer matrix could be related to the adhesion and interfacial strength between them. A typical strategy in polymeric materials to overcome with the chemical compatibility between filler and the matrix could be the use of compatibilizer additives. The additives have to be evaluated in combination with melt processing variables in order to optimize the degree of dispersion. In this study, the morphology properties of the obtained materials were analyzed in order to generate a quantification method of the degree of the dispersion. The modelling case materials in this case were halloysite as filler, and polypropylene, as polymer matrix.

Quantification methods for the degree of dispersion of nanoparticles in polymer materials represent a challenge when the morphology properties have to be correlated with the physical properties of the materials. Until now there is no simple and exhaustive method to cover all kind of applications even more if the applications are related to industry. In this study an effort to overcome those difficulties is been proposed using image analysis and the definition and quantification of morphology parameters for polymer composites prepared by melt extrusion in two stages: masterbatch and nanocomposite using tubular nanofillers. The analysis is been done considering industry resources related with low time and equipment characterization of materials.

The methodology involved the realization of a Plackett-Burman experimental design. The imaging uptake considered several magnification and combination of detectors using Optical Microscopy, and Electron Microscopy (SEM&TEM). The main results considered the morphologies of the raw materials, regarding tubular nanofillers, as well as the masterbatch and the nanocomposites prepared by melt compounding. The quantification of the morphological properties was realized using statistical methods. The measured values include agglomerates quantification by size and number, dispersed area %, agglomerated area % and so on. The information was used to calculate parameters as deagglomeration and eccentricity factors. In addition, evidences for the adhesion and interfacial strength were obtained for each material.

In summary an efficient methodology for the quantification of the degree of dispersion of composites prepared in melt was developed. The main advantages for the industry are the low time and physical resources.

The authors would like to thank to CID Centro de Investigación y Desarrollo Tecnológico for the facilities to use the polymer processing equipment as well as the microscopy characterization techniques.

Fig. 1: Fig. 1. SEM original micrograph and treated image for the composite masterbatch of Run 6 of tubular nanofiller in polymer matrix. The images were obtained using a FEI-Phillips SEM XL-30 at 20 kV and 650x using BSE detector. The dark zone is associated with the polymer and the bright zone is associated with the agglomerates of tubular nanofillers.

Fig. 2: Fig. 2. SEM original micrograph and treated image for the polymer composite of Run 9 of tubular nanofiller in polymer matrix. The images were obtained using a FEI-Phillips SEM XL-30 at 20 kV and 650x using BSE detector. The dark zone is associated with the polymer and the bright zone is associated with the agglomerates of tubular nanofillers.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-7-P-6065 Effect of TiO2 nanoparticles morphology on the photodegradation of nanocomposite films with polyethylene matrix.

Jardim P. M.1, Merlo A. F.1, Pereira A. F.1
1Federal University of Rio de Janeiro COPPE/UFRJ
pjardim@metalmat.ufrj.br

Among the various semiconductors, TiO2 is considered to be an almost ideal photocatalyst since it is relatively inexpensive, chemically stable and its photogenerated holes are highly oxidizing. In this context, nanoestructured TiO2 has been playing an increasing role in photocatalytic applications where crystal structure, size (surface area) and shape (exposed surfaces) are important. In particular, anatase has been proven to show the best performance among all the TiO2 crystallographic phases.
Photodegradation of polymers has been gaining attention as a useful way to decompose solid polymers in open air and avoid environmental pollution. Encouraging results have been reported in the literature for the photodegradation of nanocomposite plastic films containing TiO2 and polymeric matrices. However, there is no report on the evaluation of the effect of TiO2 nanoparticles morphology on the photodegradation of nanocomposite plastic films containing TiO2.
In the present work TiO2 nanocrystals were synthesized starting from hydrogen trititanate nanotubes (H-TTNT), obtained by the alkaline hydrothermal method. The H-TTNT material was submitted to thermal treatments at 550oC, 650oC and 750oC for 2 h and analyzed by means of X-Ray Diffraction and Transmission Electron Microscopy. Four nanocomposite films were produced with polyethylene matrix and 5% of TiO2 based nanomaterial: H-TTNT treated at 550oC, 650oC, 750oC and commercial TiO2 P-25. It was also produced pure polyethylene films for comparison. The photodegradation of these films was evaluated by means of measuring the weight reduction under UV radiation. The films containing P-25 and H-TTNT treated at 550oC showed the highest degradation rate. H-TTNT treated at 550 and 650oC contain only anatase while H-TTNT treated at 750oC exhibited ~8% of rutile, determined by Rietveld refinement of XRD results. H-TTNT treated at 550oC showed mainly nanorods (Fig.1) with diameters below 10nm while H-TTNT treated at 750oC contains particles in the range of 20 to 100nm and many particles with the anatase equilibrium crystal shape (Fig. 2). The higher performance for polymer degradation observed for nanocomposites containing H-TTNT treated at 550oC was tentatively attributed to the nanorods high energy surface facets.

The authors thank the Brazilian agencies CNPq and CAPES for its financial support.

Fig. 1: H-TTNT heat treated at 550oC.

Fig. 2: H-TTNT heat treated at 750oC.
↑ Go to the Table of content ↑

MS-8. Semiconductors and materials for information technologies

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-8-IN-1611 In-Situ Observation of Filamentary Conducting Paths in ReRAM Materials

PARK G. S.1, PARK S. Y.1, Li X. S.1
1Samsung Advanced Institute of Technology, Samsung Electronics Co., Ltd., Suwon-Si, 443-803, Republic of Korea
gs8144.park@samsung.com

Electrically induced resistive switching in metal-insulator-metal structures is a subject of increasing scientific interest, because it is one of the alternatives that satisfies current requirements for universal non-volatile memories. However, the origin of the switching mechanism is still controversial. There have been numerous attempts to identify the origin of resistance changes in various resistive random access memory (ReRAM) materials and thereby understand the switching mechanism associated with the behaviors of oxygen vacancies [1] and the metal ions [2]. Here, we introduce the observation and identification of conducting paths in the solid electrolyte-based and oxide-based resistive switching devices under different switching conditions using a unique in-situ probing technique inside TEM in conjunction with the high spatial resolution of EELS and EDS.

To understand switching behaviors in a solid electrolyte memory composed of Cu-doped GeTe sandwiched between a Cu BE and a TE, we performed in-situ TEM observations at various voltages and measured the corresponding I–V characteristics. Figure 1a shows an in-situ I–V scan. Starting from the high resistance state, we applied a negative voltage up to –0.8 V, and then applied a positive voltage of + 0.4 V. Cross-sectional Z-contrast STEM images were obtained after each voltage application (Figs. 1b–e). After applying –0.8 V, the multiple filaments become strengthened (Fig. 1d). Subsequent application of a positive voltage annihilates the filaments (Fig. 1e) [3]. We also constructed the oxide-based ReRAM device using a cross-sectional sample of the Pt/SiO2/Ta2O5–x /TaO2–x/Pt structure to enable real-time observation of the voltage-induced structural changes in the conduction paths (Figs. 2a, b). The switching behavior of the ReRAM device inside the TEM was confirmed by in-situ measurements of the I–V characteristics at various voltages. This device sample exhibited reversible bipolar resistance switching behavior between the LRS and HRS by DC I–V sweeps (Fig. 2c). Comparison of Z-contrast STEM images taken at the same location under LRS (Fig. 2d) and HRS (Fig. 2f) unambiguously exhibited the nanoscale filament formation in the SiO2 layer; it was ~ 1.5-2 nm in width and 0.6-1.5 nm in length (Fig. 2e) [4].

These in-situ studies provide crucial information for understanding the dynamics of filament formation and annihilation processes. We expect that the in-situ experimental technique can further expand its applications in a variety of nonvolatile memory system.

References
[1] R. Waser et al. Adv. Mater. 21, (2009) 2632.
[2] Y. Yang et al. Nat. Commun. 732, (2012).
[3] S. J. Choi et al. Adv. Mater. 23, (2011) 3272.
[4] G. S. Park et al. Nat. Commun. 2382, (2013).

 

Fig. 1: In-situ observations of voltage-induced changes in microstructure. a, In-situ I-V scan. b-e, Cross-sectional Z-contrast STEM images obtained after voltage applications of 0, -0.4, -0.8 and +0.4 V, respectively.

Fig. 2: In-situ observation of the conducting paths. a, Schematic of the in-situ experimental setup. b, Cross-sectional STEM image when the Pt-Ir tip approached the top Pt electrode. c, A series of I–V measurements. d-f, In-situ STEM images in LRS (d) and HRS (f). Magnified images of nanoscale filaments (e) obtained from the red boxed areas (A and B) in d.
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-8-IN-1975 Correlation of Optoelectronic and Transport Properties of GaN/AlN Nanowires with Polarity and Crystal Structure

den Hertog M. I.1, Gonzalez-Posada F.2, Songmuang R.1, Rouviere J. L.2, Gayral B.2, Monroy E.2
1Institut Neél CNRS/UJF UPR2940, Grenoble, France, 2CEA-Grenoble, INAC / SP2M, Grenoble, France
martien.den-hertog@grenoble.cnrs.fr

Nitride based semiconductor nanowires (NWs) are promising candidates for many device applications ranging from electronics and optoelectronics to energy conversion and spintronics. The optoelectronic properties of nitride NWs are heavily affected by the strong spontaneous and piezoelectric polarization fields induced by the polarity and strain, combined with the effect of surface states. A correlated study gives insight into the precise effect of polarity, heterostructure and surface states on optoelectronic and transport properties of a single NW.

In this work, we correlate the crystal structure and heterostructure measured by scanning transmission electron microscopy (STEM) and the luminescence and photodetector performance of defect-free GaN-AlN NW heterostructures on the level of a single NW, paying particular attention to the impact of the measuring environment on the electronic transport and photocurrent dynamics. The effects of GaN/AlN heterostructure engineering and surface states are discussed.

GaN NWs are grown by plasma-assisted MBE on Si(111) [1]. They have a length of 1.2–1.5 μm and a diameter of 30−80 nm. Individual NWs were dispersed on electron transparent Si3N4 membranes and contacted using e-beam lithography.

Using aberration-corrected annular bright field (ABF) and high angle annular dark field (HAADF) STEM, we identify the NW growth axis to be the N-polar [000−1] direction (Fig. 1). The electrical transport characteristics of the NWs are explained by the polarization-induced asymmetric potential profile and by the presence of an AlN/GaN shell around the GaN base of the wire. The AlN insertion blocks the electron flow through the GaN core, confining the current to the radial GaN outer shell, close to the NW sidewalls, which increases the sensitivity of the photocurrent to the environment and in particular to the presence of oxygen. The desorption of oxygen adatoms in vacuum leads to a reduction of the nonradiative surface trap density, increasing both dark current and photocurrent [2].

References

[1] R. Songmuang, T. Ben, B. Daudin, D. Gonzalez, and E. Monroy, Nanotechnol. 21, 295605 (2010).

[2] M. I. den Hertog, F. González-Posada, R. Songmuang, J.-L. Rouviere, T. Fournier, B. Fernandez, and E. Monroy, Nano Lett. 12, 5691 (2012).

Financial support from the French FMN-SMINGUE 2011, the French CNRS and CEA METSA network, the ANR-2011-NANO-027 “UVLamp” project, the EU ERC-StG “TERAGAN” (#278428) project, and the ANR-2013-JCJC "COSMOS" is acknowledged.

Fig. 1: Left to right, top: schematic of the NW structure. HAADF STEM image of a contacted GaN NW with an AlN insertion. Zoom of the boxed region along [11-20]. Bottom: Atomic structure of GaN along [11-20] overlaid on an ABF STEM image. I−V characteristic from the same NW device measured in the air and in vacuum, in the dark and under UV illumination.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-1408 In situ electron holography study of charge distribution in high-κ charge trap memory

Yao Y.1, Li C.2, Huo Z.3, Liu M.4, Gu C.5, Duan X.6, Wang Y.7, Gu L.8, Yu R.9
1Beijing National Laboratory of Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China, 2Laboratory of Nano-Fabrication and Novel Devices Integrated Technology, Institute of Microelectronics, Chinese Academy of Sciences, Beijing 100029, People’s Republic of China
yaoyuan@iphy.ac.cn

Charge trapping memory with high-κ dielectric substituting floating gate as the charge capture layer is a candidate for the next generation storage devices since it radically improves the retention force, weakens the coupling effect and the charge leakage. The elusive spatial charge distribution in the CTM plays an important role influencing the program/erase speed and the retention force. Therefore, it is critical to map the charge distribution in the dielectric layers to clear the trapping mechanism of CTM and improve the design and fabrication process to ensure the performance of the future memory devices. I-V and C-V measurements are the usual characterization tools to deduce the charge distribution along vertical direction of the charges with the appropriate models. But these methods concerning models only depict the effective charge distribution with a poor spatial resolution and moreover, these indirect electrical investigations can be disturbed so easily by the measurement environment or parameters that controversial results were reported in the literatures.
Electron holography is a powerful means to image the electrostatic potential distribution because the charges in the sample can alter the phase of the penetrated electron wave and such phase disturbance can be retrieved from the electron interference patterns. In situ electron holography in TEM has been adapted here to map the vertical and lateral charge distribution within the CTM simultaneously. Benefited by the high resolution electron holography, the charge trapping process traced under different gate biases indicates unambiguously that the electrons penetrate through the HfO2 layer and aggregate beneath the interface between HfO2 and Al2O3 films.

It was supported by State Key Development Program for Basic Research of China (Grant Nos. 2010CB934202, 2012CB932302 and 2013CB932904), NNSFC (Grant No. 10974235 and 11274365).

Fig. 1: (a) The low magnification image of the CTM sample. (b) The normalized I-V curves measured on the wafer and in TEM. (c) The high magnification image of the polycrystalline HfO2 layer. (d) The phase image of the unbiased CTM sample. The dashed lines in (c) and (d) indicate the position of grain boundary. Scale bar: (a) 10 nm, (b) 5 nm, (c) 5 nm.

Fig. 2: Projected charge density maps under different bias. (a) 5 V, (b) 6 V, (c) 7 V, (d) 8 V, (d) 9 V. The dashed lines indicate the position of grain boundary, as shown in Fig. 1(c) and (d). Scale bar: 5 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-1683 Structure and magnetism in strained Ge1−x−ySnxMny films grown on Ge(001) by low temperature molecular beam epitaxy

Prestat E.1,2,3, Barski A.1, Bellet-Amalric E.1, Jacquot J. F.1, Morel R.1, Tainoff D.1, Jain A.1, Porret C.1, Bayle-Guillemaud P.1, Jamet M.1
1INAC, CEA and Université Joseph Fourier, 17 rue des Martyrs, 38054 Grenoble, France. , 2Karlsruher Institut für Technologie (KIT), Laboratorium für Elektronenmikroskopie, D-76128 Karlsruhe, Germany, 3School of Materials, University of Manchester, Manchester M13 9PL, UK
eric.prestat@manchester.ac.uk

For spintronics, ferromagnetic semiconductors are of great interest because they allow the injection of spin polarized current into a non-magnetic semiconductor. However, diluted magnetic semiconductors with a Curie temperature (Tc) below room-temperature, are not usable in current spintronic devices. One alternative route to fabricate room temperature ferromagnetic semiconductors is to use the spinodal decomposition to form high Tc nanostructures, as demonstrated in the GeMn system [1]. This GeMn system appears as a promising candidate to be used in spintronic devices, especially because of its perfect compatibility with silicon technologies.

In this presentation, we report on the structural and magnetic properties of GeSnMn films grown on Ge(001) by low temperature molecular beam epitaxy using high resolution X-ray diffraction (HRXRD), high resolution transmission electron microscopy (HRTEM), electron energy loss spectroscopy (EELS) and superconducting quantum interference device (SQUID). Chemical mechanical wedge polishing was used to obtain high quality samples with flat and large surfaces free of damages and almost free of amorphous layers. HRTEM, STEM-EELS observations were performed on a FEI Titan3 Ultimate 80-300 microscope operating at 200 kV and equipped with probe-side and image-side aberration-correctors.

Similar to Mn-doped Ge films, GeMn nanocolumns of a few nanometers in diameter are formed during the growth, as revealed by the HRTEM images in figure 1a-b and the EELS elemental map (figure 3a-c). Sn map (figure 2c) shows that the matrix exhibits a GeSn solid solution while there is a Sn-rich GeSn shell around GeMn nanocolumns. The interface Ge/GeSnMn is perfectly coherent as demonstrated by HRTEM (figure 1a) and HRXRD (figure2b), leading to a pseudomorphic growth of the GeSnMn layers. The out-of-plane lattice distance can be monitored by the Sn concentration, as shown by HRXRD in figure 2a.

Electron diffraction patterns exhibit the presence of the forbidden (200)- and (020)-Bragg reflections in the GeSnMn layer -figure 2f. These diffracted peaks have a peculiar cross-like feature oriented along the [110] and [110] directions. We show how the GeSn shells provide these reflections.

The magnetization in GeSnMn layers is higher than in GeMn films (figure 1e). This magnetic moment enhancement is independent of the Sn concentration and thus the strain state around the Mn-rich nanocolumns. However, the Sn-rich shell, which is formed around the nanocolumns, could change the electronic structure of Mn atoms in the nanocolumns, which could explain the magnetization enhancement [2].

[1] M Jamet et al, Nature Materials 5 (2006), p. 653
[2] E Prestat et al, Applied Physic Letters 103 (2023), 012403

Fig. 1: HRTEM images of a GeSnMn layers on Ge substrate:(a) in cross-section and (b) in plane view.

Fig. 2: (a) θ–2θ X-ray diffraction spectra: the position of the peaks between 65° and 66° of the θ-2θ X-ray diffraction spectra corresponding to the GeSnMn epitaxial depends on the Sn content (b) X-ray map around the (-1-15) Bragg peak. (c) Magnetization versus temperature of GeMn and GeSnMn layers.

Fig. 3: (a-c) Ge, Mn and Sn elemental map obtained by EELS. (d) Mn versus Sn composite map showing the Sn shell around the Mn-rich nanocolumns. (e) Electron diffraction pattern in the [001]-zone axis.(f) Magnified image of the forbidden (200)-Bragg reflection showing the peculiar cross-like feature of the diffracted peak.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-1702 Investigation of 3D Strain in FinFETs by Nano Beam Diffraction Parallel and Perpendicular to the Trenches

Favia P.1, Witters L.1, Mitard J.1, Collaert N.1, Bender H.1
1Imec, Leuven, Belgium
paola.favia@imec.be

The FinFET architectures exploit the third dimension to improve scalability and higher performance per chip area with respect to planar FET. In this work we investigate 3D strain in FinFETs by measuring the Ge/SiGe lattice mismatch across and along the fin direction for two different process steps in the device fabrication. The first step includes the growth of thin strained Ge layer on top of a SiGe strain-relaxed buffer (SRB) blanket wafer on which shallow trench isolation (STI) processing is applied. The second step consists of a replacement metal gate (RMG) dummy gate patterning, extension implantation, spacer formation and SiGe source and drain (S/D) epitaxial growth. Strain is measured by nano beam diffraction (NBD) on a TECNAI 300kV microscope. The TEM specimens are prepared by FIB across and along the fin as shown in Fig. 1a, b, and c. For the NBD line profiles the electron beam is scanned perpendicular to the Ge surface and the reference is acquired in the SRB SiGe buffer. In Fig. 2a the across and vertical SiGe/Ge mismatches are shown for the first fabrication step, i.e. up to STI processing. The values in both directions are very similar indicating either relaxation or uniaxial strain in the SiGe fin. In order to avoid this ambiguity, it is necessary to investigate the mismatch along the fin getting therefore access to the 3D strain information. The correspondent NBD results along the fin direction are shown in Fig. 2b. While the vertical mismatch values are, consistently with results in Fig. 2a, close to 0.02, the mismatch along the fin is close to zero indicating that the Ge is strained in this direction. We can conclude that the Ge fin is uniaxially strained along the fin. Similar results, shown in Fig. 3a,b are obtained for the second processing step up to the S/D epitaxial growth. In this case as well, the device appears to be strained along the fin indicating that strain is maintained during the subsequent S/D epitaxial processing. Similar strain results are obtained for fins under or outside the gate. With this study we affirm the importance, for 3D devices such as FinFETs, of investigating the mismatch along and across the structure in order to obtain 3D information on strain. The NBD technique gives information on strain in the plane perpendicular to the direction of the electrons therefore the only way to investigate the third dimension is either by preparing the TEM specimen in both direction, i.e. across and along the fin, or being able to rotate the specimen by 90 degrees along the fin while still being able to orient the specimen along zone axis for NBD data processing. This last possibility is a work in progress as some technical difficulties, such as specimen preparation and specimen alignment, need to be addressed.

Fig. 1: (a) SEM image of the device indicating the directions across and along the fin. HAADF-STEM across (b) and along (c) the fins.

Fig. 2: (left) SiGe/Ge mismatch across the fin and (right) along the fin for the first process step. Uniaxial strain is found along the fin.

Fig. 3: (left) SiGe/Ge mismatch across the fin and (right) along the fin for the second process step. Also in this case uniaxial strain is found along the fin indicating that strain is maintained during the subsequent S/D epitaxial processing.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-1869 Active dopant mapping from plasmon peaks by STEM EELS

Delaye V.1, Serra R.1, Cooper D.1
1CEA, LETI, MINATEC Campus, 17 rue des Martyrs, 38054 GRENOBLE Cedex 9, France.
vincent.delaye@cea.fr

Dopant engineering for the sub-28 nm nodes of CMOS technology is currently receiving a great deal of attention in the semiconductor industry. Indeed, the fluctuation of the position and the concentration of dopants is the main source of variability in the performance and may limit the ultimate dimensions of devices that can be achieved. Advanced characterization is thus required to study and optimize the doping process. Electron holography is the first characterization technique developed for mapping active dopants at the nanometer scale. This technique combined with a specimen preparation using FIB allows the positions of the junctions in a transistor to be measured in order to determine the effective channel length [1]. The total concentration of arsenic or phosphorus as dopants in silicon is also possible in a TEM using EELS or EDS [2]. Low loss EELS experiments also allow the detection of substitutional boron atoms in doped amorphous silicon layers used for photovoltaic applications [3]: the energy shift of the silicon volume plasmon peak is accurately measured and this shift is related to valence electronic density and varies linearly with dopant concentration. In this study, this technique is implemented successfully for the first time on crystalline silicon. The specimen analyzed in this study was grown on (001) silicon using RPCVD with closely spaced highly doped with boron nanometer layers (Fig. 1a). SIMS profile exhibits a maximum concentration of 2.5×1021 at/cm3 while 1×1019 at/cm3 is measured in the substrate (Fig. 2a). A 100 nm thick lamella was then prepared using a FEI Strata FIB at 8 kV to minimize damage. High resolution HAADF STEM image and low energy-loss spectra data cube were acquired at 80kV using a Cs image and probe corrected FEI Titan Ultimate microscope with a GATAN Quantum ER energy filter. The energy resolution reached with the monochromator and a dispersion of 0.025 eV/ch was less than 0.2 eV. A 520x80 pixels map (0.13 nm/pixel) was acquired with a 100 pA probe and a 0.7 ms exposure time for a total acquisition time of 70 seconds. Zero loss and silicon volume plasmon peak energy positions were determined for each spectrum using the Digital Micrograph NLLS fitting routine and subtracted to give the effective plasmon peak energy related to active boron concentration. A 70 meV offset is measured between highly doped layer and the silicon substrate (16.85 eV). Fig. 2b shows the good linearity between EELS and boron concentration (SIMS measurements). Figs. 1b to 1d illustrate how to improve the sensitivity by averaging.
[1] D. Cooper et al. Ultramicroscopy, 110, 5 (2010)
[2] R. Pantel et al., Transmission Electron Microscopy in Micro-Nanoelectronics, Wiley (2013)
[3] M. Duchamp et al., J Appl Ph, 113, 093513 (2013)

This work has been financially supported by the Recherche Technologique de Base (RTB). The experiments were performed on the Nanocharacterisation Platform (PFNC) at MINATEC Campus.

Fig. 1: a) STEM dark field image of the analyzed area (520x80 pixels), boron doped layers appear bright b) 16.85 eV silicon plasmon peak energy offset c) after binning by two the results (260x40 pixels) d) after binning by four the results (130x20 pixels)

Fig. 2: a) Boron concentration measured by SIMS and plasmon energy offset cumulated (80) profile for the seven first layers b) Linear relation between SIMS (logarithmic scale) and EELS using measurements starting from the substrate to the highest boron concentration of the first layer
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-1883 Characterization of single photon emitter with cathodoluminescence signal

Meuret S.1, Tizei L. G.1, Auzelle T.4, Blazit J.1, Tencé M.1, Chang H.3, Daudin B.4, Treussart F.2, Zobelli A.1, Kociak M.1
1Laboratoire de physique des Solides, Orsay, France, 2Laboratoire Aimé Cotton, Orsay, France, 3Institute of Atomic and Molecular Sciences, Taipei, Taiwan, 4CEA-CNRS Group “Nanophysique et Semiconducteurs”, Grenoble, France
sophie.meuret@u-psud.fr

The interest for single photon emitters (SPE) has tremendously grown over the last decades, due to their possible application in quantum information. Famous SPE are, for example, quantum dots of InAs/GaAs or NV centers in diamond. A SPE emits only one photon at the time, and therefore it is a natural candidate for solid quantum bits. The usual way to characterize them is to perform an intensity interferometry experiment (Hanbury Brown and Twiss (HBT)). Such an experiment measures the autocorrelation function g(2)(τ) of emitters. The g(2)(τ) function of a SPE presents a dip at very short delay (g(2)(0) < 1), a phenomenon called anti-bunching. Here, we used a unique home-made set-up of cathodoluminescence (CL) in a scanning transmission electron microscope (STEM) coupled to an HBT experiment allowing nanometer resolution. The g(2)(τ) obtained with our STEM-CL set-up is called hereafter CL−g(2)(τ). The details of the experiment are explained in [1,2] and in figure 1. Two HBT set-up have been built, one working in the visible range with two single photon avalanche detectors and the other working in the near UV range using photomultiplers.

As a proof of principle, we will present results on NV centers acquired with the first set-up. The CL−g(2)(τ) acquired on a nano diamond is shown in figure 2-b) and presented in detail in [2]. We can clearly see a dip at short time delay, proving the possibility to study SPE with fast electrons (60 keV). Then we will present a new UV-SPE in hexagonal Boron nitride studied with the second set-up, showing that characterizing SPE with fast electron can open new horizon on quantum information device. In order to go further in the understanding of this new technics, we will see that even if the interaction mechanisms of photoluminescence (PL) and CL-STEM are close enough to give the same emission spectra [3], they may lead to huge differences in their g(2)(τ) function, called respectively PL−g(2)(τ) and the CL−g(2)(τ). Indeed the interaction of electrons with mater produces a plasmon which will decay into multiple electron-hole pairs at the gap energy (Eg e-h), while the PL-photon mater interaction produces only one Eg e-h. Thus, if there is more than one SPE in the sample, one electron can excite simultaneously multiple centers leading to the synchronization of emission and thus to the emission of packets of photons. Therefore if the number of excited center is above ten the CL- g(2)(τ) function will presented a huge bunching effect (g(2)(0) > 1) in stark contrast to the expected flat PL-g(2)(τ) function (g(2)(0) = 1) see figure 3.

[1] Zagonel and al., Nano Letters 11, 568-73 (2011)

[2] Tizei and al., PRL 110, 153,604 (2013)

[3] Mahfoud and al., J. Phys. Chem. Lett., 4090-94 (2013)

This work has received funding from the European Union Seventh Framework Programme under Grant Agreement ESTEEM2 and also from the French Ministry of Defense through a grant from the Direction Générale de l’Armement.

Fig. 1: Sketch of the set-up. a) the STEM. b) Transmitted electrons are used to produce ADF and BF images. The light coming out of the sample is sent to an optical fiber which can either go to a spectrometer c) or the autocorrelation experiment d). In c) one can see the emission spectrum of an NV center in diamond and the emission map, filtered at 570 nm.

Fig. 2: SPE studied in a STEM [2]. a) The ADF image of the diamond. b) Associated experimental g(2)(τ). The dip of the blue curve is lower that the dip of the red one, respectively acquired with an excitation on the area marked by the blue and red square in a). This shows that the g(2)(τ) is sensitive to excitation variation of at least 100 nm resolution.

Fig. 3: The bunching effect. Continuous lines are the experimental results from CL−g(2)(τ) of a nano-diamond (size ≈ 100 nm, containing a few hundreds of NV centers). The excitation current ranges from 1.6 pA to 137 pA. On the inset, PL-g(2)(τ) with excitation on the same sample (but not the same diamond) for two different excitation powers.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-1963 In-situ propagation of a metal-semiconductor phase in silicon and germanium nanowires observed by transmission electron microscopy

El hajraoui K.1, Den hertog M. I.1, Zeiner C.3, Mongillo M.2, Prager A.2, Lugstein A.3, Rouvière J. L.2
1Institut Néel-CNRS, Grenoble, France, 2CEA-Grenoble, Grenoble, France, 3Vienna University of Technology, Vienna, Austria
khalil.el-hajraoui@neel.cnrs.fr

Semiconductor nanowires (NWs) are promising candidates for many device applications ranging from electronics and optoelectronics to energy conversion and spintronics. However, typical NW devices are fabricated using electron beam lithography and therefore source, drain and channel length still depend on the spatial resolution of the lithography. In this work we show fabrication of NW devices in a transmission electron microscope (TEM) where we can obtain atomic resolution on the channel length using in-situ propagation of a metallic phase in the semiconducting NW. The corresponding channel length is independent on the lithography resolution. We show results on semiconducting NW devices fabricated on two different electron transparent Si3N4 membranes: a planar membrane and a membrane where devices are suspended over holes and we demonstrate a real-time observation of the metal diffusion in the semiconducting NW. First we describe the process of making lithographically defined reliable electrical contacts on individual NWs dispersed on a membrane. Second we present first results on in-situ propagation of a metal-semiconductor phase in Ge NWs by joule heating [1] while measuring the current through the device. Three different metals are used as contacts: platinum, copper and aluminum. Different phenomena can occur in PtSi, AlGe and CuGe [2] NWs during phase propagation. Furthermore we study the crystalline structure of the different phases and the diffusion mechanisms of the different metals.
[1] M. Mongillo, P. Spathis, G. Katsaros, P. Gentile, M. Sanquer and S. De Franceschi, ACS Nano, 5, 7117-7123 (2011).
[2] T. Buchhart, A. Lugstein, Y. J. Hyun, G. Hochleitner and E. Bertagnolli, Nano. Lett, 9, 3739-3742 (2009).

Financial support from the French ANR for the ̎ COSMOS ̎ project is acknowledged. We thank J-L. Thomassin, B. Fernandez and T. Fournier for their technical support.

Fig. 1: TEM image of suspended Ge NW device connected by Cu pads showing the procedure of the joule heating for the in-situ propagation.

Fig. 2: TEM image of Ge NW after the in-situ propagation showing the progression of the CuxGey phase.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-2115 Nanobeam precession electron diffraction for high precision strain analysis in ultra-scaled semiconductor devices

Bernier N.1, Rouvière J. L.2, Cooper D.1, Nguyen P.1,3, Barraud S.1, Vigouroux M.1, Audoit G.1, Lafond D.1, Delaye V.1
1CEA, LETI, Minatec campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France., 2CEA, INAC, Minatec campus, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France., 3SOITEC, Parc Technologiques des Fontaines, 38926 Bernin, France.
nicolas.bernier@cea.fr

The advances in strain engineering must be supported by improvements in local strain characterization techniques to address the simulation, design and fabrication challenges faced by the semiconductor industry. Though TEM is the method of choice for nanoscale measurement of strain, the existing techniques suffer from serious limitations such as the poor strain sensitivity of 10-3 for high-resolution imaging or the difficulty of using dark field electron holography to analyze silicon-on-insulator (SOI) devices due to the miscut between the region of interest and the substrate. Recently, Rouvière et al. [1] introduced the use of precession electron diffraction (PED) for strain measurement with 1 nm spatial resolution and 2 x 10-4 sensitivity. In this study we demonstrate that strain can be precisely measured by PED in aggressively scaled devices. For this purpose, PED is compared to geometrical phase analysis (GPA) on HAADF STEM images and Finite Element simulations on 11.5-nm-wide channel SOI device and sub-10 nm SiGe nanowire.
The HAADF images and PED patterns have been acquired on a FEI Titan Ultimate microscope equipped with two Cs-correctors and an X-FEG source operated at 200 kV. The PED patterns have been recorded on a 2k x 2k Gatan CCD camera using a precession speed of 0.1 s, a semi-convergence angle of 2.4 mrad, a precession angle of 12.7 mrad and a beam size of ~ 2 nm (measured on the sample with the precession activated). For both techniques: (i) the deformation is measured relative to the Si substrate, (ii) non-pertinent areas of strain mappings are removed based on either the amplitude image of the inverse filtered Fourier transform for GPA or the virtual HAADF image computed from diffraction patterns for PED.
As seen from Figs. 2(a-c), the strain (ε002 and ε220 along the growth and in-plane directions, respectively) and rotation (θ) maps measured by PED and GPA on the SOI device shown in Fig. 1(a) are in good agreement. They both give evidence of a fully relaxed state. More importantly, the strain profiles (Fig. 2(d)) measured along the channel prove that the noise is significantly reduced using PED, allowing the strain state in small areas, e.g. source-drain (S/D), to be precisely determined. The higher strain level measured in S/D is consistent with the Ge enrichment in S/D measured by EDX (Figs. 1(c-d)). Figs. 3(e-f) demonstrate that high precision and high spatial resolution strain distributions can be acquired by PED on a sub-10 nm SiGe nanowire. As seen from Figs. 3(b-c), GPA applied to the same device provides mappings composed of high strain field fluctuations which make the interpretation of the strain state in nanowires difficult.
[1] J.L. Rouvière et al., Appl. Phys. Lett. 103 (2013) 241913.

This work has been financially supported by the Recherche Technologique de Base (RTB). The experiments were performed on the Nanocharacterisation platform at MINATEC Campus.

Fig. 1: (a) HAADF STEM image of the SOI device, (b) magnified image indicated in (a) by the blue rectangle, (c) Ge and (d) Si EDX mappings (see the color legends for the concentration in at. %)

Fig. 2: (a-c) Comparison between the different strain and rotation maps measured by PED and GPA on the SOI device shown in Fig. 1 (the substrate is not shown for better visualization), (d) strain profiles acquired along the channel and S/D. Note the significant noise reduction using PED

Fig. 3: (a) HAADF STEM and (d) virtual HAADF computed from the PED patterns acquired on the SiGe nanowire; (b-c) GPA and (e-f) PED measured strain mappings
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-2521 Observation of charge distributions in semiconductor nanostructures using comprehensive TEM techniques

Li L.1
11Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan, 430074, China
luying.li@hust.edu.cn

Epitaxial germanium QDs embedded in boron-doped silicon have been studied using off-axis electron holography to estimate the number of holes associated with a single QD. Holes were confined near the base of the pyramidal, 25-nm-wide Ge QDs. About 30 holes were localized to the investigated dot. For comparison, the average number of holes confined to each Ge dot was found to be about 40, using a C-V measurement. [1] Hole accumulation in Ge/Si core/shell NWs has been observed and quantified using off-axis electron holography and other electron microscopy techniques. HAADF scanning transmission electron microscopy images and off-axis electron holograms were obtained from specific NWs. The excess phase shifts measured across the NWs indicated the presence of holes inside the Ge cores. [2]

Homogeneous ZB/WZ heterostructural junctions have been successfully synthesized in ZnSe nanobelts, and polarity continuity is determined through aberration-corrected HAADF imaging. The hypothesized saw-tooth-like potential profile is directly revealed at the nanoscale using off-axis electron holography. With the exclusion of the other possible contributions, spontaneous polarization is identified as the predominant factor causing the experimental profile. [3] Polytype heterocrystalline structures within InAs nanopillars are characterized by multiple TEM techniques. The electric field related to spontaneous polarization within the ZB region is revealed at nanometer scale using off-axis electron holography, and the measured value of spontaneous polarization for WZ-InAs is close to published results. Through probe-corrected HAADF imaging, strain-induced variations of local spontaneous polarization are determined with atomic resolution. Moreover, spontaneous polarization values along the interface normal are calculated and possible explanations are provided. The strain-induced variations of spontaneous polarization along the interface normal would provide valuable information for tailoring charge distribution in semiconductor nanostructures and for fabrication of future devices. [4]

References
[1] Li L., Ketharanathan S., Drucker J. and McCartney M. R.,  Appl. Phys. Lett., Vol. 94, No. 23, (2009), pp 232108.
[2] Li L., Smith D. J., Dailey E., Madras P., Drucker J. and McCartney M. R.,  Nano Lett., Vol. 11, No. 2, (2011), pp 493-497.
[3] Li L., Jin L., Wang J., Smith D. J., Yin W., Yan Y., Sang H., Choy W. and McCartney M. R.,  Adv. Mater., Vol. 24, No. 10, (2012), pp 1328-1332.
[4] Li L., Gan Z., McCartney M. R., Liang H., Yu H., Yin W., Yan Y., Gao Y., Wang J. and Smith D. J.,  Adv. Mater., Vol. 26, No. 7, (2014), pp 1052-1057.

This study was supported by MOE Doctoral Fund, the Fundamental Research Funds for the Central Universities, the National Science Foundation of China, SRF for ROCS, SEM. Luying Li thanks S. Ketharanathan, E. Dailey, P. Madras, J. Drucker, L. Jin, J. Wang, W. Yin, Y. Yan, H. Sang, W. C. H. Choy, Z. Gan, H. Liang, H. Yu, Y. Gao, D. J. Smith, and M. R. McCartney for their contributions to this study.

Fig. 1: Fig. 1(a) Electron hologram of individual Ge quantum dot with [110] projection embedded in Si [001] substrate. (b) Phase image of the Ge quantum dot. (c) Phase image of Ge/Si core/shell nanowire with the area used for HAADF intensity line profile labeled, and the result is shown by white squres in (d).

Fig. 2: Fig. 2(a) Phase image from region including WZ/ZB/WZ junctions, and the box region used for profiling is shown in color. (b) Phase shift profile of the box region labled in (a). (c) Probe-corrected HAADF image of the InAs nanopillar including multiple stacking disorder, regions of WZ structure are highlighted by yellow background.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-2549 High-Resolution Transmission Electron Microscopy Imaging and Raman Spectroscopy of Two-Dimensional Transition-Metal Dichalcogenides

Reifler E. S.1, Nuhfer N. T.1, Towe E.1
1Carnegie Mellon University, Pittsburgh, PA, USA
ereifler@andrew.cmu.edu

We present a study of some structural and optical characteristics of two-dimensional transition-metal dichalcogenides. The study includes aberration-corrected high-resolution transmission electron microscopy (HRTEM) imaging and Raman spectroscopy measurements of molybdenum disulfide (MoS2), molybdenum diselenide (MoSe2), and tungsten diselenide (WSe2) films. The measurements examine the in-plane crystal structure and vibrational modes of these materials. Our results contribute several intrinsic characteristics for these materials, and thus enable further investigations of their potential for applications.

Samples for the HRTEM experiments were prepared directly from natural, bulk MoS2 and from synthetic bulk MoSe2 and WSe2 single crystals to minimize introducing defects that might arise from the usual transfer of the samples from a separate substrate onto a TEM grid. The imaging work is carried out in an image-aberration-corrected FEI Titan G2 80-300 transmission electron microscope that is capable of atomic resolution. Micrographs of monolayer MoS2, few-layer MoSe2, and WSe2 are shown, respectively, in Figures 1(a) through 3(a). Using these direct images and an analysis of their Fourier transforms, one can identify the hexagonal crystal structure of the films. Furthermore, it becomes trivial to extract the in-plane lattice parameters from the direct images; these agree with values that are theoretically calculated [1].

Several monolayer and few-layer MoS2, MoSe2, and WSe2 samples were prepared for Raman spectroscopy to investigate the vibrational modes of the films [2]. The Raman measurements were performed using a Renishaw inVia Raman system equipped with a 532-nm laser as an excitation source; this system has a resolution of ~1cm-1. By monitoring the peak spectral location of the Raman modes, it was possible to determine the approximate number of layers in few-layer samples of the dichalcogenides—similar to what is done in graphene [3]. Each sample of MoS2, MoSe2, or WSe2 exhibits two characteristic Raman modes; these are indicated in Figures 1(b) through 3(b). A correlation and analysis of these modes as a function of sample thickness provide useful insight into the layer-to-layer interactions in the materials.

These experimental results provide a useful data set for understanding some of the intrinsic properties of two-dimensional transition-metal dichalcogenides.

 

References:

[1] Wilson and Yoffe, Advances in Physics, 18:73, 193-335 (1969).

[2] K.S. Novoselov, et al., Science 306, 666-669 (2004).

[3] A.C. Ferrari, et al., Phys. Rev. Lett. 97, 187401 (2006).

Fig. 1: (a) HRTEM image of monolayer MoS2, including the extracted in-plane lattice constant. The fast Fourier transform of the area outlined by the white dashed box is inset. (b) Raman spectra of monolayer, few-layer, and bulk MoS2, with vibrational modes A1g and E12g labeled. Dashed lines indicate the approximate spectral location of the bulk peaks.

Fig. 2: (a) HRTEM image of few-layer MoSe2, including the extracted in-plane lattice constant. The fast Fourier transform of the area outlined by the white dashed box is inset. (b) Raman spectra of monolayer, few-layer, and bulk MoSe2, with vibrational modes A1g and E12g labeled. Dashed lines indicate the approximate spectral location of the bulk peaks.

Fig. 3: (a) HRTEM image of few-layer WSe2, including the extracted in-plane lattice constant. The fast Fourier transform of the area outlined by the white dashed box is inset. (b) Raman spectra of monolayer, few-layer, and bulk WSe2, with vibrational modes A1g and E12g labeled. Dashed lines indicate the approximate spectral location of the bulk peaks.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-2767 Direct Mapping of Strain State in epitaxial InGaN/GaN Multilayers using Dark-field Inline Electron Holography

Lee J.1, Song K.1, Koch C. T.2, Jung W.1, Tyutyunnikov D.3, Kim J.1, Park C.1, Aken P. A.3, Oh S.1
1POSTECH, Pohang, Republic of Korea, 2Ulm University, Ulm, Germany, 3Max-Planck-Institute for Intelligente Systeme, Stuttgart, Germany
jklee89@postech.ac.kr

The structural basis of modern light emitting diodes (LEDs) is InxGa1-xN/GaN multiple quantum well (MQW). In conventional LED MQWs grown along the c-axis of the wurtzite structure, which corresponds to the polar direction of the crystal structure, the epitaxial strain arising from the pseudomorphic straining of each layer induces a piezoelectric polarization. The induced piezoelectric field has been considered as a major cause for the degradation of internal quantum efficiency and also as a dominant mechanism for the efficiency droop. Therefore, the accurate measurement of strain is important for the strain engineering of LEDs.
In the present study, we measured the two-dimensional (2-D) lattice strains in pseudomorphically grown polar InGaN/GaN MQW structure using dark-field (DF) inline electron holography [1, 2]. The results demonstrate that the in-plane lattice matching between the two layers is accomplished by predominant (compressive) straining of thinner InGaN QWs (2.5 nm in thickness) to fit onto thicker GaN QBs (10 nm). This is quite understandable in the scheme of classical thin film mechanics of an epitaxial multilayer system [3]. However, the out-of-plane strain map revealed not only InGaN QWs but also GaN QBs are strained in opposite sign by the nonzero out-of-plane stresses arising from the constrained tetragonal distortion of InGaN by adjoining GaN layers, which results in amplification of the polarization gradient and consequently the polarization charges at the interfaces.
In an InGaN/GaN MQW grown with the nonpolar (a-plane) orientation, the strain maps revealed much more complicated distributions than those of polar system. As shown in Fig. 1, the in-plane lattice of active layers (InGaN QW (10 nm in thickness) and GaN QB (12 nm)) appeared to be pseudomorphically strained to the bulk substrate as a whole, via the local +/- strain undulation with a periodicity comparable to the film thicknesses (λ~ 12 nm) (Fig. 1c). Also, from the out-of-plane strain map (Fig. 2), it was confirmed that both InGaN QW and GaN QB, also p- and n-GaN were strained adjacent to their respective interfaces in opposite sign, indicating that not only InGaN QW but also GaN QB are not perfectly rigid when they establish the lattice coherency across the interfaces of this system. Possibly because of this unique strain distribution, the strain in the QW and QB layers shows a binodal distribution with double peaks (Fig. 2). We will further discuss the strain undulation phenomenon in the scheme of strain energy minimization. Importantly, the strain undulation seems to potentially induce a piezoelectric polarization along the non-polar growth direction.

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2011-0029406).

Fig. 1: a. Dark-field TEM image showing the active quantum well structure of a non-polar In0.1Ga0.9N/GaN LED, and 2-D in-plane lattice strain map. b. Enlarged strain map of In0.1Ga0.9N/GaN well region. c. Profile obtained within upper QW (white dotted box in b), showing local +/- undulation.

Fig. 2: a. 2-D out-of-plane lattice strain map of a non-polar In0.1Ga0.9N/GaN LED. b. Profile obtained along the growth direction from n-GaN to p-GaN. QWs and QB are strained in opposite signs, showing double-peak shape. Inset shows exponential fitting to the largely compressed region of bulk GaN near the interface with double-well region.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-3268 Strain Analyisis by Nano-Beam Electron Diffraction using millisecond frames of a direct electron pnCCD detector

Müller K.1, Ryll H.2, Ordavo I.2, Ihle S.2, Huth M.2, Simson M.3, Zweck J.4, Volz K.5, Soltau H.2, Potapov P.6, Strüder L.2, Schowalter M.1, Mahr C.1, Erben D.1, Rosenauer A.1
1Universität Bremen, Bremen, Germany, 2PNSensor GmbH, München, Germany, 3PNDetector GmbH, München, Germany, 4Universität Regensburg, Regensburg, Germany, 5Phlipps Universität Marburg, Marburg, Germany, 6GLOBALFOUNDRIES Dresden Module 1, Dresden, Germany
mueller@ifp.uni-bremen.de

Carrier mobilities or composition of nanostructures such as metal-oxide semiconductor field effect transistors (MOSFET) are tightly connected with local strain. Due to the uniqueness of Bragg's equation, nano-beam electron diffraction (NBED) is one of the most accurate tools for strain quantification with a precision in the 10-4 range. Strain analysis by NBED (SANBED) has recently improved drastically by using convergent STEM probes to yield sub-nm spatial resolution [1], and by precessing the STEM beam [2]. But still a major problem is the limited speed of contemporary, slow-scan CCDs with frame times around 100ms.

We introduce a scintillator-free, low-noise and radiation-hard pnCCD detector [3,4] with a detection quantum efficiency (DQE) close to 1, combined with an ultrafast readout-hardware working at 1kHz and above. We first investigated the InxGa1-xNyAs1-y/GaAs layers shown coloured in Fig.1a. The strain profile obtained from the 004 reflection recorded on a conventional Gatan CCD with 0.5s frame time is shown black. As depicted in Fig.1b, the pnCCD camera yields the same strain profile with equal precision of 0.07% at half the frame time of 0.2s. This is remarkable since each 300keV electron deposits its energy in up to 10 camera pixels, causing a large but isotropic point spread as shown in Fig.2. Next we used pnCCD frame times of 1ms only to obtain the profile in Fig.1c, agreeing with a-b while exhibiting a slightly lower precision of 0.13%, but speeding up the acquisition by a factor of 200. Fig.1d shows a strain profile calculated from the 008 reflection which is highly sensitive to strain but also very weak. The high DQE of the pnCCD allows for an improvement of strain precision to up to 0.04%.

Secondly 2D strain mapping was performed at a GexSi1-x/Si MOSFET shown in Fig.3. Parts a-b correspond to strain maps in the red region of the inset with 45x60=2700 scan pixels of the STEM beam and diffraction patterns recorded on a Gatan CCD with an overall acquisition time of 10min. Although sparsely sampled, the effect of the S/D-stressors is clearly observed with compressive lateral and tensile vertical strain of 2 and 3% below gate G, respectively. The high potential of the pnCCD camera is seen from Fig.3c-d, where we scanned over the yellow square in Fig.3a, sampled at 256x256=65536 pixels. With a frame time of 4ms for each diffraction pattern, the whole acquisition took only 4min while still resolving lateral and vertical strain maps with the GeSi/Si interface and a dislocation marked dashed in c-d. The precision achieved here was 0.1%, determined from a strain map in pure Si.

[1] Microsc. Microanal. 18 (2012) p.995

[2] Appl. Phys. Lett. 103 (2013) p.241913

[3] Appl. Phys. Lett. 101 (2012) p.212110

[4] Rev. Sci. Inst. 68 (1997) p.4271

Fig. 1: (a) STEM HAADF image (coloured) of InxGa1-xNyAs1-y/GaAs layers and strain profile along [001] measured from the 004 reflection by NBED using a Gatan UltraScan CCD. (b-c) Same as (a) but a scintillator-free pnCCD camera with (b) 200ms and (c) 1ms frame time was used. (d) As in (b) but strain was measured from the 008 reflection.

Fig. 2: Direct electron detection using the pnCCD camera at a primary energy of 300keV. At this relatively high energy, signal electrons are generated in traces of up to 10 pixels length. However, an FFT of a large number of single events shows the isotropy of this point spread, allowing a recognition of the 004 disc with high precision [1,3].

Fig. 3: (a-b) Strain map in the red region of the inset, showing a MOSFET with source/drain/gate S/D/G. The 333 reflection was recorded on a Gatan UltraScan CCD at 45x60 scan positions rastered in 10min. (c-d) Strain map in the yellow region. Here, 222 was recorded on a pnCCD with the STEM beam scanning over 256x256 pixels in 4min. Dashed: dislocation.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-8-O-3343 Heterojunctions in monolayer semiconductors

Sanchez A. M.1, Peters J. J.1, Wu S.2, Huang C.2, Xu X.2, Cobden D. H.2, Beanland R.1
1University of Warwick,, Gibbet Hill Road, Coventry, UK, 2University of Washington, Seattle, USA
a.m.sanchez@warwick.ac.uk

The fascinating mechanical and electrical properties of graphene, absent in bulk graphite, have encouraged researchers to look for other two-dimensional (2D) materials. The transition-metal dichalcogenides (TMD), of general form MX2, have a layered structure with just van der Waals forces between the layers. The properties of bulk TMD range from insulators (i.e. HfS2) to metals (i.e. VSe2). Monolayers of semiconducting TMD exhibiting a direct band gap have promise for an extensive range of applications in electronics and optics.

Single layers of TMD compounds can be obtained by exfoliation of bulk material, both mechanical and chemical, but new growth techniques will be needed to produce structures with real control over optical and electrical properties (e.g. bandgap engineering). A whole new research field is being developed in the growth of these materials, analogous to the development of heteroepitaxial semiconductor growth. Here, we examine the very first single crystal heterojunction in a monolayer material, produced in MoSe2/WSe2.

The samples were grown by physical vapor transport and characterised using atomic resolution transmission electronic microscopy. Imaging and EDX analysis was carried out in a doubly-corrected ARM200F (80-200kV).

An atomic resolution Annular Dark Field (ADF) scanning transmission electron microscope (STEM) image of a WSe2/MoSe2 heterojunction is shown in Fig. 1(a). The intensity contrast is sufficient to distinguish atoms on the triangular sublattice of the honeycomb containing transition metals. We examine the transition metal lattice and Se lattice separately, allowing true atom-by-atom characterisation of the boundary between the two materials. A histogram of the intensities at these sites (Fig. 1 (b)) has two separate peaks, which we plot using a color-scale where the center of the Mo peak is red and that of the W peak is blue. A histogram of the intensities at the chalcogen sublattice sites is superposed in green. Fig. 1(c) shows the inferred identities of the atoms in the same area. These maps show that, the substitution of transition metals happens rapidly while the Se lattice shows no change at the interface. This shows that the heterostructure lattice has crystalline perfection. Fig. 1(d) shows the intensity along a particular line normal to the interface passing through both M and X sublattices, illustrating how the three kinds of atom can be readily distinguished. A few defects (missing one Se atom or missing both Se) were also observed.

XX (DE-SC0008145) and DC (DE-SC0002197) are supported by DoE, BES, Materials Science and Engineering Division. SW acknowledges a UW CEI fellowship. WY is supported by Hong Kong RGC  (HKU705513P), Hong Kong UGC (AoE/P-04/08) and a Croucher Innovation Award. XX acknowledges the Research Corporation through a Cottrell Scholar Award. AMS thanks the SCRA and the HEFCE Strategic Development Fund.

Fig. 1: Figure 1 | Atomic analysis at the interface. (a) ADF STEM image of a selected interface area. (b) Histogram of intensities on the metal sublattice sites (red-blue colorscale) and the chalcogenide sublattice sites (green). (c). Intensities at the metal sublattice sites for a selected area. (d), Intensity plot of a line cut across the interface
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2466 The core-shell structure of dysprosium-doped BaTiO3 ceramics

Park D.1,3, Markus K.2, Souza R.2, Martin M.2, Mayer J.1,3, Weirich T.1
1Central Facility for Electron Microscopy, RWTH Aachen University, Germany, 2Institute of Physical Chemistry, RWTH Aachen University, Germany, 3Ernst Ruska Centre for Microscopy and Spectroscopy with Electrons (ER-C), Research Centre Jülich, Germany
park@gfe.rwth-aachen.de

BaTiO3 (BTO) ceramics are commonly used in multilayer ceramic capacitors (MLCCs) to increase capacitance. In spite of the relatively high permitivity of BTO, its permitivity varies in a non-linear manner with temperature and shows a rapid increase in the vicinity of the ferroelectric transition temperatures. To produce temperature stable capacitors, BTO ceramics are doped by rare-earth elements together with various additives. By doping rare-earth ions, the permitivity is flattened over a wide range of temperatures, leading to a more stable temperature dependency of the permitivity [1]. In particular, dysprosium (Dy)-doped BTO shows some significant improvements for capacitor applications [2]. Since these improvements are directly linked with the microstructure, we started an investigation on the doping behavior of Dy in BTO ceramics by transmission electron microscopy (TEM).

Energy filtering transmission electron microscopy (EFTEM) was mainly applied to reveal the spatial distribution of Dy and the incorporation of Dy ions into the perovskite structure of BTO. Elemental distribution maps based on the three-window technique show strong diffraction contrast, disturbing the interpretation of a spatial distribution of elements [3]. However, jump-ratio images based on the two-window technique contain less diffraction contrast and are thus an alternative when the former approach yields unreliable results. In this study, both methods are compared and discussed in detail.

As shown in Fig. 1, the jump-ratio images clearly show a core-shell structure of the Dy-doped BTO grains. Ti-L2,3 and Ba-M4,5 maps show that the substitutions of Dy on both Ti and Ba sites occur at the shell regions. The Dy-M4,5 map confirms it again showing higher contrast only at the shell regions. This result is also supported by electron energy-loss (EEL) spectra measured at the core and shell regions (Fig. 2). It is assumed that this amphoteric character of Dy results in a decrease in the leakage current and prolongs the life time of the capacitors [2].

At the triple points and the grain boundaries, two types of secondary phases with distinct chemical composition can be identified by jump-ratio maps (Fig. 1, see arrows with different colors in the Ti-L2,3 map). Several Dy-enriched grains were also observed in the specimen as shown in Fig. 2 (c) and (d).

References
1. Kishi, H. et. al., Japanese Journal of Applied Physics (2003), 42, 1-15
2. Sakabe, Y. et. al., Japanese Journal of Applied Physics (2002), 41, 5668
3. Grogger, W.et. al., Phys. Stat. Sol. (a), Wiley Online Library (1998), 166, 315-325

Fig. 1: The jump-ratio maps for (a) Ti-L2,3 , (b) Ba-M4,5 , (c) Dy-M4,5 , and (d) O-K edge of the BaTiO3 ceramic. The core-shell structure is clearly visible in the jump-ratio maps. Regions with the secondary phases of distinct chemical composition are indicated by arrows of different color in the Ti jump-ratio map.

Fig. 2: (a) The high angle annular dark field (HAADF) image of the core-shell structure, (b) EEL spectra at the core (blue) and shell (green) region. The spectra are aligned at the Ti-L3 edge. (c) The HAADF image of the secondary phase of the Dy-rich grain and (d) the EEL spectrum for the Dy-rich grain.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1439 Strain relaxation mechanisms at interfaces in III-As and III-Sb heterostructure nanowires by atomic resolution STEM

de la Mata M.1, Magen C.2, Shtrikman H.3, Caroff P.4,5, Arbiol J.1,6
1Instituto de Ciencia de Materiales de Barcelona, ICMAB-CSIC 08193 Bellaterra, Spain, 2Laboratorio de Microscopías Avanzadas, Instituto de Nanociencia de Aragon-ARAID, Universidad de Zaragoza, 50018 Zaragoza, Spain, 3Braun Center for Submicron Research, Department of Condensed Matter Physics, Weizmann Institute of Science, Rehovot 76100, Israel, 4Institut d’Électronique, de Microélectronique et de Nanotechnologie, UMR CNRS , 5Department of Electronic Materials Engineering, Research School of Physics and Engineering, The Australian National University, Canberra, 6Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, CAT, Spain
mmata@icmab.es

Semiconductor nanowires (NWs) have attracted huge attention during last years. Due to their wide range of applicability and modified physical properties when compared to planar structures, they are used in fields such as nanoelectronics, optoelectronics or biosensing [1]. Also they can be used as platform for the study of condensed matter physics [2]. Their synthesis allows for the combination of highly mismatched materials, otherwise not achievable. On this way, it is possible to combine different materials in a NW to create axial as well as radial heterostructures.
III-V semiconductors have been extensively synthesized and probed in this context, especially arsenide combinations. On the other hand, antimonides are of extreme interest as among them are the highest hole mobility (GaSb) and the narrower band gap and the highest electron bulk mobility (InSb) III-Vs materials, extending the operability range of the related devices.
The final NW properties will be influenced by the material quality, closely related to the presence of strain as a consequence of combining different materials. Through careful inspections of the crystal structures and thanks to the employ of aberration corrected STEM techniques, we are able to study the mechanisms that allow for the strain relaxation at atomic scale in these NWs in three different cases: i) radial InAs/GaAs NWs; ii) axial InAs/InSb NWs; and iii) axial GaAs/GaSb NWs. Flat or bended interphases were found depending on the material combinations, but in all of the studied systems, the partial/total relaxation takes place through the formation of misfit dislocations. These defects could be directly identified and analyzed by means of geometrical phase analyses (GPA) [3]. Given the spatial resolution achieved in aberration corrected STEM, we can also resolve the dumbbells and identify the elemental constituents within the crystalline structure, allowing a polarity study in the combined materials and through the interfaces [4,5]. Then, it is possible to get a deeper understanding on the heterostructural properties and their direct influence on the electronic behavior.

References:
[1] M. de la Mata, et al., J. Mat. Chem. C, 1, 4300 (2013)
[2] M. Heiss, et al., Nature Materials, 12, 439 (2013)
[3] M. J. Hÿtch, et al., Ultramicroscopy, 74, 131 (1998)
[4] M. de la Mata, et al., Nano Letters, 12, 2579 (2012)
[5] M.I.B. Utama, M. de la Mata, et al., Adv. Funct. Mat., 23, 1636 (2013)

MdlM would like to acknowledge CSIC for the JAE-PreDOC scholarship

Fig. 1: a) General HAADF STEM view of the InAs/InSb axial heterostructured NW. b) GPA deformation map obtained in the same region. c) Rotation map obtained by GPA. d) HAADF STEM image of the core-shell InAs/GaAs NW. e) GPA deformation map obtained in the same region. f) Corresponding Rotation map.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1479 Structural and chemical study of colloidal II–VI semiconductor quantum dots by aberration-corrected STEM

Patriarche G.1, Pedetti S.2, Nasilowski M.2, Tessier M. D.2, Bouet C.2, Cassette E.2, Malher B.2, Dubertret B.2
1Laboratoire de Photonique et de nanostructures, CNRS, UPR 20, route de Nozay, 91460 Marcoussis, France, 2Laboratoire de Physique et d’Etude des Matériaux, CNRS, Université Pierre et Marie Curie, ESPCI, 10 rue Vauquelin, 75005, Paris, France
gilles.patriarche@lpn.cnrs.fr

Complex colloidal semiconductor quantum dots such as core/shell or core/crown nanoplatelets can now be synthesized. Achieving these heterostructures has improved greatly the optical properties of the colloidal quantum dots. The optical properties of these colloidal quantum dots will depend not only on the morphology of the heterostructure (in particular the size and the shape of the core), the chemical nature and the presence of a gradient at the interfaces, but also on the elastic deformation inside the quantum dots due to lattice mismatch between the materials. The study of these heterostructures by aberrations corrected Scanning Transmission Electron Microscopy (STEM) provides access to their structure until the atomic scale. High Angle Annular Dark Field STEM images allow direct access to the atomic structure of the nanoparticles, and the contrast of the columns of the atoms is linked on their chemical nature ("Z-contrast” images). From these atomic resolution STEM images, it is possible to establish the strain fields of the heterostructure. Moreover, we can achieve correspondingly chemical quantitative analysis by STEM-EDX with a spatial resolution of about 1nm. We show studies realized on CdSe/Cd(Zn)S core/shell nanoplatelets [1,2] and core/crown CdSe/CdS [3] and CdSe/CdTe.

[1] Core/Shell Colloidal Semiconductor Nanoplatelets
B. Malher, B. Nadal, C. Bouet, G. Patriarche, B. Dubertret
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY 134 (2012) 18591-18598

[2] Colloidal CdSe/CdS Dot-in-Plate Nanocrystals with 2D-Polarized Emission
E. Cassette, B. Mahler, J.-M. Guigner, G. Patriarche, B. Dubertret, T. Pons
ACS NANO 6 (2012) 6741-6750

[3] Efficient Exciton Concentrators Built from Colloidal Core/Crown CdSe/CdS Semiconductor Nanoplatelets
M. D. Tessier, P. Spinicelli, D. Dupont, G. Patriarche, S. Ithurria, B. Dubertret
NANO LETTERS 14 (2014) 207-213

This work was supported by the French National Agency for Research (ANR), project "SNAP", ANR-12-BS10-011

Fig. 1: HAADF-STEM image of a colloidal CdSe/Cd0.7Zn0.3S core/shell platelet vertically aligned following the <001> axis and the full-field strains (εxx, εyy and εxy) mapped by using the geometric phase analysis (GPA). The core/shell structure is elastically matched in the plane (100) of the platelet and pseudomorphically relaxed along the <100> direction.

Fig. 2: Line profile extracted from the εxx strain map, the value is averaged over a width of about 10nm. We measure a deformation of about 16% along the <100> direction. The difference between the lattice parameters of the two materials is about 8% (the composition of the shell Cd0.7Zn0.3S has been established by quantitative EDX analysis)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1498 Electron nanodiffraction and fluctuation electron microscopy of phase-change materials

Bornhoefft M.1,2, Saltzmann T.3, Benke J.6, Voyles P. M.5, Simon U.3,4, Wuttig M.4,6, Mayer J.1,2,4
1Central Facility for Electron Microscopy, RWTH Aachen University, Germany, 2Ernst Ruska-Centre, Forschungszentrum Jülich GmbH,Germany, 3Institut für Anorganische Chemie, RWTH Aachen University, Germany, 4JARA - Fundamentals of Future Information Technologies, 5Department of Materials Science and Engineering, University of Wisconsin, USA, 6I. Institute of Physics, RWTH Aachen University, Germany
bornhoefft@gfe.rwth-aachen.de

We investigate phase-change material samples with nanodiffraction and fluctuation electron microscopy (FEM) combined with scanning transmission electron microscopy (STEM). Both methods rely on illuminating the areas of interest of the sample by a small (2 nm probe size) and almost parallel (convergence angle smaller than 2 mrad) electron probe. This creates nanodiffraction patterns with the spatial resolution defined by the probe size. We also extract information about the nanometer scale medium range atomic order (MRO) of amorphous materials by calculating the variance of the diffracted intensities in many nanodiffraction patterns a technique called STEM-FEM (Voyles & Muller,2002). In the present work nanodiffraction is used to identify crystalline regions of Sb2Te3 samples. The STEM-FEM technique is used to gather information about the MRO of AgInSbTe (AIST) samples.


In order to understand the reaction mechanisms during the wet chemical synthesis of hexagonal Sb2Te3 platelets, we investigated intermediate stages of the reaction with nanodiffraction. By scanning the intermediate products with the electron probe and collecting nanodiffraction patterns at the same time, it is possible to identify the crystalline areas of the platelets. Understanding the reaction mechanism can help to improve the synthesis to create smaller (sizes below 50 nm) Sb2Te3 platelets. These platelets may have promising applications in possible nonvolatile memory devices.


AIST is a potential phase-change material for building nonvolatile data storage devices. A deep understanding of the crystallization kinetics of AIST is needed, because the crystallization speed is the limiting factor of the writing speed of possible memory devices. The MRO of the amorphous phase of AIST could play an important role in understanding the difference of crystallization speeds of as-deposited and melt-quenched AIST (Lee et al.). The normalized variance is calculated by doing FEM in STEM mode in a Titan-STEM.


References
Voyles, P.M., & Muller, D.A. (2002). Fluctuation microscopy in STEM. Ultramicroscopy 93, 147-159

Lee, Bong-Sub et al., J. R. (2009). Observation of the role of subcritical nuclei in crystallization of a glassy solid. Science, 326(5955), 980–984

The authors gratefully acknowledge funding from the DFG in the framework of the SFB 917 “Nanoswitches”.

Fig. 1: (A) STEM angular darkfield image of a Sb2Te3 platelet. The orange box marks the region scanned by nanodiffraction. The blue cross marks the position of the electron probe on a crystalline region ((B) nanodiffraction pattern). The red cross marks the position of the electron probe on an amorphous region ((C) nanodiffraction pattern) .

Fig. 2: Variance of as-deposited AIST plotted versus the diffraction vector k. The peaks in the plot show a strong MRO in as-deposited amorphous AIST capped with ZnS-SiO2.
Fig. 3:
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1530 An attempt to visualize dopant distribution in Si by low-voltage SEM-EBIC

Tanaka S.1, Niwa T.2, Tanji T.1
1EcoTopia Science Institute, Nagoya University, Japan, 2Department of Electronics, Nagoya University, Japan
s-tanaka@esi.nagoya-u.ac.jp

Due to the shrinkage of modern semiconductor devices, there is a great need for dopant profiling techniques. Electron-beam-induced current (EBIC) technique involves scattering of the incident electrons and diffusion of the generated excess minority carriers, therefore, the EBIC technique is usually considered as a low spatial resolution technique. However, the generation volume of the excess minority carriers can be reduced by using low-energy electron beam. Also, the diffusion length can be shortened considerably by making use of the surface recombination effect. When very thin samples are used, the surface recombination effect should be very effective in reducing the diffusion length. In this paper, we used such conditions to visualize a dopant distribution in silicon (Si).

Fig.1 shows schematically the principle of our experiment. A semiconductor with high and low dopant concentration regions is assumed here. A Schottky electrode is formed on one surface and ohmic electrode is formed on the other surface. An electron beam is incident from the Schottky electrode side and scanned over the surface. We want EBIC to vary with the depletion layer width, so the accelerating voltage is adjusted such that the scattering of the incident electrons occurs mostly inside the depletion of the low dopant concentration region, and mostly outside of the depletion region of the high dopant concentration region as shown schematically in this figure. In the low dopant concentration region, most of excess minority carriers are generated in the depletion region and they are driven by the built-in electric field, so a large EBIC is expected. In the high dopant concentration region, the excess minority carriers generated outside the built-in electric field can be suppressed by the surface recombination effect, i.e., by using a thin sample.

We used a p-type Si for these experiments. Prior to the EBIC experiment, concentration profile shown in fig.2 was made by ion implantation. Then, it was glued face-to-face and processed to be cross-sectional thin sample. Schottky (Ti) and ohmic (Al) contacts were made on the surfaces. Figs 3(a) and 3(b) are SEM and EBIC images, respectively, taken at 3 kV. Dark bands seen along the original surfaces correspond to the ion-implanted region, where the carrier concentration is higher than the other region, therefore, smaller EBIC is expected. Although very faint, two narrow dark bands can be seen along the original surface in an edge region. To show this clearly, a line profile measured between the arrows is shown in fig. 4. The minimums occur at about 0.3 µm and 1.0 µm from the surface, which corresponds to the maximums of the dopant concentrations.

This work was supported by JSPS KAKENHI Grant Number 25420285.

 

Fig. 1: Schematic representation of the principle of visualization of dopant distribution by EBIC.

Fig. 2: Simulated acceptor (B) concentration profile as a function of sample surface.

Fig. 3: (a) SEM and (b) EBIC images taken at 3 kV.

Fig. 4: Line profile measured between the arrows in fig. 3(b). About 120 nm width was averaged.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1572 GaN nanowires seeded by Al droplets on Si (111) : control of polarity and chronology of their elongation

Largeau L.1, Galopin E.2, Gogneau N.1, Travers L.1, Glas F.1, Harmand J. C.1
11Laboratoire de Photonique et de Nanostructures (LPN), CNRS, Route de Nozay, 91460 Marcoussis, France, 2IEM, Cité scientifique, avenue Poincaré, 59652 Villeneuve d'Ascq Cedex
ludovic.largeau@lpn.cnrs.fr

Catalyst-free GaN nanowires (NWs) are known for the high quality of their crystal structure. They are intensively investigated worldwide to fabricate improved optoelectronic devices. Plasma-assisted molecular beam epitaxy (MBE) is a well-established technique to elaborate these NWs. The control of their crystal polarity is an important issue. Indeed, in such wurtzite crystals, a large number of properties such as growth kinetics, impurity or dopant incorporation and direction of piezoelectric field are driven by the crystal polarity.

While it is well established that N-rich conditions are necessary to form self-catalyzed GaN NWs, several possible routes are used to initiate their growth. In our process, we use a small amount of Al to form AlN platelets at the Si surface as evidenced by grazing incidence X-Ray diffraction (GIXRD) and scanning electron microscopy (SEM). We observe that these AlN platelets act as pedestals for the subsequent growth of GaN NWs. Consequently, the GaN NWs adopt the polarity of the AlN pedestals. Convergent beam electron diffraction (CBED) and chemical etching [1] reveal a single N-type polarity for all the NWs. We propose that the particular nucleation mechanism of the AlN pedestals is responsible for this N-polarity.

Finally, we use thin AlN layers as time markers inside the NWs to investigate the chronology of their formation. We observe at the atomic scale the shape and position of these AlN markers, longitudinally and along a radial cross-section by high-angle annular dark field scanning transmission electron microscopy (HAADF STEM) using a Cs-probe aberration corrected STEM Jeol 2200 FS. We deduce the kinetics of NW elongation which indicates that diffusion of Ga adatoms along the NW sidewalls contributes to the NW axial growth. At longer growth duration, shell formation around the initial cores is evidenced. Their growth mechanism is revealed by the morphology of the markers: bunches of monolayers are formed at the bottom of the NW and propagate along its sidewall facets, toward the top facet. The core and shell compete with each other in collecting Ga adatoms from the sidewalls. In general, the shell grows faster than the core and finally they merge with each other [3]

1. L. Largeau, E. Galopin, N. Gogneau, L. Travers, F. Glas, J.C. Harmand, Cryst. Growth & Design, 12, 2724 (2012).

2. E. Galopin, L. Largeau, G. Patriarche, L. Travers, F. Glas, J.C. Harmand, Nanotechnology 22, 245606 (2011)

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1584 Microscopic Characterization of Ferroelectric Field Effect Transistors with a Nanoimprinted Gate Dielectric

Cai R.1, Kassa H. G.1, Nysten B.1, Jonas A. M.1
1Bio & Soft Matter, Institute of Condensed Matter and Nanosciences, Université catholique de Louvain, Croix du Sud 1/L7.04.02, B1348 Louvain-la-Neuve, Belgium
ronggang.cai@uclouvain.be

  Ferroelectric polymers such as poly(vinylidene fluoride-co-trifluoroethylene), P(VDF-TrFE), are attracting renewed interest for use in organic non-volatile memory devices, e.g., in capacitance arrays, memory diodes or ferroelectric field effect transistors (FeFETs). In FeFET devices, the gate dielectric is ferroelectric. Therefore, the accumulation of charges at the interface between the semiconductor and the gate dielectric is not only modulated by the gate voltage, but also by the state of polarization of the ferroelectric polymer. The performance of a FeFET is significantly influenced by the parameters of the ferroelectric gate dielectric : a lower coercive field Ec corresponds to lower operation voltages, while a larger remnant polarization Pr induces more conducting charges and results in a higher on/off current ratio.
  We have previously reported that, when confining the crystallization of P(VDF-TrFE) in the nanocavities/nanochannels of a mold, preferred crystallographic orientation and a significant increase of crystal perfection ensue, with as a result a decreased coercive field.1, 2 However, no report shows how to exploit this beneficial effect in a real memory FeFET device. In this communication, we integrate nanoimprint lithography technology (NIL) in the fabrication process of the FeFET, and characterize the resulting device architecture in an atomic force microscope, using a conductive cantilever as gate electrode. The structure of a nanoimprinted FeFET and the experimental configurations for the characterization of the FeFET are shown in Figure 1. The ferroelectric property of nanoimprinted P(VDF-TrFE) in the device was characterized by piezoresponse force microscopy. We found that, by using a nanoimprinted P(VDF-TrFE) gate, the coercive field of P(VDF-TrFE) is significantly reduced in the device; as a consequence, the voltage needed to obtain the maximum remnant polarization can be decreased by ca. 30% in the device (Figure 2). This leads to a decreased operating voltage for the memory application, which is of interest for the realization of low voltage, all organic FeFET memories.

REFERENCE
1. Hu, Z.; Tian, M.; Nysten, B.; Jonas, A. M. Nat Mater 2009, 8, (1), 62-67.
2. Kassa, H. G.; Cai, R.; Marrani, A.; Nysten, B.; Hu, Z.; Jonas, A. M. Macromolecules 2013, 46, (21), 8569-8579.

Fig. 1: Figure 1: (a) The structure of a nanoimprinted FeFET. In the case of a reference FeFET, imprinted P(VDF-TrFE) was replaced by a continuous P(VDF-TrFE) film with the same thickness as the nanoimprinted one. (b,c) Experimental configurations for (b) applying Vgs to polarize the P(VDF-TrFE) and (c) applying Vds to measure Ids.

Fig. 2: Figure 2: Polarization induced current Ipol verse gate voltage Vgs curve of a reference FeFET and a nanoimprinted FeFET. The tip electrode scanning size for both FeFETs was 50 µm x 50 µm. The current values of both FeFETs are normalized to have the same P(VDF-TrFE) working area for better comparison.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1632 Investigations Focused on the Local Composition Determination of Dilute Nitride Quaternary Material Systems Grown on Si-substrates

Wegele T.1, Beyer A.1, Zimprich M.1, Volz K.1
1Philipps-Universität Marburg, Faculty of Physics and Materials Science Center, Marburg, Germany
tatjana.wegele@physik.uni-marburg.de

The epitaxial growth of multi-component semiconductor layers such as Ga(NxAsyP(1-x-y)) enables the improvement of laser and transistor devices because of the tunable band gap and lattice constant. However, many physical aspects of the formation such complex materials are still unknown and the determination of the chemical composition as well as understanding of the local effects pose a true challenge for an investigator.
In order to improve optical properties according to the earlier experiments [1] post-growth annealing is applied to the multi-quantum well (QW) heterostructures. The look inside the material and especially the advantage of the high resolution attainable in scanning transmission electron microscope (STEM) can answer the question, what influence the annealing treatment has on the specimen on the atomic scale. We investigated Ga(NxAsyP(1-x-y))-quantum wells in the Si-based laser structures. The investigations were performed using a double Cs-corrected JEOL JEM 2200 FS. The annular dark-field STEM-images of the annealed specimens reveal local structural changes in the Ga(NxAsyP(1-x-y))-QWs, that were not observed in the specimens without a thermal treatment. In order to understand and to explain the nature of these changes as well as a possible reason of their appearance a series of the high resolution STEM-images were acquired for different detector angular ranges. The intensities in the experimental images were evaluated using in-house written software. To prove the interpretation of the experimental results several simulations based on the absorptive potential [2] and frozen phonon [3] methods were carried out and compared with the experimental contrast.

[1] Annealing Experiments of the GaP Based Dilute Nitride Ga(NAsP), B. Kunert, D. Trusheim, V. Voßebürger, K. Volz, and W. Stolz, Phys. Stat. Sol. (a) 205, No. 1, 114–119 (2008).
[2] A Practical Approach for STEM Image Simulation Based on the FFT Multislice Method, K. Ishizuka, Ultramicroscopy, 90(2-3), 71-83 (2002).
[3] Incoherent Imaging of Zone Axis Crystals with ADF STEM, R. L. Loane, P. Xu, J. Silcox, Ultramicroscopy, 40, 121-138 (1992).

We acknowledge support of the DFG in the framework of the GRK 1782.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1634 Investigation of gallium phosphide antimonide grown on exactly oriented (001) silicon substrate

Ott A.1, Beyer A.1, Jandieri K.1, Ruiz Perez A.2, Kunert B.2, Stolz W.1,2, Volz K.1
1Philipps-Universität Marburg, Faculty of Physics and Materials Science Center, Marburg, Germany, 2NAsP III/V GmbH, Marburg, Germany
otta@staff.uni-marburg.de

Antimonide-based materials, such as gallium phosphide antimonide (GaPSb), that are grown highly mismatched on silicon substrates have interesting applications in high electron mobility III/IV channel layers on Si (001) substrates. As there are no III/IV semiconductors with high electron mobility which can be grown lattice matched on Si, different buffer layers have to be studied and the defect formation within these layers has to be understood in detail to optimize the layer structures for later device application.

To avoid antiphase boundaries penetrating through the Ga(PSb) layer, a gallium phosphide (GaP) layer is grown between the silicon substrate and the Ga(PSb). The antiphase boundaries created by the growth of a polar material on the non-polar silicon substrate annihilate within the GaP [1]. The mismatch between GaP and Ga(PSb) is between zero to twelve percent, depending on the composition. The strain induced by the high mismatch should be relaxed by misfit dislocations at the interface. However, dark-field and high resolution TEM have shown that the bulk contains many other defects like stacking faults, twins and threading dislocations. High resolution TEM and HAADF (high-angle annual dark-field) scanning transmission microscopy investigations of the interface using a double Cs-corrected JEOL 2200FS (S)TEM have revealed that the misfit dislocations mainly are Lomer dislocations and 60° dislocations pairs. Molecular dynamics simulations with Stillinger-Weber potentials have been used to model the structure of the dislocations theoretically. The crystal model has been used as input to simulate the HAADF images with the multislice algorithm and frozen phonon simulation. This contribution shows that HAADF imaging in combination with molecular dynamics simulation is very suitable for defect characterization at the interface of strained materials. TEM is very useful to gain insight on the crystal structure of GaPSb and other metamorphic buffer layers so that their growth can be optimized.

[1] GaP-nucleation on exact Si (001) substrates for III/V device integration, K. Volz, A. Beyer, W. Witte, J. Ohlmann, I. Németh, B. Kunert, W. Stolz, Journal of Crystal Growth 315 (1) (2011), pp. 37 – 47

Fig. 1: High resolution HAADF image of the GaP/GaPSb interface with misfit dislocations and stacking faults.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1644 Characterization of the interface structure of (GaIn)As and Ga(NAs) grown on GaAs

Han H.1, Beyer A.1, Gries K. I.1, Wolfgang S.1, Kerstin V.1
1Philipps-Universität Marburg, Faculty of Physics and Materials Science Center, Marburg, Germany
han.han@physik.uni-marburg.de

Ternary (GaIn)As as well as Ga(NAs) materials are widely applied in the fields of glass fiber communication, solar cells, electronic and optoelectronic industries [1]. The physical properties of quantum wells can be significantly influenced by both the chemical composition and the interface morphology, whereas the latter can be controlled by the growth temperature and by the introduction of growth interruptions. As a result, it is of great importance to characterize the crystallographic interface structures for technological applications. In the present work, we are mainly focused on the characterization of the interface structure of quantum wells using atomic force microscopy (AFM) and transmission electron microscopy (TEM).
The (GaIn)As and Ga(NAs) quantum wells were grown with metal organic vapor phase epitaxy (MOVPE) on GaAs (001) substrate at temperatures of 525°C and 625 °C with different growth interruption times (0s, 20s, 40s, 120s). After growth of each quantum well, both smooth and island-like structures were observed with AFM as shown in Fig.1. To analyze the crystallographic structure and composition of the islands, high resolution TEM was utilized to investigate the interface between the ternary materials and the GaAs from both, [010] and [110], directions. TEM results, giving quantitative information from the cross-section of the samples, will be correlated with AFM data, which shows the surface of quantum wells. The results suggest that there is a characteristic island structure depending on growth conditions. The islands exhibit a height of about two atomic layers and a width of smaller than 10 nm. Hence, HRTEM in combination with AFM provides a good method to obtain information of the interface structures of quantum wells.

References:
[1] K. Volz, et.al. Doping, electrical properties and solar cell application of GaInNAs, in: A. Erol (Ed.), Dilute III–V Nitride Semiconductors and Material Systems, Springer, Berlin, Heidelberg, 2008, pp. 269–404.

We gratefully acknowledge financial support of the DFG in the frame work of SFB1083.

Fig. 1: Fig. 1 AFM micrograph of an interior Ga(NAs) interface; the island-like structures are marked by the white circles.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1648 Structural characterization of GaSb-based heterostructures grown on Si

Bahri M.1, Largeau L.1, Mauguin O.1, Patriarche G.1, Madiomanana K.2, Rodriguez J. B.2, Cerutti L.2, Tournié E.2
1Laboratoire de Photonique et Nanostructures (LPN), CNRS-UPR20, Route de Nozay, 91460 Marcoussis, France, 2Institut d'Electronique du Sud (IES), Université Montpellier 2, CNRS-UMR 5214, Place Eugene Bataillon , 34095 Montpellier cedex 5, France
mounib.bahri@lpn.cnrs.fr

Monolithic integration of Gallium Antimonide (GaSb) heterostructures on Silicon (Si) is a promising road for producing efficient optoelectronics devices (lasers diodes [1], integrated photonic circuit...).
Three problems are to be overcome for growing such heterostructures on Si: First, the lattice mismatch between GaSb and Si (12.2%) which generates a high density of dislocations. Second, the growth of polar semiconductors on non-polar ones causes antiphase boundaries (APBs). Finally, the 3D growth mode of GaSb on Si which is due to the surface energy difference between the substrate and the films.
To understand and reduce the defects density, we have investigated the structural properties of GaSb grown on Si by Molecular Beam Epitaxy (MBE) using the two complementary techniques X-Ray Diffraction (XRD) and Scanning Transmission Electron Microscopy(TEM/STEM).
We have observed a 2D array of misfit dislocations at the interface GaSb/Si [2]. Coupling Grazing incidence XRD and STEM observations, we confirmed that this array is formed of a pure 90°-type dislocations (Lomer-type). The Geometrical Phase Analysis [3] (GPA) shows a well localized stress field around misfit dislocations. In addition to dislocations, we have reduced the twins density from 77 twins/µm to 11 twins/µm (measured on cross-section along a <110> direction near the interface between the layer and substrate) with optimizing surface treatment. We have used a vicinal Si substrates with different angles of miscut to suppress APBs but those defects are always present. AlSb buffer layer was also used to ameliorate the crystalline quality of GaSb and evolve from a 3D islands growth mode to a 2D one [4]. In addition to the 3D islands growth of AlSb buffer, we have identified (using EDX) an AlSb wetting layer at the interface. We are now investigating an alternative route based on thermal treatment to prepare the substrate and reduce the APBs density.
[1] J.R. Reboul, L. Cerutti, J.B. Rodriguez, P. Grech, and E. Tournié ,Appl. Phys. Lett. 99, 121113 (2011).
[2] S. Hosseini Vajargah, M. Couillard, K. Cui, S. Ghanad Tavakoli, B. Robinson, R. N. Kleiman, J. S. Preston, and G. A. Botton, Appl. Phys. Lett. 98, 082113 (2011).
[3]M. J. Hÿtch, J.-L. Putaux, J.-M. Penisson, NATURE 423, 270-273 (2003).
[4] Y. H. Kim, J. Y. Lee, Y. G. Noh, M. D. Kim, S. M. Cho, Y. J. Kwon, J. E. Oh, Appl. Phys. Lett. 88, 241907 (2006).

We are grateful to the ANR for funding support. Project reference: OPTOSI-ANR-2012-BS03 002.

Fig. 1: STEM-HAADF image of GaSb/Si with AlSb buffer layer.

Fig. 2: TEM micrograph illustrating an example of twin and APBs.

Fig. 3: STEM-BF micrograph of interfacial misfit dislocations on GaSb/Si.

Fig. 4: Strain map parallel to the interface (εXX).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1749 Electron Microscopy and optical properties Of PbGeSe Thin Films Chalcogenide Semiconductors

ElMandouh Z. S.1, ElMeleegi H. A.2
1Prof.Dr
znmandouh@yahoo.com

Pb20GexSe80-x thin films have been prepared on amorphous substrates by vacuum deposition. Transmission electron micrographs shows the effect of Ge addition on the morphology of particles of Pb20GexSe80-x thin films. Measurements of optical spectra has been done for Pb20GexSe80-x in the visible to near IR region as a function of composition and morphology. The composition of the system was checked with EDX. The optical constants; refractive index(n) , and the extinction coefficient (k) were determined from transmission and reflection spectra. Absorption coefficient (α) could be calculated from transmission data, the direct energy gap could be calculated from the relation between (α2) and (α2/3)vs. energy (hv) in e.V. Direct energy gaps were found to increase with decreasing Ge content. The direct gap were found to range from 1.2 to 2.1 e.V. The real and imaginary dielectric constant were calculated and related their values to concentration of germanium. The optical conductivity was determined for all concentrations of germanium in Pb20GexSe80-x. This system is promising candidates for up to date applications such as the production of a new generation of p-n junctions, photovoltaic cells, transistors, and solar cells. These applications is depending on thermoelectric properties as Pb20GexSe80-x where x = 20% for p-type and x = 25% for n-type. Other applications that depends on thermoelectric power is possible such as heat generators and energy conversion applications. Also the thermoelectric analysis were included such as Figure of Merit, thermoelectric conversion efficiency, and the Fermi energy level changes upon heating.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-1983 Investigations of segregation phenomena in highly strained Mn-doped Ge wetting layers and Ge quantum dots embedded in silicon

Prestat E.1,2,3, Porret C.1, Favre-Nicolin V.1, Tainoff D.1, Boukhari M.1, Bayle-Guillemaud P.1, Jamet M.1, Barski A.1
1INAC, SP2M, CEA and Université Joseph Fourier, 17 rue des Martyrs, 38054 Grenoble, France , 2Karlsruher Institut für Technologie (KIT), Laboratorium für Elektronenmikroskopie, D-76128 Karlsruhe, Germany, 3School of Materials, University of Manchester, Manchester M13 9PL, UK
eric.prestat@manchester.ac.uk

Mn-doped Ge quantum dots (QDs) embedded in Si are particularly interesting since their small size and the confinement effects may affect the electronic structure, the spin interactions and thus their ferromagnetic properties. Room-temperature and electric-field-controlled ferromagnetism were demonstrated in self-assembled Mn0.05Ge0.95 QDs [1].
Highly strained Mn-doped Ge wetting layers (WLs) and QDs embedded in Si have been prepared by Molecular Beam Epitaxy via Stranski-Krastanow growth mode on Si. High resolution scanning transmission electron microscopy (HRSTEM) imaging and Electron energy loss spectroscopy (EELS) spectrum imaging were performed at 200 kV on a FEI Titan3 Ultimate fitted with probe- and image-side aberrations correctors. EELS measurements were done in STEM mode using a Gatan Quantum spectrometer. TEM specimens were prepared using chemical mechanical wedge polishing to obtain clean and damage-free specimen.
In this presentation we report on Mn diffusion and the formation of Mn-rich precipitates in highly strained few monolayer thick Ge WLs and nanometric size Ge QDs heterostructures embedded in silicon. In the Ge/Si system Mn always precipitates and the size and the position of Mn-rich precipitates depend on the growth temperature (figure 1). At high growth temperature manganese strongly diffuses from germanium to silicon. By decreasing the growth temperature manganese diffusion is reduced [2]. In the Ge QDs system grown at low temperature Mn precipitates are detected, not only in partially relaxed Ge QDs but also in fully strained Ge WLs, between dots, as shown by the figure 2 [2]. Mn precipitates are identified by EELS (figure 2e) and three of them are indicated by white arrows in figure 2a.
In the GeMn system, the growth has to be performed at low temperatures (< 150 °C) to incorporate Mn and avoid the formation of Ge3Mn5 clusters [3,4]. Nevertheless, lateral segregation is still observed and leads to the formation of Mn-rich nanocolumns, i.e. elongated nanostructures parallel to the growth direction [4]. The nucleation of these GeMn nanocolumns required a critical thickness of 4 nm, which was explained by a subsurfactant epitaxial growth with Mn atoms occupying subsurface interstitial sites [4,5]. In this work we demonstrate that it is possible to incorporate Mn into extremely thin strained Ge layers. This feature is likely due to the electronic structure modification in these few Ge layers grown in compressive strain on silicon [6].

[1] F Xiu et al, Nat. Mater. 9 (2010), p. 337.
[2] E Prestat et al, APL 104 (2014).
[3] M Jamet et al, Nat. Mater. 5 (2006), p. 653.
[4] T Devillers et al, PRB 76 (2007), p. 205306.
[5] C Zeng et al, PRL 100 (2014), p. 066101.
[6] T Dietl, Nat. Mater. 5 (2006), p. 673.

Fig. 1: Cross-sectional STEM-EELS of Mn-doped Ge WLs growth at different temperatures and different Mn concentrations. (a) High angle annular dark field (HAADF) image of the four layers: the two lowest are grown at 220 °C and the two at the top are grown at 380 °C. (b) Ge map and (c) Mn map obtained by EELS. In (d) Mn (green) versus Ge (red) composite map.

Fig. 2: Plane view (a) Bright field- and (b) HAADF-STEM images of one Ge(Mn) QDs layer grown on Si substrate. (c-f EELS analysis of two Ge(Mn) QDs. (c) HAADF-STEM image acquired simultaneously than the EELS SI. (d) Ge map (e) Mn map and (f) Si map obtained by EELS. The arrows in (a) indicate Mn precipitates.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2116 Reaction of Ni with p-InGaAs at 350°C RTP

Lábár J. L.1, Menyhárd M.1, Gurbán S.1, Hoummada K.2, Ghegin E.3,4, Nemouchi F.3
1Research Institute for Natural Sciences of the HAS, Institute for Technical Physics and Materials Science, Konkoly-Thege M. út 29-33, H-1121 Budapest, Hungary, 2IM2NP, UMR CNRS 6137, 142 Av. Escadrille Normandie Niemen 13397 Marseille, France, 3CEA, LETI, Grenoble, France, 4STMicroelectronics, Crolles, France
labar.janos@ttk.mta.hu

Although nickel has long been used as a contact material to InGaAs, details of low temperature phase formation are not perfectly explored yet. In a recent publication formation of a single Ni4InGaAs2 phase with hexagonal structure was described and epitaxial growth to the InGaAs substrate at 250°C was claimed [1]. We confirm the formation of the hexagonal structure here, however we report on the formation of a polycrystalline Ni6InGaAs2 reaction layer at 350°C.

A 300 nm thick lattice matched p-(In0.53Ga0.47)As layer was grown epitaxially on InP substrate. 20 nm Ni was deposited on it and covered with 7 nm TiN protecting film. The samples were RTP annealed for 60 s at 350°C in nitrogen atmosphere to induce the solid state reaction between Ni thin film and the InGaAs layer. The reaction product was studied by Auger Electron Spectroscopy (AES) depth profiling, cross sectional TEM and Atom Probe Tomography (APT). The AES analysis used 5 keV primary electron energy, 20 nA beam current with a diameter of 40 µm. For the depth profiling Ar+ ions of 1 keV energy were applied and the angle of incidence with respect to the surface normal was 80o. All specimens were rotated during sputtering. The Ar pressure was 2.5*10-7 torr. The TEM lamellae were prepared by Ar+-ion milling (10 keV, 2 mA) till perforation, followed by 2 keV ion milling to remove damaged layer. Both BF and HRTEM images and selected area electron diffraction patterns were recorded in a JEOL 3010, operated at 300 keV. The GATAN camera in the GIF Tridiem was used for imaging and a GATAN Orius camera in the JEOL 3010 recorded the diffraction patterns. Fast Fourier transforms (FFT) of the HRTEM images were also analyzed identically to diffraction patterns.

The reaction layer is about 55‑60 nm thick polycrystalline film (Fig 1). Its composition is close to Ni6InGaAs2, as measured by AES. The same composition was also determined by APT. Examining both the individual grains one-by-one in the HRTEM image and of the FFTs from different grains in that HRTEM, we see that the growth is not epitaxial in general, since all grains have different orientations. As an example, a HRTEM image including 3 grains and the FFT for one of the grains indexed with the ProcessDiffraction program [2] are shown in Fig. 3 and Fig.4, respectively. For the indexing we used the hexagonal structure published in [1] but allowed for a mall tolerance in the measured d-values, because our XRD measurements showed that the measured diffraction lines were slightly shifted from the values reported in the literature. The small change in the lattice parameters may be connected to the different composition.

[1] Ivana et al., J. Vac. Sci. Technol. B 31(1) 012202‑1 -012202‑8 (2013).

[2] Lábár J.L., Ultramicroscopy 103(3) 237-249. (2005)

N. Szász is acknowledged for the preparation of the TEM lamellae

Fig. 1: Polycrystalline reaction layer. The interface to the InGaAs is close to planar at large scale however, non-perfect planarity is obvious from the undulations following grains at the bottom interface. Layer thickness is 55 nm±10 nm.

Fig. 2: AES depth profile of the in-depth distribution of elements in the reaction layer. It is seen that the composition is almost constant along the depth of the layer and it is close to Ni6InGaAs2, a value also measured with APT.

Fig. 3: HRTEM image with 3 grains of the reaction product layer all differently oriented. All 3 grains were successfully indexed as hexagonal Ni4InGaAs2 phase allowing for a small tolerance in the d-values.

Fig. 4: Central part of the FFT from the middle grain (Grain2) in Fig. 3. Indexed as Ni4InGaAs2 allowing for a small tolerance in the d-values. Successful indexing of several such patterns from different orientations indicate that the crystal structure must be the one published in [1] with slightly different lattice parameters.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2330 Texture and microstructure characterization by Precession Electron Diffraction (PED) technique for advanced semiconductor technologies

Valery A.1, Pofelski A.1, Clément L.1, Rauch E. F.2
1STMicroelectronics, 850 rue Jean Monnet, F-38920 Crolles, France, 2SIMAP/GPM2 laboratory, 101 rue de la Physique, 38402 Saint Martin d'Hères, France
alexia.valery@st.com

Manufacturing semiconductor devices involves dealing with polycrystalline materials at different steps of the fabrication process. As the size of devices reduces constantly, microstructure and texture of such materials can be a source of variability in devices performance. That is why advanced characterization is essential to ensure the development of new technology (28nm and 14nm) nodes. Electron Backscatter Diffraction (EBSD) analysis known as a SEM-based technique to determine grains orientation has shown limitations in resolution for the characterization of material microstructure.
New perspectives are offered with a recent Automated Crystal Orientation and phase Mapping (ACOM-TEM) technique developed for TEMs [1]. This EBSD-TEM like attachment (also known as ASTAR tool from NanoMEGAS [2]) provides maps with a nanometer resolution. An example realized on an integrated device is represented in Figures 1(b) and (c). While Figure 1(b) shows the different phases present in the device scanned area, Figure 1(c) reveals the orientation of every grain found in each of these materials.
The interest of ASTAR is first illustrated with a failure analysis case realized on the poly-Silicon gate microstructure of a 28nm device technology. Coupling this technique with others TEM based techniques (morphology and chemical analysis) has enabled the understanding of the specific failure mechanism which will be presented.
More challenging are materials presenting grains sizes of less than 20nm, like the Tungsten used to fill contacts in 14nm FDSOI process flow. To tackle such problems, it is necessary to fully define, characterize and optimize parameters affecting the quality of the results. As the ACOM approach always ends with the solution, which corresponds to the highest matching index for each point of the area scanned [1], it is of importance to consider the so-called reliability parameter. This factor is a measure of the uniqueness of the solution. This is illustrated with Figure 2 that concerns a p-MOS shared contact filled with Tungsten. The orientation map (Figure 2(a)) is supplemented by the reliability values (Figure 2(b)). Reliability is retrieved in grey scale, black areas representing the poorest values. Parameters such as probe size, diffraction camera length, and TEM lamella thickness through the excitation error used in the simulated diffraction templates were adjusted to maximize the reliability map. Benefit of precession to improve the reliability [1] was also verified in the present case. During this study, all the experiments were carried out using a FEI TECNAI FEG TEM operating at a high voltage of 200 keV.

References:
[1] Edgar F. Rauch et al., Z. Kristallogr. 225 (2010) 103-109
[2] NanoMEGAS - http://www.nanomegas.com/

Fig. 1: Fig. 1 (a) STEM VLAADF image from a device cut in cross section. (b) ASTAR’s phase map highlighting different polycrystalline materials (poly-Silicon, Nickel Silicide, Tungsten, Copper), and its (c) corresponding orientation mapping.

Fig. 2: Fig. 2 (a) ASTAR's orientation map of shared (left one) and single (right one) contacts in Tungsten combined with its reliability map. (b) Corresponding ASTAR’s reliability map with values scaled from 1 to 20% on a grey scale.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2155 Combined study of the evolution of composition, strain, and luminescence in InGaN thin films

Pantzas K.1,2,3, Patriarche G.2, Sundaram S.3, Kociak M.4, Cherkashin N.5, Troadec D.6, Ougazzaden A.1,3
1Georgia Institute of Technology, GT-Lorraine, Metz, France, 2Laboratoire de Photonique et de Nanostructures, CNRS UPR 20, Marcoussis, France, 3UMI 2958 GT-CNRS, Metz, France, 4Laboratoire de Physique du Solide, Université Paris XI, CNRS UMR 8502, Orsay, France, 5CEMES, CNRS UPR 29, Toulouse, France, 6Institut d'Electronique, de Microélectronique et de Nanostructures, CNRS UMR 8520, Villeneuve Dasq, France
konstantinos.pantzas@lpn.cnrs.fr

Over the past decade, considerable attention has been given to the growth of InGaN epilayers for photovoltaic applications. Such applications require the growth of epilayers with high indium contents, but, most importantly, with a thickness of over 100nm. At such thicknesses material quality has been shown to rapidly deteriorate, and the epilayers become rough, compositionally inhomogeneous, and highly defective. In a recent contribution, the present group proposed that by periodically inserting ultra-thin GaN layers during the growth of the InGaN epilayer, one could suppress the fluctuations in the indium composition, and thereby produce high quality epilayers that meet the requirements for photovoltaic applications. An experimental demonstration of the improvement that could be obtained using this semi-bulk growth process was given with InGaN epilayers containing 8% indium. In the present contribution, the most advanced transmission electron microscopy techniques are combined for the first time to evaluate the improvement obtained in semi-bulk InGaN epilayers grown by MOVPE. The epilayers used in this study contain 16% indium, double that of the previous contribution for a similar thickness. Particular attention is given to the strain and luminescence of these epilayers. More specifically, electron holography in a transmission electron microscope (HoloDark) was used to map the deformation in both the growth and the in plane directions with nanometric resolution. The results show that the semi-bulk InGaN epilayers are pseudomorphically strained on the underlying GaN substrate (see Fig.1). Furthermore, cathodoluminescence in a scanning transmission electron microscope (STEM-CL) was used to map the luminescence of these epilayers at the nanometer scale. A single, sharp emission is observed throughout the epilayers where the compositionnal fluctuations have been successfully suppressed (see Fig.2). These results serve to showcase the potential of semi-bulk InGaN for optoelectronic applications. Potential pathways to demonstrating epilayers with higher indium contents are discussed.

The authors kindly acknowledge support from the French CNRS RENATECH network and from the French METSA network.

Fig. 1: (left) HAADF STEM image of the semibulk InGaN epilayer ; (middle) Mapping of thedeformation along the c axis ; (right) mapping of the deformation along the a axis.

Fig. 2: (left) HAADF-STEM image of the zone mapped in STEM-CL ; (middle) Observed luminesencepeak ; (right) mapping of the intensity of the observed peak
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2196 Self-assembly of Al:ZnO doped nanopyramids studied by transmission electron microscopy

Javon E.1, Gaceur M.2, Margeat O.2, Ackermann J.2, Bals S.1, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium, 2Aix Marseille Université, CNRS, CINaM UMR 7325, 13288, Marseille, France
elsa.javon@uantwerpen.be

Self-assembly of nanostructures has recently gained increasing interest in the context of technological applications of nanoscale devices since it generates new collective properties [1] and leads to new nanomaterials that yield versatile functionalities [2]. ZnO nanocrystals attract great attention because of their applications for solar cells, photocatalysis, UV lasing or chemical sensing. Since their properties highly depend on morphology and size, it is of great importance to study the (3D) structure of the nanoparticles. In this study, we investigated the influence of the Al-doping on the shape and the coupling of ZnO nanopyramids by advanced Transmission Electron Microscopy (TEM).
Since the self-assembly should be characterized in 3 dimensions, HAADF-STEM is combined with electron tomography. Figure 1 illustrates the 3D reconstruction of a couple of ZnO nanopyramids and highlights their faceting. The coupling occurs between the two flat bases, which are not in direct contact to each other and is even stronger for particles that yield a pronounced pyramid shape. A random misalignment for each couple is found in addition to a concave shape of both surfaces at the base of the pyramids. Identification of the surface planes was performed by a combination of electron tomography and aberration corrected HAADF-STEM images (Figure 2.a) whereas high resolution TEM images (Figure 2.b) highlight the presence of ligands molecules. The base (0001) surfaces are very flat (except for the convex surface) but the {1-101} edges show elementary steps.

The coupling of the nanopyramids might be related to the intrinsic polarity along the [0001] direction of the ZnO nanoparticles. It is likely that the polarity of the NPs would lead to the preferential coupling of bases with different polarization. To study this effect, we combined HAADF-STEM and (ABF)-STEM imaging which allows mapping of the lightest elements such as hydrogen and oxygen together with heavier elements. The results presented in Figure 3 show that the Zn atoms are oriented toward the base and O points toward the tip leading to a O-polarization. Also for other nanopyramids the same observation was made. We therefore conclude that apparently, the strength induced by the ligands is stronger in comparison to the effect introduced by polarization.


[1] Z. Tang, N. Kotov, One-dimensional assemblies of nanoparticles: preparation, properties and promise, 2005.
[2] S. Kinge, M. Crego-Calama, and D. N. Reinhoudt, Self-assembling NPs at surfaces and interfaces, chemphychem, 2008

Acknowledgments: The authors acknowledge financial support from European Research Council (ERC Advanced Grant # 24691-COUNTATOMS and ERC Starting Grant #335078-COLOURATOMS)

Fig. 1: Volume rendering of the electron tomography tilt series HAADF-STEM projections oriented in three different positions.

Fig. 2: (a) High resolution STEM-HAADF images of a Al-doped ZnO nanopyramid in [11-20] zone axis and their corresponding Fourier transform (spectra). (b) HRTEM images of a ZnO couple of nanopyramids in the [11-20] zone axis.

Fig. 3: (a) Atomic resolution aberration corrected HAADF-STEM detail of the structure in a zoomed region of the edge of the nanopyramid. (b) Atomic resolution aberration corrected ABF-STEM detail in the same area as in (a). (c) Temperature colored detail of the HAADF-STEM image (green) superimposed with the ABF-STEM image (red) of the same region.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2279 The Study of the Structural and Vibrational Properties of Bismuth Oxides (Bi2O3) Synthesized from Tannic Acid

Ascencio-Aguirre F. M.1, Zorrilla-Cangas C.2, Herrera-Becerra R.2
1Posgrado en Ciencia e Ingeniería de Materiales, Instituto de Física, UNAM. Circuito Exterior, Ciudad Universitaria, Coyoacán, 04510, México., 2Departamento de Materia Condensada / Instituto de Física UNAM. Circuito de la Investigación Científica Ciudad Universitaria CP 04510 México.
fascencio@fisica.unam.mx

Bi2O3 is a material with potential applications at a nanometric level which range from catalysis, radiation dosimetry, the field of medicine, etc. In this project, a method of synthesis from “Green Chemistry” previously used and reported [1] is used to form Bi2O3 nanoparticles. Like in other cases, tannic acid plays the role of reducing agent and stabilizer with which the result is a fast and inexpensive method, compared to other synthesis methods, and with low environmental impact, in the production of nanoparticles. The crystalline phases of Bi2O3 obtained at a nanometric level are characterized by the microscopy used in this project.
Nanoparticles of Bi2O3 were obtained by reducing bismuth nitrate pentahydrate (Bi(NO3)3*5H2O) in a solution at a concentration of 3 mM, with the help of tannic acid (C76H52O46) at a concentration of 0.45 mM. The variation of the method consists on freezing the Bi2O3 nanoparticle solutions with liquid nitrogen and the following process of lyophilization[2]. The process was repeated for different values of pH in the solutions with the purpose of finding the best conditions for synthesis. The characterization process was performed on a transmission electron microscope (TEM) JEOL 2010 model and the spectroscopy studies were performed with a Micro-Raman ThermoScientific.
In the “HRTEM” images, figure 1A, different phases of Bi2O3 nanoparticles were found, preferably β-Bi2O3 and γ-Bi2O3, figure 1B and figure 2A. In the “HAADF” images, different forms of regular shape and size were found.  However, the particles do not show spherical symmetry. The granulometry studies show a good distribution in size with an average particle of (11±5) nm. The Raman spectroscopy, figure 2B confirms the formation of α-Bi2O3 particles in the following frequencies: (87, 121, 307 y 459 cm-1), which match previous reports from sources[3].
We can conclude that we have a new method in the synthesis of Bi2O3 nanoparticles with a low cost and low environmental impact using (Bi(NO3)3*5H2O) as a precursory agent and (C76H52O46) as reducing agent and stabilizer of said nanoparticles.

[1]  R. Herrera-Becerra, J. L. Rius, y C. Zorrilla, «Tannin biosynthesis of iron oxide nanoparticles», Applied Physics  A. (2010).
[2] Abdelwahed, W, G Degobert, S Stainmesse, y H Fessi. «Freeze-drying of nanoparticles: Formulation, process and storage considerations». Advanced Drug Delivery Reviews (2006).
[3] A.J Salazar-Pérez, M.A Camacho-Lopez, R.A. Morales . F. Ureña, and Jesus Arenas Alatorre. «Structural evolution of Bi2O3 prepared by thermal oxidation of bismuth nanoparticles. » Superficies y Vacío ( 2005).

Our gratitude to Roberto Hernández Reyes for his aid with the Electronic Microscope at IFUNAM and the financial support from DGAPA with grant PAPIIT IN105112.

Fig. 1: HAADF image and particles size distribution.

Fig. 2: TEM image showing some particles.

Fig. 3: HRTEM image for γ-Bi2O3 fase.

Fig. 4: Raman espectra.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2371 Characterization of an Fe-ZnO nanorod by STEM-EELS

Baik H. S.1, Yang M.2, Kim M. S.3, Park J. C.4, Min B. K.5
1Korea Basic Science Institute, Seoul, Korea, 2Korea Basic Science Institute, Kangneung, Korea, 3Samsung Electronics, Suwon, Korea, 4Gumi Electronics & Information Technology Research Institute, Gumi, Korea, 5Yeungnam University, Gyeongsan, Korea
baikhs@kbsi.re.kr


Fe-ZnO  is a candidate material in the field of room temperature magnetic semiconductors and dilute magnetic semiconductors(DMS). However, to accomplish DMS, the localized magnetic phase has to be formed inside the semiconductor. Many studies have been reported over the past 15 years; however, it was difficult to produce but also to analyse even if it is done[1]. In this study we focus on these dual difficulties, but specifically on the analytical difficulty.

Fe-ZnO nanorods have been fabricated using colloidal synthesis by a wet chemistry method[2] which can control the length and diameter of the nanorod product. This method has been successfully applied to synthesis of Co-ZnO, but not yet in the case of Fe-ZnO. We first synthesized ZnO:Fe by boiling Fe-spearate, Zn-spearate and Na- oleate in a solution of 1-octadecene. After mixing Fe-spearate and Zn-spearate at the specified
ratio, various morphologies of nanorods were formed along the doping concentration during the synthesis reaction(Figure 2). In order to detect the Fe inside of ZnO, we utilized an aberration-corrected scanning transmission electron microscope (Jeol ARM 200CFG) combined with electron energy loss spectroscopy(Gatan inc). Magnetic characteristics of Fe-ZnO nanorods were evaluated with the SQUID technique.


Figures 2 and 3 present TEM micrographs, magnetic characteristics and STEM-EELS sepctra. Various aspect ratios of nanorods can be found through the TEM images. The length and diameter range of our nanorods are 50 ~ 100 nm and 10 ~ 20 nm, respectively. Magnetic characteristics of the 100-nm nanorod were measured and evidenced ferromagnetism. There is considerable Z-contrast changing along the long axis of the nanorod, which suggests rod composition variation, By Z-contrast mechanism there is more Zn atom in the brighter region and smaller in dark region. The intensity ratio of Fe L2,3 is a useful tool for evaluating chemical state. In the dark region, the increasing Fe L2 partial ratio from Fe L3 suggests that Fe oxidation state is also increasing[3]. On the other hand, Zn spectrum becomes shapeless in the dark region, probably due to the lattice distorsion. In order to estimate the exact chemical state of Fe, we applied "Hartree-Slater and modified double-step hydrogenic continuum models" to remove backgound from the Fe L23 edge spectrum. Furthermore, if Fe is substituted for Zn in the nanorod to form the magnetic phase, a lot of Zn vacancies can be formed, and HRSTEM reveals strong strain in the dark contrast region. It is necessary to calculate the defect formation energy and electronic structure variation in details. 

[1] T. Dietl et al. Science 2000, 287, 1019
[2] Yang Y et al. Am Chem Soc. 2010, 132(38), 13882-94
[3] Schmid, H.K. et al. W. Micron 2006, 37, 426?432

This work was supported from KBSI project T34520.

Fig. 1: Schematic description of Fe-ZnO synthesis

Fig. 2: A, B, C: Various aspect ratio of Fe-ZnO nanorods, D: magnetic characteristics of the 100-nm nanorod

Fig. 3: STEM image(left) and core Loss  spectra obtained from brighter and darker region(right)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2427 Electrical Characteristics of Resistive Switching Devices Observed by TEM and Atom Probe Tomography

Lee J. H.1, 2, Cha E. J.1, Chae B. K.1, Kim J. J.2, Lee S. Y.2, Hwang H. S.1, Park C. G.2, 3
1Department of Materials Science and Engineering, Pohang University of Science and Technology, Pohang, Republic of Korea, 2Semiconductor Business, Samsung Electronics, Hwasung, Republic of Korea, 3National Institute for Nanomaterials Technology, Pohang, Republic of Korea
jaylee@postech.ac.kr

Metal-Insulator-Transition (M-I-T) is the basis of electrical or thermal-driven phase change of oxide layer. Some oxides can change their conductivity from insulator to metal above certain current density. Although M-I-T is not fully understood at present, it can be used as a switching device for the solution of sneak leakage problem. In order to apply the bipolar switching materials as the active layer of Resistive-switching Random Access Memory (RRAM), selection device which can minimize the sneak leakage current is needed. Among various candidates, we chose Nb-oxide for the selection device because of its compatibility with semiconductor structure. We have elucidated the mechanism of M-I-T of the amorphous NbO2 layer by using in-situ Transmission Electron Microscopy (TEM) technique combined with Atom Probe Tomography (APT).
At first, we have prepared the sandwich-type stack consisting of top-electrode (TE)/Nb-oxide/bottom-electrode (BE) as shown in Fig. 1. Then, I-V curves were measured by using the in-situ probing system which can reveal the microstructural change simultaneously. APT was sequentially used to achieve compositional change in three-dimensions.
During the mild operation of Icompliance < 10 μA, forming voltage was gradually approached to stable level of Vth = 0.6 V, while a threshold switching (TS) was distinctly measured. Reverse bias could reset forming step. No crystallization was, however, observed during the operation. This means that the TS of amorphous Nb-oxide is not caused by distinct crystallization but by charge injection. APT analysis after switching operation could reveal that cyclic fatigue could lead to the compositional change of Nb-oxide layer into the insulating NbO2 and metallic NbOx. This compositional redistribution is likely to enhance the reliability of the device.
On the contrary, the severe operation of Icompliance > 100 μA caused the filament formation of NbO2 layer. Conductive paths in the NbO2 layer were competitively formed by intense operations. The present result demonstrates that the resistive switching of NbO2 relies on different mechanisms with respect to applied current. This could be confirmed by the followed in-situ heating experiment and electron energy loss spectroscopy analysis.

This research was supported by Samsung Electronics Co., Ltd. The authors thank NINT for supplying the analysis equipment.

Fig. 1: Image for the in-situ probing experiment. Switching layer consisting of TE/Nb-oxide/BE stack is shown in inset image. The Nb-oxide layer could be sustained amorphous phase even after cyclic I-V measurements.

Fig. 2: Tip-shaped sample prepared by using FIB for the combinatory analysis of electrical probing and APT. After I-V measurement, sample was moved to APT chamber to analyse the elemental distribution of Nb-oxide.

Fig. 3: Threshold voltage alteration during M-I-T cycles. Compliance current was set to 0.5 μA.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2439 Ge nanostructure heteroepitaxy on a crystalline LaAlO3(001) substrate

Campos A. P.1, Ospina C. A.2, Mortada H.3, Rossi A. L.4, Dentel D.3, Bischoff J. L.3, Derivaz M.3, Werckmann J.5
1Divisão de Metrologia de Materiais, Instituto Nacional de Metrologia, Qualidade e Tecnologia -Inmetro, Duque de Caxias-RJ, Brazil, 2Brazilian Nanotechnology National Laboratory, Campinas, Brazil, 3Institut de Science des Matériaux de Mulhouse, Mulhouse, France, 4Centro Brasileiro de Pesquisas Físicas, Rio de Janeiro, Brazil, 5Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de Metrologia - Inmetro, Qualidade e Tecnologia, Duque de Caxias-RJ, Brazil
apcampos@inmetro.gov.br

Embedding nanocrystals (NC) in non-volatile flash memories are promising devices for computers, mobile phones or USB keys. The insertion of a semicondutor (SC) in an insulating matrix requires the elaboration of complex "oxide/SC/oxide/Si(001)" heterostructures, and the control of the associated successive growth steps. It’s in this context that we have studied the Ge initial growth mechanisms on LaAlO3(001), a crystalline oxide with a high dielectric constant (high-k material). In a previous work [1, 2], it has been shown the chemical and structural properties obtained in-situ, by X-ray photoelectron spectroscopy, X-ray photoelectron diffraction, electron diffraction (LEED and RHEED) and transmission electron microscopy (TEM). 10 monolayers (ML) Ge have been deposited at 600°C by molecular beam epitaxy on a c(2 x 2) reconstructed LaAlO3(001) surface. In these conditions, islands can be observed due to a lower LaAlO3(001) surface free energy. Some of them exhibit a preferential relationship in their heteroepitaxy (Fig 1), where the Ge(001) planes are parallel to the LaAlO3(001) ones, but rotated by 45° in the [001] direction, i.e. Ge<110>// LAO<100>. In this presentation we turned our attention to other types of NC on the LAO(001)-c(2x2) surface. We show that several growth modes are actually present:
i) NC formation supported by a Ge wetting layer of 1 atomic plane (Fig 2). ii) NC, usually twinned with a coherent (11-1) Σ3 twin plane, as shown in figure 3 where Ge (112) // LAO (001), and Ge <110> // LAO <100>. In this case a chemical process of relaxation occurs at the interface characterized by the formation of a mixed ML of Ge-La. iii) Between the NC (fig.4), the presence of a germanium layer consisting of a few atomic ML is observed.
This diversity of growth modes is the result of an almost instantaneous crystallization of the germanium surface at 600°C, in which lattice parameter distortions and interfacial energy are involved. In order to promote this diversity, we have probably to consider a sequenced process, but quasi instantaneous, in which there is a change in the surface energies.

[1] Didier Dentel et al., Acta Materialia 60 (2012) 1928-1936
[2] Jean-Luc Bischoff et al., Physica Status Solidi A (2012) 1-6

Fig. 1: Round shaped Ge NC (Restored wave function phase image)

Fig. 2: Stranski-Krastanov growth mode (STEM dark field)

Fig. 3: Twin and chemicalrelaxed mixed Ge-La monolayer (STEM dark field)

Fig. 4: Ge layer (Restored wave function phase image)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2472 Mechanism of twin suppression in Bi2Se3 thin films

Tarakina N. V.1, Schreyeck S.1, Luysberg M.2, Grauer S.1, Schumacher C.1, Karczewski G.1,3, Brunner K.1, Gould C.1, Buhmann H.1, Dunin-Borkowski R. E.2, Molenkamp L. W.1
1Experimentelle Physik III, Physikalisches Institut and Wilhelm Conrad Röntgen-Research Centre for Complex Material Systems, Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany, 2Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute 5, Forschungszentrum Jülich, D-52425 Jülich, Germany, 3Institute of Physics, Polish Academy of Sciences, Al. Lotników 32/46, 02-668 Warsaw, Poland
nadezda.tarakina@physik.uni-wuerzburg.de

Since the theoretical prediction [1] of surface states in Bi2Se3 and their subsequent experimental confirmation by means of ARPES measurements [2], there have been no transport measurements unambiguously showing the presence of surface conductivity in Bi2Se3. Defects like twinning, Se vacancies, mosaicity twist and tilt, are known to influence transport properties.
The goal of the present work was to reveal the origin of the formation of different structural defects in Bi2Se3 thin films. We conducted a detailed study of layers grown by MBE on InP(111)A and -B terminated flat and rough substrates. This choice of substrate reduces the formation of mosaicity twist sufficiently due to an almost perfect lattice match (0.2%) between InP and Bi2Se3.
Bismuth selenide layers grown on flat InP(111)B were found to have a "poor-crystalline quality" interface layer, which consists of crystalline domains with a thickness of one quintuple layer (QL). They are not always perfectly aligned to the substrate; misalignment occurs in areas where domains meet and try to merge. There are two difficulties in this process. First, the ‘flat’ InP(111)B substrate (RRMS = 0.1 nm) is not atomically flat, but has diatomic surface steps with a height of 3.38 Å (0.35 QL). Second, even if two nucleation points form on a perfectly flat area, there is always a chance of twin formation, depending on how the second layer of the QL is formed (A−B−C or A−C−B). Because of both reasons we conclude that "2D information" passed on to the film by a flat substrate is not sufficient for realizing the controlled growth of Bi2Se3.
AFM, XRD and STEM measurements of Bi2Se3 grown on a rough InP(111)B substrate (RRMS = 2.1 nm) reveal the absence of twin domains and a high-quality interface. Since the sides of the hollows are higher than the height of a QL (9.6 Å), they behave as additional {1-11} surfaces, so that both the substrate surface and the side surface of the hollow define the alignment of the QL layers and the stacking within a QL, providing the "3D information" that results in the unique layer stacking. Similar experiments performed using Fe-doped InP(111)A substrates showed the same tendency; the only difference between A and B terminated substrates was the particular family of twin domains that was suppressed by roughness.
The suppression of twins results in a reduction of the carrier density up to 89% compared to values obtained for twinned Bi2Se3 layers.

[1] H Zhang et al., Nature Phys. 5 (2009), p. 438.
[2] D Hsieh et al., Nature 460 (2009), p. 1101.

This work has been funded by the EU ERC-AG Program (project 3-TOP), by The Helmholtz Virtual Institute for Topological Insulators (VITI) and the Bavarian Ministry of Sciences, Research and the Arts.

Fig. 1: Cross-sectional HAADF-STEM images of the interface between a Bi2Se3 film and a flat InP(111)B substrate. Small vertical arrows mark positions of twin boundaries, formed perpendicular to the substrate; horizontal arrows and kinks in zig-zag lines mark positions of twin boundaries, formed parallel to the substrate. 

Fig. 2:  Cross-sectional HAADF-STEM image of an interface region of a Bi2Se3 film grown on a rough InP(111)B substrate with a simulated image inserted. A difference in contrast between the experimental HAADF-STEM image and the simulated image is present since roughness has been not included in the simulation.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2487 High-resolution studies of ZnO layers deposited by atomic layer deposition

Pécz B.1, Baji Z.1, Horváth Z. E.1, Lábadi z.1, Kovács A.2
1MTA TTK MFA Research Centre for Natural Sciences, HAS, Konkoly-Thege u. 29-33, 1121 Budapest, Hungary, 2Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Peter Grünberg Institute, Forschungszentrum Jülich, D-52425, Germany
pecz.bela@ttk.mta.hu

ZnO is one of the wide bandgap semiconducting materials and also the most widely used transparent conductive oxide (TCO) layer in solar cells. Very conform layers can be prepared by atomic layer epitaxy (ALD). This promises that even structured solar cells can be covered by this method in which the layer is formed in many cycles, as only a monolayer thick material is deposited in a single cycle. Here we study the structure of ALD ZnO layers deposited on various single crystalline substrates using conventional and high-resolution transmission electron microscopy (TEM) as well as by X-ray diffraction (XRD).
Diethyl Zinc (DEZ) and water were used as precursors for growth of ZnO on single crystalline sapphire, GaN, SiC and diamond substrates. The growth was carried out between 150°C and 300°C without any buffer layer and the nominal thickness of the layers is 40 nm.
The layers were characterised by TEM using a Philips CM20 conventional microscope operating at 200 kV and a spherical aberration (Cs) corrected FEI Titan for high-resolution imaging at 300 kV. All of the samples in cross-sectional and in-plane geometries have been thinned by conventional Ar ion milling at 10 kV with a final polishing at low voltages (<1 keV). XRD was used to determine the orientation of the grown layer. The X-ray and TEM results will be compared in this study.
The ZnO layer on sapphire was found polycrystalline. Fig. 1 shows the overview of the whole grown layer in cross-section. The contrast of the ZnO layer clearly shows the polycrystalline nature of the layer, which was supported by electron diffraction patterns as well. Fig. 2 is a high resolution Cs-corrected TEM image showing grains in the ZnO layer with different orientations. The fact, that efforts to grow single crystalline ZnO on single crystalline sapphire failed can be explained by the large misfit and by the low deposition temperature.
ZnO deposited on GaN was found to be single crystalline in both XRD and high-resolution TEM analyses due to the low misfit. However, local TEM investigations revealed some small regions of ZnO on GaN, which are misoriented.
Long columnar domains can be seen in the ZnO layer grown on diamond at 300°C. The ZnO layer is composed of two domains, both of them have the c-axis parallel to the surface normal and the two domains are twisted around the c-axis by 30°.

The support of the Hungarian OTKA grant (K108869) and from the European Union under the contract for an Integrated Infrastructure Initiative (ESTEEM2) are kindly acknowledged.

Fig. 1: Cross-sectional TEM image showing the whole ZnO layer.

Fig. 2: High-resolution TEM image of the sapphire/ZnO interface region showing the polycrystalline ZnO.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2492 Evaluation of the Carbon-doped GeSbTe by Atom Probe Tomography(APT) combined with Transmission Electron Microscopy(TEM)

Chae B. G.1, Lee J. H.1, 2, Kim Y. T.1, Lee B. H.3, Gu G. H.2, Lee S. Y.2, Park C. G.1, 3
1Pohang University of Science and Technology, Pohang, Korea, 2Samsung Electronics, 3National Institute for Nanomaterials and Technology, Pohang, Korea
cbkls2002@postech.ac.kr

GeSbTe (GST) is one of the most attractive materials used for the Phase-change Random Access Memory (PRAM) because of the high ION/IOFF ratio and rapid phase-transition between amorphous and crystalline states. The phase stability of GST layer, however, should be strengthened in order to assure ten year’s reliability of devices. That is, cyclic read /write operations can lead to atomic migration and phase separation resulting in the failure of devices. In order to enhance the resistance to thermal and electrical stresses, doping of light elements is conventionally adapted to GST. In this study, a carbon-doped GST (C-GST) was prepared and evaluated the migration behavior of GST layer by using sequential experiments of in-situ TEM and atom probe tomography (APT).
The stack of TiSiN(heater, 10nm)/C-GST (undoped or carbon-doped, 30~100 nm)/TiN(top electrode, 30nm) was deposited on the Si substrate by using PVD. At first, the phase-changing behavior of the FIB-prepared device sample was investigated. By using in-situ TEM probing, the gap with two orders of magnitude was achieved between High Resistance State (HRS) and Low Resistance State (LRS). Then, the same specimen was loaded to an APT, in order to investigate the exact movement of elements corresponding to the electrical fatigue. Femtosecond-pulsed UV laser (100kHz, 343nm) was utilized for the assistance of the field-evaporation of the sample.
The resistance of undoped-GST was rapidly dropped by cyclic I-V measurement and finally stuck to the low resistance state as shown in figure 4. At the same time, the local contrast of undoped-GST layer was continuously changed. According to AP analysis, this contrast change was due to the phase separation of GST into GeSb-rich phase and Te-rich phase. On the contrary, no significant migration was observed at the C-GST sample. However, atom probe tomographic image obtained after I-V measurement reveals that GeTe cluster was formed in the C-GST layer. Since the GeTe materials is also phase-changing material with acceptable performance, the GeTe clustering can be allowed from the device performance point of view.

Fig. 1:  TEM image of needle-like GST stack. Cyclic I-V curve could be simultaneously measured in a TEM.

Fig. 2: Data retention measurements of GST stack. Resistance of undoped GST is gradually decreased and stuck at LRS. C-GST keeps the high resistance up to 103 cycles.

Fig. 3: APT Elemental map of C-GSTbefore cyclic I-V measurement. Orange dots represent GeTe. No significantclustering was observed.

Fig. 4: APT Elemental map of C-GST after cyclic I-V measurement. GeTe clusters were randomly distributed in the C-GST layer.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2584 Kinked Si nanowires controlled by twins

He Z.1, Nguyen Hung T.2, Pribat D.3
1State Key Laboratory for Advanced Metals and Materials, University of Science & Technology Beijing, Beijing 100083, China, 2Institute of Chemistry & Materials Science, 17 Hoang Sam, Ha Noi, Vietnam, 3Department of Energy Science, Sungkyunkwan University, Suwon 440-746, Korea
hezhanbing@gmail.com

Kinked Si nanowires (KSiNWs) have potentials for nanoelectronics in integrated electronic devices. The turning angles of the KSiNWs, however, are difficult to be controlled because of the limited understanding to the mechanisms of the kinks. We will give a talk about the growth mechanisms of the KSiNWs grown by CVD. By using transmission electron microscopy, the connection of the adjacent components of KSiNWs were studied in details. We noticed that all the Si segments have {111} twin boundaries (TBs). More importantly, these twins are closely related with the kinks. As we know, multiple twins are quite common in semiconductor nanowires and the growth mechanisms of the twins in Si, InP,  nanowires etc., whether the twin boundaries are normal or perpendicular to the axes, have been widely discussed elsewhere. Therefore, we focus on the kinks rather than twins. Although the turning angles of the KSiNWs are changeable, the atomic layers of the close Si segments are connected epitaxially. We found that coherent {111} twins are crucial not only for the kinked angles, but also for the epitaxial growth. The corresponding geometric analysis was carried out to explain the turning angles based on TEM observation.

We conclude that multiple twins in Si nanowires are classified into three types: 1) with {111} TBs parallel to; 2) normal to; 3) inclined to the axes of Si nanowires. We detected 71°, 90°, 109°, 125°, and 158° turning angles, which lie with the combination of different types of twins in the connected Si segments. The TBs running along the length of the wire is a necessary conditon for the formation of 90° turning angles. The atomic layers at the connected area of the kinks are continuous because {111} planes are equivalent from the crystallographic point of view.

 

Acknowledgements

ZH thanks the support from the funding of University of Science and Technology Beijing, China (06111022) and the Fundamental Research Funds for the Central Universities, China.

Fig. 1: General view of KSiNWs. (a) the top view SEMimage of the KSiNWs. (b), (c), the side view SEM images from the up, and lowpart respectively. The kinked areas were marked by pink arrowheads foremphasis. (d)-(f), bright-field TEM images to show some typical KSiNWs. g, aHAADF-STEM image to display the catalyst nanoparticle (shown as white) on thetip.

Fig. 2: Details of a L-shaped KSiNWs. (a) a bright-field TEM image taken ata low magnification. ( b) Abundant thin lines lie on the both segments with thelines parallel to each other. (c) Selected area electron diffraction patternindicating the characterization of {111} twins. (d)-(f) Twins in differentparts revealed by HREM images.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2606 Tunnel conductivity switching in a single nano floating gate memory induced by scanning tunnelling microscopy

Gambardella A.1, Preziosi M.2, Cavallini M.1
11) Consiglio Nazionale delle Ricerche-Istituto per lo studio dei Materiali Nanostrutturati (CNR- ISMN), via P. Gobetti 101, 40129 Bologna, Italy., 22) University of California, Santa Barbara Electrical & Computer Engineering Harold Frank Hall
a.gambardella@bo.ismn.cnr.it

Non-volatile flash memory devices based on floating gate memories and memristors are the most likely candidates for the post-CMOS technology in nanoscale memory-bit cells and computation devices1. The use of nanoparticles (NPs) as novel architectures, named nano-floating gate-memories (NFGM)2, is of enormous interest due to advantages in tunability of charge trapping sites - which can be controlled by the size and nature of the NPs-, making them ideal candidates for new flash memory devices compatible with plastic electronics2. However, while these systems are quickly approaching the stage of industrial application, some important problems remain unsolved, such as the limits of spatial resolution where the switching occurs, and the role of proximity to the interface of isolated NPs which has never been directly observed nor understood.
We demonstrated3 that a reversible switching can be induced in the tunnel conductivity at the level of a single NP in a NFGM constituted by 50 nm large-Cobalt NPs embedded into a TiO2 matrix, by applying appropriate voltage pulses using a scanning tunnelling microscopy (STM) tip. The positive pulses inject holes into the NP, which becomes positively charged, and its proximity to the surface works as a floating gate, inducing a band bending towards a lower energy states due to the electrons in the TiO2 being attracted by the charged NP, while the surface morphology remains unaltered. These states are present in the thin layer in between the STM tip and the surface become accessible as empty states for tunnelling from the STM tip at energy below the conduction band of TiO2. The injection of electrons in a positively charged NP discharges the NP, thereby resetting the system. We show how this procedure can be used on a template of embedded NPs to create a template entire regions We performed our experiments at room temperature using prototypical materials which are commercially available and whose processing is well established. Our study demonstrates the switching in tunnel conductivity in single NP and provides useful information for the understanding mechanism of resistive switching.

1. J. Borghetti et al. Nature 464, 873-876 (2010).
2. M. Kang, K.-J. Baeg, D. Khim, Y-Y. Noh, Y-Y. & D-Y. Kim, Adv. Funct. Mater. 23, 3503-3512 (2013).
3. A. Gambardella, M. Prezioso and M. Cavallini, Scientific Reports 4, 4196- (2014)

We thank Zhara Hemmatian, Denis Gentili and Vittorio Morandi for sample preparation and SEM measurements, Tobias Cramer for the useful discussion and the help to model the process and Laurel L. McClure for editing the manuscript. A.G. was supported by EU-FP7 project NMP3-LA-2010-246102 (IFOX).

Fig. 1: Surface Topography and Tunnel Conductivity before and after the tip conditioning (+5V, 500ms). The Cobalt nanoparticles embedded into the semiconducting TiO2 matrix (left) switch to a new state becoming detectable as changes in the tunnel conductivity maps, while surface morphology remains unaltered (right).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2613 Size-dependent structural quality of InAs Nanowires Grown by Molecular Beam Epitaxy

Zhang Z.1, Lu Z.2, Chen P.2, Xu H.1, Guo Y.1, Liao Z.1, Shi S.2, Lu W.2, Zou J.1,3
1Materials Engineering, The University of Queensland, St. Lucia, Queensland 4072, Australia, 2National Laboratory for Infrared Physics, Shanghai Institute of Technical Physics, Chinese Academy of Sciences, 500 Yu-Tian Road, Shanghai 200083, People's Republic of China, 3Centre for Microscopy and Microanalysis, The University of Queensland, St. Lucia, Queensland 4072, Australia
z.zhang6@uq.edu.au

III-V nanowires have attracted great interest due to their potential applications in optoelectronics and nanoelectronics.1 For nanowires to be practically useful, controlling the structural quality is critical. Here we report the size-dependent structural quality of Au induced InAs nanowires grown on the GaAs {111}B substrate in a molecular beam epitaxy (MBE) reactor. The morphological, structural and chemical characteristics of the grown nanowires and their corresponding catalysts were investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).
Fig.1 shows SEM and TEM investigations of InAs nanowires. Fig.1a is a typical tilted SEM image in which nanowires, in which nanowires have varied diameters. TEM investigations were performed on the nanowires with different diameters. Figs. 1b and 1d show the high-resolution TEM images of a typical thick nanowire (~45nm) and a typical thin nanowire (~30nm), both near the nanowire tops, respectively. As can be seen, the thick nanowire has many planar defects along its growth direction, while the thin nanowire contains no lattice-defects. To understand this phenomenon, energy dispersive spectroscopy (EDS) analysis was preformed to determine the composition of the catalysts and their underlying nanowires. Figs. 1b and 1d show EDS spectra of a thick nanowire and a thin nanowire, respectively. Our quantitative analysis of the EDS results indicates both nanowires are InAs, and the catalyst’s composition of the thick nanowire contains ~16 at.% In and ~84 at.% Au, while the catalyst’s composition of the thin nanowire contains ~39 at.% In and ~61 at.% Au.
Based on these experimental results, the following growth behaviour of nanowires can be proposed. When Au nanoparticles are formed from its thin film, different sizes of Au nanoparticles cab be formed. With introducing the In source, In atoms diffuse into the Au nanoparticles to form Au-In alloyed particles. Due to different In diffusibility in Au nanoparticles with difference sizes, smaller Au nanoparticles may quickly absorb more In to form Au-In catalysts with a high In concentration, while larger Au nanoparticles tend to absorb relatively less In. Since nanowire growth at high supersaturation favors wurtzite structure, InAs nanowires induced by small catalysts with a high In concentration and therefore high In supersaturation2 have defect-free wurtzite structure, while defected InAs nanowires are induced by larger catalyst with a low In supersaturation.
References
1 H. Xia, Z. Y. Lu, T. X. Li, P. Parkinson, Z. M. Liao, F. H. Liu, W. Lu, W. D. Hu, P. P. Chen, H. Y. Xu, J. Zou, and C. Jagadish, Acs Nano 6, 6005 (2012).
2 F. Glas, J-C. Harmand, and G. Patriarche, Phys. Rev. Lett. 99, 146101 (2007).

This work has been supported by the Australian Research Council. 

Fig. 1: (a) Tilted SEM image showing thick and thin nanowires. (b, d) High-resolution TEM images taken from the top of thick nanowire and thin nanowire, respectively. (c,e) EDS spectra of catalyst and its underlying nanowire from thick and thin nanowire, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2633 Field mapping in the TEM by off-axis electron holography.

Cooper D.1, Rouveire J. L.2
1CEA-LETI, Minatec, 17 rue des Martyrs, 38054 Grenoble, Cedex 9, France, 2CEA-INACI, Minatec, 17 rue des Martyrs, 38054 Grenoble, Cedex 9, France
david.cooper@cea.fr

In this presentation we will show how off-axis electron holography is routinely used in the semiconductor industry for mapping the presence of active dopants [1] and strain with nm-scale resolution. The need for site specifivity means that focused ion beam (FIB) milling is indispensible for the preparation of specimens. In this presentation we will discuss the problems and advantages of using the FIB for both dopant and strain mapping by electron holography.

Figure 1 shows STEM images of two different FIB-prepared fully processed semiconductor devices with and without spacers which are used to control the diffusion of dopants under the gate. The devices have been electrically tested so that the holography results can be compared to these tests and simulations. Maps of the electrostatic potential distribution arising from the presence of active dopants have been acquired by off-axis electron holography. Figure 2 shows four different boron doped pMOS devices. Device A has a wide spacer and B has a narrow spacer. The effect of the width of the spacer can be directly seen in the potential map and the active dopants have diffused underneath the gate in this case degrading the electrical properties of the device. Devices C and D show that by changing the energy and dose of the dopant implants, the electrostatic potential distribution can be tuned to provide excellent electrical properties [2].

Dark field electron holography [3] can also be used to measure the strain in the same devices. Figure 3 shows a STEM image and strain maps for the pMOS device strained using a SiN CESL. Strain maps for the inplane and growth direction are shown. Here very low values of strain are expected and dark holography can easily detect the distribution of strain under the gate. We will comapre the results obtained by dark field electron holography to other TEM-based strain mapping techniques.

[1] Rau et al. Phys Rev Lett 82, 2614 (1999)
[2] Cooper et al. Semi Science and Tech 28 125013 (2013)
[3] Hytch et al. Nature 453, 1086 (2008)

DC and thanks the ERC for the starting grant “Holoview”. These experiments were performed on the platform nanocharacterisation at Minatec (PFNC).

Fig. 1: STEM images of the pMOS devices showing the different spacer thicknesses.

Fig. 2: Electrostatic potential distributions for four different devices examined by off-axis electron holography. The spatial resolution in the potential maps is 5 nm.

Fig. 3: (a) STEM image of a pMOS device (b) strain map for the in plane and (c) growth directions. The presence of dislocations can be observed in the in plane region.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2664 TEM studies of high aspect ratio surfaces coated with thin dielectric films by atomic layer deposition

Schindler P.1,4, Logar M.1, Usui T.1, Provine J.3, Karnthaler H. P.4, Prinz F. B.1,2
1Stanford University, Dept. of Mechanical Engineering, Stanford, CA, USA, 2Stanford University, Dept. of Material Science and Engineering, Stanford, CA, USA, 3Stanford University, Dept. of Electrical Engineering, Stanford, CA, USA, 4University of Vienna, Physics of Nanostructured Materials, Vienna, Austria
peter.schindler@stanford.edu

Key pieces of modern day technology such as the dynamic random-access memory (DRAM) keep following the trend of down-sizing. This requires the dielectric layer that is a crucial component in these structures to reduce its thickness [1]. As downscaling continues the thickness of dielectric components reaches a limit where electrons can tunnel through the dielectric. To overcome this limitation high-k dielectrics such as HfO2 [2], TiO2 or BaTiO3 are investigated. Coating high aspect ratio substrates with a dielectric increases the capacitance of DRAMs by the factor of the aspect ratio. Morphology and conformity of this coating layer play a crucial role for the quality and functionality of a DRAM. We report on a transmission electron microscopy (TEM) study of thin TiO2 layers deposited on a high aspect ratio trench-like substrate.
The key enabling technique to coat high aspect ratio surfaces pinhole–free is atomic layer deposition (ALD) [3]. Two self-limiting reactions of precursors with the surface ensure good uniformity of layers with precise control of the thickness. In addition, a variant of ALD based on O2 plasma as oxidant rather than water vapour is used. This plasma-enhanced ALD (PEALD) has the advantage of lowering the temperatures that are necessary for the precursor ligands to react with the surface. Both, ALD and PEALD were applied to coat an amorphous Si substrate that features a trench-like structure with TiO2 using TDMA-Ti precursor at 75°C at a chamber temperature of 240°C.
Fig. 1 shows TEM micrographs of the trench structure (in a cross-sectional view) covered with 100 cycles of TiO2 using ALD and PEALD, respectively. The micrograph (e) gives an overview of the trench substrate. (a) and (b) show the top part while (c) and (d) show the lower part of the trenches. For (a) and (c) ALD was applied whereas for (b) and (d) PEALD was used for the deposition. PEALD yields better step coverage (bottom thickness divided by top thickness) of 88% compared to 79% for ALD.
In the HRTEM micrograph in Fig. 2 the atomic structure of TiO2 is resolved revealing that PEALD unlike ALD induces crystallization at the same chamber and precursor temperature. In the inlay image of Fig. 2 the simulated diffraction pattern of the anatase phase of TiO2 is compared to the SAD pattern yielding good agreement. Diffraction spots indicated by an arrow correspond to the brookite phase. Both phases have been observed in HRTEM as well. It is planned to investigate how the parameters during PEALD (temperature, plasma power and purging times) influence crystallization.
1. H. Wong and H. Iwai, Microelectron. Eng. 83 (2006)
2. T. Usui, et al., Appl. Phys. Lett. 101 (2012)

3. S. M. George, Chem. Rev. 110 (2010)

Fig. 1: TEM of high aspect ratio trenches are shown in cross-sectional view. (a) and (b) show top end of trenches coated with TiO2 using thermal ALD and PEALD, respectively. (c) and (d) show bottom end of trenches coated with TiO2 using ALD and PEALD, respectively. (e) TEM at lower magnification illustrates the high aspect ratio.

Fig. 2: HRTEM confirming crystalline structure of TiO2 deposited with PEALD on top end of the trench structure (cf. Fig. 1 (b)). Inlay shows SAD spots compared to the simulated diffraction rings of the anatase phase of TiO2. Diffraction spots corresponding to the brookite phase are indicated by arrows.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2675 Investigation of GaAs/AlGaAs heterostructure core-shell nanowires by aberration corrected STEM

Zheng C. L.1, Wong-Leung J.2, Kauko H.3, Zhu Y.1, Dwyer C.4, Van Helvoort A.3, Gao Q.2, Tan H. H.2, Jagadish C.2, Etheridge J.1
1Monash University, Victoria, Australia, 2The Australian National University, Canberra, Australia, 3Norwegian University of Science and Technology, Trondheim, Norway, 4Forschungszentrum Juelich, Juelich, Germany
changlin.zheng@monash.edu

Recently high quality GaAs/AlGaAs quantum well heterostructures based on core multishell nanowires were fabricated by metal organic vapour phase epitaxy (MOCVD) [1]. The nanowires show excellent optical properties including extremely high quantum efficiency, intense emission for extremely low submicrowatt excitation. Calculations suggest that the optical properties depend critically on the morphology and composition of the GaAs quantum wells [1]. In order to fully understand the structure-property relationship and to optimize the growth process, it is necessary to determine the local atomic arrangement and composition with high spatial resolution. In this work we use aberration corrected scanning transmission electron microscopy (STEM) to investigate the cross-sectional structure and morphology of the nanowires [2]. Several structural features, including the width of the quantum well were found to have a 3-fold rotational symmetry about the <111> growth axis. By using atomic resolution high angle annular dark field (HAADF) STEM, the crystal polarity was determined directly from the asymmetric intensity distribution in the dumbbell structure along the [110] projection and further linked to the three-fold symmetry of the heterostructure morphology about the nanowire growth axis. These results indicate that the two-dimensional vapour-solid (VS) growth of the nanowire multishells depends strongly on the polarity of the nanowire sidewall facets, which determine the surface energies and surface reconstruction of the facets, and hence driving the anisotropic growth of the heterostructures [2].

The aluminium composition of GaAs/AlGaAs heterostructures was measured by quantitative HAADF-STEM. The intensity of HAADF images was normalized to the incident beam intensity and transferred to an absolute scale to compare with simulated HAADF image intensities. The STEM simulation uses multislice calculations incorporating thermal diffuse scattering via a frozen phonon approach. By comparing the simulated electrons intensity with the experimental results, the HAADF-STEM images were translated into aluminium composition maps with high spatial resolution as shown in Fig. 2 [3].

[1] M. Fickenscher, T. Shi, H. E. Jackson, L. M. Smith, J. M. Yarrison-Rice, C.L. Zheng, P. Miller , J. Etheridge, B. M. Wong, Q. Gao, S. Deshpande, H. H. Tan and C. Jagadish, Nano letters 2013 13 (3): 1016-1022.

[2] C. Zheng, J. Wong-Leung, Q. Gao, H. H. Tan, C. Jagadish, and J. Etheridge, Nano Letters 2013 13 (8), 3742-3748.

[3] H. Kauko, C. L. Zheng, Y. Zhu, S. Glanvill, C. Dwyer, A. M. Munshi, B. O. Fimland, A. T. J. van Helvoort, and J. Etheridge, Appl. Phys. Lett. 103, 232111, (2013).

Funding is acknowledged from the Australian Research Council Grants LE0454166. The authors acknowledge access to Australian National Fabrication Facilities (ANFF).

Fig. 1: HAADF-STEM image of an AlGaAs/GaAs nanowire cross-section viewed along the ⟨111⟩ direction. Several morphological features, including the different thickness of the AlGaAs rich bands, the different size of the corners of AlGaAs QW as well as the GaAs cap layer and the tapered GaAs QW, all show three-fold rotational symmetry around the growth axis.

Fig. 2: (a) High resolution STEM-HAADF image of GaAs/AlGaAs quantum well heterostructures and (b) the corresponding Al composition mapping by quantitative STEM.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2703 Analysis of structural defects in three-dimensional Ge crystals grown on (001)-Si substrates.

Arroyo Rojas Dasilva Y.1, Erni R.1, Gröning P.2, Isa F.3, Isella G.3, Kreiliger T.4, von Känel H.4
1Electron Microscopy Center, EMPA, Swiss Federal Laboratories for Materials Science and Technology, Dübendorf, Switzerland., 2Advanced Materials and Surfaces, EMPA, Swiss Federal Laboratories for Materials Science and Technology, Dübendorf, Switzerland., 3L-NESS and Politecnico di Milano, Como, Italy., 4Solid State Physics Laboratory, ETH, Zurich, Switzerland.
Yadira.Arroyo@empa.ch

Ge/Si heterostructures show many advantageous properties making them promising candidates for building blocks in various applications, such as microprocessors, high performance photo- and X-ray detectors and Ge lasers. However, Ge layers grown on Si substrates show a high density of crystal defects due to the large lattice mismatch of 4.2 % between Si and Ge as well as cracks at thicknesses beyond a few microns. One of the ways to solve these problems is to grow dense arrays of three-dimensional Ge crystals on deeply patterned Si wafers by Low Energy Plasma Enhanced Chemical Vapor Deposition (LEPECVD)1. By using this innovative approach, epitaxial and thermal strains can be confined close to the heterointerface1.The threading dislocations (TDs) affecting the electrical and optical properties of the material are the main defects yet to be eliminated. For this reason, it is important to know which kind of TDs are present. Two different samples have been analyzed, with Si pillar base width of 5 µm and 15 µm, and Ge height of 8 µm and 30 µm, respectively (Fig.1). The characterization of the Ge crystals has been performed using transmission electron microscopy techniques such as two beam conditions and weak-beam imaging, high-resolution TEM (HRTEM) and scanning TEM (STEM).

TEM analysis shows that the Ge/Si interface is not smooth. Due to the lattice mismatch and the rough interface several dislocations originate at the heterointerface (up to 1x109 cm-2); many of them end at the faces of the pillars reducing the TD density towards the crystal surface. In addition to TDs laying in (111) planes, which exit through the sidewalls of tall Ge crystals, we also find vertical TDs traversing the crystals from the interface right to the surface along the [001] direction2. These dislocations are not visible under g=(004) diffraction condition, they have edge character with g=1/2[1-10] (Fig.2). Apart from these dislocations, TDs running along the [1-10] direction are observed with b=1/2<110> as well as partial dislocations with b=1/3<111> and b=1/6<112>. Dislocations with b=1/2[011] are also observed, they are not visible at g=(11-1) and g=(02-2) reflections.

In summary, the present work confirms the presence of the dislocation network and addresses the types of dislocations in Ge/Si crystal such as the TD running along [001] and [1-10], and other kinds of dislocations found in the crystals. The characterization of the different dislocation types, revealing their origin and density in Ge/Si crystals, will help to further understand the properties of these complex semiconductor structures and will outline ways of optimizing their epitaxial growth.

1 Falub C. V. et al. Science, 335 , 1330-1334, 2012.

2 Marzegalli A. et al. Adv. Mater., 25, 4408-4412, 2013.

Fig. 1: Ge crystals epitaxially grown on (001)-Si pillars: (a) SEM image of 8 µm tall Ge crystals on (001)-Si pillars with 5 µm base width. (b) TEM image revealing TDs under g=(2-20) and g=(004) diffraction conditions. (c) TD labeled by a black arrow is not visible under g=(004) diffraction condition.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2794 Epitaxial high-k dielectrics: ternary rare-earth based oxides

Wendt F.1, 2, Schäfer A.1, 2, Mantl S.1, 2, Hardtdegen H.1, 2, Mikulics M.1, 2, Lenk S.1, 2, Barthel J.2, 3, Schubert J.1, 2, Luysberg M.3, 4
1Peter Grünberg Institute 9, Forschungszentrum Jülich GmbH, Jülich, Germany, 2JARA, Fundamentals of Future Information Technology, Germany, 3Ernst Ruska Center, Forschungszentrum Jülich GmbH, Jülich, Germany, 4Peter Grünberg Institute 5, Forschungszentrum Jülich GmbH, Jülich, Germany
f.wendt@fz-juelich.de

Ternary rare-earth based oxides are promising candidates for gate insulators in e.g. high electron mobility transistors due to their wide band gap (> 5 eV) and large permittivity (>24). Here we report on oxides, such as GdScO3 or LaLuO3, for which epitaxial growth could be achieved on GaN (0001) [1]. Such crystalline, high-k epitaxial layers potentially enable epitaxial overgrowth and hence offer new pathways towards 3D integration. Therefore a careful determination of the layers’ structural properties including interface abruptness and dielectric properties is essential.

The ternary rare-earth based oxides were deposited by pulsed laser deposition (PLD) using a KrF excimer laser (wavelength 248 nm, pulse width 20 ns, fluence 2.5 J/cm2) at between 620 °C and 740 °C at which crystalline growth occurs. X-Ray diffraction (XRD) and electron diffraction (ED) experiments were carried out to determine the structure of the crystalline phase. For high-resolution analyses of the interface, wedge shaped TEM specimens with a wedge of 3° were prepared by mechanical grinding with the MultiPrepTM polishing system from Allied High Tech Products. Subsequently Ar ion milling at 4.5 keV and 6° angle was employed for 5 min. High resolution images were obtained with an aberration corrected FEI Titan 80-300 TEM. The dielectric properties of the films are determined by capacitance-voltage measurements.

The studies revealed a novel hexagonal structure opposed to the well-known orthorhombic phase. This is demonstrated in the diffraction pattern of GdScO3/GaN(0001) shown in Figure 1. Clearly, pseudomorphic growth has taken place. The lattice constants deduced from XRD, a=0.360 nm and c=0.595 nm, agree within the margin of errors with the values measured by ED. Because of the large mismatch between GdScO3 and GaN of 12 % structural defects appear within the layers (not shown). Figure 2 displays the interface between GdScO3 and GaN in atomic resolution. The interface is atomically sharp. Local variations in contrast arise due to changes in crystal tilt, which typically arise from structural defects. Complete results of GdScO3 and LaLuO3 will be presented. The dielectric properties of the films are determined by capacitance-voltage measurements. In the case of GdScO3 a permittivity of 27 is measured for a 16 nm thick film, making this novel crystalline material promising for high k dielectrics.

References

[1] A. Schäfer et al, submitted to Semiconductor Science and Technology (2014)

Thanks to P. Bayle-Guillemaud and N. Mollard from the CEA Grenoble for the introduction to the MultiPrep.

Fig. 1: Diffraction pattern of GdScO3/GaN. The encircled spots are indexed with respect to the hexagonal lattices of GaN (red) and GdScO3 (blue).

Fig. 2: High resolution TEM image of GdScO3 grown onto GaN. The growth direction [0001] is marked by arrows.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2808 FIB/TEM characterization of Si/Ge/Sn alloys

Benedetti A.1, Stefanov S.2, Chiussi S.2
1C.A.C.T.I, Vigo University, Campus Universitario, Vigo (Spain), 2Dpto. Fısica Aplicada, E.E. Industrial, Vigo University, Campus Universitario, Vigo (Spain)
abenedetti@uvigo.es

In recent years, GeSn has emerged as a valuable candidate for the convergence of Si based microelectronics and photonics.

However, the extreme low solubility of Sn and the large (14.7%) lattice mismatch between the elements rendered the use of conventional MBE and CVD growth methods problematic. On the other hand, a very promising alternative to the conventional MBE and CVD growth methods is given by pulsed laser induced epitaxy (PLIE), in which epitaxial GeSn is achieved through fast non-equilibrium solid-liquid-solid phase transitions1.

In order to assess the quality of the PLIE-grown GeSn films, a full characterisation in terms of cristallinity and morphology, as well as of Sn concentration and strain distribution, is required. 
In this work, several TEM as well as FIB based techniques are employed in this sense. HREM and BF/DF imaging are used to analyse crystallinity (Figs. 1,2) and defect distribution of the materials and STEM/EDS analysis to determine Sn distribution across the layers.
On the other hand, Geometrical Phase Analysis (GPA) provides, by analysing the phase variations between different regions, a detailed map of local distortion as well as revealing the presence of defects.

In particular, we focused on the relaxation mechanism of the initially pseudomorphic GeSn layers.
Finally, we present a preliminary analysis of the local distortion of Kikuchi lines, and hence the local strain fields, in PLIE-grown layers, obtained by Electron BackScattered Diffraction (EBSD) in a FIB. An example of EBSD pattern from the surface of a plain GeSn layer is shown in Fig.3.

References

1 - S. Stefanov, J. C. Conde, A. Benedetti, C. Serra, J. Werner, M. Oehme, J. Schulze, D. Buca, B. Hollander, S. Mantl, S. Chiussi

Laser synthesis of germanium tin alloys on virtual germanium

Appl. Phys. Letters 100, 104101 (2012)

Fig. 1: Fig. 1 - BF image of the whole structure

Fig. 2: Fig.2 – Cross sectional HREM image of the top surface of a GeSn layer, showing very good crystallinity.

Fig. 3: Fig. 3 – EBSD pattern from the surface of a GeSn layer
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2815 Strain determination by CBED in Si-rib structures for photonic devices

Balboni R.1, Bolognini G.1, Corticelli F.1, Ferri M.1, Mancarella F.1, Marini D.1 2, Montanari B. G.3
1CNR, Istituto IMM, Bologna, Italy, 2Università degli Studi di Bologna, Bologna, Italy, 3Laboratorio MIST-ER, Bologna, Italy
balboni@bo.imm.cnr.it

Strained silicon opens interesting perspectives for photonic applications, since the modification in the crystal structure symmetry can give rise to non-linear effects. The deposition of a straining layer on top of a silicon waveguide can break the silicon lattice inversion thus enabling significant linear electro-optic effect [1] or second-harmonic generation [2]. It is therefore important to monitor the stress induced by the process steps used to define the structures and to check the validity of the stress model adopted in the computer simulations of the process itself. In this work the Convergent Beam Electron Diffraction (CBED) technique was used to estimate the lattice deformation induced by a silicon-nitride film deposited on micrometer-scale silicon rib structures.

The manufacturing process consisted in the deposition of low-temperature silicon oxide on Si wafers, followed by photolithography and selective removal through reactive-ion etching. An Si3N4 film was then deposited on the structures, inducing a significant strain inside the silicon ribs. In the analysed samples, shown in Fig. 1, the rib height and width were 450 nm and 2 μm respectively, while the deposited Si3N4 thickness was 375 nm. TEM samples thinned along the [110] orientation were analysed in the [230] projection in a Tecnai F20T transmission electron microscope operated at 200 kV and in the STEM mode; the lamella thickness was estimated about 350 nm. HOLZ lines patterns were recorded in a matrix of points using the procedure reported in [3] and analysed with the ASAC software [4]. The resulting stress configuration was also simulated using a finite-element method.

An overall compressive strain was measured, as induced by the deposited Si3N4. Fig. 2 shows a comparison of the measured and simulated strain component εZZ. Both results behave symmetrically with respect to the rib width with a reduced compressive strain at the centre. Fig. 3 shows the same results for the εXZ shear strain component: as expected, both curves show an antisymmetric behaviour with respect to the rib centre, which is directly reflected in the diffraction pattern features (see insets in Fig. 3). Although the simulation seems to slightly underestimate the crystal deformation, the overall agreement can be considered good.

In conclusion, the combination of process simulation and CBED strain measurement results proved to be effective in predicting the optical behaviour of strained crystal silicon structures.

[1] R. S. Jacobsen et al., Nature, vol. 441, pp. 199-202, May 2006.

[2] M. Cazzanelli et al., Nature Materials, vol. 11, pp. 148-154, Feb 2012.

[3] A. Armigliato, R. Balboni and S. Frabboni, App. Phys. Lett. 86 (2005), p.63508.

[4] http://stream.bo.cnr.it/docs/iTEM_Solution_ASAC.pdf

Fig. 1: ADF STEM image of Silicon rib section. Foil normal is [110] while vertical direction is [001]. The Si3N4 appears brighter in the image than the silicon substrate.

Fig. 2: Experimental (dots+bars) and simulated (squares) of the εZZ strain component across the Si rib (along [-110] direction) at a height of 65 nm with respect to the rib bottom floor.

Fig. 3: Experimental (dots+bars) and simulated (squares) of the εZZ strain component, same conditions of Fig. 2. In the insets a portion of the CBED patterns, registered at the points indicated by the arrows, show an antisymmetrical behaviour.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2819 TEM investigations of InxGa1-xAs quantum dots in GaP

Selve S.1, Niermann T.2, Stracke G.3, Simke J.1, Strittmatter A.3, Bimberg D.3
1Technical University Berlin, Center for Electron Microscopy (ZELMI), Straße des 17. Juni 135, 10623 Berlin, Germany, 2Institut für Optik und Atomare Physik, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany, 3Institut für Festkörperphysik, Technical University Berlin, Hardenbergstraße 36, 10623 Berlin, Germany
soeren.selve@tu-berlin.de

For optoelectronic applications, InGaAs quantum dots (QDs) in GaAs are a well-known materials system with various advantageous properties. However, the integration of GaAs-based devices with standard silicon based technologies remains challenging, because of the large lattice mismatch between these materials. Within the III-V semiconductors GaP allows a pseudomorphic growth on Si due to the small lattice mismatch of only 0.4%. But GaP is an indirect semiconductor, thus not very suitable for optoelectronic applications. Calculations show [1] that InGaAs-QDs in a GaP matrix allow radiative direct transitions under certain composition, size and strain conditions. Such QDs are a promising way to combine their advantageous optoelectronic properties with the structural advantages of GaP.
By metalorganic vapor-phase epitaxy, InGaAs-QDs were successfully grown within GaP. Prior to the InGaAs, a few monolayer (ML) thick GaAs-layer was deposited onto the substrate [2]. After the InGaAs-layer, the growth was interrupted for several seconds to allow for QD-formation. The subsequent deposition of a few ML GaAs facilitates a further strain engineering of these dots. A strong increase in the photoluminescence intensity of these structures indicate the switching from indirect to direct optical transitions within these dots [3].
As the quaternary InGaAsP system is zink blende structure, the strong composition dependence of the {200}-structure factor (Fig. 1) can be exploited to investigate the structural properties of these dots within the TEM. Therefore, all samples were prepared as cross-section along <100> zone axis. For instance, zone-axis HRTEM combined with Fourier filtering of the (020)-coefficient reveals the shape of a truncated pyramid, which is typical for QDs (Fig. 2).
Furthermore, fourier-filtered micrographs under two-beam conditions can be used to obtain further details of the structure. For instance, a comparison of a conventional (200) darkfield (intensity of diffracted beam) with the amplitude of the (200) image Fourier coefficient (amplitude of complex product between direct and diffracted beam) reveals an InP-enriched layer, 7 nm above the InGaAs-layer (Fig. 3).
This enriched layer could be attributed to In segregation during growth.


References:
[1] C. Roberts, et al., Electronic, optical, and structural properties of (In,Ga)-As/GaP quantum dots. Physical Review B 86, 205316 (2012)
[2] G. Stracke, et al., Growth of In0.25 Ga0.75 As quantum dots on GaP utilizing a GaAs interlayer. Applied Physics Letters 101, 223110 (2012)
[3] G. Stracke, et al., Indirect and direct optical transitions in In0.5Ga0.5As/GaP quantum dots. Submitted

The authors acknowledge support from the DFG within SFB 787.

Fig. 1: Composition dependency of the {200} structure factor in Volts. Calculated using the isolated atom approximation and Doyle&Turner atom form factors.

Fig. 2: Real part of Fourier-filtered (020) reflection of HRTEM micrograph (a.u.). The filtered intensity is proportional and sensitive to compositional changes.

Fig. 3: Comparison of contrasts in conventional (200) darkfield micrograph (left) and Fourier-filtered (200)-coefficient of image under two-beam conditions-revealing an InP-enrichment due to segregation above the InGaAs-layer.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2843 On the influence of the sample preparation and analysis method of TEM bismuth telluride

Milagres T.1, Balzuweit K.1,2, Nascimento V. B.1, Ladeira L. O.1, Soares E. A.1, Carvalho V. E.1
1(1) Physics Department of the Federal University of Minas Gerais (UFMG), 2(2) Center of Microscopy of the Federal University of Minas Gerais (CM-UFMG)
thaismilagres20@gmail.com

Bismuth Telluride is a very well known thermoelectric material with relatively high coefficients at room temperature. Recently, interest in bismuth telluride has been renewed as scientists measured it as a 3D topological insulator. Bismuth telluride is a relatively easy material to obtain and different compositions are being studied both as bulk material and as thin films.

The main aim of the study is to compare the structure of bulk and surface of Bridgman grown crystals using both transmission electron microscopy (TEM and HRTEM) and surface characterization techniques as Low Energy Electron Diffraction (LEED).

Crystals of Bi2Te3 were Bridgman grown in a sealed quartz ampoule in a directional resistance oven at a temperature of 600oC. The crystals were slowly cooled down to room temperature and the quartz ampoule broken to retrieve the grown crystal. Conventional X-ray diffraction showed patterns compatible with a single crystal along the sample except for the starting point, which was discarded. Low loss energy measurements were performed and also showed patterns compatible with a single crystalline sample.

The grown crystals were cleaved and fixed on a substrate for LEED. The samples for TEM were prepared in two ways: cleaved pieces were crushed in an agate mortar and deposited directly onto a holey carbon copper grid. Thin cleaved slices were chosen and mounted directly onto a sample holder and cut into thin slices with a Leica UC6 Ultramicrotome with a diamond knife into water. The thin slices were fished out and deposited onto a holey carbon coated TEM grid. A second ultramicrotome sample with a thinner cut was also made.

The samples were analyzed at a 200kV transmission electron microscope (Tecnai G2-20 from FEI). The LEED samples were cleaved then sputtered with 0.5 keV Ar+, annealed for 60 minutes at 473 K and cooled down to 223 K before measurement.

The four diffraction patterns show a quite distinct behavior of the different preparation methods and analysis.

The authors would like to acknowledge the Center of Microscopy at the Universidade Federal de Minas Gerais (www.microscopia.ufmg.br) for providing the equipment and technical support for experiments involving electron microscopy.

Fig. 1: Diffraction pattern of the crushed sample

Fig. 2: Diffraction pattern of the ultramicrotome cut sample

Fig. 3: Diffraction pattern of the thinner ultramicrotome cut sample

Fig. 4: Low loss diffraction pattern
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2872 Amorphization of Indium Phosphide by High Energy Gold Ion Bombardment

Khalil A. S.1
1Tabbin Institute for Metallurgical Studies (TIMS), Cairo, Egypt
askhalil2004@yahoo.com


Irradiation of single crystalline compound semiconductors with energetic heavy ion beams produces disorder in the irradiated crystal lattices. Indium Phosphide (InP) is an important technological III-V compound semiconductor used in space solar arrays and variety of electronic devices. In this investigation, both electron transparent and bulk samples cut from InP (001) wafer were bombarded by 200 MeV Au+ ions (~ 1MeV/amu) at fluencies ranging from 5x1010 to 1x1014 ion/cm2 in order to study the amorphization process of the InP (complete atomic disorder of the original InP crystal lattice). As shown in figure 1 for thin foil samples observed by TEM, the progression of amorphization is followed with increasing the fluence of irradiating Au+ ions. Complete amorphization occurs at fluence of < 5x1013 ion/cm2 as confirmed by the inset showing the selected area electron diffraction (SAD) pattern were the diffused rings and the absence of any diffraction spots are synonymous with amorphous state. This observation was further supplemented by Rutherford Backscattering Spectrometry (RBS/C technique) for the bulk InP samples which showed that at irradiation fluence of 1x1014 ion/cm2 the sample surface was completely amorphous. Generally, amorphization in swift heavy ion irradiated compound semiconductors like InP can best be described by a combination of both hetergenous mechanism (direct impact amorphization were each ion creates an amorphous ion track) and homogenous mechanism (defect accumulation were each ion creates an agglomeration of point defects). Models have been developed wherein an impinging ion can produce both a combination and coexistence of both amorphous ion tracks and point defect-rich zones which, when overlapping, convert the crystal to amorphous state [1]. By invoking the Hecking model [2] for the accumulation of damage we showed that the amorphization process proceeds by the accumulation and overlap of ion tracks.
In this model an indicator of the amorphization process are the values of relative disorder (∆χ)min defined as:

(∆χ)min = (Yirradiated - Yunirradiated) / (Yrandom - Yunirradiated)

Where Y is the measured RBS/C yields. The values (∆χ)min give an estimate of the relative amount of disorder in the crystal and reaches unity for a complete amorphous state. Plotting (∆χ)min versus the ion fluence as shown in figure 2 we conclude that amorphization favours the homogenous mechanism [3]. This was confirmed by HRTEM observations which show that individual ion track cores are not amorphous as shown in figure 3.

References

[1] W.J. Weber, Instruments and Methods B, 166/167 (2000) 98-106.
[2] N. Hecking et al, Nuclear Instruments and Methods B, 15 (1986) 760-764.
[3] A.S. Khalil, PhD Thesis, Australian National University, Australia (2007).

Fig. 1: Figure 1: TEM micrographs for three different irradiating fluencies. In (a) for 5x1010ion/cm2, the ion tracks are well separated and in (b) overlap leads to amorphous pockets at 1x1013 ion/cm2. These will grow with increasing ion fluencies till complete amorphization occurs at ion fluence of 1x1014 ion/cm2 (c).

Fig. 2: Figure 2: The plot of (∆χ)min versus Au+ ion fluencies, the best fit can be described by the Hecking model which gives more weight to homogenous mechanism parameter σs rather than the heterogeneous mechanism parameter σa.

Fig. 3: Figure 3: HRTEM of an ion track core, the continuation of lattice fringes implies that the core is not amorphous.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2877 Mechanism of the Ti-assisted Al-induced layer exchange (Ti.AlILE)

Kraschewski S. M.1, Butz B.1, Gannott F.2, Zaumseil J.2, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Department Werkstoffwissenschaften, Universität Erlangen-Nürnberg, Germany, 2Lehrstuhl für Polymerwerkstoffe (LSP), Department Werkstoffwissenschaften, Universität Erlangen-Nürnberg, Germany
simon.m.kraschewski@ww.uni-erlangen.de

Al-induced layer exchange (AlILE) is an effective route to fabricate polycrystalline seed layers from amorphous Si (a-Si) for solar cell and transistor applications. The process utilizes the tuneable nucleation and growth of crystalline Si (c-Si) in nano-crystalline Al (Fig. 1). This means, heating a stack of a-Si/diffusion barrier/Al at a temperature of around 450 °C leads to the diffusion of Si into the Al layer followed by the sparse crystallization of Si at some Al grain boundaries and the subsequent in-plane growth of those crystallites. Thereby Al is replaced by Si (change of stacking order). This results in Si grain sizes up to several tens of micrometres. A crucial role for this reaction plays the barrier layer between Si and Al, which is intendedly induced in order to tailor the diffusion kinetics (larger Si grain sizes). Usually, the barrier layer is formed by oxidizing the surface of the Al prior to deposition of a-Si. Recently, it has been shown that with oxidized Ti as a barrier layer the resulting Si grain sizes can be increased up to 250 µm [1]. However the underlying mechanism is not clarified.

In our study, layers of Al (45 nm), Ti/TiOx (2-5 nm) and a-Si (85 nm) were subsequently deposited onto quartz glass using e-beam PVD. The Ti layer was either natively oxidized in air (Ti/native TiOx) or TiOx was directly deposited (PLD-TiOx). In situ heating light microscopy was performed in order to pre-characterize the process kinetics of the samples (growth of c-Si). For TEM studies a FEI TITAN3 80-300 was used. In situ TEM heating experiments with a DENSsolutions heating holder were conducted to follow reaction-phase formation and Si crystallization. It turned out in our experiments that the complete oxidation of Ti is important to generate secondary-phase free c-Si layers. The in situ heating experiments further revealed that, in the case of only natively oxidized Ti (remaining metallic Ti in the vicinity of the Al layer), the metallic Ti forms secondary phases with Al and Si at grain boundaries of the Al layer (brighter crystallites in Fig. 2b). The most important result is, independent of the complete oxidation of the deposited Ti, that the TiOx is unintendedly reduced by the underlying Al during heat treatment. This causes the formation of aluminium oxide and metallic Ti, which can further react to secondary phases in the upper layer (Fig. 3b). EDXS and SAED show that the secondary phases exhibit TiAl3-type structure and consist of Ti, Al and Si. Our results indicate that the Ti.AlILE process is similar to a conventional AlILE process where native aluminium oxide is utilized as the barrier layer, simply due to the fact that such an oxide forms upon reduction of the TiOx.

[1] Antesberger et al., J APPL PHYS 112, (2012)

The authors gratefully acknowledge financial support by the DFG via research training group 1896 and the cluster of excellence EXC 315. DENSsolutions is acknowledged for providing a sample heating system.

Fig. 1: Ti.AlILE process using a) native oxidized Ti and b) PLD-TiOx: Starting at 350 °C, in case of a), the unoxidized Ti forms a secondary phase in the bottom layer (cf. 2b). In both cases, a) and b), later Al reduces TiOx, releasing Ti, which forms a secondary phase in the upper layer and Si crystallizes as in conventional AlILE.

Fig. 2: Ti.AlILE process with natively oxidized Ti: ADF-STEM planview images (the a-Si layer on top is not recognizable): a) before heat treatment (cf 1a) and b) after 30 min at 450 °C showing secondary phases (brighter crystallites) in the original Al layer containing Ti, Al and Si (preferentially formed at grain boundaries of Al).

Fig. 3: Reduction of TiOx by Al: ADF-STEM cross-sectional images of a) unreacted and b) reacted region of a sample with PLD-TiOx (30 min at 450 °C). While the TiOx layer is clearly discernable in a) (bright contrast), it has disappeared after reaction due to the reduction of the TiOx by Al. This causes the formation of secondary phases in the upper layer.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-2953 Alkali metal-TCNQ organic semiconducting charge transfer complex materials on textile as 3D templates as flexible electronic devices

Ramanathan R.1, Field M. R.2, Bansal V.1
1NanoBiotechnology Research Laboratory, RMIT University, GPO Box 2476, Melbourne, VIC 3000, Australia, 2RMIT Microscopy and Microanalysis Facility, RMIT University, GPO Box 2476, Melbourne, VIC 3000, Australia
matthew.field@rmit.edu.au

Metal-organic semiconducting materials that are based on the charge transfer complexes of 7,7,8,8-tetracyanoquinodimethane (TCNQ) have gained significant attention due to interesting opto-electronic properties, which have generated opportunities for fabricating organic nanostructured optical and electronic devices[1]. Among the various metal-organic complexes, majority of recent efforts have focused on investigating CuTCNQ and AgTCNQ[2]. Notably, although alkali metal-TCNQ complexes such as LiTCNQ, NaTCNQ and KTCNQ also have interesting electrical charge transportation properties, these materials have not received intensive scrutiny for device fabrication, particularly due to the high reactivity of alkali metals and therefore significant challenges associated with their fabrication[3].
From nanomaterial templating perspective, textiles belong to a class of versatile materials that are typically porous, flexible, and are woven into a 3D matrix. These interwoven structures are relatively stable, rich in surface area and exhibit hierarchical ordering [4]. The true potential of textiles can be further realised by using them as microtemplates for directly growing nanomaterial and/ or adding interesting functionalities. In this context, the use of textiles as templates to directly grow metal-TCNQ organic semiconductor charge transfer complexes offers unique advantages in terms of low-cost yields, flexibility, ease of synthesis and robustness. More importantly, the good absorbent property of cotton-based textiles allows high binding of ionic reactant species to textile fibres by simple solution immersion. This research outlines a simple ‘dip-grow’ approach to synthesise alkali-metal TCNQ nanostructures on a 3D textile template and demonstrate their capability for opto-electronics and gas sensors.

V. Bansal thanks the Australian Research Council for research funding through the Discovery and Linkage grant schemes. V.Bansal also acknowledges the support of the Ian Potter Foundation to establish a multimode spectroscopy facility at RMIT University, which were used in this study. Theinstrument and technical support from the RMIT Microscopy and Microanalysis Facility is duly acknowledged.

Fig. 1: SEM images of the uniformly decorated high aspect ratio (~100) nanorods of 10-50 µm length KTCNQ arrays grown around textile fibres in radial symmetry. The absence of microrods on untreated substrate ascertains that the observed structures are indeed alkali metal-TCNQ.

Fig. 2: SEM images of NaTCNQ arrays grown around textile fibres. The absence of microrods on untreated substrate ascertains that the observed structures are indeed alkali metal-TCNQ.

Fig. 3: An I-V curve of K-TCNQ in the presence and absence of solar-light. A pronounced hysteresis in the I-V characteristics was observed due to switching from a high to low resistive state
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-3000 Dependence of microsrtucture in AlN thin films on the annealing temperature for sapphire substrate

Kuwano N.1, Jesbains K.1, Akiyoshi R.2, Hayashi K.2, Soejima Y.2, Itakura M.2, Miyake H.3, Hiramatsu K.3
1MJIIT, Universiti Teknologi Malaysia, Kuala Lumpur, Malaysia, 2Kyushu University, Kasuga, Japan, 3Mie University, Tsu, Japan
noriyuki.kuwano.577@m.kyushu-u.ac.jp

Aluminum nitride (AlN) crystallizes in wurtzite structure (P63mc) with the lattice parameters of a=0.311 nm and c=0.498nm. AlN is a very promising material for application not only to surface acoustic wave (SAW) devices and optoelectronic devices for very short wavelength regions but also substrate materials. In the course of research on metal-organic vapor phase epitaxy (MOVPE) of AlN, it was found that the crystallinity of AlN depends upon the annealing temperature of sapphire substrate. In this work, detail characterization was conducted to clarify the origin of its annealing temperature dependence.

Prior to the deposition of AlN, a sapphire (0001) substrate (0.2 degree off toward m-direction) was annealed at Tan=1150 - 1350oC for surface-cleaning. Thereafter, AlN was deposited by MOVPE first at 1200oC to be 100nm in thickness and then overgrown at 1500oC. Thin-foil samples were made with a focused ion beam (FIB) mill (Hitachi. FB-2000K), followed by finishing with an argon-ion mill. TEM observation was performed with a JEM-2000EX microscope (JEOL). SEM observation and EBSD analysis were also carried out (Zeiss, Ultra55) for the AlN specimens without any prior treatment such as coating or etching.

It was found from the X-ray rocking curve (XRC) measurement that the twisting of AlN along c-axis is around 3 degree for Tan > 1250oC, while very little twisting occurs for Tan < 1250oC. Fig. 1 shows the results of TEM observation for the AlN specimens of Tan=1350oC and 1225oC. One can see that in both specimens, threading dislocations (TDs) have been formed and run upward. The densities of dislocations with screw and edge components are roughly estimated to be approximately 109 cm-2 and 1010 cm-2, respectively. In the specimen of Tan=1350oC, TDs are straight in shape and form a columnar structure of AlN. Fig.2 shows SEM and EBSD results of the specimens. In the SE (out-lens) image of the specimen of Tan=1350oC shown in (a), one can see pits and steps. In the SE (In-lens) image (b), dark winding line-contrast is recognized. The locations of the line contrast coincide with those of steps in (a). The grain reference orientation deviation (GROD) map (the maximum deviation~3o) shown in (c) indicates clearly that the dark line contrast corresponds to the boundaries of twisting domains. For the specimen of Tan=1225oC, on the other hand, pits and step are seen in the SE-image (Out-lens) shown in (d), but dark line contrast is not recognized in (e), and the GROD map in (f) does not indicate the existence of twisting domains. These results suggest that low-angle grain boundaries can be observed in SE image (In lens). The origin of the contrast is not clarified.

This work was partly supported by FRGS/2/2013/SG06/UTM/01/2-18865 from MOHE, Malaysia.

Fig. 1: CTEM results for the cross section of AlN/sapphire. (a)(b)(c) Specimen of Tan=1350oC, (d)(e)(f) Specimen of Tan=1225oC, (a)(d) Bright field image; zone axis illumination // [01-10], (b)(e) Dark field image; two-beam condition g=0002, (c)(f) Dark field image; two-beam condition g=2-1-10.

Fig. 2: SEM and EBSD results for the top surface of AlN.   Acceleration voltage is 2 kV. Direct magnification =30k.  (a)(b)(c) Specimen of Tan=1350oC, (d)(e)(f) Specimen of Tan=1225oC, (a)(d) SE image (out-lens), (b)(e) SE image (In-lens), (c)(f) Grain reference orientation deviation (GROD) map; Maximum deviation angle= 3o

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-3096 Polarity-Related Growth Mechanism of Branched GaN Nanostructures with InGaN Quantum Wells

Zamani R. R.1, Oppo C. I.1, Müller M.2, Karbaum C.2, Bertram F.2, Christen J.2, Malindretos J.1, Rizzi A.1
1IV. Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany, 2Institut für Experimentelle Physik, Otto-von-Guericke-Universität Magdeburg, Magdeburg, Germany
reza.r.zamani@gmail.com

III-nitrides are important compounds in solid state lighting and display systems as they are appropriate semiconductor materials for light-emitting diodes (LEDs) with high efficiency and functionality. However, further improvement in material quality and structure design is needed. In particular, the polarization issue in the III-N wurtzite (WZ) heterostructures has been extensively investigated. Growth directions other than along the usual polar c-axis are necessary for the extension of the emission spectrum from the UV-blue towards the green with high efficiency. The LEDs active region is based on GaN/InGaN multi quantum well (MQW) structures, as both material quality and In incorporation are critical issues when growing on non-polar or semipolar directions in planar devices.1 The growth of InGaN/GaN MQWs on different facets of nanostructures might be a promising approach for the extension of the emission spectrum.2

Here, Ga-polar GaN nanocolumn arrays3 with InGaN quantum wells are grown by molecular beam epitaxy (MBE) and the growth mechanism is studied. Interestingly, the nanocolumns branch out under particular conditions. Branching is attributed to the matters of polytypism, and polarity in the structure.4 It is well-known that c-planes with N polarity have the highest potential for growth of GaN nanocolumns compared to all other polar, semipolar, and non-polar facets.5 In the case of Ga-polar nanocolumns studied in this work, the number of structural defects increases, while In is incorporating. This eventually leads to the formation of a tetrahedron-like zinc-blende GaN on top of the column, which provides three free N-polar facets ({111} planes) and acts as a seed for the growth of three branches. It is also observed that the particular growth condition, together with In presence stimulate the growth of non-polar facets. While the branches grow in N-polar [0001] direction, they laterally expand. This expansion, which can also be seen in parasitic N-polar nanowires grown on the substrate mask material (Mo), is significantly higher than the case of pure GaN with normal growth conditions, because the lateral growth in nonpolar direction is almost equal to the growth in [0001] N-polar direction.

Moreover, cathodoluminescence (CL) in SEM and TEM is performed in order to study the luminescence properties of the branched nanostructures with high spatial resolution.

References

1 S. P. Denbaars, et al, Acta Materialia 61, 945 (2013)

2 C.Y. Cho, et al, Appl. Phys. Lett. 93, 241109 (2008)

3 Urban et al, New J. Phys. 15, 053045 (2013)

4 R.R. Zamani et al, ACS Nano 8, in press (2014), DOI: 10.1021/nn405747h

5 S. Li, A. Waag, J. Appl. Phys. 111, 071101 (2012)

The authors acknowledge the European Union Seventh Framework Program under ‘nanowiring’, with grant agreement number 265073.

Fig. 1: (a) SEM micrograph of a Ga-polar GaN/InGaN nanocolumn array; the inset show a bird-view of a branched nanocolumn. (b) STEM image showing the structure of the branched and non-branched nanocolumns, (c) EELS compositional maps showing Ga in red and In in green; (d) TEM image of a non-branched WZ nanocolumn and (e) the corresponding power spectrum
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-3138 Dark field holography strain analysis of buried AlAs/oxide stressor layers

Kießling F.1, Niermann T.1, Schulze J. H.2, Strittmatter A.2, Schliwa A.2, Pohl U. W.2, Lehmann M.1
1TU Berlin, Institut für Optik und Atomare Physik, Straße des 17. Juni 135, Sekr. ER 1-1, 10623 Berlin, Germany, 2TU Berlin, Institut für Festkörperphysik, Hardenbergstr. 36, Sekr. EW 5-2, 10623 Berlin, Germany
felix.kiessling@physik.tu-berlin.de

Side-controlled quantum dot (QD) growth is a promising solution for single-photon sources. The nucleation of self-organized QDs can be influenced by a buried oxide stressor formed by a partially oxidized AlAs layer beneath a GaAs layer [1]. The length differences of Al-O and Al-As bonds form a locally varying strain field modifying the free energy of the GaAs (001) surface.
We used dark field electron holography (DFH) [2] in Lorentz mode to directly measure the strain distribution over a large field of view. The resulting geometric phase contains the strain gradient which can be directly obtained for uniform thick specimen [3].
For specimen preparation, focused ion beam (FIB, FEI Helios Nanolab 600) etching was used to fabricate a specimen with a smooth thickness of about (110 ± 10) nm. Images were recorded by FEI Titan Berlin Holography Special 300 kV TEM at the TU Berlin.
The examined specimen was a linear wedge-shaped mesa test structure with a buried oxidized AlAs layer [4]. The layer sequence from substrate to the surface comprises only the stressor structure of partially oxidized AlAs and a GaAs spacer layer. The stressor structure consists of a sandwich of Al0.9Ga0.1As/AlAs/Al0.9Ga0.1As which was laterally oxidized from the mesa edges by water vapor [5] forming an oxide aperture (1.08 ± 0.02) µm wide (fig. 1). The bright contrast region on both sides corresponds to the oxidized part of the AlAs layer.
Figure 3 shows the strain distributions as obtained from DFHs of (111) and (111). In all four images, strain-free GaAs substrate region below the AlAs were used to quantify the strain in the corresponding maps. Directly above the edges of the oxidization front symmetric to the opening, compressive strain in growth direction (εzz) (fig. 3a) and tensile strain in-plane (εyy) (fig. 3b) in the same area is detectable. The shear component (fig. 3c) is not detectable and the strong rotation (fig. 3d) exhibits an anti symmetrical behavior.
The behavior of our analysis corresponds well with calculated strain fields based on continuum elastic models [4] in the area of the aperture opening (fig. 2). The measured local tensile surface strain up to 0.5 % is by a factor of 4 smaller than the calculated one. This results from specimen relaxation, preparation artifacts and averaging due to the specimen tilt of about 15°. Such tensile surface strain can influence the QD nucleation in the aperture area.

1. A. Strittmatter et al., Applied Physics Letters 100 (2012) 093111.
2. M. Hÿtch et al., Nature 453 (2008) 1086.
3. M. Hÿtch et al., Journal of Physics: Conference Series 241 (2010) 012027.
4. A. Strittmatter et al., physica status solidi (a) 209 (2012) 2411.
5. K. D. Choquette et al., IEEE Journal of Selected Topics in Quantum Electronics 3 (1997) 916.

This work is supported by the DFG Collaborative Research Centre 787 ‘Semiconductor Nanophotonics’.

Fig. 1: a) Sketch of linear wedge-shaped mesa with position of TEM lamella, b) bright field conventional TEM overview of the mesa showing the edges of the aperture. The oxidized layer is visible as bright contrast in the AlGaAs layer sandwich.

Fig. 2: Calculated line scans of surface strains εxx + εyy across a circular mesa of 3 µm diameter exhibiting different aperture sizes; the horizontal line marks unstrained GaAs [4]. The magenta colored line is the central moving average of nine data points (27 nm width) from line scan in Figure 2b increased by a factor of 4 for better comparison.

Fig. 3: Local strain of the lattice in percent with a) strain of in growth direction εzz (002), b) strain in lattice direction εyy (220), c) shear component εzy, and d) the rotation component ωzy. Scale bar corresponding to the color in a)-d).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-3317 Investigating the structural properties of multi-layers InAs quantum dots structures using scanning transmission electron microscopy

Landi S. M.1, Senna C. A.1, Pires M. P.2, Souza P. L.3
1Instituto Nacional de Metrologia, Qualidade e Tecnologia, DIMAT, RJ, Brasil, 2Universidade Federal de Rio de Janeiro, IF, RJ, and DISSE-INCT, Brasil, 3Pontifícia Universidade Católica de Rio de Janeiro, LabSem, RJ, and DISSE-INCT, Brasil
smlandi@inmetro.gov.br

Self-assembled semiconductor quantum dots (QDs) are of interest for a wide range of optoelectronic applications that include infrared photodetectors, lasers and optical amplifiers [1]. As the structural properties of the QDs, such as their morphology and chemical composition, determine their optical and electronic properties, accurate techniques for understanding of these parameters are required.
In this communication we describe the results of an aberration-corrected scanning transmission electron microscopy (STEM) study in combination with X-ray energy dispersive spectrometry (EDS) to investigate the structural properties of multiple layers of InAs QDs grown on InGaAs and capped with InP. We analyzed the strain field of QDs that determines the nucleation in the subsequent layers. STEM/EDS elemental maps show the effect of this strain field on the composition of the InGaAs layer, suggesting a interdiffusion between In and Ga atoms. We also use atomic resolution STEM to identify local changes of lattice distances. The samples were grown by metal-organic vapor phase epitaxy on a (100) InP substrate. The cross sectional specimens for TEM analysis were prepared by focused ion beam technique (FIB). HRTEM and STEM/EDS analysis were performed using a Cs-probe corrected Titan 80-300 operating at 300kV.

[1] M. H. Degani, M. Z. Maialle, P. F. Farinas, N. Studart, M. P. Pires, P. L. Souza, Multiple-photon peak generation near the ~10 μm range in quantum dot infrared photodetectors, J. Appl. Phys. 109 (2011) 064510–1-8

This work was partially support by the CNPq

Fig. 1: High resolution STEM image of an InAs quantum dot.

Fig. 2: (a) HAADF image of stacked QDs. (b) – (f) STEM/EDS elemental maps of the marked area: (b) HAADF, (c) As, (d) In, (e) Ga and (f) P.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-3337 Electron tomography and aberration-corrected TEM and STEM study of the polarity of colloidal wurtzite CdSe nanopyramids used in assemblies and as seeds for CdS pod growth

Brescia R.1, Bertoni G.1,2, Turner S.3, Ghosh S.1, Arciniegas M. P.1, Manna L.1
1Nanochemistry, Istituto Italiano di Tecnologia, via Morego 30, IT 16163 Genova, Italy, 2IMEM-CNR, Parco Area delle Scienze 37/A, IT 43124 Parma, Italy, 3EMAT, University of Antwerp, Groenenborgerlaan 171, BE 2020 Antwerp, Belgium
rosaria.brescia@iit.it

Several protocols have been reported in the last few decades for the production of colloidal CdSe nanocrystals (NCs) with the wurtzite crystal structure. In particular, NCs elongated along the c-axis were subjected to studies both aimed at calculation of equilibrium configurations (e.g., [1]) and at the experimental determination of the polarity (e.g., [2]). These results report of NCs often showing different endings at the opposite <0001> directions, with flat ends pointing towards the [0001] direction. This is due to the preferential binding of the most common surfactants to the Cd ions and to the consequent higher stability of (0001) facets, terminating with Cd atoms exposing one dangling bond.
In this contribution, CdSe NCs were obtained using a mixture of two different Cd precursors (CdCl2 / CdO=50% wt.) and a lower amount of phosphonic acids (hexylphosphonic acid and octadecylphosphonic acid) compared to the standard CdSe nanorod synthesis. The synthesis results in monodispersed wurtzite CdSe nanopyramids (NPs, Fig. 1) with a {0001} equilateral base facet (side length = 20 nm) and three lateral {01-12} isosceles sides (lateral edges = 16 nm). The NP shape is directly evidenced by HAADF tomography volume reconstruction (Fig. 2a). Preliminary results of aberration-corrected HRTEM imaging in negative CS conditions [3] and exit wave reconstruction (EWR) show phase variations in few Cd-Se dumbbells (ZCd=48, ZSe=34), suggesting the NP base as corresponding to (000-1) planes (Fig. 1 b-c). These analyses were carried out on an image-corrected Jeol JEM 2200FS microscope operated at 200 kV. A further preliminary proof comes from annular bright field (ABF) imaging, obtained on a probe-corrected FEI Titan “cubed” microscope (operated at 200 kV, see Fig. 1d).
The unusual polarity of these CdSe NPs is mainly related to the use of CdCl2 as a Cd precursor, as Cl- is known to form complexes with phosphonic acids [4], therefore limiting their amount for NC surface passivation. This effect is also enhanced by the relatively low amount of phosphonic acids employed during the process. The detailed knowledge of the faceting of these peculiar NCs will be useful to elucidate the seeded growth of wurtzite CdS branches starting from the NPs (Fig. 2b). The knowledge of the polarity of the NP base will be fundamental also to explain the formation of peculiar patterns based on these NCs upon addition of particular surfactants (Fig. 2c).

[1] L. Manna et al., J. Phys. Chem. B 109, 6183 (2005)
[2] G. Bertoni et al., ACS Nano 6, 6453 (2012)
[3] K. Urban et al., Phil. Trans. R. Soc. A 367, 3735 (2009)
[4] M. R. Kim et al., ACS Nano 6, 11088 (2012)

The authors acknowledge the European Commission FP7 Integrated Infrastructure Initiative ESTEEM2 for partially funding this research project.

Fig. 1: (a) Overview HAADF image of CdSe NPs. (b) HRTEM image of a NP ([10-10] z.a., CS=-40 μm, defocus=11 nm). (c) EWR phase for the same NP (magnified portion). Some columns with higher phase are directed towards the apex (NP sketch in the corner). (d) Filtered ABF images of a NP portion, showing intensity variations in few dumbbells ([10-10] z.a.).

Fig. 2: (a-b) HAADF tomography-based volume reconstructions (isosurface views) of (a) two views of a CdSe NP and (b) a CdSe/CdS branched NC obtained by seeded growth starting from a CdSe NP. (c) BF-TEM image of a region showing four-leaved clover assemblies of the NPs obtained upon addition of particular surfactants.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-3339 TEM analysis and electrical probing on thin TEM lamellas of CBRAM stacks

Seidel F.1,2, Richard O.1, Bender H.1, Hantschel T.1, Goux L.1, Jurczak M.1, Vandervorst W.1,2
1imec, Kapeldreef 75, B-3001 Leuven, Belgium, 2KUL, IKS, Celestijnenlaan 200d - bus 2418, B-3001 Leuven, Belgium
seidelf@imec.be

Conductive bridging RAM (CBRAM) is an emerging nonvolatile memory concept and alternative to RRAM, PCM or MRAM, offering lower energy consumption, higher speed and better 3D implementation opportunities. Despite research efforts, there are still two major obstacles to be overcome: the cycle-to-cycle and device-to-device variability. One cannot tackle these problems without examining the functioning of such device structures at the atomic level. TEM observation of the conductive bridge, also referred to as filament, is challenging due to localization problems and FIB preparation influences. One can avoid these issues by direct electrical contacting the electron transparent TEM lamellae. This requires the ability to create a stable electrical contact on a < 100 nm thin TEM lamella without destroying it.

In this work we prepared TEM lamellae from a blanket layer stack containing a 5 nm Al2O3 electrolyte and created a top contact by depositing Pt using the FIB beam. As bottom contact the TEM Cu grid was used. To lower the contact resistance, a 100nm thick platinum layer is sputtered over the specimen prior to the final thinning process. After thinning, the lamellae are cut into several cells allowing multiple filament creation within one lamella (fig. 2). In order to contact these small areas we rely on a so-called nanoprobing setup implemented in a SEM. The setup is based on four independent probes with fine mechanical control as they are piezo-driven, providing sub-nm precision for landing on the TEM lamellae contacts.

Basically the probe-contact area can now be used to impose the required I-V cycling of the dielectricum. However one does observe that the contacting resistance dominates the entire system such that the resistance drop from a filament bridging the Al2O3 layer cannot be observed. To overcome this problem the quality of the contact resistance is improved by replacing the commercially available tungsten probes which have a thin oxide on their surface causing the high resistance, with conductive diamond tips. These probe are in-house fabricated, oxide free and gave a good electrical contact with substantially decreased tip wear (fig. 1).The IV’s obtained with these nanoprobes, show a gradual SET process, with a resistance change from the high to low resistance state of 100 X (fig. 3). It is shown that the cells on these lamellae can be reset to their original high resistance state at least 5 times. In order to elucidate the material changes related to the SET/Reset process, TEM/STEM and chemical EDS analysis are performed in a Tecnai and Titan microscopes, on the structures before/after the switching (fig. 4).

Felix Seidel acknowledges the Institute for Promotion of Innovation by Science and Technology in Flanders (IWT) for his Ph.D. fellowship.

Fig. 1: Figure 1: SEM image during the nanoprobing with diamond tips. Probe P1 functions as bottom electrode contact on the Pt coated Cu grid and P2 as top electrode on the ion Pt. Inset: stack scheme, FIB-cut and Pt backcontact. Red line indicates position of expected filament.

Fig. 2: Figure 2: STEM image of the sample before switching. FIB cuts reach into silicon and divide the specimen into cells, which can be tested independently. Inset: HR-TEM of switching layers. If a positive bias is applied on the Cu, Cu+ ions bridge through the Al2O3 layer towards the Si.

Fig. 3: Figure 3: IV cycling on a single cell of the stack on the thinned lamella allows to set and reset the device several times.

Fig. 4: Figure 4: EDX map showing the distribution of elements.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-3402 In-situ TEM with electrical characterisation of tapered InAs nanowires with Ohmic contacts

Zhang C.1,2, Xu Q.2, Zandbergen H. W.2
1School of Computer, National University of Defense Technology, Changsha 410073, China, 2Delft University of Technology, Kavli Institute of Nanoscience, 2628 CJ Delft, the Netherlands
h.w.zandbergen@tudelft.nl

Semiconductor nanowires have attracted much interest due to their outstanding properties as building blocks in nanoelectronic devices [1]. Among these, InAs nanowires with a smaller band gap and higher electron moblility exhibits particular potential for high performance transistors, memory, interconnects and sensors. Since a high stability of these nanowires (NW) is required, it is important to perform failure tests. There are some reports on in-situ TEM with electrical failure tests [e.g. 2-5] on the electrical properties as well as the failure tests of semiconductor nanowires, but these were done with an in-situ STM tip in a TEM holder. In this set-up the electrical contact of the STM tip with NWs is realized by moving the STM tip to contact one side of the NWs However, it is difficult to control the contact quality as well the contact resistance between the NWs and STM tip. We have developed an alternative set-up allowing the investigation of the electrical properties as well as breakdown of tapered InAs nanowires with Ohmic contacts using a homemade in-situ TEM biasing holder. Furthermore, by having more than two contacts on the nanowire we could also measure the contact resistance of the applied contacts (except for the most outer contacts), which showed that the contact were Ohmic. By measuring a number of segments of a tapered nanowire we determined that the electrical resistivity is constant (~10-2Ω·cm) for nanowires with diameter larger than 120nm and gradually increases for thinner sections. The electrical breakdown started in the position close to the cathode side, and starts with electromigration of In, followed by the sublimation of arsenic. The critical current density for breakdown was about 106 A/cm2. The set-up for electrical measurement is shown in Fig. 1(a). The electrodes deposited on silicon nitride membrane consist 5 nm Cr and 95 nm Au with a width of 500 nm and separated by 4 µm from each other. Then of 500nm wide 5 µm long gaps were created by drilling holes on the membrane using a FIB. Next, a single tapered nanowire was transferred onto the electrodes by using an ex-situ nano-manipulator. Finally platinum was deposited on top of the joints between the nanowire and the electrodes with FIB to ensure a good contact quality. REFERENCES 1. S. W. Nam, et al, Science 336, 1561(2012) 2. T. Westover, et al, Nano Lett. 9, 257 (2009). 3. Z. Xu, D. Golberg, and Y. Bando, Nano Lett. 9, 2251 (2009). 4. K. Davami, et al, Chem. Phys. Chem. 13, 347 (2012) 5. J. Zhao, H.Y. Sun, S. Dai, Y. Wang, and J. Zhu, Nano Lett. 11, 4647 (2011).

This work was supported by the ERC project 267922–NEMinTEM and the National Natural Science Foundation of China (Grant No. 61106084 and 61332003). C. Zhang and H. Wang are grateful for the support of China Scholarship Council.

Fig. 1: (a) Scheme of the in-situ chip. The tapered InAs nanowire is connected to five electrodes on a SiN membrane. Each of the electrodes is 500 nm wide and separated by 4µm. Slits are present on the SiN membrane, allowing TEM of the NWs suspended over these slits. (b) bright field TEM image of tapered InAs nanowires with Ohmic contacts.

Fig. 2: (a) HAADF image of one InAs nanowire after breakdown, particles were found close to the anode, while the breakdown happened in a position close to the cathode. (b)-(d) EDX maps of the nanowire at low magnification, which shows clearly the particles close to the anode are rich in indium and oxygen.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-3461 Microstruture characterization of Ti/Al/Ti/Au and Ti/Al/Mo/Au based ohmic contacts on AlGaN/GaN Heterostructures

Chandran N.1, Kolaklieva L.2, Dhanasekaran V.1, Kakanakov R.2, Sall M.1, Polychroniadis E. K.1
1Department of Physics, Aristotle University of Thessaloniki, Thessaloniki - 54124, Greece, 2Central Laboratory of Applied Physics, Bulgarian Academy of Sciences, 59, St. Petersburg Blvd, 4000 Plovdiv, Bulgaria
naren@physics.auth.gr

The multilayer metallization schemes of Ti/Al/Ti/Au and Ti/Al/Mo/Au ohmic contacts are prepared at identical techniques and conditions but altered only the barrier metals as Ti and Mo with same thickness of 30 nm. Scanning Transmission electron microscopy (STEM) and associated analytical technique are used to study the interface microstructure of the metalizations which are annealed at 800 °C. The study is focused on understand the diffusion behavier of the barrier layer at identical conditions.

Fig.1 shows the cross sectional STEM images of Ti barrier based multilayer as Ti/Al/Ti/Au metallization scheme. In this metallization scheme has obviously observed in copious amount of Au inter-diffused through 30nm Ti barrier during the annealing. Thick layer of 8-10 nm has formed on the AlGaN layer. The considerable amount of Au has penetrated through AlGaN by forming tubular protrusion at regular interval. In this Au layer was segregated out in AlGaN/GaN interface. The boundary range could not be observed between Ti/Al/Ti layers and it has to be formed other phases such as Au2Ti, Al3Ti and Au2Al at in and out of diffusion layers . TiN phase is formed adjacent to Au rich thick structure and it may be a key factor for low resistance. HRSTEM image is shown in the figure 3 and it is represented that the thick layer possesses TiN, AlN along with Au.The high rate of diffusion through Ti barrier layer indicates its ineffectiveness of prevention to Au and Al diffusions at an annealing temperature of 800 °C. The larger region of TiN phase formation at the contact area is used to promote the electron transport. However, accumulation of high occupancy Au rich thick layer could be degrading its efficiency. Whereas in fig.2 shows Ti/Al/Mo/Au scheme, Mo has higher melting point than other metals, claimed to be a potential diffusion barrier due to its excellent contact resistivity. But, in this case the larger amount of Mo grains was combined as Au-Mo region due to their low solubility to Au. The Au rich thick layer was measured about 6 – 10 nm at the region of below the Au-Mo layer as shown in the Fig 3. Also HRSTEM image is indicated to absence of Al or Ti on this layer. It is indicated that the consumption of GaN layer is much lower than Ti barrier. The higher ratio of Al and Ti mixture surface is observed at outer-diffusion compared with inter-diffusion. This degrades the formation of TiN layer at contact region. The lowest contact resistivity of 4x10-6 Ω.cm–2 for a Ti barrier and 7x10-6 Ω.cm–2 for a Mo barrier is obtained at a Ti/Al ratio of 0.43. We conclude that Ti remains better barrier layer for this condition but the segregation Mo particles did not allow Au layer to form binary phase with Al and Ti and also effected formation of critical TiN phase.

The work is financially supported by Marie Curie Actions under the framework of the project “NetFISiC” No. 264613

Fig. 1: Low magnification STEM image of Ti/Al/Ti/ Au multilayer ohmic contact.

Fig. 2: Low magnification STEM image of Ti/Al/Mo/ Au multilayer ohmic contact

Fig. 3: HHRSTEM image at the Au rich contact layer with multiple phases on Ti/Al/Ti/ Au ohmic contact

Fig. 4: HRSTEM image at the Au rich layer shows GaN phase on Ti/Al/Mo/ Au ohmic contact
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-5731 TEM investigation of semipolar GaN grown on Si(001) offcut substrates using AlN and 3C-SiC buffer layer

Sorokin L. M.1, Kalmykov A. E.1, Myasoedov A. V.1, Bessolov V. N.1, Kukushkin S. A.2
1Ioffe Physical-Technical Institute, Russian Academy of Sciences, St. Petersburg, 194021 Russia, 2Institute for Problems of Mechanical Engineering, Russian Academy of Sciences, St. Petersburg, 199178 Russia
aekalm@mail.ioffe.ru

Gallium nitride (GaN) has been recognized as a promising material for high-performance light-emitting devices. Commercial nitride semiconductors are grown on single-crystal substrates with (0001) oriented surfaces. However, electrostatic fields generated by the spontaneous and piezoelectric polarization along [0001] axis in wurzite GaN layers lower the luminous efficiency of the devices. One way to increase the efficiency is use of GaN layers grown on semipolar planes [1]. Mostly, GaN-based devices are fabricated using silicon carbide or sapphire substrates. These substrates are expensive and insulating and are not available in large diameter. On the other hand, silicon is inexpensive material and has reasonable thermal and electrical conductivity. Si(001) substrate is preferable for GaN growth since Si(001) single crystal is the material of the electronic silicon industry.
In this work results of TEM investigation of thick (up to 15 µm) semipolar GaN layers grown on 1.5-inch Si(001) offcut substrates with 3C-SiC and AlN buffer layers are presented. The offcut angles were 0°, 4° and 7° toward the Si [110] direction (samples 1, 2 and 3). The growth of GaN layer was realized by hydride-chloride vapor-phase epitaxy (HVPE) on AlN/3C–SiC/Si template. The SiC and AlN buffer layers were formed by solid-phase epitaxy [2] and HVPE respectively without any prior masking and etching of silicon substrate.
It has been established that GaN layer of sample 1 consists of oriented wurzite grains. The [2-1-10] axis of all GaN grains is parallel to [110] directions of silicon substrate. The [0001] axis of the majority of GaN grains is near parallel to <111> Si so the angle between GaN [0001] axis and normal to substrate is equal approximately 52°. Grains contain basal plane stacking faults with a density varying from grain to grain within 2×105 - 3×106 cm-1.
In the case of samples 2 and 3 structure of the GaN layer changed significantly. The layer is a single crystal and the density of stacking faults decreased considerably (fig. 1). Unusual orientation relations were revealed in these heterostructures: the GaN [0001] axis is still near parallel to Si<111>, however, the angle between AlN[0001] and GaN[0001] is ~1.5° (fig. 2).

References
1. A. E. Romanov _ T. J. Baker, S. Nakamura, J. S. Speckb. Strain-induced polarization in wurtzite III-nitride semipolar layers// J. Appl. Phys. 100, 2006, p. 023522(1-10)
2. Kukushkin S.A., Osipov A.V. A new method for the synthesis of epitaxial layers of silicon carbide on silicon owing to formation of dilatation dipoles // J. Appl. Phys., 113, 2013, P. 024909(1-7)

TEM investigation was made on the equipment of the Joint Research Centre «Material science and characterization in advanced technology» (Ioffe Institute, St. Petersburg, Russia).

Fig. 1: Cross-sectional TEM image of sample 2.

Fig. 2: SAED pattern registered from the area shown in fig. 1
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-5804 Electroluminescence imaging of defects in GaN HEMT structures

Priesol J.1, Šatka A.1, 2, Sládek Ľ.1
1Institute of Electronics and Photonics, Slovak University of Technology, Ilkovičova 3, 812 19 Bratislava, Slovakia, 2International Laser Centre, Ilkovičova 3, 841 04 Bratislava
juraj.priesol@stuba.sk

This contribution deals with the detection and analysis of electroluminescence (EL) emitted by depletion mode (normally-on) InAlN/GaN high electron mobility transistors (HEMTs) at room temperature and high drain-source voltages. Contacting of examined transistor structures was performed under an optical microscope equipped with color CCD camera and thermoelectric cooling system; for further details about the developed low-light imaging setup see [1]. It has been observed that EL signal comes predominantly from the area along the gate fingers, which is consistent with observations made by other groups [2], [3]. EL maps captured at gate-source voltage VGS = 0 V and at drain-source voltages VDS in the range from 20 to 30 V revealed inhomogeneities in intensity of emitted EL signal (Fig. 1). This is clearly evident for VDS below 25 V and particularly for right gate electrode, where the area with weaker luminescence is marked in Fig. 1f by dotted line. Such EL quenching is directly linked to that part of transistor, where the intensity of electric field is suppressed due to the local electric breakdown or due to leakage currents in the vicinity of the gate terminal. It has been found, that some electroluminescence arises also from the area near the edge of the drain contact pad (marked by arrow in Fig. 1f). In this region, a large amount of EL signal intensity varies significantly over time, which is most likely caused by local charging and consequent discharging of the traps represented by metastable states in the energy gap of the semiconductor. Such time-instability of EL intensity has been observed also in the vicinity of the gate fingers, which is easily noticeable in Fig. 1e in the form of a bright spot highlighted by arrow; the rest of captured maps do not show any evidence of increased luminescence in this area. Similar results have been observed also by mapping of currents induced by electron beam (EBIC) in scanning electron microscope [4]. Obtained results will be discussed in context of the quality assessment of such advanced semiconductor devices.

 

References
[1] Priesol, J. et al, In Proceedings of ADEPT, 2nd International Conference on Advances in Electronic and Photonic Technologies, June 1-4, 2014, Tatranská Lomnica, Slovakia; accepted, in print
[2] Lossy, R. et al, Phys. Stat. Sol. C 6, No. 6, 1382–1385 (2009)
[3] Meneghini, M. et al, IEEE Trans. Device Mater. Rel. 13 (2), 357–361 (2013)
[4] Kováč, J. et al, Microelectronics Reliability 52 (7), 1323–1327 (2012)

 

This work was supported by the Slovak Research and Development Agency under the contract APVV-0367-11 and by the Scientific Grant Agency, No. 1/0921/13. Also the support from the STU project LUMIGaN is kindly acknowledged.

Fig. 1: Image of HEMT captured by optical microscope with marked gate G, source S and drain D electrodes (a) and corresponding EL maps captured at VGS = 0 V and VDS = 30 V (b), 27.5 V (c), 25 V (d), 22.5 V (e), and 20 V (f).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-5886 Quantitative HRTEM analysis of epitaxial perovskite multilayer on Si(001) single crystal substrate

Ghica C.1, Negrea R. F.1, 2, Teodorescu V. S.1, Maraloiu V. A.1, Nistor L. C.1, Chirila C.1, Pintilie L.1
1National Institute of Materials Physics, Magurele, Romania, 2University of Bucharest, Faculty of Physics, Magurele, Romania
cghica@infim.ro

Multilayers based on perovskite ferroelectric materials (PbZrxTi1-xO3, BaTiO3, etc.) are intensively studied for the development of ferroelectric random-access memories (FeRAM) or the fabrication of artificial multiferroic devices. High quality epitaxial perovskite multilayers are currently grown by pulsed laser deposition on substrates with perovskite structure and reduced lattice mismatch, like SrTiO3 (STO). The possibility of integrating such perovskite multilayer devices onto silicon chips is of high practical interest. Given the structural difference between Si (diamond cubic structure, aSi= 0.543 nm) and the perovskite layers to be grown on top (pseudocubic lattice parameter ap around 0.39÷0.41 nm), the characterization of the microstructural aspects regarding the growth of the perovskite layers on Si substrates deserves a special attention.
This work contains a quantitative HRTEM analysis of the epitaxial growth of PZT52/48/SrRuO3/SrTiO3 multilayer onto Si(001) substrate. A cross-section specimen has been prepared for TEM by mechanical thinning followed by ion milling at 6o angle of incidence and 4 kV acceleration voltage using a Gatan PIPS installation. The TEM/HRTEM investigation has been performed on a JEM ARM 200F electron microscope operated at 200 kV.
The selected area electron diffraction pattern reveals the epitaxial growth of the STO, SRO and PZT layers. Despite the presence of the amorphous SiO2 layer, there is a clear orientation relationship between the STO layer and the Si substrate: (001)STO || (001)Si and (010)STO || (110)Si.
The mismatch between the (110)Si planes (d110=0.1916 nm) and (010)STO (d010=0.1953 nm) with respect to Si is of 1.9%, considering the bulk values. The pseudocubic lattice constant for the PZT layer measured on the electron diffraction pattern is 0.404 nm, which makes a 3.6% mismatch with respect to the STO lattice parameter. The lattice fringe deformation across the HRTEM micrograph has been analyzed using the Geometrical Phase Method. The HRTEM lattice fringe deformation with respect to (220)Si planes has been mapped out. The numerical values across the map can be read out on the line profile pointing from the substrate to the PZT layer. The measured values of fringe deformation correspond to the bulk values.
Our quantitative HRTEM study indicates that the STO lattice is fully relaxed due to the presence of the amorphous SiO2 layer at the interface, while the PZT layer shows an in-plane compression across the first 10 nm at the interface with the SRO layer. The strained region inside PZT exhibits clusters of dislocations for strain relaxation, as indicated by the geometrical phase image.

The authors acknowledge UEFSCDI and ROSA for financial support through the PN-II-ID-PCE-2012-4-0362, PN-II-ID-PCE-2011-3-0268 and STAR 94/2013 projects.

Fig. 1: TEM image displaying the PZT/SRO/STO/Si multilayer structure and the corresponding SAED pattern from a large area including the Si substrate and the deposited multilayer.

Fig. 2: HRTEM image showing the epitaxial growth of the SRO and PZT layers on the STO layer.

Fig. 3: (a) HRTEM image of the PZT/SRO/STO/Si sample; (b) Corresponding power spectrum; (c) geometrical phase image corresponding to the selected spot 220Si; (d) map of the lattice fringe deformation with respect to the (220)Si lattice planes; (e) averaged line profile of the lattice fringe deformation along the black arrow on map (d).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-5976 TEM Characterization of Low Temperature Gallium Nitride Layer grown under different Nitridation conditions

Muraleedharan K.1, Sabyasachi Saha2, Krishna YGR3, Banerjee D.3, Raghavan S.4
1Technical Core Group, Defence R&D Organisation, New Delhi, India , 2Electron Microscopy Group, Defence Metallurgical Research Laboratory, Kanchanbagh, Hyderabad, India, 3Department of Materials Engineering, Indian Institute of Science, Bangalore, India, 4Centre for Nano Science and Engineering, Indian Institute of Science, Bangalore, India
muralee15oct@gmail.com

Gallium and other Group-III Nitrides are the materials of choice for various semiconductor and optoelectronic applications. The large tunable band gap of nitride alloys starting from InN (1.9eV), GaN (3.4eV) and AlN (6.2eV) in conjunction with its material properties would enable the bandgap engineering by appropriate alloying for fabrication of various high power and high frequency devices.


Growth of bulk GaN is difficult due to the extreme requirements of temperature and pressure. Due to the unavailability of bulk GaN wafers, GaN based devices have to be fabricated on foreign substrates such as sapphire, silicon and silicon carbide etc. Growth on these lattice and thermally mismatched substrates lead to formation of various defects and high dislocation densities (~109-1010/cm2).


This present study is aimed at understanding the microstructural evolution of Gallium Nitride during the early stages of growth on c-plane sapphire, which in-turn may further enhance our understanding on the genesis of dislocations in the group-III Nitride materials. For the current work GaN growth runs have been carried out in a MOCVD reactor (AIXTRON make) using the standard two step growth recipe under different nitridation temperatures (TN = 530, 800 and 1100oC). This study aims to investigate the early stages of growth and understand the nature of the low temperature (LT-GaN), its morphology and defect structure as it only acts as the nucleation site for successive GaN layers grown above it. Microstructural characterization of the LT-GaN layer under different nitridation temperatures has been carried out.


TEM and High Resolution TEM investigations have revealed the formation of LT-GaN islands on sapphire. Different orientation relationships and various defects have also been observed in the LT-GaN layer under different nitridation temperatures. These will be discussed in detail in the paper.

Authors thank the Defence R&D Organisation and Indian Institute of Science, Bangalore for funding. Permission from Director, DMRL to publish these results are gratefully acknowledged. Authors also thank Dr R Muralidharan, Director, Solid State Physics Laboratory, Delhi, India for encouragement.

Fig. 1: TEM Bright Field Micrograph showing the LT-GaN islands grown on c-plane sapphire nitirded at 530oC

Fig. 2: (a) HRTEM Micrograph (TN=800oC) in ZA [ 11-20] Sapphire and [10-10] GaN; and (b) HR micrograph (TN=1100oC) in ZA [10-10] Sapphire and [11-20] GaN.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-5977 HR STEM study of InGaAs/InAlAs hetrostructures and interfaces morphology

Presniakov M. Y.1, Trunkin I. N.1, Vasiliev A. L.1, 2, Galiev G. B.3, Pushkarev S. S.3, 4
1National Research Centre “Kurchatov Institute”, 123182 Moscow, Russia, 2A.V. Shubnikov Institute of Crystallography, Russian Academy of Sciences, 119333 Moscow, Russia, 3Institute of Ultrahigh Frequency Semiconductor Electronics, Russian Academy of Sciences, 117105 Moscow, Russia, 4National Nuclear Research University “MEPHI”, 115409 Moscow, Russia
mpresniakov@gmail.com

Metamorphic heterostructures with InAlAs/InGaAs/InAlAs quantum well on GaAs substrates are the promising materials for the fabrication of high performance microwave devices (high electron mobility transistor – HEMT). Metamorphic HEMT heterostructures on GaAs substrates have slightly inferior structural quality – surface morphology and structural defects density as compared to InP-based HEMT. The heterostructures were grown on (1 0 0) oriented and (1 0 0) + 2° vicinal GaAs substrates. The structural perfection of a composite quantum well was investigated (fig.1, 2) by aberration corrected (probe Cs corrected) TEM/STEM TITAN 80-300. The structural parameters of the layers and the interfaces including flatness were studied by high-resolution HAADF STEM. By using the intensity histogram filtration, it was found, that the interfaces between the bottom part of the In enriched layers and the top part of metamorphic buffer (MB) layers were flat and abrupt. The dislocation density was estimated by weak beam dark field imaging method. The dislocations of different types were observed and some of them were associated with stacking faults. The estimated dislocation density in the MB was 5x109 cm-2. In all samples these dislocations were uniformly distributed over the whole MB and close to the inverse layer their density decreases to 5x108 cm-2. After the inverse step and smoothing layer, and close to the active region of the heterostructure the dislocation density decreased further by two orders of magnitude The heterostructures with nano-inserts of InAs in the quantum well on InP substrates will be considered in this work (fig.3). It was found, that the influence of the quality of the quantum well heterointerfaces on the electron mobility is not the dominant factor.
The work carried out as part of the contract with the State Ministry of Education № 14.ВВВ.21.0009.

Fig. 1: HR HAADF STEM images of the QW area of the(a) “(100)” sample and (b) “(100) + 2°” sample

Fig. 2: The histograms of HR HAADF STEM signal intensity of the QW area and the corresponding interpolating functions

Fig. 3: STEM image of QW and corresponding intensity histogram 
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-6002 Plan-view CBED analysis of crystal polarity in core-shell GaN rods

Dieker C.1, Tessarek C.2, Christiansen S.2,3, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Universität Erlangen-Nürnberg, Erlangen, Germany, 2Max Planck Institute for the Science of Light, Günther-Scharowski-Str. 1, 91058 Erlangen, Germany, 3Helmholtz Zentrum für Materialien und Energie, Hahn-Meitner-Platz 1, 14109 Berlin, Germany
christel.dieker@fau.de

GaN rods grown by MOVPE on sapphire are attracting considerable interest because of their potential use in optoelectronic devices. Self-catalyzed, mask-free growth of GaN rods with high aspect ratio and low defect densities has been demonstrated [1-3]. The crystal polarity of GaN plays a key role in the growth process and strongly affects the morphology and faceting of the rods. In particular, rods composed of strands with opposite polarity and distinct surface facets are frequently observed, either in side-to-side [1] or core-shell [2] configuration. In this contribution we demonstrate that CBED is perfectly suited for polarity analysis of GaN core-shell rods both in cross-section and plan-view geometry.

Fig. 1 summarizes polarity analysis in cross-section geometry [2]. Freestanding rods with diameters ranging from 500 to 1500 nm extend out of the substrate as can be seen from the bright field image. The {0002} dark field image of an individual rod reveals the different polarities of core and shell by exploiting polarity sensitive contrast. Furthermore, the surface facets are different. While the shell possesses a flat facet the core shows inclined facets which form a cone protruding from the top surface. This enables CBED analysis in thin parts at the tip and sidewall of the rod yielding Ga and N polarity of the core and shell, respectively [2].

Fig. 2 summarizes polarity analysis in plan-view geometry. After scratching off GaN rods from the sapphire substrate and transferring them onto a Si wafer FIB lift out has been used for fabrication of a thin TEM lamella perpendicular to the rod axis. Fig. 2b shows a bright field image of a rod section viewed against the growth direction. In order to reveal the inversion domain boundary and enable polarity determination the sample has been tilted from [0001] counterclockwise by 31.6° about the vertical axis to [2,-1,-1,3]. The projected crystal structure of a rod with Ga polarity is depicted in Fig. 2c and the corresponding CBED pattern calculated for a slice with 95 nm thickness using JEMS [4] is shown in Fig. 2f. Comparison with the experimental CBED patterns Figs. 2d and 2e unambiguously proves that the core and shell possess Ga and N polarity, respectively, in agreement with the result obtained in cross-section geometry.

The plan-view polarity analysis cannot only be applied to core-shell GaN rods but is also be expected to be very useful for the investigation of inversion domains in thin GaN films.

[1] Tessarek et al., Jpn. J. App. Phys. 52 (2013) 08JE09.

[2] Tessarek et al., J. App. Phys. 114 (2013), 144304.

[3] Tessarek et al., Cryst. Growth Des. 14 (2014), 1486.

[4] P. A. Stadelmann, Jems Electron Microscopy Software (1999-2012), java version 3.7624U2012, CIME-EPFL, Switzerland.

Financial support by the DFG through the Cluster of Excellence EXC315 "Engineering of Advanced Materials" is gratefully acknowledged.

Fig. 1: Core-shell GaN rods with different polarities in the core and shell region. The CBED analysis shows that the core and shell possess Ga and N polarity, respectively (from [2]).

Fig. 2: CBED polarity analysis in plan view geometry confirming the polarity obtained in Fig. 1 (see text for details).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-8-P-6006 Switching behavior of single Ag-TCNQ nanowires: an in situ Transmission Electron Microscopy study

Ran K.1, Rösner B.2, Butz B.1, Fink R.2, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), University of Erlangen-Nürnberg, Erlangen, Germany, 2Physical Chemistry II, University of Erlangen-Nürnberg, Erlangen, Germany
ke.ran@ww.uni-erlangen.de

Unique properties, such as high density of charge carriers and electric field-induced switching behavior are found in one-dimensional nanostructures of metal-tetracyanoquinodimethane (M-TCNQ).[1] For the case of Ag-TCNQ, reversible phase transition can be induced by electric field applied along the TCNQ ∏-∏ stacking direction, resulting in resistive switching with an on-off ratio reported to be as high as 104. A proposed mechanism suggests that, a partial neutral species of Ag and TCNQ form during the transition, provide additional conduction channels and increase the material conductivity substantially. Studies based on large-area Ag-TCNQ nanowires (NWs) provided useful information.[2] However, there is still lack of detailed studies from individual NWs, where the NW size and electric behavior can be correlated, and much more intrinsic qualities of a particular NW can be expected.
Using in situ Transmission Electron Microscopy (TEM), we are able to investigate the switching behaviors of individual Ag-TCNQ NWs. For the in situ study the NWs were grown directly on a Au TEM grid, as shown in Figure 1a. Figure 1b depicts one unit cell of the orthorhombic Ag-TCNQ phase II structure [3] along different axes. Typical TEM image and diffraction pattern from a single Ag-TCNQ NW are shown in Figure 1c and 1d. Generally, a NW growth direction along [100] is observed. In situ electric measurements were performed using an STM-TEM holder from Nanofactory Instruments AB in combination with the aberration-corrected Titan3 80-300 microscope at the University of Erlangen-Nürnberg. The W tip and the Au TEM grid serve as the two electrodes as schematically shown in the inset of Figure 2a.
In our study, for up to 30 individual NWs with different sizes, phase transition and resistive switching have been successfully induced. The typical I-V behavior of a single NW is shown in Figure 2a: starting from a state with low conductivity, and then switched on once the electric field reaching a certain point. The differentiation of current over bias, shown as inset in lower right of Figure 2a, as well suggests a sudden increase in the NW conductivity. Together with the large current passing through the NW after switched on, Joule heating becomes an issue which can easily lead to the NW breaking down. Figure 2b shows a NW switched on and surviving a current rang up to ~100 nA. However, it broke down at the center under the same bias sweep, but with current compliance increased to ~1 µA. This failure indicates that, heat dissipation should be taken into consideration for achieving high performance devices.

Reference
[1] Potember P. S. et al., Appl. Phys. Lett., 34, 1979, 405.
[2] Xiao K. et al., Adv. Funct. Mater., 18, 2008, 3043.
[3] Shields L. J. Chem. Soc. Faraday Trans. 2, 1985, 1.

The authors gratefully acknowledge financial support by the DFG through the Research Training Group GRK1896 and the Cluster of Excellence EXC 315.

Fig. 1: Figure 1. (a) TEM image of Ag-TCNQ NWs grown directly on a Au TEM grid. (b) A unit cell of Ag-TCNQ along a-axis (top) and c-axis (bottom). (c) TEM image of an individual AgTCNQ NW. (d) Diffraction pattern from the NW shown in (c), taken along [001] zone axis.

Fig. 2: Figure 2. (a) I-V behavior from a single NW, by sweeping the bias 10 V→-10 V→10 V. Current compliance of 1 µA was applied. Upper left inset shows the NW, and the electric measurement setup. Lower right inset is dI/dV from 0 V→-10 V. (b-c) Repeating the bias sweep 0 V→-10 V→ 0 V for a same NW with 100 nA and 1 µA current compliance respectively.
↑ Go to the Table of content ↑

MS-9. Defects in materials and phase transformations

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-9-IN-3031 Nanostructured shape memory alloys: Phase transformations and martensitic interfaces studied by TEM

Waitz T.1, Peterlechner M.1,2, Gammer C.1,3, Schmidt V.2, Tsuchiya K.4, Chakif M.5, Mangler C.1, Schindler P.1,6
1University of Vienna, Faculty of Physics, Vienna, Austria, 2Westfälische Wilhelms-Universität Münster, Institut für Materialphysik, Münster, Germany, 3National Center for Electron Microscopy, Lawrence Berkeley National Laboratory, Berkeley, CA , USA, 4Research Center for Strategic Materials, National Institute for Materials Science, Tsukuba, Japan, 5Chair of Applied Laser Technology, Ruhr-Universität Bochum, Bochum, Germany, 6Stanford University, Dept. of Mechanical Engineering, Stanford, CA, USA
thomas.waitz@univie.ac.at

Shape memory alloys (SMA) show unique properties based on a martensitic phase transformation. The martensitic structures and interfaces control shape memory and superelasticity. Crystal size at the nanoscale can strongly impact martensitic phase transformations. The present work reports recent results of transmission electron microscopy (TEM) studies of phase transformations, defect structures, and martensitic interfaces of nanostructured SMA obtained by different processing routes.

Bulk nanocrystalline SMA alloys can be processed by methods of severe plastic deformation (SPD). As shown by TEM bright field (BF) and dark field (DF) images, SPD of ferromagnetic NiMnGa SMA yields strong grain refinement. Using TEM diffraction, concomitant disordering of the tetragonal martensite and a phase change to a face centred cubic structure is observed. Upon heating, the ordered Heusler austenite forms similar to that of the coarse grains. However upon cooling, TEM lattice fringe images and selected area diffraction shows that in the small grains a highly defective structure of modulated 14M martensite arises.

Highly localized processes of amorphization in NiTi-based and CuZr-based SMA subjected to SPD were analysed. Figure 1a shows SPD processed CuZr. While the density of dislocations is rather low, numerous deformation bands intersect the twinned martensite. Bands that extend several hundreds of nm while their width is several nm only frequently show intersections and bifurcations. HRTEM (cf. Figure 1b) and nanodiffraction reveal that bands contain an amorphous phase. Opposed to models predicting localized amorphization by accumulation of dislocations at twin boundaries, the bands are rather intersecting them. While SPD amorphization of NiTi is almost complete, TEM shows a high density of nanocrystalline debris. Upon heating, debris acting as nuclei strongly impacts crystallization kinetics. Figure 2 shows the results of TEM in-situ heating yielding a rather high nucleation rate while the rate of growth is very small causing nanocrystallization.

Strong size effects on martensitic phase transformations can arise by the high surface-to-volume ratio of freestanding nanocrystals. Figure 3a to c shows TEM images of core-shell nanospheres processed by femtosecond laser ablation of NiTi targets. A single laminate of twinned martensite is observed in smaller martensitic cores of the nanospheres (cf. Figure a and b) while larger cores can host self-accommodated groups of different martensitic laminates.

Financial support by the Austrian COMET Programme is acknowledged.

Fig. 1: Nanoscale amorphization of CuZr. (a) TEM BF image of twinned Cm martensite containing numerous amorphous nanobands. Frequently bands mutually intersect (e.g. near A) and show branching (near B). (b) HRTEM image. A nanoband inclined to the (001) twin boundaries (dashed lines) runs parallel to (1-10) lattice planes (full lines). (BD=[110]).

Fig. 2: Nanocrystallization of NiTi. TEM in situ heating (temperature of 377°C). (a) BF images of nanocrystals embedded in the amorphous matrix. (b) After 51 min of heating new nanocrystals (marked by arrows) nucleated isolated from each other. (c) Diameter of four nanocrystals measured as a function of time showing constant rates of growth.

Fig. 3: TEM images of core-shell nanospheres processed by laser ablation of NiTi targets. The core contains NiTi with a martensitic B19´ structure. (a) and (b) Nanoparticles with a core diameter of 45 and 75 nm, resp., containing a single laminate of twinned martensite. (c) Nanoparticle with a core of 83 nm diameter containing at least two laminates.
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-9-IN-6084 STEM-Based Characterization of Defects and Precipitates in Structural Materials

Smith T. L.1, Bowers M. L.1, McAllister D. A.1, De Graef M.2, Mills M. J.1
1Department of Materials Science and Engineering, Center for Electron Microscopy and Analysis (CEMAS), The Ohio State University, Columbus OH 43210, USA, 2Department of Materials Science and Engineering, Carnegie Mellon University, Pittsburgh PA 15213. USA
mills.108@osu.edu

Advancements in our ability to characterize deformation mechanisms and precipitate structures finer length-scales are providing new insights into the governing deformation mechanisms and precipitation processes in several important commercial alloy systems. These advancements are based upon developments using STEM-based approaches to imaging and chemical analysis using EDS.

Diffraction contrast STEM imaging holds significant advantages over conventional TEM (CTEM) imaging of defects [1-3]. The advantages include the suppression of auxiliary contrast effects (bend contours, etc.) and the ability to image in thicker specimens than is practical using conventional TEM at standard operating voltages. Additionally, CTEM visibility rules such as those for stacking faults and “g•b analysis” for dislocations also remain in STEM mode provided the convergence condition and detector geometry are configured appropriately, as in Figure 1 showing a dislocation analysis for a Ni-base superalloy. High contrast, bright-field images along low index zone axes can also be formed – a mode which is not feasible with CTEM. These conditions will be described and image simulations that support these conclusions using the CTEM Soft program will also be presented.

High resolution high angle annular dark field (HAADF) imaging and ChemiSTEM compositional analysis has provided new insights into microstructure development and deformation processes in the commercial superalloy 718. Atomic-scale imaging using HAADF (Figure 2a) is extremely effective for revealing the morphology and size of these precipitates, where the Nb-rich γʹʹ (DO22 structure) phase appears with enhanced intensity relative to the FCC matrix. The highly planar {010} faces bounding the γʹʹ phase are interfaces with the γʹ phase. While Z contrast from the L12 ordering in the γʹ phase is indistinguishable from the matrix, this conclusion is unambiguously supported by ChemiSTEM EDS mapping, as shown in Figure 2b. Deep insight into the mechanisms of plastic deformation have also been gained through HAADF imaging, which in this alloy has advantages relative to diffraction contrast imaging due to the large strain fields of the precipitates that tend to obscure the dislocation structures.

References
[1]P.J. Phillips, M.J. Mills, and M. De Graef, Philosophical Magazine 91 (2011) 2081-2101.
[2] P.J. Phillips, M.C. Brandes, M.J. Mills, and M. De Graef Ultramicroscopy 111 (2011) 1483-1487.
[3] P.J. Phillips, M. De Graef, L. Kovarik, A. Agrawal, W. Windl, M.J. Mills, Ultramicroscopy, 116 (2012) 47–55.

Support from the following program is gratefully acknowledged: the National Science Foundation, Division of Materials Research, under the GOALI program DMR-0907561 (for MLB and MJM), the GE University Strategic Alliance Program (for TLS) and the Metals Affordability Initiative program (for DAM).

Fig. 1: Example of a dislocation analysis in a Ni-base superalloy. Shown are zone axis [001] bright field and two dark field images using g020 and g200. Inset region shows two different families of dislocations.

Fig. 2: (a) HAADF STEM image on <110> zone showing a composite γʹʹ/γʹ particle. (b) ChemiSTEM™ EDS map showing net intensity of Nb and Al peaks which enables discrimination of γʹ and γʹʹ particles.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-1620 Chemical analyses of Ga segregation at Pb nanoparticles using ChemiSTEM Technology

Moros A.1, Lazar S.2, Rösner H.1, Wilde G.1
1Institut für Materialphysik, Universität Münster, Wilhelm-Klemm-Straße 10, D-48149 Münster, Germany, 2FEI Company, Achtseweg Noord 5, 5600 KA Eindhoven, The Netherlands
rosner@uni-muenster.de

Al-Pb composites consisting of nanometer-sized Pb inclusions embedded in a polycrystalline Al matrix serve as model systems for size-dependent melting studies [1]. To analyze the impact of the surrounding matrix on the melting and solidification temperatures of the Pb nanoparticles, Ga was added to the matrix within the solute solution regime. The alloying of Ga expands the lattice of the Al matrix and thus reduces the mismatch between matrix and particle. Although Ga is immiscible with Pb, the melting and solidification of the Pb nanoparticles was affected by the Ga addition. A large undercooling of the solidification onset of up to 100K with respect to melting was found with increasing Ga content [2]. Chemical analyses (EDX) were performed using a double-corrected, monochromated Titan 60-300 with ChemiSTEMTM technology. The results of this investigation are shown in the Figures. The Pb nanoparticles were oriented along the <110>-direction of the fcc matrix and imaged by HAADF-STEM (Fig. 1). The chemical analysis shows that Ga segregates at the particle-matrix hetero-interface (Fig. 2). The Ga concentration has been traced in the form of a profile across the Pb nano-particle (see boxed area in Fig. 2). It shows peaks in the Ga signal at the interfaces to about 7.4 at.% (Fig. 3). The level across the Pb nanoparticle is about 1.2 at.% higher on average than the surrounding matrix concentration of 4.7 at.% due to the other Ga enriched interfaces which are projected in plan view. The width of the Ga enriched zone around the Pb nanoparticle is about 2.2 nm. This Ga enriched zone acts as a buffer between the nanoparticle and the nominal Al94Ga6-matrix inhibiting the nucleation of the liquid Pb. It has been shown that vacancies play a key role in accommodating the misfit strain upon solidification of nanoparticles embedded in Al [1,3]. Presumably the increased number of vacancies available around the Pb nanoparticles relative to the matrix lead to an accelerated Ga diffusion in this region and thus to the observed Ga segregation. The Ga segregation, in turn, leads to solid solution hardening of the buffer phase, which then becomes more rigid with the Ga increase. This hinders an elastic accommodation of the mismatch strain from the matrix side. The scenario of a suppressed nucleation upon cooling is hence explicable since the liquid Pb nanoparticles contract upon solidification and thus bring a negative pressure on the Pb particles. Thus the undercooling of the Pb nanoparticles can be explained by thermodynamics according to Clausius-Clapeyron.

[1] E. Johnson, H.H. Andersen, U. Dahmen, Microsc. Res. Techniq. 64 (2004), p. 356.
[2] A. Moros et al. to be published.
[3] L.H. Zhang, E. Johnson, U. Dahmen, Acta Mater. 53 (2005) p.3635.

 

Support by the DFG under the grant WI 1899/14-1 is gratefully acknowledged.

Fig. 1: HAADF-STEM image of a faceted Pb nanoparticle embedded in an Al94Ga6-matrix viewed along the <110>-direction. Six out of fourteen interfaces are imaged in edge-on conditions.

Fig. 2: EDX Ga map using a probe current of 450 pA and an acquisition time of 454 s.

Fig. 3: Al, Ga and Pb profiles corresponding to the boxed area in Fig.2.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-1805 First experimental study of nanoscale plasticity mechanisms in nanocrystalline Pd thin films under hydrogen cycling

Amin-Ahmadi B.1, Idrissi H.1,2, Malet L.3, Delmelle R.2, Godet S.3, Pardoen T.2, Proost J.2, Schryvers D.1
1Electron Microscopy for Materials Science (EMAT), University of Antwerp, Belgium, 2Université catholique de Louvain, Institute of Mechanics, Materials and Civil Engineering, Louvain-la-Neuve, Belgium, 3Université Libre de Bruxelles, Matters and Materials Department, Belgium
behnam.amin-ahmadi@uantwerpen.be


Thin Pd membranes constitute an enabling material in hydrogen permeation and sensing applications. Due to hydriding, the initial volume of the Pd structure expands by about 10% due to the α→β-phase transformation, which induces a large plastic deformation within the material [1,2]. In the present work, nanoscale plasticity mechanisms activated in sputtered nanocrystalline (nc) Pd thin films subjected to hydriding cycles at different hydrogen pressures have been investigated for the first time using advanced TEM. The in-situ measurement of the evolution of the internal stress during hydriding of the nc Pd films shows that this internal stress increases rapidly in the compressive direction, and gradually reaches a constant value of 120 MPa tensile stress for the α-phase transformation and 920 MPa compressive stress for the β-phase transformation (Figure 1a). The automated crystallographic orientation mapping in TEM (ACOM-TEM) before and after hydriding did not reveal significant changes of the grain size and the crystallographic texture, excluding grain boundary mediated processes as dominant hydrogen induced plasticity mechanisms.
Figures 1b and 1c show HRTEM images of ∑3 {111} coherent twin boundaries (TBs) in Pd films before and after hydriding to the β-phase, respectively. In these figures, clear loss of the coherency of these boundaries can be observed. Such a feature is due to the interaction of coherent TBs with lattice dislocations generated during hydriding. This can be confirmed by the local g-maps shown in the same figures and demonstrating a clear increase of dislocation density after hydriding to the β-phase. However, significant changes of dislocation density or the coherency of coherent TBs have not been observed in Pd films hydrated to the α-phase. These results confirm that hydrogen induced plasticity is mainly controlled by dislocation activity at higher hydrogen pressures. Surprisingly, an fcc→9R phase transformation at Σ3 {112} incoherent TBs (Fig. 2) as well as a high density of stacking faults (SFs) (Fig. 2b) have been observed after hydriding to the β-phase indicating a clear effect of hydrogen on the stacking fault energy of Pd. Such observations also suggest that hydrogen atoms remain trapped at the defect cores after dehydriding. Our findings provide precious information for the validation of atomistic simulations on the interaction of hydrogen with extended defects and for better understanding of the effect of hydriding on the macroscopic mechanical properties of nc metallic thin films.

References

1. B. Amin-Ahmadi, H. Idrissi, R. Delmelle, T. Pardoen, J. Proost, D. Schryvers, Appl. Phys. Lett. 071911, 102 (2013).
2. H. Idrissi, B. Amin-Ahmadi, B. Wang, D. Schryvers, Phys. Status Solidi B, 1–14 (2014).

Fig. 1: Figure 1. a) Evolution of the internal stress in nc Pd during hydriding cycle b) HRTEM micrograph of a CTB in the as-deposited Pd film. A local g-map is shown in the upper left. c) HRTEM image showing the loss of coherency of a ∑3{111} CTB after hydriding to the β-phase. The local g-map from the dashed square is shown as upper-right inset.

Fig. 2: Figure 2. a) HRTEM image of 9R band embedded in the Pd matrix after hydriding to the β-phase. b) HRTEM image of hydrated Pd film in the β-phase showing a 9R band at a Σ3 {112} incoherent TB and several SFs indicated by arrowheads. The lower left inset shows the shift in the position of the {111} planes in the SF indicated by dashed lines.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-1872 Investigating the pressure in helium nanobubbles in silicon through in situ Energy-Filtered TEM

Alix K.1, David M. L.1, Lucas G.2, Alexander D. T.2, Pailloux F.1, Pizzagalli L.1, Hébert C.2
1Institut Pprime, CNRS/Université de Poitiers, 86962 Futuroscope-Chasseneuil, France, 2Interdisciplinary Centre for Electron Microscopy (CIME), EPFL, CH-1015 Lausanne, Switzerland
kevin.alix@univ-poitiers.fr

The properties of nano-scale defects such as bubbles in materials are extensively studied for both current and potential future purposes. Those range from the mechanical effects of alpha-particle irradiation in nuclear reactor walls, to the study of plasmonics and fluids at the nanometric scale. The creation of He bubbles in Si and other semiconductors is particularly interesting for their potential applications for electronics, such as their ability for gettering or the Smartcut™ process.

Our purpose is to improve the understanding of the processes governing the evolution of those bubbles during thermal annealing by studying their inner fluid pressure and density, which are predominant factors in their behavior during growth.

So far, spatially-resolved EELS has been shown to be a powerful tool for elemental quantification. But the intense probe used for this technique results, for our systems, in the desorption of He, making consecutive experiments on any single bubble difficult. Furthermore, this forbids studying bubbles during in situ annealing. Here the method uses EFTEM instead of STEM for spectrum acquisition, in order to greatly reduce the local irradiation intensity and to facilitate sample drift detection as well (see Figs. 1-3). This, combined with the measurement of the density-related He1s→2p transition-energy shift (Fig. 3), provides a means to determine the density in the bubbles.

In our He-implanted Si samples, bubbles range from approximately 20 to less than 5 nanometers in diameter (Fig. 1). Initial results from EFTEM have allowed us to establish conditions and procedures for the acquisition, treatment and extraction of data from the samples. Specifically, optimal parameters were determined for signal quality versus acquisition time. The energy filtering aberrations and sample drift can now be corrected simultaneously, by a procedure which has been coded in-house and shows good results on both aspects. Noise reduction is necessary, and a statistical treatment is applied to the data (MSA) for further signal improvement. Finally, the spectra are deconvolved for multiple scattering, and the He K-edge is extract and fitted(Fig. 3). Pixel per pixel, density and pressure maps can now be obtained over several bubbles simultaneously (Fig. 4), and mean pressure and size can be extracted for each one. While this procedure was being implemented, in situ thermal annealing experiments were performed, clearly showing bubbles emptying between room temperature and 800°C, and movement and shape alteration in the same range. Acquisitions were performed with various annealing temperature and time steps, allowing for the detailed study of the bubbles' behavior relative to those parameters as well as their proximity with one another.

Fig. 1: Filtered image at 17+-0,5eV, around Si plasmon, with largest bubbles clearly visible.

Fig. 2: Filtered image at 23+-0.5eV, around He K-edge, same area as Fig. 1.

Fig. 3: Single spectrum acquired via EFTEM, and post-treatment extracted signal. He K-edge clearly visible at ~23eV, behind Si plasmon at ~17eV.

Fig. 4: Density map obtained from same area as Fig. 1 showing density variations between and across bubbles. Data truncated below 90nm-3 for clarity.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-2092 Dislocation deformation under high compressive stress in GaN growth

Yang M.1, Baik H. S.1, Yang C. W.2
1Korea Basic Science Institute, 2Sungkyunkwan University
yangmino72@gmail.com

GaN is a wide and direct band gap semiconductor emitting blue light. The wide single crystal substrates for device manufacturing have been obtained heteroepitaxially through chemical vapor deposition on sapphire, and they contain lots of dislocations. Dislocations have been observed lying parallel to c-axis, whose core structures do not change significantly with the film growth. But when Si(111) used for the heterosubstrate, the dislocations undergo structural changes because they are under high compressive stress. It is derived from the large thermal mismatch between GaN and Si(111).
For the present study, GaN on Si(111) was prepared by a metal-organic vapor phase epitaxy (MOVPE) method. [1] The atomic configurations were investigated with a 300keV scanning transmission electron microscope (STEM) (ARM200F, JEOL) equipped with a high angle annular dark field (HAADF) detector. And to analyze the strain distribution around the dislocation, geometry phase analysis (GPA) was applied to the HRSTEM images. [2]
We have investigated structural changes occurred in the threading edge dislocation (TED) among the types of threading dislocations in the GaN/Si(111) as it is the majority. TEDs are inclined toward <1-100> and <2-1-10>. [Fig. 1(c)] The inclination could be understood by “surface-mediated climb” mechanism suggested by Follstaedt. et. al. [3] The TED core shifted to a compressive region and deformed to a structure consisting of two partial edge dislocations. (Not shown here) The partial dislocations introduced local stacking fault (SF). The core of the partial dislocation has structures of distorted (1-100) surfaces, which are different from those found in conventional TED. The SF between the cores is suspected of having strong metallic Ga bonds, which could be serious electrical defects.

[1] Kai Cheng, M. Leys, S. Degroote, M. Germain, and G. Borghs, Appl. Phys. Lett. 92, 192111 (2008)
[2] A. K. Gutakovskii, A. L. Chuvilin, S. A. Song, Bulletin of the Russian Academy of Sciences Physics 71(10),1426 (2007)
[3] D. M. Follstaedt, S. R. Lee, P. P. Provencio, A. A. Allerman, J. A. Floro,and M. H. Crawford,Appl. Phys. Lett.87, 121112 (2005)

This work was supported from KBSI project T34520.

Fig. 1: WBDF TEM images of (a) g=1-100 and (b) g=0002 show inclined TEDs with an angle of ~17º. The [0001] plane-view exhibits TEDs inclined toward <1-100> and <2-1-10>. Circles indicate TED inclined to <1-100>.

Fig. 2: (a) GPA applied to [0001] HRSTEM images with two orthogonal diffraction spots of g=11-20 and g=1-100 in the FFT images. The map of inter-planar distance for (b) g=11-20 and (c) g=1-100.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-2168 In-situ TEM study of Zn2SiO4 nanotube formation by Kirkendall effect

Tripathi S.1, Ravishankar N.1
1Materials Research Centre, Indian Institute of Science, Bangalore, India
shalinitripathi2307@gmail.com

Hollow Zn2SiO4 has evoked significant interest as a multifunctional material with applications in various domains ranging from cathode ray tubes, electrocatalysis, luminescent devices and selective metal ion absorption. Among various methods for fabrication of hollow structures, the classic one is Kirkendall process, exploiting differences in the diffusion rates of different species. In this study, we synthesize Zn2SiO4 by exploiting Kirkendall effect involving the reaction between ZnO nanorods and a chemically-synthesized SiO2 shell. Using in-situ heating in the TEM, we have monitored the nucleation and coalescence of the Kirkendall voids leading to tubular structure in the SiO2-coated ZnO nanorods. We compare the mechanism of formation of the nanotubes with results from in-situ heating in the TEM and samples heated in air as well as under reducing conditions. While heating the samples in air leads to microstructural evolution that is similar to the in-situ experiments, there is a significant difference in the samples heated under reducing conditions. Heating the ZnO@SiO2 nanostructures in a reducing atmosphere (95% Ar + 5% H2) leads to the formation of amorphous silica nanotubes owing to etching of ZnO in H2 atmosphere. Thus, this understanding of the relevant diffusion processes for the diffusion couple at nanoscale presents a general conceptual platform to fabricate different multifunctional one-dimensional hollow nanostructures. The fabricated Zn2SiO4 nanotube show excellent toxic metal ions (titanium and uranium) adsorption property and also serve as good cathode materials for Li-ion batteries.

The electron microscopes are a part of the Advanced Facility for Microscopy and Microanalysis (AFMM) at the Indian Institute of Science.

Fig. 1: Fig. 1. (a) schematic of microstructure evolution under different conditions; (b), (c) and (d) are TEM images showing nanotube formation from ZnO@SiO2 on annealing in air; (e) Comparison of the XRD patterns of the ZnO@SiO2 core@shell structure, as synthesized and after heating the same in air at 900oC, thus resulting into rhombohedral Zn2SiO4 phase

Fig. 2: Fig. 2. (a-f) are results of in-situ heating showing the hollow structure formation; (g) is SAED pattern of intermediate showing mixed phase of silicate (marked in red) and ZnO; (h) HRTEM showing Kirkendall void

Fig. 3: Fig. 3. (a) BF TEM image showing SiO2 nanotubes formed on heating ZnO@SiO2 in reducing atmosphere; (d-g) are STEM elemental mapping results from the region (b-c) showing no presence of Zn, further confirming etching of ZnO in H2 atmosphere
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-2288 Probing atomic scale dynamics with Z-contrast STEM

Pennycook T. J.1,2, Jones L.1, Pettersson H.3, Nicolosi V.3, Nellist P. D.1,2
1Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH, UK, 2SuperSTEM Laboratory, Daresbury, WA4 4AD, UK, 3Trinity College Dublin, College Green, Dublin 2, IR
timothy.pennycook@materials.ox.ac.uk

Aberration correction has enabled atomic resolution at low accelerating voltages in scanning transmission electron microscopy (STEM). The continuing improvement of the resolution of STEM instruments not only provides clearer images, but also enables atomic scale information to be recorded more rapidly. Rapid scanning provides numerous benefits. With a single rapidly scanned image it is often possible to outrun damage in beam sensitive materials. Recording a series of rapidly scanned images provides a means of removing motion blur. Instead of recording a single image with a long dwell time, one can drift correct a series of rapidly scanned images and then average them for a sharper image. Such series can however also be used to record dynamic processes in action. The dynamics could be triggered with a change in temperature, however using specially designed heating or cryogenic sample holders can compromise the stability of the microscope and reduce resolution. An alternative is to use the electron beam itself to provide the energy needed to overcome the energy barriers involved in dynamic processes. As we show, if the beam is gentle enough and scanned sufficiently rapidly, useful information about dynamic processes can be revealed. Dynamic processes such as solid-state chemical reactions are ubiquitous in materials science. They are relied upon for applications ranging from materials synthesis to device operations. For instance, electrochemical energy systems rely on ion exchanges to store or release energy. Furthermore in devices such as batteries these exchanges often necessitate not only the transport of ions but also phase changes which involve their own transformation dynamics. We demonstrate the use of Z-contrast annular dark-field electron microscopy to directly observe the atomic scale dynamics of a manganese oxide phase change. The energy of the electron probe is used to transform Mn3O4 into MnO. By recording a time series of rapidly acquired atomic resolution images we uncover the detailed motions of the atomic columns as the phase front advances. Furthermore, the atomic number contrast of the Z-contrast images allows changes in the occupancy of the atomic columns to be quantified. The results illustrate the advantages of applying STEM to study dynamic processes. Z-contrast imaging offers interpretability and the ability to quantify changes in occupancy while EELS offers additional robust compositional and chemical information.

Research sponsored by the UK Engineering and Physical Sciences Research Council through the UK National Facility for Aberration-Corrected STEM (SuperSTEM). The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3).

Fig. 1: HAADF image of phase front between MnO (top) and spinel Mn3O4 (bottom).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-2347 Atomic-scale confirmation of polar antiphase boundaries in nonpolar materials

Wei X. K.1,2, Tagantsev A. K.1, Kvasov A.1, Jia C. L.2,3, Du H. C.2,4, Roleder K.5, Setter N.1
1Ceramics laboratory, EPFL-Swiss Federal Institute of Technology, Lausanne 1015, Switzerland, 2Peter Grünberg Institute and Ernst Ruska Center for Microscopy and Spectroscopy with Electrons, Research Center Jülich, 52425 Jülich, Germany, 3International Centre of Dielectric Research, Xi'an Jiaotong University, Xi'an 710049, China, 4Gemeinschaftslabor für Elektronenmikroskopie (GFE) RWTH Aachen, Ahornstraße 55, 52074 Aachen, Germany , 5Institute of Physics, Silesian University, 40007 Katowice, Poland
xiankui.wei@epfl.ch

     Recently, inherent interfaces in perovskite oxides have initiated a fascinating and challenging research subject due to their broad prospects in developing new generations of electronic devices. The typical paradigms include electronic conductivity and large photovoltaic effect at ferroelectric domain walls, e.g., in BaTiO3 [1] and in BiFeO3 [2] respectively, polarity at twin boundary of CaTiO3 [3], and ferromagnetism at antiphase boundary (APB) of Mn-doped hexagonal BaTiO3 [4]. These domain boundaries are particularly interesting because they can be created, annihilated, rewritten and displaced electrically inside the materials, which lead to agile functionalities at nanoscale.
     Previously, theoretical investigation on SrTiO3 has predicted that at temperatures below ~ 40 K, spontaneous polarization and ferroelectricity can be developed in easy and hard boundaries, respectively [5], which make these APBs attractive for practical future applications, if this will happen at room temperature. Using negative spherical-aberration imaging (NCSI) technique in an aberration-corrected TEM, the development of spontaneous polarization (PS ≈ 14 uC/cm2) inside an easy boundary has been experimentally confirmed in antiferroelectric PbZrO3 at room temperature. As result of breaking of Pb cation ordering and octahedral rotation across the boundary, lattice parameter is found to strongly change perpendicular to the boundary plane. First-principles calculations confirm the polarity at the APB. Considering their straight geometry and small sizes, the strain-free APBs are expected to stimulate further interests of both fundamental and applied research in the future.

References
[1] T. Sluka et al, Nat. Commun. 4, 1808 (2013).
[2] S.Y. Yang et al, Nat. Nanotechnol. 5, 143 (2010).
[3] S. Van Aert et al, Adv. Mater. 24, 523 (2012).
[4] X.-K. Wei et al, Appl. Phys. Lett. 102, 242910 (2013).
[5] Alexander K. Tagantsev et al, Phys. Rev. B 64, 224107 (2001).
[6] X.-K. Wei et al, Nat. Commun. 5, 3031 (2014).

The authors thank funding from the EU 7th Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure InitiativeI3) and from the European Research Council under the EU 7th Framework Programme (FP7/2007-2013) / ERC grant agreement No [268058] Mobile-W. The Swiss National Science Foundation is acknowledged for additional financial support [Grant number 200020 140539/1].

Fig. 1: Fig. 1. Domain boundary types in tetragonal SrTiO3 (space group I4/mcm, octahedral rotation symbol a0a0c-). (a) Easy boundary: the octahedral rotation c-axis is orthogonal to the boundary plane. (b) Hard boundary: the octahedral rotation c-axis is parallel to the boundary plane.

Fig. 2: Fig. 2. (a) Morphology of the APBs in PbZrO3. (b) HRTEM image of PbZrO3 along [001] direction. The schematic unit cells are overlapped on the image. Yellow circles: Pb columns, green circles: Zr/O columns, red circles: oxygen columns. The schematic unit cells illustrated at both sides show the p-phase shift of the APB.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-2442 Atomic Structure of antiphase nanodomains in Fe-doped SrTiO3 films used for resistive switching memory (ReRAM) applications

Du H.1, 2, Jia C.1, 3, Mayer J.1, 2, Lenser C.3
1Ernst Ruska-Centrum für Mikroskopie und Spektroskopie mit Elektronen, Forschungszentrum Jülich GmbH, Jülich, 52425, Germany, 2Gemeinschaftslabor für Elektronenmikroskopie (GFE), RWTH Aachen, Aachen, 52074, Germany, 3Peter Grünberg Institut, Forschungszentrum Jülich GmbH, Jülich, 52425, Germany
h.du@fz-juelich.de

Strontium titanate (SrTiO3) has been used as a model material to understand the underlying mechanism of resistive switching phenomena for ReRAM applications.[1,2] It has been widely accepted that lattice defects play an essential role in the resistive switching processes in SrTiO3.[2,3] Therefore, a profound knowledge of the microstructures of a large variety of defects is a prerequisite for thoroughly understanding the microscopic switching mechanism. In this study, we have investigated antiphase nanodomains in Fe-doped SrTiO3 films by high resolution scanning transmission electron microscopy (STEM).

Randomly distributed dark contrast features with size up to about a few nanometers were observed in both the cross-section and plan-view bright field images of the Fe-doped SrTiO3 film (Fig. 1). High-resolution cross-section HAADF images recorded along [100] and [110] zone axes reveal that these dark contrast features are antiphase nanodomains formed by half unit cell shifting along the [011] direction with respect to the surrounding film (Fig. 2). Plan-view HAADF imaging shows that the antiphase boundaries appear to be composed of edge-shared TiO6 octahedra with a local Ti enrichment (Fig. 3). The edge-shared TiO6 octahedra are commonly seen in TiO2, such as anatase and rutile. The observed antiphase boundaries therefore differ from those of the Ruddlesden-Popper phases,[4] which are with an A-site atom enrichment.

Since antiphase nanodomains were not observed in Fe-doped SrTiO3 single crystals, Fe-doping alone is not sufficient for the formation of antiphase nanodomains. A reasonable explanation for the formation of the antiphase nanodomains appears to be atomic scale chemical inhomogeneities or fluctuations during the film growth causing local Ti-enrichments, which in turn induce the formation of antiphase nanodomains.

The identified defect structures offer great potential for tailoring the electronic properties of SrTiO3. By deliberately controllable chemical fluctuations, a similar formation of Antiphase nanodomains in other perovskite oxides is conceivable.

References

[1] Waser, R.; Aono, M. Nat. Mater. 2007, 6, 833.

[2] Waser, R.; Dittmann, R.; Staikov, G.; Szot, K. Adv. Mater. 2009, 21, 2632.

[3] Szot, K.; Speier, W.; Bihlmayer, G.; Waser, R. Nat. Mater. 2006, 5, 312.

[4] Ruddlesden, S. N.; Popper, P. Acta Cryst. 1958, 11, 54.

This work has been supported by the DFG (SFB 917, Project Z2). The authors thank Cong Zhang for providing the single crystal samples, Doris Meertens, Wilma Sybertz, and Maximilian Kruth for preparing the TEM lamellae, Juri Barthel for permitting to use the Dr. Probe software for STEM simulation, and Lothar Houben and Chris Boothroyd for training in operating the FEI Titan-PICO microscope.

Fig. 1: Cross-section a) and plan view b) BF-TEM images of a Fe-doped SrTiO3 film grown on a Nb-doped SrTiO3 substrate.

Fig. 2: Cross section HAADF images recorded along a) the [100] and b) the [110] zone axes. Defect areas were marked by rectangles. Solid circles: matrix lattice; open circles: antiphase lattice (green: Sr, yellow: Ti). Scale bar: 1.0 nm.

Fig. 3: Plan view HAADF image of an antiphase nanodomain imaged aong the [001] zone axis. Edge-on areas were marked by rectangles, which was used for modeling and simulation. Structure models of the marked antiphase boundary and anatase are shown for comparison (green: Sr, yellow: Ti, red: O). Scale bar: 1.0 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-2636 TEM investigation of the elementary plasticity mechanisms in TRIP/TWIP Ti-12 wt.% Mo alloy

Idrissi H.1,2, Marteleur M.2, Prima F.3, Jacques P. J.2, Schryvers D.1
1Electron Microscopy for Materials Science (EMAT), University of Antwerp, Groenenbor-gerlaan 171, B-2020 Antwerp, Belgium, 2Université catholique de Louvain, Institute of Mechanics, Materials and Civil Engineering, Place Sainte 2, B-1348 Louvain-la-Neuve, Belgium, 3Laboratoire de Physico-Chimie des Surfaces, Groupe de Métallurgie Structurale, Chimie-ParisTech, Rue Pierre et Marie Curie, 75005 Paris, France
hosni.idrissi@uantwerpen.be

During the past few decades, interest in titanium alloys has continuously increased due to their combination of properties such as high strength, low density, biocompatibility and good corrosion resistance. However, both their low ductility and their lack of strain-hardening limit their use in advanced applications where superior combinations of strength and ductility are required. Recently, an electronic design approach for the development of a new family of titanium alloys exhibiting a combination of high ductility and improved strain-hardening rate has been proposed and exemplified in the binary Ti–12 wt.%Mo grade [1]. The chemical formulation of such alloys was designed following the Morinaga model based on the cluster DV-Xα method by mapping electronic parameters Bo (bond order) and Md (d-orbital energy) [2]. This map is of great interest since it can be used as a tool to design new titanium alloys exhibiting specific improved performances.

Based on this approach, as-quenched Ti-12 wt.% Mo alloy with athermal ω phase has been designed to exhibit simultaneous transformation induced plasticity (TRIP) and twinning in-duced plasticity (TWIP) effects in order to improve mechanical properties of the as-quenched β phase [1]. Mechanical tests exhibit a work hardening rate never reached before in Ti alloys (Figure 1a) [3]. The monotonic raising of the strain-hardening rate reaches a maximum value of ~ 2000 MPa from the elastic limit to ε = 0.1, the early stage of the plastic deformation. Electron back scattered diffraction (EBSD) has shown the occurrence of {332} <113> twinning and β→α” stress-induced martensitic transformations during tensile loading (Figures 1b and 1c). On the other hand, high resolution transmission electron microscopy (HRTEM) revealed the presence of {112} <111> nanotwins (Figure 2a) accompanied with stress-induced ω phase (Figure 2b). The ω phase was found to nucleate on the (211) mechanical twin boundaries. The nanoscale mechanisms controlling the formation and the interaction of phase boundaries and twin boundaries have been investigated using TEM techniques including aberration corrected TEM and in-situ TEM micro/nanomechanical testing. Special efforts have been also made to elucidate the elementary mechanisms controlling the interaction of deformation dislocations (especially screw dislocations) with the stress induced interfaces. Finally, the role of these fundamental mechanisms in the remarkable mechanical properties exhibited by the Ti alloy used in the present study is discussed.

[1] M. Marteleur et al., Scripta Materialia, Vol 66, Issue 10, 2012, pp. 749-752
[2] M. Abdel-Hady et al., Scripta Materialia. Vol 55, Issue 5, 2006, pp. 477-480
[3] F. Sun et al., Acta Materialia, Vol 61, Issue 17, 2013, pp. 6406-6417

Fig. 1: Figure 1: (a) Tensile true strain/true stress curve is shown by the black line. The strain-hardening rate, dσ/dε, is plotted in black circles and the smoothed curve is shown in red. (b) EBSD map showing {332} <113> twins (orange) in β matrix (blue) in sample deformed at ε = 0.05. (c) EBSD map showing two variants of α” phase (blue and green).

Fig. 2: Figure 2: HRTEM images taken along [110] in sample deformed at ε = 0.007, showing (a) {112} <111> mechanical twin lamella. Note the high strain field at the tip of the twin due to the presence of twinning partial dislocations stopping in the β matrix and (b) stress-induced ω phase nucleating on the (211) plane of the twin lamella.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-3021 Charge-Density Wave and Domain-Contrast Reversals in aThree-Dimensional Material System Observed by TransmissionElectron Microscopy

Ming-Hao Lee1, Cheng-Hsuan Chen 1, Ming-Wen Chu1
1Center for Condensed Matter Sciences, National Taiwan University, Taiwan
d95222022@ntu.edu.tw

The charge-density wave (CDW) is an electronic ground state with broken translational symmetry brought by correlated electron-phonon interactions. Decades of studies on the subject have established that a highly anisotropic band structure is essential for the emergence of this ground state and is characteristic to a wide spectrum of low-dimensional materials. Indeed, CDW has been largely found in one-dimensional (1D) and two-dimensional (2D) material systems such as NbSe3 and TaSe2. The existence of CDW in three-dimensional materials is usually not expected. It is not found until recently that the tetragonal rare-earth transition-metal silicide system with three-dimensional crystallographic structure R5T4Si10, where R is Dy, Ho, Er, Tm, and Lu, and T = Ir and Rh, can exhibit CDW phase transitions. Here, we report the investigations of CDW in Dy5Ir4Si10 at different temperatures using transmission electron microscopy (TEM) techniques including electron diffraction (Figure 1) and dark-field superlattice imaging. Incommensurate superlattice diffraction spots along c axis were observed in the electron diffraction patterns (Figure 2) when the sample was cooled below the well-known CDW transition temperature at ~208 K. CDW becomes commensurate with further cooling and configurations of CDW dislocations imaged by the dark-field technique convincingly show that the CDW phase transition is accompanied by a concomitant cell-doubling structural phase transition. Intriguingly, the cell-doubling transition is featured by an inversion-symmetry breaking observed by further convergent beam electron diffraction. A disparity in the CDW modulation vectors, q+ and q-, readily arises, breaking the CDW principle that the associated modulations should remain invariant upon spatial inversion. Upon dark-field imaging using respective q+ and q-, we surprisingly observed the contrast reversal of the CDW domains (Figure 3), a phenomenon largely undocumented in the past. The potential linking of this discovery to the emergent chiral CDW, which allow the breakdown of the spatial-invariant principle, was discussed.

Fig. 1: Electron diffraction pattern along the [100] zone axis obtained at 100 K, which shows the commensurate satellite spots of charge-density wave modulations characterized by q+ = (0, 0, ¼) and q- = (0, 0, –¼). The room-temperature pattern along the same projection is also shown (inset).

Fig. 2: [100]-zone-axis diffraction pattern taken at the same sample region to Figure1 at otherwise 190 K,showing the emergence of incommensurate satellite peaksas indicated by the white circle.

Fig. 3: (a) and (b), the superlattice dark-field TEM images obtained from the respective commensurate q+ = (0, 0,¼)and q- = (0, 0, –¼) vectors in Figure 1 as a result of the symmetry breaking along c axis upon the CDW modulation.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-3148 Thermal-activated Cation Exchange Reactions between Ionic Nanocrystals

Casu A.1, Genovese A.1, Falqui A.1,2
1Department of Nanochemistry, Istituto Italiano di Tecnologia, Genova, Italy, 2King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
alberto.casu@iit.it

Cation exchange reactions have been recently demonstrated to be a powerful route to synthesize inorganic nanocrystals (NCs) with controlled size, shape, and crystal phase. In this kind of reactions the cations within the crystal structure of ionic NCs can be replaced with other cation species, while the sublattice of anions remains in place [1].
This study concentrates on transmission electron microscope (TEM) experiments of cation exchange and diffusion reactions between CdSe and Cu2Se nanocrystals via thermal annealing. We are be able to fully convert nanorods and nanowires of CdSe with hexagonal structure into Cu2Se nanorods and nanowires with cubic structure, directly at subsolidus condition (i.e. not in solution or passing through melt status) by thermal activation performed in-situ (Figure 1), as confirmed by selected area electron diffraction (SAED) patterns analysis. TEM and energy filtered TEM (EFTEM) analysis revealed that Cu species interdiffusion and concomitant Cd sublimation reactions are activated when mixture of nanorods or nanowires of CdSe and nanoparticles of Cu2Se are annealed together under high vacuum condition up to 400°C. The as-obtained cation exchange preserves shape and textural relation of nanoparticles during thermal reaction, aside from an anisotropic partial dissolution of hexagonal wurtzite nanorods and nanowires, as shown from high angle annular dark field (HAADF) scanning TEM (STEM) imaging. These thermal instability and consequent preferential dissolution processes are considered to be activated by intra-crystalline diffusion of Cu species from Cu2Se nanoparticles into hexagonal CdSe nanorods and nanowires lattices which trigger out-diffusion and sublimation of Cd. The mixture of CdSe and Cu2Se nanocrystals is therefore an example of thermally unstable system, despite the separate components showing to be stable under the same conditions.

[1] Robinson, R. et al. Science 317 (2007) 355.

Fig. 1: In-situ annealing experiment of CdSe nanorods and Cu2Se nanoparticles. Zero loss filtered image, corresponding EFTEM maps of Cu and Cd, SAED patterns with integrated diffractogram acquired at room temperature (a-c) and 400°C (d-f). Scale bars 50 nm.

Fig. 2: In-situ annealing experiment of CdSe nanowires and Cu2Se nanoparticles. a) zero loss filtered image; EFTEM maps, Cu-green, Cd-blue acquired at room temperature (b) and 400°C (c); d-e) HAADF images acquired at 400°C showing anisotropic partial dissolution of nanowires over the time at beginning (d), 4 (e) and 8 minutes (f). Scale bars 50 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-9-O-5698 Assessing phase stability and lattice misfit in Co-base superalloys at elevated temperatures by in situ TEM heating experiments

Eggeler Y. M.1, Müller J.1, Fries S. G.2, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Department for Material Science, Friedrich Alexander Universität Erlangen-Nürnberg, 91058 Erlangen, Germany, 2Interdisciplinary centre for advanced materials simulation (ICAMS), Department of scale bridging Thermodynamic and Kinetic Simulation (SKTS), Ruhr-Universität Bochum, 44801 Bochum, Germany
yolita.eggeler@fau.de

Co-based alloys, of a composition of Co-12Al-9W, form a stable two phase γ/γ’ microstructure at 900 °C [1]. This microstructure is morphologically identical to the microstructure of Ni base superalloys and promises greater temperature capability due to the higher melting point of Co compared to Ni. γ’ cubes, consisting of the L12 crystal structure are coherently embedded in a solid solution fcc (A1) γ matrix. In contrast to Ni-base superalloys the lattice constant of the γ’ phase is larger than the one of the γ matrix corresponding to a positive lattice misfit. To ensure precipitate hardening at temperatures, which are relevant to practical applications, 700-1100 °C, as experienced in gas turbine applications, the stability of the γ/γ’ phases is of fundamental importance.

In this study the stability of the γ and γ’ phase as well as the lattice misfit in Co based alloys was investigated. Employing in situ heating in the transmission electron microscope (TEM) the dissolution of the γ’ precipitates is directly observed. Small tertiary γ’ precipitates in the channels start to diminish at a temperature of about 800 °C and are fully dissolved at 850 °C. The volume fraction of the γ channels increases at the expense of the γ’ precipitates, Figure 1. During heating, the different thermal expansion of γ and γ’ and the redistribution of alloying elements changes the lattice parameters and therefore the resulting lattice mismatch [2]. In contrast to lattice misfit measurements by x-ray diffraction (XRD) [3], where the contributions from γ and γ’ are often difficult to separate, the diffraction intensities of the two phases can be clearly distinguished in selected area electron diffraction patterns. Combined with in situ heating experiments this enables the determination of the local lattice misfit at various temperatures up to 950 °C .

With the γ and γ’ volume fractions, evaluated from the images, and the lattice parameters at different temperatures, a thermodynamic assessment using the software Thermo-calc , and TCNI6 database (www.thermocalc.com), shows to be in good agreement, as illustrated for the volume fraction in Figure 2.

[1] J. Sato et al, Science (2006), vol.312, p. 90

[2] Yu.N. Gornostyrev et al, Scripta Materialia (2007), vol.56, p. 81-82

[3] F.Pyczak et al, Materials Science & Engineering A (2013), vol.571, p. 13-18

The authors gratefully acknowledge the collaboration with Prof. Pollock from the University of California in Santa Barbara, GE, NSF, and the DFG priority program SFB-TR 103 for financal support.

Fig. 1: Series of TEM dark field micrographs recorded during in situ heating of a Ni containing Co-base alloy. The arrow marks the region, where the tertiary γ’ precipitates show contrast at 708 °C and 801 °C until no contrast is observed at 854 °C, where the tertiary γ’ precipitates are fully dissolved.

Fig. 2: The evolution of phases with temperature for the Ni containing Co-base superalloy shown in Figure 1 calculated with the software Thermo-calc. Dots represent the experimentally obtained volume fraction of the γ’ phase (blue) and the γ phase (red) during in situ heating.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-1622 Simultaneous strain and chemical mapping applied to a Ni-based superalloy

Mueller J.1, Niekiel F.1, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Erlangen, Germany
julian.mueller@ww.uni-erlangen.de

Strain analysis with high spatial resolution is important to understand and tune e.g. mechanical or electrical properties of materials and devices. Averaging scattering methods, like neutron and x-ray scattering [1], are well established to measure strain, but have the drawback of no (or almost no) spatial information. Spatially resolved TEM techniques (CBED [2], GPA), on the other hand, often suffer from the limited number of data points or from the small volume which can be probed.

Here we present a method to map strain by CBED on a very local scale. EDX spectra are recorded simultaneously, which enables to correlate structural information from the CBED patterns to the chemical information.

While there are well established tools to quantify EDX spectra, the evaluation of a large number of CBED patterns associated with such a mapping fueled the development of an automized procedure. As sketched in Fig. 1, the CBED patterns are first binarized using a variety of image filters. A Hough transformation is applied on the binarized image to roughly find the lines. In the next step these lines are refined on the original CBED pattern minimizing the intensity along the line. The distances of particular intersection points are calculated to characterize the local strain state of the material.

In principle, this technique is not limited to a particular material system or microstructure. However, it best reveals its benefits in investigations of strain at interfaces, in layered structures or of strain distributions due to chemical gradients. In the example depicted in Fig. 2, a γ/γ’ microstructure of a Ni based superalloy (ERBO1C) was investigated in which cuboidal γ’ precipitates (L12) are coherently embedded in a γ solid solution matrix. Due to a small lattice misfit (<0.5%), the γ channels are weakly tetragonally distorted. This small distortion has a large influence on nucleation, mobility and annihilation of interface dislocations as well as on the directional coarsening of the microstructure during creep deformation.

For the mapping in Fig.1 an area of 200 nm x 200 nm of a γ/γ’ microstructure has been selected. In b) and c) the chemical distribution of Ni and Cr is shown. By using the technique described above, the lattice parameter axx, ayy and the tetragonal distortion axx/ayy are evaluated (d)-f)). It can clearly be seen that both orientation dependent lattice parameters vary in the perpendicular γ channels, while in the γ’ precipitate no significant difference is observed. The direct comparison of the chemical and structural mapping demonstrates the good agreement of both data sets.

[1] R. Gills et al., Appl. Phys. A (2002) 47: 1446C.

[2] C. Schulze and M. Feller-Kniepmeier, Mater. Sci. Eng. (2000) 281: 204H.

The authors gratefully acknowledge the collaborative research center SFB/TRR 103 and the DFG training research group 1229 for financial support.

Fig. 1: Sketch of the automized CBED pattern evaluation procedure: The CBED pattern a) is binarized in the first step to roughly find the lines via Hough transformation b). These are then optimized on the original image minimizing the intensity along the lines c) to calculate the distances of particular intersection points to characterize the strain state.

Fig. 2: a) STEM image of a γ/γ’ microstructure. The mapped area is highlighted with a red rectangle. b) and c) show the Ni and Cr distribution of the mapped area from EDXS. d) and e) show the orientation dependent lattice parameter determined from CBED patterns, the tetragonal distortion is plotted in f).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-1726 Transformations through pseudomorphosis of asbestos minerals in thermally processed asbestos-containing materials investigated through SEM/EDS and micro-Raman spectroscopy: implications for recycling of hazardous wastes.

Viani A.1, Gualtieri A.2, Mácová P.1, Pollastri S.2
1Centrum Excelence Telč, ÚTAM AV ČR, Batelovská 485-6, 58856 Telč, Czech Republic, 2Dipartimento di Scienze Chimiche e Geologiche, Università di Modena e Reggio Emilia, Via Sant’Eufemia 19, I-41121 Modena, Italy
viani@itam.cas.cz

Asbestos-containing materials, extensively used in the past in many European countries, are now considered hazardous wastes of great concern. It has been proved that inertization can be obtained via thermal treatment above 1100 °C. This solution relies upon the scientific evidence that all asbestos minerals at high temperature transform into stable crystalline silicates via a solid state recrystallization process [1]. Asbestos fibres preserve the same external crystal habit although a complete modification of the structure at a molecular scale occurred. This phenomenon is called pseudomorphosis. With increasing the temperature of the thermal treatment above 650-750 °C, the transformation sequence of chrysotile asbestos predicts the crystallization of forsterite (Mg2SiO4) and enstatite (MgSiO3 ) [1]. In a system high in Ca, such as cement-asbestos, crystallization of cement phases such as larnite (Ca2SiO4), ferrite (ideally Ca4Al2Fe2O10), and Al-,Ca-,Mg-rich silicates, such as akermanite (ideally Ca2MgSi2O7) and merwinite (ideally Ca3MgSi2O8), occurs. In this work, analytical and spectroscopic techniques coupled with microscopy allowed for the study of individual residual pseudo-morphosed fibre bundles, in cement-asbestos samples heat treated at 1200 °C. Phases detected were mainly monticellite (CaMgSiO4) or akermanite. They likely formed through the reactions: CaO + MgSiO3 (en) -> CaMgSiO4 (mtc), and CaMgSiO4 (mtc) + CaO + SiO2 -> Ca2MgSiO7 (ake). This suggests that, although transformation reactions occurred largely at the solid state, a substantial mobilisation of Ca and Mg resulted. Such a process is essential for the attainment of the bulk mineralogical composition predicted by the phase diagrams in the system CaO-MgO-SiO2 [2]; however, because of crystallization under non equilibrium conditions, departures from the expected bulk phase composition are still observed. This study contributes to the definition of factors conditioning the recycling of transformed cement-asbestos as secondary raw material [2-3].

[1] Cattaneo A, Gualtieri AF and Artioli G. (2003) Kinetic study of the dehydroxylation of chrysotile asbestos with temperature by in situ XRPD. Physics and Chemistry of Minerals, 30, 177-183.
[2] Viani A, Gualtieri AF. (2014) Preparation of magnesium phosphate cement by recycling the product of thermal transformation of asbestos containing wastes. Cement and Concrete Research, 58, 56-66.
[3] Viani A, Gualtieri AF. (2013) Recycling the product of thermal transformation of cement-asbestos for the preparation of calcium sulfoaluminate clinker. Journal of Hazardous Materials, 260, 813-818.

Research supported by the project CZ 1.05/1.1.00/02.0060 from the European Regional Development Fund and the Czech Ministry for Education, Youth and Sports

Fig. 1: Example of asbestos fibres in a cement-asbestos sample after thermal treatment at 1200 °C. Pseudomorhosis is evidenced by the extensive recrystallization in what was initially an association of asbestos fibres.

Fig. 2: Micro Raman spectrum of the residual pseudo-morphosed fibre bundle shown in the inset picture. Main Raman bands of merwinite are reported.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-1785 Visualizing the room temperature plastic deformation of single crystal magnesium carried out through unit-cell-reconstruction

Liu B. Y.1, Shan Z. W.2
1Xi’an Jiaotong University, Xi’an, China
boyuliu07@gmail.com

Dislocation and deformation twinning are traditionally known to be plasticity carriers of crystalline materials at room temperature. By using in-situ TEM mechanical testing technique (Fig. 1), here we report that the plasticity of a specially orientated single crystal magnesium is carried out neither by dislocation nor by deformation twinning, but through a direct reconstruction of the unit cell, which results a twinning-like reorientation without a traditional defined twinning plane[1].
Deformation twinning is a major mode of plastic deformation for magnesium and its alloys, due to their very limited number of slip systems. But twinning mechanisms for such metals are much less understood. In our experiments, when the submicron-sized single-crystal magnesium was compressed normal to the prismatic ({10-10}) plane or stretched normal to the basal ({0002}) plane, a twinning-like reorientation was produced. However, the rotation angle of the lattice was around 90 degree, rather than the theoretical value of 86.3 degree. Moreover, the boundary largely deviated from the twinning plane that is a crystallographic plane mirroring the parent and the twin lattices. Most surprisingly, in one sample, the sweeping boundary produced a sizable plastic strain of tetragonal compression character instead of simple shear. Aberration-corrected TEM observations revealed that the boundary between the parent lattice and the “twin” lattice was composed predominantly of semi-coherent basal-prismatic interfaces instead of the {101-2} twinning plane. We proposed and demonstrated that the migration of this boundary was dominated by the movement of these interfaces undergoing basal-prismatic transformation via unit-cell-reconstruction (Fig. 2). Under the external stress, the basal plane would be transformed into the prismatic plane and vice-versa. As a result, the boundary could migrate, which led to the growth of one grain with the expense of the other one, and finally produced a considerable plastic strain (~6%). The propagation of such boundary may be less sensitive to precipitates that are traditionally important for age hardening. The reported novel plastic deformation mode in magnesium may have implication for the alloy design.

We thank Dr. Bin Li (MSU, USA), Dr. Jian Wang (LANL, USA), Dr. Xi-Yan Zhang (CQU, China), Dr. Chun-Lin Jia, Dr. Ju Li, Dr. Jun Sun and Dr. Evan Ma (XJTU, China) for their great contribution to this work.

Fig. 1: Figure 1 Experimental setup. The in-situ test was conducted on a Hysitron PicoIndenter 95 holder inside a TEM (JEOL 2100F). The sub-micron sized sample and the diamond tip were fabricated by FIB. Red and blue color of the sample schematic represent to two grains.

Fig. 2: Figure 2 Schematics of dislocation (left), twin (mid) and unit-cell-reconstruction.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-1789 Dislocation slip-twin interactions and nucleation of twins in a Mg-Zn alloy

Singh A.1, Somekawa H.1, Mukai T.2
1Structural Materials Unit, National Institute for Materials Science, Tsukuba 305-0047, Japan, 2Department of Mechanical Engineering, Kobe University, Kobe 657-8501, Japan
alok.singh@nims.go.jp

Twinning is a very important deformation mechanism in hexagonal metals, such as magnesium. The most commonly observed twin is {10-12} type twin, followed by {10-11} type twin, depending on the direction of stress. {10-11} type twin is often followed by a re-twinning of {10-12} type, forming a double twin, which also leads to fracture. Complex interactions between dislocation slip and twins occur in hexagonal systems. Nucleation of twins is also a subject of intense investigations. Atomistic simulations show that nucleation of {10-12} twins most likely occur on grain boundaries, especially with low angle misorientations [1]. In the present work twin-dislocation interactions and twin nucleation have been examined in a deformed Mg-Zn alloy.

An extruded Mg-2.4at%Zn alloy with grain size of 1-3μm was fractured by three-point bending test. A specimen below the crack surface was sampled by focused ion beam technique, and studied by a JEOL 4000EX transmission electron microscope, operated at 400 kV.

Complex structure of twinning and slip was observed beneath the crack surface. Relatively away from the surface, an array of {10-11} type twins was observed, with width of about 200 nm. Diffraction contrast showed extensive basal and prismatic slips. Dislocation pile-ups occurred on the twin boundaries. Twins re-twinned to form {10-11}-{10-12} double twins. Repeated twinning with the help of slip activity led to finer twin domains, with final sizes of about 50 nm, whose boundaries were no longer planar. Three domains A, B and C related by double twinning are shown in Fig. 1. Basal slip activity in B, and its interaction with B-C twin boundary is observed. 

Nucleation of twinning was observed on single tilt grain boundaries (STGB), such as in Fig. 2. Grains P and Q are tilted about 24-29° about <11-20> zone axis. An array of dislocations forming a 2° low sub-boundary (at Q’) interacts with the STGB, creating a twin nucleus N, which makes a {10-11} type twin with the matrix grain Q. This nucleation process is an experimental example of that shown for {10-12} twin by simulation [1].

An array of {10-11} type nano-sized twins of less than 50 nm in size was observed in the matrix with high level of basal and prismatic slip activity [2]. These are first examples of nucleation of {10-11} type twins, mediated by dislocations. These dislocation-twins interactions and nucleation of twins will be described and discussed in the presentation.

References:
[1] J. Wang et al., Scripta Mater. 63 (2010) 741.
[2] A. Singh, H. Somekawa and T. Mukai, Philos. Mag., 2014, doi: 10.1080/14786435.2013.869052

The authors are grateful to Ms. M. Isaki at NIMS for technical assistance.

Fig. 1: Nano-domains related by twinning, all oriented along a <11-20> zone axis. Lines on basal planes are drawn. A and B are related by {10-12} type twinning (ideal angle 86°). B and C are related by {10-11} twinning (ideal angle 56°). B and D are related by {10-12} twinning. A 16° boundary exists between C and E. A-B-C make a double twin.

Fig. 2: Nucleus N of a twin on single tilt grain boundary between grains P and Q. All are oriented along a <11-20> zone axis. A array or a pile-up of dislocations is observed at Q'.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-1801 EBSD Characterization of Stress-Induced ω Phase and Twinning in Metastable β Ti-V Alloys

Wang X.1, Xing H.1, Sun J.1
1Shanghai Key Laboratory of Advanced High-temperature Materials and Precision Forming, School of Materials Science and Engineering, Shanghai Jiaotong University, Shanghai 200240, PR China
jsun@sjtu.edu.cn

Recently, stress-induced products such as α″, ω phase and twinning and their effect on mechanical properties of titanium base alloys have attracted considerable attention. It has been revealed that the structural stability of the β phase significantly influences the plastic deformation mode in metastable β-Ti based alloys. In this work, stress-induced products in a series of metastable β-type Ti-xV (x=16, 18 and 20 wt%) binary alloys after tensile tests were characterized by EBSD. The stress-induced ω phase and twinning and their corresponding Schmid factors were correlated with the structural stability of the Ti-V binary alloys.

The β-type Ti-xV (x=16, 18 and 20 wt%) binary alloys were prepared by arc melting in vacuum and were hot-rolled and subjected to solution treatment at 1123 K followed by water quenching. The initial microstructure of Ti-V binary alloys is single β phase. They were tensile-tested at room temperature and a strain rate of 2×10-4 s-1. The samples for EBSD measurements were electro-polished in a solution of 5% perchloric acid and 95% methanol at −30 °C and 50 V. The tensile direction of samples was set to be parallel to the RD in EBSD measurements. The EBSD maps were taken by JEOL 7000F SEM equipped with Oxford HKL. Fig. 1(a, b) are the EBSD orientation and phase maps of Ti-16V alloy, respectively. Many stress-induced plate-like features can be observed within grains and identified as stress-induced ω phase. The habit plane of ω phase is {-5502}ω//{332}β determined by the lattice correlation boundary method. Supposing the activated growth direction of ω phase is {332}<113> in the β phase, the corresponding Schmid factors fall into a range between 0.44 and 0.48 as shown in Fig. 1(c). Fig. 2 (a, b) are the orientation and phase maps of Ti-18V alloy. The plates with mis-orientation angle of 50.5° along the <110> direction correspond to {332}<113> twins in the β phase. Additionally, stress-induced ω phases can be observed in the grain. Fig. 2(c) shows that the Schmid factors of {332}<113> deformation twins are between 0.43 and 0.50 and stress-induced ω phase is 0.47. The deformation twins are activated easily in comparison with stress-induced ω phase. Fig. 3(a) shows the orientation map of Ti-20V alloy, where all the plates were identified as {332}<113> deformation twins. The Schmid factors of {332}<113> twins are between 0.44 and 0.46 as shown in Fig. 3(b).The structural stability of β phase increases with increasing the content of V in Ti-xV binary alloys. Based on EBSD results, it can be concluded that the plastic deformation mechanism transforms from stress-induced ω phase to {332}<113> deformation twinning with increasing of the structural stability of β phase in Ti-xV binary alloys.

This research is financially supported by the NSFC under Contract no. 51371113 and 51101099 and by the STCSM under Contract no. 13dz2260300.

Fig. 1: Fig. 1 EBSD orientation map (a), phase map (b) and Schmid factors (c) of Ti-16V alloy.

Fig. 2: Fig. 2 EBSD orientation map (a), phase map (b) and Schmid factors (c) of Ti-18V alloy.

Fig. 3: Fig. 3 EBSD orientation map (a) and Schmid factors (b) of Ti-20V alloy.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-1922 Superhard WB3+x with ordered interstitial-boron solution

Cheng X. Y.1, Zhang W.1, Chen X. Q.1, Niu H. Y.1, Liu P. T.1, Du K.1
1Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences
kuidu@imr.ac.cn

Typical ultrahard or superhard materials would require three-dimensional bonding networks commonly consisting of high densities of strong covalent bonds, atomic constituents and valence electrons. Currently, the most powerful way to yield the high densities of these factors is the synthesis under high pressure conditions. However, in recent years transition metal borides have attracted extensive interests because of superior mechanical properties and ambient-condition synthesis without the need of high pressure. Among those borides, the W-B system has attracted particular attention since the report on WB4 with a measured superhardnees, Hv of about 46.3 GPa, as the highest measured hardness among the borides mentioned above. Although the superhardness of WB4 has been again confirmed experimentally and interpreted theoretically, subsequent first-principles calculations show contradictions on high-pressure experimental findings and theoretical predictions. Therefore, this tungsten boride still needs further clarification.

Because boron is a weak electronic scatterer, it is impossible to refine the accurate structure of WB3+x from XRD patterns of powder samples. Nevertheless, the aberration-corrected HRTEM image provides a powerful tool to directly visualize the light mass elements (i.e., oxygen and boron). For the hP16-WB3 structure, between any two dense boron lines there exists a tungsten-atom line, which consists of a repeated unit of every two tungsten atoms separated by a void. However, aberration-corrected HRTEM images reveal that the voids are partially occupied in the WB3/WB3+x boundary (Fig. 1e) and fully occupied in the WB3+x region (Fig. 1f) by extra boron atoms. As further evidence, the extra atoms can be also found in the aberration-corrected HRTEM images in Fig. 1j and 1k projected along the [0001] direction. This suggests that WB3+x can be considered as defective hP16-WB3 in which the extra x boron atoms occupy the interstitial sites in the tungsten layers.

By means of aberration-corrected high-resolution transmission electron microscopy experiments and in combination with variable-composition evolutionary algorithm coupled with density functional theory, we have studied and characterized the composition, structure and hardness properties of WB3+x (x < 0.5). The results rationalize the seemingly contradictory high-pressure experimental findings and suggest that the interstitial boron atom is located in the tungsten layer and vertically interconnect with four boron atoms, thus forming a typical three-center boron net with the upper and lower boron layers in a three-dimensional covalent network, which thereby strengthen the hardness.

[1] Zhang et al, Phys Rev Lett 106 (2011) 165505.

[2] Cheng et al, Appl Phys Lett, 103 (2013) 171903.

This work was supported by NSFC of China (Grant Numbers: 51221264, 51390473, 51074151, and 51174188) as well as Beijing Supercomputing Center of CAS (including its Shenyang branch) and Vienna Scientific Clusters. This work made use of the resources of the Beijing National Center for Electron Microscopy.

Fig. 1: The aberration-corrected HRTEM image of the superhard WB3+x. (a) The projection of the hP20-WB4. (b) The AC HRTEM image of WB3+x and their boundaries. (c, h) The projections of hP16-WB3. (d),(e), (f) and (i), (j), (k) The AC HRTEM images along the [11-20] and [0001] directions, respectively. (g) and (l) The projections of defective WB3+x.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-1925 Multiscale characterizations of martensitic transformation  in Ti-Ni shape memory alloys

Nishida M.1, Soejima Y.2, Mitsuhara M.1, Inamura T.3
1Department of Engineering Science for Electronics and Materials, Faculty of Engineering Science, Kyushu University, Fukuoka, Japan, 2Department of Applied Science for Electronics and Materials, Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, Fukuoka, Japan, 3Precision and Intelligence Laboratory, Tokyo Institute of Technology, Yokohama, Japan
nishida@asem.kyushu-u.ac.jp

The microstructure of the martensite in shape memory alloys is characterized by the combination of multiple habit plane variants (HPVs) to minimize the elastic strain energy upon transformation, which is so-called self-accommodation (SA). In the present study, the SA morphology of the B19’ martensite is systematically investigated by SEM, CTEM and STEM. The SEM used was Cal Zeiss ULTRA 55. The STEM used was JEM-ARM200F equipped with a spherical aberration corrector for electron optic system. The surface relief morphology of wide area in polycrystalline bulk specimen was observed by SEM. The crystallographic aspects of each HPV was determined by electron diffraction experiments with CTEM. The interface structure between HPVs was investigated by HAADF-STEM.
There are twelve pairs of the minimum SA unit consisting of two HPVs with V-shaped morphology connected to a {-1-11}B19’ Type I variant accommodation twin. It is found that an ideal SA morphology consists of three V-shaped units, i.e., a total of six HPVs, clustered around one of the <111>B2 poles with hexagonal shape as shown in Fig. 1. Triangular and parallelogram SA morphologies are also observed. The triangular morphology consists of a V-shaped unit and third HPV. Although there are four candidates of the third variant in the triangular SA morphology, specific two HPVs are only confirmed. The parallelogram morphology consists of two V-shaped units, i.e., a total of four HPVs [1]. The variant selection rule and the number of possible HPV combinations in each of these self-accommodation morphologies are established. It is revealed that there are four kinds of characteristic HPVs interface to complete the SA morphologies mentioned above. The HAADF-STEM observations well agree with the prediction of crystallographic aspects of the interfaces from the phenomenological theory of martensite crystallography and the geometrically nonlinear theory [2, 3].
In-situ cooling and heating SEM observations and three dimensional SA morphologies are also discussed. The evidence of thin foil effect in in-situ TEM observations will be provided

References
[1] M. Nishida, T. Nishiura, H. Kawano, T. Inamura, Philos. Mag. 92 (2012) 2215-2233.
[2] M. Nishida, E. Okunishi, T. Nishiura, H. Kawano, T. Inamura, S. Ii, T. Hara, Philos. Mag. 92 (2012) 2234-2246.
[3] T. Inamura, T. Nishiura, H. Kawano, H. Hosoda, M. Nishida, Philos. Mag. 92 (2012) 2247-2263.

This work was partly supported by a Grant-in-Aid for Scientific Research on Innovative Areas, “Bulk Nanostructured Metals”, No. 25102707, from MEXT, Japan and “Strategic Young Researcher Overseas Visits Program for Accelerating Brain Circulation”, No. R-2408 from JSPS, Japan.

Fig. 1: SEM image of reverse transformation surface relief showing ideal six HPVs cluster around [111]B2 in Ti-51.0 at% Ni alloy.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2103 Atomic-Resolution HAADF/ABF-STEM Observation of Radiation-Induced Defects in Ceria

Takaki S.1, Yasuda K.1, Yamamoto T.1, Matsumura S.1, Ishikawa N.2
1Department of Applied Quantum Physics and Nuclear Engineering, Kyushu University, Japan, 2Nuclear Science and Technology Directorate, Japan Atomic Energy Agency, Japan
syo@nucl.kyushu-u.ac.jp

Oxide ceramics with fluorite structure have potential applications in the field of nuclear-technology, such as to inert matrix fuels and transmutation targets, because of their exceptional resistance to radiation damage with energetic particles. In the environment of those fuel/target applications, high-density electronic excitation will be induced along the path of fission fragments, resulting in the formation of columnar defects of ion tracks. Understanding of the atomic structure of ion tracks is indispensable to clarify of the microstructure stability of the fuel/target materials under the radiation environment. This paper reports the atomic structure of ion tracks in CeO2 exposed by high-density electronic excitation under the irradiation of swift heavy ions through atomic-resolution HAADF- and ABF-STEM observations.

Sintered polycrystals of CeO2 specimens were irradiated with 200 MeV Xe ions at Tandem accelerator facility in Japan Atomic Energy Agency (JAEA) at an ambient temperature to fluence ranging from 3×1011 to 1×1014 ions⋅cm-2. High-density electronic excitation, whose electronic stopping power is 30 keV/nm, is induced at the surface region of specimens, at which microstructure observations were prrformed in the present study. Imaging and analytical TEM and scanning TEM (STEM) techniques were applied to the ion-irradiated specimens by using JEOL ARM-200F at HVEM Laboratory of Kyushu University to understand the structure of ion tracks in an atomic scale.

A low-magnification HAADF-STEM image in ceria from an end-on direction shows black-dot contrast of ion tracks as shown in Fig. 1. A line analysis of the signal intensity reveals that the atomic density of Ce cations inside the ion tracks decreases significantly. A high resolution HAADF-STEM image of an ion track in ceria (Fig. 2) shows that the crystal structure of Ce-cation column is retained even at the core region of ion tracks. It is also noted that the base-line signal intensity increases especially around the core damage region of the ion track for a size of about 4nm. An ABF-STEM image of the identical ion track shown in Fig. 2 showed that O-anion columns is preferentially blurred and/or disappeared at the core damage region of the ion track. An intensity profile across the ion track shows that the intensity of the O-anion signal is significantly reduced at the core damage region for 4 nm in diameter. Those results clearly show that the oxygen sublattice in fluorite structure is significantly disordered at the core damage region of ion tracks. The atomic structure of ion tracks implies the formation of vacancies or small vacancy clusters inside the ion tracks.

This work was partly supported by a Grant-in-Aid for Scientific Research (C) (#25420692) by Japan Society for Promotion of Science.

Fig. 1: Low magnification HAADF-STEM image of CeO2 irradiated with 200 MeV Xe ions to a fluence of 1×1014 ions⋅cm-2, which was taken from the ion-irradiated direction to show ion tracks from an end-on condition.

Fig. 2: High resolution HAADF STEM image of CeO2 taken from the [001] direction including an ion track (located at the center of the micrograph) formed under 200 MeV Xe ion irradiation to a fluence of 3×1012 ions⋅cm-2. Inserted is an atomic model of CeO2 superimposed on the HAADF-STEM image from the [001] direction.

Fig. 3: ABF-STEM image for the identical region shown in Fig. 2 (a). Magnified images of the peripheral region (b) and the core damage region of the ion track (c) are also presented.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2286 CIGS on steel substrate: Analysis of interlayer reactions using electron microscopy techniques

DONZEL-GARGAND O.1, THERSLEFF T.1, FOURDRINIER L.2, LEIFER K.1
1Uppsala university, Uppsala, Sweden, 2AC&CS - CRM group, Liege, Belgium
oldo@angstrom.uu.se

Depositing CIGS solar-cells on steel-substrates would allow fabrication of light flexible cells. This specificity would also give access to a roll-to-roll deposition process, i.e. a production-line process that would eventually lead to a cheaper final product. But electrical performance of this kind of cells is degraded by inter-reactions between the thin film multilayer and the steel substrate [1, 2]. To limit this drawback, a barrier layer is added between the steel substrate and the back contact layer [2]. Nonetheless, elemental diffusion may occur anyway.
Consequences on the electrical properties of the cell of such a phenomenon are well described in the literature [3], but no direct observation of the altered layer has been shown. These reactions occur under the surface as deep as several hundred nanometers and maybe extremely local, what makes their analysis non-trivial. The purpose of this work is to use electron microscopy techniques to study, on the nano-scale, reactions and products between the steel substrate and the active CIGS multilayer.
The analysis uses two different samples elaborated by sputtering, then annealed at 550°C for 15mn under Se atmosphere. The first sample is [Steel + Ti + Mo + Se] (Figure 1), and the second is [Steel + Ti + Mo + CIGS]. The analyses have been obtained using Transmission Electron Microscopy.
SEM imaging and EDS mapping reveals surface defects spaced by several microns as well as strong changes of sample composition in the micron size defects. In order to understand the nanometric origin of this diffusion process, targeted cross-sections of the defects were prepared using the Focused Ion Beam in-situ lift-out method. TEM-BF and HAADF imaging on the first sample revealed crystals resulting from reaction between steel and Se (“C” figure1). Structural analysis using electron diffraction combined with EDS elemental mapping confirmed the formation of a second phase consisting of hexagonal CrSe whereas Fe is largely absent in these grains.
Though such diffusion processes can be blocked by thick amorphous layers, the interaction between steel and thinner layer remains important for the development of future functional steel surfaces. Correct understanding of interlayer reaction may allow to control defects, thus improving the final solar-cell efficiency.

[1] P. Jackson et al. (2004), Contamination of Cu(In,Ga)Se2 solar cells by metallic substrate elements
[2] Wuerz, R. et al. (2009). CIGS thin-film solar cells on steel substrates, doi:10.1016/j.tsf.2008.11.016
[3] F. Pianezzi*et al. (2012). Electronic properties of Cu (In, Ga) Se2 solar cells on stainless steel foils without diffusion barrier. doi:10.1002/pip.1247

Fig. 1: STEM High Angle Annular Dark Field (Left) and TEM Bright field (Right).

Fig. 2: Selected Area Diffraction patterns (aperture diameter 400nm, position "C" figure 1); experimental (left), simulated (right), representation of the CrSe 3D-crystal orientation (center)

Fig. 3: STEM _EDS mapping [Blue: Cr K-edge, Yellow: Se K-edge, Red: Fe K-edge] (Left); RBG compiled image (Right)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2315 In-situ TEM study of the microstructural evolution in an aged Ti-50.3 at.% Ni shape memory alloy

López G. A.1, López-Ferreño I.2, Ruiz-Larrea I.1, Lopez-Echarri A.2, Breczewski T.1, San Juan J.2, Nó M. L.1
1Applied Physics II, University of the Basque Country, Bilbao, Spain, 2Condensed Matter Physics, University of the Basque Country, Bilbao, Spain
gabrielalejandro.lopez@ehu.es

Near-equiatomic NiTi shape memory alloys have attracted widespread interest for applications due to their excellent properties [Prog. Mater. Sci. 50(2005)511]. One of the main characteristics ruling potential applications is the transformation behavior, what can be notably affected by several thermomechanical treatments like post-deformation annealing, thermal cycling or aging [Scr. Mater. 45(2001)153; Mater. Sci. Eng. A 332(2002)25; Acta Mater. 50 (2002)4255]. In fully solution-annealed NiTi, the B2 austenite transforms directly into B19’ martensite, but the presence of Ni3Ti4 coherent precipitates and internal stresses can lead to the formation of the intermeadiate R-phase [Prog. Mater. Sci. 50(2005)511]. The austenite to R-phase transformation presents unique properties like narrow thermal hysteresis or high stability during cycling [Prog. Mater. Sci. 50(2005)511, Mater. Sci. Eng. A 332(2002)25] and for these reasons to understand correctly this transformation is very promising. Only few works on this transition were reported and two- or multi-stage transformations involving the formation of B19’ martensite were claimed [Scr. Mater. 69(2013)545; Scr. Mater. 72-73(2014)21]. One essential issue to be solved in order to take advantage of the R-phase transition is the B19’ suppression [Scr. Mater. 72-73(2014)21]. In the present work, in-situ TEM was used to provide direct evidence of the microstructural evolution in an aged Ti-50.3 at.% Ni alloy.

TEM slices cut from the TiNi wire were assembled together in a DSC holder to perform 75 cycles from -100 to 100°C at a heating/cooling rate of 10°C/min. Electron transparent foils were prepared by electropolishing. In order to interpret the peaks observed in the DSC measurements (see Fig. 1), the sample was investigated at different temperatures after cooling/heating in-situ in the TEM.

An image taken at 25°C upon direct transformation is shown in Fig.2. Selected area diffraction patterns (SAD) were acquired in the areas indicated by numbers. At this temperature, clear evidence of the typical R-phase spots at 1/3<110>B2 positions was only observed near grain boundaries (GB) (area 3). As following a sequence, at -10°C evidence for martensite variants were also observed in areas with a high concentration of defects (as area 2 in Fig.2). Finally, at -180°C the alloy completely transformed (Fig.3).

The phases involved in the observed multi-stage transformation were characterized in all cases (see Fig.4 as example). The starting microstructure is critical to determine the nucleation of the martensite variants and the DSC peaks are related to that.

This work was supported by the MAT2012-36421 and Consolider-Ingenio CSD2009-00013 projects from the Spanish Ministry, and by the Consolidated Research Group IT-10-310 and by the ACTIMAT-2013 from ETORTEK programs from the Basque Government.

Fig. 1: DSC measurement after 75 cycles between -100°C and 100°C. The peak temperatures as well as the martensite and austenite start and finish temperatures are indicated for reference.

Fig. 2: Bright field image taken at 25°C after cooling from 100°C near a GB.

Fig. 3: a) and b) Bright field images acquired at -180°C showing the material completely transformed.

Fig. 4: a) and b) SAD patterns acquired along the [111]B2 zone axis; characteristic R-phase spots at 1/3 <110>B2 positions and spots at 1/2 <110>B2 positions together with streaks typical for the twinned B19' are evident.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2417 Martensite Formation in Austenite Related to Deformation Twins in Adjacent Ferrite Grains in Duplex Stainless Steel

Kildahl P.1, Karlsen M.1 2, Aursand M.2, Hjelen J.1
1Department of Materials Science and Engineering, Norwegian University of Science and Technology, Trondheim, Norway, 2Statoil Research Centre, Rotvoll, Trondheim, Norway
jarle.hjelen@material.ntnu.no

Charpy impact testing has been performed in order to provoke twinning in a UNS S31803 duplex stainless steel. Bright field optical microscopy of samples deformed at temperatures below -40°C revealed deformation twins which appear to continue into adjacent austenite grains. Samples deformed at -46°C and -70°C have been examined by EBSD. To be able to examine the same region of interest by optical microscopy and EBSD, the final sample preparation was polishing with OP-U suspension.

Figure 1 shows a bright field optical image of a sample deformed at -70°C. Two deformation twins in the ferrite phase are apparently penetrating the subjacent austenite grain. In the EBSD orientation map shown in Figure 2, a change in orientation (color change) for the elongated features relative to the twins is observed at the phase boundary. This misorientation is 31.5°. Both of the deformation twins have a {112} twinning plane and <111> twinning direction. This well-known bcc twinning mode, can be presented as a rotation of ±60° about a common <111> axis.

Figure 3 is showing a phase map were bcc and fcc structures are red and green, respectively. It is observed that the elongated features inside the austenite, and the deformation twins in the ferrite, all have a bcc structure. It should also be noted that the image quality map in Figure 4, is darker for the austenite phase, while bright for the ferrite phase, including the twins. This indicates a higher degree of deformation in the austenite lattice, especially for the elongated features.

The orientation relationship, between the austenite grain (fcc) and the elongated feature (bcc), can be presented as a rotation of 90° about a common <112> axis. This is in accordance with the orientation relationship as proposed by Kurdjomov-Sachs (K-S) for the crystallographic relation that connects the parent and the product orientation during the diffusionless γ (fcc) to αM (bcc) transformation. This indicates that the elongated features observed in connection with the deformation twins are deformation-induced martensite. An equal relationship also applies for the sample deformed at -46°C. Rotated data sets, with (001)[100] austenite orientation, for samples deformed at -46°C and -70°C has revealed two martensite variants in accordance to theoretical K-S variants; type II.6 and III.3, respectively [1].

In samples deformed at temperatures below -40°C, elongated features with martensitic structure are observed in the austenite. These features are in connection with the deformation twins in the ferrite phase. A general increase in deformation twin density was observed for decreasing temperatures, with a transition temperature at -40°C.

 

Reference:

[1] M. Karlsen et al. Metallurgical and Material Transactions A, 40A:310-320, 2009

The authors wish to acknowlegde Senior Engineer, PhD Yingda Yu for providing technical support and guidance during the EBSD analysis.

Fig. 1: Bright field micrograph of specimen polished with OP-Ususpension. The lighter grains are austenite phase and the darker matrix is the ferrite phase, with deformation twins visible as dark bands. Inside the marked area, two parallell bands are crossing the phase boundary.

Fig. 2: EBSD orientation map of the delimited region in Figure 1. The two parallell bands change color (orientation) when crossing the phase boundary. The misorientation is 31.5°. The angle axis pair between the yellow austenite grain and the pink band is a rotation of 90° about a common <112> axis.

Fig. 3: Phase map showing the ferrite phase (bcc) in red and austenite phase (fcc) in green. It is observed that the elongated features in connection with the deformation twins have bcc-structure.

Fig. 4: Image quality map shows light twins inside the bright ferrite phase. In contrast, the austenite grain is darker than the ferrite grain. The elongated features appear even darker. Areas with dark colors indicate a higher degree of deformation in the lattice.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2443 EBSD study of Cu-Al-Ni single crystal martensites and their orientation relationships

Egido N.1, San Juan J.2, López G. A.1, Nó M. L.1
1Applied Physics II, University of the Basque Country, Bilbao, Spain, 2Condensed Matter Physics, University of the Basque Country, Bilbao, Spain
nora.egido@ehu.es

Shape Memory Alloys (SMAs) are intelligent materials which are able to change their own shape in response to a variation in the outer temperature or to an applied tension over the material. These outstanding properties which are known as superelasticty, pseudoelasticity and shape memory effect are the consequence of the thermo-elastic Martensitic Transformation (MT). MT in Cu-Al-Ni takes place between a high temperature β3 phase which is cubic, and low temperature phases β´3(monoclinic) or γ´3 (orthorhombic), depending on the alloy composition[1].

The Phenomenological Theory (PT) had been used to calculate the habit plane between austenite and martensite, as well as the possible twinning relationships between the different variants of martensite. These theoretical predictions were initially verified by optical microscopy, and the corresponding Orientation Relationships (OR) were determined by the back reflection Laue method. Most recently Transmission Electron Microscopy (TEM) and Electron Back-Scattered Diffraction (EBSD) have been used to confirm the PT on SMAs. But in spite of the fact that the EBSD is a powerful and fast method for orientation determination, barely any study has been focused on the systematic use of EBSD for martensite characterization on Cu-based SMAs.

Taking everything into account, in this work it is proposed EBSD as an efficient tool for the orientation determination of the martensite lathes and also for the characterization of the interface planes between martensites in Cu-Al-Ni SMAs. In order to achieve this goal, first a systematic and fast method for indexing martensites on Cu-Al-Ni SMA single crystal is proposed, where predictions of the interfaces of the variants are also given based on the self-accommodating groups. Second, directions and distances in degrees have been determined to reach the edge-on condition in the TEM for each OR between martensites for a proper characterization of the interface. This method has been tested on TEM samples from the bulk specimen where the characterization had been performed, obtaining a suitable match between the predictions and the TEM results.

[1] V Recarte, R B Pérez-Sáez, E H Bocanegra, M L Nó, J San Juan. Metall Mater Trans A 33, (2002) 2581

[2] T Saburi, C M Wayman, K Takata, S Nenno. Acta Metall 27, (1978) 979

[3] M L Nó, A Ibarra, D Caillard, J San Juan. Acta Mater 58, (2010) 6181 

This work was supported by the Spanish Ministry of Economy and Competivity, MICINN projects MAT2012-36421 and the Consolider-Ingenio CSD2009-00013, the consolidated Researchch Group IT310-10 fom the Education Department and by the ACTIMAT-2013 from ETORTEK programs of the Basque Government.

Fig. 1: Orientation determination of the martensites by EBSD, the notation used is the one described by Saburi[2]. The PT predicts that the interface for this case is (402)3´//(-40-2)4//(100)β3. For the β´3- γ´3 case, the basal planes of both phases are parallel and come from the (-202)β3 as was observed previously by insitu TEM[3]. 

Fig. 2: Edge on TEM SAD pattern of the same interface described in the Fig1. Thus it has been proved that the interface predicted by the EBSD in conjunction with the   PT is the correct one.

Fig. 3: Atomic 3D real space of the martensites in the same conditions than the diffraction pattern and EBSD maps. The arrows give the shift direction of atoms to become in lattice coincidence.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2585 Characterisation of Neutron Irradiated Gallium Arsenide

Janse van Vuuren A.1, Olivier E. J.1, Neethling J. H.1
1Centre for HRTEM, Physics Department, Nelson Mandela Metropolitan University, Port Elizabeth, South Africa
arnojvv@gmail.com

Space based solar cells are exposed to high doses of radiation from electrons, protons, γ-rays, X-rays and also neutrons [1, 2]. The resulting radiation damage leads to the eventual degradation of these solar cells. It is therefore important to understand and predict the radiation effects on the properties of these materials. GaAs is a well suited material for solar cells deployed in space, due to its intrinsic semiconducting properties [3, 4]. A review of the relevant literature indicates that the effects of neutron irradiation on GaAs have not been studied in detail. The aim of this investigation is the characterisation of the microstructure of GaAs irradiated with a spectrum of fast and thermal neutrons to a dose of 1x1020 neutrons/cm2. Irradiation is followed by annealing in the temperature range from 600 to 1000 °C. The evolution of the microstructure was investigated by using conventional and high resolution transmission electron microscopy (TEM and HRTEM). Conventional TEM revealed a high density of dislocation loops in the unannealed neutron irradiated GaAs (Fig. 1). The presence of these small loops indicates that annealing occurred during the neutron irradiation process, facilitating the agglomeration of point defects. The loop diameters increased after annealing at 600 °C (Fig. 2) and 800 °C (Fig. 3). The dislocation loops which have {110} habit planes were found to be of interstitial nature. This finding is in agreement with earlier studies on proton bombarded and 1 MeV electron irradiated GaAs where interstitial loops on {110} planes became visible after annealing at 500 °C [5]. It was found that the dislocation loop density produced by a dose of 1x1020 neutrons/cm2 in GaAs, is similar to that produced by a proton dose of ~1x1016 protons/cm2 at their projected range [6]. High resolution (aberration corrected) TEM and STEM of samples annealed at 600 °C confirmed the nature of the dislocation loops (not shown) and also revealed the presence of defects that were not observed before by conventional TEM (Fig. 4). These defects were found to be two-layer nano-twins on the {111} plane. An explanation for the origin of the nano-twins in neutron irradiated and annealed GaAs wil be presented.

References
[1] M. Hadrami et al., Sol. Energy Materials and Solar Cells 90 (2006) 1486
[2] A.F. Meftah et al., Renewable Energy 34 (2009) 2422
[3] T.V. Torchynska, G.P. Polupan, Semiconductor Phys., Quantum Electronics & Optoelectronics 5 (2002) 63
[4] C.S. Solanki and G. Beaucarne, Energy for Sustainable Development 11 (2007) 17
[5] J.H. Neethling, Proc. 13th Internat. Congress on EM, Paris, 2A (1994) 101
[6] J.H. Neethling and H.C. Snyman, J. Appl. Phys. 60(3) (1986) 941

Fig. 1: A bright field TEM micrograph of neutron irradiated GaAs as-irradiated.

Fig. 2: A bright field TEM micrograph of neutron irradiated GaAs annealed at 600 °C.

Fig. 3: A bright field TEM micrograph of neutron irradiated GaAs annealed at 800 °C.

Fig. 4: An unfiltered HAADF STEM micrograph of a double-layer nano-twin in neutron irradiated GaAs annealed at 600 °C.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2587 Phase transformations in the nitrocarburizing surface of carbon steelsrevisited by microstructure and property characterizations

Chen W. L.1, Wu C. L.1, Chen J. H.1, Ni S.2
1Hunan University, Changsha, China, 2Central South University, Changsha, China
cuilan-wu@163.com

Ferritic nitrocarburizing is a widely employed industry process, by which a strengthened nitrocarburized surface can be formed on mechanical tools or parts made of steels. Mainly composed of the ε-Fe2-3(C,N) and the γ′-Fe4(C,N) carbonitride phases as well as the α-Fe phase, the nitrocarburized surface has featured microstructures and properties that are directly related to the phase transformations occurred in the surface layers. Thus far the following phase transformation sequence for the surface has generally been accepted: α-Fe + N/C → ε → γ′. In the present work, these phase transformations were systematically revisited by microstructure and property characterizations in association with a controlled nitrocarburizing process by which the microstructures and properties of the surface are adjustable. A Siemens D5000 XRD, a JSM 6700F SEM, a JEOL HRTEM operating at 300kV and a JSM JXA-8230 electron probe microanalyzer (EPMA) equipped with a wavelength dispersive spectroscopy (WDS) were used for microstructure examinations. An instrumented nanoindenter (CSM UNHT) and a standard Vickers hardness tester were employed for property measurements. Our study demonstrates that to fully understand the microstructure and the property of a nitrocarburized surface, the following phase transformation sequence has to be adopted: α-Fe + N/C→ γ-N/C + N/C → γ′ + N/C → ε, in which the existence of a transitional austenite phase containing N/C-atoms (γ-N/C) has to be assumed and the γ′-phase actually forms prior to the ε-phase.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2641 Correlating the atomic and electronic structures of CaTiO3:Pr long persistence phosphors with nanoscale spatially-resolved cathodoluminescence.

Bocher L.1, Aguirre M. H.2, Otal E. H.3, Kociak M.1
1Laboratoire de Physique des Solides, CNRS-UMR 8502, Université Paris-Sud, 91405 Orsay, France, 2Dept. of Physics Condensed Matter and Laboratory of Advanced Microscopy, Institute of Nanoscience of Aragón, University of Zaragoza, Spain, 3DEINSO-Dept. of Solid State Research CITEDEF–CONICET Buenos Aires and UTN-Santa Cruz Regional Faculty, Río Gallegos, Argentina
laura.bocher@u-psud.fr

The development of new persistent luminescent phosphors in solid state lighting research is a challenge for advanced flat-panel applications. Persistent phosphors involve two types of active centers, i.e. emitters that are capable of emitting radiation after being excited and traps which rather store excitation energy and release it gradually to the emitters. Whereas the emission wavelength of a persistent phosphor is mainly determined by the emitter, the persistence intensity and time are determined by the trapping states generally associated with lattice defects or dopants.

The long persistence of CaTiO3:Pr phosphors is based on the luminescence of the rare earth atoms dispersed in the perovskite lattice, more specifically due to the 1D2–3H4 transition at 615 nm, i.e. close to the “ideal red”. Despite numerous studies dedicated on the enhancement of the afterglow efficiency at the mascroscopic level, no clear mechanism of the afterglow process have been suggested to date. A recent study on the incorporation of CaO excess in the perovskite lattice demonstrates an increase of the persistent luminescence [1]. The well-known defect chemistry of CaTiO3 suggests the formation of Ruddlesden-Popper (RP) planar faults or layers intergrowth due to CaO excess. Besides, Ti and/or O vacancies can be induced by the incorporation of CaO in excess. These chemical inhomogeneities in the matrix may have a stronger influence on the persistence luminescence since they can act as traps and increase the decay time, but no direct evidence has been revealed so far.

Here we combine electron-based spectroscopies as an unique approch to correlate the structural/chemical heterogeneities in the CaTiO3 lattice with their optical response probed at the nanoscale. We use aberration-corrected electron microscopes coupled to high-resolution energy electron-loss spectroscopy (EELS) for imaging and identifying down to the atomic scale the different structural defects in the perovskite matrix. Besides, we map at the nanoscale the spatial distribution of the spectrally-resolved cathodoluminescence (CL) using a home-made optical spectrometer coupled to a STEM-VG microscope [2]. Hence the local Pr distribution on the cationic sites of the perosvkite lattice as well as the inter-valence charge transfer mechanism are further investigated.

[1] E. H. Otal et al. Optical Materials Express, 2 (2012) 405

[2] L. Zagonel et al. Nanoletters, 11 (2011) 568

The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative - I3)

Fig. 1: a) HAADF intensity map of the probed crystallite, b) extracted CL spectra (over 40*40 nm2) at two distinct sites: (i) with solely the red emission and (ii) with a self-trapped exciton emission additionnally to the red one, c) and d) maps of the excition emission and red emission in the host material, respectively.

Fig. 2: a) large view STEM-HAADF image from an edge of the same crystallite studied in Fig. 1 a), b) STEM-HAADF image over the planar structural defect with the scanned region of interest, c) and d) Ca-L2,3 and Ti-L2,3 maps, respectively, indicating the planar defect as an extra single CaO plan typical of Ruddlesden-Popper defect in perovskite lattice.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2692 Microstructural and optical characterization of grain boundaries in GaN films grown on polycrystalline graphene layers

Yoo H.1, Yoon S.1, Chung K.1, Yi G. C.1, Kim M.1
1Seoul National University, Seoul, Republic of Korea
yoohb86@snu.ac.kr

 Hybrid systems of inorganic semiconductor films and graphene have recently opened up a new field of transferable optoelectronics.1 Particularly, GaN films grown on chemically vapor-deposited (CVD) graphene makes it possible to take a step toward practical application.2 Nevertheless, polycrystallinity of the CVD graphene have resulted in the formation of high-angle grain boundaries in the GaN films which have rarely been reported. Investigation of defects such as grain boundaries is essential since they play a significant role in determining physical properties of the system. In this report, we examined the optical properties of grain boundaries in conjunction with microstructure and electronic structure characterization to discuss the origin of those optical properties.
 Optical properties of the grain boundaries in GaN films were investigated by combination of electron backscatter diffraction (EBSD) and cathodoluminescence (CL) analyses. The position of grain boundaries was located in scanning electron microscope by detecting EBSD pattern where CL analysis was conducted subsequently, revealing that the grain boundaries in the GaN films act as non-radiative recombination sites (Fig.1).
In order to further investigate the origin of the optical properties of the grain boundaries, we first found the atomic configurations of those grain boundaries using an aberration corrected scanning transmission electron microscope (STEM). Atomic configuration of the grain boundaries showed different arrangements depending on the misorientation angles of each grain. For example, a coincident site lattice boundary formed at specific misorientation condition was composed of a periodic array of open core structures (Fig.2). Mostly, constituent core structures of grain boundaries were similar to previously reported dislocation core structures. In some cases, however, grain boundaries exhibit non-periodic arrangement of certain types of core structures that have rarely been reported.
 Based on the atomic configurations investigated by STEM analysis, we performed density functional calculations of the grain boundaries to probe the local electronic structures which are highly related to optical properties. Furthermore, variation of local electronic structure induced near the grain boundaries was investigated experimentally by detecting electron energy loss near edge fine structure, making it possible to discuss the origin of non-radiative optical characteristics of the grain boundaries.
 To this end, we characterize the optical properties of the grain boundaries in GaN films, and we expect that the origin of them could be identified by atomic and electronic structure characterization.
1 H. Yoo et al., Adv. Mater. 24 515 (2012)
2 H. Yoo et al., Appl. Phys. Lett. 102 051908 (2013)

Fig. 1: SEM image, EBSD mapping images, and CL mapping image of GaN thin films. (a) SEM image of the GaN thin films for EBSD and CL analyses. EBSD inverse pole figure maps in (b) the normal direction and (c) the transverse direction. (d) CL panchromatic image obtained from the same region where EBSD analysis was conducted.

Fig. 2: Fourier-filtered Cs-corrected STEM image of coincident site lattice grain boundaries in GaN thin films
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2706 Synthesis and characterisation of mackinawite (FeS) and influence of Cu-doping on phase and structure transformations

Zavašnik J.1, Rečnik A.1
1Jožef Stefan Institute, Ljubljana, Slovenia
janez.zavasnik@ijs.si

Iron sulphides play important role in natural processes and are widely used as geochemical indicators. Despite extensive research in last decades many questions about this highly-complex system remain unanswered. Although it is generally accepted that Fe-sulphides nucleate from the amorphous precipitates [1], the exact understanding of the subsequent phase transformations becomes complicated by metastable transient phases, extensive solid solutions and unquenchable polymorphs. To explore some of these issues we synthesized mackinawite-like near-amorphous precipitate from Fe-chloride, sulphur and diethanolamine using ultrasonic irradiation. This “first precipitate” was further solvothermally treated at temperatures up to 200 °C; to preserve the intermediate metastable phases, small amount of Fe in starting reagents was substituted with Cu. For characterisation of the products we employed XRD (PW1710, Philips Analytical B.V., Germany) and various TEM techniques (JEM-2100, Jeol Inc., Tokyo, Japan).

The cell parameters of fresh undoped FeS correspond to the reference values for layered FeS (mackinawite) [2], while doping with Cu results in the expansion of the unit cell along the c-axis, proportional to the amount of Cu in the starting composition [3]. Incorporation of Cu between the (001) layers of mackinawite strengthens the structure and enhances its crystallinity. The incorporation of transition metals in the structure is a temporary process and these interactions strongly influence the pathways for the subsequent phase transitions (Fig. 2). While during solvothermal treatment undoped FeS transforms directly to pyrite (FeS2), the Cu-rich mackinawite transforms into different Cu-Fe-S phases, depending on the amount of Cu absorbed in the initial precipitate: large amounts of Cu results in the formation of bornite and chalcopyrite, while at low Cu concentrations a mixture of Cu-rich mackinawite and cubic FeS [4] is obtained, which are not observed in pure Fe-S system (Fig. 1). The investigation of such product showed that structural defects can be preserved during structural transformation.

References:

[1] Benning LG, Wilkin RT, Barnes HL (2000) Reaction pathways in the Fe–S system below 100°C. Chemical Geology 167

[2] Csákberényi-Malasics D, Rodriguez-Blanco JD, Kovács Kis V, Rečnik A, Benning LG, Pósfai M (2012) Structural properties and transformations of precipitated FeS. Chemical Geology 294-295

[3] Morse JW, Arakaki T (1993) Adsorption and coprecipitation of divalent metals with mackinawite (FeS). Geochimica et Cosmochimica Acta 57

[4] Zavašnik J, Stanković N, Arshad SM, Rečnik A (2014) Sonochemical synthesis of mackinawite and the role of Cu addition on phase transformations in the Fe-S system. Journal of Nanoparticle Research 16

This work was supported by the Slovenian Research Agency under Grant No. 1000‐10‐310072 and by the Seventh Framework Programme of the European Commission: ESTEEM2 (Enabling Science and Technology for European Electron Microscopy), contract number 312483.

Fig. 1: The near-amorphous first precipitate, obtained with sonochemical synthesis, transforms during solvothermal treatment into different (Cu, Fe)-S phases, depending on the initial amount of Cu in the starting reagents, while low amounts of Cu propagates a mixture of Cu-rich mackinawite and cubic FeS.

Fig. 2: Incorporation of Cu between S-S layers of mackinawite structure during sonochemical synthesis results in expansion of unit cell in c-axis and has a significant influence on phase transformation of Fe-sulphides during further solvothermal treatment.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2750 HAADF-STEM, EELS and DFT study of (111) twins in MgAl2O4 spinel

Rečnik A.1, Drev S.1, Dražić G.2, Komelj M.1, Daneu N.1
1Department for Nanostructured Materials, Jožef Stefan Institute, Ljubljana, Slovenia, 2Laboratory for Materials Chemistry, National Institute of Chemistry, Ljubljana, Slovenia
aleksander.recnik@ijs.si

The structural study of (111) twin of natural spinel crystals from Mogok (Burma) suggested that tetrahedrally coordinated Mg2+ ions, linked to the twin-forming hcp stacking, are probably replaced by Be2+. It has been also proposed that incorporation of Be during crystal growth triggers tropochemical twinning in spinel and the formation of modulated compounds of the taaffeite BexMgyAl2(x+y)O4(x+y) homologous series [Daneu et al. 2007]. This hypothesis has been experimentally supported by liquid-phase assisted reactive sintering of primary oxides, where the addition of BeO triggered abundant twinning of spinel, whereas no twins formed in pure Al2O3–MgO system [Drev et al. 2013]. Atomic-resolution HAADF-STEM images of (111) twin boundaries indicated a distinctive dark contrast at the boundary tetrahedral sites (Fig. 1), but spectroscopic evidence for a single atomic layer of Be was still lacking to confirm the tropochemical origin of twinning. To produce such an evidence, we designed analytical strategy for accurate measurement of Be on the twin boundary, using a probe-corrected field-emission gun transmission electron microscope with the scanning unit (Jeol-ARM). First, EELS spectra were collected form synthetic chrysoberyl (BeAl2O4) and taaffeite (BeMg3Al8O16) samples, where Be2+ ions are in a similar structural environment as in the (111) twin boundary in spinel. The material is quite sensitive to high electron doses and spectral imaging (ESI) was out of question. As opposed to the (111) twin boundary in spinel, where we have only one situation per crystal, in taaffeite structure there are multiple situations where the analytical approach could be tested. Therefore the spectra were collected from (0001) planes of taaffeite using short acquisition times and a narrow rectangular window of approx. dimensions of 20 x 0.2 nm2, containing only Be atoms in hcp layers of the structure. The width of the stripe was limited by the neighboring Al-rich kagome layers, which could cause overshadowing of Be-K edge (111 eV) by the tails of the Al-L2,3 edge (79 eV). To avoid collecting energy losses from neighboring atomic layers the spectra were recorded form thin crystal parts. The same analytical approach was then implemented on the (111) twin boundaries in spinel, where the presence of Be was successfully confirmed. Based on experimental HAADF-STEM images supercell models containing 336 atoms were constructed for simulations and twin-boundary energy calculations within the frame of density-functional theory (DFT). The calculated boundary energy for Mg2+ occupying the interfacial tetrahedral sites is one order of magnitude larger than if the sites are occupied by Be2+. Continued in Acknowledgement ...

... Comparison of the twin boundary energy to bulk spinel suggests that the twinning is more favorable than a fault-free structure (Fig. 2), which explains anisotropic growth of (111) twins after their nucleation [Drev et al. 2013].

References:
[1] Daneu N, Rečnik A et al: Phys Chem Min 34 (2007) 233-247.
[2] Drev S, Rečnik A, Daneu N: CrystEngComm 15 (2013) 2640-2647. 

Fig. 1: Z-contrast image of (111) twin with EELS spectra for chrysoberyl, taaffeite and (111) twin boundary in spinel. Fig. 2: Comparison of simulated and experimental HAADF-STEM images of (111) twin. DFT calculations indicate that Be-rich boundary has the lowest energy. Intensity maxima correspond to atomic columns of overlaid models.
Fig. 2:
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2809 Direct Detection of Spontaneous Polarization in Wurtzite GaAs Nanowires via Differential Phase Contrast Microscopy

Bauer B.1, Hubmann J.1, Wild J.1, Reiger E.1, Bougeard J.1, Zweck J.1
1Institute for Experimental and Applied Physics, University of Regensburg, Regensburg, Germany
johannes1.wild@physik.uni-r.de

Semiconductor Nanowires (NWs) are known to exhibit crystal structures that can differ from the stable bulk crystal structure. In GaAs, which has Zinc-Blende (ZB) structure in the bulk, NWs can also crystallize in Wurtzite (WZ) crystal structure which – in contrast to ZB – can show spontaneous polarization (SP) due to the lower symmetry of the WZ crystal lattice. The SP occurs along the crystallographic (0001) axis which corresponds to one of the cubic {111} axes and arises as virtual “sheets“ of alternatingly charged planes perpendicular to the (0001) axis. We will show a first direct evidence of the SP in WZ-GaAs together with a quantified measurement of its strength.

We use Differential Phase Contrast (DPC) microscopy in a FEI Tecnai F30 scanning TEM to detect the SP. As electrons pass the sample perpendicular to the (0001) axis they get deflected by the elctric field generated by the charged planes which act like a series of capacitor plates. By using a position sensitive four quadrant detector we can measure this deflection and thus visualize the effect of the SP [1]. The system was calibrated to allow quantification of the electric fields that deflect the electron beam.

In fig. 1 we show measurements from the tip of a GaAs NW where the crystal structure changes from WZ to ZB. As expected the charge distribution (fig. 1(C)) differs significantly between ZB and WZ crystal structure where the latter reveals an oscillating behavior while the former is zero despite some noise. In addition it can be seen that also twin defects in the ZB where the stacking order reverses from ABC to CBA show significant charging (fig. 2).

As quantitative measurement of SP are not possible directly on the WZ structure we use an arrangement of two closely related twin defects which can be treated like a plate capacitor filled with a dielectric. By measuring the electric field difference between inside and outside this structure we can calculate SP to be 0.0027(6) C/m² which is in very good agreement with theoretical estimations [2].

[1] Lohr et al., Ultramic., 117 (2012), 7.

[2] Belabbes et al., Phys. Rev. B, 87 (2013), 035305.; Jahn et al., Phys. Rev. B, 85 (2012), 045323.

Our work is funded by the DFG via Grants SFB 689 “Spin Phenomena in Reduced Dimensions” and No. 957 “Focused Researcher’s Group: PolarCoN FOR”.

Fig. 1: Overview (A) and HRTEM (B) of the NW tip region. (C) Charge distribution map with superimposed line scan profile showing the difference in charge density between WZ (oscillating behavior) and ZB (only noise) structure.

Fig. 2: (A) Overview over two twin defects in the ZB region of a NW. (B) HRTEM of one twin boundary showing the stacking reversal. (C) Charge distribution map revealing one positive and one negative charged layer at the twin boundaries.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-2983 Indium incorporation, interfacial properties, and strain relaxation at InGaN interlayers grown by PAMBE

Bazioti C.1, Kehagias T.1, Papadomanolaki E.2, Iliopoulos E.2, 3, Dimitrakopulos G. P.1
1Physics Department, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece, 2Microelectronics Research Group, Physics Department, University of Crete, P.O. Box 2208, 71003 Heraklion-Crete, Greece, 3IESL, FORTH, P.O. Box 1385, 71110 Heraklion-Crete, Greece
gdim@auth.gr

Indium adatom mobility plays a crucial role in defining the final configuration of InGaN nanostructures. This behavior is particularly important in the effort to obtain high alloy content quantum wells that can operate with high internal efficiency, towards advanced optoelectronic and photovoltaic applications. The advantages of plasma assisted molecular beam epitaxy (PAMBE) regarding metastability and suppression of phase separation have not been fully exploited so far in this regard.
We consider PAMBE-grown InGaN interlayers grown on (0001) GaN/Al2O3 templates, with various thicknesses from 1 nm up to 40 nm, in heterostructures comprising 100 nm GaN spacers (Fig. 1). Growth of InGaN was performed under the same conditions, i.e. at 450oC under slightly metal rich conditions. Such conditions have been shown to produce 40% average indium contents when InGaN was grown in epilayer form, albeit through phase separation phenomena. In the present case we focused on elucidating such phenomena at early stage and on understanding the influence of strain relaxation. Observations were performed using transmission electron microscopy (TEM). The employed techniques were diffraction contrast and high resolution TEM (HRTEM), taking care to minimize beam exposure as well as the ion milling time during sample preparation. Nanoscale strain measurements were performed using geometrical phase analysis (GPA).
Under the given growth conditions, the critical InGaN thickness for strain relaxation was determined experimentally at 5 nm, through the observation of threading dislocation (TD) emanation, as shown in Fig. 1. The elastic and plastic strain components of the total misfit were determined. Thin InGaN interlayers exhibited a sharp GaN/InGaN interface followed by a rough InGaN/GaN interface. Although the first GaN monolayers after the InGaN layers were grown at the InGaN growth temperature in order to limit indium interdiffusion, the average indium distribution was found to be graded, peaking close to the GaN/InGaN interface and reducing gradually as shown in Fig. 2(a). The indium incorporation was determined to increase with increasing interlayer thickness under the same growth conditions, exceeding 30% after the 5 nm layer thickness, as shown by the attained lattice strain measurements [e.g. Fig. 2(b)]. Compositional inhomogeneities associated to strain fluctuations were studied in association to the defect structure and the degree of plastic relaxation.

This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: THALES.

Fig. 1: Cross sectional bright field TEM image showing a heterostructure comprising InGaN interlayers with increasing nominal thickness and 100 nm GaN spacers. Emanation of TDs connected to misfit dislocations is observed after the 5 nm thick interlayer (arrow).

Fig. 2: HRTEM images of InGaN interlayers with superimposed GPA maps of the relative d-spacing of (0002) planes. (a) A ~2 nm layer with strain grading after the GaN/InGaN interface and peak (0002) mismatch >1.5%. (b) A partially relaxed layer of ~7 nm thickness with (0002) mismatch of ~ 4%. (Arrows indicate the directions of the line profiles.)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3071 HRTEM/STEM characterization of interfaces and strain-related distortions in epitaxial perovskite heterostructures

Negrea R. F.1, 2, Ghica C.1, Teodorescu V. S.1, Maraloiu V. A.1, Nistor L. C.1
1National Institute of Materials Physics, Magurele, Romania, 2University of Bucharest, Faculty of Physics, Magurele, Romania
raluca.damian@infim.ro

Most of the currently studied artificial multiferroic systems are based on epitaxial multilayers grown onto SrTiO3 (001) single crystals (STO), using SrRuO3 (SRO) epitaxial layers as bottom electrode. In the ferroelectric heterostructures, the microstructural characteristics of the deposited thin films as well as the interfaces between the ferroelectric layers and the electrodes play a fundamental role in the electrical behavior of the heterojunction (polarization hysteresis loops, C-V and I-V characteristics). Atomic scale structural and chemical characterization plays a major role in further understanding the extrinsic contributions to the electrical characteristics. We have used a Cs probe corrected JEM ARM 200F electron microscope to investigate the interface structure and strain driven structural distortions in the PZT/SRO/STO and BTO/SRO/STO systems, where PZT stands for PbZr0.2Ti0.8O3 and BTO for BaTiO3. Pulsed Laser Deposition (PLD) has been used for the deposition of the epitaxial SRO, PZT and BTO layers onto STO(001) substrates.

We have performed Scanning Transmission Electron Microscopy (STEM) and Electron Energy Loss Spectroscopy (EELS) for the atomic resolution characterization of the SRO-PZT and SRO-BTO interfaces (Figure 1). The atomic interdiffusion at the interface has been studied using the Z contrast in STEM imaging by High-Angle Annular Dark Field and Annular Bright Field. Our studies reveal that an atomic interdiffusion occurs across a region of up to 7 atomic planes around the SRO-PZT and SRO-BTO interfaces. Atomic scale EELS – Spectrum Imaging (EELS-SI) reveals the nature and position of the atomic species at the interface.

SAED patterns from areas including the SRO layer exhibit faint diffraction spots appearing in positions which are not allowed by the reflection conditions in the space groups of SRO, PZT, BTO or STO. FFT of the corresponding HRTEM micrographs prove that the concerned spots are generated from nanometric areas inside the SRO layer. We have performed a quantitative HRTEM study to measure and map the strain fields inside the SRO layer at the nanometric scale. Our study, supported by quantitative image processing by the Geometrical Phase Method and image simulation, clearly proves a strain-driven monoclinic distortion in nanometric domains inside the thin SrRuO3 epitaxial layers, thus explaining the presence of the diffraction spots in forbidden positions in the SAED patterns.

The authors acknowledge UEFSCDI for financial support through the PN-II-ID-PCE-2012-4-0362 and the PN-II-ID-PCE-2011-3-0268 projects.

Fig. 1: (a) HAADF – STEM image of SRO - BTO interface; (b) Intensity profile along green arrow through the atomic columns in image (a); (c) Atomic resolution EELS – SI at the SRO - BTO interface.

Fig. 2: (a) HRTEM image of an epitaxial SRO thin film sandwiched between the STO(001) substrate and the PZT layer; (b) FFT corresponding to area 1 in HRTEM micrograph; (c) Supplementary spots appearing in the FFT corresponding to area 2 in HRTEM micrograph.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3108 Precise IDB-type identification by phase shift in HRTEM imaging

Koukoula T.1, Kioseoglou J.1, Kehagias T.1, Georgakilas A.2, Komninou P.1
1Physics Department, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece, 2Microelectronics Research Group, IESL, FORTH, P.O. Box 1385, GR-71110 Heraklion Crete, and Department of Physics, University of Crete, P.O. Box 2208, GR-71003 Heraklion Crete, Greece
tkouk@auth.gr

Inversion domain boundaries (IDBs), bounding two neighboring domains with opposite polarity, are extended defects that may have detrimental implications in the performance of III-Nitride active layers, where the IDB* and the Holt type IDB models are observed by Transmission Electron Microscopy (TEM) methods. In particular, the high formation energy Holt IDB has been found to be electrically active by inducing electronic states in the band-gap, while the IDB* is inactive. Therefore, it is crucial to unambiguously determine the type of IDBs in order to appreciate their influence on the electronic properties of III-Nitrides heterostructures-nanostructures.
Here, we present a concise methodology, based on Geometrical Phase Analysis (GPA), in order to precisely identify the type of the observed IDBs, by inspecting the phase shift P(r) between adjacent inverse polarity domains in the phase images of High-Resolution TEM (HRTEM) micrographs. This means that for a specific type of IDB, the phase in two neighboring inverse polarity crystals would be shifted by a certain value that corresponds to the translation vector of the IDB. Depending on the diffracting conditions, P(r) is ±3π/4 and ±π/4 for the Holt and IDB* models, respectively, when the 0001 reflection is used. The corresponding values for the 0002 reflection are ±3π/2 and ±π/2. An interesting case is that the rigid body translation between the two models (±1/2[0001]), introduces an ambiguity in the identification of the specific IDB model, when the 0002 reflection is used. Following an extensive series of through focus and thickness HRTEM image simulations, it was found that the phase shift of the Holt model is strongly influenced by the imaging conditions (defocus and thickness values of the specimen). Thus, in order to irrefutably identify a Holt IDB, the 0001 reflection should be used.
The above methodology was implemented to identify the related IDB model between a GaN nanoisland (NI) and a GaN nanowire (NW), grown on Si(111) by MBE [Fig. 1(a)]. The corresponding phase images are shown in Figs. 1(b) and (c), using the 0002 g vector and the 0001 g vector of the Fast Fourier Transform (FFT), respectively, while line profiles of the phase images perpendicular to the IDBs are given in Figs. 1(e) and (f). Fig. 1(d) is the phase image using the 0002 vector of a simulated Holt IDB [inset in Fig.1 (a)]. The GaN NI is used as reference region in both cases and even though identification of the IDB type is unclear when using the 0002 g vector [Fig. 1(f)], this ambiguity is raised employing the 0001 g vector [Fig. 1(e)], which reveals the Holt type character of the IDB.

This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: THALES, project “NanoWire”.

Fig. 1: (a) HRTEM image of an IDB between a GaN NI and a GaN NW. HRTEM image simulation of a Holt IDB is given as inset (b) & (c) Corresponding phase images using the 0002 g vector and the 0001 g vector in the FFT, respectively. (d) Phase image using the 0002 g vector of a simulated Holt IDB (e) & (f) Line profiles of the phase images.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3119 In-situ Transmission Electron Microscopy Study of Precipitation in AA3003 Aluminium Alloy

Poková M.1, Cieslar M.1
1Charles University in Prague, Faculty of Mathematics and Physics, Department of Physics of Materials, Ke Karlovu 5, 121 16, Prague 2
pokova@karlov.mff.cuni.cz

Aluminium alloys belong to the most widely used metallic materials in many industrial applications. Final properties of the alloy are highly dependant on the thermo-mechanical treatment during manufacturing and the resulting microstructure. Thus detailed knowledge of the microstructure evolution during processing is required for tailoring the final product.
Aluminium alloy from AA3003 series with main alloying elements Mn, Fe and Si was prepared by twin-roll casting in industrial conditions. This material was further subjected to severe plastic deformation by equal channel angular pressing (ECAP). During ECAP the billet is pressed through two channels of equal cross-section, which intersect at an angle of 90°. The equivalent strain imposed to the material after one pass is ε~1. Such deformation leads to fragmentation of the grains and ultimately to formation of ultra-fine grained structure [1].
The objective of the present work was to evaluate the role of ECAP on the precipitation kinetics during isochronal annealing. Three materials were compared – as-cast material, material after one ECAP pass and material after four ECAP passes. Measurements of relative electrical resistivity changes were combined with in-situ heating in transmission electron microscope JEOL 200FX working at 200 kV. The in-situ observation enabled detailed study of the precipitates evolution during the whole temperature range.
At lower annealing temperatures the recovery of dislocation substructure took place. During annealing up to 300 °C the first precipitates of α-AlMnFeSi phase [2] started to form. Their size increased with further annealing but above 450 °C the smaller ones dissolved back to the solid solution. The precipitation started first in the material after four ECAP passes, last in the as-cast material, which was not subjected to deformation. It is known that in this type of alloy the precipitates form preferentially on grain boundaries [3]. As the number of the grain boundaries increases with the imposed deformation, the precipitation was facilitated in the materials subjected to ECAP.

[1] M. Poková and M. Cieslar: In-situ TEM study of the role of pre-annealing on the microstructure development of an AA3003 aluminium alloy subjected to ECAP. Kovové Materiály (2014) in press.
[2] M. Karlík, T. Mánik and H. Lauschman: Influence of Si and Fe on the distribution of intermetallic compounds in twin-roll cast Al-Mn-Zr alloys Journal of Alloys and Compounds 515 (2012) 108-113.
[3] M. Poková, M. Cieslar and J. Lacaze: The Influence of Silicon Content on Recrystallization of Twin-Roll Cast Aluminum Alloys for Heat Exchangers. Acta Physica Polonica A 122 (2012) 625-629.

The authors gratefully acknowledge the financial supports of grants GAUK 1428213, GAČR P107-12-0921 and SVV-2014-269303.

Fig. 1: The evolution of electrical resistivity during isochronal annealing (derivation of relative changes). The peaks in positive values correspond to precipitation, the negative ones correlate with particles dissolution. “Init” denotes material after twin-roll casting, “1P” and “4P” materials after one and four passes in ECAP, respectively.

Fig. 2: Micrographs from in-situ TEM heating with a rate 50 °C/50 min. The evolution of microstructure of the alloy after four ECAP passes – recovery of dislocation substructure, grain growth, precipitation of α-AlMnFeSi phase and its dissolution back to solid solution.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3126 Microstructure Processes During In-situ Tensile Test in UFG Copper

Král P.1, Petrenec M.2, Dvořák J.1, Svoboda M.1, Sklenička V.1
11. Institute of Physics of Materials, ASCR, Zizkova 22, 616 62 Brno, Czech Republic, 22. TESCAN ORSAY HOLDING, a.s., Libušina tř. 21, 623 00 Brno, Czech Republic
martin.petrenec@tescan.cz

Experiments were conducted to investigate deformation-induced processes during in-situ tensile test at elevated temperature. The billets of coarse-grained copper were processed by equal-channel angular pressing (ECAP) at room temperature using a die that had an internal angle of 90° between the two parts of the channel and an outer arc of curvature of ~ 20°, where these two parts intersect [1]. The pressing speed was 10 mm/min. To obtain an ultrafine-grained (UFG) material, the billets were subsequently pressed by route Bc by 8 ECAP passes to give the mean grain size ~ 0.7 µm (Fig. 1a).

The constant strain-rate test in tension was performed at 473 K using testing GATAN stage Microtest 2000EW with EH 2000 heated grips which is configured for in-situ electron back scatter diffraction (EBSD) observations. Microstructure was examined by SEM-FEG TESCAN MIRA 3 XM equipped by EBSD detector HKL NordlysMax. The tension test was interrupted by fast stress reductions after different deformation step and observation of microstructure changes was performed.

Despite of a considerable interest in ECAP processing method, there are not many works documenting microstructure evolution and changes during creep testing and determining creep mechanisms of ultrafine-grained materials processed by ECAP. It was found that creep resistance of UFG pure Al and Cu is considerably improved after one ECAP pass in comparison with coarse grained material, however, further repetitive pressing leads to a noticeable deterioration in creep properties of ECAP material [2,3] Recently it was observed the coarsening of the grains in microstructure of ECAP copper during creep at elevated temperature [4]. It was suggested that creep behaviour is controlled by storage and dynamic recovery of dislocations at high-angle boundaries [4,5].

In the present work was found that ultrafine-grained microstructure is instable and significant grain growth has already occurred during heating to the testing temperature (Fig. 1b). Static recrystallization during heating led to the formation of high fraction of special boundaries ∑3 and ∑9 (Fig. 1d). The tensile deformation at 473 K led to the additional grain growth (Fig. 1c) and during tensile testing (Fig. 2) the nucleation and subsequent growth of cavities were observed.

References:

[1] Valiev R Z, Langdon T G, Progr. Mater. Sci. 51 (2006) 881

[2] Kral P, Dvorak J, Seda P, Jäger A, Sklenicka V, Rev. Adv. Mater. Sci. 31 (2012) 14

[3] Dvorak J, Sklenicka V, Kral P, Svoboda M, Saxl I, Rev. Adv. Mater. Sci 25 (2010) 225 [4] Blum W, Dvorak J, Kral P, Eisenlohr F, Sklenicka V, Mater. Sci. Eng. A 590 (2014) 423

[5] Blum W, Dvorak J, Kral P, Eisenlohr F, Sklenicka V, J. Mater. Sci. 49 (2014) 2987

Financial support for this work was provided by the Czech Science Foundation under Grant P108/11/2260.

Fig. 1: In-situ observation of microstructure after a) 8 ECAP passes, b) subsequent heating to the test temperature, c) ε ~ 0.1 at 473 K and d) distribution of misorientation angle

Fig. 2: The dependence of true stress vs. true strain for copper processed by 8 ECAP passes
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3146 Copper sulfide nanocrystals with tunable composition

Genovese A.1, Casu A.1, Falqui A.1,2, Xie Y.1
1Department of Nanochemistry, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova, Italy, 2King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
alessandro.genovese@iit.it

Localized surface plasmon resonances (LSPRs) arise from the interaction of electromagnetic radiation with free charge carriers in nanoscale metals, which leads to coherent charge oscillations at optical frequencies. LSPRs of metallic nanocrystals (NCs) have drawn great attention due to their various applications. However, the occurrence of LSPR is not restricted only to nanostructured metals, but is also observable in various other nanomaterials, e.g. the binary copper chalcogenides. The interesting properties of Cu2-xS, a well-known p-type semiconductor exhibiting stoichiometry-dependent bandgap [1], make its NCs appealing in very diverse fields. Thus, in recent years various studies on plasmonic behavior and different synthetic approaches with controlled Cu stoichiometry were proposed. NCs with compositions comprised between the limiting cases represented by CuS and Cu2S (i.e. covellite and chalcocite, respectively) can be routinely made, although each one with a different synthetic procedure. This makes it difficult to compare their physical properties, since each sample has its own geometrical parameters and type of surface passivation.
Our approach, based on the reaction of the as-synthesized covellite NCs with a Cu(I) complex at room temperature, allows to access several stoichiometries in colloidal copper sulfide NCs, starting from CuS (covellite) NCs, up to Cu2S. Thus, starting from a common sample, by this approach it is possible to access a wide range of compositions of NCs and study variations in their structure and plasmonic response: from the metallic covellite, with a high density of free carriers, up to Cu2S NCs with no localized surface plasmon resonance (Figure 1). In all these NCs the valency of Cu in the lattice stays close to +1, while the mixed -1/-2 valency of S in covellite gradually evolves to -2 with increasing the Cu content, i.e. sulfur is progressively reduced. The addition of copper to covellite NCs is similar to the intercalation of metal species in layered transition metal dichalcogenides (TMDCs), i.e. the dichalcogenide bonds holding the layers are progressively broken to make room for the intercalated metals, while their overall crystal structure does not change much (Figure 2). However, differently from TMDCs, the intercalation in covellite NCs is sustained by a change in the redox state of the anion framework. Furthermore, the amount of Cu incorporated in the NCs upon reaction is associated with the formation of an equimolar amount of Cu(II) species in solution, so that the reaction scheme can be written as: CuS + 2γCu(I) → Cu1+γS + γCu(II).
[1] Liu et al. Thin Solid Films, 431-432, (2003), 477.

The research leading to these results has received funding from the European Union’s Seventh Framework Programme FP7/2007-2013 under grant agreement n. 240111 (ERC Grant NANOARCH).

Fig. 1: A, B) optical absorbance spectra and XRD patterns from CuS to Cu2S NCs. For Cu2S, the black pattern (at RT) is compatible with a mix of low temperature chalcocite and anilite, the green one (after a thermal treatment at 150°C) is compatible with hexagonal chalcocite. C) Crystal structure projections of covellite, anilite, hexagonal chalcocite.

Fig. 2: HRTEM images of single Cu1+γS (0≤γ<0.8) NCs showing the (11-20) lattice planes with d-spacing ranging between 1.89 Å (Figure 2A, covellite) and 1.98 Å (Figure 2D, high temperature chalcocite). E) 1D-integrated diffraction patterns obtained from SAED collected on the same samples
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3152 The Impact of Dislocations in InGaN/GaN quantum-well structure

Yoon S.1, Yoo H.1, Kwon Y. K.2, Kim M.1
1School of Materials Science & Engineering, Seoul National University, Seoul 151-744, Korea, 2Department of Physics and Research Institute for Basic Sciences, Kyung Hee University, Seoul 130-701, Korea
smyoon25@gmail.com

       InGaN is an industrially important material used for active layers in the quantum-well structure for enhanced (blue) light emission in optoelectronic applications. InGaN/GaN multi quantum-well structure is known to have excessive density of threading dislocations, which could significantly influence the luminescence characteristics. Accordingly, there have been many discussions about the effects of the dislocations on optical properties. Some argue that the dislocations would not have much effect on the luminescence characteristics since the quantum dotlike regions formed by the phase segregation of InGaN enhance quantum confinement effects and these effects dominate the luminescence characteristics1, while others state that dislocations can give rise to In diffusion and In/Ga intermixing, which finally result in the breakdown of the quantum-well structure and the degrade of the luminescence performance2. Even though extensive researches on dislocations in InGaN layers have already been reported, these researches have progressed mostly based on various experimental findings. Recently, demands for systematic studies based on quantum mechanical calculations have been increasing to understand the atomistic role of dislocations on optical properties of InGaN layers.

       We have investigated the effect of dislocations on the segregation of Indium atoms on InGaN layers by comparing the interaction energy between Indium and dislocations by replacing Indium atoms for various Ga sites. The first principles calculations based on the density functional theory have been carried out using the VASP pseudopotential code to study the role of dislocation in the InGaN. We used the functional of the local-density approximation for the exchange-correlations and ultrasoft pseudopotential method for descriptions of core electrons. In order to model the dislocation of InGaN, we used 2√3x7x2 wurtzite GaN supercell of 224 atoms. We confirmed that an Indium atom located in the vicinity of the dislocation core was energetically much more stable (Fig. 1). Additional Indium atoms are also preferred to be located around the core, which indicates that the dislocation could play a role in drawing Indium atoms and thereby cause the indium segregation (Fig. 2). Moreover, these calculations indicate that the Indium atoms around dislocations could be possibly cause red-shift effect in the luminescence characteristics (Fig. 3).

Reference :
1 I. H. Ho and G. B. Stringfellow, Applied Physics Letters 69, 2701 (1996).
2 N. Duxbury, U. Bangert, P. Dawson, E. J. Thrush, W. Van Der Stricht, K. Jacobs, and I. Moerman, Applied Physics Letters 76, 1600 (2000).

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF 2013034238 and NRF 2011-0016188)

Fig. 1: Most stable configuration of GaN dislocation structure containing one indium atom. Green and purple atoms indicate Gallium and Indium, respectively. Dotted polygons signify the core of GaN edge dislocations (closed core).

Fig. 2: Most stable configuration of GaN dislocation structure containing three indium atoms. The numbered atoms will be used for the local density of states (LDOS) analysis of Fig. 3. It should be noticed that the supercell structure is constructed with two layers of unit cell.

Fig. 3: LDOS of five atoms in the structure of Fig. 2. LDOS of In1 shows the possibility of the red-shift effects in the luminescence characteristics. The bandgap of bulk GaN was estimated as 2.5eV in our calculation conditions.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3181 Colloidal Au2Cd Alloy-CdSe Nanocrystal Heterostructures For Plasmonic Applications

Casu A.1, Genovese A.1, Guardia P.1, Falqui A.1,2
1Department of Nanochemistry, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova, Italy, 2King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
alberto.casu@iit.it

Up to now only a few synthetic routes for the colloidal synthesis of intermetallic NCs were published [1], despite the recent intensive study on properties of these materials.[2] In this context, we report the synthesis of colloidal heterostructures made of a core of Au2Cd alloy surrounded by a shell of CdSe via a one-pot approach. Gold NCs, acting as seeds, were converted to AuCd alloy NCs as intermediate step. The formation of a CdSe shell was triggered as soon as a Se precursor was injected in the solution containing the AuCd NCs. One peculiarity of our synthesis method is that the Cd atoms employed for the formation of the shell seem to be supplied by the AuCd alloy NCs with minor or no contribution from residual Cd species present in solution. This assumption is supported by the fact that the composition of the core varied from AuCd to Au2Cd upon CdSe shell growth, then the shell growth stopped spontaneously. Therefore, both the initial AuCd core synthesis (from Au NCs to AuCd NCs) and the following shell growth are self-limited by the formation of Au-Cd alloy NCs with two compositions that are particularly stable also in the bulk, namely AuCd and Au2Cd. The AuCd NCs were therefore acting as a reservoir of Cd atoms. This synthesis approach results in a drastic reduction or total absence of byproducts (i.e. CdSe), while yielding more symmetric core/shell structures with variable core size.
Metal-semiconductor NC heterostructures are model systems for understanding the interplay between the localized surface plasmon resonances in the metal domain and the relaxation of the excited carriers in the semiconductor domain. Since the spectral features of the individual Au2Cd and CdSe domains in our core/shell NCs overlap in the visible range, in order to better understand the hot electrons relaxation dynamics of these systems we recorded transient absorption spectra by pumping either below (800 nm)or above the CdSe bandgap (400 nm). We obtained similar spectral shape and lifetime for the plasmon peak in both cases, which indicates that most of the photons were absorbed by the Au2Cd core. By fitting the decay of the transient absorption signal at the plasmon peak position for different pump power levels, we extracted a low-power limit of half a picosecond for the hot electrons relaxation lifetime. Almost the same relaxation time was found for the core/shell NCs and for the AuCd NCs seeds used for their synthesis. This indicates that in the Au2Cd/CdSe core/shell NCs the relaxation of hot charge carriers is determined mainly by their interaction with the bulk phonons, while surface modes seem to play a minor role.

1 Chen, W.; Yu, R. et al. - Angew. Chem. Int. Ed. 2010, 122, 2979–2983

2 Zeng, J.; Huang, J. et al. - Adv. Mater. 2010, 22, 1936–1940.

Fig. 1: a,b)TEM images of Au and AuCd alloy NCs. c)HRTEM image of AuCd NCs. d)HAADF STEM image of AuCd NCs and their EDX spectrum with the Cd and Au peaks. Cu peaks come from Cu TEM grid. The chemical composition is consistent with an atom ratio Au:Cd of 1:0.94. e)Static absorption spectra of Au and AuCd alloy NCs. f)XRD pattern of AuCd alloys NCs.

Fig. 2: TEM image of Au2Cd/CdSe core/shell NCs. b)HRTEM image of a single Au2Cd/CdSe core/shell. c)HAADF STEM image of Au2Cd/CdSe core/shell NCs. EDX line profile (red line) exhibits Au, Cd, and Se distribution across the cores and the shells. d)Static absorption spectra of Au, AuCd and Au2Cd/CdSe samples. e)XRD pattern of the Au2Cd/CdSe sample
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3265 TEM in-situ heating of Bi nanoparticles embedded in a Zn matrix

Peterlechner M.1, Song T. E.1, Wilde G.1
1University of Münster, Institute of Materials Physics, Wilhelm-Klemm-Str. 10, 48149 Münster
martin.peterlechner@uni-muenster.de

Melting and crystallization of small particles are of scientific interest due to size effects, and being studied since decades. However, nanoparticles embedded in a matrix show additional contributions to their melting behaviour caused by the interfaces [1]. In the present study, Bi was processed as nanoparticles embedded in a Zn matrix. The aim of the work is to understand the contributions of the interface structure to the overall melting behaviour of the nanoparticles.
Samples of embedded nanoparticles were made using melt-spinning. The as processed bands of Zn with embedded Bi nanoparticles were ion-milled using acceleration voltages between below 3.5 kV. Scanning electron microscopy (SEM) and focused ion beam (FIB) imaging was accomplished using a Zeiss EsB1540. TEM was made using a Zeiss Libra 200FE with an corrected in-column Omega filter and an aberration-corrected Titan 80-300. In-situ TEM heating experiments were conducted using a Hitachi H800.
Figure 1 shows an FIB image of the as processed Zn-Bi sample. The surface layer of the thin melt spun ribbons was removed by the FIB Ga beam to obtain an oxide free surface showing strong channelling contrast of the Zn grains. Bi nanoparticles show up dark. A detailed TEM analysis was made to obtain a size distribution. In Figure 2a) a TEM bright-field image is shown. Bi nanoparticles show an elongated morphology with an c/a-ratio of ~1.8. The volume weighted size distribution of the embedded nanoparticles has a mean about 13 nm. It should be emphasised, that Bi particles have a sharp interface along their elongated direction (Zn{0,0,1}||Bi{1,0,2}), whereas other facets are not pronounced. To gain insights into the melting behaviour, TEM in-situ experiments were conducted. Figure 3 shows TEM bright-field images taken at one selected area during the heating experiment. Figure 3a) is taken at room temperature (RT) before the heating; a few nanoparticles are numbered. During heating, nanoparticles melt and get mobile, as observed at a nominal temperature of 220°C (cf. Figure 3b)). After heating up to 270°C and subsequent cooling back to RT the number and position of several Bi nanoparticles has changed (cf. Figure 3c).
However, in agreement with previous studies it was possible to process Bi nanoparticles in a Zn matrix. The size of the nanoparticles was about 13 nm in the short direction (named a) with an c/a-ratio of ~1.8. Based on the presented in-situ heating experiments it can be concluded, that in the Zn-Bi system already below the bulk melting point of 271°C nanoparticles are molten and show mobility. Moreover, Ostwald ripening was observed.

1. Couchman, P.R.P. & Jesser, W.W.A., 1977. Thermodynamic theory of size dependence of melting temperature in metals. Nature, 269(6), pp.481–483.

We kindly acknowledge the help of Di Wang and Christian Kübel at the Karlsruhe-Nano-Micro-Facility (KNMF) at KIT for help with the Titan experiments.

Fig. 1: FIB image of the as prepared Zn-Bi. Channelling contrast arises showing Zn grains with a size of several micrometres. Bi particles occur with dark contrast, homogenously distributed in grains and some at grain boundaries. Additional contrast arises along grain boundaries, where Bi is spreading over the sample surface and into the Zn matrix.

Fig. 2: TEM of the as-processed Zn-Bi melt-spun ribbons. In a) a TEM bright-field image shows elongated nanoparticles. The long- and short- axes are denoted  by c and a as indicated. A size distribution of the short axis a is plotted in b). The mean of the volume weighted size distribution of a is 13 nm and the c/a-ratio is ~1.8.

Fig. 3: In-situ TEM experiment of embedded Bi nanoparticles. Bright-field images were taken at different temperatures during the experiment, in a) at RT, b) at ~220°C and in c) at RT after heating up to 270°C. Selected nanoparticles are numbered. During the heating nanoparticles moved and aggregated.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3308 High resolution transmission electron microscopy study on mixed phase Ge nanoparticle on off axis 4H-SiC.

Chandran N.1, Frangis N.1, Alassaad K.2, Ferro G.2, Polychroniadis E. K.1
1Department of physics, Aristotle university of Thessaloniki, Thessaloniki - 54124, Greece, 2Universite Claude Bernard Lyon 1, 69622 Cedex, France
naren@physics.auth.gr

Germanium islands growth on SiC has attracted much attention for the development of optoelectronics in SiC technology. The epitaxial relationships between Ge nano-islands and SiC were well studied by reflection high-energy electron diffraction (RHEED), HRXRD and STM etc [1]. It has found that the Ge islands does not form always single crystalline nature and possess several orientations related to the substrate. These may be exhibited with defects, different structural phases and inclusions etc., depending on growth techniques and conditions.

In the present work, Ge islands were deposited on 8 degree off axis 4H-SiC by the CVD technique. The Ge nanoparticles were characterized by high resolution transmission electron microscopy (HRTEM).

The characterization shows that in general (but not always) Ge nano-particles grow with {111}Ge//(0001)SiC. We have presented in this work, HRTEM studies of a Ge nano-particle with different orientation than the above mentioned one. A HRTEM image of this nano-particle taken along [-1100]SiC is shown in fig.1. It can be identified that the particle reveals several phases. Fig. 2 represented the filtered and magnified images of selected areas of interest such as A, B, C and E, D, F are corressponding to Fast Fourier Transforms (FFT’s). Using the SiC lattice parameters as an internal standards, the three marked areas can be characterized. In fig. 2A the configuration of the lattice planes and measured d-spacings are identified that Ge nanoparticle oriented along along [-112]Ge. In fig. 2B two orientations of Ge are found with electron beam parallel to [-112]Ge and [-110]Ge respectively. The respective planes are identified in fig. 2B for the corresponding zone directions and are compatible with FFT as shown in fig. 2D. A similar case is also identified in the area of fig. 2C. The estimated lattice parameters for the area with the [-110] orientation is indicated the presence of Si, which is rather unexpected. Also the same results have been obtained from the corresponding FFT (Fig. 2F). The extra periodicity has shown in the figure 2C can most probably be attributed to the overlapping between Ge and Si. The SiC substrate should have played major role to form Si. The possible reason for this could be that at higher temperature (1500⁰ C) the Ge could promote the wetting of topmost layer of the Si rich surface and should increase the surface energy and the dangling bonds. During cooling processes, the Ge solidifies (800–900 ⁰C) reduces the surface energy and dangling bonds. At this time the remaining dangling bonds helps lead the formation either Si or GeSi particle[2]
[1] K. Aït-Mansour et.al Applied Surface Science 241 (3-4), 403 (2005)
[2] K. Aït-Mansour et.al Journal of Physics D: Applied Physics 40 (20), 6225 (2007)

The work is financially supported by Marie Curie Actions under the framework of the project “NetFISiC” No. 264613

Fig. 1: A HRTEM image of a mixed phase Ge nano-particle on off axis 4H-SiC. Three different areas (A, B, C) are chosen to identify the different phases

Fig. 2: Fig 2: HRTEM images (A, B, C) and the corresponding FFTs (D, E, F). In area A a single Ge nanocrystal imaged along [ ̅112] is seen. In area B two Ge nanocrystals imaged along [ ̅112] and [ ̅110] are found. In area C the presence of Ge (imaged along [1 ̅12]) and Si (imaged along [ ̅110]) is detected.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3417 Scanned Electron Diffraction Studies of Self-assembled Monolayers

Aloni S.1, Altoe V.1, Katan A.2, Martin F.2, Salmeron M.2
1Molecular Foundry Altoe, Lawrence Berkeley National Laboratory, Berkeley, CA 94706, 22Materials Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94706
saloni@lbl.gov

Organic electronic devices are modern technologies based on charge transport properties of molecular assemblies. However the promises of low cost fabrication technologies are overshadowed by extreme dependence of the electronic properties on their internal structure, which is difficult to determine. While X-ray scattering techniques provide high-resolution structural information of organic monolayers they lack the spatial resolution to study domain structure. On the other end scanning probe techniques provide lattice periodicity information but are unsuitable for study of large areas. We have combined the advantages of electron diffraction based techniques, with a low dose, scanned parallel beam and automated diffraction analysis to study structure of organic self assembled monolayers.

Organic molecules were dissolved in chloroform and deposited on Langmuir-Blodgette trough. Molecules were assembled at the air-water interface and transferred to variety of electron transparent substrates such as, amorphous continuous and lacey carbon, silicon nitride and silicon oxide membrane 10-20nm thick. Small variations in sample preparation alter the molecular film thickness and morphology all films were pre screened by AFM. As with many organic materials, electron beam damages the monolayers with rates that depend on the nature of the support and the structure of the monolayer. Thus all structural information was obtained with a total dose of less then 50 e-/Å2.

The morphology of typical monolayer varies between a continuous 50-100 µm films for stearic acid and DH5TBA, .5-2 µm islands for pentathiophene buteric acid derivatives and small 100nm domains for TSB (see Fig. 1). All the molecules organize into a herringbone arrangement with two molecules per unit cell described in 2 dimensions by the p2gg space group. Their diffraction pattern exhibits the expected lack of (0,1) and (1,0) reflections and are of a=5-6.5 Å(±0.1 Å) and b=7-9 Å (±0.1 Å)in size for the short and long lattice parameters, respectively.Discrete dark field images (see fig. 2) provide insight into local variations in the films crystallinity and quality. Comparison of the change of lattice parameters as function of small changes in molecule structure or sample preparation provides valuable insight into the monolayer formation mechanisms (see Fig. 3D).

On our experimental setup the resolution is limited by the radiation hardness of the sample and is few 60-90nm for these monolayers. However, improvement in data acquisition techniques, mainly better and faster data acquisition combined with more sensitive cameras will allow acceding 10nm resolution.

References: [1] Hendriksen, B.L.M.; et al. Nano Lett. 2011, 11, 4107-4112. [2] Altoe V.; et al. Nano Lett. 2012,12,1295 (2012)

This work was performed at the Molecular Foundry at LBNL, and was supported by the Office of Science, Office of Basic Energy Sciences of the DOE under contract NO. DE-AC02-05CH11231.

Fig. 1: Dark field (HAADF) STEM images showing the morphology of 5TBA (A), D5TBA,  (B), DH5TBA (C), and TSB (D) supported on TEM grids. Scale bar: 500nm. (E) Correlation between the orientation of the long axis of the radial D5TBA monolayer domains and the [0,1] lattice direction. (F) Models of the molecules used in this study.

Fig. 2:  a) Diffraction patterns from 4 areas depicted by the blue rectangle in e. b) AFM cross-section of two 5TBA islands on a TEM grid, c) top (upper) and side view (lower) of the crystalline structure, d) simulated kinematic diffraction pattern, e)  HAADF image of the with yellow arrows point in the (0,1) crystallographic direction.

Fig. 3: Analysis of the crystallographic information from a 5TBA monolayer. (A) HAADF image, scale bar: 500nm. (B) Orientation of the domains . (C) Discrete dark field images scattered into the {1,1},{0,2},{1,2} and {1,3} reflections.  (D) Graphic representation of the changes in lattice parameters  of the self assembled thin films studied here. 
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-3530 Microscopy of Phase Transformations in Commercial Aluminium Alloys

Solange P. Y.1, Benjamin H.1, Rosario A. M.2
1Universidad de Oriente. Instituto de Investigaciones en Biomedicina y Ciencias Aplicadas. Departamento de Ciencia de los Materiales. Laboratorio de Caracterización de Materiales. Cumaná, Sucre –Venezuela., 2Universidad de Oriente. Instituto de Investigaciones en Biomedicina y Ciencias Aplicadas. Departamento de Ciencia de los Materiales. Laboratorio de Caracterización de Materiales. Cumaná, Sucre –Venezuela., 3Universidad de los Andes. Departamento de Física. Laboratorio de Análisis Químico Estructural de Materiales (LAQUEM). Mérida, Venezuela
dupar99@gmail.com

Commercial aluminium alloys (AA) 6000 (Al-Mg-Si) and 3000 (Al-Mn) series, are mainly made for extrusion and rolling respectively. In industry, the casting alloys are homogenized to dissolve and / or transforming the rich second phase particles in Fe and producing a fine and homogeneous grain structure and new intermetallics, which results in increased ductility and improved properties for the extrusion and rolling, respectively. In this work commercial aluminium alloys made by the Venezuelan company CVG-ALCASA were used to characterize the transformation β→α of these intermetallics during homogeneization of the alloys Al-Mg-Si (6063) and Al-Mn (3003), apprecianting that they change their shape, composition, crystalline structure, size and distribution. In the case of the AA 6063 the monoclinic β-(AlFeSi) phase is transformed to a cubic β-(AlFeSi) phase. Meanwhile, in AA3003 alloy the orthorhombic β-(AlFeSi) phase is transformed in phase α-(AlFeMnSi), cubic. Homogenization heat treatment was conducted for the AA6063 at 560 °C (range of β→α transformation) for 4 hours and for the AA3003 alloy heat treatment temperature was 500 °C for 1 hour. In both cases, the cooling was conducted at a rate of about 200 °C/hour. Transmission electron microscopy (H-600 TEM operated at 100 KV) and scanning electron microscopy in the secondary electron emission mode (SEM S-2500) coupled to an energy dispersive spectrometer (EDS) Thermo Noran was used. In the case of the AA6063 (ALMgSi), TEM observations allowed to establish the kinetics and possible mechanism of β→α transformation. It was observed that the particles α-(AlFeSi) of globular morphology nucleate on the β-(AlFeSi) rod-like phase (Figure 1). During processing, the β-phase intermetallics are gradually replaced by a dispersion of particles α (Figure 2). Subsequently, islands of α particles grow and are rounded at the expense of the β phase remaining (Figure 3). Microstructural observations of the AA3003 alloy revealed that during transformation β→α, the β phase is consumed by the growth of α phase, resulting in a duplex β//α combination. In the case, the β→α transformation takes place through the eutectoid decomposition of the β-(AlFeMn) phase, but requires silicon diffusion from the matrix along the interface between β and α. The SEM image of the alloy AA3003 (Figure 4) shows the growth of α phase at the expense of β phase, which is confirmed by the corresponding EDS spectrum. Because the α phase has higher contrast than the β phase, the SEM images can be used to demonstrate key features of the transformation.

This research was supported by IIBCAUDO of the University of Oriente.

Fig. 1: Nucleation mechanism of α-(AlFeSi) particles from the β-(AlFeSi) phase.

Fig. 2: TEM micrograph of the process of growing-strangulation.

Fig. 3: TEM micrograph showing the separation of the new phase α-AlFeSi.

Fig. 4: SEM micrograph showing the growth mechanism of α-(AlFeMnSi) phase from the β-(AlFeMn) phase.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-5704 Comparison of Cs-corrected HRTEM/HRSTEM images of {001} platelets in diamond with two possible structural platelet models

Neethling J. H.1, Olivier E. J.1, Naidoo S. R.2, O'Connell J. H.1, Kroon R. E.3
1Centre for HRTEM, Nelson Mandela Metropolitan University, Port Elizabeth, South Africa , 2DST/NRF Centre of Excellence in Strong Materials, WITS University, Johannesburg, South Africa , 3Physics Department, University of the Free State, Bloemfontein, South Africa
jan.neethling@nmmu.ac.za

Planar defects in diamond known as platelets on the {001} planes are found in type Ia diamonds which contain nitrogen impurities [1,2]. Humble proposed a model for the diamond platelet consisting of a double layer of carbon atoms [3]. More recently, Miranda and co-workers proposed a new model for the microscopic structure of platelets in diamond which forms by a shearing process [1]. The core of the platelet defect is a double layer of threefold coordinated sp2 carbon atoms embedded in the sp3 diamond matrix [1]. The model proposed by Humble [3] is one of the earlier carbon interstitial models favoured by Goss and co-workers [4]. It is the purpose of this paper to establish whether Cs-corrected high resolution (scanning) transmission electron microscopy (HRSTEM/ HRTEM) imaging of platelets in type Ia diamond can determine which of the two models, Miranda and co-workers [1] or the earlier carbon interstitial model of Goss and co-workers [4], are in better agreement with the experimental results. HRTEM specimens were prepared by using a Helios Nanolab 650 focused ion beam (FIB) SEM and investigated in a double Cs-corrected JEOL JEM-ARM200F HRTEM.

Fig.1(a) is a HRTEM image of a {001} platelet in diamond viewed edge-on. Fig. 1(b) and (c) show typical but different HAADF STEM images of {001} platelets viewed edge on. The atomic structure of the {001} platelet model proposed by Miranda and co-workers [1] is different when viewed along the two perpendicular <110> directions. A similar type of inequivalence of the projected platelet structures along the <110> and <1-10> directions is present in the Humble [3] model favoured by Goss and co-workers [4]. Supercells containing the platelet defect in diamond (Fig. 2) were constructed using the atomic positions from the models of Miranda et al. [1] and Humble [3]. The difference in HAADF STEM platelet images of two different platelets shown in Fig. 1(b) and (c) is consistent with the platelet models shown in Fig. 2 when viewed along two perpendicular <110> directions. It is impossible to tilt the diamond foil through 90º in the HRTEM in order to image a {001} platelet along two perpendicular <110> directions, hence images of different platelets were recorded since it is likely that some will be viewed along the <110> and others along the <1-1 0> directions according to the indexing convention adopted by Miranda et al. [1] and Goss et al. [4] and shown in Fig. 2. Comparisons of HRTEM/HRSTEM images and image simulations of the platelet models shown in Fig. 2 will be presented.

References

[1] C.R. Miranda, et al., Phys. Rev. Lett. 93, 265502 (2004).
[2] J. Bruley, Phil. Mag. Lett. 66, 47 (1992).
[3] P. Humble, Proc. R. Soc. London, A 381, 65 (1982).
[4] J.P. Goss, et al., Phys. Rev. B 67, 165208 (2003).

 

The financial support of the Department of Science and Technology and the National Research Foundation are gratefully acknowledged.

Fig. 1: (a) HRTEM image of a {001} platelet in diamond viewed edge-on. (b) and (c) show typical but different HAADF STEM images of {001} platelets viewed edge on. Beam direction = <110>. Scale bar: 1.26 nm

Fig. 2: Supercells containing the platelet defect in diamond constructed using the atomic positions from the models of Miranda et al. [1] (a and b) and Humble [3] (c and d). The vertical direction is <001> for all the platelets and the viewing directions are <110> for (b) and (c), and <1-1 0> for (a) and (d).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-5705 Formation and growth mechanism of indium-decorated inversion domain boundaries in one-dimensional zinc oxide nanostructures

Schaan G.1, Lotz M.1, Schmid H.1,2, Mader W.1
1Institute of Inorganic Chemistry, University of Bonn, Römerstraße 164, D-53121 Bonn, 2JEOL (Germany) GmbH, Oskar-von-Miller-Straße 1A, D-85386 Eching b. München
gschaan@uni-bonn.de

Materials based on zinc oxide with additions of other transition and main group metal oxides offer a broad range of applications such as varistors, transparent conducting oxides (TCOs), gas sensors and dye-sensitized solar cells. They have good semiconducting and optical properties at a low price and easy availability.[1] In order to fully comprehend the physical properties of these materials, it is crucial to gain an understanding of the atomic arrangement and the formation mechanisms of their unique structural features, i.e. basal and pyramidal inversion domain boundaries (IDBs).
ZnO nanowires (NWs) were grown on fused silica substrates via a thermal evaporation method and a metal-seeded growth mechanism.[2] Doping of said NWs with indium was performed by spin-coating with an organic solution of indium nitrate. For thermal decomposition of the solution droplets and subsequent reaction of the In2O3 particles with the NW surface, the specimen was then annealed inside a furnace in air. Conventional TEM bright field imaging was conducted with Philips/FEI CM30 T and CM300 FEG-UT microscopes operated at 300 kV. HAADF and BF/ABF STEM imaging at high resolution was performed using an advanced analytical TEM/STEM system (JEOL JEM-ARM 200CF equipped with a cold FEG and probe Cs corrector).[3]
The aforementioned synthesis method yields NWs of different growth directions. Type-<10-11> NWs are favourable for TEM investigations due to their distinct morphology. It can be shown that the formation of In-decorated basal IDBs occurs largely at {10-11} facets of the NW surface and especially at boundaries between {0001} and {10-11} facets. These IDBs permeate the ZnO NW at a rate of up to 2.7 nm min-1.
High-resolution HAADF imaging reveals that {0001} facets of the nanostructure are not terminated by the basal IDB proper, but by the unreactive oxygen-terminated -c face of a 2- layered ZnO slab, leading to an energetically favourable surface (Fig. 1). The higher Z contrasts observed in cation columns in the vicinity of {10-11} facets indicate that they are enriched with In compared to the surrounding ZnO domain, while also being terminated by a single-layer slab of ZnO.
In atomic-resolution ABF STEM images of incomplete IDBs, a discrete boundary between the basal IDB and 2 {0002} lattice planes of ZnO is observed. At the pyramidal IDB, a displacement of cations is clearly visible while the anion lattice remains virtually unaffected. (Fig. 2) We conclude that the pyramidal IDB acts as a "conveyor belt" for indium cations towards the end of the incomplete basal IDB.

[1] D. P. Norton et al., Mater. Today 6 (2004), 34-40.
[2] H. Simon, T. Krekeler, G. Schaan, W. Mader, Cryst. Growth Des. 13 (2013), 572-580.
[3] H. Schmid et al., Ultramicrosc. 127 (2013), 76-84.

Fig. 1: Annular bright-field (ABF, left) and high-resolution high-angle annular dark field (HAADF, right) STEM images in <2-1-10> orientation. Fully developed basal and pyramidal IDBs can be observed. {10-11} and {0001} facets are terminated by one and two layers of ZnO respectively.

Fig. 2: High-resolution ABF STEM image in <10-10> orientation with a superimposed model of the crystal structure showing considerable displacement of cations at the pyramidal IDB.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-5715 HRTEM and EELS study of C+ implanted and annealed diamond

Olivier E. J.1, Neethling J. H.1, Naidoo S. R.2, Nshingabigwi E. K.2,3, Janse van Vuuren A.1
1Centre for HRTEM and DST/NRF Centre of Excellence in Strong Materials, Nelson Mandela Metropolitan University, Port Elizabeth 6031, South Africa., 2. DST/NRF Centre of Excellence in Strong Materials and School of Physics, University of the Witwatersrand, WITS 2050, Johannesburg, South Africa, 3Department of Physics, National University of Rwanda, P.O. Box 117, Huye, Rwanda.
jolivier@nmmu.ac.za

The transformation of diamond to graphite by ion implantation and annealing has been reported by several authors [1,2]. Previous studies done on the carbon implantation of diamond using the Cold Implant Rapid Anneal technique have indicated that if the critical dose of about 5.2 × 1015 carbon ions is exceeded, diamond transforms to graphite [2]. Below the critical dose, recrystallization of the implanted layer into the original diamond structure is seen [3]. This paper present results of a high resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy (EELS) investigation of room temperature, single energy (150 keV) C implanted diamond (synthetic type IIa, <100>) using a dose (1,5x1016 C+/cm2) exceeded the critical dose as defined above. The implanted layer was studied by cutting the diamond into several pieces and annealing each piece at different temperatures of up to a 1000 oC. HRTEM specimens were prepared by using a Helios Nanolab 650 FIB/SEM and investigated in a double Cs-corrected JEOL JEM-ARM200F HRTEM equipped with a Quantum Gatan Image Filter.

Fig. 1 is a bright-field scanning TEM (STEM) image of carbon implanted and annealed diamond, showing the implanted region extending from the diamond surface to the interface between the damage layer and diamond substrate. Inserted is a selected area diffraction pattern obtained from the layer showing amorphous rings. Fig. 2 shows an EEL spectrum of the C k-edge obtained from the implanted layer. Although the π* peak is an indication of the sp2 character of the carbon layer, the overall EEL spectrum in Fig. 2 is characteristic of highly disordered or amorphous carbon [4]. Further results of the structural evolution of the implanted layer with annealing will be presented.

This investigation illustrates the value of HRTEM and EELS to determine the nature of defects generated by ion implantation in diamond and the micro- and nanostructure of the implanted layer. These techniques are also very useful for the determination of the critical implantation and annealing conditions for the transformation of diamond to graphite.

References

[1] RA Spits et al, Nucl. Instr. and Meth. in Phys. Res. B 85 (1994) p. 347.

[2] EK Nshingabigwi et al, Proceedings of the South African Institute of Physics (2011) p. 711.

[3] TE Derry et al, Nucl. Instr. and Meth. in Phys. Res. B 267 (2009) p. 2705.
[4] H Daniels et al, Phil. Mag. 87 (2007) p. 4073.

The authors gratefully acknowledge funding from the Department of Science and Technology, the National Research Foundation and Sasol.

Fig. 1: Bright STEM image of the implanted layer after implantation. Inserted is a selected area diffraction pattern of the implanted layer

Fig. 2: EEL spectrum of the implanted layer showing an amorphous carbon character
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-5738 STEM observations of indium segregation in InAlN epitaxial layers

Borysiuk J.1, Sobczak K.1, Kaminska A.1, Jezierska E.2, Yamamoto A.3, Schenk D.4, Suski T.5, Zytkiewicz Z. R.1
1Institute of Physics, Polish Academy of Sciences, Warsaw, Poland, 2Faculty of Materials Science and Engineering, Warsaw University of Technology, Warsaw, Poland, 3Department of Electrical and Electronics Engineering, University of Fukui, Fukui, Japan, 4SOITEC Specialty Electronics, Villejust, France, 5Institute of High Pressure Physics, Polish Academy of Sciences, Warsaw, Poland
borysiuk@ifpan.edu.pl

Indium segregation in high In content InAlN layers grown on GaN/sapphire templates was investigated by scanning transmission electron microscopy (STEM). The chemical nonuniformity of the layers was determined from energy dispersive X-ray analysis (EDX) and high-angle annular dark-field (HAADF) imaging. Low In content samples show no indication of a preferential incorporation of indium, proving that compositionally uniform layers and structures could be grown. For high content of indium, above 20 at % on average, the growth becomes unstable, leading to the preferential incorporation of In at some orientations [1,2]. The V-shaped In-rich structures were observed, resulting in the preferential indium segregation at the sides of the structures (Fig. 1). Indium atoms segregation was usually visible as a bending of lattice planes in InAlN structure. A detailed, structural model of strained defects, based on high-resolution TEM (HRTEM) observations was proposed. The model assumes coherent substitution of aluminum by indium in Al lattice sites which leads to the a-plane compressive stress accumulation. Such stress leads to the vertical strain by upward motion of the neighboring lattice sites and elongation of the c-lattice constant locally. Theoretical (simulated) HRTEM images, taking into account the In concentration variations were generated using JEMS software [3]. The changes of the contrast intensity between different atomic columns with variable In content were compared with the experimental data.

[1] G. Perillat-Merceroz, G. Cosendey, J.-F. Carlin, R. Butté, N. Grandjean, J. Appl. Phys. 113 (2013) 063506
[2] P. Vennégués, B.S. Diaby, H. Kim-Chauveau, L. Bodiou, H.P.D. Schenk, E. Frayssinet, R.W. Martin, I.M. Watson, J. Cryst. Growth 353 (2012) 108
[3] P.A. Stadelmann, Ultramicroscopy 21 (1987) 131

This work was financed by the resources of National Science Center (Poland) allocated by decisions: DEC-2011/03/B/ST5/02698 and DEC-2012/05/B/ST3/03113.

Fig. 1: HAADF and TEM cross-section images of InAlN layers with indium content of 25% (a,c) and 28% (b,d), respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-5912 Atomic structure analysis of MgO Near-Sigma 5 (310)[001] Symmetrical Tilt Grain Boundary

Saito M.1, Inoue K.2, Wang Z.2, Kotani M.2, Ikuhara Y.2, 3
1JEOL, Tokyo, Japan, 2Tohoku University, Sendai, Japan, 3The University of Tokyo, Tokyo, Japan
msaito@sigma.t.u-tokyo.ac.jp

Real materials including ceramics and minerals are, in general, of polycrystalline nature, and the prevailing presence of internal interfaces between grains, i.e., grain boundaries (GBs) often influences significantly their mechanical, electrical and physical properties. Magnesium oxide (MgO) is one of the best characterized oxide materials in terms of GBs and defects, and is often considered as a model oxide system owing to its simple rocksalt structure (with both the Mg and O atoms octahedrally coordinated) [1-2].

In this work, we investigated the microstructures of "near-Σ5" GB (Σ indicates the degree of geometrical coincidence at a GB ) in MgO in order to understand how the misalignment of tilting angles from the exact Σ5 orientation can modify GB structures at the atomic scale. Also it remains unknown whether impurities are segregated to the near-Σ5 GB and how such segregation can drive GB structure change and thus modify material property.

Here, we apply a bicrystal technique to fabricate a symmetrical tilt near-Σ5 GB with a bonding-angle deviation of ~1.7±0.1° from the exact Σ5 orientation, i.e., from the (310) plane. (S)TEM observations were performed by JEOL JEM-2010F (200 kV) and JEM-2100F (with Cs-corrector, 200 kV). Finally, we interpreted the GB local structure via mathematical approach based on O-lattice theory [3].

As a result, dark-field (DF) images showed periodically aligned edge dislocations on the boundary in order to compensate lattice mismatch due to misalignment. Annular bright field (ABF) images also revealed that the near-Σ5 GB comprises an alternating array of six normal Σ5 GB structural units and one deformed Σ17 GB structural unit, and importantly the Ca and Ti impurities are selectively segregated to the Σ5 units, while they are absent at the Σ17 units [4]. This near-Σ5 GB with tilting angle of 35.3° is mathematically equivalent to Σ533 GB on (22 7 0) plane. According to dissociation rule expected by O-lattice theory, this kind of GB with high Σ value can easily dissociate into two low Σ GB with special structure units, i.e., six Σ5 GB units and one Σ17 GB unit. This mathematical expectation is completely same as the obtained experimental result. The detailed will be reported.

[1] Z. Wang, M. Saito, K. P. McKenna, L. Gu, S. Tsukimoto, A. L. Shluger, and Y. Ikuhara, Nature 479, 380-383 (2011).

[2] M. Saito, Z. Wang, S. Tsukimoto, and Y. Ikuhara, Journal of Materials Science 48, 5470-5474 (2013).

[3] W. Bollmann, "Crystal defects and crystalline interface", Springer-Verlag, New York (1970).

[4] M. Saito, Z. Wang, and Y. Ikuhara, Journal of Materials Science, 49, 3956-3961 (2014).

This work was conducted in part at the Research Hub for the Advanced Nano Characterization and the “Nanotechnology Platform” at the Univ. of Tokyo supported by the MEXT of Japan and also at the Grant-in-Aid for Scientific Research (C) (grant no. 23560817) and the IKETANI and IZUMI Science Foundation for financial supports.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-6026 Characterization of Planar Defects in Sn-Doped ZnO Nanowires

Cai R. F.1, 2, Hsieh C. Y.1, Chang M. T.1, Lai M. W.1, Lo S. C.1, Chen F. R.2
11. Dept. of Electron Microscopy Development and Application, Material and Chemical Research Laboratories, Industrial Technology Research Institute (ITRI), 22. Department of Engineering and System Science, National Tsing Hua University, Taiwan (R.O,C.)
renfong@itri.org.tw

ZnO nanowires have been widely applied for fabricating functional optoelectronic devices such as LEDs, solar cells and thin film transistors. Elemental doping is an effective method to modify the properties of ZnO. Previous studies have shown that the doped atoms produce planar defects inside ZnO nanowires [1-2]. To understand the mechanism of this phenomenon, a comprehensive study of dopant induced defects within ZnO nanowires is necessary. Because annular dark field (ADF) and annular bright field (ABF) imaging techniques in Cs-corrected STEM can provide ultra-high resolution and very informative images, it is recognized as a powerful strategy to investigate the doping-induced defect structure [3].
In present study, we obtained the microstructural defect details of Sn doped ZnO (SZO) nanowires with probe forming Cs- corrected STEM. ADF and ABF detectors were used for the image acquisition. The inner-outer detection semi-angles of ADF and ABF detectors were 68-170 mrad and 11-23 mrad, respectively. The crystal structure of the sample nanowires were identified as wurtize SZO based on electron diffracting technique. Figure 1 shows the HAADF and ABF images of the planar defect in which the atoms of Sn, Zn and O were labelled with overlaid spheres. It was found that the planar defect in SZO nanowire is inversion domain boundary. In addition, according to EELS line scanning, Sn impurities were found preferring to incorporate in the inversion domain boundary. The twin boundary were also found in different SZO nanowires. Figure 2 shows the twin boundary in SZO nanowire. From the FFT of the HAADF image, it indicated the twin boundary is parallel to (1 -1 0 -3) plane, and the angle between upper and bottom crystal (0 1 -1 0) planes is about 150 degrees. It is interesting that the ordering variation in intensity of HAADF image on the twin boundary was observed. The right hand side of the dumbbell atoms is brighter than left hand side on the twin boundary. This phenomenon was not found in the inversion domain boundary, the result was shown in figure 3. Because the intensity of HAADF image is proportional to Z1.7 [4], it is supposed that the intensity variation on the twin boundary might be resulted from different dopant concentration.

[1] Y. Ding et al., Phys. Rev. B, vol. 70, no. 23, p. 235408, Dec. 2004.
[2] Y. C. Park et al., Appl. Phys. Lett., vol. 102, no. 3, p. 033103, 2013.
[3] S. Pennycook and P. Nellist, Scanning transmission electron microscopy: imaging and analysis. 2011.
[4] O. L. Krivanek et al., Nature, vol. 464, no. 7288, pp. 571–574, 2010.

The authers gratefully thank Prof. H. Kurata and Dr. Y. H. Wu for their insightful comments.

Fig. 1: Inversion domain boundary in SZO nanowire. (a) HAADF image. (b) ABF image and supposed atomic structure overlaid on the images.

Fig. 2: Twin boundary in SZO nanowire. (a) HAADF image and FFT of ROI-1. (b) HAADF image and the supposed atomic structure overlaid on the image in the twin boundary.

Fig. 3: Line profile in the planar defect. (a) Inversion domain boundary. (b) Twin boundary.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-9-P-6041 MICROSTRUCTURE OF DISSIMILAR WELDING: CARBON STEEL A36 AND AUSTENITIC STAINLESS STEEL E309L-E308L

Zamora Rangel L. z.1, Aguilar Torres A. T.2, Diaz Perez L. D.3, Sandoval Jimenez A. s.4, Muciño Gutierrez O. M.5
1Instituto Nacional de Investigaciones Nucleares. Departamento de Tecnología de Materiales, México.
luis.zamora@inin.gob.mx

Keywords: RPV, Cladding, BWR, SMAW, HAZ.

Recently, considerable research in the welding of austenitic stainless steels has again been developed by use for nuclear and the other energy applications. Austenitic Stainless Steel has been widely used a weld filler to join the low-alloy steel reactor pressure vessel (RPV).
The chemical compositions of low alloy steel A36 contain 0.22% C, 0.014% S and 0.015% P, and the weld filler austenitic stainless steel E308L contain 19.07%Cr, 9.99% Ni, 0.026% C, 0.534% Si, and the E309L, 23.65% Cr, 12.49% Ni, 0.028% C, 0.421% Si. The welding conditions were chosen based on the construction specifications of the nuclear power plants. The weld deposit a coating of three layers of stainless steel, the firth pass with E309L electrode and others two steps with E308L electrode on a carbon steel plate type A36(figure 1),simulating the properties of the inner lining of a vessel (cladding) of the BWRs reactor . Plate type A36, with dimensions 28.09 x 10.63 x 1.26 cm, were welded under a constrained conditions using shielded metal arc welding (SMAW)(75 A and 22 V),interpass temperature ≤ 90°C.
The metallographic examination of the weld were realized by optical microscopy and scanning electron microscopy (SEM), energy dispersive X ray (EDX), using the microscopes JEOL JEM 6010LV. The specimens were cut from center area of the weld metal (10 mm X 10 mm X 10 mm).The transverse section of the weld were polished, etched with 3 pct Nital for 15 seconds the carbon steel A-36, and the weld metal, heat-affected zone (HAZ) with aqua regia, and then examined [1]. As a result of welding, these materials may, depending on composition, solidify with a structure different. One problem with fusion welding of these materials is their susceptibility to solidification cracking. Cracks have been found in various regions of the weld zone with different orientations in the weld zone (figure 2), such as centerline cracks, transverse cracks, and micro cracks in the underlying weld metal or adjacent or HAZ. The general structure of the first weld pass consists of elongated columnar dendrites at the melt pool, where heath transfer is highly directional, and more equiaxed cellular dendrites in the middle with slower cooling (figure 3 and 4).
The Vickers hardness in the range of 165-220 HV, was increase of the steel plate to third weld pass [2].

REFERENCES
[1] ASM Handbook, Metallography and Microstructures, Volume 9[2] L. Zamora R., Aguilar Torres J.A, Díaz Pérez L., Sandoval Jiménez A. “Análisis microestructural de recubrimiento de acero inoxidable austenítico sobre placa de acero al carbón A-36” XXII Congreso Técnico Científico ININ-SUTIN, Centro Nuclear de México, Diciembre 2012.

The authors are greatly to C. Jorge Perez del Prado of the Department of Materials Technology for this work is gratefully acknowledgements.

Fig. 1: Carbon Steel Plate A-36 and austenitic stainless steel Weld metal.

Fig. 2: Carbon steel A36 and stainless steel 304 at 500X.

Fig. 3: Interface Carbon steel A36 and stainless steel, 500X

Fig. 4: Interface Carbon steel A36 and stainless steel
↑ Go to the Table of content ↑

MS-10. Porous and architectured materials

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-10-IN-1593 Porous structures in 2 and 3 dimensions

Van Tendeloo G.1, Turner S.1, Bals S.1
1EMAT, University of Antwerp, Belgium
staf.vantendeloo@uantwerpen.be

Metal-organic frameworks (MOFs) are a new class of porous materials, consisting of metal ion centers linked together by organic linkers to create crystalline porous networks. MOFs have received a great deal of attention because of their high specific surface areas and pore volumes, applicable in gas (H2) storage, catalysis, and photovoltaics.1 For specific applications MOF crystals can be loaded with catalytically active materials like Pd, Au, Cu and Ru in the form of nanoparticles, small clusters or single atoms, leading to a heightened activity in e.g. olefin hydrogenolysis and methanol synthesis or with semiconductor nanomaterials like GaN or ZnO for improved optical properties.2

Characterizing these delicate materials is far from trivial. Most of the common characterization techniques like X-ray diffraction of nitrogen adsorption for this type of materials offer global information only. However, in the case of nanostructured and/or -sized systems or upon loading of the frameworks, local structure information is of pivotal importance. Transmission electron microscopy is ideally suited for this, as it can provide structural information down to atomic resolution. MOFs can however be considered as soft matter and are therefore very sensitive to electron beam illumination, making TEM investigation of MOFs challenging.

One of the first MOFs studied by TEM was MIL-101 (Cr). The relatively stable nature of this MOF made pore imaging by HRTEM feasible3 Most other commonly-used MOF materials are significantly less stable to electron beam illumination. Improved TEM technology including low-voltage techniques, cooling holders, beam-blanking systems and fast-readout cameras however do allow pore imaging of far less stable MOFs like MOF-5 and ZIF-8.4 Advanced TEM techniques like tomography make it possible to determine the distribution of nanoparticles within the MOF framework, as in the recent example of hydrogen storage material Pd@COF-102.5 Combining imaging with spectroscopy in the electron microscope allows distinguishing between chemical species within the frameworks and measuring bonding at the local scale.1

Studying porous materials in 3 dimensions at the atomic scale is even more challenging. Again, beam damage is the limiting factor. We applied low-dose aberration corrected HAADF-STEM to zeolite crystals loaded with Ag. A careful analysis of HAADF-STEM images acquired along different zone-axes enabled us to propose a 3D model for the positions of the Ag ions.

1Turner S. et al. (2008) Chemistry of Materials, 20, 5622-5627.

2Esken D. et al. (2011) JACS, 133, 16370-16373.

3Lebedev O. I. et al. (2005) Chemistry of Materials, 17, 6525-6527.

4Wiktor C. et al. (2012) Microporous and Mesoporous Materials, 162 131-135.

We acknowledges financial support from the ERC Advanced Grant “Countatoms” (GVT), the ERC Starting Grant “Colouratoms” (SB) and FWO support (ST).

Fig. 1: Fig. 1: a) High resolution image of an intact cubic MOF-5 crystal (FT inset). 4 b) Enlarged image of region in the white box in a) with image simulation. c) Fourier filtered image with image simulation d) Simulated HRTEM image e) Structure of MOF-5; the white stripes in d) correspond to the terephthalate linkers of MOF-5, the pores are in grey.

Fig. 2: Fig. 2. a) Tomographic reconstruction of Pd@COF-102.  The Pd nanoparticles are rendered in gold, the COF framework in soft off-white. b) Orthoslice through the 3D reconstruction of a loaded Pd@COF-102. The particles visible as white dots are clearly present in the whole crystals without any preferential distribution in size or position.

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-10-IN-6086 Crossover Plasticity Mechanisms in Nano Single- and Polycrystalline Metals: By in situ atomic scale mechanical microscopy

Han X. D.1, Zhang Z.2, Wang L. H.1, Yue Y. H.1, Ma E.3, Chen M. W.4, Zhang Y. F.1, Zheng K.1, Mao S. C.1, Liu P.1, Deng Q. S.1
1Beijing University of Technology, China, 2Zhejiang University, China, 3Johns Hopkins University, USA, 4Tohoku University, Japan
xdhan@bjut.edu.cn

We developed in situ atomic scale mechanical microscopy (In Situ: ASM) for investigation of the deformation dynamics of materials with simultaneous atomic scale imaging. The In situ TEM mechanical testing techniques are generally applicable to regular TEM samples by tensile experiment with simultaneous double tilt, and thus the ability of obtaining atomic scale imaging about the deformation dynamics of the deformed materials. We show several examples of the atomic scale deformation dynamics of Ni, Pt, Au and Cu as well as Cu-Zr metallic glass. It was revealed that these materials show unusual large strain elasticity, size-dependent elasto-plastic transitions and the unusual plasticity behaviors at nano-scale. For example, the cross-over plasticity mechanisms from partial dislocation to full dislocation and twins were discovered in deformation of nano single crystalline Cu samples. A long standing puzzle and uncertainties of grain rotation in ultra-small nano-sized polycrystalline materials (grain size less than 8nm) was demonstrated through this in situ: ASM. It was uncovered that the dislocation climb takes care of the grain rotation behaviors in small grain systems. These results shed lights in understanding the interesting mechanical behaviors and the related dislocation activities of the metallic materials at small scale and useful for designing new materials with strength and ductility as well as those applications in micro- and nano- electronics and mechanics.

Reference:

[1] NANO LETTERS, 9, 2471 (2009)

[2] NANO LETTERS, 11, 3151 (2011)

[3] NANO LETTERS, 12, 4045 (2011)

[4] NATURE COMMUNICATIONS, 1, 24, DOI: 10.1038/ncomms1021, 2010

[5] NATURE COMMUNICATIONS, 3, 609, DOI: 10.1038/ncomms1619, 2012

[6] NATURE COMMUNICATIONS, 4, 2413 | DOI: 10.1038/ncomms3413, 2013

[7] NATURE COMMUNICATIONS, 5, 4402, DOI: 10.1038/ncomms5402, 2014

[8] PHYS. REV. LETTS., (2010)

[9] SCIENCE, 339, 1191 (2013)

[10] SCIENTIFIC REPORTS, 2, 852 (2012), DOI: 10.1038/srep00852

[11] ACAT MATER., 59, 6511 (2011)

[12] ACTA MATER. 61, 4689 (2013)

[13] NANO LETTERS, 12, 4595 (2012)

[14] NANO LETTERS, 12, 4045 (2012)

[15] APPL. PHYS. LETTERS, 101, 233109 (2012)

[16] APPL. PHYS. LETTERS, 104, 013111 (2014)

This work was supported by the National 973 Program of China (2009CB623700), the National Outstanding Young Investigator Grant of China (10825419), the Key Project of C-Natural Science Foundation (50831001),  the Beijing High-level Talents (PHR20100503), the Beijing PXM201101420409000053, the Natural Science Foundation (11004004) and the Beijing 211 Project.

Fig. 1: Experimental Set-up for in situ ASM, see reference [7, 8] et al.

Fig. 2: In situ atomic scale dynamic observation: Grain Rotation through grain boundary dislocation climb. See reference [7].
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-10-O-1558 Mesoporosity in photocatalytically active oxynitride single crystals characterized by electron tomography, HREM, nanobeam diffraction and EELS

Pokrant S.1, Cheynet M.2, Irsen S.3, Maegli A.1, Erni R.4
1Laboratory of Solid State Chemistry and Catalysis, Empa, Dübendorf, Switzerland, 2SIMAP, PHELMA-Campus, Grenoble, France, 3Electron Microscopy and Analysis, caesar, Bonn, Germany, 4Electron Microscopy Center, Empa, Dübendorf, Switzerland
simone.pokrant@empa.ch

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-10-O-2548 High resolution imaging of ZIF-8 metal-organic framework structure with a low-dose electron counting direct detection camera

Pan M.1, Czarnik C. M.1, Pan Y. C.2, Sougrat R.2, Li K.2, Han Y.2, Ciston J.3
1Gatan, Inc., California, USA, 2King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia, 3National Center for Electron Microscopy (NCEM), LBNL, Berkeley, California, USA
mpan@gatan.com

Metal-organic frameworks (MOF) have attracted increasing attention in recent years due to their vast potential in clean energy and environmentally relevant applications such as hydrogen storage, gas separation and purification, and catalysis of materials. ZIF-8 has demonstrated its ability in separating propylene/propane gas mixtures by kinetic adsorption. The crystalline structures of MOF are very fragile under electron beam illumination (much more sensitive than zeolites or inorganic mesoporous materials). Therefore it is technically challenging to image the nanoporous structures of MOFs at high resolution.

Direct detection of high energy electrons with an active CMOS (complimentary metal-oxide semiconductor) sensor is replacing the traditional CCD (charge-coupled device) cameras for many performance demanding applications in electron microscopy such as imaging beam sensitive specimens. The detection quantum efficiency (DQE) can be improved significantly by counting individual electrons that reach the image sensor and setting the residual sensor background to zero, thus allowing images with increased signal-to-noise ratio (SNR) to be captured under extremely low electron dose.

Figure 1 is a low magnification TEM image showing small size (30 – 100 nm) ZIF-8 particles. Structural damage was evident by the fast fading of diffraction rings even under a low intensity parallel electron beam. Usually high order diffraction spots disappeared first. The critical dose for spots with lattice spacing 0.3 nm or smaller was estimated to be in the range of ~ 20 - 30 e-/pixel.

To capture high resolution images, we employed the K2 direct detection camera operating in electron counting mode. The electron dose rate was set to be ~ 1 e-/pixel/sec. Figure 2 is a counted image of a ZIF-8 particle oriented in [111] zone axis recorded at 300 kV. The TEM magnification was 43 kx and the total electron dose is ~3 e-/pixel. This particle retains well-defined hexagonal shape and a high degree of crystallinity. Figure 3 is the fast Fourier transform (FFT) image of this particle that clearly shows high resolution information transfer of 0.3 nm or beyond in all directions.

We have demonstrated that direct detection camera with electron counting is capable of recording high resolution images of MOF specimens with a dose of a few electrons per pixel.

The authors wish to thank Karen Bustillo at NCEM, LBNL for her assistance in this work.

Fig. 1: Low magnification TEM image of ZIF-8. Particle size ranges from 30 to 100nm.

Fig. 2: High resolution electron counted image of a ZIF-8 particle recorded with K2 direct detection camera at 300 kV and TEM magnification at 43 kx. Total electron dose is ~ 3 e-/pixel. The image shows exceptional SNR for the given electron dose.

Fig. 3: FFT image of the particle in fig. 2 showing high resolution information transfer of 0.3 nm or beyond in all directions.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-10-O-3231 Structure determination of novel zeolite structures utilizing electron microscopy and electron diffraction

Willhammar T.1, Sun J.1, Wan W.1, Oleynikov P.1, Zhang D.1, Zou X.1, Moliner M.2, Corma A.2
1Berzelii Centre EXSELENT on Porous Materials and Department of Materials and Environmental Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden, 2Instituto de Tecnología Química (UPV-CSIC), Universidad Politécnica de Valencia, E-46022 Valencia, Spain
tom.willhammar@mmk.su.se

Structure determination of new zeolite materials is essential in order to understand and design the synthesis and develop new applications. Because of small crystal size and possible disorder, structure determination of many zeolite materials is a huge challenge. Recent advances in electron microscopy and electron crystallography have facilitated the study of these materials greatly. The power of these methods is here shown by the structure determination of two new zeolite structures, ITQ-39[1] and ITQ-38[2].

Electron crystallography has two mayor advantages over conventional x-ray diffraction. Firstly we can now acquire single crystal diffraction data from crystals smaller than 50 nm utilizing the Rotation Electron Diffraction (RED) method [3]. With this data in hand important information about the structure such as unit cell, symmetry etc. can be obtained, if the structure is ordered it can be obtained ab-initio from the RED data. The second benefit of electron microcopy is the possibility to obtain high resolution transmission electron microscopy (HRTEM) images. Recently a new method to reconstruct the projected potential from a through-focus series of HRTEM images, called QFocus, has been developed.[4] Such an image reveals the local arrangement of atoms and channels in the material and is of great importance in the study of disordered materials.

The structure of the new zeolite ITQ-39 could not be determined by conventional methods due to the very small crystal size, 30x30x500 nm, and severe disorder. By collecting RED data from a crystal of ITQ-39 the presence of stacking disorder could be observed as lines of diffuse scattering, Fig. 1. Also twining could be seen. By collecting structure projection images along two perpendicular directions the aforementioned features could be confirmed and furthermore the channels and smaller ring could be observed, see Fig. 1. Crystallographic structure factors were extracted from ordered regions of the structure projection images. These regions were as small as 5x10 nm. By merging the structure factors from the two images, a 3D potential map could be constructed and the atomic structure could be determined, Fig. 2. It turned out that the material is a new zeolite family built from a random intergrowth of three different polytypes.

Electron crystallography has later been applied also to a second novel zeolite ITQ-38. It has shown to be a method that can be applied more generally and has a large potential for structure determination of zeolites as well as for other materials.

References
[1] Willhammar, T et al., Nature Chem. 4  188–194 (2012).
[2] Moliner, M et al., J. Am. Chem. Soc., 134 6473–6478 (2012).
[3] Zhang, DL et al., Z. Kristallogr.  225 94-102(2010).
[4] Wan, W et al. Ultramicroscopy 115 50 (2012).

Fig. 1: In the (a) h0l and (b) 0kl slices from the RED data the twinning and the stacking disorder can be identified, structure projection images along (c) [010] and (d) [100] directions reveals the local arrangement of the atoms and the disorder.

Fig. 2: The three dimensional electrostatic potential map reconstructed from the structure projection images (blue) and the framework structure determined from is (yellow).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-10-O-3344 Electron tomography investigation of a novel etching process yielding hollow structures in Cu2-xSe(core)/Cu2-xS(shell) nanocrystals exposed to oxidizing environments

Brescia R.1, Miszta K.1, Bertoni G.1,2, Prato M.1, Marras S.1, Xie Y.1, Kim M. R.1, Manna L.1
1Nanochemistry, Istituto Italiano di Tecnologia (IIT), via Morego 30, IT 16163 Genova, Italy, 2IMEM-CNR, Parco Area delle Scienze 37/A, IT 43124 Parma, Italy
rosaria.brescia@iit.it

Electron tomography has proven to be a unique tool for the elucidation of nanoscale mechanisms that cannot be clarified by simple two-dimensional imaging. In this contribution a novel etching process, induced by exposure of colloidal Cu2-xSe(core)/Cu2-xS(shell) bullet-in-rod nanocrystals (NCs) to oxidative environments, is analyzed via a combination of TEM-related techniques, with a key role played by electron tomography. The progressive addition of an oxidizing agent (namely, cupric chloride, CuCl2, in solution with methanol) leads to extraction of electrons and Cu+ ions from the NCs suspended in toluene. The process has been followed by exposure of the NCs to increasing amounts of CuCl2 (η = molar ratio of CuCl2 to Cu+ ions in the NCs). Surprisingly, the NC region initially dismantled is the Cu2-xSe core rather than the relatively thick (about 3 nm) outer Cu2-xS shell (Fig. 1 a-c). HAADF-STEM tomography-based volume reconstruction of the weakly etched NCs (η=2) evidences in most cases a hollow morphology (Fig. 2i). Only a small fraction of the NCs exhibits empty channels connecting the central void to the shell surface at the same etching stage (Fig. 2ii). This demonstrates that Cu+ ions extracted from the NCs into the solution must have diffused from the core through into the shell. This mechanism is substantially different from the classical Kirkendall effect [1], in which the formation of hollow structures is due to the different diffusion rates of two species across an interface. In this case the out-diffusion of a single species (Cu+ ions) is demonstrated.
Further addition of CuCl2 to the Cu2-xSe/Cu2-xS NCs leads to dismantling of the shell, after the core region has been emptied (Fig. 2iii-iv). A central porous region is left with CuSxSe1-x composition. Selected area electron diffraction (SAED) patterns and HRTEM analyses show an evolution of the shell material from monoclinic Cu2S (α-chalcocite) to a covellite (hexagonal CuS)-like structure, which shows higher stability by surviving till later stages of the oxidation process (Fig. 1 d-h).
The etching mechanism presented here is triggered both by the stronger tendency of Cu2-xSe over Cu2-xS to oxidation and by the fast Cu+ ion diffusion in copper chalcogenides. This novel process for the fabrication of copper chalcogenide hollow crystals can be employed in the production of nanostructured crystals for applications in catalysis, energy storage, plasmonics and medicine.

[1] Y. Yin et al., Science 304, 711 (2004)

Fig. 1: (a-c) HAADF images of NCs obtained at increasing η and corresponding longitudinal EDS line scans. (d) Azimuthally integrated SAED patterns showing structural evolution of NCs. (e,f) HRTEM image and corresponding Fourier transform of the tip (shell) region of an initial NC and (g,h) same for a CuSxSe1-x porous NC at late etching stage.

Fig. 2: Top: HAADF images of selected Cu2-xSe/Cu2-xS NCs observed after addition of CuCl2 (η=2 for i-ii, η=4 for iii-iv). The lower panels show (a) the isosurface rendering of the corresponding reconstructed volume (WBP+SIRT), (b) a longitudinal cut-through, and (c,d) transversal sections, with planes perpendicular to the elongation direction.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-1574 Mesoscale ordering of porous CNT compacts and its effects on conductance

Gnanasekaran K.1, de With G.1, Friedrich H.1
1Laboratory of Materials and Interface Chemistry, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, The Netherlands
k.gnanasekaran@tue.nl

The performance of any hierarchically structured material composed of functional particles is not only governed by the intrinsic properties of the particles but also strongly influenced by the morphology of the 3D network that is formed. For instance, few experimental and theoretical studies show that besides a dependence on the intrinsic properties of carbon nanotubes, the good electrical and mechanical properties also depends on the 3D organization of the particle network such that the contact properties can be controlled by changing the network topology [1, 2]. The main factors that influence these changes in any network are the aspect ratio, polydispersity, bending flexibility, tortuosity and attractive interactions of the particles [2]. Here we report that the electrical properties of rod-shaped CNT particles can be optimized by changing the morphological arrangement of the nanofillers forming the 3D network. To this end the self-organization of CNT’s of different average lengths and polydispersity into porous 3D compacts was studied. Our experimental findings show a twofold increase in electrical conductivity for the optimized network resulting from the synergetic assembly of optimized CNT populations into hierarchically structured materials. Mixture of varying CNT lengths introduces a competing length distribution in the system which alters the self-organized packing and results in higher conductivity at certain specific configuration as shown in the Fig. 1. The volume fraction normalized conductivity plot also indicates that the global variation in packing density of the CNT compacts is independent of the electrical conductivity. Large-area HAADF-STEM imaging (~100 μm2) (Fig. 2) at nanometer resolution of thick sections (~500 nm) followed by quantitative image analysis (Fig. 3) was used for analysis. This analysis shows a correlation between the local packing variations and the average length of the CNTs which could be used as a criterion to obtain such an optimized network. The entire work reveals the significant influence of the particle size distribution on network connectivity during the optimization of the conductive properties of CNT systems. The direct quantitative capability of HAADF-STEM imaging, along with the large area analysis at nanometer resolution, gives a unique upper hand for understanding many problems on self-assembly and hierarchical ordering which extends the electron microscopy towards a bulk characterization technique.

References

1. Hermant MC, Klumperman B, Kyrylyuk AV, van der Schoot P, Koning CE. Soft Matter. 2009;5(4):878-85.
2. Kyrylyuk AV, Hermant MC, Schilling T, Klumperman B, Koning CE, van der Schoot P. Nat Nano. 2011;6(6):364-9.

The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7-MC-ITN) under grant agreement No. 264710. The authors would like to thank the Directorate-General for Science, Research and Development of the European Commission for financial support of the research.

Fig. 1: Volume fraction normalized electrical conductivity plotted as a function of average aspect ratio. Aspect ratio is varied by mixing three different CNT systems - A, B and C at appropriate ratios.

Fig. 2: Stitched HAADF-STEM micrographs revealing the local packing variations in CNT network topology.

Fig. 3: Segmentation of CNT network based on local packing variations.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-1615 Atomic Scale Analysis of Cs+ Captured within Zeolitic Framework

Yoshida K.1, Toyoura K.2, Matsunaga K.1,2, Nakahira A.3, Kurata H.4, Ikuhara Y. H.1, Sasaki Y.1
1Nanostructures Research Laboratory, Japane Fine Ceramics Center, Nagoya, Japan, 2Department of Materials Science & Engineering, Nagoya University, Nagoya, Japan, 3Graduate School of Engineering, Osaka Prefecture University, Sakai, Japan, 4Institute for Chemical Research, Kyoto University, Uji, Japan
kaname_yoshida@jfcc.or.jp

Zeolites have great potential for applications in various fields. Especially, application to management of leaks of radioactive waste such as 137Cs attracts attention in Japan. However ion-exchange behavior within zeolites have been poorly understood. In order to clarify this issue, precise structural information of adsorbed cations in the zeolite is inevitable. In our study, cation-exchanging mechanism of Cs+ within NaA zeolite was considered precisely. In order to understand where adsorbed cations are in the zeolitic nanocavities, we employed aberration corrected high-angle annular dark-field scanning transmission electron microscope (AC-HAADF-STEM) and aberration corrected high-resolution transmission electron microscope (AC-HRTEM). Ab initio molecular dynamics (AIMD) simulations were also performed, in order to understand the atomic-level dynamics of Cs+ and Na+ within the zeolitic nanocavities.

Cs-exchanged zeolite was prepared by immersion of NaA zeolite into 5000 mg/L of nonradioactive CsCl aqueous solution for 12 hours. FIG. 1 shows already-known crystal structure of NaA [1]. There are three kinds of Na+ sites within NaA. In this study, sites at the center of single six-membered ring (S6R) and single eight-membered ring (S8R) are named Na1 and Na2 respectively. Na2 site is slightly shifted from the center of S8R and forms symmetrically equivalent four positions which have occupancy of ca. 25%. That is, a Na+ cation at Na2 site exists in either of four positions in a S8R. Another extra site of Na3 is located at the inside of the α-cage, but occupancy of Na+ at this site is considerably low (6.6%). FIG. 2 shows the AC-HAADF-STEM images of NaA and Cs-exchanged NaA, which were taken by a JEOL JEM2100F. The results of AC-HAADF-STEM observations exhibit that Cs+ cations were exchanged with Na+ cations only at Na2 site. Precise locations of Cs+ cations captured at Na2 site were evaluated by AC-HRTEM observations performed on a JEOL JEM2200FS. Comparison of simulated images with experimental image (FIG. 3d) exhibits that Cs+ cations captured at Na2 sites are exactly located on a center of S8R, in contrast to Na+ cations. The difference in the cation sites in the S8R between Na+ and Cs+ were also confirmed from the trajectories in the present AIMD simulations. This may also be an indication that Cs+ is firmly ‘captured’ at the center of S8R. Lower leaching from zeolite could be effective for long-term storage of Cs+. To improve ion-exchange capability of zeolite, it is essential to choose the suitable framework whose cavities fit to ionic radius of cation.

References

[1] J. J. Pluth and J. V. Smith, J. Am. Chem. Soc. 102 (1980) 4704.

All authors acknowledge Prof. Y. Ikuhara at the University of Tokyo and JFCC for proposing the collaboration between the authors and a great contribution to start this work.

Fig. 1: Crystal structure of NaA zeolite. (a) [001]-projection of NaA zeolite, (b) Schematic modelof α-cage and (c) Schematic model of β-cage.

Fig. 2: Experimental AC-HAADF-STEM images. (a) Raw image of NaA, (b) filtered image and structural model of NaA, (c) raw image of Cs-exchanged NaA and (d) filtered image and structural model of Cs-exchanged NaA.

Fig. 3: Experimental AC-HRTEM images. (a) Raw image of NaA, (b) filtered image and structural model of NaA, (c) raw image of Cs-exchanged NaA and (d) filtered image and structural model of Cs-exchanged NaA.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-1616 Plasmonic Nanopores Observed by Scanning Transmission Electron Microscope Cathodoluminescence

Sannomiya T.1, Junesch J.2, Shi J.1, Nakamura Y.1, Yamamoto N.1
1Tokyo Institute of Technology, Tokyo, Japan, 2ETH Zürich, Zürich, Switzerland
sannomiya@mtl.titech.ac.jp

Substance transport plays an essential role in biological processes, such as inter cellular communication. To investigate such transport phenomena, synthesized solid state membranes with nanosized pores, so called nanopores, can be utilized. Nanopore membranes are also useful for filtering devices or catalytic electrodes. We fabricated ultrathin nanopore membranes out of various materials by combined colloidal lithography and film transfer technique. The pore size could be reduced down to 10 nm scale by additional material deposition. The fabricated nanopores were characterized by transmission electron microscopy as well as optical transmission spectroscopy. Short-range ordered (SRO) Au nanopores showed plasmonic resonance in the Vis-NIR wavelength range due to the coupling between the pores through surface plasmon polariton. Optical measurement showed that the nanopore membranes with hydrophobic surfaces were able to support water on only one side of the membrane, which is important for high performance biosensors or medium separation. To analyze the inter-pore optical coupling of the plasmonic nanopores, we applied aberration corrected scanning transmission electron microscope (STEM) with cathodoluminescence (CL) detection system. CL spectrum of the SRO plasmonic nanopores showed consistent spectral features as the optical extinction spectra. As expected, SRO structure showed inhomogeneous optical coupling between the pores with their local resonances depending on the inter-pore distances. We also found a symmetric coupling mode of nanopores at long wavelengths than the anti-symmetric coupling mode when pores are located close to each other. Such anti-symmetric mode was observed even for a connected pore pair.

This research was supported by JST CREST.

Fig. 1: TEM image of pores fabricated in a suspended AlN/Au/AlN membrane.

Fig. 2: Cathodoluminescence mapping of short-range ordered plasmonic nanopores in a suspended AlN/Au/AlN membrane, measured at the wavelengths of 700 and 800 nm. Horizontally polarized light has been detected.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-1710 How to image the atomic structure of Ag-exchanged FAU-Y and FAU-X zeolites by using aberration corrected transmission electron microscopy

Altantzis T.1, Coutino-Gonzales E.2, Martinez G. T.1, Roeffaers M. B.2, Hofkens J.2, Béché A.1, Bals S.1, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium , 2Laboratory of Photochemistry and Technology, K.U. Leuven, Celestijnenlaan 200 F, B-3001 Leuven, Belgium
thomas.altantzis@uantwerpen.be

Zeolites are crystalline microporous aluminosilicate minerals with numerous industrial and scientific applications, including catalysis, preparation of advanced materials and nuclear processing. Transmission Electron Microscopy (TEM) is a very powerful technique to characterize the structure and composition of such materials at the atomic scale. For the case of cation exchanged zeolites, it is of crucial importance to determine the atomic positions of the cations in the zeolitic framework. Such knowledge enables one to understand and optimize the connection between the structure and the properties of the materials. Recently, high angle annular dark field scanning transmission electron microscopy (HAADF-STEM) was used to image Ag cations in the LTA framework.1 HAADF-STEM images yield an intensity which scales with the atomic number Z and the thickness of the sample.2 Therefore, the Ag cations will appear with higher intensity in comparison to the elements in the zeolitic framework (Si, Al and O).
Unfortunately, zeolites are extremely sensitive to damage caused by the electron beam, especially in the case of a relatively low Si/Al ratio. The use of aberration corrected TEM at relatively low electron dose, however, does enable one to obtain high resolution images. Here, we combine monochromated, aberration corrected HAADF-STEM with a careful image analysis, the so-called template matching technique, in order to determine differences in the 3 dimensional positions of Ag cations present in FAU-X and FAU-Y. For both samples, which have an fcc structure, HAADF-STEM images were therefore acquired along the three main zone axes [100], [110] and [111] and compared with the results from X-ray diffraction (XRD).3,4 This is illustrated in Figure 1 and Figure 2 for the [110] zone axis. This enabled us to propose a 3D model for the atomic structure of Ag-exchanged FAUY and FAUX zeolites (Figure 3).

1. A. Mayoral et al. Angew. Chem. Int. Ed., 2011, 50, 11230-11233

2. S. J. Pennycook. Annual Review of materials Science, 1992, 22, 171-195

3. Butikova et al. Kristallografiya, 1989, 34, 1136-1140

4. Eulenberger et al. J. Phys. Chem., 1967, 71, 1812-1819

The authors acknowledge financial support from European Research Council (ERC Advanced Grant # 24691-COUNTATOMS and ERC Starting Grant #335078-COLOURATOMS)

Fig. 1: High resolution HAADF-STEM image of Ag exchanged FAU-X zeolite, acquired along [110]. The inset presents the results of the averaging procedure of the template matching, using 50 templates.

Fig. 2: a) Comparison between the averaged image and the XRD model (overlay)3 for the FAU-X along the [110] zone axis. Inconsistencies between the experimental image and the XRD model are indicated by yellow circles in Figure a. b) Comparison between the averaged image and the refined model (overlay).

Fig. 3: Proposed model for FAU-X sample, oriented along the [110], [100] and [111].
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-1771 Si/Al ratio control of zeolita beta

Urbina de Navarro C. .1, Ortega N.1, López C.1, García L.1
1Universidad Central de Venezuela, Caracas, Venezuela
caribay.urbina@ciens.ucv.ve

Zeolite beta is a solid of interest due to its acidic properties and unique structure with a framework of three-dimensional system of interconnected 12 membered ring channels. Structure of zeolite beta has an intergrowth of three polymorphs A, B and C. It has a tortuous channel with a pore opening of 5,6 Å x 6,5 Å and a straight channel with a pore opening of 5,7 Å x 7,5 Å. It is usually synthesized by hydrothermal method in the presence of tetraethylammonium hydroxide (TEAOH) as organic template under high pH conditions. The synthesis of pure zeolite beta is determined by different factors such a time, temperature and Si/Al ratio. For example, depending of the Si/Al ratio it can be synthesized pure zeolite beta phase or a mixed of zeolite beta phases with impurities of other type of zeolite. In this work, zeolite beta was synthesized by varying the Si/Al ratio from 13 to 50. The source of aluminum (Al2(SO4)3) was dissolved in water and a source of silica (SiO2) was added in tetraethylammonium and potassium chloride (KCl). The mixture was stirred and placed in a Parr reactor under crystallization temperature of 170 °C during 24 hours. The solid was filtered, washed and dried at 70 °C, followed by calcination at 500 ° C for 5 hours. The solid obtained were characterized by XRD and SEM. The XRD pattern of the synthesized solid shows characteristic peaks of zeolite beta at 7.6° and 22.4° and new peaks around 14º, 21º and 23º. These new peaks can be associated with zeolite ZSM-12, this suggest that the synthesized solid may consist of a mixture of zeolite beta phase and zeolite ZSM-12. Characterization by Scanning Electron Microscopy (Figure 1) shows different morphologies such as aggregates of microcrystals corresponding to zeolite beta and crystals of hexagonal habit corresponding to zeolite ZSM-12. The characterization by XRD and SEM suggest that the synthesis conditions used in this work allowed obtaining zeolite beta with impurities of zeolite ZSM -12.

This research was supported by FONACIT 20010001442

Fig. 1: Reference zeolite beta

Fig. 2: Si/Al 13

Fig. 3: Si/Al 25

Fig. 4: Si/Al 50
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-1823 Structural characterization of empty and ion exchanged natural HEU-type zeolites

Filippousi M.1, Turner S.1, Katsikini M.2, Pinakidou F.2, Kantiranis N.3, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium, 2Solid State Physics Section, Physics Department, Aristotle University of Thessaloniki, GR-54124, Thessaloniki, Greece, 3Department of Mineralogy-Petrology-Economic Geology, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
maria.filippousi@uantwerpen.be

Natural zeolites have attracted worldwide interest for use in a broad range of applications, even though they may contain more impurities compared to the synthetic ones. Since natural zeolites are abundant in nature, they are easily available and environmental friendly. HEU-type zeolites (clinoptilolite and heulandite; monoclinic crystal structure, space group C2/m, cell parameters a≈17.7, b≈17.9 and c≈7.4 Å [1]) are the most abundant minerals on earth exhibiting a zeolite structure, making them low-cost industrial minerals with several commercial applications. [2]To date, the main technique to study the structure of natural HEU-type zeolites has been single crystal X-ray diffraction. [2] However, XRD collects structural information from a large area to reveal an average structure, and therefore does not provide local information. In order to gain more information on the pure HEU-type zeolite at a local level, low voltage aberration corrected TEM was used in this study.In this work, we report the first characterization of the atomic structure of the HEU-type zeolite through use of low voltage aberration corrected transmission electron microscopy under low dose conditions (Figure 1). The sensitivity of this type of zeolite to the electron beam is in the typical range for natural zeolites and therefore the described measuring procedures for TEM imaging of intact HEU-type zeolites are applicable to many other sensitive, natural zeolites. Using low-voltage, aberration-corrected transmission electron microscopy at low-dose conditions, the interaction of loaded natural zeolites with Ag+ and Zn2+ ions is also studied. In the case of HEUAg, the presence of Ag clusters within the HEU zeolite structure as well as larger Ag nanoparticles at the surface of HEUAg has been confirmed. The larger Ag nanoparticles are found to be preferentially positioned at the zeolite surface, while the small Ag clusters are embedded in the HEU channels (Figures 2 a,c,e). For HEUZn, no large Zn(O) nanoparticles are present, instead, the HEU channels are evidenced to be decorated by small Zn(O) clusters (Figures 2 b,d,f). Finally, in order to characterize the Zn2+ environment, we resorted to EXAFS measurements. The results demonstrate that the zinc cations in HEUZn arrange in the form of small octahedral oxo-complexes.

1. N. Döbelin, T. Armbruster, Am. Mineral. 88 (2003) 527.

2. A. Godelitsas, T. Armbruster, Microporous Mesoporous Mater. 61 (2003) 3.

GVT and MF acknowledge funding from the ERC grant N°246791 under the 7th Framework Program (FP7) COUNTATOMS. This work is also performed within the framework of the IAP-PAI.

Fig. 1: (a) HRTEM image of the HEU-type zeolite along [010]. A simulated image at Δf = -150 nm and thickness 67 nm is presented as an inset. (b) A model of the HEU-type zeolite along [010] overlaid on the magnified image simulation.

Fig. 2: HAADF-STEM images of (a) HEUAg and (b) HEUZn. 3D representation of the reconstructed volume of the (c) HEUAg and (d) HEUZn. Orthoslices through the reconstructed volume of the (e) HEUAg sample and (f) HEUZn sample, the bright regions correspond to Zn(O)clusters positions, which are located in the middle of the volume.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-2053 Twinning in the structure of zeolite ITQ-39

Kapaca E.1, Willhammar T.1, Wan W.1, Zou X.1, Moliner M.2, Gonzalez J.2, 3, Martinez C.2, Rey F.2, Corma A.2
1Inorganic and Structural Chemistry, Berzelii Centre EXSELENT on Porous Materials, Department of Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden., 2Instituto de Tecnologia Quimica (UPV-CSIC), Universidad Politecnica de Valencia, Consejo Superior de Investigaciones Cientificas, Valencia, Spain, 3Escuela de Ciencias Quimicas, Universidad de Colima, Colima, Mexico
elina.kapaca@mmk.su.se

Zeolites are aluminosilicates and have wide industrial applications in catalysis, adsorption and separation due to their large surface areas and well-defined pores with molecular dimensions. The structure of an aluminosilicate zeolite ITQ-39 has been determined by electron crystallography and is one of the most complex zeolite ever solved. ITQ-39 is an intergrowth of three different polymorphs that are built from the same layer but with different stacking sequences and the structure also contains twinning. ITQ-39 consists of a three-dimensional intersecting pairwise 12-ring and 10-ring pore system that makes it promising catalyst for converting naphtha into diesel fuel. Although the structure has been solved there is still a lot left to understand about the disorder that occurs in it. For the further studies high-resolution electron microscopy (HRTEM) images along [010] were applied and twinning was investigated using structure projection reconstruction from through-focus series (Q-Focus). In a particle consisting of several triangular shaped ITQ-39 crystals twinning perpendicular to the c*-axis was observed. Twinning was confirmed between different crystals in one particle, also in a local or large area within a crystal. In the structure twinning mainly is observed as a change of direction of pairwise 12-ring channels and there are several options how they can be rearranged (Fig. 1). Twin boundaries can occur as a single twin plane, several twin planes following each other or disordered boundaries (Fig. 2). Structure model of twinned crystal was built and applied for all three polymorphs.

The project is supported by the Swedish Research Council (VR), the Swedish Governmental Agency for Innovation Systems (VINNOVA) and the Knut & Alice Wallenberg Foundation through the project grant 3DEM-NATUR and a grant for purchasing the TEM.

Fig. 1: The rearrangement of pairwise 12-ring channels in the twinned crystal of zeolite ITQ-39 displayed in the structure projection reconstructed from through-focus series of HRTEM images along [010].

Fig. 2: The disordered twin boundary (a) and the single twin plane (b) observed in the structure projection reconstructed from through-focus series of HRTEM images along [010].
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-2449 Electron microscopy study of supercritical fluidselectrodeposition of mesoporous silica templates

Kashtiban R. J.1, Beanland R.1, Sloan J.1, Cummings C.2, Robertson, C.2, Richardson, P.2, Hector A. L.2, Smith D. C.2, Bartlett P. N.2
1University of Warwick, Coventry, UK, 2University of Southampton, Southampton, UK
r.jalilikashtiban@warwick.ac.uk

The importance and sophistication of materials deposition techniques can be seen in the cost of silicon fabrication facilities used to produce integrated circuits which are measured in billions of dollars. There now exists a wide range of deposition technologies. Vacuum techniques such as thermal evaporation, sputtering, chemical vapour deposition and molecular beam epitaxy are particularly suited to the deposition of high quality thin films. Whilst materials deposition is universal in technology, specific techniques can be found in particular applications in which their unique characteristics can surpass other techniques. For instance, as supercritical fluids have no surface tension and improved mass transport over liquids they have advantages over other deposition techniques for high aspect ratio trenches and pores [1].
Recent studies have shown that it is possible to electrodeposit a range of materials, such as Cu, Ag and Ge, from various supercritical fluids to grow nanowires. There are many reasons for the interest in nanowires for applications. These include at the simplest level the possibility of further miniaturisation of existing electronic and data storage components [2-3]. The proximity of the surface to the whole of the bulk of the nanowire makes nanowire chemical and biochemical sensors capable of single molecule sensitivity. In the field of thermoelectric materials, nanowires show increased figure of merit over bulk materials because of decreased photonic thermal
conductivity. Other routes by which nanowires could contribute to energy efficiency and generation include photovoltaic nanowire devices and piezoelectric nanowires [4-5]. The fact that nanowires are less susceptible to defects means that it may be possible to produce ultra-strength materials from them. Most of the near-to-market applications use nanowires with diameters in excess of 10nm, however initial investigations of ultrathin nanowires [6] suggest that these may be even more exciting in the medium term. In this contribution we will present results of materials recently prepared by supercritical fluid electrodeposition (SCFED) in mesoporous silica templates, with pore sizes as low as 3 nm. We use various electron microscopy techniques including high resolution transmission electron microscopy (HRTEM), high angle annular dark field scanning TEM (HAADF-STEM) and Energy Dispersive X-ray Spectrometry (EDS) in a doubly aberration-corrected electron microscope.

[1] JM Blackburn et al, Science 294 (2001) 141
[2] W Lu and CM Lieber, Nat. Mater. 6 (2007) 841
[3] R Skomski, J. Phys.: Condens. Matter 15 (2003) R841
[4] CJ Vineis et al, Adv. Mater. 22 (2010) 3970
[5] B Tian, TJ Kempa and CM Lieber, Chem. Soc. Rev. 38 (2009) 16
[6] T Zhu and J Li, Prog. Mater. Sci., 55 (2010) 710

We thank the EPSRC for funding SCFED project.

Fig. 1: TEM micrograph of 3 nm mesoporous silica pores

Fig. 2: EDS STEM elemental maps of Ge filled mesoporous silica film
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-2495 Atomic resolution analysis of beam sensitive ordered porous materials

Mayoral A.1, Anderson P. A.2, Coronas J.3, Sanchez M.4, Diaz I.4
1Laboratorio de Microscopías Avanzadas (LMA), Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, Zaragoza, Spain, 2School of Chemistry, University of Birmingham, Edgbaston, Birmingham, United Kingdom, 3Chemical and Environmental Engineering Department and Nanoscience Institute of Aragon (INA) Universidad of Zaragoza, 50018 Zaragoza (Spain), 4Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain
amayoral@unizar.es

With the recent advances in transmission electron microscopes, mainly through the implementation of spherical aberration correctors, sub-angstrom resolution is becoming easily achievable overcoming the challenge of lateral resolution (especially for higher voltages). Our next efforts therefore, effort should now be devoted into optimizing the different modes of observations, improvement of detectors, and in-situ experiments among many others. One of the main challenges from the electron microscopy and materials science point of view is the observation of beam sensitive materials. For the cases of knock-on damage, lowering the accelerating voltage is the solution (as happens for carbon based materials). On the other hand if radiolysis causes the bond disruption there is not a straightforward approach, and thus dose as well as voltage has to be taken into account. Zeolites and zeotypes suffer from this problem and the final image resolution has always suffered from this effect. In this work, we present the data that can be acquired by using Cs-corrected STEM under various conditions. Many zeolites have been observed including the most beam sensitive one, LTA type (Si/Al = 1), figure 1a, in its bare form and loaded with silver ions, whose structure has been analyzed, figure 1b[1, 2]. Due to the benefits of ADF detectors, which provide direct observations of heavier elements, metals contained in zeolites are more easily identified, but also, structural defects can be studied more clearly through this method, as in the case of the titanosilicate ETS-10[3], figure 2.
The results presented here, prove the effectiveness of this method in acquiring true atomic-column resolution images which will provide new information on guest species within the zeolite cavities as well as observing the structural defects with unprecedented resolution. In addition, metal organic framework (MOF) materials, which have also been analysed, will be presented with virtual atomic resolution of the metals allowing for structure identification in direct space.

References

[1] A. Mayoral, T. Carey, P. A. Anderson, A. Lubk, I. Diaz, Angew. Chem. Int. Ed. 50 (2011) 11230–11233.
[2] A. Mayoral, T. Carey, P. A. Anderson, I. Diaz, Micropor. Mesopor. Mater. 166 (2013) 117-122.
[3] A. Mayoral, J. Coronas, C. Casado, C. Tellez, I. Díaz, Chem. Cat. Chem. 5 (2013) 2595–2598.

The authors would like to acknowledge the ESTEEM2 and Talem program for funding. MSS and ID acknowledge funding from MINECO (MAT2012-31127 project).

Fig. 1: Cs-corrected STEM HAADF images of a) as-synthesized Na zeolite A along the [001] with the simulated image inset. b) dehydrated Ag zeolite A with a ball and stick model inset, where silver appears in grey, oxygen in red and silicon and aluminum in dark and light blue.

Fig. 2: Cs-corrected STEM HAADF analysis of ETS-10. a) Atomic resolution image along the [110] zone axis. b) Lower magnification image along the same orientation where some defects are marked by circles. c) GPA analysis rotational map of b), where the reference area is marked by a dashed rectangle.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-2534 3D Multiscale characterization of silica aerogel composites

PERRET A.1,2, FORAY G.1, ROIBAN L.1, MASENELLI-VARLOT K.1, MAIRE E.1, ADRIEN J.1, YRIEIX B.2
1Université de Lyon, INSA-Lyon, UCBL, CNRS, MATEIS - 69621 VILLEURBANNE Cedex, France, 2EDF R&D MMC Department, Avenue des Renardières - Ecuelles, 77818 MORET-SUR-LOING cedex, France
anouk.perret@insa-lyon.fr

The reduction of energy wastage has become a global concern together with the development of green energies and the increase of energy efficiency. In France, the national objectives on the reduction of the rejections of greenhouse gases bring to the necessity of a thermal renovation for 75 % of the buildings. As the requirements for old and new buildings increase their standards, design thinner and more efficient insulation materials is of great and increasing interest. New insulating materials with thermal conductivities lower than still dry air (25 mW/(m.K)), such as based silica aerogel products (15 mW/(m.K)), recently developed [1], are an interesting choice to answer those new functionalities.
In our study, silica aerogels (porosity > 80%, specific surface > 600 m²/g) are available as granular materials and bound stiff composite boards (aerogels / latex) [2]. The optimization of these materials requires understanding the link between their microstructure, their thermal conductivity and their mechanical behavior. On the one hand, the abundance of mesopores (50 nm) guarantees the low thermal conductivity of the final composite whereas the total porosity (> 90 %) can lead to a low mechanical resistance of the aerogel. On the other hand, the binder used to design and shape the composite leads to a stronger mechanical behaviour (cohesion of the grains of silica aerogels between them). However, the binder has a tremendous conductivity and it will induce an unwanted increase of the overall thermal conductivity even at low volume fractions. An optimization of the proportions and the respective morphologies of both phases must thus be proposed to reach simultaneously the targeted thermal and mechanical properties.

The objective of the presentation is to propose a three-dimensional characterization from the nanometer scale to the millimeter scale of the composite aerogel / latex board by coupling various microscopy techniques. The mesoporous network within an aerogel grain will be studied by Electron Tomography in a TEM (figures 1 and 2). The grain pile-up will be investigated at the micron scale by X-ray tomography (figure 3). As far as the composites are concerned, the formation of a binder network will be followed by wet-STEM in ESEM and the final microstructure will be characterized in 3D using the previously mentioned techniques.

1. A. Bisson , A.Rigacci., D.Lecomte, P.Achard, Effective thermal conductivity of divided silica xerogel beds. Non crystalline solids, 2004. 350: p. 379-384.
2. B.Yrieix , B.Morel., G.Foray , A.Bogner, Patent FR 2975691 WO2012168617 : Matériau super-isolant à pression atmosphérique à base d'aérogel.

The authors acknowledge ADEME and METSA for financial support, Prof. Ovidiu ERSEN for fruitful discussions.

Fig. 1:  Silica particle: Projection extracted from the TEM tilt series at 0°.

Fig. 2: Silica particle: 3D model of one silica aerogel particle derived from electron tomography experiments.

Fig. 3: X-Ray tomographic slice showing the aerogel grain pile-up. Aerogel grain size D50 = 800µm, resolution = 15µm image size=15 mm*15 mm.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-2540 Structural analysis of Cu nanowires deposited into porous Al2O3 via supercritical fluid electrodeposition

Marks S.1, Kashtiban R.1, Smith D.2, Cook D.2, Cummings C.2, Beanland R.1, Sloan J.1
1University of Warwick, Coventry, United Kingdom, 2University of Southampton, Southampton, United Kingdom
s.r.marks@warwick.ac.uk

Aqueous electrodeposition has been used to successfully synthesise nanowires inside porous substrates for nearly a decade[1]. Porous Anodic Aluminium Oxide (AAO) is a good substrate chosen for its high pore density, uniform pore distribution and controllable pore size. This nanowire growth technique has allowed for investigation of a wide range of nanowire diameters, 15 – 150nm[2]. But aqueous electrodeposition is limited as it becomes unstable at the lower range of pore diameters, <15nm. We are developing a new electrodeposition technique using supercritical fluids[3], a phase of matter at a temperature and pressure above its critical point that incorporates a combination of liquid and gas properties. In the supercritical fluid phase a substance has no surface tension therefore allowing it to fill small pores and allowing for electrodeposition.
This technique has been used to create Cu nanowires with a diameter of 15nm inside a porous AAO substrate. Cross sections of the sample were prepared using a JEOL 4500 FIB/SEM with a cross section lift out technique and planview samples via conventional hand polishing and ion polishing under liquid nitrogen cooling using a Gatan Precision Ion Polishing System (PIPS). These were then imaged using a JEOL 2000 TEM, JEOL 2100 TEM/STEM and a JEOL ARM200F TEM/STEM.
Cross section samples preparation via the FIB rendered a different image of the sample showing scattered discontinuous copper wires as shown in Figure 1a. This is a strange result suggesting that the samples may be naturally forming this way or may be heat sensitive with the Cu nanowires being affected by heating during the FIB milling periods. All planview samples were created using heat free methods and liquid nitrogen cooling during PIPS.
Analysis of the planview samples using EDS confirmed Cu nanowires had formed inside AAO templates. TEM imaging showed a good filling rate within samples of 85%, with an average Cu diameter of 14nm, it also highlighted deformations among the filled pores with some pores combining and a range of nanowire circularities from elliptical to circular (Figure 1b). The planview analysis also highlighted a structural defect that can form within the AAO creating pockets of channels running through the sample perpendicular to the direction of growth (Figure 2a). Atomic resolution imaging of the wires showed that the wires have a [101] preferred orientation (Figure 2b).
We will present analysis of Cu nanowires prepared by SCFED and compare them with SCFED Te nanowires.

[1] D Routkevitch et al Journal Of Physical Chemistry 100 (1996) 14037 – 14047.
[2] X Lin et al Solid State Communication 151 (2011) 1708-1711.
[3] P Bartlett et al ChemElectroChem 1 (2014) 187-194.

The EPSRC for funding my PhD.

Fig. 1: Figure 1. a) Cross section of FIB produced sample, crystalline deformed non uniform wires identified by diffraction pattern. b) Low magnification planview image, high rate of filling easily observable along with a range of different pore sizes.

Fig. 2: Figure 2. a) Planview image of AAO structural defect, instead of growing from bottom up they are forming perpendicularly to the growth direction. b) Atomic resolution imaging of a Cu nanowire with the (101) orientation clearly visible.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-2564 Low Dose Microscopy of Porous Prussian Blue Analogue Materials for Hydrogen Storage

Calderon H. A.1, Huerta A.1, Reguera E.2, Kisielowski C.3
1Departamento de Física, ESFM-IPN, Mexico DF, 2CICATA LEGARIA, IPN, Mexico DF, 3JCAP-NCEM, LBNL Berkeley Ca, USA
hcalder@esfm.ipn.mx

Prussian blue analogues (PBA) form a family of materials that are used as prototype of porous solids for Hydrogen storage. Their porous framework is related to systematically arranged vacancies in the building block, [Mn(CN)6]n-6. The accepted structural model for these materials (cubic ) assumes a random vacancy distribution, which results in a 33 % of framework free volume. Nevertheless recent spectroscopic and structural evidences show that such a model is a limiting case. Vacancies remain ordered or at least partially ordered and thus another structural model needs to be considered i.e, a model (also cubic). In such a model, the accessible framework volume reaches 50 %. For copper PBA conclusive clue on an ordered vacancies system has been obtained and the hydrogen storage capacity for this material is the highest within the series [1]. High-resolution electron microscopy (HREM) appears as an excellent tool to shed light on the vacancy distribution in PBA and related solids. The TEAM 05 microscope is used at 80 KV. Focal series are used to apply the exit wave reconstruction (EWR) procedure and obtain phase and amplitude images from low dose experimental images. This is done in order to minimize beam sample interaction. As reference material, the rhombohedral phase of the Zn analogue is used where the porous framework is not related to the existence of vacant sites.. Figure 1 shows a nanoparticle of Cu containing PBA material. The particle has different crystalline regions that are enlarged in Fig. 1b-c together with some FFT spectra. The dose rate in use is 200 e-/Å2s. Ordered vacancies are very apparent in this structure (see arrows). The basic cubic structure can be also seen according to the different projections in the powder particle. For instance, Figs. 1b-c show a projection along [011] although each blob is formed by a number of intensity maxima. This is most likely the center of charge in the structure formed by light atoms (C and N, mainly). Fig. 1c shows a grain boundary and also details of the center of charge in the structure. Fig. 2a shows a view of the Zn containing PBA material at a dose rate of 420 e-/Å2s. Figs. 2 b-d show phase images of the same area but with a reduced dose of 115 e-/Å2s. These phase images show details of the atomic arrangement and the center of charge as well. In addition they suggest rather small cavities or arrangements of vacancies in a disordered manner. Starting from the nanopore apparently two layers with different Z position overlap producing a slight displacement in X,Y directions and give rise to the image when the projection is considered. The obtained results are conclusive on the presence of a system of ordered vacancies for copper PBA.

HAC acknowledges support from IPN (COFAA-SIP), CONACYT (FOINST. 75/2012), ICyT and LBNL. NCEM- LBNL is gratefully acknowledged for the use of the TEAM 05 microscope.

Fig. 1: (a) Cu rich PBA material powder nanoparticle imaged with an electron dose rate of 200 e-/Å2s. (b-c) enlargement of selected areas in (a) and corresponding FFTs.

Fig. 2: (a) Zn containing PBA material at a dose rate of 420 e-/Å2s. (b,c) Phase images (40 image focal series at a dose rate of 115 e-/Å2s) after EWR around a nanopore in the Zn containing PBA material and corresponding FFTs.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-2693 Investigation of the bonding behavior and chemical stability of silica-based nanotubes and their 3D mesocrystals

Dennenwaldt T.1,2, Sedlmaier S. J.1,3, Binek A.1, Schnick W.1, Scheu C.1,2
1Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 Munich, Germany, 2New address: Max Planck Institute for Iron Research, Max-Planck-Str. 1, 40237 Düsseldorf, Germany, 3New address: Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford OX1 3QR, UK
t.dennenwaldt@mpie.de

Recently, we reported a template-free synthesis for amorphous silica-based nanotubes (SBNTs) through a solid-state chemistry approach.1 The SBNTs are amorphous in terms of diffraction and assemble to ordered 3D hyperbranched mesocrystals (Fig. 1). In the present study, the bonding behavior and the elemental distribution of these SBNTs were investigated at the nanoscale using energy dispersive X-ray diffraction (EDX) and electron energy loss spectroscopy (EELS) in a transmission electron microscope (TEM). The analysis of the walls and the interior of the SBNTs revealed chemical homogeneity along the SBNTs. A comparison of energy-loss near-edge structures (ELNES) of the Si-L2,3-edges of the SBNTs with the two amorphous bulk reference materials SiO2 and Si3N4 showed that the bonding situation of Si in the SBNTs is a mixture of the one in SiO2 and Si3N4 exhibiting a tetrahedral coordination with mostly O and N as bonding atoms (Fig. 2). By analyzing the low loss region of the EEL spectra the band gap energy was determined at 5.7 ± 0.2 eV and the plasmon maximum at 23 ± 0.2 eV indicating that the electronic structure of the SBNTs is indeed dominated by a mixture between SiO2 and Si3N4. In line with these results the SBNTs show a higher stability to extreme pH conditions than pure amorphous SiO2 nanotubes2 which is attributed to the incorporation of nitrogen as well as phosphorus. A correlation between nitrogen incorporation and enhancement of the basicity is known from the literature.3 The increased chemical stability of SBNTs is promising for potential applications in e.g. nanofluidic systems.

1 Sedlmaier, S. J.; Dennenwaldt, T.; Scheu, C.; Schnick, W. Template-Free Inorganic Synthesis of Silica-Based Nanotubes and Their Self-Assembly to Mesocrystals. J. Mater. Chem. 2012, 22, 15511.
2 Hu, K.-W.; Hsu, K.-C.; Yeh, C.-S. pH-Dependent Biodegradable Silica Nanotubes Derived from Gd(OH)3 Nanorods and Their Potential for Oral Drug Delivery and MR Imaging. Biomaterials 2010, 31, 6843.
3 Wang, J.; Liu, Q. Structural Change and Characterization in Nitrogen-Incorporated SBA15 Oxynitride Mesoporous Materials via Different Thermal History. Microporous Mesoporous Mater. 2005, 83, 225.

We kindly acknowledge C. Minke (Department of Chemistry, LMU) for the SEM analysis.

Fig. 1: Fig. 1. Scanning electron microscopy (SEM) images of SBNTs showing a) a 3D assembly and b) a zoom-in view of a network.

Fig. 2: Fig. 2. Comparison of the ELNES of the amorphous bulk compounds Si3N4 (blue), SiO2 (red) and of an individual SBNT (green). a) Si-L1,2,3- edges, b) N-K-edges and c) O-K-edges.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-3042 Amorphous Silicon-coatings with high amount of closed porosity

Schierholz R.1, 2, Lacroix B.1, Caballero-Hernández J.1, Godinho V.1, Duchamp M.3, Fernández A.1
1Instituto de Ciencia de Materiales de Sevilla, Calle Américo Vespucio 49, 41092 Sevilla, Spain, 2Institute of Energy and Climate Research: Fundamental Electrochemistry (IEK-9), Forschungszentrum Jülich, Ostring O 10, 52425 Jülich, Germany, 3Ernst Ruska-Centre for microscopy and Spectroscopy with Electrons, Forschungszentrum Jülich, Nordring N9, 52425 Jülich, Germany
r.schierholz@fz-juelich.de

Porous silicon coatings with reduced refractive index could are of interest as anti reflective coatings for photovoltaics. We present coatings of amorphous silicon coatings with a high amount of porosity deposited by glancing angle magnetron deposition under Helium atmosphere [1]. The sputtering parameters provide control on the porosity. To get an estimate of the Helium pressure inside the pores and correlate this to the deposition parameters we performed (scanning) transmission electron microscopy ((S)TEM) and electron energy loss spectroscopy and do the analysis of STEM spectrum images with our own MATLAB code to compare different methods for the Helium density extraction from EEL-spectra. One method refers to the blue-shift [2] of the 1s to 2p transition from its free atom value at 21.218 eV [3].

n(He) = C*ΔE+D

This method was applied by several authors and a wide range of values for the factor C were obtained also some authors add a constant D so we choose two extreme values C = 0.015 eV nm³ [4] and C = 0.015 eV nm³ 0.044 eVnm³ [5]. For this reason the second method based on the intensity of the EELS-signal

n(He) = I(He)/(I(ZLP)*σ(He)*d)

is used for cross checking. This method requires a background substrations since the signal is sitting on top of the plasmon peak [6]. Again some uncertainties are introduced by the tabulated values for the cross-section σ and the pore diameter d measured from the image. The obtained microscopic values are compared to results from proton backscattering and ellipsometry. As a reference sample for those macroscopic measurements a “dense” coating deposited under Argon atmosphere is chosen.

References

[1] V. Godinho et al. Nanotechnology, 24 (2013), 275604.

[2] W. Jäger et al. Radiation Effects, 78 (1983), 315-325

[3]H. G. Kuhn Atomic Spectra, London: Longmans (1962), p.132

[4] M.-L. David et al. Applied Physics Letters, 98, (2011), 171903

[5] D. Taverna et al. Phys. Rev. Lett., 100 (2008) 035301

[6] C. A. Walsh et al. Philosophical Magazine A, 80 (2000) 1507

This work was supported by the EU 7FP (project Al-NanoFunc CT-REGPOT-2011-1-285895), the CSIC (project 201060E102), the Spanish Ministry MINECO (projects CSD2008–00023 and CTQ2012-32519) and Junta de Andalucía (TEP217 and PE2012-TEP862). The authors also acknowledge the collaboration with the Ernst Ruska-Centre of the Forschungszentrum Jülich within the ER-C proposal A-084.

Fig. 1: TEM-image of the “dense” coating deposited under Ar- atmosphere.

Fig. 2: TEM-image of the porous coating deposited under He-atmosphere.

Fig. 3: Deconvoluted EEL-spectra of the coatings deposited under Ar-atmosphere and He-atmosphere. For the latter two spectra, one for the matrix and one at a pore center, are displayed.The Helium signal at about 22 eV can be recognized for the pore position.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-3184 Microstructural characterization of artificially aged mortars with different additives using mercury intrusion porosimetry and scanning electron microscopy

Petráňová V.1, Nunes C. L.1, Niedoba K.1, Křivánková D.1, Valach J.1
1Institute of Theoretical and Applied Mechanics AS CR, v.v.i., Prague, Czech Republic
petranova@itam.cas.cz

Abstract

Scanning electron microscopy (SEM) and mercury intrusion porosimetry (MIP) are two suitable methods to study macroscopic properties of materials at the microstructural level. MIP is a common method for the quantification of the total volume of open pores and distribution of pore sizes. Additional structural and chemical information can be obtained by combining MIP results and SEM imaging coupled with energy-dispersive X-ray detector (EDX) [1]. A set various mortar types (L - lime-, LO - lime with oil-, LM - lime with metakaolin- and LMO - lime with metakaolin and oil mortar) prepared 6 months prior to testing was selected for the investigation. The set was divided into two groups. The first group of specimens was immersed in 3 wt % NaCl solution at room temperature for 8 hours and the second one was immersed only in deionized water to provide reference data for comparison. After 15 immersion cycles the specimens were desalinated and dried. Thin sections of the reference, salt and water aged specimens were then investigated using MIP and SEM. The data from MIP show increasing amount of micropores between sizes 0.05 and 0.1 µm in the LM salt aged mortar. Similar effect was observed in the L water aged mortar with increasing amount of micropores within the range 0.1 and 0.3 µm. However, the data obtained for the other mortars with oil (LO and LMO) show decreasing volume of micropores between sizes 0.02 and 0.1 µm. Microscopic analysis revealed that the most fractured sample was the L mortar because of shrinkage during drying while the remaining mortars (LO, LM and LMO) were mostly undamaged. EPMA (electron probe microanalysis) enabled the determination of the CaO content in the carbonated and less carbonated areas in the original not-aged mortars. The density of the binder on less carbonated zones was lower than in carbonated areas as well as the content of CaO which decreases in 5.4 wt % in L, 7.8 wt % in LO and 13 wt % in LM mortar. LMO specimens didn’t show any interfaces between carbonated and non-carbonated areas, therefore it wasn’t possible to establish differences in the CaO content. Subsequently, changes on the amount of CaO were measured on the unaged and aged specimens. Water and salt ageing tests decreased the CaO content of the LMO mortar and in the less carbonated areas of the L mortar. The investigation using MIP, SEM and EPMA methods showed that the mortars were structurally influenced only at the nanoscale level. From the chemical point of view, the most resistant mortar types to ageing were the LO and LM mortars which showed the same content of CaO as in the not aged specimens.

References

[1] Stefanidou M. Methods for porosity measurement in lime-based mortars. Const Buil Mat 24 (2010) 2572-2578.

The research has been supported by Czech science foundation (project no. P105/12/G059) and by RVO: 68378297.

Fig. 1: Carbonated binder in the LM mortar.

Fig. 2: Less carbonated binder in the LM mortar.

Fig. 3: The interface between less carbonated and carbonated areas in the L mortar.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-3213 Electron microscopy study of metal-dielectric nanostructures based on porous silicon applied as Surface-Enhanced Raman Scattering substrates.

Bejtka K.1, Virga A.2, Giorgis F.1,2, Rivolo P.2, Descrovi E.2, Angelini A.2, Novara C.2, Ricci A.1, Geobaldo F.2, Pirri C. F.1,2, Chiodoni A.1
1Istituto Italiano di Tecnologia, Center for Space Human Robotics, Torino, ITALY, 2Politecnico di Torino, Department of Applied Science and Technology, Torino, ITALY
katarzyna.bejtka@iit.it

Surface-enhanced Raman scattering (SERS) is a sensitive technique allowing vibrational spectra from individual molecules to be measured. It is a high-sensitive label-free detection method in materials science, biophysics, medical diagnostics, and molecular biology. Clusters of silver and gold nanoparticles (NPs), generally used in colloidal solution, represent the most efficient types of SERS-active substrates exhibiting the largest enhancement effects. In such systems, the Raman efficiency is strictly correlated to the spatial arrangement of the nanoparticle clusters, characterized by “hot-spots” where the collocation of nanoparticles is optimized in dimer/trimer assemblies.
The present work presents the morphological and structural characteristics of complex metal-dielectric nanostructures consisting of Ag nanoparticles (Ag NPs) synthesized within a mesoporous silicon matrix [1,2]. The deep understanding of the morphology and distribution of the nanoparticles is necessary to further interpret the contribution to plasmonic resonances in the visible-near-infrared range.
The specimens were characterized by Field Emission Scanning Electron Microscopy (FESEM), Transmission Electron Microscopy (TEM), HRTEM, tomography, STEM and EDX. The cross-section lamellas for TEM investigation were prepared by Focused Ion Beam (FIB), using the sample as prepared, without any embedding in polymeric matrix.
Ag NPs have been synthesized through the impregnation of mesoporous Si in AgNO3 aqueous solution. The synthesis kinetics allows the growth of particles with size within and/or beyond the pore diameter, and their density and average size are strictly dependent on the redox-synthesis parameters. The morphology of the samples was initially characterized by FESEM: the size distribution and the density of the Ag NPs were evaluated (shown in Figure 1).
The morphology of the structure in cross-section was further investigated by TEM, revealing that crystals of various sizes and distribution in terms of distances and penetration are formed within the pores of the mesoporous silicon matrix. Figure 2 shows an example were the penetration depth was of about 400nm. The reconstruction by TEM tomography provides further insight on the distribution of nanoparticles in 3D.
The correlation between the nanostructure morphology analyzed by SEM and TEM microscopies and the optical response of the SERS substrates will be shown.

Authors V.A., Gi.F., R.P., D.E., A.A., N.C. and Go.F. acknowledge the funding from the Italian Flagship Project NANOMAX and the Italian FIRB 2011 NEWTON.

Fig. 1: FESEM images of Ag NPs obtained by immersion of p-Si in AgNO3 solution with different temperature/dipping time/salt concentration.

Fig. 2: FIB lamella of p-Si filled with the Ag NPs. (a) Bright Filed STEM, (b) HAADF STEM.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-3437 Advanced Electron Microscopy to Characterize the Hierarchical Three-Dimensional Morphology of Porous Solids

Stoeckel D.1,2, Kübel C.3,4, Chakravadhanula K. V.3,4, Hormann K.2, Prang R.3, Scherer T.3,4, Smarsly B. M.1, Tallarek U.2
1Justus-Liebig-University, Giessen, Germany, 2Philipps-University, Marburg, Germany, 3Karlsruhe Institute of Technology (KIT), Institute of Nanotechnology, Karlsruhe, Germany, 4Karlsruhe Institute of Technology (KIT), Karlsruhe Nano Micro Facility, Karlsruhe, Germany
cvskiran@kit.edu

Hierarchical porous silica in monolithic form is used in chromatography and heterogeneous catalysis as an alternative to other fixed bed structures. Their distinctly bimodal pore size distribution is essential to the performance of silica monoliths as solid support. A continuous network of macropores (~2 µm) enables liquid transport at high flow rates without necessitating high pressures, while the nanometer-sized mesopores provide the high surface area for sufficient contact between chemicals and the stationary phase (for separation or catalysis) immobilized on the fixed bed.

Beyond a superficial characterization of the two pore categories, the monolith’s disordered pore structure remained largely unknown until recently, when the macropore space has been accessed by confocal laser scanning microscopy [1]. For the much smaller mesopores nothing beyond pore size data form bulk methods, e.g. mercury intrusion porosimetry and nitrogen physisorption is available at present. However, an accurate and quantitative morphological characterization would be needed to tailor the morphological properties of mesoporous adsorbents for their intended use.

Here we demonstrate the reconstruction and characterization of the complete morphology of a silica monolith, from macropores to mesopores, by combining FIB slice&view techniques and high-resolution STEM tomography. Based on the three-dimensional reconstruction we performed a comprehensive statistical analysis to extract key structural parameters relevant to mass transport at the macropore and mesopore level. The reconstructed model is also the starting point for simulations of flow, mass transport [2], sorption and reaction which aim at a fundamental understanding of the morphology-transport relationships of hierarchically structured, disordered materials as a basis to improve morphological features responsible for separation efficiency and catalytic activity [3].

References

[1] S. Bruns, T. Hara, B. M. Smarsly, U. Tallarek, J. Chromatogr. A 2011, 1218, 5187-5194.

[2] D. Hlushkou, S. Bruns, A. Seidel-Morgenstern, U. Tallarek, J. Sep. Sci. 2011, 34, 2026-2037.

[3] D. Hlushkou, K. Hormann, A. Höltzel, S. Khirevich, Seidel-Morgenstern, A.; Tallarek, U.; J. Chromatogr. A 2013, 1303: 28-28.

Fig. 1: Characterization of hierarchical structure combing FIB-SEM and HAADF-STEM tomographic techniques to build a 3D model
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-3453 Morphological and chemical characterization of sandstone from Constantine, North-East of Algeria.

Benguedouar M.1, Bouchear M.1, Benabbas C.2,3
1Materials Sciences and Applications Research Unit, Department of Physics, Faculty of Exact Sciences, Constantine 1 University, Algeria, 2Geology and Environment Laboratory, Department of Earth Sciences, Faculty of Earth Sciences, of geology and Planning, FSTGAT, Constantine 1 University, Algeria, 3Management Institute and Urban Technology, Constantine 3 University, Algeria
mouniabenyamina@gmail.com

The north-east area of Constantine has a very complex geological site.
The variety of sedimentary rocks such as sandstone in abundance represents a big importance in the industry and road infrastructure.
Aggregates are the major constituents of concrete and typically occupy a large proportion of its volume. The concrete’s properties are mainly influenced by the quality of the aggregates.
Sandstone is a widespread aggregate resource and it is increasingly being used in concrete construction around the world.
The geological properties of this sedimentary rock are fairly diverse such as quartzite aggregate that may produce a range of hardened concrete properties. Therefore, it is important to study and characterize the aggregate to obtain predictable concrete properties.
The Environmental Scanning Electron (ESEM/EDS) and Optical Microscopy are to study the morphological aspect of the existing phases. X- Ray Diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FT-IR) analysis of sandstone are required to investigate the structural properties of them.
The SEM morphology coupled with EDS analysis shows what has been observed by previous techniques, confirms the existence of elements proportions.
The electron microscopy observations show that the extracted samples (Fig. 1) have morphology with angular grains of quartz with an average size from 10 to 30 μm. One can observe that these grains are covered with fine particles of calcite.
The optical observations reveal the presence of different oxides and the intergranular phyllosilicates such as: the Montmorillonite (Na, Ca)0,3(Al, Mg)2Si4O10 or the Kaolinite ( Al2Si2O5 ) as minor elements.
The XRD spectra reveal the present phases, that confirmed by using the Infrared spectroscopy (Fig.2) through their vibrational modes.
The combination of these advanced techniques, which were not designed with the purpose of answering geological or environmental questions, can generate complementary of geological materials and opening up new approaches in the study of porous geomaterials.
All authors thank, the Process Engineering Laboratory, University of Bejaia and Birine Nuclear Center, Ain Ouassera, Algeria for their collaboration.
 

All authors thank, the Process Engineering Laboratory, University of Bejaia and Birine Nuclear Center, Ain Ouassera, Algeria for their collaboration.
 

Fig. 1: SEM morphology of sandstone (X 1600): angular grains of quartz size around: 10 to 30 [μm], covered with fine particles and the grains of calcite.

Fig. 2: We can note that the Sandstone are mainly composed of quartz (1429, 1082, 778, 694, 461 cm-1). Low Intensity peaks can be seen 3500 and 4000 cm-1 corresponding to oxygen-hydrogen bonds HOH, which indicate the presence of clay type compounds.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-3515 Multidomain analysis of porous silica media

Thersleff T.1, Sjöström C.2, Norberg P.2, Magnusson J.2, Larsson A.3, Leifer K.1
1Department of Engineering Sciences, Uppsala University, Box 534, 75121 Uppsala, Sweden, 2Svenska Aerogel AB, Strömmavägen 2, 80309 Gävle, Sweden, 3SP Technical Research Institute of Sweden, Box 5607, SE-114 86 Stockholm, Sweden
thth@angstrom.uu.se

Porous silica, also known as Aerogel, is an exciting material due to its light weight, molecular filtration properties, and low thermal conductivity. These three characteristics make Aerogel materials particularly well-suited for use in vacuum insulated paneling for interior construction purposes. Exploiting these properties however requires a comprehensive understanding of the porous network spanning multiple spatial domains ranging from the sub-millimeter to nanometer. One technique capable of probing all of these spatial domains simultaneously is electron microscopy; however, due to the electrically insulating nature of Aerogel powders, this has traditionally been a challenging technique to apply.
In this work, we employ ultra-low dose Scanning Electron Micrsocopy (SEM) imaging techniques and Focused Ion Beam (FIB) cross-sections to extract morphological information from Aerogel powders under compression from multiple spatial domains. The low-dose SEM was performed on a Merlin field emission SEM from Carl Zeiss company operated at acceleration voltages down to 100 V and currents of down to 100 pA. FIB cross-sections were prepared on a Strata DB235 FIB from FEI company and viewed in backscattered electron mode at low voltage.
Figure 1 shows a low-dose SEM image from nanometer-sized Aerogel agglomerates acquired using an acceleration voltage of 100 V and current of 100 pA. Such images enable the visualization of the agglomerate surfaces of with unprecedented spatial resolution and allow us to probe their surface morphology. Figure 2 shows a FIB cross-section on a larger Aerogel agglomerate. The mesoporous structure is revealed in this manner and statistics can be applied to the pore sizes and shapes. An ex-situ compression series was performed and the effect of compression on the mesoporous metrics are quantified and interpreted within the framework of thermal transport in porous silica media.

Fig. 1: Figure 1 - High resolution SEM image of nanometer-sized Aerogel agglomerates acquired with an acceleration voltage of 100 V and current of 100 pA. False color is used to enhance contrast.

Fig. 2: Figure 2 - FIB cross-section of an Aerogel agglomerate revealing its mesoporous structure.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-3519 New Analytical Developments for powder characterization

Brackx E.1, Pages S.1, Dugne O.1, Tissot N.1, Cabie M.2, Podor R.3, Lahaye M.4, Remy E.5
1CEA, DEN, DTEC, SGCS, LMAC, 30207 Bagnols sur Cèze, France , 2CP2M Université d’Aix Marseille III, Faculté des Sciences de Saint Jérôme, 13397 Marseille, France , 3ICSM UMR 5257 – CEA / CNRS / UM2 / ENSCM, 30207 Bagnols sur Cèze, France , 4Placamat, Université de Bordeaux 1, 33608 Pessac CEDEX, France, 5CEA, DEN, DRCP, SECA, LCAR, 30207 Bagnols sur Cèze
emmanuelle.brackx@cea.fr

Powders and divided solids are widely used in industry as intermediate or finished products in many fields: foods, cosmetics, construction, pharmaceuticals, transport, electronics and, of course, nuclear energy. Optimizing their use requires control of processing based on an understanding of the phenomena involved (sintering, chemical reactivity, purity, etc.). Modeling and understanding these phenomena require data and characteristics that may be difficult to obtain.
The present work cites examples illustrating different physicochemical characterization techniques suitable for analysis of uranium based powder samples (oxide, carbide, fluoride and metallic) in the raw state or after preparation (ion polishing) used in nuclear cycle.
The first section discusses dimensional characterization (particle size, morphology, porosity) by means of image analysis and preparation techniques: SEM, TEM, FIB, electron and X ray tomography with possible 2D and 3D image analysis. Morphological data in the form of the powder size and shape can be obtained with new developments in 2D analysis of processed SEM images. Among the imaging techniques only image analysis by SEM, FIB tomography (Figure 1), and MRI are considered to be porosimetry techniques covering a wide range of pore sizes. FIB tomography also provides data on the powder specific surface area.
The second part of this work describes elemental chemical characterization. Low-voltage EDS spectroscopy on polished powder cross sections is used to map (Figure 2) the distribution of phases and to detect possible impurities in the solid grains and agglomerates. The contribution of this type characterization to understanding the reaction mechanisms of phase transformations (especially solid-gas reactions) is illustrated for the hydrofluorination of uranium dioxide. Sample preparation by mounting followed by ion polishing, optimization of the EDS analysis parameters, and the contribution to solid-gas reaction models are discussed in this study. Complementary surface analyses as Auger spectrometry allows powder surface oxidation.
The third section describes the characterization of the chemical reactivity of powders. These studies are performed by high-temperature in situ treatment in an environmental SEM, and allow observation of the powder morphology transformation.
The examples discussed concern materials used in nuclear fuel fabrication processes.

Fig. 1: Illustration of the potential of FIB-SEM tomography for the characterization of porous media. Visualization of closed pores labeled in different colors (a), Pore size distribution determined after skeletonization of porous matrix.

Fig. 2: X-ray EDS mapping of UO2 powder partially transformed into UF4. Red and green phase represents respectively Oxygen and Fluor.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-3525 A novel structure of aluminium phosphate zeolite sovled by RED (Rotation Electron Diffraction)

Peng F.1, Chen H.1, Sun J.1
1Inorganic and Structural Chemistry and Berzelii Centre EXSELENT on Porous Materials, Department of Materials and Environmental Chemistry, Stockholm University, Arrhenius Laboratory, Stockholm SE-106 91, Sweden.
fei.peng@mmk.su.se

Keywords: aluminium phosphate zeolite, seven-member ring, electron diffraction

Normally, X-ray diffraction (XRD) has been used to get the structure of the crystalline materials for a long time. Compared to XRD, electron diffraction has two advantages. The size of the crystals can be as smaller as 5nm*5nm*5nm and the phase information can be obtained from high-resolution transmission electron microscopy (HRTEM) images [1]. The rotation electron diffraction (RED) method [2] makes the structure determination by electron crystallography more convenient and effective.
Aluminophosphate (APO) molecular sieves were synthesised by the U.S. company (Union Carbide) in 1980s [3]. Here, we report a new structure, an aluminium phosphate zeolite with special seven-member ring. For this new material, the crystal is not big enough to be characterized by traditional methods such as single X-Ray diffraction. The RED (Rotation Electron Diffraction) method has been used here to determine the structure of this aluminium phosphate zeolite. The data of RED has been shown in Figure 1. The structure of this APO zeolite has been solved and been shown in Figure 2.

[1]Willhammar, T., Yun, Y. & Zou, X. Structural Determination of Ordered Porous Solids by Electron Crystallography. Adv. Funct. Mater. 24, 182–199 (2014).
[2]Wan, W., Sun, J., Su, J., Hovmöller, S. & Zou, X. Three-dimensional rotation electron diffraction: software RED for automated data collection and data processing. J. Appl. Cryst. 46, 1863–1873 (2013).
[3]Wilson S., Lok B. USP: 4310440, 1982.

Fig. 1: Figure 1 a) Overview of the diffraction data collected by RED. b) Along a* axis in reciprocal space. c) Along b* axis in reciprocal space. d) Along c* axis in reciprocal space. (Reflection condition: h00, h=2n; 0k0, k=2n; h0l, h=2n)

Fig. 2: Figure 2 The structure solved by using Shelx. a) Along a axis; b) Along b axis; c) Along c axis; (Unit cell: a = 8.257 Å, b = 9.910 Å, c= 7.605 Å, α= 90.02°, β=106.87°, γ = 90.01° Space group: P21/a)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-5746 Formation and characterization of the epoxy resin - porous glass interphases

Ostrowski A.1, Byrne H. J.2, Sanctuary R.1
1Laboratory for the Physics of Advanced Materials, University of Luxembourg, Luxembourg, 2FOCAS Institute, Dublin Institute of Technology, Dublin, Ireland
aleksander.ostrowski@uni.lu

Investigation of the polymer interphases is an emerging field nowadays. In many cases interphases determine the functionality of a system. There is a great demand for exploration of fundamental understanding of the interphases and elucidation of their formation, dimensions dependent on various influencing factors, change of functional properties, etc. Porous glasses are particularly interesting materials as they can represent inverse nanocomposites, where the interconnected pores with dimensions of nanometer scale are filled with a reactive polymer. Furthermore, confined reactive polymers are able to react within the pores of porous glasses. This is of particular interest in the polymer research as the reaction kinetics may be strongly driven by the confined environment.

The epoxy resin system used in this experiment is applied on porous glass and penetrates its pores with an extent dependent on the pore size, temperature and epoxy components mixing ratio. Developed over the recent time challenging sample preparation procedure allowed to produce very smooth epoxy - porous glass cross-sections. It included formation of interphases, followed by production of cross-sections and their polishing: 1st step - manual, 2nd step - ion beam etching.

In this study, combined AFM - Raman microspectroscopy was used to investigate the epoxy-porous glass interphases. It allowed for morphological studies and chemical differentiation between different regions at the cross-section and determination of the degree of cure of epoxy system in the porous glass.

The present project is supported by the National Research Fund, Luxembourg

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-5782 EFTEM and EDX analytical electron tomography of silica alumina supports for catalysts

Roiban L.1, Lepinay K.1,2, Lorut F.2, Gay A. S.3, Sorbier L.3, Ersen O.4, Epicier T.1,5
1University of Lyon, MATEIS, INSA de Lyon/Université Lyon I, 69621 Villeurbanne Cedex, France., 2STMicroelectronics, 850 rue Jean Monnet F-38926 Crolles Cedex, France., 3IFP Energies nouvelles, Rond-point de l'échangeur de Solaize, BP 3, 69360 Solaize, France., 4IPCMS, Université de Strasbourg, 23 rue du Loess, BP 43, 67034 Strasbourg Cedex 2, France, 5University of Lyon, IRCELYON, Université Lyon I, 2, Av. A. Einstein, 69626 Villeurbanne Cedex, France.
lucian.roiban@insa-lyon.fr

In the last two decades, tilted tomography in a TEM has been developed as an essential requirement for the three dimensional (3D) quantification of nanomaterials [1,2]. During the tilt series acquisition, a projection of the area of interest is recorded at each angle over a large angular amplitude, the final resolution along the Z axis being directly related to the maximal tilting angle. Getting 3D chemical information in the classical modes, i.e. bright field (BF) and Scanning TEM in the High Angle Annular Dark Field mode (STEM-HAADF) is generally delicate or impossible: the chemical information is provided by the mass-thickness differences between all phases, which cannot be properly measured in BF-TEM, and are delicate to quantify in STEM-HAADF. This is especially the case of compounds constituted by chemical phase with close masses and having similar same atomic density. A possible solution is to perform spectroscopic analysis simultaneously during the tilt series, either Energy Filtered TEM (EFTEM) [3,4] or STEM Energy-dispersive X-ray spectroscopy (EDX) tomography [3,5]. In this general context both EFTEM and STEM EDX tomography were employed in the study of a silica alumina support for catalysts composed of 50% SiO2 and 50% Al2O3. Amorphous silica-alumina are mesoporous materials characterised by a moderate acidity and are preferentially employed as catalyst support in hydrocarbon reactions such as hydrocracking of Vacuum Gaz Oil fraction or Fisher-Tropsch waxes [6,7]. The same sample prepared by the co-gel method was studied in a way to compare the chemical quantification provided by these techniques. The results (Fig. 1 and 2) show that silica (in green) is forming the core of the sample and that the surface is essentially covered by alumina (in red). Quantitative measurements indicate that alumina covers abound 80% of the surface of the grain even if the sample is formed by equal quantities of Al2O3 and SiO2.

References: [1] P.A. Midgley, R.E. Dunin-Borkowski, Nature Mat., 8 (2009) 271-280; [2] T. Epicier, chap. 3 in ‘Imagerie 3D en mécanique des matériaux’, ed. J.Y. Buffière, E. Maire, Hermès - Lavoisier, Paris, (2014); [3] G. Möbus, R.C. Doole, B.J. Inkson, Ultramicroscopy, 96 (2003) 433-451; [4] L. Roiban, L.Sorbier, C. Pichon, P. Bayle-Guillemaud, J. Werckmann, , M. Drillon, O. Ersen, Microsc. Microanal. 18 (2012), 1118–1128; [5] K. Lepinay, F. Lorut, R. Pantel, T. Epicier, Micron 47 (2013) 43-49;[6] G. Busca, Acid Catalysts in Industrial Hydrocarbon Chemistry Chemical Reviews 107 (2007), 5366-5410; [7] C. Marcilly, Catalyse acido-basique. Application au raffinage et à la pétrochimie. Volume 2. Technip Editions: Paris (2003)

The authors acknowledge CIFRE, ST Microelectronics and IFPen for financial support

Fig. 1: EFTEM tomography (200 kV, JEOL2100F), a) cross section through the chemical map volume parallel to the XY plane, where silica is represented in green and alumina in red; b) model computed from the reconstructed volume: the silica forms the core of the sample covered by alumina.

Fig. 2: STEM-EDX tomography (120 kV, FEI Osiris): a-b) cross sections through the reconstructed chemical models of silica (green) and alumina (red); c) outer surface of the sample showing that alumina is covering the main part of the grain.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-10-P-5827 Microstructural response of in situ formed nickel-graphite composites to phase separation

1 Arina V. Ukhina, 1 Dina V. Dudina, 1 Boris B. Bokhonov, 2 Alexander G. Anisimov, 2 Vyacheslav I. Mali
1 Institute of Solid State Chemistry and Mechanochemistry SB RAS, Kutateladze str. 18, Novosibirsk, 630128, Russia 2 Lavrentiev Institute of Hydrodynamics SB RAS, Lavrentiev Ave. 15, Novosibirsk, 630090, Russia
dina1807@gmail.com

Using Scanning Electron Microscopy, we study how the nickel-graphite composites (Fig.1) obtained from nickel-amorphous carbon powder mixtures containing 50 vol. % Ni by graphitization-accompanied Spark Plasma Sintering (SPS) respond microstructurally to phase separation. The in situ graphitization implies intimate contact between nickel and graphite; the latter forming by a dissolution-precipitation mechanism. The treatment of the composites ― removal of nickel by dissolution in acid and graphite by annealing in air ― was aimed at creating porous structures. The burnout of graphite during annealing of the composites in air proceeded parallel to the formation of NiO. The removal of graphite, however, did not lead to the formation of structures with uniform porosity distribution. The morphological outcome of the oxidation treatment appears to be dependent on the relative density of the Ni-C compacts as well as on the presence of Ni(C) solid solutions. During oxidation, the presence of carbon in the solid solution results in the evolution of CO thus preventing the formation of a continuous NiO film. A longer annealing of the compacts in air led to the formation of NiO structures shown in Fig.2. The NiO-based compact that formed did contain porosity; however, that porosity was inherited from the nickel-graphite composite that was not fully densified; in addition, the hollow NiO structures contained closed porosity. The NiO network formed as a result of nickel oxidation simultaneous with oxidation of carbon did not structurally repeat the pre-existing nickel network. A conclusion was drawn that in the microstructural evolution accompanying the burnout of carbon from the nickel-graphite composites, sintering of NiO, rather than porosity creation, dominates. By dissolving nickel from the SPS-ed specimens in acid, porous pellets were obtained that perfectly retained their shape and consisted of graphite platelets with diameters ranging from 0.3 to 2 μm and a thickness of 0.2 μm. The porous graphite formed from mechanically milled mixture was of uniform structure. In the networks formed from the non-milled nickel-amorphous carbon mixtures, pores 4-5 μm in diameter were present (Fig.3). The platelets of porous graphite obtained from the compacts consolidated in the solid state were smaller than those of the graphite crystallized in contact with molten nickel.

Fig. 1: Microstructure of the SPS-ed nickel-graphite composite, SPS-temperature 1000°C.

Fig. 2: NiO structures evolved during oxidation of the SPS-ed nickel-graphite compact during annealing in air.

Fig. 3: Porous graphite obtained by phase separation in the nickel-graphite SPS-ed composite.
↑ Go to the Table of content ↑

MS-11. Amorphous and disordered materials, liquid crystals, quasicrystals

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-11-IN-2573 Icosahedral and Crystal-like Medium-Range Order in Zr-Cu-Al Bulk Metallic Glasses

Zhang P.1, Maldonis J.1, Besser M.2, Kramer M. J.2, Melgarejo Z. H.1, Stone D. S.1, Voyles P. M.1
1Materials Science and Engineering, University of Wisconsin-Madison, Madison, WI USA, 2Ames Laboratory, Ames, IA USA
voyles@engr.wisc.edu

We have derived models for the structure of Zr-Cu-Al bulk metallic glasses (BMGs) from hybrid Reverse Monte Carlo (HRMC) simulations combining fluctuation electron microscopy (FEM) data and an empirical interatomic potential [1]. The FEM data constrains the nanoscale, medium-range order of the models, and the potential constrains the physical and chemical short-range order. Figure 1 shows the structures formed in a model of Zr50Cu45Al5 by the inclusion of the FEM data: chains of icosahedral nearest-neighbor clusters and more spherical clusters with crystallographically-allowed four- and six-fold rotational symmetry. A model based only on the potential does not have these structures and does not agree with the FEM data.


Figure 2(a) shows FEM data V(k) for Zr50Cu35Al15, which is a poorer glass former than Zr50Cu45Al5. It contains peaks at 0.37 Å-1 and 0.41 Å-1, identified as arising from icosahedral-like and crystal-like medium-range order respectively from HRMC simulations. Compared to Zr50Cu45Al5, the as-quenched state has stronger crystal-like order and weaker icosahedral-like order. The icosahedral-like order increases with minimal annealing (10 minutes at 0.83Tg), consistent with the behavior of Zr50Cu45Al5, but the crystal-like order is comparatively more stable. Figure 2(b) shows the medium-range correlation length Λ derived from variable resolution (VR) FEM experiments [2], in which the probe size is systematically varied from 1.3 to 11 nm. The decay in V(k = 0.37 or 0.41 Å-1) as a function of probe size gives Λ for the icosahedral- and crystal-like structure types respectively. Minimal annealing significantly increases Λ for the icosahedral-like order and decreases it for the crystal-like order.


The correlations between structure and glass-forming ability for the two alloys suggest that icosahedral medium-range order as well as icosahedral short-range order favors glass formation, and that more stable crystal-like order disfavors it. Detailed HRMC modeling of Zr50Cu35Al15 will also be discussed.


References
1. Jinwoo Hwang, Z. H. Melgarejo, Y. E. Kalay, I. Kalay, M. J. Kramer, D. S. Stone, and P. M. Voyles Phys. Rev. Lett. 108, 195505 (2012)
2. J. M. Gibson, M. M. J. Treacy, and P. M. Voyles, Ultramicroscopy 83, 169 (2000).

This project was supported by the US National Science Foundation (CMMI-1232731 and DMR-1205899O and Department of Energy (DE-AC02-07CH11358).

Fig. 1: Zr50Cu45Al5 HRMC model: (a) Crystal-like cluster, with the atoms colored blue to red by their local five-fold symmetry (b) 3D reciprocal space of the crystal-like cluster showing 4- and 6-fold rotational symmetry, (c) edge-on view of an icosahedral chain showing 2-fold symmetry, (d) an icosahedral chain.

Fig. 2: Zr50Cu35Al15 FEM data as a function of annealing at 0.83Tg (673 K). (a) V(k, R = 2 nm) showing peaks associated identified as icosahedral- and crystal-like order. (b) Variable-resolution FEM medium-range order correlation lengths Λ for the two structure types as a function of annealing.
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-11-IN-2672 Electron Microscopy of Quasicrystals – the State of the Art

Abe E.1
1University of Tokyo, Tokyo, Japan
abe@material.t.u-tokyo.ac.jp

Quasicrystals represent aperiodically ordered form of solids with symmetries long thought forbidden in nature. As stated with special emphasis in the Noble Lecture by Prof. Shechtman, the quasicrystal discovery is definitely the victory of electron microscopy – the first icosahedral stereogram was build up with a series of electron diffraction patterns from a tiny grain [1], and the following high-resolution electron microscope image indeed confirmed a unique aperiodic order that cannot be consistent with twinning of periodic crystals. Almost 30 years after these early electron microscopy works, we are now in the era of aberration-corrected electron microscopy that realizes a remarkable resolution of sub-Ångstrom scale. In the talk, I will describe the microscopic view of quasicrystals using state-of-the-art scanning transmission electron microscopy [2,3], providing several striking details that have been veiled in the quasicrystal structures. I should emphasize that electron microscopy is still an important, essential tool to answer the longstanding key questions “Where are the atoms? And why do quasicrystals form?”

References

1) D. Shechtman et al, “Metallic Phase with Long-Range Orientational Order and No Translational Symmetry” Phys. Rev. Lett. (1984) 53, 1951–1954.

2) E. Abe, “Structure of Quasicrystals” in Scanning Transmission Electron Microscopy, edited by S. J. Pennycook & P. D. Nellist, pp. 583-614: Springer New York (2011).

3) E. Abe, “Electron microscopy of quasicrystals – where are the atoms?” Chem. Soc. Rev. (2012) 41, 6787–6798

Fig. 1: Ultrahigh-resolution HAADF-STEM images taken by aberration-corrected 300 kV-STEM; decagonal quasicrystals of Al72Ni20Co8 (top) and Al70Mn17Pd13 (bottom). (Reproduced from Abe (2012) (Ref.2) with permission).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-11-O-1451 Transmission Electron Microscopy Characterization of Al-based Quasicrystal and Approximant Thin Films

Garbrecht M.1, Olsson S.1, Birch J.1, Hultman L.1, Eriksson F.1
1Thin Film Physics Division, IFM, Linköping University, SE-581 83 Linköping, Sweden.
magnus.garbrecht@liu.se

Quasicrystals and their associated approximant phases have drawn increased scientific attention during the past decade for the purpose of fundamental research as well as their possible applications as tribological coatings, thermal barrier thin films, solar absorbers, and low friction coatings [1,2]. Owing to their infinitely large unit cell, the determination of the local atomic structure of a quasicrystal is a challenging task to be performed by (scanning) transmission electron microscopy ((S)TEM). However, an associated approximant phase with comparable physical properties such as hardness, wear resistance, thermal - and electric conductivity, and friction shares the local atomic arrangement and thus allows conventional structure analysis to be performed [3].
We showed in the past that thin films of the Ψ-Al62.5Cu25Fe12.5 quasicrystalline phase can be prepared by annealing a multilayer stack on an Al2O3 (sapphire) substrate [4]. Furthermore, the α-approximant with nominal composition close to the icosahedral quasicrystalline phase can be grown similarly by high-vacuum magnetron sputtering of individual Al, Cu, and Fe layers onto Si(100) substrates, yielding a total thickness of 400 nm [5]. Figure 1 shows HAADF-STEM micrographs of the cubic α-approximant phase that was formed by Si diffusion from the substrate into the thin films activated by subsequent annealing to temperatures close below 450°C, where the quasicrystal phase starts to form. In parallel, the corresponding icosahedral Ψ-quasicrystalline phase was grown on Al2O3 (0001) as thin films in the same fashion (Figure 2).
In a similar way, Al-Cu-Co quasicrystalline and approximant thin films were grown on sapphire, and an Al-Cu-Co-Si quasicrystalline phase as a thin film on silicon substrates [6].
We present a detailed analysis on the quality and resulting microstructure of the Al-based quasicrystalline and approximant thin films employing high-resolution (S)/TEM, EDX, and electron diffraction methods, and discuss covering clusters and tiling analyses. All experiments were conducted at Linköping’s double-corrected and monochromated FEI Titan3 60-300 microscope equipped with a Gatan Quantum ERS GIF, high brightness XFEG source, and Super-X EDX detector, operated at 300 kV.

1. J-M. Dubois, S.S. Kang, A. Perrot, Mater. Sci. Eng. A 179/180 (1994) 122.
2. J-M. Dubois, Useful Quasicrystals, World Scientific Publ., Singapore, 2005.
3. U. Mizutani, T. Takeuchi and H. Sato, J. Phys.: Condens. Mat. 14 (2002) 767.
4. S. Olsson, F. Eriksson, J. Birch, and L. Hultman, Thin Solid Films 526 (2012) 740.
5. S. Olsson, F. Eriksson, J. Jensen, M. Garbrecht, J. Birch, and L. Hultman, Thin Solid Films, 550 (2014) 105.
6. S. Olsson, M. Garbrecht, J. Birch, L. Hultman, and F. Eriksson, J. Mater. Res., subm., 2014.

We acknowledge the Knut and Alice Wallenberg (KAW) Foundation for the Electron Microscope Laboratory in Linköping.

Fig. 1: HAADF-STEM micrographs of an α-approximant thin film with nominal composition Al55Si7Cu25.5Fe12.5 grown on Si(100) in [111] (a) and [110] (b) zone axis.

Fig. 2: HRTEM micrograph of the icosahedral Ψ-quasicrystalline phase with nominal composition Al62.5Cu25Fe12.5, grown as a thin film on Al2O3 (0001), viewed along the 5-fold [100000] (a), and 3-fold [222000] incident beam direction (b).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-11-O-1794 Systematic mapping of icosahedral short-range order in a melt-spun Zr36Cu64 metallic glass using scanning electron nano-diffraction

Liu A. C.1,2, Neish M. J.2,3, Bourgeois L.4,1, Ott R. T.5, Kramer M. J.5,6, de Jonge M. D.7
1Monash Centre for Electron Microscopy, Monash University, Clayton, Australia, 2School of Physics, Monash University, Clayton, Australia, 3School of Physics, University of Melbourne, Parkville, Australia, 4Department of Materials Engineering, Monash University, Clayton, Australia, 5Division of Materials Science and Engineering, Ames Laboratory, Ames, USA, 6Department of Materials Science and Engineering, Iowa State University, Ames, US, 7Australian Synchrotron, Clayton, Australia
amelia.liu@monash.edu

Understanding the structure of disordered materials remains a challenge. In the aberration-corrected scanning transmission electron microscope (STEM) intense, coherent, quasi-parallel and nanometre-sized beams may be achieved allowing diffracted information to be obtained from small volumes.  Electron nano-diffraction (END) patterns from thin metallic glass specimens contain angular correlations related to symmetries in nearest-neighbour polyhedral clusters [1]. Examining the persistence of these angular correlations in a scanned array of END patterns allows a measure of the medium-range order in the material [1]. We apply this novel technique to understanding the excellent glass formability of ZrxCu100-x glasses [1].

Scanned arrays of END patterns from a melt-spun Zr36Cu64 glass were obtained in a Titan3 80-300 FEGTEM (Fig. 1 a) and b)). Subtle angular symmetries in the END patterns were detected by calculating the angular autocorrelation function (Fig. 1 c) and d)). The autocorrelation function was decomposed into a Fourier Cosine series at each scattering vector magnitude and the symmetry intensities in each pattern were measured (Fig. 1 e)) and mapped as a function of scanned distances to examine the extent of any order (Fig. 2).

We statistically analysed the incidence of two-, six- and ten-fold symmetries in the SEND patterns and found that these compare favourably to those expected for a random ensemble of icosahedra, consistent with many modeling studies.

Fig 2. shows the 2-, 6- and 10-fold symmetry maps for both the experimental glass and a model glass structure. The only correlation length that extends beyond the probe diameter is the experimental two-fold map, demonstrating that the glass has extended order. The MRO consistent with this trend is face-sharing or interpenetrating icosahedral clusters in which the 2-fold symmetry axes align, but the 5- and 6-fold do not. The correlation length in the 2-fold map corresponds to four face-sharing or seven interpenetrating icosahedra.

Using scanning END and a novel analysis of the angular correlations in END patterns we determine that the S-MRO in Zr36Cu64 is consistent with efficiently packed icosahedral clusters [1], suggesting a structural basis for glass formability.

[1] A. C. Y. Liu, M. J. Neish, G. Stokol, G. A. Buckley, L. A. Smillie, M. D. de Jonge, R. T. Ott, M. J. Kramer, and L. Bourgeois, Phys. Rev. Lett., 110, 205505 (2013).

ACL acknowledges the Science Faculty, Monash University. TEM was performed in the MCEM.  Specimens were prepared at Ames Laboratory, funded by the US DoE (Office of Science–Basic Energy Sciences) Contract No. DE-AC02-07CH11358.

Fig. 1: a) HAADF reference image of glass with scanned area b) END pattern c) angular cross-correlation function and d) profile at 4 nm-1 e) magnitude of 0-12-fold symmetry intensities

Fig. 2: Maps of symmetry intensity from array of END patterns for model glass (a),c),e)) and experimental glass (b),d),f)) and corresponding radially averaged, two-dimensional autocorrelation functions.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-11-O-2456 Microstructure evolution in phase separating Al-based amorphous alloys

Kim K. C.1, Kim C. I.1, Kim W. T.2, Kim D. H.1
1Department of Materials and Science Engineering, Yonsei University, Seoul, Korea, 2Deptartment of Optical Engineering, Cheongju University, Cheongju, Korea
dohkim@yonsei.ac.kr

Phase separation can occur when there is at least one atom pair with positive enthalpy of mixing or there is a large difference in enthalpy of mixing between the atom pairs in metallic glass forming systems. By selecting a proper pseudo-binary section of the miscibility gap in the multi-component system, droplet-type microstructure formed by nucleation and growth mechanism or interconnected-type microstructure formed by spinodal decomposition can be obtained in the as-solidified microstructure. Thermodynamic calculation of miscibility gap allows the formation of full spectrum of the microstructure from interconnected type to droplet type microstructure. Phase separation phenomena can provide some advantages in utilizing metallic glasses, for example, introduction of complex shape forming by using the two-step glass transition phenomena or synthesis of porous glass by leaching out one of the two separated amorphous phases. In the present study, we have investigated the microstructural evolution in phase separating Al-based amorphous alloys. The double halo rings in the electron diffraction pattern and nm scale interconnected STEM image obtained from the as-melt-spun alloys indicate that two different amorphous phases form during solidification. With heat treatment below the glass transition temperature, the scale of phase separation increases. Detailed investigation on the crystallization behavior reveals that a metastable single crystalline phase forms at early stage of crystallization. However, at later stage, equilibrium phases replace the metastable phase.

This work was supported by the Global Research Laboratory Program of the Korean Ministry of Education, Science and Technology.

Fig. 1: STEM image and diffraction pattern showing phase separation in Al-Mn-Ge amorphous alloy

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-11-O-3423 Analysis of structural order in Fe1-xZrx thin amorphous films

Leifer K.1, Xie L.1, Kocevski V.2, Rusz J.2, Hjorvarsson B.3, To Baben M.4, Sun T.5, Zaluzec N. J.6
1Applied Materials Science, Engineering Science, Uppsala University, Uppsala, Sweden, 2Materials Theory, Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden, 3Materials Physics, Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden, 4Inst. Werkstoffchemie, RWTH Aachen, Germany, 5Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Argonne Illinois 60439, USA, 6Electron Microscopy Center, Materials Science Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne Illinois 60439, USA
klaus.leifer@angstrom.uu.se

Amorphous materials are among the most intriguing to analyse with microscopies since single atom positions cannot yet be resolved. Whereas the pair correlation function gives information about next neighbor distances from evaluation of diffraction patterns, variance plots in fluctuation electron microscopy (FEM) were shown to be sensitive to small structural changes in the amorphous material. In metallic alloys amorphisation is often induced by adding larger atoms B to a transition metal A. When exceeding a critical concentration xc of atom B in A1-xBx, the alloy will transform from the crystalline to the amorphous phase. Thus, it is likely that the structure of the amorphous material depends on the concentration difference x- xc.

With this idea in mind, we have analysed Fe1-xZrx samples as a function of x (x=0.1-0.29), where the x is chosen to be above xc. The films were grown by sputter deposition where the amorphous Fe1-xZrx was 14nm thick. The films were analysed by FEM in plan view geometry in the STEM geometry of FEM. Our data were obtained from the quantitative evaluation diffraction patterns contained from a full scanning transmission FEM (STFEM) data set. We have optimized the acquisition such that oxidation of the sample, the influence of cladding Al0.7Zr0.3 layers did not impact the analysis.

The variance as a function of q-vector was extracted from such diffraction patterns and subsequently, the size of structurally coherent clusters was determined (Fig. 1). The structural coherence length depends only weakly on x. In addition, correlographs, computed from the data such as in ref [3] show a clear increase of structural order with decreasing x.

In order to understand how the FEM data are related to the structure of the amorphous films, we have simulated the amorphous structure by melting a supercell using classical molecular dynamics and considering an embedded atom model interatomic potential. We calculated the FEM diffraction patterns for both, perfectly amorphous structures as well as for structure models containing crystalline clusters of various sizes and orientations (Fig. 2). We observe changes in the variance of micro-diffraction patterns and correlate them with experimental findings.

[1] M. M. J. Treacy and K. B. Borisenko, Science 335, 950, 2012.

[2] A. Liebig, P. T. Korelis, H. Lidbaum, G. Andersson, K. Leifer, B. Hjörvarsson, Phys. Rev. B 75, 214202, 2007.

[3] J. M. Gibson, M. M. J. Treacy, T. Sun, N. J. Zaluzec, Phys. Rev. Lett. 105, 125504, 2010.

We gratefully acknowledge support from STINT and Swedish Science Council.

Fig. 1: Typical variance diffraction pattern (left) and variance plot (right) of the Fe0.81Zr0.19 sample.

Fig. 2: FEM diffraction pattern calculated from the model of amorphous FeZr.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-1776 In situ TEM observation of Crystal-to-Amorphous-to-Crystal (C-A-C) transition in Cr2Ti

Nagase T.1, 2, Anada S.1, Yasuda H.1, Mori H.1
1Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Japan, 2Division of Materials and Manufacturing Science, Graduate School of Engineering, Osaka University, Japan
t-nagase@uhvem.osaka-u.ac.jp

In situ TEM observation of Crystal-to-Amorphous-to-Crystal (C-A-C) transition in Cr2Ti was reported focusing on the temperature dependence and the crystallization behavior. Figure 1 shows In situ TEM observation of the change in BF images and corresponding SAD patterns of Cr2Ti stimulated by MeV electron irradiation at 103 K (a) and room temperature of 298 K (b) [1]. At 103 K, a crystalline-to-amorphous (C-A) transition occurred under the irradiation (a1→a3), resulting in the formation of an amorphous single phase. Further irradiation, an amorphous-to-crystalline (A-C) transition occurred as the subsequent structural change (a3→a5) and bcc solid solution single phase was formed. This type of amorphization-crystallization phase transition was called a crystalline-to-amorphous-to-crystalline (C-A-C) transition. At 298 K, black dot contrast appeared in BF image after 60 s irradiation (b2). After irradiation for 180 s (b3), nano-granular contrast appeared at the center of the irradiated area, as seen in the BF image, and discontinuous Debye rings began to appear in the SAD pattern. Upon further irradiation, the nano-granular contrast changed to conventional polycrystalline contrast in the BF images due to bcc solid solution formation (b3→b5). Thermal crystallization of a Cr-Ti amorphous phase obtained by C-A transition and the irradiation induced crystallization during C-A-C transition was also investigated for clarifying the origin of C-A-C transition.

[1] S. Anada, T. Nagase, H. Yasuda, H. Mori: J. of Alloys and Compounds, 579, 646-653 (2013).

This work was supported in part by Grants for Excellent Graduate Schools, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Fig. 1: Figure 1 In situ TEM observations of the change in BF images and SAD patterns in Cr2Ti intermetallic compounds stimulated by MeV electron irradiation at 103 K at a dose rate of 3.7 × 1024 m-2s-1 (a) and at 298 K at a dose rate of 6.7 × 1024 m-2s-1 (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-1835 Investigations of amorphous Polymer-Derived Ceramics by Pair Distribution Function obtained from Electron Diffraction in TEM

Hapis S.1, Kleebe H. J.1, Rohrer J.1, Riedel R.1, Mu X.2, Van Aken P. A.2
1Technical University Darmstadt, Darmstadt, Germany, 2Max-Planck Insitute for Intelligent Systems, Stuttgart, Germany
hapis@geo.tu-darmstadt.de

This study is focused on the sensitivity of the final ceramic microstructure to the molecular structure of the precursor. Bulk ceramics with different polymers were synthesized and annealed at different temperatures. Subsequent examinations were carried out with Transmission Electron Microscopy (TEM), including High Resolution Transmission Electron Microscopy (HRTEM) and the calculation of the Pair Distribution Function (PDF) from electron diffraction patterns of the predominantly amorphous matrix. Afterwards the experimental PDF are compared to calculated PDFs obtained from structure models determined via classical Molecular Dynamics simulation.

The Pair Distribution Function is a powerful technique to investigate the short range order in disordered materials like the amorphous Polymer-Derived Ceramics up to ~1400°C. The PDF is already well established for example in X-ray or neutron diffraction but the advantage of the PDF from electron diffraction is that a very small sample volume can be analyzed in conjunction with the corresponding image information (HRTEM). Therefore, this technique is used for a better understanding of the crystallization behavior of these ceramics. By now, it is known that the onset of crystallization in boron-containing ceramics is shifted towards higher temperatures; however, the resulting crystal-size differs significantly and the amorphous matrix shows distinctly different features. The relationship between the molecular architecture and chemistry on the amorphous nature of the pre-ceramic and, in addition, its influence on the resulting crystallization behavior will be discussed.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-1914 High-resolution radial distribution functions of several forms of pure amorphous silicon measured using tilted-illumination selected area electron diffraction

Liu A. C.1,2, Gorecki A.2, Haberl B.3, Bradby J. E.3, Williams J. S.3, Petersen T. C.2
1Monash Centre for Electron Microscopy, Monash University, Clayton, Australia, 2School of Physics, Monash University, Clayton, Australia, 3Department of Electronic Materials Engineering, The Australian National University, Canberra, Australia
amelia.liu@monash.edu

Pure amorphous silicon (a-Si) is a model tetrahedral amorphous material that can be created by ion implantation and indentation [1]. These forms exist in small volumes, making measurements using transmission electron microscopy (TEM) an excellent option.  We demonstrate high-resolution radial distribution measurements of implanted and indented a-Si measured using tilted-illumination selected area diffraction in the TEM.

At advanced x-ray and neutron facilities, large scattering angles can be achieved to yield high-resolution RDFs in bulk homogeneous specimens. Fixed apertures in the TEM and finite dynamic range of electron detectors can limit the accessible range of diffraction angles, and the resolution of the RDF. However, by sequentially tilting the incident TEM illumination and increasing corresponding acquisition times, large angles can be accessed, resulting in RDF resolutions comparable to those from neutron and x-ray measurements [2] yet from sub-micron areas.

Several forms of as-prepared and annealed a-Si were prepared as detailed previously [1].  Selected area diffraction patterns were obtained in a JEOL JEM 2100F operated at 200 kV.  The dark-tilt was  incrementally increased to access larger diffraction angles. By increasing the exposure time for higher angle diffraction patterns the Poisson noise contribution was kept below 15% across the whole range sampled (2sin(θ)/λ = 3.3-3.7 Å-1). The diffraction patterns were spliced together [2] (Fig. 1) and analysed using RDFTools, a free software package [3].

Fig. 2 shows the RDFs measured for the as-implanted a-Si and the relaxed implanted a-Si films [2]. Average coordination numbers for the as-implanted and relaxed specimens were measured to be 3.7±0.3 and 3.9±0.3, respectively. Bond angles of 109±0.5o and 110±0.6o were measured for the as-implanted and relaxed a-Si [2].

Tilted illumination selected area diffraction can produce high resolution RDFs of amorphous materials in small volumes to distinguish subtle pair correlation differences between specimens with different treatments. However, in striking contrast to other amorphous networks, the structural re-arrangements of ion-implanted a-Si due to a relaxation anneal are subtle.


[1] B. Haberl, A. C. Y. Liu, J. E. Bradby, S. Ruffell, J. S. Williams, and P. Munroe, Phys. Rev. B, 79, 155209 (2009).
[2] A. Gorecki, T. C. Petersen and A. C. Y. Liu, Microscopy and Microanalysis, 20, 50, (2014)
[3] D. R. G. Mitchell and T. C. Petersen, Microsc. Res. Tech. 75, 153, (2012).

ACL gratefully acknowledges the support of the Science Faculty and the Monash Centre for Electron Microscopy (MCEM), Monash University. TP acknowledges support from the MCEM. The authors acknowledge use of facilities within the MCEM. JEB acknowledges an ARC QEII.

Fig. 1: a) Dark-field tilt is used to extend the angular range of a diffraction pattern. b) Diffracted intensities are spliced together, so that the noise level (c)) is below 15% of signal across the whole range.

Fig. 2: Reduced radial distribution function of as-implanted and relaxed ion implanted a-Si.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-1918 Chemical ordering of Co and Ni in crystalline approximants related to Al-Co-Ni decagonal quasicrystals, studied by Cs-corrected STEM with EDS

YASUHARA A.1, Hiraga K.2
1JEOL Ltd., Tokyo, Japan, 2Tohoku University, Sendai, Japan
ayasuhar@jeol.co.jp

    Six-types of decagonal quasicrystals (DQCs) and some crystalline approximants have been found in Al-Co-Ni alloys which have a wide range of compositional ratios of Ni/Co and a nearly constant Al composition of approximately 70 at %. It is considered that various structures of the DQCs are stabilized by chemical ordering of Co and Ni.　The arrangements of transition-metal (TM) atoms have been determined by Cs-corrected HAADF-STEM observations [1-3]. However, the study of the chemical ordering in Al-Co-Ni DQCs is difficult in HAADF or ABF STEM observations, because atomic number difference between Co and Ni is only one, resulting in very low contrast between these atomic columns in STEM images. Recent developments of an X-ray detector with large solid angle and an ultrafine and intense probe realized with a Cs-corrector enable us to perform atomic-resolution chemical analysis. Our intention in this paper is to detect the chemical ordering of Co and Ni in Al-Co-Ni crystalline phases, which are closely related to the structure of the Al-Co-Ni DQCs, by the atomic-resolution EDS. However, this method has been considered to be difficult for some materials, which are easily damaged by intense electron irradiation in STEM. To reduce the electron dose on a specimen, we have tried to obtain an EDS map formed by integration of several EDS maps from fresh areas. The maps from the areas were obtained by periodic sample shifts, which are determined by translational vectors of a and c, where a and c are lattice parameters in the unit cell of the crystalline phase. It should be noted that this method applicable to the crystalline phases that have periodic arrangements.
    Figure 1 shows atomic-resolution EDS maps of a W-(AlCoNi) crystalline phase, taken with a newly developed silicon drift detector (SDD), installed on an aberration corrected microscope (JEM-ARM200F). The chemical ordering of Co and Ni is clearly seen in the map, shown in Fig. 1(d), which is a superimpose map of Co and Ni.

[1] A. Yasuhara, K. Saito and K. Hiraga, in: Aperiodic Crystals (Proc. of Aperiodic 2012 Edited by. S. Schmid, R. L. Withers and R. Lifshitz, Springer 2013) p. 219-224.
[2] K. Hiraga and A. Yasuhara, Mater. Trans. 54 (2013) 493-497.
[3] K. Hiraga and A. Yasuhara, Mater. Trans. 54 (2013) 720-724.

Fig. 1: Figure 1 HAADF-STEM image (a), atomic-resolution EDS maps of Co and Ni (b, c) and a superimposed map (d) for the W-(AlCoNi) crystalline phase. The locations of atomic clusters are indicated by circles in images. Note that the chemical ordering of Co and Ni occurs at regions between the Co-rich atomic clusters and Ni-rich regions.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-1920 Microstructural characterization of Al-based composites reinforced with Al-Cu-Fe quasicrystalline phase

Wolf W.1, Coimbrão D. D.1, Gallego J.2, Aliaga C. R.1, Travessa D.3, Cardoso K. R.3, Bolfarini C.1, Kiminami C. S.1, Afonso C. R.1, Botta W. J.1
1Federal University of São Carlos, São Carlos, SP, Brazil, 2São Paulo State University, Ilha Solteira, SP, Brazil, 3Federal University of São Paulo, São Paulo, SP, Brazil
fdiego@ufscar.br

Since the discovery of quasicrystalline (QC) phases more than one hundred different quasicrystalline alloys have been observed and large efforts have been made in order to understand and apply the unique properties presented by these materials. Due to their structure, QC alloys have high hardness, high elastic modulus, low thermal and electric conductivity and good corrosion resistance. However, these alloys are brittle at room temperature and as consequence their application as structural component is limited. On the other hand, the use of such alloys as reinforcing phase in a metal-matrix composite is a potential field of application for the QC materials.
In the present work, hot extrusion was used to produce aluminum-based composites reinforced with Al65Cu20Fe15 (at.%) QC alloy. The QC alloy was fabricated by arc melting, submitted to mechanical alloying (MA) and then to a subsequent heat treatment to obtain a single phase QC-powder. MA was also used to produce the mechanical mixture of Al and the QC alloy (10% of QC-phase in wt.%) where the mixed powders were ball milled during 5 h in a planetary high-energy mill with rotating speeds of 200 and 600 rpm, respectively. The powders were then hot extruded at 420 °C. The consolidated samples were analyzed by transmission electron microscopy (TEM) using a FEI TECNAI G2 F20.
X ray diffraction patterns (not shown here) of the alloy powder that was used as the reinforcement phase in the composite confirmed that the single QC phases was obtained after the heat treatment at 700 °C. Figure 1 shows the selected area electron diffraction patterns (SAED) obtained from the particles in the hot extruded composite, confirming the icosahedral structure of the QC phase, which was stable during production of the Al-QC composite.
Figure 2 shows bright field and dark field STEM micrographs of the composites fabricated with mixing velocity of 200 rpm. Such mixing condition did not produce a good dispersion of the QC phase, which remained mostly in the grain boundaries. Figure 3 shows bright field and dark field STEM micrographs of the composites fabricated with mixing velocity of 600 rpm. These micrographs reveal a much better and finer dispersion of the QC-phase in the Al matrix, with the particles distributed in the interior of the grains. Torsion tests for both composites indicated equivalent tensile strength of 130 MPa for the composite with the coarser distribution of the QC phase (Fig. 2) and 200 MPa for the composite with finer distribution of the QC phase (Fig. 3). Therefore the composite with finer particles presented a substantial increase on the mechanical strength, and the attractive values of tensile strength were associated with the uniform dispersions of the QC-phase inside the grains.

The authors gratefully acknowledge the financial support of the Brazilian institutions FAPESP, CNPq, CAPES and FINEP.

Fig. 1: SAED pattern of the QC-phase in hot extruded composite, with 2, 3 and 5-fold symmetry, confirming the icosahedral symmetry of the reinforcement phase.

Fig. 2: STEM bright field and dark field images of the composite fabricated with powder mixing velocity of 200 rpm; the QC particles are coarse and preferentiality located at the grain boundaries.

Fig. 3: STEM bright field and dark field images of the composite fabricated with powder mixing velocity of 600 rpm; the QC particles are finer, with a uniform dispersion preferentiality located inside the grains.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-2032 Induced-Assembly of M13 Bacteriophage Arrays Using Carbon Thin Films

Moghimian P.1, Srot V.1, Rothenstein D.2, Facey S. J.3, van Aken P. A.1
1Stuttgart Center for Electron Microscopy, Max Planck Institute for Intelligent Systems, Stuttgart, Germany, 2Institute for Materials Science, University of Stuttgart, Stuttgart, Germany , 3Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany
moghimian@is.mpg.de

Fabrication of well-ordered and defect-free two dimensional (2D) structures on the nanoscale is of technological importance in energy storage and electronic devices [1].  The biologically-inspired processes, in which the biological entities (e.g. phages) serve as 2D scaffolds for the directed synthesis of a range of inorganic nanostructures, are essential for designing the next-generation multicomponent materials [2]. In order to induce in-situ growth of uniform and homogenous inorganic nanostructures, bio-assisted synthesis methods are utilized as promising tools to attain smooth organic-inorganic hierarchical layers. To this end, not only preparation of a densely-packed monolayer of biological entities onto specific surfaces is inevitable, but also achieving a directionally ordered pattern of the biological entities, extending in a long length range is crucial to enhance the functional properties of the final product. It has been reported that the surface chemistry, roughness and the state of hydrophobicity play important role in the surface protein adsorption and their further assembly [3]. In this study, filamentous wild-type (WT) M13 bacteriophages were deposited from a viral solution (< 5 mg/ml) on amorphous carbon (a-C) and silicon oxide (SiOx) surfaces. Using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), we show that the a-C surface induces the assembly of M13 phages into parallel arrays. Figure 1 shows a bright-field TEM (BF-TEM) image of disordered phages immobilized on the SiOx surface. The trend of the system was towards the formation of an isotropic phase. However, viral particles show a high degree of alignment along a common axis on a-C surface as per nematic liquid crystalline model (see Figure 2). The M13 phage particles were found to have a high affinity for incorporation into the closely-ordered pattern onto a-C. Interestingly, the aforementioned architecture can be obtained by applying phage solution on the surface without employing nanoparticle assembly methods such as dip coating or convective assembly. Our strategy is to facilitate the immobilization process in a highly ordered manner, and to attain a fully covered surface with densely-packed and highly-oriented M13 phage viral particles in a long-range basis. Such closely-ordered pattern can further function as templates to nucleate highly uniform and smooth inorganic layers.

References

[1] L. Shen, N. Bao, Z. Zhou, P. E. Prevelige and A. Gupta, Journal of Materials Chemistry, 2011, 21, 18868-18876.

[2] S. W. Lee, C. B. Mao, C. E. Flynn and A. M. Belcher, Science, 2002, 296, 892-895.

[3] T. Berlind, P. Tengvall, L. Hultman and H. Arwin, Acta Biomaterialia, 2011, 7, 1369-1378.

Financial support by the DFG for funding SPP 1569 is gratefully acknowledged. The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under grant agreement n°312483 (ESTEEM2).

Fig. 1: BF-TEM image of randomly distributed WT M13 phages immobilized on a SiOx membrane from a droplet of M13 viral solution, depicting a highly disordered and web-like structure. The inset shows the 2D Fourier transform (FT) image of the corresponding BF-TEM image.

Fig. 2: BF-TEM image of WT M13 phages closely-packed and oriented on a-C support film from a droplet of M13 viral solution, showing the 2D alignment along a common direction. The inset shows the 2D FT image of the corresponding BF-TEM image.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-2138 Production of Bulk Cu36Ti34Zr22Ni8 Amorphous Alloy by Ball Milling and Hot Compaction: Phase Separation in the Solid State.

Jorge Jr A. M.1,2,3, Medeiros B. B.1, Nogueira R. P.3, Bolfarini C.1
1Departamento de Engenharia de Materiais, Universidade Federal de São Carlos, 13565-905 São Carlos, SP, Brazil, 2SiMap Laboratory CNRS, INPG – UJF, Grenoble, BP75, 38402 St-Martin d'Hères, France, 3LEPMI CNRS, INPG – UJF, Grenoble, BP 75 - 38402 Saint Martin d'Hères, France
moreira@ufscar.br

The low ductility and brittle fracture at room temperature extremely limit the application of bulk metallic glasses (BMGs) as structural materials. Heterogeneous microstructures combining glassy matrix and crystalline particles improve their ductility and strength by delaying the fast propagation of shear bands. Recently was found that cold rolling of Cu60Zr20Ti20 BMG leads to the occurrence of phase separation in the solid state, which may be an effective way to strengthen the BMG. However, the size of these BMGs is limited to 8 mm. The aim of this work was to produce phase separated BMG by milling ingots of as cast Cu36Ti34Zr22Ni8 for 16 h, followed by hot compaction inside the supercooled liquid region of the powders and bulks with a diameter of 14 mm and 20 mm in length were produced. Ni was chosen because it can improve the glass forming ability by substituting Cu and avoiding the formation of Cu51Zr14. Melt spun ribbons were produced by melt spinning for comparison. Characteristic temperatures were determined by differential scanning calorimetry (DSC). The fine structure of the specimens was examined by transmission electron microscopy (TEM) at a voltage of 200 KV using an FEI-TECNAI G2F20 microscope. Ribbon and bulk samples were thinned by ion milling under conditions such that damage is avoided in the structure during thinning. Structural characterization by synchrotron radiation (not shown here) revealed the amorphous character of ribbons and powders, and some nanocrystallization in the bulks after hot compaction. Fig. 1a shows DSC of powders and ribbons, where is possible to observe the changes in thermal behavior. The increase of temperature means worst glass forming ability, but, from the point of view of powder metallurgy, this means thermal stability that may be good for final mechanical applications. The amorphous character of ribbons can be observed in Figs. 1b and 1c in whose inset was already possible to observe a non-homogeneous distribution of the second phase (1 nm). Figs. 2a-d show images of bulk samples, where is possible to observe homogeneously distributed second amorphous phase (darker regions), which are really composed by 2 nm second phase and amorphous matrix (Fig. 2b). Those darker regions are embedded in an amorphous matrix (Fig. 1c). Some nanocrystallization is also possible to be observed in Fig. 1d, which is mainly occurring in the second phase. After annealing of bulks, this was confirmed in Figs. 1e-f. In conclusion, it is possible to produce phase separation in the solid state by milling, there was a change in the thermal behavior leading to thermal stability of bulks, but this decreased the processing window leading to small amount of nanocrystals during hot pressing, which mainly occurred in the second phase.

The authors acknowledge the Brazilian funding agencies FAPESP under the grant number 2012/13179-6 and CNPq for their financial support.

Fig. 1: (a) DSC comparing the thermal behavior of powders and ribbons. (b) TEM BF image of ribbon and inset showing the amorphous state. (c) Higher magnification of Fig. b and HRTEM in the upper right side showing non homogeneous phase separation.

Fig. 2: (a) BF image of bulk: homogeneous phase separation (darker regions) and inset showing the amorphous state. (b) HRTEM of a region in 2a: second phase regions embedded in amorphous matrix. (c) Amorphous matrix in 2a. (d) nanocrystallized regions (e-f) Bulk after annealing confirming that crystallization is mainly occurring in the second phase.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-2206 Alterations in density along the propagation direction of shear bands in a metallic glass determined by correlative analytical TEM

Schmidt V.1, Rösner H.1, Peterlechner M.1, Voyles P. M.2, Wilde G.1
1Institute for Materials Physics, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany, 2Materials Science and Engineering, University of Wisconsin-Madison, 1509 University Ave, Madison, WI53706, USA
vitalij.schmidt@uni-muenster.de

Deformation flow in metallic glasses is restricted to narrow areas called shear bands. They are associated with structural changes with respect to the surrounding amorphous matrix. Shear bands are widely believed to contain more "free volume" than the surrounding, undeformed glass. Higher free volume means lower atomic density, which is consistent with lighter contrast in high-resolution TEM images [1]. As part of a comprehensive study of the local density and the structure of shear bands using the signals of HAADF-STEM, EELS, EDX and nanobeam diffraction [2], we show that shear bands in Al88Y7Fe5 metallic glass switch back and forth between having lower density than the matrix to having higher density than the matrix as they propagate. The correlative data allows calculation of the specimen foil thickness from low-loss EEL spectra and subsequently determination of density changes by the ratio of the HAADF-STEM intensities, ∆ρ=(I2-I1)/I1 , where Δρ is the difference in density, I1 and I2 are the HAADF intensities of different areas within comparable thicknesses. EDX and nanobeam diffraction provide information about chemical composition and medium-range order respectively.

Melt-spun and subsequently cold-rolled Al88Y7Fe5 ribbons were prepared for TEM by electro-polishing. HAADF-STEM reveals shear bands with contrast changes from bright to dark and vice versa along their propagation direction accompanied by slight deflections. Figure 1 shows a representative example of such a shear band. The orientation and size of the shear band are examined. For example the propagation direction was unambiguously determined by the presence of the bifurcation. Comparing different segments to the surrounding matrix shows an alteration in the HAADF intensity and thus in density [3]. Figure 2 displays such changes for a part of the shear band. Slight changes in composition were also found for the dark and bright parts of the shear band as well as an increase in medium-range order.

[1] P.E. Donovan, W.M. Stobbs, Acta Metall. 29 (1981) 1419,

[2] H. Rösner et al. accepted in Ultramicroscopy.

[3] V. Schmidt to be published.

We kindly acknowledge financial support by the DFG via SPP 1595 (Topological engineering of ultra-strong glasses) and we thank the Ernst Ruska-Centrum, Jülich, for the use of the Titan 80-300 STEM.

Fig. 1: A HAADF-STEM image of cold-rolled Al88Y7Fe5 showing a shear band with several contrast changes along its propagation direction (from left to right).

Fig. 2: Alterations in HAADF-STEM intensity in the shear band (Fig. 1) relative to the surrounding matrix along the propagation direction. Dark and bright parts of the shear band are indicated by the grey and white background, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-2209 TEM STUDY OF Short range order in SbVO4

Vilanova-Martínez P.1, Hernández-Velasco J.1, Agulló-Rueda F.1, Landa-Cánovas A. R.1
1Instituto de Ciencia de Materiales de Madrid, ICMM-CSIC. Sor Juana Inés de la Cruz, 3; 28049 Madrid, Spain
palomavilanova@icmm.csic.es

~SbVO4 is a key element in the catalysis of propane to acrylonitrile. The system maintains the rutile structure during the whole existence range accommodating the non-stoichiometry in a "soft way", i.e., without extended defects, which are so common in other rutile systems. The great structural flexibility exhibited, very important for its catalytic performance, involves cation vacancies, changes of the oxidation state of vanadium (V4+, V3+, and Sb5+), long range ordering, structural modulations, short range ordering, etc. [1,2] In this work we study the structure of one of the key points of the system, the composition limit Sb1.0V1.0O4.

The samples have been prepared by heating Sb2O3 and V2O5 in an equimolar ratio (1:1) at 800C under argon atmosphere in two runs of 12 hours with a careful grinding in the interval. The sample has been characterized by powder X-ray diffraction in a Powder Diffractometre Bruker D8 Advance with Cu Kα radiation with rapid detector (lynxeye). Electron diffraction experiments were carried out in a JEOL 2000FXII transmission electron microscope with a double-tilt holder. The sample has also been characterized by neutron diffraction experiments (Instruments D1B-ILL, Grenoble and E6, E9 at HZB, Berlin), magnetic susceptibility measurements, DSC calorimetry and Raman spectroscopy. The sample exhibits a basic rutile powder X-ray diffraction pattern without extra characteristics. TEM exhibits very crystalline crystals with a typical shape of tetragonal prisms capped at both ends. However, electron diffraction shows the presence of intense diffuse lines that, after careful tilting experiments, are demonstrated to be two-dimensional wavy diffuse sheets. Notice that the diffuse intensity lines are absent in the [001] zone axis due to the fact that the diffuse intensity sheets are perpendicular to the [001] direction. While these diffuse sheets seem to be very straight in zone axes such as [110] and [101], they reveal a wavy nature when tilting away from these and specially at the [100] zone axis. This diffuse intensity condense in a two fold superlattice when the sample is prepared under nitrogen atmosphere. Neutron diffraction and magnetic susceptibility measurements reveal magnetic ordering at TN < 50K from the ordering of vanadium magnetic moments [3].

[1] A.R. Landa-Cánovas, J. Nilsson, S. Hansen, K. Staahl and A. Andersson. J. Solid State Chem. 116, 369-377 (1995). [2] Angel R. Landa-Cánovas, F. Javier García-García, Staffan Hansen. Catalysis Today 158 (2010) 156. [3] J. Hernández-Velasco, J. García-García, A.R. Landa-Cánovas. Microsc. & Microanal. 18, 95-96 (2012).

The authors thank the Spanish Government (project MAT2011-27192) for financial support.

Fig. 1: SAED patterns of SbVO4 crystals orientated along different zone axes. The sharp diffraction maxima belongs to the basic rutile lattice. The diffuse lines between the Bragg maxima are caused by SRO phenomena.

Fig. 2: a) HRTEM processed image of a crystal of SbVO4 oriented along the [100] zone axis showing the presence of SRO. b) FFT of the original image
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-2465 Structural changes induced by high energy ball milling of a magnetite-graphite-molybdenum disulphide blend

Häusler I.1, Dörfel I.1, Peplinski B.1, Rooch H.1, Österle W.1
1BAM Federal Institute for Materials Research and Testing
ines.haeusler@bam.de

Automotive brake systems show a wide range of possible variations of brake pad formulations. Despite of that, the microstructures of the tribofilms which form during the braking and which are essential for the tribological properties of brakes are analog. All films show essentially a similar composition, namely: iron oxide (main component) as well as graphite and metal sulphides as minor components. Furthermore, the nanocrystalline structure is a main characteristic of such films. To establish a basis for systematic investigations of tribo-film properties as a function of the composition, a model system was prepared by mixing and ball milling of the three essential components. For characterization of the ball-milled powders X-ray diffraction (XRD) and transmission electron microscopy (TEM) were applied.

XRD investigations showed that the diffraction pattern of the powder blend was drastically changed in the result of that ball milling: The position, intensity and profile of some diffraction lines changed substantially, other disappeared completely. In particular, the Bragg reflections of MoS2 and graphite are no longer detectable. Obviously, the ball milling caused a break-down of the crystal structure of the initial components and led to their chemical reaction. Therefore, TEM methods are used to clarify: Where are the missing components and what happened during ball milling?

The samples for the TEM investigations were prepared in two steps. The ball milled powder was rubbed onto a SiC disc which contained cracks. Subsequently, a cross-sectional target preparation of a powder-filled crack was performed, using FIB technique by a Quanta 3D device. Fig. 1 shows a dark-field scanning TEM image (DF-STEM) of such a TEM lamella.

The structural and chemical properties of the powder mixture in the crack were analysed using a TEM/STEM JEOL JEM 2200FS (FEG, 200 kV, UHR pole piece, in-column energy filter), equipped with a LN2 free energy dispersive X-ray SD detector from Bruker Company (XFlash® 6T, Energy resolution 128 eV, detector size 30 mm2) and an ASTAR system (NanoMegas Company) for scanning nano-beam electron diffraction (SNBED) including the ACOM software for calculations of phase and orientation maps. A DigiScan system (Gatan Company) for scanning TEM (STEM), two dark-field detectors as well as a bright field detector and a 1k slow-scan CCD camera (Gatan) complete the system. For simulations of HREM images the software JEMS [Stadelmann] was used.

The TEM investigations identified magnetite nanocrystallites, embedded in a matrix of MoS2 and graphite which is partly amorphous, partly crystalline with a loss of translation symmetry in [00.1] direction, representing the van der Waals linkage of this structure of hexagonal layers.

Fig. 1: Cross-sectional DF-STEM view of the thinned lamella showing the powder mixture in a ceramic substrate crack

Fig. 2: HRTEM image of the powder mixture in the crack of the SiC disc (cf. Fig. 1; FIB lamella).

Fig. 3: MoS2; a) Structure in [2-1.0] orientation; b) Experimental HRTEM detail (see marked areas in Fig. 2) with intensity profiles which demonstrate the variations of distances of lattice fringes; c) Simulated HRTEM images of unstrained MoS2 for different thicknesses (Scherzer focus) using JEMS [Stadelmann]
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-2772 The study of disorder and lattice dynamics using electron diffraction

Eggeman A. S.1, Illig S.2, Hua X.3, Grey C. P.3, Sirringhaus H.2, Midgley P. A.1
1Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Rd, CB3 0FS, UK, 2Cavendish Laboratory, University of Cambridge, 19 JJ Thomson Ave, Cambridge CB3 0HE, UK, 3Department of Chemistry, University of Cambridge, Lensfield Rd, CB2 1EW, UK
ase25@cam.ac.uk

Although many of the physical properties of materials are strongly dependent on the structure's atomic disorder, direct measurement of such disorder can be difficult, especially where the materials are nanostructured or as thin films. However the small (nm-sized) probe available in a TEM, together with the sensitivity of electron diffraction intensities to even small atomic displacements makes it an ideal method to analyse the disorder in modern materials.

One of the major difficulties for accurately simulating electron diffraction patterns from disordered structures is the need to include dynamical scattering. Using GPU processing, a version of the multislice code has been developed to allow these dynamical simulations to be performed in a time-effective manner [1]. This has enabled a number of studies to be performed on different materials systems that exhibit disorder and lattice vibrations.

The first of these described here is on TIPS-pentacene, a high performance organic semiconductor [2]. Here a lattice vibration arising from a transverse displacement of the conductive pentacene molecule was determined from electron diffraction patterns (shown in Figure 1a) together with a refinement of the ensemble of pentacene fragment displacements (shown in Figure 1b). This provided direct evidence in support of molecular dynamics simulations, which allowed interpretation of the reported transport properties. This has led to the investigation of a family of related pentacene derivatives, such as TMTES-pentacene, with additional significant lattice vibrational modes. An example of the more complex diffuse scattering in this material is shown in Figure 1c, with a dynamical simulation shown in Figure 1d).

Another disordered system under study is lithium vanadate [3]. This is a layered oxide into which lithium ions can intercalate, making it a candidate material for high density lithium ion battery electrodes (replacing the current graphite electrodes that offer very poor stored charge density). There are a number of static disorder mechanisms occurring in the material (a typical electron diffraction pattern is shown in Figure 2a), involving both the vanadium oxide lattice as well as the lithium distribution through the material. Preliminary simulations to identify these different diffuse features (for example in Figure 2b) Show how this approach will be used to describe the complete structure of this material.

[1] A. S. Eggeman, A. London & P. A. Midgley, Ultram. 134 (2013), 44-47
[2] A. S. Eggeman et al. Nature Materials, 12 (2013), 1045-1049
[3] A. R. Armstrong et al. Nature Materials, 10 (2011), 223-229

The authors would like to acknowledge financial support from ERC grant 291522-3DIMAGE and the 7th Framework Programme of the EC: ESTEEM2, contract no. 312483.

Fig. 1: Electron diffraction patterns indicating diffuse scattering from a) TIPS-pentacene with b) dynamical scattering simulations. c) Experimental diffraction from TMTES-pentacene with d) scattering simulations

Fig. 2: a) Experimental diffraction pattern recorded from lithium vanadate parallel to [010], b) dynamical simulations of the diffuse streaks (indicated by dotted lines) found in this pattern.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-3137 Study of TiXSi1-X thin film polycrystalline and metallic glasses by transmission electron microscopy and atomic force microscopy

Siqueira C. A.1, Bettini J.1
1Brazilian Nanotechnology National Laboratory, Campinas, São Paulo, 13083970, Brazil 1
jefferson.bettini@lnnano.cnpem.br

Bulk metallic glasses formed by supercooling the liquid state of metallic alloys have potentially superior mechanical properties than those of crystalline materials, such as high strength and large elastic strain. The main issue for metallic glass is the formation rapid shear banding, significantly reducing their structural applications. Recently, it has been demonstrated this issue can be contoured when the size is reduced [1, 2]. Thermal evaporation is used as a way to growth thin films, and in specific cases, it can be used to growth of metallic glasses. In this work, thin films of Ti:Si alloys were produced by thermal evaporation. An extensive study of this material was performed using transmission electron microscopy (TEM) and atomic force microscopy (AFM). Thin Ti:Si alloys support films for electron microscopy were prepared by coating standard EM grids with evaporated films floated off mica. The mechanical stability of films was followed by eye when the film floated in the water and also checking the cover of the film in the TEM grid. We have grown Ti:Si alloys where the composition was varied from 5% of Ti to 95% of Ti. The films composition was double-checked by Energy Dispersive Spectroscopy (EDS) and Electron Energy Loss Spectroscopy (EELS). Transmission electron image and electron diffraction were used to check the crystallinity of the films (Figure 1). Ti:Si alloys films presented a polycrystalline structure or metallic glass structure depending on the composition. The roughness surface and thickness of Ti:Si films were measured by Atomic Force Microscopy (AFM). At room temperature, the specific resistance of the films was followed by four-probe method. Finally, the oxidation of films was measured by EELS (Figure 2).

References:

[1] Dongchan Jang and Julia R. Greer, Transition from a strong-yet-brittle to a stronger-and-ductile state by size reduction of metallic glasses, Nat. Materials, 9, 215 (2010).

[2] J. H. Luo, F. F. Wu, J.Y. Huang, J. Q. Wang, and S. X. Mao1, Superelongation and Atomic Chain Formation in Nanosized Metallic Glass, PRL 104, 215503 (2010).

We would like to thank LCS for the use of AFM equipment, Dr. Carlos Cesar Bof Bufon for four-probe measurements and CNPq for the financial support no. 482978/2011-2.

Fig. 1: Selected Area Electron Diffraction of a) Ti95Si5 thin film and b) Ti70Si30 tin film. Ti95Si5 sample showed polycrystalline diffraction pattern and Ti70Si30 an amorphous pattern.

Fig. 2: EELS Spectrum of a) Ti L edge and O K edge of Ti95Si5 tin film and b) Si L edge of Ti70Si30 thin film. Ti95Si5 sample showed a caracteristic EELS Spectrum of TiO2 while Ti70Si30 showed a Si pure.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-11-P-5724 Production of Fe-based amorphous alloys coatings

Afonso C. R.1, Gargarella P.1, Bolfarini C.1, Botta W. J.1, Kiminami C. S.1
1Department of Materials Engineering (DEMa), Universidade Federal de São Carlos (UFSCar)
conrado@ufscar.br

Bulk metallic glasses present high mechanical strength and good resistance to sliding and abrasive wear and corrosion [1]. This characteristic, in association with the very high values tensile strength up to 4 GPa for Fe-based bulk glassy alloys indicating that coatings can represent good application’s opportunities for metallic glasses. In the present work, we chose the glass formers Fe60Cr8Nb8B24 , Fe72Nb4Si10B14 and Fe43.2Co28.8B19.2Nb4Si4.8 (%at) alloys to produce coatings over mild steel plate substrates using powder flame spray (PFS) and spray forming processes [2]. Nitrogen atomized powders of Fe60Cr8Nb8B24 alloy with spherical morphology in the size range < 45 µm were used for the PFS process. Fe43.2Co28.8B19.2Nb4Si4.8 glassy matrix composite coatings were produced as well by pre-placed laser cladding on AISI 1020 steel. The microstructure of the powders and coatings were characterized by X-ray diffraction (XRD), scanning (SEM) and transmission electron microscopy (TEM) and differential scanning calorimetry (DSC). Dry sand/rubber wheel apparatus were used to evaluate wear behaviour of the amorphous coating produced by PFS process. The PFS coatings presented high fraction of amorphous phase with a layered structure, high porosity (~10%) and low oxidation level. The pre-placed coating formed micrometric-sized dendrites of the ductile α-(Fe,Co) phase homogeneously dispersed in a glassy matrix as a result of convection effects during the processing together with iron borides formed in the coating resulted in hardness of 1045 HV. Fe72Nb4Si10B14 spray formed 1mm thick coating showed porosity around 5% with almost fully amorphous structure, according to Figures 1 and 2 (SEM and TEM analysis). The low volume loss after the wear tests indicated a good wear resistance. The present results suggest that pre-placed laser cladding, spray forming and powder flame spray are promising processing routes to fabricate Fe-based glassy and nanocrystalline coatings for industrial applications.

The authors would like to thank FAPESP (São Paulo State Research Foundaton) for the finantial support thriugh the Project # 2012/18429-0, and the company Petrobras.

Fig. 1: SEM-BSE micrographs showing the microstructure of the PFS coating using atomized powder of Fe60Cr8Nb8B24 alloy showing (a) general view of the microstructure with amorphous and crystalline particles and, in detail (b) nanocrystalline FeNbB intermetallics embedded in the remaining amorphous phase.

Fig. 2: TEM micrographs in bright field (BF) mode of coatings for Fe60Cr8Nb8B24 alloy obtained by PFS coating (C1) showing (a) general microstructure with nanocristals embedded in an amorphous matrix, (b) ring shaped selected area diffraction (SAD) patterns of typical of nanocrystalline structure.
↑ Go to the Table of content ↑

MS-12. Magnetic, superconducting, ferroelectric and multiferroic materials

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-12-IN-1463 Exploring Oxides at Atomic Scale

Arbiol J.1,2, Guzman R.1, de la Mata M.1, Magen C.3, Queralto A.1, Belarre F. J.1, Coll M.1, Gazquez J.1, Varela M.4,5, Puig T.1, Obradors X.1
1Institut de Ciència de Materials de Barcelona, ICMAB-CSIC, 08193 Bellaterra, Spain, 2Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain, 3Laboratorio de Microscopías Avanzadas, Instituto de Nanociencia de Aragon-ARAID, Universidad de Zaragoza, 50018 Zaragoza, Spain, 4Universidad Complutense de Madrid. Madrid 28040, Spain, 5Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA
arbiol@icrea.cat

The structural properties of oxide materials at the nanoscale have acquired an increasing interest in the last decade due to the wide range of newly improved applications. Metal oxides can be redesigned at the atomic scale creating interfaces or heterostructures that may lead to novel or improved functionalities (magnetic, superconducting, ferroelectric…). In this way, atomic resolution microscopy plays an important role in order to understand the influence that changes in structure and chemistry (strain, presence of defects, composition, oxidation state) may have on the physical properties of these materials.
In the present work, we choose two examples where by making use of aberration corrected scanning transmission electron microscopy (STEM) in its different working modes, we show state of the art analyses on different oxide systems. On one hand, we analyze the interaction of spontaneously segregated oxide nanoparticles (BaZrO3 and Ba2YTaO6) randomly distributed within YBa2Cu3O7 (YBCO) superconducting nanocomposite films. It is interesting to study the role of these nanoparticles on the generation of defects and incoherent interfaces with associated strain, which at the same time influence the flux pinning efficiency of these HTS superconductors. In this way, we have been able to unambiguously identify the atomic structure of the individual defects, their intrinsic self-assembling behavior as well as their interaction (Fig. 1) [1,2].
On the other, we analyze the case of CeO2 nanostructures. CeO2 has been deeply studied for its widespread range of applications: It is employed in solid oxide fuel cells (SOFTs) as ionic conductor; the variable oxidation state (OS) of cerium (Ce4+↔ Ce3+) makes ceria a suitable catalyst; but it also has interesting ionic conductivity and dielectric properties; and it is commonly used as buffer layer for oxide superconductors. Thus, it is important to determine the oxidation state at atomic scale, as well as localizing the position of the oxygen atoms (Fig. 2). We have used a combination of low angle annular dark field (LAADF), annular bright field (ABF) and STEM-EELS to study the presence of ordered oxygen vacancies in partially reduced CeO2 nanostructures (such as nanowires and nanopyramids).

References:

[1] A. Llordes, et al., Nat. Mater., 11, 329 (2012)
[2] R. Guzman, et al., Appl. Phys. Lett., 102, 081906 (2013)

FP7-NMP-LA-2012-280432 (EUROTAPES). ORNL supported by US DoE MSED. UCM supported by ERC StG STEMOX. MdlM and AQ with JAE Pre-Doc scholarships by CSIC & European Social Fund.

Fig. 1: a) Atomic resolution HAADF image of an YBCO-BZO interface along the [001] direction. The enlarged areas correspond to the YBa2Cu3O7 (Y123) and Y2Ba4Cu8O16 (Y248) structures. b) Atomic models for the Y123 and Y248 structures along [001]. c) FFT filtered image of marked zone in a), showing the Y123-Y248 transition.

Fig. 2: a) Atomic resolution ABF STEM image obtained at a CeO2/LaAlO3 interface. b) Atomic model of the interface including the presence of Oxygen vacancies (Ce3+). c) ABF STEM image simulation.
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-12-IN-1873 Atomic scale views of charge transfer processes in ferromagnetic/superconducting complex oxide interfaces

Salafranca J.1,2, Rincon J.2,3, Tornos J.1, Leon C.1, Santamaria J.1, Dagotto E. R.2,4, Pennycook S. J.5, Varela M.1,2
1GFMC, Universidad Complutense, 28040 Madrid, Spain , 2Materials Science & Technology Div., Oak Ridge Natl. Lab., Oak Ridge, TN 37831, USA , 3Center for Nanophase Materials Sciences, Oak Ridge Natl. Lab., Oak Ridge, TN 37831, USA , 4Dept. of Physics & Astronomy, Univ. of Tennessee, Knoxville, TN 37996, USA , 5Dept. of Materials Science & Engineering, Univ. of Tennessee, Knoxville, TN 37996, USA
mvarelaxx1@gmail.com

A variety of physical phenomena can be found in complex oxide interfaces due to the presence of competing interactions with similar characteristic energies. In particular, ferromagnetic/superconducting (FM/SC) heterostructures based on combining a colossal magnetoresistant manganite such as La2/3Ca1/3MnO3 (LCMO) with a high Tc superconducting cuprate like YBa2Cu3O7-δ (YBCO) have attracted much attention. These heterostructures allow studying the interaction between superconductivity and magnetism in strongly correlated systems. Also, the competition between electrostatic effects and orbital physics can give rise to exotic electronic reconstructions. It has been reported that electronic charge can be transferred from the manganite to the cuprate [1,2], inducing a net magnetic moment in the Cu atoms as well as changes in orbital occupation [3-5]. In this talk we present a study of the structure, chemistry and electronic properties of oxide FM/SC interfaces combining electron microscopy with theoretical calculations. By means of atomic resolution scanning transmission electron microscopy and electron energy-loss spectroscopy (EELS), we find that the interfaces display high structural quality and are chemically sharp (Fig. 1). Through the analysis of the EELS fine structure, we can produce maps of the transition metal oxidation state profile across the interface. These maps suggest a non-monotonic modulation of the d-orbital occupancy across the layers, resulting from a transfer of electrons into the cuprate. Model calculations will be used to explain these profiles in terms of the competition between standard charge transfer tendencies (due to band mismatch), strong chemical bonding effects across the interface, and chemical disorder with different characteristic length scales. Research at ORNL supported by the U.S. Dept. of Energy, Basic Energy Sciences, Materials Sciences & Engineering Division, and through the Center for Nanophase Materials Sciences, sponsored by the Scientific User Facilities Division, DoE-BES. JSal was supported by the ERC Starting Investigator Award STEMOX and Juan de la Cierva JCI-2011-09428. Research at UCM supported by Spanish MICINN/MINECO through MAT2011-27470-C02 and Consolider Ingenio 2010 - CSD2009-00013 (Imagine), and by CAM grant S2009/MAT-1756 (PHAMA). Computations supported by the National Center for Supercomputing Applications (US DoE, contract no. DE-AC02-05CH11231).

[1] M. Varela et al., arXiv:cond-mat/0508564.

[2] S. Yunoki et al., Phys. Rev. B 76, 064532 (2007)

[3] J. Chakhalian et al., Nature Phys. 2, 244 (2006)

[4] J. Chakhalian et al., Science 318, 1114 (2007)

[5] J. Salafranca and S. Okamoto, Phys. Rev. Lett. 105, 256804 (2010)

[6] C. Visani et al., Phys. Rev. B 84, 060405 (2011)

Fig. 1: Figure 1: Z-contrast image of a LCMO/YBCO/LCMO trilayer. The inset shows the result of overlaying EELS maps using normalized integrated intensities for the Mn L2,3 (red), Ba M4,5 (blue), and La M4,5 (green) edges, on a matching scale. Data from a Nion UltraSTEM100, at 100 kV, equipped with a Gatan Enfina spectrometer. Adapted from ref. [6].
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-12-IN-2027 Strain-driven multiferroic thin films and their bi-phase reversibility: a TEM study at atomic resolution

Ke X.1, Zhang J.2,3, Ramesh R.3,4, Van Tendeloo G.1
1EMAT (Electron Microscopy for Materials Science), University of Antwerp, Groenenborgerlaan 171, Antwerp, 2020, Belgium, 2Department of Physics, Beijing Normal University, Beijing, 100875, China, 3Department of Physics, University of California, Berkeley, 94720, USA, 4Department of Materials Science and Engineering, University of California, Berkeley, 94720, USA
xiaoxing.ke@uantwerpen.be

BiFeO3 (BFO) has been one of the most widely studied multiferroic materials in the past decade. Compared to its bulk crystal with a rhombohedral structure, heteroeptaxially constrained thin film of BFO has demonstrated a significant increase in its intrinsic electric polarization (~60µC/cm2). The enhancement of the polarization is attributed to its high sensitivity to small changes in lattice structure. Further development in coherent heterostructure of BFO/LAO has therefore put its strain-sensitivitiy into application and successfully produced distortive perovskite structure. pseudo. Pseudo-rhombohedral (R-phase) and super-tetragonal BFO (T-phase) thin films display a high spontaneous polarization up to ~148 µC/cm2. Adding to its outstanding multiferroic behavior the large difference in c/a ratio between R- and T-phase BFO has attracted attention for potential use as energy storage in shape memory device. Although the diversity and multi-functions of BFO phases present an opportunity to implement thin film devices, its stimulus-response and reversibility under harsh conditions remain to be investigated.

In this abstract, we focus on in-situ study of stain-driven BFO thin film where its R-T phase transformation is tuned via strain distribution and temperature control. Focused Ion Beam (FIB) is used to tailor strain distribution at BFO/LAO interface at two dimensions as in-plane epitaxial strain provides driving force for distortive perovskite structures. When the strain is retained in one dimension (LAO//[100]) on purpose and stepped-released along its perpendicular direction (LAO//[010]), interlaced R-T phases are reserved when lamella thickness is above 300nm. Nevertheless, when thickness is reduced down to below ~100nm, only R-phase remains as evidenced by TEM. In addition, it is noticed that the strain-released R-phase presents different lattice structure near the LAO interface. Since ferroic properties are a sensitive function of the relative positions of anions and cations in the structure, HRSTEM is performed to investigate the structure at atomic resolution and with picometer precision. These data can cast light on the interface engineering to alter the polarization.

The reversibility between R-T phases is demonstrated by in-situ TEM. A pure R-phase BFO film at room temperature is transformed into pure T-phase at 400°C in a reversible manner. The c/a ratio difference between T-phase and R-phase gives a maximum full strain of up to ~14% vertical to the thin film interface. As dislocations are crucial to ferroelectric properties including domain walls pinning etc., the possibilities of introducing dislocations during thermal-induced phase transition are studied by in-situ HRSTEM down to an atomic scale.

X. Ke and G. Van Tendeloo acknowledge the ERC Advanced Grant No. 246791-COUNTATOMS.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-1425 TEM studies on RMnO3 multiferroic materials

Zhang Q H.1, Tan G T.2, Wang L J.1, Gu L.1, Hirata A.3, Chen M W.3, Jin C Q.1, Yao Y.1, Wang Y G.1, Duan X F.1, Yu R C.1
1Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 10090, China, 2Department of Physics, Beijing Normal University, Beijing 100875, China, 3WPI Advanced Institute for Materials Research, Tohoku University, Sendai, 980-8577, Japan
rcyu@aphy.iphy.ac.cn

Multiferroic materials, displaying simultaneously ferromagnetism and ferroelectricity, have recently attracted growing interest due to their intriguing physical properties and potential applications.1,2 In this presentation, we show our transmission electron microscopy results of RMnO3 multiferroic materials. Using state-of-the-art aberration-corrected annular-bright-field and high-angle annular-dark-field scanning transmission electron microscopy, we investigated the structure of multiferroic vortex domains in YMnO3 at atomic scale. Two types of displacements were identified among six domain walls; six translation-ferroelectric domains denoted by α+, γ−, β+, α−, γ+ and β−, respectively, were recognized, demonstrating the interlocking nature of the anti-vortex domain. We found that the anti-vortex core is about four unit cells wide. We reconstructed the vortex model with three swirling pairs of domain walls along the [001] direction. Two types of 180 degree domain walls, i.e., the transverse and the longitudinal domain walls are identified, which is in consistency with the interlock between ferroelectric and structural translation domain wall predicted previously.3 These wall structures are different from the polarization inversion in conventional ferroelectrics. These results4-6 are very critical for the understanding of topological behaviors and unusual properties of the multiferroic vortex. In addition, we found a new ferroelectric phase induced by oxygen vacancy ordering. We proposed a proper structure model and examined its correctness.

Rererences

1 A. J. Freedman and H. Schmid, Magnetoelectric Interaction Phenomena in Crystals, Gordon and Breach: London (1975).

2 G. Srinivasan, E. T. Rasmussen, B. J. Levin, and R. Hayes, Phys. Rev. B, 65 (2002) 134402.

3 T. Choi, Y. Horibe, H. T. Yi, Y. J. Choi, W. D. Wu, and S. W. Cheong, Nat. Mater. 9 (2010) 253.

4 Q. H. Zhang, L. J. Wang, X. K. Wei, R. C. Yu, L. Gu, A. Hirata, M. W. Chen, C. Q. Jin, Y. Yao, Y. G. Wang, X. F. Duan, Phys. Rev. B (R), 85 (2012) 020102.

5 Qinghua Zhang, Guotai Tan, Lin Gu, Yuan Yao, Changqing Jin, Yanguo Wang, Xiaofeng Duan, Richeng Yu, Scientific Reports, 3 (2013) 2471.

6 Qinghua Zhang, Sandong Guo, Binghui Ge, Peng Chen, Yuan Yao, Lijuan Wang, Lin Gu, Yanguo Wang, Xiaofeng Duan, Changqing Jin, Banggui Liu, and Richeng Yu, J. Am. Cer. Soc., DOI: 10.1111/jace.12747.

This work was supported by 973 (2012CB932302) and NNSF of China (11174336). The sample was from Pengcheng Dai’s group at UT/Rice supported by the US DOE, BES (DE-FG02-05ER46202).

Fig. 1: HAADF image of the anti-vortex domains. The domain walls are marked by red dotted lines and the red circle is are used to mark the region of the vortex core.

Fig. 2: The reconstructed model of the vortex domains along [001] direction. The red dotted lines and the circle indicate the locations of domain walls and the core of the vortex, respectively. The yellow circles with a dot and the blue circles with a cross represent Yup and Ydown atoms. The green circles represent Y atoms at the paraelectric position.

Fig. 3: Type-Ⅰand type-Ⅱ domain walls along the [010] direction including all the four kinds of domain walls.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-1466 HRTEM Study of Topological Vortex-like Domain Pattern in Multiferroic Hexagonal Manganite YMnO3 by Cs-corrected TEM

Zhang X.1,2, Yu Y.1,2
1School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China, 2Beijing National Center for Electron Microscopy, Beijing 100084, China
xzzhang@tsinghua.edu.cn

    Multiferroic hexagonal manganite YMnO3 (YMO) has attracted extensive attention owing to its vortex-like domain patterns with ferroelectric, magnetic, and structural correlations and the resulting attractive physical properties. The complicated domain pattern of YMO has long been discovered. However, little is known about the real structure and origin of this domain pattern. Recent studies have shown that on the micro-scale, the typical domain pattern in YMO has a cloverleaf shape, and six interlocked ferroelectric and structural antiphase domain walls merge into a vortex core. However, the configuration of the domain pattern is still controversial. To gain insight into this problem, researches on nanoscale, particularly on the atomic-scale are necessary.

    Using Cs-corrected transmission electron microscopy, we demonstrate the atomic details of a topological vortex-like domain pattern in multiferroic hexagonal manganite YMnO3. We have demonstrated an example of the topological vortex-like domain pattern of YMO on the atomic-scale. The vortex-like pattern with domain configuration of α+, β-, α+, β-, α+, β- is revealed. We point out that distinguishing of six ferroelectric domains in the vortex-like pattern is not the sufficient condition to determine whether this pattern is a real vortex or not (Fig.1). The antiphase relationship must be carefully checked. Besides, the existence of domain walls (DWs) in two-dimensional projection is also a crucial point to be considered when discussing the vortex-like domain pattern. Our atomic detailed observations push forward the understanding of the intriguing vortex-like patterns in hexagonal manganites. Moreover, configurations of two kinds of interlocked DWs are revealed with the help of atomistic simulation (Fig.2). The antiphase domain boundary I (APBⅠ) and ferroelectric domain boundary (FEB) are overlapped while the antiphase domain boundary II (APBⅡ) and the FEB are separate on the atomic-scale, and surface hexagon of the hexagonal unit cell in the vicinity of the DW boundary suffers from slightly distortion. As more and more theoretical work are begin to focus on the interlocked DWs recently, such extensive investigations of the DWs can provide a reference for future theoretical studies. Finally, we should emphasize that all these fascinating results can be useful not only for this specific material but also as a guideline to envision domain behavior of other hexagonal multiferroics. The present study can throw further light on understanding of structure-property relation in multiferroic hexagonal manganites.

Reference: Y. Yu, et al., Appl. Phys. Lett. 103, 032901 (2013).

This work is supported by the National Science Foundation of China and the Ministry of Science and Technology of China.

Fig. 1: (a) Hexagonal crystal unit cell of YMO (red: Y, green: Mn, blue: O). (b) Atomic projection of YMO in the [110] zone axis. (c) HRTEM image of a multi-domain region. Areas with different polarization directions are indicated by arrows and different types of DWs are indicated by different color dot lines.

Fig. 2: (a), (b) The atomic displacements of Y ions near the APBⅠ + FEB [region Ⅰ in Fig. 1c] and APBⅡ + FEB [region Ⅱ in Fig. 1c], respectively. (c), (d) The [110] atomic structural model of APBⅠ + FEB and APBⅡ + FEB, respectively. (e), (f) Upper panels are the close-ups of the immediate vicinity of the DW boundary in (a) and (b), respectively.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-1524 Characterization of synthetic Fe-oxide nanoparticles: microstructure and magnetic properties

Lari L.1,2, Kepaptsoglou D. M.3, Nedelkoski Z.1,2, Moeen Uddin G.1,2, Wen T.4, Booth R. A.4, Oberdick S. D.4, Majetich S.4, Lazarov V. K.1,2
1Department of Physics, University of York, Heslington, York, YO10 5DD, UK , 2York-JEOL Nanocentre, University of York, Heslington, York, YO10 5BR, UK, 3SuperSTEM Laboratory, STFC Daresbury Campus, Keckwick Lane, Daresbury WA4 4AD, UK, 4Department of Physics, Carnegie Mellon University, Pittsburgh, PA, USA
leonardo.lari@york.ac.uk

Magnetite nanoparticles (NPs) are attracting a lot of attention due to their potential applications that range from data storage to biomedical applications such as hyperthermia. The main challenge in the field of magnetite NPs is controlling their structure, size and stoichiometry, parameters that ultimately determine their magnetic properties.
The different behaviour of the magnetic properties of Magnetite Nanoparticles has previously been associated to differences in sample size distributions and/or the deviation from stoichiometric bulk Fe3O4.
In this work we study NPs synthetized using three different methods based on high temperature decomposition followed by organic surfactant coating, [1-3] NPs synthesis methods.
Magnetic measurements of the three samples show quite different magnetic behaviours despite being monodispersed samples. All three methods produce NPs and based on selected area electron diffraction and x-ray diffraction, all produce magnetite with the possibility of a small maghemite fraction. Despite this, the measured magnetic properties are quite different.
Magnetization curves at 10 K and 5 T field gives magnetization values of 81 ± 12 emu/g for Sun method particles 12.3 ± 2.9 nm in diameter and drastically lower values for Hyeon and Calvin method, 39 ± 4 emu/g and 37 ± 1 emu/g, respectively. High resolution scanning transmission electron microscopy shows that the Colvin method particles have significant amount of stacking faults such as antiphase domain boundaries and twinning defects. These defects when present in thin films magnetite [4], can change the relative proportion of ferromagnetic and antiferromagnetic interactions, which consequently modify their magnetic properties, mainly due to the increase of antiferromagnetic interactions across the defects. The presence of similar type of defects in NPs can also potentially modify strongly their magnetic properties, and it is a possible reason for the huge variation of their magnetic properties that depend strongly of the preparation method.

[1] S. Sun, et al., J. Am. Chem. Soc. 126, 273 (2004)
[2] J. Park, et al. Nature Mater. 3, 891 (2004)
[3] W. W. Yu, et al, Chem. Comm. 20, 2306 (2004)
[4] D. T. Margulies, et al., Phys. Rev. Lett. 79, 5163 (1997)

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-1560 Observation and analysis of skyrmions and their helicity in composition-spread chiral helimagnets

Shibata K.1, Yu X. Z.2, Hara T.3, Morikawa D.2, Kanazawa N.1, Kimoto K.3, Ishiwata S.1, Matsui Y.3, Tokura Y.1,2
1The University of Tokyo, Tokyo, Japan, 2RIKEN Center for Emergent Matter Science (CEMS), Wako, Japan, 3National Institute for Materials Science, Tsukuba ,Japan
shibata@cmr.t.u-tokyo.ac.jp

A magnetic skyrmion is a topologically-stable spin vortex structure (Fig. 1a). Recently skyrmions and their hexagonally crystalized phase, skyrmion crystal (SkX), have been confirmed to form in chiral-lattice helimagnets by small-angle neutron scattering and Lorentz microscopy1,2. Skyrmions have recently attracted much attention due to their intriguing electromagnetic properties, therefore controlling skyrmions and skyrmion lattice itself, such as the size and helicity, spin swirling direction, becomes the important topic to be studied.
Here, we report on the size and helicity in composition-spread chiral-lattice helimagnets Mn1−xFexGe with using combined observational and analytical techniques of TEM3. We observed magnetic structures in each composition sample with Lorentz microscopy, and analyzed composition, crystal chirality and thickness by energy dispersive X-ray spectrometry (EDX), convergent-beam electron diffraction (CBED), and electron energy-loss spectroscopy (EELS), respectively.
We observed spatial distribution of skyrmion size and its dependence on local composition by Lorentz microscopy and EDX (Fig. 1b-d). Furthermore, we analyzed skyrmion helicity and crystal chirality with utilizing Lorentz microscopy and CBED in each microcrystal to reveal composition dependence of their correlations (Fig. 2).
We found through these analyses that the skyrmion size and the helical modulation period show non-monotonous variation with the composition x, accompanying a divergent behavior around x = 0.8, where the correlation between magnetic helicity and crystal chirality is reversed (Fig. 3).
The underlying mechanism is the continuous variation of spin-orbit interaction strength, accompanying sign change depending on composition in the metallic alloys.
1, S. Mühlbauer et al., Science 323, 915 (2009),
2, X. Z. Yu et al., Nature 465, 901 (2010),
3, K. Shibata et al., Nature Nanotechnology 8, 723-728 (2013).

The authors thank N. Nagaosa, S. Seki, T. Kurumaji and Y. Okamura for helpful discussions. This study was supported by a Grant-in-Aid for Scientific Research (grant no. 24224009) from MEXT, and by the Funding Program forWorld-Leading Innovative R&D on Science and Technology (FIRST Program).

Fig. 1: Dependence of skyrmion size on x, obtained in a microcrystal of Mn1-xFexGe with varying composition (x≈0.7). (a) Schematic of the magnetic-moment configuration in a skyrmion. (b) Lorentz TEM image of skyrmions (area A). (c) Composition (x) map obtained by STEM-EDX (area A). (d) x dependence of skyrmion size in area A and area B (not shown here).

Fig. 2: Over-focused Lorentz TEM images of magnetic helix and skyrmion, and CBED disk patterns used for determination of crystal chirality obtained for nominally x=1.0, 0.9 and 0.7 samples. L and R represent left-handed and right-handed crystal chirality, respectively, as determined by comparison between experimental and calculated patterns.

Fig. 3: Composition dependence of the magnetic period and the correlation between crystal chirality and magnetic helicity. The sign of correlation is described by different background colors.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-1709 3 dimensional elemental mapping of epitaxially grown Fe-Co nanodumbbells

Altantzis T.1, Liakakos N.2, Gatel C.3, Blon T.2, Lentijo-Mozo S.2, Lacroix L. M.2, Respaud M.2, Soulantica K.2, Bals S.1, Van Tendeloo G.1
1EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020, Antwerp, Belgium , 2INSA, UPS, CNRS, LPCNO, Université de Toulouse, 135 avenue de Rangueil, 31077, Toulouse, France, 3Centre d’Elaboration de Matériaux et d’Etudes Structurales (CNRS), 29, rue Jeanne Marvig, 31055, Toulouse, France
thomas.altantzis@uantwerpen.be

New developments in the field of nanotechnology drive the need for advanced quantitative characterization techniques in three dimensions that can be applied to complex nanostructures. Such nanostructures are often composed of different compounds, since the co-existence of different materials on the same nano-object increases its functionalities, or changes its original properties thanks to a synergy between the constituents. When magnetically “soft” Fe cubes grow on magnetically “hard” Co nanorods, the overall effective magnetic anisotropy is drastically reduced compared to the bare Co nanorod.

Here, we investigated the 3 dimensional (3D) structures of Fe-Co hybrid nanodumbbells using electron tomography. In materials science, electron tomography measurements are typically performed in High Angle Annular Dark Field Scanning Transmission Electron Microscopy (HAADF-STEM) mode (Figure 1).1 We can clearly observe that cubes with concave facets are attached on a nanorod. Although the morphology of the nanodumbbells is clear, the small difference in atomic number Z between Co and Fe does not allow to distinguish between the two components in the final 3D reconstruction. New developments concerning the design of EDX detectors enabled us to combine EDX and electron tomography,2 leading to a 3D visualization of the chemical structure as presented in Figure 2. In this figure, Fe is presented using a transparent visualisation and the segregation between Fe and Co is clear. The Co nanorods penetrate into the Fe nanostructures and a limited amount of Fe has been deposited on the Co rod close to the Fe cube. It must be noted that this information could not be obtained from conventional HAADF-STEM reconstructions. Therefore, the use of EDX tomography will be of great potential in the investigation of a broad range of complex nanostructures.

1. S. J. Pennycook. Annual Review of materials Science, 1992, 22, 171-195

2. P. Schlossmacher et al. Microscopy Today, 2010, 18, 14

The authors acknowledge financial support from European Research Council (Grant COUNTATOMS and Grant COLOURATOMS) and the European Union Seventh Framework Programme - ESTEEM2.

Fig. 1: a) HAADF-STEM image of the dumbbell, which is part of the tilt series. b, c and d) 3D representation of the reconstructed volume presented along different viewing directions. The distribution of Fe and Co in the structure cannot be distinguished.

Fig. 2: a, b and c) 2D EDX maps from the same particle revealing the distribution of the elements in the structure. d) 3D representation of the reconstructed volume by using EDX-tomography. We clearly see the segregation between Fe (green) and Co (blue).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-1708 Energy-filtered nano-beam electron diffuse scattering of relaxor ferroelectrics PMN and PMN-xPT

Pacaud J.1, Saïdi W.1, Withers R.2, Dkhil B.3, Zuo J M.4
1Institut Pprime, UPR CNRS 3346, University of Poitiers, 11 Ave P et M Curie, 86962 Chasseneuil, France, 2Em. Pr., Research School of Chemistry, Australian National University, Canberra, Australia, 3Laboratoire Structure, Propriétés et Modélisation des Solides, UMR CNRS 8580, Ecole Centrale Paris, France, 4Material Science and Engineering, University of Illinois at Urbana Champaign, USA
jerome.pacaud@univ-poitiers.fr

PbMg1/3Nb2/3O3 (PMN) and its solid solution (1-x) PbMg1/3Nb2/3O3 -(x)PbTiO3 (PMN-xPT) are relaxor ferroelectrics which have attracted attention in the last few decades because of their very interesting dielectric and piezoelectric properties and have since be two of the most extensively studied. All the previous studies emphasized the role of the local structural fluctuations leading to local changes in symmetries [1] due to displacements of ions in the unit-cell. This behavior is quite universally known in the perovskite family and is driving most of its properties.
We studied PMN and PMN-xPT by electron diffuse scattering using an in-column energy filter and Imaging-Plates as detector. This set-up is particularly well suited for this kind of investigation due to the high sensitivity for very low signal. Compared to the neutron, electron diffraction has the advantage of two dimensional recording of diffuse scattering and eventually sensitivity to charge ordering but quantitative analysis is limited due to the complication of multiple scattering and the lack of sufficient energy resolution for the study of inelastic phonon scattering. We found evidences for streaks of intensity along the [110]* direction as previously found in PbZr1/3Nb2/3O3 (PZN) with neutron diffraction [2] (Fig. 1). Moreover, weak diffuse scattering sheets can be observed along (111)* reciprocal planes showing the existence of correlations along the [111] directions of the direct lattice. Figure 2 shows a diffraction pattern taken along [02-1] zone axis presenting both diffuse features. This sheets of diffuse scattering can be related to the displacement of Pb ions along the diagonals of the cube found by simulation [3] but greatly complexify the analysis of the shape of the diffuse intensity.
This study shows that, taking into account the limitations previously stated, electron diffuse scattering can be an invaluable tool for investigating local fluctuations of the structure in such systems.

[1] K.-H. Kim, D.A. Payne, J.M. Zuo, Phys. Rev. B, 86, 184113 (2012)
[2] T.R. Welberry, D.J. Goossens, M.J. Gutmann, Phys. Rev. B, 74, 224108 (2006)
[3] M. Pasciak, T.R. Welberry, J. Kulda et al. Phys. Rev. B, 85, 224109 (2012)

Fig. 1: Left: Section of neutron diffuse scattering in PZN from [2]. Right: [001] zone axis energy filtered electron diffraction pattern on Imaging Plate (this study).

Fig. 2: [2-10] zone axis electron diffraction pattern showing the <110>* streaks and the {111}* sheets of diffuse scattering. The small dots are other <110>* streaks crossing the Ewald sphere.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-1737 Thickness dependent atomic structure and microsctructures of supertetragonal multiferroic BiFeO3 thin films

Pailloux F.1, Couillard M.2,3, Saidi W.1, Fusil S.4, Bruno F.4, Garcia V.4, Carrétéro C.4, Jacquet E.4, Bibes M.4, Barthélémy A.4, Botton G. A.4, Pacaud J.1
1Institut Pprime, UPR3346 CNRS-University of Poitiers, France, 2CCEM, McMaster University, Hamilton, Canada, 3NRC Canada, Ottawa, Canada, 4UMPhy CNRS/Thales, Orsay, France
frederic.pailloux@univ-poitiers.fr

BiFeO3 thin films grown on LaAlO3 substrates exhibit a giant c/a ratio driven by the in-plane epitaxial stress imposed by the substrate leading to a so called supertetragonal phase. Previous structural studies [1] have shown that this behavior applies to film thicknesses ranging from few unit cells to several tens of nanometers. Deeper analysis of the thicker films reveals the coexistence of a mixture of phases which are most probably promoted by the stress relaxation as they are not observed in the thinner ones [2]. Despite numerous studies on this topic, the real atomic structure of BiFeO3 remains under debate.

We revisit the atomic structure and microstructure of these supertetragonal phases of highly strained epitaxial BiFeO3 thin films. Quantitative atomic resolution scanning transmission electron microscopy is used to directly image the atomic positions. Electron energy loss spectroscopy is further employed to reveal subtle electronic structure features.

The monoclinic Cm phase suggested by electron diffraction and predicted by ab initio calculations is evidenced by annular bright field imaging (fig.1). The relative positions of Bi, Fe and O atoms in the BiFeO3 unit cell have also been probed to compare them with the structural models proposed in the literature by ab initio calculations [3] confirming the reorganization of the unit-cell with the transformation of the oxygen octahedron in a square-based pyramid; this structure being nano-twinned in thicker films (fig. 2). Monochromated EELS experiments have subsequently been carried out to investigate the O-K and Fe-L23 edges. For the thinner films, the O-K fine structures experience changes from the interface to the surface of the film. Multilinear fit of the data set with specific fingerprints was employed in order to map the fine structures. The map reveals a modification of the crystal field resulting in a distortion of FeO5 pyramids described above by the underlying symmetry imposed by the LAO substrate.

Interpreted in a framework of antiferrodistortive distortions coupling with the substrate, these results point towards a phase near the interface closer to the P4mm purely tetragonal phase [4].

Our results emphasize the need for quantitative microscopy to investigate the subtle structure of these complex functional materials.

[1] H. Béa et al., Phys. Rev. Lett., 102 (2009), 217603.

[2] I.C. Infante et al., Phys. Rev. Lett., 107 (2011), 237601.

[3] O. Diéguez et al., Phys. Rev. B., 83 (2011), 0940105.

[4] F. Pailloux et al. Phys Rev B, to be published

Part of this work was carried out at the CCEM, McMaster University/NSERC.

This work was supported by ANR Oxitronics project.

Région Poitou-Charente is acknowledged for financial support.

Fig. 1: (a) magnified area of an ABF micrograph of the T-like phase: SNR has been improved through noise filtering by multivariate statistical processing. Arrows indicate the oxygen atoms. (b) The super‑tetragonal monoclinically distorted Cm unit-cell projected along the [001] direction (a=9.475 Å, b=7.580 Å) superimposed on (a).

Fig. 2: (a) HAADF-STEM image of a BFO//LAO interface. (b) Tilt map of the planes perpendicular to the film/substrate interface, showing the presence of nano-twins on the left-hand side of the map.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-1797 Analysis of Magnetic Devices by Spin-polarized Scanning Electron Microscopy (Spin SEM)

Kohashi T.1, Motai K.1, Nishiuchi T.2, Hirosawa S.2,4, Maruyama Y.3
1Central Research Laboratory, Hitachi, Ltd., Hatoyama, Saitama 350-0395, Japan, 2Magnetic Materials Research Laboratory, Hitachi Metals, Ltd., Osaka 618-0013, Japan, 3HGST Japan, a Western Digital company, Odawara 256-8510, Japan, 4Present address: National Institute for Material Science, Tsukuba 305-0047, Japan
teruo.kohashi.fc@hitachi.com

Various magnetic devices, such as permanent magnets and recording devices, have made progress in their performances and still been studied intensively. To keep up with the improvement of these devices, measurement techniques should also be further developed. Since spin SEM can observe magnetic domain structures, it has been used to study magnetic devices[1,2]. However, to study upcoming subjects in magnetic device, unprecedented measurements are being demanded. In this paper, two new challenges are presented to meet these demands.
The first challenge is to measure magnetization at the grain boundaries of a NdFeB sintered magnet. Magnetism at the grain-boundary phase has a significant effect on the coercivity of the magnet; therefore, it has been intensively studied [3-5]. The grain-boundary phase is, however, very thin, typically 2 nm. It is therefore difficult to measure its magnetization separately from that inside grains. Accordingly, taking advantage of the short probing depth (< 1 nm) of spin SEM, the magnetization in the grain-boundary phase was measured. Spin-SEM images were taken on the fractured sample surface, as shown in Figs. 1( (a) before and (b) after surface milling by Ar ions). The dashed circles show the same grain with intense contrast, which is supposed to be fractured inside the grain. Other areas have weak contrasts in (a) and strong contrast in (b), because the grain-boundary phase, which covers the fractured surface in (a), was removed by the milling, and then the magnetization inside the grain was revealed in (b). The spin-polarization data from the grain-boundary phase were analyzed, and it is concluded that the grain-boundary phase has substantial magnetization and is ferromagnetic.
The other is to observe the change in magnetization in the magnetic shield of a HDD recording head. To achieve higher recording density and to write a small bit, the behavior of the magnetic shield around the main pole in the recording head should first be understood. Accordingly, in the present study, a system for applying a current was installed in the sample stage of spin SEM to activate the head. Magnetic-domain images of the magnetic shield were obtained by changing the current applied to the head (Fig. 2). These images confirm that the direction of the magnetization in the shield was moved considerably by switching the polarity of a current of 20 mA.
References
[1]T. Kohashi et al., J. Electron Microsc.; 59, 43(2010).
[2]T. Kohashi et al., J. Mag. Soc. Jpn. 33, 374(2009).
[3]H. Sepehri-Amin et al., Acta Mater. 60, 819(2012).
[4]T. Nakamura et al., Proc. of 22nd Int. Workshop on Rare-Earth Permanent Magnets and their Applications, 230(2012).
[5]Y. Murakami et al., The 37th annual conference on MAGNETCS in Japan, 5p-B2(2013).

Fig. 1: Spin-SEM images of fractured surface of NdFeB sintered magnet: (a) before and (b) after ion milling.

Fig. 2: Spin-SEM images of the magnetic shield in a HDD recording head. Applied currents are +20 mA (left) and -20 mA(right). Colors in the images show the magnetization direction by the color wheel shown below.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-1824 Lorentz TEM study on nanometric magnetic bubbles in Ru-doped bilayered manganites

Morikawa D.1, Yu X. Z.1, Kaneko Y.1, Tokunaga Y.1, Nagai T.2, Kimoto K.2, Arima T.1,3, Tokura Y.1,3
1RIKEN Center for Emergent Matter Science (CEMS), Wako, Japan, 2National Institute for Materials Science (NIMS), Tsukuba, Japan, 3The University of Tokyo, Tokyo, Japan
d-morikawa@riken.jp

Recently, nanometric magnetic textures have been extensively investigated for new data storage devises [1,2]. The control of magnetic anisotropy plays an important role for the creation of magnetic bubbles (kinds of magnetic texture). To realize nanometric bubbles, we target the ferromagnetic compound La1.2Sr1.8Mn2O7, which has easy-axis type magnetic anisotropy by the substitution of Ru for Mn [3,4]. We have observed nanometric magnetic bubbles accompanied by stripe domains at zero magnetic field.
Figures 1 (a) and 1 (b) show the schematic crystal structure and magnetization curves at 5 K of bilayered manganese oxide La1.2Sr1.8(Mn1-yRuy)2O7 (y = 0.1), respectively, indicating that the compound has tetragonal crystal structure (I4/mmm) and easy-axis type magnetic anisotropy. To reveal the magnetic configurations, we carried out Lorentz transmission electron microscopy (TEM) observation by using a transmission electron microscope JEM-2100F operated at an accelerating voltage of 200 kV. The magnetic field was applied perpendicular to the thin sample by using the objective lens. Figure 2 shows Lorentz TEM Fresnel images of La1.2Sr1.8(Mn1-yRuy)2O7 with (a) y = 0.07 and (b) y = 0.1 with an over-focused condition and zero electric current on objective lens. We have found that nanometric magnetic bubbles are formed, and the type of bubble depends on the Ru doping level. The thin plate of the y = 0.07 compound shows the so-called Types I and II magnetic bubbles. Type I magnetic bubble is found in the red rectangular area in Fig. 2 (a). Figure 2 (c) shows the schematics of magnetic bubble and the expected contrast of Lorentz TEM Fresnel image with the over-focused condition for Types I and II. On the other hands, only Type II magnetic bubbles were obtained for the y = 0.1 thin sample. Lorentz TEM images of La1.2Sr1.8(Mn1-yRuy)2O7 with y = 0.05, 0.07, 0.1, 0.15 indicate that the two types of magnetic bubbles coexist in the y = 0.05 and 0.07 sample, while both of the thin plates for the y = 0.1 and 0.15 show only Type II magnetic bubbles. The y-dependence is attributable to the change in magnetic anisotropy with Ru doping.
[1] N. Nagaosa and Y. Tokura, Nat. Nanotechnol. 8, 899 (2013).
[2] X. Z. Yu et al., Nat. Commun. 5, 3198 (2014).
[3] Y. Onose et al., Appl. Phys. Lett. 86, 242502 (2005).
[4] X. Z. Yu et al., J. Magn. Magn. Mater. 302, 391 (2006).

The authors would like to thank Ms. W. Z. Zhang (NIMS) and Materials Characterization Support Unit (RIKEN CEMS) for experimental supports. This study was supported by Founding Program for World-Leading Innovative R&D on Science and Technology (FIRST program), the JSPS Grant-in-Aid for Scientific Research, (No. 24224009), and Nanotechnology Platform (No. A-13-NM-0156) of the MEXT, Japan.

Fig. 1: (a) Schematic of the crystal structure of bilayered manganese oxide La1.2Sr1.8(Mn1-yRuy)2O7 (0 ≤ y ≤ 0.15). (b) Magnetization curves of a La1.2Sr1.8(Mn1-yRuy)2O7 (y = 0.1) single crystal at 5 K.

Fig. 2: Lorentz TEM Fresnel images of the magnetic bubbles for (001) La1.2Sr1.8(Mn1-yRuy)2O7 crystals at 6 K: (a) y=0.07, (b) y=0.1. (c) Schematics of Type I and Type II magnetic bubbles and their over-focused Lorentz TEM Fresnel images.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2007 Atomic level evolution of lattice variation and strontium redistribution in Sr-δ-doped La2CuO4

Wang Y.1, Sigle W.1, Baiutti F.2, Gregori G.2, Logvenov G.2, Maier J.2, van Aken P. A.1
1Max Planck Institute for Intelligent Systems, Stuttgart, Germany, 2Max Planck Institute for Solid State Research, Stuttgart, Germany
yi.wang@is.mpg.de

Superconductivity in copper oxides arises when a parent insulator compound is doped beyond some critical concentration [1]. In the case of La2CuO4 (LCO), high-Tc superconductivity is obtained either by substituting La3+ with Sr2+ or by inserting interstitial O2- [2]. Recently, by using atomic layer-by-layer oxide molecular beam epitaxy, we have fabricated Sr-δ-doped LCO multilayered structures, in which some atomic layers of LaO have been fully substituted by SrO layers, and by varying the spacing between the LCO and SrO layers high-Tc superconductivity (~ 40 K) has been obtained. In this contribution, the local variation of in-plane and out-of plane atomic lattice parameters and strontium redistribution in the Sr-δ-doped LCO multilayers on LaSrAlO4 (LSAO) substrate was investigated using a JEOL ARM 200CF scanning transmission electron microscope (STEM) equipped with a cold field-emission electron source, a probe corrector, a large-solid-angle SDD-type EDX detector, and a Gatan GIF Quantum ERS spectrometer. The microscope was operated at 200 kV, a semi-convergence angle of 30 mrad, and 90 - 370 mrad and 11-23 mrad collection angles were used to obtain high angle annular dark-field (HAADF) and annular bright-field (ABF) images.

Figure 1 shows the crystal structure model of LCO (a) and LSAO (b) and their epitaxial orientation relationship, where the atomic positions are assigned in the HAADF (c) and ABF (d) images. Figure 2a represents a typical cross-sectional HAADF image from the substrate to the vacuum, where no structure defects are observed. Atomically resolved HAADF and ABF images, which were simultaneously acquired of the Sr-δ-doped region, are presented in Figure 2b and 2c. The local lattice and oxygen octahedral distortion were evaluated by the image analysis.

A detailed study on the Sr redistribution at the interface was performed by atomic resolution HAADF imaging in combination with EDX and EELS, as shown in Figure 3. Due to the difference in atomic number (ZSr=38, ZLa=57), the atomic columns dominated either by La or Sr give rise to different contrast in the HAADF image. In the Sr-δ-doped region the atomic column intensity is significantly lower than in pure LCO. An averaged image intensity profile along the growth direction shows that in the Sr-δ-doped region the image intensity has a relatively sharp intensity drop followed by a slowly increasing intensity pointing to an asymmetric distribution of Sr along the growth direction. This asymmetric Sr distribution is confirmed by Sr-L EDX and Sr-L2,3 EELS (insert in Fig.3) line-scan profiles.

[1] P.A.Lee, N.Nagaosa, and X.G.Wen, Rev.Mod.Phys. 78 (2006) 17.
[2] B.O.Wells, et al., Science 277 (1997) 1067.

The research leading to these results has received funding from the European Union Seventh Framework Programme [FP/2007-2013] under grant agreement no312483 (ESTEEM2). U. Salzberger is particularly acknowledged for TEM specimen preparation.

Fig. 1: The projected crystal structure of (a) LCO and (b) LSAO. Atomic-resolution HAADF (c) and ABF (d) images showing the orientation relationship between LCO and LSAO.

Fig. 2: (a) HAADF image of Sr-δ-doped LCO on LSAO. Enlargement of simultaneously acquired (b) HAADF and (c) ABF images of the Sr-δ-doped area (Schematically highlighted by the yellow box).

Fig. 3: HAADF image of Sr-δ-doped LCO. The inset shows the integrated Sr-L EDX and Sr-L2,3 EELS line profiles.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2048 Strain analysis of PZT thin films by aberration-corrected HRTEM and dark-field electron holography (DFEH)

Denneulin T.1, Wollschläger N.2, Österle W.2, Hÿtch M. J.1
1CEMES CNRS, 29 rue Jeanne Marvig, 31055 Toulouse, France., 2BAM, Unter den Eichen 87, 12205 Berlin, Germany.
thibaud.denneulin@cemes.fr

Piezoelectric materials have a large number of applications in ferroelectric memories and microelectronics devices [1]. In particular, piezoelectronic transistors are foreseen as a new alternative to the metal–oxide–semiconductor devices [2]. They exhibit high speed and low power consumption thanks to the properties of piezoelectric and piezoresistive materials. Consequently there is a growing need for the characterization of strain in piezo thin films.
Here we have investigated a 100 nm thick Pb(Zr0.2,Ti0.8)O3 layer (PZT) epitaxially grown on a SrTiO3 substrate (STO) using transmission electron microscopy (TEM) strain measurement techniques. Dark-field electron holography (DFEH) [3] was used to provide strain maps with ≈500 nm field-of-view and 6 nm spatial resolution. DFEH was operated in aberration-corrected Lorentz mode on a multiple biprism Hitachi HF-3300 microscope (I2TEM-Toulouse). High-resolution TEM (HRTEM) was conducted on an aberration-corrected FEI F20 Tecnai microscope in order to observe the structure of the ferroelectric domains. Strain maps with 2 nm resolution and 120 nm field-of-view were obtained by geometrical phase analysis (GPA).
Fig. 1(a) is a TEM image of the sample prepared by focused ion beam. Fig. 1(b,c) shows the growth εzz and the in-plane εxx deformation maps obtained by DFEH. The deformation is defined relatively to the STO substrate. The εzz deformation in the PZT layer is 5-6% while εxx is ≈1%. Considering the lattice parameters of tetragonal PZ0.2T given in the literature (c = 0.414 nm and a = b = 0.394 nm), the measurements are coherent with a fully relaxed state. This can be related to the high number of dislocations at the interface. Localized variations due to the dislocations can be observed on the shear and the rotation maps (Fig. 1(d,f)). Inclined domains can also be seen but higher resolution is needed to interpret the information. Fig. 2(a,b) is a HRTEM image showing an a-domain separated by 90° domain walls from the surrounding c-domains. The rotation map (Fig. 2(c)) obtained by GPA shows that the lattice is tilted by ≈2.7° across the domain wall. Similarly, the εxx and εzz strains (Fig. 2(d,e)) vary by ≈5% across the wall which is coherent with the parameters given above. Strain gradients similar to those observed in PbTiO3 [4] were also evidenced and this will be discussed during the presentation.
In the next experiments we plan to combine DFEH and GPA with in-situ biasing to investigate the piezoelectric effect and the switching of the ferroelectric domains.

[1] M Dawber et al, Rev. Mod. Phys. 77 (2005), 1083–1130
[2] DM Newns et al, Adv. Mater. 24 (2012), 3672–3677
[3] MJ Hÿtch et al, Nature 453 (2008), 1086–1089
[4] G Catalan et al, Nat. Mater. 10 (2011), 963–967

This work received financial support from the European Union under the Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative Reference 312483-ESTEEM2 and the European Metrology Research Programme (EMRP) Project IND54 Nanostrain. The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union.

Fig. 1: (a) TEM image of a Pb(Zr0.2,Ti0.8)O3 (PZT) layer grown by epitaxy on a SrTiO3 (STO) substrate. (b) εzz growth, (c) εxx in-plane and (d) εxz shear strain maps obtained by dark-field electron holography. (e) Strain profiles extracted from the maps along the [100] growth direction. (f) Lattice rotation map.

Fig. 2: (a) HRTEM image of the PZT layer showing an inclined a-domain. (b) Enlargement of the image in the region defined by a dashed rectangle. (c) Rotation map and (d-g) strain field calculated by geometrical phase analysis. An average filter of 2 nm has been applied to reduce the noise.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2073 Electron holographic tomography reveals the three-dimensional magnetic field distribution of Co2FeGa Heusler nanowires

Simon P.1, Wolf D.2, Sturm S.2, Lichte H.2, Levin A. A.1, Qian J.1, Fecher G. H.1, Wang C.1, Felser C.1
1Max-Planck-Institut für Chemische Physik fester Stoffe, Nöthnitzer Str. 40, 01187 Dresden, Germany, 2Institute of Structure Physics, Triebenberg Laboratory, Technical University of Dresden, Zum Triebenberg 50, 01328 Dresden Zaschendorf, Germany
Paul.Simon@cpfs.mpg.de

The ferromagnetic Heusler compounds, with a L21 ordered structure, emerge as fascinating multifunctional materials exhibiting appealing physical properties that are interesting to versatile research fields such as spintronics [1], magnetic shape memory [2], and topological insulators [3,4]. Specifically the predicted half-metallicity in many Heusler compounds enable them as promising electrodes (spin injection sources) for spintronic devices based on giant magnetoresistance or magnetic tunnel junction. The properties of Heusler compounds may be enhanced by accessing the nanoscale-size regime [5]. The unique one-dimensional geometry of nanowires has been reported to give birth to novel structural and physical properties. For example, in the regime of semiconductor spintronics, nanowire morphologies have been used to demonstrate fundamental spintronics functionality such as tunneling spin injection and two-terminal spin-valve in heterojunctions. We report the first synthesis of L21 ordered Co2FeGa nanowires using SBA-15 silica as the templates. The well-ordered Heusler structure of Co2FeGa nanowires are verified by X-ray diffraction and extended X-ray absorption fine structure. We employ electron holographic tomography to study the local magnetic configurations of free-standing Co2FeGa Heusler nanowires [6-8]. The magnetic dipole stray fields of Co2FeGa nanowires are observed by electron holography indicating single magnetic domains. The internal and outward magnetic inductions flux distribution of the nanowires are visualized by electron holography and the magnetic phase shifts give rise to intrinsic magnetic induction magnitude lying in the range of 0.4 - 0.7 T as lower limit taking into account the shielding effect of the stray field, the induction reaches a value of up to 1.1 T. By acquiring electron holographic tomography, the three-dimensional distributions of the x-component of electrostatic and magnetic field inside the nanowire are mapped for the first time.

[1] C. Felser, G.H. Fecher, Spintronics: From Materials to Devices (Springer Netherlands 2013)

[2] K. Bhatacharya, S. Conti, G. Zanzotto, J. Zimmer, Nature, 428, 55 (2004)

[3] S. Chadov, X.L. Qi, J. Kübler, G.H. Fecher, C. Felser,S.C. Zhang, Nat. Mater. 9, 541 (2010)

[4] X.L. Qi, R. Li, J. Zang, and S.C. Zhang, Science 323, 1184 (2009)

[5] W. Lu and C.M. Lieber, Nat. Mater. 6, 841 (2007)

[6] G. Lai, T. Hirayama, A. Fukuhara, K. Ishizuka, T. Tanji, A. Tonomura, J. Appl. Phys. 75,4593 (1994)

[7] R.E. Dunin-Borkowski, T. Kasama: Proc. Micr. Microanal. 10, 1010–1011 (2004)

[8] D. Wolf, A. Lubk, F. Röder, H. Lichte, Curr. Op. Sol. State & Mater. Sci. 17, 126 (2013)

 Financial support by the DFG is gratefully acknowledged (Project TP 2.3-A in research unit FOR 1464 ‘ASPIMATT’).

Fig. 1: Figure 1. Imaging of magnetic stray fields of the Co2FeGa dipole: (a) reconstructed phase of hologram. (b) Phase 14 times amplified. (c) Short wire of 300nm length placed horizontally on Y-shaped bars of a lacey grid surrounded by vacuum. The wire is visualized with complete dipole stray field. (d) Phase 20 times amplified.

Fig. 2: Electrostatic 3D potential of Heusler nanowire.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2170 Insulating ferromagnetic LaCoO3-δ films: the role of ordered oxygen vacancies

Biskup N.1,2,8, Salafranca J.1,2, Mehta V.3, Suzuki Y.3,4, Pennycook S.5, Pantelides S.6,7,2, Varela M.2,1
1Dept. de Fisica Aplicada III & Instituto Pluridisciplinar, Univ. Complutense de Madrid. Spain., 2Materials Science and Technology Div., Oak Ridge Natl. Laboratory, USA., 3Dept. of Material Science and Engineering, Univ. of California, USA., 4Dept. of Applied Phys. & Geballe Lab. for Advanced Materials, Stanford University, USA., 5Dept. of Materials Science and Engineering, Univ. of Tennessee, Knoxville, TN, USA, 6Dept. of Physics and Astronomy, Vanderbilt Univ., USA., 7Dept. of Electrical Engineering and Computer Science, Vanderbilt Univ., USA., 8Centro Nacional de Microscopia Electronica, Madrid, Spain
biskupneven@yahoo.com

The ordering of oxygen vacancies constitutes a new avenue towards finding novel physical phenomena and new functionalities in transition metal oxides. Ordered arrays of vacancies may not only enhance or suppress existing properties, but also lead to collective states absent in the perfect crystallographic phase, or in samples with randomly distributed defects. Along these lines, epitaxial strain in thin films can be used to tune ordering phenomena not present in the bulk, which may result in unusual properties. For example, epitaxial LaCoO3 (LCO) thin films under tensile strain, e.g., grown on SrTiO3, are ferromagnetic (FM) at low temperatures, while the bulk material is non-magnetic [1]. The origin of the observed FM ordering has been debated extensively on the basis of theoretical calculations and complementary experimental data. Competing interactions of comparable magnitude permit Co atoms to present low-spin (LS), intermediate spin (IS) or high-spin (HS) [2,3]. Different types of Co spin states and ordering have been proposed in strained films, but the layers have been assumed stoichiometric and no O deficiency has been considered [4,5]. Here, we demonstrate, using atomic resolution electron microscopy and electron energy-loss spectroscopy (EELS), that epitaxial LCO thin films contain ordered arrays of oxygen vacancies (Figure 1). The epitaxial strain is relaxed through the local lattice expansion at ordered oxygen-deficient atomic planes. The vacancies lead to excess electrons in the Co d-states and thus to the charge order of Co ions, as demonstrated by EELS through the Co L23 ratio. Density-functional calculations show a spin state ordering not present in bulk that results in a net magnetic moment [6]. 
[1] D. Fuchs et al., Physical Review B 75, 144402 (2007).
[2] J. B. Goodenough, Journal of Physics and Chemistry of Solids 6, 287 (1958).
[3] M. A. Señaris-Rodriguez, J. B. Goodenough, Journal of Solid State Chemistry 116, 224 (1995).
[4] W. S. Choi et al., NanoLetters. 12, 4966 (2012).
[5] J. Fujioka et al., Physical Review Letters 111 027206 (2013).
[6] N. Biškup et al., Physical Review Letters 112, 087202 (2014).

U.S. DoE, BES, Materials Sciences & Engineering Div., and ORNL’s Center for Nanophase Materials Science; ERC St. Invest. grant #239739 STEMOX and Juan de la Cierva program; U.S. DoE, Div. of Materials Sci. & Eng. (contracts# DE-AC02-05CH11231 and DE-SC0008505); U.S. DoE Grant #DE-FG02-09ER46554 and the McMinn Endowment; Natl. Center for Supercomputing Applications (U.S. DoE).

Fig. 1: Atomic resolution EELS mapping of the LCO film. High resolution Z-contrast image with O K map as the inset. The profile of O K map (yellow graph) shows the O vacancy ordering. Bottom right: Model showing the magnetic ordering, according to DFT calculations. Green arrows denote the spin orientation for the high spin Co. Adapted from reference [6]
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2182 Atomically resolved EELS study of B-site ordering in a La2CoMnO6 thin film

Egoavil R.1, Gauquelin N.1, Béché A.1, Verbeeck J.1, Jungbauer M.2, Huhn S.2, Moshnyaga V.2, van Tendeloo G.1
1EMAT, Department of Physics, University of Antwerp, Antwerp, Belgium, 2Physikalisches Institut, Universität Göttingen, Göttingen, Germany
ricardo.egoavil@uantwerpen.be

Double perovskites like a ferromagnetic insulating La2CoMnO6 have received considerable attention because of surprising multiferroic properties [1]. One of the most striking examples is that the local ordering of the Co/Mn cations plays an important role in the magnetic properties of this material. A ferromagnetic transition arises from the local interaction of the Co-O-Mn chains [2]. The absolute valence and spin states for Co and Mn in ordered and disordered phases are however not fully understood [3,4]. XAS experiments suggest that ordered phases involve Co2+ and Mn4+ states, whereas the disordered phase – Co3+ and Mn3+ [3]. We performed a complete atomic scale TEM characterization, including a detailed analysis in reciprocal space of the Bragg reflection corresponding to the Co/Mn ordering. A procedure was implemented to estimate the density of ordered and disordered regions in the La2CoMnO6 thin film grown on SrTiO3(111) substrate by metalorganic aerosol deposition technique. Atomically resolved EDX maps directly show the ordered and disordered regions in accordance with EELS data from the same regions. The fine structure of the Co-L2,3 and Mn-L2,3 edges indicates subtle differences between ordered and disordered phases in good agreement with XAS experiments [3]. In the disordered phase the valence state is estimated to be a mixture of 2+ and 3+ for Co, and for Mn a mixture of 3+ and 4+ states, while in the ordered phase Co2+ and mainly Mn4+. These local observations provide a better understanding of electronic interactions between Co and Mn and their importance for the ferromagnetic transition in double perovskites, like La2CoMnO6, and similar materials.

[1] Yi Qi Lin and Xiang Ming Chen, J. Am. Ceram. Soc., 94 [3], 782–787 (2011).
[2] H. Wadati, D.G. Hawthorn, T. Z. Regier, M. P. Singh, K.D. Truong, P. Fournier, G. A. Sawatzky, Chemical and Material Science, 49, 114-115 (2009).
[3] Santu Baidya and T. Saha-Dasgupt, Phys. Rev. B 84, 035131 (2011).
[4] M. P. Singh, K. D. Truong and P. Fournier. Appl. Phys. Lett. 91, 042504 (2007).
[5] M. P. Singh, S. Charpentier, K. D. Truong, and P. Fournier. Appl. Phys. Lett. 90, 211915 (2007).

The European Union under FP7-IFOX, ERC starting grant VORTEX, Integrated Infrastructure Initiative-ESTEEM2 and the scientific research Flanders FWO project are gratefully acknowledged for financial support.

Fig. 1: Fig.1: (a) HAADF overview image and (b,c) atomically resolved EDX maps of two different regions in the La2CoMnO6 film, showing the Co/Mn distribution of the ordered (1) and disordered (2) phases. The EDX color maps demonstrate the cation ordering in a direct way. (d,e) ELNES of Mn and Co showing subtle differences related to valency changes.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2193 Lattice expansion and polarization induced by dipole at LaAlO3 (or CaTiO3)/SrTiO3 interfaces

Choi J. K.1, Lee J. H.2, Baek S. H.2, Moon S. Y.2, Kim J. S.2, Park J. H.2,3, Hwang C. S.2,3, Choi J. H.2, Chang H. J.1
1Advanced Analysis Center, Korea Institute of Science and Technology, Seoul, Korea, 2Electronic Materials Research Center, Korea Institute of Science and Technology, Seoul, Korea, 3Department of Materials Science and Engineering and Inter-university Semiconductor Research Center, Seoul National University, Seoul, Korea
almacore@kist.re.kr

The recent growth technique enabling fabrication of oxide heterostructure with atomically abrupt interfaces between dissimilar materials allows us to investigate novel physics of emergent phenomena arising at the interface. One of the striking examples is the recent discovery of two-dimensional electron gas at oxide interface: electrically conducting layer is formed at the insulating LaAlO3 (LAO) and SrTiO3 (STO) interface, which has raised interest in the interface structure of LAO/STO. Recently developed spherical aberration corrected imaging techniques in (scanning) transmission electron microscopy is powerful tool to study the interface structure enabling mapping the atomic position and measuring the displacement of atomic column in cross-section plane within an error below an Å in a real space regardless of film thickness.

In this work, epitaxial 30 nm STO or CaTiO (CTO) films were grown on TiO2-terminated (001) SrTiO3 substrates by pulsed laser deposition (PLD) and subsequently, 5-nm- thick LAO layer was deposited on the STO/STO and CTO/STO structure under the same deposition condition. We utilized Z-contrast imaging in aberration-corrected scanning transmission electron microscopy to measure the lattice distance change across the interface at picometer level. Cross-sectional samples for STEM analysis were prepared by mechanical thinning, precision polishing, and ion milling (PIPS 691). STEM images and EEL spectra were collected using a Cs-corrected microscope Titan S 80-300 operated at 300 kV equipped with a Gatan Quantum 966 spectrometers.

The lattice distances along c-direction between A-site cations, that is Sr-Sr, Ca-Ca and La-La distances, were measured from the displayed image (35×10 unit cells) of HAADF image of a LAO/STO (Fig. 1(a)) and a LAO/CTO thin films (Fig. 1(b)) grown on STO in [100] pseudocubic orientation. Remarkably, out-of-plane lattice distance dc increases at the STO/LAO interface through 3 unit cells and falls down reaching to LAO bulk value in the graph averaging the distances over 10 rows parallel to the interface. This abrupt expansion of unitcell along out-of-plane direction at LAO/STO interfaces was expected experimentally and theoretically in previous reports, and its origin was to be octahedral distortion or electrostrictive effect in the polar LAO layer as well as intermixing. Similar lattice expansion in c-direction was also observed in CTO/LAO interfaces. The origin of this unitcell expansion or cation displacement will be discussed based on the first principles calculations results.

The authors thank Y.W. Jeong for important contributions in TEM sampling and observation. This research was sponsored by the KIST Institutional Program (2V2920, 2E24001, 2E24070).

Fig. 1: STEM HAADF image of (a) a LAO/STO and (b) CTO/STO thin film grown on STO substrate taken along the crystallographic [100]STO direction. The graphs show strain along c-direction in STO (or CTO) and LAO as function of distance from a reference plane in the lower STO (or CTO) layer by averaging the data obtained from 10 rows along the interface.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2474 Electron doping and structure of interfaces and defects in multiferroic heterostructures with ferroelectric barrier

Marinova M.1, Gloter A.1, March K.1, Carrétéro C.2, Yamada H.2,3, Garcia V.2, Barthélémy A.2, Bibes M.2, Colliex C.1, Stéphan O.1
1LPS, Bât. 510, CNRS UMR 8502, Université Paris Sud XI, 91405 Orsay, France, 2Thales RT, 1 Avenue A. Fresnel, 91767 Palaiseau, France, 3AIST, Tsukuba 305-8562, Japan
maya.marinova@univ-lille1.fr

The ferroelectric (FE) control of ground states in multiferroic nanostructures based on strongly correlated oxides is an important task in solid state physics and spintronics. Recently a ferroelectrically induced modulation of resistivity [1] and carrier densities [2] has been shown for nanostructures between the multiferroic BiFeO3 (BFO) and the Mott insulator CaMnO3 (CMO). The strong doping sensitivity of CMO makes it susceptible to electronic state transitions induced interfacially by the large polarization of BFO. Understanding the specific properties of this and other similar oxide-based heterostructures, requires an accurate estimation of small charge modulations, strain, presence of impurities, cationic inter-diffusion, interface structure and defects, ferroelectricity, appearance of dead layers.
In this study, we address at the atomic level the electronic and structural properties of Ca1-xCexMnO3 (CCMO)/BFO (x=0, 2, 4 at% Ce) nanostructures by STEM-EELS. A HAADF image and a Ce concentration profile are given in Fig. 1(a) and (c). Upon doping with tetravalent Ce4+ partial mixed valence appears in the manganite. The changes in the oxidation state are revealed by a fine chemical shift on the Mn-L3,2 edges, Fig. 2(a). Analyzing the atomically-resolved Mn-L3 edges, we therefore propose a method to evaluate small changes in the electron density at the scale of a single unit cell for the different Ce doping levels, Fig. 2(b). This permits to estimate charge densities at the interfaces between the BFO and the CCMO layers and between the substrate and the CCMO layers, Fig. 1(b). Using theoretical calculations, the estimated two-dimensional electron gas at the BFO/CCMO interface is interpreted in terms of electrostatic doping and polar discontinuities. The as-grown interfacial termination planes are important for the stabilization of the direction of the FE polarization and for the accumulation of carriers. In addition, the STEM-EELS data reveal the presence of a dead layer at the substrate/CCMO interface related to different structural defects [3].
Further such effects as interface structure, polar discontinuity and appearance of defects will be discussed in the context of other important multiferroic heterostructures such as LaNiO3/BiFeO3 and (La,Sr)MnO3/BaTiO3/FeRh.
References
[1] H.Yamada, V.Garcia, S.Fusil, S.Boyn, M.Marinova, A.Gloter, S.Xavier, J.Grollier, E.Jacquet, C.Carrétéro, C.Deranlot, M.Bibes, A.Barthélémy, ACS Nano 7, 5385 (2013).
[2] H.Yamada, M.Marinova, P.Altuntas, A.Crassous, L.Bégon-Lours, S.Fusil, E.Jacquet, V.Garcia, K.Bouzehouane, A.Gloter, J.E.Villegas, A.Barthélémy, M.Bibes, Sci. Rep. 3, 2834 (2013).
[3] M.Marinova, A.Gloter, C.Carrétéro, H.Yamada, V.Garcia, K.March, O.Stéphan, A.Barthélémy, M.Bibes and C.Colliex, submitted.

This work was supported by the French Agence Nationale de la Recherche NOMILOPS project (ANR-11-BS10-0016), 7th framework EU program ESTEEM2 (grant No. 312483) and the ERC Grant FEMMES, No. 267579.

Fig. 1: (a) A HAADF image of the heterostructure YAlO3/CCMO/BFO (x=0, 2, 4 at% Ce) (b) The excess charge per unit cell at the interface between the YAlO3 and CMO (on the left side) and between the CCMO and the BFO(on the right side). (c) Ce concentration profile for the same heterostructure.

Fig. 2: (a) ELNES spectra of Mn-L3,2 edges of the undoped, 2 at% Ce and 4 at% Ce doped layers at 640 eV, after background subtraction, where a small chemical shift is evident upon Ce4+ substitution. (b) EEL spectra for undoped, 2 at% Ce and 4 at% Ce doped layers, in the energy range between 600 and 950 eV, where Mn-L3,2 and Ce-M5,4 edges are seen.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2532 Magnetic microstructure in stress-annealed Fe73.5Si15.5B7Nb3Cu1 soft magnetic alloys

Kovács A.1, Pradeep K. G.2, Li Z. A.3, Herzer G.4, Raabe D.2, Dunin-Borkowski R. E.1
1Ernst-Ruska Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Forschungszentrum Jülich, D-52425 Jülich, Germany, 2Max-Planck-Institut für Eisenforschung, Düsseldorf, Germany, 3Faculty of Physics and Center for Nanointegration (CeNIDE), University of Duisburg-Essen, D-48047, Duisburg, Germany, 4Vacuumschmelze GmbH & Co. KG, Hanau, Germany
a.kovacs@fz-juelich.de

The unique physical and magnetic properties of Fe-Si-Nb-Cu-B alloys [1], such as their low coercivity and high saturation magnetization combined with near-zero magnetostriction, make them attractive for high-frequency applications. Furthermore, their magnetic properties can be tailored by applying a magnetic field or stress during annealing, resulting in uniaxial anisotropy. Here, we study the nanostructure and magnetic domain state of stress-annealed Fe73.5Si15.5B7Nb3Cu1 using atom probe tomography (APT) and transmission electron microscopy (TEM). A 600 MPa stress was applied to selected samples during rapid annealing for 4 s [2], resulting in strong uniaxial anisotropy perpendicular to the stress direction, as confirmed using bulk measurements performed using a superconductive quantum interference device magnetometer. X-ray diffraction and APT studies revealed that the samples comprised 80 vol.% of a crystalline Fe3Si phase with a DO3 structure and 20 vol.% of an amorphous matrix that was enriched in B and Nb, as shown in Fig. 1 [3]. The Fe3Si grain size in the present samples was measured to be (10±3) nm, while Cu clusters were observed to form with sizes of ~6 nm. Specimens were prepared for TEM examination from rapid-annealed Fe73.5Si15.5B7Nb3Cu1 ribbons using an FEI Helios Nanolab 600i dual-beam focused ion beam (FIB) workstation. Structural studies of the samples using TEM revealed a polycrystalline microstructure without any detectable crystallographic texture, as shown in Fig. 1 (b). Fresnel defocus images and off-axis electron holograms were recorded using an FEI Titan TEM operated at 300 kV in magnetic-field-free conditions (< 0.5 mT) with the conventional microscope objective lens switched off. Off-axis electron holograms were recorded using an electrostatic biprism located close to the selected area aperture plane of the microscope. Figure 2 (a) shows a magnetic domain wall (DW) pattern comprising near-perfect 180° and 90° DWs in stress-annealed sample. Figure 2 (b) shows a Fresnel defocus image and off-axis electron holography results obtained from the intersection of a 180° DW with two 90° DWs. The insets show equally-spaced cosine phase contours around the intersection of the DWs in reconstructed phase images recorded from the same region of the specimen. The width (full-width-at-half-minimum) of the divergent contrast arising from a 180° DW in a defocus series of images, extrapolated to zero defocus, provides a value of (53±10) nm, as shown in Fig. 2 (c). Direct measurements from the corresponding phase profiles in electron holograms provided upper limits of (49±3) and (94±3) nm for the widths of the 180° and 90° DWs (not shown).

The authors acknowledge financial support from the German Research Foundation, the Helmholtz Association and the European Research Council.

Fig. 1: Figure 1. (a) APT spatial distribution map and (b) BF TEM image of Fe73.5Si15.5B7Nb3Cu1 that had been annealed at 695 °C for 10 s in the presence of a stress of 600 MPa. The electron diffraction pattern in (b) shows that the grains have random orientations.

Fig. 2: Figure 2. (a, b) Fresnel defocus image recorded 1 mm overfocus. The double-headed white arrow in (a) indicates the applied stress direction. The insets are reconstructed phase images. The step in phase between adjacent contours is 2π radians. (c) Width of the divergent contrast arising from the 180° DW measured as a function of defocus.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2757 Mapping oxygen and lanthanum sub-lattice distortions in (001)-, (110)- and (111)-oriented LaAlO3/SrTiO3 interfaces hosting 2DELs

Gazquez J.1, Herranz G.1, Scigaj M.1, Dix N.1, Sanchez F.1, Fontcuberta J.1, Varela M.2, 3
1Institut de Ciència de Materials de Barcelona, Bellaterra, Spain, 2Materials Science and Technology Division, Oak Ridge National Laboratory, Oak Ridge, USA, 3Dpt. Fisica Aplicada III, Universidad Complutense de Madrid, Madrid, Spain
jgazqueza@gmail.com

At interfaces in epitaxial films strain, chemistry, and symmetry can change, interact, and enable new functionalities. A paradigmatic case is the (001)-oriented LaAlO3/SrTiO3 (LAO/STO) heterostructure, where a confined two-dimensional electron liquid (2DEL) at the interface between these two oxide band-insulators is found. Yet, with the recently uncovered 2DELs for other growth orientations, (110)- and (111)- oriented LAO/STO interfaces [1,2], it has been realized that the response of such interfaces to built-in potentials may be more complex than previously thought, and that the polar discontinuity scenario might not be enough to account for the metallic state at the LAO/STO interface. We have used aberration corrected scanning transmission electron microscopy (STEM) in combination with electron energy loss spectroscopy (EELS) to probe in real space, with atomic resolution, how these interfaces (see figure 1) counter the effect of existing potentials along different orientations. The mapping of the oxygen sub-lattice showed the presence of different distortions in (001)-oriented interfaces, which depends on LAO thicknesses, and is compatible with the appearance of large electrostatic potentials because of the polar character of this interface. In contrast, in the (110)-oriented interface, which in the ideal non-reconstructed limit should not have any polar discontinuity, there is a clear absence of polar distortions across the (110)LAO/STO interface. On the other hand, the (111)-interfaces do not show distortions and appear to be much more diffuse and much less sharp than (001) and (110) orientations, showing an increased number of misfit dislocations, which in turn degrade their transport properties.

[1] G. Herranz et al., Scientific Reports 2 758 (2012)
[2] A. Annadi et al., Nature communications 4 1838 (2013)

Work at ORNL U.S. supported by the U.S. DOE-BES. Research at UCM supported by the ERC Starting Investigator Award. JG acknowledges the Spanish Government RyC-2012-11709 contract.

Fig. 1: From left to right, high resolution Z-contrast images of (001), (110) and (111)-oriented LAO/STO-interfaces. All images were acquired along the [110] zone axis with a 5th order aberration corrected Nion UltraSTEM 200 operated at 200 kV.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2802 Enhanced Polarization Propagation by Mixing Polar and Nonpolar Phases in Piezoelectric Oxides

Choi S. Y.1, Kim S. D.1, Choi M.1, Rhyim Y. M.1
11 Korea Institute of Materials Science, Changwon, Republic of Korea
youngchoi@kims.re.kr

Properties of new functional materials strongly depend on the composition and atomic structure down to the level of single atoms, and thus characterization at the atomic scale has been a key technology in materials science. Recently, we found via aberration-corrected scanning transmission electron microscopy (STEM) that the giant electro-strain was induced by the polar core – nonpolar shell model wherein electric field-induced strain can be enhanced by the electric field induced polarization propagations from the polar core to nonpolar shell region, as described in Fig. 1, offering a new mechanism to achieve large electromechanical coupling in non-Pb based ceramics. It has been expected that the propagation of polarization can be promoted by the mixture of polar and nonpolar phases such as in relaxor ferroelectric materials; or by using the nanocomposite materials embedding the ferroelectric nanoparticles, each of which exhibits the flexible single domain. As polarization configuration at the interface between polar and nonpolar phases in nanocomposite have a completely behave different with that of interior domains, peculiar types of polarization configuration, such as nanoscale rotational vortices, can be dominant in the nanometric dimension, where the large strain effect should be necessarily considered.

Since there is still poor discussion about the details of polarization behavior in those peculiar materials systems, we utilize aberration-corrected STEM to determine the local polarization giving rise to atomic displacement (Fig. 2) and also in-situ TEM technique to dynamically observe the polarization by biasing the electric field or mechanical stress (Fig. 3). We successfully analyzed the electrical response when a single crystalline BaTiO3 nanoparticle was mechanically compressed by a conductive indenter. Moreover, ~10 nm-BaTiO3 particles, consisting of polar core and nonpolar shell, exhibit the flexo-polarization behavior, contrary to the conventional prediction that BaTiO3 nanoparticles undergo the phase transition from ferroelectric to paraelectric. In this study, by combining the HAADF-STEM and in-situ TEM skills, the surfacial charge and strain have a crucial impact on the formation of unusual domain structures, suggesting plausible flexoelectricity in the piezoelectric oxides.

This work was supported by the Global Frontier R&D Program (2013-073298) on Center for Hybrid Interface Materials (HIM) funded by the Ministry of Science, ICT & Future Planning. (2013M3A6B1078872)

Fig. 1: Unipolar S-E curve for our polar-nonplar composite ceramic, which shows unpoled core-shell structures (I), generation of polarization in the cores by poling (II), and polarization propagation to the nonpolar shell region (III) under applied fields. The dashed line represents a poling process.

Fig. 2: (a) DF-STEM image for the core-shell structured grains in the sample. ABF-STEM images of (b) core region and (c) shell region in a CaZrO3-modified (KNaLi)NbTiO3. The brighter spots (red arrow) indicate the B-site column; the less bright spots (yellow arrow) indicate the A-site column.

Fig. 3: Cross-sectional view of the indentation process of a BaTiO3 nanoparticle.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2803 Nanoscale probe of magnetism, orbital occupation, and structural distortions in iron-based superconductors

Cantoni C.1, Idrobo J.1, Pennycook S. J.2
1Oak Ridge National Laboratory, Oak Ridge, TN, USA, 2University of Tennessee, Knoxville, TN, USA
cantonic@ornl.gov

Using aberration-corrected scanning transmission electron microscopy (STEM) coupled with electron energy loss spectroscopy (EELS) we established the validity of a method for comparing local magnetic moments that can be adopted for many Fe- and transition-metal-compounds, and used in the future to map the local magnetic moment with sub-nanometer spatial resolution. We have applied the analysis of the Fe-L2,3 ratio to a large class of iron-based superconducting (FBS) and parent compounds (some of which are imaged in Fig. 1) and showed that for these materials, the L2,3 ratio calculated with the Pearson’s method has a linear relationship with the local magnetic moment (LMM) of Fe. This local characterization of the LMM, together with information on the total 3d band occupancy and the ratio between the number of holes in 3d5/2 and 3d1/2, allowed us to clarify the nature of the puzzling LMM behavior in iron-based superconductors and relate this to different orbital occupancies. Moreover, although the common understanding has been that both long-range and local magnetic moments decrease with doping, we find that, near the onset of superconductivity, the LMM increases and shows a dome-like maximum near optimal doping, where no ordered magnetic moment is present. The Fe LMM couples to atom displacements in 122 arsenides, which give rise to nanometer-size twin domains, lowering the symmetry of the crystal, as shown in Fig. 2. These are remarkable direct observation of magnetoelastic interactions in iron-base superconductors as well as evidence of the importance of magnetic fluctuations in superconductivity.

Research supported by the Materials Sciences and Engineering Division Office of Basic Energy Sciences, U.S. Department of Energy.

Fig. 1: High angle annular dark field STEM images ofvarious iron-based compounds in different projections. a, PrFeAsO, [100]. b, Ca0.85Pr0.15Fe2As2,[100]. c, Fe0.99Te0.45Se0.55, [100]. d, TlFe1.6Se2,[100]. e, TlFe1.6Se2, [110] of √5 × √5 supercell. f, BaFe1.92Co0.08As2,[001]. g, CaFe2As2, [111]. h, TlFe1.6Se2, [110].

Fig. 2: Evolution of lattice distortions with dopinglevel and local Fe magnetic moment in Ba(Fe1-xCox)2As2; 0 ≤ x ≤ 0.16.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2840 Effect of cationic ordering on magnetic properties in Sr2FeReO6 double perovskites

Lim T. W.1,2, Kim S. D.1, Rhyim Y. M.1, Yun J. D.1, Choi S. Y.1
1Advanced Characterization and Analysis Group, Korea Institute of Materials Science, South Korea, 2Departmeent of Nano science and Engineering of Kyungnam Univercity, South Korea
i9moneya@kims.re.kr

Ferromagnetic double perovskites (A2BB’O6, A=alkaline earth metals and B/B’=transition metals) have recently attracted great attention due to their presumed half-metallicity as well as high curie temperature. To this end, Sr2FeReO6 (SFRO), one of typical double perovskites, is being actively studied with this purpose. The magnetic structure of A2BB'O6 is known to originate from the ordered arrangement of parallel Fe3+ (3d5, S=5/2) magnetic moments, antiferromagnetically coupled with Re5+ (5d2, S=1) spins, and therefore the properties of the material depend on their ordering in B-sites. In this study, the cationic ordering was controlled by excessive Re amount and their magnetic properties have been investigated as a function of cationic ordering. Furthermore, what the cationic disordered defects, so called antisite (AS) defects, look like was examined by aberration-corrected STEM.

0, 5, 10, and 15 wt%-Re-excess SFRO powders were prepared via a conventional solid state reaction for 24hr and annealed at 1000℃ for 20min in Ar. The prepared powders were sintered by Spark Plasma Sintering (SPS) at 1150℃ for 20min in Ar. XRD results confirm that cationic ordering of Fe and Re, which is one of major parameters to affect the magnetic properties in SFRO and the secondary phases are not observed in all samples (Fig. 1(a)). As shown in Fig. 1 (b), the height of (011) and (110) peaks appear to be variable by amount of excess-Re and thus cationic ordering percentages are found to be~ 75% (0%-Re), ~ 80% (5%-Re), ~ 90% (10%-Re) and ~ 95% (15%-Re) by calculating the ratio of (011) and (110) peak height and XRD simulation. Since the Ms of SFRO are deteriorated by the cationic disordering, Ms of 15wt%-Re-excess SFRO sample is the largest value amongst the prepared samples. These results directly demonstrate that excess of Re effectively enhance the cationic ordering in SFRO and accordingly the magnetic properties as well, as shown in Fig. 1 and 2. Lower Ms and higher Hc in the 0wt% Re sample reveal that the ferromagnetic alignment of Fe and Re atoms is interfered by the Fe/Re disordering. Aberration-corrected STEM results of Fig. 3 show that high degree of Fe/Re ordering maintains in 15wt%-Re-excess SFRO sample whilst cationic-disordered regions are observed in the 0wt% Re sample. This STEM observation implies that AS defects inferred by cationic disordering exhibit clustered rather than randomly scattered.

This work was supported by the Global Frontier R&D Program (2013-073298) on Center for Hybrid Interface Materials (HIM) funded by the Ministry of Science, ICT & Future Planning. (2013M3A6B1078872)

Fig. 1: Figure 1 (a) XRD patterns of SFRO-xRe with 0wt%≤x≤15wt% (b) Zoom in from 19° to 21°

Fig. 2: (a) measured magnetic property via VSM byincreasing amount of Re (b) The change of the magnetic saturation (Ms) and AS defect

Fig. 3: High-angle annular dark-fieldscanning transmission electron microscopy (HAADF-STEM) images from (a)SFRO-0wt% Re sample (b) SFRO-15wt% Re sample.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2886 TEM measurements of Cr diffusion into the Cu stabilizer in Cr-plated Nb3Sn superconducting wires with reduced Cu residual resistivity ratio.

Bartova B.1, Alknes P.2, Bordini B.2, Cantoni M.3, Devred A.4, Ballarino A.2, Bottura L.2
1CERN, European Organization for Nuclear Research, Engineering Department, CH-1211 Geneva, Switzerland, 2CERN, European Organization for Nuclear Research, Accelerator Technology Department, CH-1211 Geneva, Switzerland, 3Centre Interdisciplinaire de Microscopie Electronique, CIME, Ecole Polytechnique Federale de Lausanne, CH-1015 Lausanne, Switzerland, 4ITER Organization, Bat 519, CEA Cadarache, 13108 St. Paul-lez-Durance, France
barbora.bartova@cern.ch

Some of the bronze-route Nb3Sn wires produced for the ITER Toroidal field magnets have values of the Cu Residual Resistivity Ratio (RRR) lower than the specifications (RRR>100) when reacted with the longest ITER heat treatment cycle (high temperature plateau 200 hours at 650°C). The low RRR value was suspected to be due to the presence of the Cr plating.
Apart from an extensive RRR measurements on 98 samples conducted at CERN a detailed TEM investigation was carried out. The main motivation for TEM study was the verification of solid solution Cr diffusion into the Cu stabilizer together with the presence of Cr rich precipitates. In our study heat treated sample (200 hours at 650°C) together with non-annealed sample (0 hours) were characterized. TEM lamellas were prepared by FIB Zeiss NVision 40 CrossBeam. Lamella of 30 microns width was prepared for the 200 h sample in order to measure the Cr content far from the Cr-Cu interface. Three windows of 7.5 microns were thinned down to electrons transparency leaving 4 thicker supports in-between to avoid undesirable bending of the sample, see Fig.1. Cr plating was milled away completely to avoid any influence on final EDS results. TEM measurements were conducted on FEI Tecnai Osiris equipped with Super-X SDD EDX detectors. Measurements focusing on characterization of precipitates present in the sample were conducted at INSA Lyon using JEOL 2010F Field Emission TEM.
Large numbers of Cr rich precipitates were found in the Cu stabilizer as well as at the Cu/Cr interface. Two different types of precipitates were found. Globular precipitates at the Cr/Cu interface with sizes up to 100 nm were identified as Cr23C6. Needle-like precipitates with the sizes up to 200 nm are present up to 13 microns from Cr/Cu interface. EDS measurements suggest CrxSy type with an average at% ratio close to 1:1. From HRTEM imaging pure Cr and CrxSyOz types were also confirmed.
Point EDS measurements in Cu stabilizer were done systematically along the length of the lamella to measure Cr content. Since the solid solution limit reported for Cr in Cu varies from 0.008 at% to 0.1 at% [1] the detection limit of EDS TEM is close to the expected values. Table 1 shows that experimental Cr level varies from 0.03 to 0.01 at.% up to 15.6 microns.
Measured values of Cr would explain degradation of RRR. Position of the precipitates also confirmed that Cr diffused up to 15 microns far from the Cr/Cu interface. Although the EDS measurements are at the edge of detection limit, another technique is not accessible without the standard of CuCr with ppm level of Cr.

[1] D. Chakrabarti and D. Laughlin. The Cr-Cu system. Journal of Phase Equilibria, 5:59-68, 1984. 10.1007/BF02868727.

Fig. 1: TEM lamella of 30 microns width was prepared at the Cr/Cu interface in order to measure Cr diffusion into the Cu stabilizer. The marker on TEM lamella support denotes Cr plating side.

Fig. 2: Bright field TEM image together with the corresponding FFT diagram (inset) shows globular precipitate of Cr23C6.

Fig. 3: Table 1 Results of EDS TEM measurements of Cr diffusion into Cu stabilizer.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-2995 Origin of strain-induced domain wall ferromagnetism in multiferroic TbMnO3 thin films

Magén C.1, Farokhipoor S.2, Daumont C. J.3, Venkatesan S.4, Snoeck E.5, Iñiguez J.6, Mostovoy M.2, Noheda B.2
1LMA-INA & ARAID, Universidad de Zaragoza, Spain, 2Zernike Institute for Advanced Materials, University of Groningen, Netherlands, 3GREMAN UMR7347, Tours, France, 4Ludwig‐Maximilians University Muenchen, Germany, 5CEMES-CNRS, Toulouse, France, 6ICMAB-CSIC, Bellaterra, Spain
cmagend@unizar.es

Strain engineering is one of the different strategies pursued to give additional or improved functionalities to thin films by means of epitaxial growth, particularly in complex oxides. The terbium manganite (TbMnO3, TMO) is an example of these materials in which epitaxial strain sets in a new functional property. TMO is a traditional multiferroic material showing antiferromagnetic (AFM) ordering below 42 K and a spin spiral state below 27 K. The latter induces the inversion symmetry breaking that causes ferroelectricity [1]. Subsequent studies of TMO have demonstrated that epitaxial growth on (100)-SrTiO3 (STO) induces a low temperature ferromagnetic (FM) state with average magnetic moments up to 1.5 μB/f.u. at 15 K. This rising ferromagnetic order is correlated with a strain-induced microstructure composed of twin domains: as thickness is reduced, a higher density of twin domain walls (DW) is found and magnetization increases. In fact, magnetization scales with the DW density [2]. In order to clarify the origin of strain-induced FM and its correlation with the DWs, we have explored the structural and chemical properties of 20-nm-thick epitaxial TMO films grown on STO (100) by pulsed laser deposition by aberration-corrected Scanning Transmission Electron Microscopy (STEM) combined with Electron-Energy Loss Spectroscopy (EELS) in a probe corrected FEI Titan at 300 kV. High-angle annular dark field (HAADF) imaging in plane view [Fig. 1(a)] and cross section [Fig. 1(b)] evidences the abundance of DWs. Particularly in cross section, the DWs are characterized by the presence of spatially ordered Tb-deficient planar defect accompanied by strong lattice distortions, as illustrated in Fig. 2(a). Atomic-resolution STEM-EELS chemical mapping shown Fig. 2(b) in demonstrates that Tb-deficient positions are in fact almost entirely occupied by Mn atoms. The electronic structure of the defect has been explored by the EELS fine structure of the O K and Mn L2,3 edges. This study reveals that the consequence of this chemical defect is a decrease of the local Mn oxidation state with respect to bulk TMO, suggesting that the Tb-by-Mn substitution induces the drastic change of the environment of Mn atoms in the defect with respect to the “bulk” Mn atoms, including the formation of oxygen vacancies. DFT+U as well as embedded cluster calculations have been performed to model the magnetic interactions in the DW, suggesting that the presence of Mn atoms in Tb positions introduces local magnetic frustration between the antiferromagnetically coupled Mn ions, at the origin of the DW FM observed in this material.

[1] Y. Kimura et al., Nature 426, 55 (2003).

[2] C. J. M. Daumont et al., J. of Phys.: Cond. Matter 21, 182001 (2009).

The authors acknowledge the European Union under the Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative Reference 312483-ESTEEM2.

Fig. 1: a) HAADF-STEM image of TbMnO3 grown on SrTiO3(100) in plane view. b) HAADF STEM image of cross sectional TbMnO3, where the DWs are marked with arrows.

Fig. 2: a) HAADF-STEM image of a DW in TbMnO3. An atomic model is superimposed where Tb is green, Mn is blue and O are in the vertices of the octaedra. b) STEM-EELS chemical mapping of a DW close to the interface with the substrate, including the integrated signals of Tb M4,5 and Mn L2,3 edges, the HAADF signal and a color mix map.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-3053 Imaging polarisation around charged antiphase boundaries in doped bismuth ferrite

MacLaren I.1, Wang L. Q.1, Salih J.1, McGrouther D.1, Stamps R. L.1, Craven A. J.1, Ramasse Q. M.2, Reaney I. M.3, Jin L.4, Kovacs A.4, Dunin-Borkowski R. E.4
1School of Physics and Astronomy, University of Glasgow, Glasgow G12 8QQ, UK, 2SuperSTEM Laboratory, SciTech Daresbury, Keckwick Lane, Warrington WA4 4AD, UK, 3Department of Materials Science and Engineering, University of Sheffield, Mappin St., Sheffield, S1 3JD, UK, 4Ernst-Ruska Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Forschungszentrum Jülich, Leo-Brandt-Straße, D-52425 Jülich, Germany
ian.maclaren@glasgow.ac.uk

Doping BiFeO3 with Ti and Nb can suppress electrical conductivity as well as altering both ferroelectric and ferromagnetic orderings [1]. Higher doping levels of 10% Ti also result in the formation of novel, negatively-charged antiphase boundaries (APBs) that polarise the surrounding perovskite matrix [2]. In this study, we investigate the polarisation and electric fields further using high-resolution STEM (HRSTEM) imaging, high resolution TEM (HRTEM) using negative Cs imaging (NCSI), differential phase contrast (DPC) STEM, and scanned diffraction.

Quantitative determination of the atomic structure using HRSTEM or HRTEM/NCSI is compared in Figure 1. The structures are qualitatively similar, but with significant quantitative differences. Specifically, the deviation of oxygen atoms from the positions in an unpolarised material is reduced in HRTEM as compared to HRSTEM. This results in reduced polarisation measured for the HRTEM, as also shown in Figure 1. The discrepancy probably arises from differences in the sample thickness, since for HRSTEM the sample was about 16 nm thick whereas the HRTEM data was taken from an area about 4 nm thick. For very thin samples, the polarisation is expected to be reduced by surface effects and by some of the electric field escaping the specimen as stray fields.

DPC STEM shows a clear signal when scanning across one of these APBS, as shown in Figure 2, peaking strongly to either side and flipping sign at the APB, consistent with the polarisation of Figure 1. Scanning diffraction, however, shows no shift of the diffraction discs, as would be suggested by simplistic interpretations of DPC data in terms of Lorentz deflections from an E-field. In fact, the data in Figure 2 forces us to adopt an alternative interpretation of the DPC signal in terms of the redistribution of intensity due to dynamical scattering in a non-centrosymmetric structure, together with almost perfect screening of the E-field by the polarisation. This is found to accord perfectly with the expectations of dielectric theory for ferroelectric and polar-ordered materials.

This investigation shows that explicit consideration of electrostatics in the sample is required when interpreting atomic or nano-resolved studies of polar-ordered materials.

[1] K. Kalantari et al., Adv. Func. Mater. 21, 3737 (2011).
[2] I. MacLaren et al., APL Materials 1, 021102 (2013).

The authors are grateful to the EPSRC (including grants EP/G069069/1, EP/G005001/1, EP/I000879/1 and EP/J009679/1, the ongoing support for SuperSTEM, and a PhD studentship for LQW), SUPA, the Alexander von Humboldt Stiftung, ESTEEM2 programme, and the DFG for supporting different portions of this work.

Fig. 1: Figure 1: Quantitative comparison of averaged repeat units for the charged antiphase boundary from HRTEM (negative Cs imaging) and from HRSTEM (HAADF and BF combined); positions marked as Fe are either Fe or Ti.  The lower graph shows the polarisation calculated from this atomic resolution data for both the HRSTEM and HRTEM data.

Fig. 2: Figure 2: Differential phase contrast STEM of a charged antiphase boundary in Ti, Nd co-doped BiFeO3 (upper part, signal direction indicated in the colour wheel).  Scanned diffraction in a line scan across one such boundary (lower part) showing a clear movement of intensity within the diffraction disc but no disc deflection.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-3240 Switching the Electrical Resistance of Ferroelectrics through Control of Charged Domain Walls

Li L.1, Jokisaari J. R.1, Melville A.2, Adamo C.2, Schlom D. G.2, Pan X. Q.1
1Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI 48109, United States, 2Department of Materials Science and Engineering, Cornell University, Ithaca, NY 14853, United States
linze@umich.edu

Charged domain walls (CDWs) in ferroelectrics, as a result of “head-to-head” or “tail-to-tail” polarization configurations, are of significant scientific and technological importance, as they have been shown to play a critical role in controlling the atomic structure, electric, photoelectric and piezoelectric properties of ferroelectric materials. The accumulation of compensating free charge that screen the bound charge at the CDW can in principle intriguer an insulator-metal transition. In this work, we use in situ transmission electron microscopy (TEM) to study the stability and dynamic behaviors of CDWs in BiFeO3 thin films. We found that the CDW can be manipulated by applying electric field, leading to the switching of electrical resistance of the ferroelectric film due to the distribution of electric charges associated with domain walls.

An epitaxial 20 nm thick BiFeO3 (BFO) film with a 20 nm thick La0.7Sr0.3MnO3 (LSMO) bottom electrode were grown on (110) TbScO3 substrates by reactive molecular-beam epitaxy (MBE). Fig. 1a shows a typical triangular 109° (vertical) /180° (inclined) domain wall junction above the BFO/LSMO interface. Applying a positive voltage results in shrinkage of the triangular domain and thus formation of a CDW with “head-to-head” polarization configuration (Fig. 1b-e). The written CDW is stable after removing the electric field (Fig. 1f). The measured current during the writing process shown in Fig. 1g suggests that, with the increasing of the length of the CDW, the local electrical resistance decreases gradually and suddenly switches to a very low value, suggesting the formation of a metallic conduction channel traversing the full thickness of the film. A subsequent low reading voltage (Fig. 1h) does not change the domain configuration (Fig. 1f), and the reading current suggests a conductive state. Applying a sufficiently large negative voltage results in expansion of the triangular domain and thus annihilation of the CDW (Fig. 2a-c). After the CDW is erased, the reading I-V curve (Fig. 2d) suggests that the ferroelectric film return to its insulating state.

In conclusion, our in-situ TEM studies show the existence of stable CDWs in BiFeO3 thin films and its configuration can be manipulated by applied electric field. It was found that the CDW can be written and erased by applying electric field, and the resulting states with and without CDWs were found to have different electrical resistance, suggesting a route to engineer ferroelectric devices.

the authors gratefully acknowledge the financial support through DOE grant DoE/BES DE-FG02-07ER46416.

Fig. 1: Electrical writing of a CDW. (a)-(f) Dark-field diffraction contrast TEM images extracted from a video showing the writing process by applying a positive bias ramp. (g) Current measured during the writing process. (h) Current measured by applying a low reading voltage after writing.

Fig. 2: Electrical erasing of a CDW. (a)-(c) Dark-field diffraction contrast TEM images extracted from a video showing the erasing process by applying a negative bias ramp. (d) Current measured when applying a low reading voltage after erasing.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-12-O-3526 Direct-write Deposition of Magnetic Nanowires in a Scanning Electron Microscope - A new fabrication route for nanomagnet logic applications

Wanzenboeck H. D.1, Gavagnin M.1, Wachter S.1, Shawrav M. M.1, Stoeger-Pollach M.2, Persson A.2, Gunnarsson K.2, Svedlindh P.2, Bertagnolli E.1
1Vienna University of Technology, Vienna, Austria, 2Uppsala University, Uppsala, Sweden
heinz.wanzenboeck@tuwien.ac.at

Scanning electron microscopy is not only indespensable for high-resolution imaging of nanowires, nanotubes or nanoparticles. Recently it has also become popular as a direct-write nanofabrication technique of achitectured nanomaterials. In a precursor gas environment the focused beam of electrons can be used for inducing a chemical vapor deposition on the nanoscale. In particular, this focused electron beam-induced deposition (FEBID) has been employed for the development of catalytic templates for nanowires, for nanoelectronic devices and for functional nanostructures such as ultrasharp AFM-tips.

This work presents the application of iron penatcarbonyl as metalorganic precursors for magnetic nanostructures. The FEBID nanofabrication of functional magnetic materials is attracting increasing attention for applications in smart magnetic sensing, data storage and nano magnetologic (NML) devices [1,2]. We have previously employed FEBID for the direct-write deposition of functional magnetic tips for MFM. In this work we will demonstrate how FEBID of iron nanostructures can be used for magnetic information processing (Fig. 1). FEBID fabricated iron nanowires exhibited a high iron content (Fe>80at.%). By magnetic force microscopy (MFM) a ferromagnetic behavior could be identified. Due to sub-µm size nanowires displayed a single domain structure. Depending on the initial external magnetisation 2 different states encoding the Boolean "0" and "1" were obtained, Such elongated single domain magnetic nanostructures are the key-elements in nanomagnetlogic (NML) technology. Conventional an advanced fully functional NML gates have been realized by FEBID (Fig. 2). Further advantage of the novel design such as a reduction of the error probability and the potential to merge several NWs in future NML constituents will be discussed.

Furthermore, using FEBID we have deposited different object geometries including lines, triangles, rectangles, pentagons and circles (Fig. 3). The magnetic domain structure was determined by the geometry of structures on the nanoscale level. Nanostructures with different rotational symmetries showed a controlled transition from the single domain to a flux closure multi-domain state in dependence on the aspect ratio.

Concluding, FEBID has been proven a successful approach for guiding the “non-structural disorder” in magnetic nanostructures. Future prospects and developments of FEBID in NML will be discussed.

We acknowledge financial support by the Austrian Science Fund (FWF) under project P24093 and European Community's Seventh Framework Program under grant agreement ENHANCE-238409. TEM analyses were carried out at the University Service Centre for Transmission Electron Microscopy, Vienna University of Technology

Fig. 1: Focused electron beam induced deposition of magnetic nanowires. (Left:) Schematic of FEBID process showing electron induced precursor decomposition (Right:) AFM topography of a lateral iron nanowire and the corresponding MFM phase shift images-  The two magnetic configurations of this single-domain wire encode the Boolean logic “0” and “1”.

Fig. 2: Magnetologic majority gate structures fabricated by FEBID (Left:) SEM micrograph of iron nanowires interacting through magnetic dipolar coupling leading to processing and transport of the digital data. (Right:) MFM topography and phase shift image of an advanced gate structure with connected helper and inputs reducing the magnetic coupling errors.

Fig. 3: Magnetic shape anisotropy of FEBID nanostructures. Samples were pre-magnetized in~ 1.80 kOe. MFM topographies (top row) of differently geometries and the corresponding MFM phase images showing the the orientation of the magnetic dipoles. Depending on geometry a transition between magnetic mono- to multidomain state is achieved.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-1426 Magnetic force microscopy investigation of arrays of nickel nanowires and nanotubes

Tabasum M. R.1, Piraux L.1, Nysten B.1
1Université catholique de Louvain, Belgium
MUHAMMAD.TABASUM@UCLOUVAIN.BE

The magnetic characteristics of NWs have been studied from various viewpoints such as magnetization reversal, magnetostatic interactions, microwave properties and calculations of their intrinsic switching field distributions (SFD).On the other hand manufacturing magnetic NTs is more difficult compared with NWs: it is the reason why their magnetic properties, magnetization reversal for instance, have not been extensively explored despite their potential advantages, such as tunable geometry and reduced magnetic materials, over NWs. In particular, interwire interactions have been proven to affect the magnetic properties of arrays of NWs, specifically their magnetization reversal process and SFD.

It has been shown that NTs exhibit core-free magnetic configuration resulting in uniform switching fields so leading to controllable magnetization reversal process. Recently, experimental researches on magnetic NTs have become an attractive field to be investigated. The knack to tune NWs/NTs geometries, interwire distance permit to control the magnetostatic energies in order to get the desired magnetic properties. However, their integration into novel devices necessitates to fully understand their properties, in particular magnetostatic interactions. Major hysteresis loops of M(H) curves provide basic understanding of the magnetic properties. However, this technique is not sufficient for in depth quantitative determination of the magnetic interactions of the nanoscopic materials entities. Magnetic Force Microscopy (MFM) has proven to be suitable for the determination of the magnetization hysteresis curves at a local scale and to gain insight into the interwire dipolar interactions.

The magnetization reversal process of Ni NWs and NTs arrays in PC template have been investigated using MFM and AGFM. The comparison of the nanoscopic magnetic force microscopy (MFM) imaging and the macroscopic hysteresis loops measurements is made, Figure 1.By comparing the magnetizations curves obtained from both techniques, it has been demonstrated that they are complimentary if one wants to get an insight of the domain configuration, dipolar coupling and the magnetization reversal process. The presented results helped us understanding their magnetization reversal. For instance the mismatch of the magnetization curves in both the cases reveals that the micromagnetic configurations inside the NWs and NTs are not coherent. NWs array demonstrated stronger magnetic interactions than the NTs arrays. These results may serve as a benchmark for comparing the behavior of NWs and NTs and their use in various applications accordingly.

Fig. 1: Comparison of the in-field MFM magnetization curves with the ones obtained by AGFM.Measurements from the array of NWs (a) and NTs (b), respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-1511 Determination of magnetic flux density of grain boundary phase in Nd-Fe-B permanent magnets

Murakami Y.1,2, Tanigaki T.2, Sasaki T.3, Takeno Y.1, Park H. S.2, Matsuda T.4, Ohkubo T.3, Hono K.3, Shindo D.1,2
1Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan, 2Center for Emergent Matter Science (CEMS), RIKEN, Wako, Japan, 3National Institute for Materials Science, Tsukuba, Japan, 4Japan Science and Technology Agency, Kawaguchi, Japan
murakami@tagen.tohoku.ac.jp

Nanostructure optimization is crucial for enhancing the coercivity of Nd-Fe-B permanent magnets, which allow a significant degree of miniaturization of electric parts because of the large energy product. Regarding the materials science/engineering of Nd-Fe-B magnets, an essential problem is to understand the magnetism of the ultrathin grain boundary (GB) phase which envelopes the individual crystal grains of Nd2Fe14B [1]. However, revealing the magnetism in the GB region (~3 nm in width) remains challenging. Here, we used electron holography to determine the magnetic flux density in the GB phase using a sintered magnet subjected to optimal annealing.

As schematically shown in Fig. 1(a), which represents the cross section of a thin-foil specimen, the phase shift of electrons was measured in the line connecting the points R and S. The thin GB phase was tilted away from the direction of incident electrons: i.e., the trace of GB phase (WGB) was approximately 110 nm in this experimental setup. The magnetic flux density in the GB phase (BGB) can be determined from Δφ, which represents the deviation of the observed phase shift (in the GB area) from the extrapolated curve assuming the absence of the GB phase [Fig. 1(b)].

Figure 1(c) shows a transmission electron microscope image of the thin-foil specimen, which contained five Nd2Fe14B grains, A-E. Using the reconstructed phase image such as shown in Fig. 1(d), we accurately measured the phase shift in the R-S line which crossed the GB (shown in yellow) between Nd2Fe14B grains A and B. The observations are plotted with light-blue dots in Fig. 2(a). Split-illumination electron holography [2] achieved the sufficient precision for measuring the phase shift of ±0.08 rad. To obtain the phase information due to the GB phase, curve fitting was carried out for area A (containing only grain A); refer to the red curve in Fig. 2(a). The deviation Δφ was plotted as a function of position along the R-S line, as shown in Fig. 2(b). The value of Δφ continued to decrease over the GB area, reaching a minimum of −0.34 rad at the border of this area. Following the simulations of Δφ, the value of BGB that explains well the observation (i.e., −0.34 rad, at the border of GB area) is ~1.0 T [Fig. 2(c)]. The result explicitly indicates that the GB phase is ferromagnetic, contrary to the traditional understanding. Our observation implies significant exchange coupling between Nd2Fe14B grains, which explains the avalanche-propagation of magnetization reversal observed in sintered magnets.

References:

[1] H. Sepehri-Amin et al., Acta Mater. 60 (2012) 819.

[2] T. Tanigaki et al., Appl. Phys. Lett. 101 (2012) 043101.

This study was supported by grants “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)” and “JST-CREST”.

Fig. 1: (a) Cross-sectional view of thin-foil specimen containing Nd2Fe14B grains (A and B), and a thin GB phase. (b) Phase shift (schematic representation) observed in the R-S line. (c) TEM image of the thin-foil specimen. (d) Reconstructed phase image of the rectangle area shown in (c). The red allows indicate the direction of magnetic flux.

Fig. 2: (a) Phase shift observed along the R-S line shown in Fig. 1(c). (b) Difference between the observations and the fitting curve, Δφ, which determined the phase shift due to the GB phase to be −0.34 rad at the border of area GB. (c) Comparison between observations and calculations (simulations) of Δφ.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-1588 Designing and probing ferroic oxide nanoobjects

Thorner G.1, Bai X.1, Anoufa M.1, Haghi-Ashtiani P.2, Karolak F.1, Bogicevic C.1, Gemeiner P.1, Guiblin N.1, Hamon A. L.2, Aubry D.2, Dkhil B.1, Kiat J. M.1, Infante I. C.1
1SPMS laboratory UMR8580 CNRS-Ecole Centrale Paris, Chatenay Malabry, France, 2MSSMat laboratory UMR8579 CNRS-Ecole Centrale Paris, Chatenay Malabry, France
paul.haghi-ashtiani@ecp.fr

The reduction of sizes in ferroic materials, and in particular ferroelectrics, is motivated by an increasing number of their industrial applications, as memory devices with enhanced storage efficiency, energy harvesters with increased anisotropic piezo-responses or photocatalysts improved activities. As a matter of fact, decreasing the thickness/size of such materials for their technological integration requires a deeper knowledge of their behavior as the boundary conditions are fundamentally changed and thus will drastically affect their physical properties. In addition, extrinsic effects including internal strains, vacancies, surface terminations, shapes, dead layers,.... make their investigation much more complex than anticipated.
With the purpose to gain a better understanding of the ferroic functional properties and their relationship to the nanoscale phenomena, transmission electron microscopy (TEM) and scanning TEM (STEM) techniques appear as very powerful tools. Here we will illustrate our findings through two particular cases of ferroic oxides, namely the model multiferroic BiFeO3 and model ferroelectric BaTiO3 synthetized as nanoobjects using different chemical routes. Firstly, we will present our results on the rhombohedral BiFeO3 (Fig. 1) nanoobjects obtained by sol-gel and hydrothermal techniques. By tuning the size of this small band gap ferroelectric, we will discuss our results on multiferroic and photoinduced properties in view of the surface termination and particle size. Secondly, ferroelectric BaTiO3 nano-objects (Fig. 2-4), as nanotori or nanocubes, have been obtained through hydrothermal synthesis in a scalable process. To unveil the topological complex crystal growth that may be involved in the spontaneous nucleation of BaTiO3 as nanotori (Fig. 2) and nanocubes (Fig. 3-4), local structure and atomic arrangement have been investigated. The nanocubes, as a second stage during the BaTiO3 growth, have been investigated by STEM tomography (Fig. 3) and EDX mapping (Fig. 4), showing the high crystalline quality of these objects, with [100]T, [010]T and [001]T terminating planes and presenting a particular internal arrangement containing nanopores. For the nanotori, two arrangements were theoretically predicted. The first one exhibits isotropic radial atomic planes and large strain, while the second is highly anisotropic, with no adaptation of the crystal structure to the torus shape. Our observations indicate that the as-synthesized nanotori grow preferentially with parallel atomic planes.
All these original results revealed at the atomic scale will be discussed in view of the theoretical predictions and the micro and macroscopic ferroic properties measured on these systems.

We acknowledge the French National Research Agency (MATMECA Equipex project) for financial support and D. Delille and A. Carlsson for their first experiments at FEI facilities.

Fig. 1: Main: TEM image at 300kV indicating the detailed surface arrangement of a BiFeO3 nanoparticle, (111)R oriented. Top left: BiFeO3 R3c sketch. Top right: Reduced Fast Fourier Transform of the TEM image.

Fig. 2: TEM image at 300kV of BaTiO3 nanotori, indicating the continuity of atomic planes through the hole.

Fig. 3: HAADF STEM image at 300kV and 70pA of BaTiO3 nanocubes. Nanocubes present rounded corners and vacuum internal regions, which can be controlled by hydrothermal synthesis conditions.

Fig. 4: HAADF STEM image (top left) and corresponding EDX mapping reconstructed from a BaTiO3 nanocube, using Ba (top right), Ti (bottom left) and O (bottom right) X-ray emission lines, at 200kV and 125pA for 10min. Scale bar: 1nm.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-1608 Orientation tuning of heterostructures in oxide thin films

Zhan Q.1, Zhu Y. M.1, Chu Y. H.2
1School of Materials Science and Engineering, University of Science and Technology, Beijing 100083, China;, 2Department of Materials Science and Engineering, National Chiao Tung University, Hsinchu, 30010, Taiwan
qzhan@mater.ustb.edu.cn

Heterostructures of transition metal oxides cover a wide range of intriguing functionalities induced by the interplays among the lattice, charge, orbital and spin degrees of freedom and offer tremendous opportunities to develop next generation electronic devices. Complex correlated oxide heterostructures with various configurations in thin film have drawn a considerable spotlight due to the strong coupling between the constituent phases and been used to tune the functionalities benefitting from their plentiful hetero-interfaces. Modification of these composite nanostructures is a fundamental topic often to be addressed.
In the present study, typical perovskite-spinel self-assembled heteroepitaxial nanostructures were chosen as the model film systems such as BiFeO3-CoFe2O4 and (La0.67Ca0.33)MnO3-NiFe2O4. Series of novel nanostructure configurations and heterointerface structures in the thin films were investigated by transmission electron microscopy and related techniques. Various configurations of included nanostructures with different orientations were demonstrated while the matrix kept the same growth direction in nanocomposite thin films, which were quite different with previous report. The different heterointerface structures between the component phases and also the substrate at an atomic scale have been investigated by high resolution transmission electron microscopy. It can conclude that many fectors especially the effect of surface energy anisotropy, strain state of matrix and atomic structure continuity on these plentiful combinations should be considered when analysis the growth mechanism of the nanostructures.
The plentiful combination forms of heterostructures and regulations on the crystallographic orientation of the constituent phases can provide more ways on tailoring degrees of freedom of complex oxide heterostructures.

This work is supported by the National Natural Science Foundation of China with Grant Nos. 51371031, 50971015 and the National Science Council, R.O.C. (NSC-101–2119-M-009–003-MY2)

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-1643 TEM investigation of grain boundaries from Nd2Fe14B hard magnets

Zickler G. A.1, Wallisch W.1, Stöger-Pollach M.2, Bernardi J.2, Üstüner K.3, Fidler J.1
1Institute of Solid State Physics, Vienna University of Technology, Vienna, Austria, 2USTEM, Vienna University of Technology, Vienna, Austria, 3Vacuumschmelze GmbH, Hanau, Germany
zickler@ifp.tuwien.ac.at

Nd2Fe14B exhibits a high magnetocristalline anisotropy and is suitable for the usage as a hard magnetic material. [1,2,3] The aim of our study is to determine the discrepancy between the theoretical and the experimental coercivity. We have investigated the role of the sintering temperature on the formation of the grain boundary phases of various sintered magnets with a standard TEM/STEM instrument (FEI TECNAI G20) and a field-emission analytical TEM/STEM (FEI TECNAI F20).
Standard methods for TEM preparation (cutting, thinning, ion milling) were used to prepare a sample parallel (p) and normal (n), respectively, to the preferable direction of the magnetisation (equal to [001] of the grains).
In Fig. 1 a TEM bright field image of two Nd2Fe14B grains from a magnet with Br =1.27 T and Hc=1630 kA/m are shown. The specimen normal and the grain boundary (GB) with 10 nm thickness are perpendicular to [001] of the right grain. This GB-phase is a characteristic attribute for a type "x" GB. In Fig.2 a TEM bright field image of a specimen with two Nd2Fe14B grains and a Nd2O3 (Mn2O3 structure) grain boundary junction is shown. The [001] direction is parallel to the specimen normal. Therefore the grain boundary is parallel to [001] and forming a type "y" GB. The nature of the observed Nd2O3 (Mn2O3) grain boundary junction phase differs from the one of the Nd-rich type “x” and “y” grain boundary phases. The corresponding lattice plains of the hard magnetic Nd2Fe14B grains are indexed in the FFT images of Fig.1 and 2. The coercive field of the investigated magnets are directly related to the morphology of the grain boundary and grain boundary junction phases.
Detailed investigations including Energy Dispersive X-Ray Spectroscopy (EDXS), Electron Energy-Loss Spectrometry (EELS) and High Annular Dark-Field STEM (HAADF-STEM) imaging will be presented.

 

References:

[1] K. Khlopkov, O. Gutfleisch, D. Eckert, D. Hinz, B. Wall,W. Rodewald, K.-H. Müller. L. Schultz, “Local texture in Nd–Fe–B sintered magnets with maximised energy density” J. Alloys and Compounds 365 (2004) 259–265.
[2] S.C. Wang, Y. Li. “In situ TEM study of Nd-rich phase in NdFeB magnet” J. Magn. Magn. Mater. 285 (2005) 177–182.
[3] G. Hrkac, T.G. Woodcock, K.T. Butler, L. Saharan, M.T. Bryan, T. Schrefl, O. Gutfleisch “Impact of different Nd-rich crystal-phases on the coercivityof Nd–Fe–B grain ensembles” Scripta Materialia 70 (2014) 35–38.

The funding from the European Community´s Seventh Framework Programme (FP7-NMP) under the grant agreement no. 309729 (ROMEO) is acknowledged.

Fig. 1: Bright field image showing a grain boundary of type "x" between two Nd2Fe14B grains. Specimen normal is perpendicular to [001].

Fig. 2: Bright Field image showing two Nd2Fe14B grains and a grain boundary junction. Specimen normal is parallel to [001]. A grain boundary of type "y" is marked.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-1899 Observation of pure ferroelectric domains

Arredondo M.1, Hart J.2, Beanland R.3, Whyte J.1, Taheri M.2, Canalias C.4, Snoeck E.5
1School of Mathematics and Physics, Queen’s University Belfast, BT7 1NN, UK, 2Department of Materials Science and Engineering, Drexel University, Philadelphia, PA, USA, 3Department of Physics, University of Warwick, Coventry, CV4 7AL, United Kingdom, 4Department of Applied Physics, Royal Institute of Technology, Roslagstullsbacken 21, 10691 Stockholm, Sweden, 5Centre d’Elaboration de Matériaux et d’Etudes Structurales (CEMES-CNRS), BP 4347, F-31055 Toulouse Cedex, France
m.arredondo@qub.ac.uk

In the last decade there has been increased interest in ferroelectrics and multiferroics, mainly motivated by the potential development of new electronic devices, where domains play a key role. This has triggered groundbreaking work on domains, aimed to domain engineering. Much of this work has been achieved by PFM and TEM, demonstrating the vast diversity and complexity of domains’ structure. Typically, ferroelectric domains and their switching mechanisms are mainly investigated by PFM, while ferroelastic domains are more commonly observed by TEM. It is highly desirable to acquire structural and dynamic information of pure ferroelectric domains by TEM. Moreover, it is even more interesting to link this to ferroelectric domain dynamics: switching, nucleation and growth. However in the structural front, so far only high-resolution Cs-corrected TEM techniques have been successful. The latter produces high-impact data but restricts the domain observation to the nanoscale regime, making it difficult to perform dynamic investigations. Therefore, we investigate ferroelectric domains in the ferroelastic domains-free KTiOPO4. Hence, making it possible to study pure domains dynamics by electrical switching. This gives us the flexibility of observing them from the micro to the nano-scale: dark field and two-beam condition. This imaging condition has shown to be able to break Friedel’s law, revealing the non-centrosymmetric nature of crystals (Fig. 1). Finally, this data is compared to PFM data for a more macroscopic analysis of the polarization (Fig. 2).

Fig. 1: Dark field imaging, revealing the ferroelectric domains.

Fig. 2: PFM data set
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-1985 Effect of substrate-induced strain on the structure of LaNiO3 thin films

López-Conesa L.1, Rebled J. M.1,2, Pesquera D.2, Sánchez F.2, Dix N.2, Magén C.3,4, Serra R.5, Casanove M. J.5, Estradé S.6, Fontcuberta J.2, Peiró F.1
1Laboratory of Electron Nanoscopies (LENS-MIND-IN2UB), Departament d'Electrònica, Universitat de Barcelona, C/Martí i Franquès 1, 08028 Barcelona, Spain, 2Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, 08153 Bellaterra, Spain, 3Laboratorio de Microscopías Avanzadas - Instituto de Nanociencia de Aragón (LMA-INA), Departamento de Física de la Materia Condensada, Universidad de Zaragoza, 50018 Zaragoza, Spain, 4Fundación ARAID, 50018 Zaragoza, Spain, 5Centre d'Elaboration des Matériaux et d'Etudes Structurales (CEMES-CNRS), 29 Rue Jeanne Marvig, Toulouse, France, 6Centres Científics i Tecnològics de la Universitat de Barcelona (CCiT-UB), C/Lluís Solé i Sabarís 1, Barcelona, Spain
llopez@el.ub.edu

LaNiO3 (LNO) is a perovskite of great importance in complex oxide electronics. Its low resistivity at room temperature and high chemical stability make it an ideal electrode candidate for many applications in complex oxide-based devices. Strain, oxygen vacancies and their mutual interplay are key aspects to understand the transport properties of these oxides1,2, since they might affect the Ni-O hybridization. In this study, we perform a thorough analysis of these aspects with high spatial resolution TEM.
We have studied LNO thin films of different thicknesses (14 nm and 35 nm) grown on several substrates that allow studying a wide range of compressive (LAO and YAO) and tensile (LSAT and STO) strain states. Aberration corrected HRTEM, HAADF-STEM, atomic resolution EELS mapping and image simulation studies have been carried out. Strain states in the films have been studied by Geometric Phase Analysis (GPA) of the high resolution images.
The presence of brownmillerite phase has been detected in the LNO strained films (figures 1 and 2). This perovskite-related superstructure occurs when oxygen vacancies order along a given crystallographic direction. Contrast modulation in HRTEM images and Z contrast in HAADF images are consistent with this vacancy ordering. Image simulations (both HRTEM and HAADF) support these findings. We report on the effect of the strain state of the film on the occurrence and orientation of the brownmillerite superstructure.
Moreover, unexpected box-like defects are found in all the films (figure 3). Defect boundaries correspond to a displacement of 1/2 of the perovskite unit cell both in the in-plane and out-of-plane directions. High resolution STEM-EELS spectrum imaging confirms a missing Ni-O plane at these boundaries. Signals from the overlapping La and Ni edges have been separated and extracted using the Blind Source Separation (BSS) method in the Hyperspy advaced signal processing toolbox.

[1] J. Chakhalian, A. J. Millis, and J. Rondinelli, Nature Mater. 11, 92 (2012)
[2] I.V. Nikulin, M.A. Novojilov, A.R. Kaul, S.N. Mudretsova and S.V. Kondrashov, Mater. Res. Bull. 39, 775-791 (2004)

We acknowledge the financial support from the Spanish Ministry of Economy and Competitivity via projects Imagine-Consolider CSD2009-2013, MAT2010-16407 and FPI and JAE predoc grants. We acknowledge the Catalan Government for financial support via project CTP2011-00018.

Fig. 1: High resolution HAADF-STEM image of LNO on LAO (-1% lattice mismatch). The brownmillerite phase is visible in the contrast modulation perpendicular to the substrate/film interface and in the superstructure spots in the FFT in the inset.

Fig. 2: HRTEM image of LNO on LSAT (+1% lattice mismatch). The brownmillerite phase is visible in the contrast modulation parallel to the substrate/film interface.

Fig. 3: HAADF-STEM image and elemental EELS maps of a defect boundary. a) HAADF reference image b) HAADF image acquired simultaneosuly to the EELS spectra. c) Ni map. d) La map. e) Composite: La in yellow and Ni in red. Displacement of 1/2 of the perovskite unit cell is visible. EELS mappings show a missing Ni-O plane in the boundary.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2036 Chemical and Defect Analysis in a ZrO2/LSMO Pillar-Matrix System

Zhou D.1, Sigle W.1, Wang Y.1, Kelsch M.1, Gao Y.2, Habermeier H.2, van Aken A. P.1
1Max Planck Institute for Intelligent Systems, Stuttgart Center for Electron Microscopy, Stuttgart, Germany , 2Max Planck Institute for Solid State Research, Stuttgart, Germany
danzhou@is.mpg.de

Recently there has been tremendous research on self-assembled vertically aligned nanocomposite thin films with two immiscible components hetero-epitaxially grown on single crystal substrates1-4. These structures have the advantages of utilizing both component functions and tuning material properties with high interface-to-volume ratio, hetero-epitaxial strain, or modifying the cation valence state.

Here we report about the characterization of self-assembled vertically aligned non-magnetic zirconium oxide (ZrO2) and ferromagnetic perovskite lanthanum strontium manganese oxide (La2/3Sr1/3MnO3, LSMO) pillar-matrix nanostructures, which are epitaxially grown on (001) single-crystalline lanthanum aluminum oxide (LaAlO3, LAO) substrate by pulsed laser deposition. With the application of electron energy-loss spectroscopy (EELS) in a probe-aberration-corrected JEOL JEM-ARM200CF, atomic resolution elemental distribution, including La, Sr, Mn, and Zr, as shown in Figure 1, and the Mn valence state variation at the interface between LSMO and ZrO2 were observed. In addition, Mn-rich walls were found connecting adjacent pillars. The crystal lattices on either side of the wall are displaced by an antiphase shift as can be seen in Figure 2. The Mn valence state in the channel was found to be decreased compared to the matrix. The wall plane is {110} or {130}. The role of the pillars and walls regarding elastic strain and local electric fields will be discussed.

The spin, charge, and orbital ordering in LSMO are extremely sensitive to local structural and elemental variations. Thus, these results provide the basis for understanding the origin of the anomalous magnetic anisotropy and modifications to the electric transport properties of LSMO by introducing non-magnetic ZrO2 pillars5-7.

References:

1. Chen et.al., Nanotechnology 2011, 22, (31).

2. Imai et.al., Acs Nano 2013, 7, (12), 11079-11086.

3. Zheng et.al., Nano Lett 2006, 6, (7), 1401-1407.

4. Liu et.al., Acs Nano 2012, 6, (8), 6952-6959.

5. Gao et.al., Prog Nat Sci-Mater 2013, 23, (2), 127-132.

6. Jin et.al., J. C. J Supercond Nov Magn 2013, 26, (5), 1621-1624.

7. Gao et.al., Solid State Commun 2013, 154, 46-50.

The research leading to these results has received funding from the European Union Seventh Framework Program [FP/2007-2013] under grant agreement no 312483 (ESTEEM2).

Fig. 1: Annular dark-field (a) and elemental distribution ((b) Sr L, (c) La M, (d) Mn L, (e) Zr L) images extracted from EELS spectrum images.

Fig. 2: (a) HAADF image of ZrO2 pillars and LSMO matrix. (b) Enlargement of a Mn-rich wall with annotations of the wall plane.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2050 EMCD-investigation of Co-Fe thin films doped with carbon by Analytical TEM

Wallisch W.1, Stöger-Pollach M.1, 2, Zickler G.1, Bernardi J.2, Giannopoulos G.3, Niarchos D.3, Fidler J.1
1Institute of Solid State Physics, Vienna University of Technology, Vienna, Austria, 2University Service Center for Transmission Electron Microscopy, Vienna University of Technology, Vienna, Austria, 3IAMPPNM, NCSR Demokritos, Athens, Greece
wolfgang.wallisch@tuwien.ac.at

For novel hard magnetic applications Co-Fe based nanostructures are proposed as potential candidate. We have studied sputtered thin Co-Fe films. One is undoped, the other one is doped with 20 at.% of Carbon. The aim of doping is to stabilize the meta-stable tetragonal phase in order to increase the magneto-crystalline anisotropy [1]. The specimen was prepared as a cross section sample for transmission electron microscopy (TEM) investigations by the focused ion beam (FIB) lift-out specimen preparation technique (Quanta 200 3D DualBeam FIB). The final thinning of the FIB prepared TEM-lamellae was done by using a Technoorg-Linda GentleMill in order to reach a sample thickness smaller than 30 nm. The microstructure was analyzed by an analytical TEM (FEI TECNAI F20) operated at 200 kV, equipped with a Gatan GIF Tridiem energy filter. The magnetic fingerprint of the samples was analyzed by detecting magnetically induced chiral electronic transitions in the TEM by employing the energy loss magnetic chiral dichroism (EMCD) technique. This way the magnetic moments of the Fe atoms can be resolved with a spatial resolution of better than 2 nm [2, 3].
A high-resolution TEM image of undoped sample was taken at 200 keV as shown in figure 1. The multilayer deposits on a single crystalline MgO substrate and its structure consists of a 2.6 nm Cr seed layer, an 29.8 nm Au50Cu50 tetragonal buffer and a magnetic layer (Co55Fe45 or Co44Fe36C20) of about 4 nm. The FIB protection layers are Pt-C and Cr layers, respectively. For the EMCD measurements a symmetric three-beam case set up was chosen. An EELS line scan of two configurations “+” and “-” recording the Fe L2,3 edge across the Fe 3 nm layer was done in the scanning mode of the TEM [3]. Finally we summed over all spectra showing the Fe-L2,3 edge for determining the EMCD signal, which is the difference of the “+” and “-” spectra (figure 2).
The magnetic structure of the carbon doped Co-Fe thin films has been confirmed by nanoanalytical TEM/STEM investigations using the EMCD technique.

References:

[1] Giannopoulos, G., et al. “Structural and magnetic properties of strongly carbon doped Fe-Co thin films” submitted for INTERMAG 2014
[2] Schattschneider, P., et al. “Energy Loss Magnetic Chiral Dichroism: A New Technique for the Study of Magnetic Properties in the Electron Microscope (invited)” Journal of Applied Physics 103, no. 7 (2008)
[3] Schattschneider, P., et al. “Detection of Magnetic Circular Dichroism on the Two-Nanometer Scale” Physical Review B 78, no. 10 (September 2008)

The funding from the European Community's Seventh Framework Programme (FP7-NMP) under grant agreement n° 280670 (REFREEPERMAG) is acknowledged.

Fig. 1: High resolution TEM images of the undoped sample. (a) Interface of the magnetic layer with a diffraction pattern (DP) of the multilayer (black Co55Fe45, green Au50Cu50 and red Cr). (b) Interface of the seed layer with a DP of the substrate.

Fig. 2: (a) High resolution TEM of the doped sample. (b) Energy-loss magnetic chiral dichroism. Fe L2, 3 edges for 3 nm Co44Fe36C20 layer measured in two configurations “+” and “-”. (c) line scan over the doped sample.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2088 Revealing domains’ hierarchy in BaTiO3

Holsgrove K. M.1, Gregg J. M.1, Arredondo M.1
1Centre for Nanostructured Media, Queen’s University, Belfast, U.K
kholsgrove04@qub.ac.uk

Ferroic materials are used for multiple device applications (transistors, memories, tunnelling barriers, etc.); their functionality is enabled by the reversible switching between equivalent states, or domains, that form to minimise the system’s free energy. Switching depends on the domains’ patterns (geometric arrangement) and their movement under external stimuli (electrical, mechanical, etc). Domain patterns strongly influence material responses and properties such as dielectric permittivity, piezoelectric coefficients and remanent polarization. In any ferroic material, the domain arrangement and size (including domain walls) depend on the boundary conditions and these are usually coupled to more than one property including strain, magnetic order and surface charge. It is known that ferroelastic domains in ferroelectrics form multirank structures, such as laminated patterns.
Recently, a new level of complexity in domain patterns has been shown [1-3], mainly due to advances in the resolution of standard techniques for domain observation, such as transmission electron microscopy (TEM) and piezoelectric force microscopy (PFM). It has been shown that a variety of domain patterns can exist simultaneously as an intricate bundle at the micro and nano-scale [4-7], indicating that one type of domain alone is not always energetically favourable and hence, a hierarchy must form in order to stabilize the system’s energy.
However, such a domain configuration is still not well understood. The fine-scale intricate mesh of domains is well below the resolution of many microscopy techniques and TEM proves to be a unique technique to shed light in this newly found domain infrastructure. In this study BaTiO3 single-crystal lamella presenting such domain hierarchy are investigated. Initial characterisation of the intricate mesh of domains suggest a complex relationship between micro-scale and nano-scale domains, with micro-scale domains occupying crystallographically defined directions. Detailed interpretation of the naturally occurring boundaries between bundles of domains will be discussed in more detail. Moreover, investigations using in-situ TEM such as heating and electrical bias will provide a greater insight into the complex dynamics (domain switching and nucleation) of this hierarchy.

[1] D. Evans et. al., Nature Commun., 4, 1534, (2013).
[2] F. Bai et al., J. Appl. Phys. 97, 054103, (2005).
[3] Catalan, G. et al., Rev. Mod. Phys., 84, 1, (2012).
[4] K. H. Kim et al., Appl. Phys. Lett., 97, 261910, (2010).
[5] Y. Ivry et al., Phys. Rev. B, 86, 205428, (2012).
[6] P. Sharma, et al., Adv. Mater., 25, 9, (2013).
[7] R. G. P. McQuaid et al, J. Phys.: Condens. Matter., 024204, 24, (2012).

We would like to acknowledge the department for employment and learning.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2114 The correlation between magnetic properties and the distribution of Heavy Rare Earths (Dy,Tb) in Nd–Fe–B-based Magnets

Sturm S.1, Zagar K.1, McGuiness P.1, Kobe S.1
1Department for nanostructured materials, Jožef Stefan Institute, Ljubljana, 1000 Slovenia
saso.sturm@ijs.si

The heavy rare earths (HREs), like Dy and Tb that partially replace the Nd in the grain-boundary diffusion process (GBDP) have a large, positive influence on the coercivity of the whole Nd–Fe–B-based magnet. In the GBDP HRE’s are deposited at the surface of already fully sintered Nd–Fe–B- magnet. These magnets are exposed to elevated temperatures allowing HRE’s to diffuse into the interior of the magnet. The improved magnetic properties of these magnets are attributed to the formation of core-shell grains with a HRE-containing shell and a Nd–Fe–B-based core. The shell, with its higher anisotropy field resulting from the HRE, hinders magnetisation reversal at the grain edges and leads to an increase in the coercivity of the magnet. The concentration gradient of HRE’s was indirectly confirmed by measuring the magnetic properties of thin slices, which were cut parallel to one of the magnet surface. It was found that the coercivity measured from the slice cut from the very central region of the magnet is still around 30% higher when compared with the untreated magnet. Although magnetic measurements clearly indicate the presence of HRE’s it is not clear what is the actual amount of HRE’s in central regions of magnets and how they are distributed in the microstructure. For that purpose a detailed analytical study was performed on two samples, Nd–Fe–B-based magnets with the addition of Dy and Tb, respectively, by applying a (scanning) transmission electron microscope (STEM/TEM UHR Jeol 2010F) equipped with an energy-dispersive x-ray spectrometer (EDXS) (LINK ISIS EDS 300) and electron energy-loss spectrometer (EELS) (Gatan PEELS 766).
Figure 1a shows a HRTEM image of a grain boundary (GB) in a Dy/Nd–Fe–B system. The analyses were carried out in the central regions of the magnet. Several EELS line-scans were acquired across the interface. The representative lines scan is shown in figure 1b. The GB is characterized by large amounts of Nd and O. The concentration of Fe in the GB region is lower compared to the adjacent bulk. EELS analysis shows clear presence of Dy located at the GB (Figure 1c). The analyses in Tb/Nd–Fe–B system were obtained from the surface regions of the magnet. In that case the EELS compositional analysis reveals Nd–Fe–B grains where Tb is homogeneously distributed within the bulk matrix, as shown in line-scan in figure 1d. The average Tb concentration normalized to Nd measured in these grains was around 30 at.%. Approximately 20 μm from the magnet surface the Nd–Fe–B grains have already developed a typical core/shell morphology. Further studies will focus in the detailed analysis of these grains. The thickness and the composition of the shell will be determined as a function of the specimen position within the magnet.

This work was financially supported by the European Union as part of the Framework 7 program, FP7-NMP-2012-SMALL-6, with the project title: Replacement and Original Magnet Engineering Options (ROMEO) and by [FP7/2007-2013] under grant agreement n°312483 (ESTEEM2)

Fig. 1: HRTEM image of a GB in Dy/Nd–Fe–B system.

Fig. 2: EELS line scan across the interface. 

Fig. 3: Dy concentration distribution across the GB. 

Fig. 4: The distribution of Tb within Nd–Fe–B grains in Tb/Nd–Fe–B system.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2176 Transmission electron microscopy study of Yb2Ti2O7

Mostaed A.1, Beanland R.1, Lees M R.1, Balakrishnan G.1
1Department of Physics, University of Warwick, Coventry, UK
a.mostaed@warwick.ac.uk

In the past two decades there has been a great interest in the magnetic behaviour of pyrochlore oxides with general formula A2B2O7, in which A is a rare-earth ion and B is a transition metal. Such materials can exhibit geometric frustration with the A and B cations ordered into separate interpenetrating lattices of corner-sharing tetrahedra [1]. Magnetic A cations can also be “stuffed” onto the nonmagnetic B sites further modifying the magnetic behaviour of these materials. For instance, Yb2Ti2O7 samples (especially single crystals) have broad features in specific heat capacity that vary in sharpness and temperature depending on the sample, indicating that the magnetic ground state may be qualitatively different in samples with different degrees of stuffing [2]. In the present work, the structure of several Yb2Ti2O7 samples (polycrystalline and single crystal) has been studied by aberration-corrected annular dark field Scanning Transmission Microscopy (ADF-STEM). The polycrystalline powders were prepared by the standard solid state synthesis method in which the constituent powders were mixed together and heated at temperatures between 1150 and 1400 ºC for 6 days. Powder X-ray diffraction was used to check the phase purity of the synthesised powders. Large single crystal samples were grown by the optical floating zone technique [3]. Atomic resolution STEM images demonstrate lattice deformation in the structure of the pyrochlores. Moreover, intensity fitting on the atomic resolution images of the network of corner sharing Yb and Ti tetrahedra, together with Electron Energy Loss Spectroscopy (EELS) studies are used to look for the presence of Yb3+ cations in Ti4+ sites. We will examine the claims of Ross et al. [2] that the variation of the magnetic ground state of the pyrochlores is as a result of random exchange bond and local lattice deformation introduced by substituting (stuffing) Yb3+ on the Ti4+ sublattice.

[1] G C Lau et al, Journal of Solid State Chemistry 179 (2006) p. 3126.
[2] K A Ross et al, Physical Review B 86 (2012) p. 174424.
[3] G Balakrishnan et al, J. Phys. Condens. Matter 10 (1998) p. L723.

Fig. 1: Fig.1. ADF-STEM image of polycrystalline Yb2Ti2O7 along [112] with overlaid Yb and Ti tetrahedra.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2207 In-situ electric-field transmission electron microscopy studies of Ba(Zr0.2Ti0.8)O3-x(Ba0.7Ca0.3)TiO3 ferroelectrics

Zakhozheva M.1, Schmitt L. A.2, Acosta M.2, Rödel J.2, Jo W.3, Kleebe H. J.1
1Institute of Applied Geosciences, Technische Universität Darmstadt, Darmstadt, Germany, 2Institute of Geo- and Material Sciences, Technische Universität Darmstadt, Darmstadt, Germany, 3School of Materials Science and Engineering, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea
Zakhozheva@geo.tu-darmstadt.de

Due to excellent dielectric, piezoelectric and mechanical properties, lead-containing ferroelectrics with perovskite structure are widely used in piezoelectric sensor and actuator applications. However, because of the toxicity of the heavy metal lead [1, 2], it is necessary to search for a suitable lead-free alternative for these materials [3].
Our studies were focused on the Ba(Zr0.2Ti0.8)O3-x(Ba0.7Ca0.3)TiO3 piezoceramic system, with x = 0.30, 0.50, 0.52 (abbreviated as BZT-xBCT). These different compositions have been synthesized by a conventional solid state reaction method. The evolution of the domain morphology in BZT-xBCT under an applied electric field was analyzed by in-situ transmission electron microscopy (TEM). For all compositions, changes in the domain structure during poling were observed. An electric field induced transformation from the multi-domain to the single-domain state was observed (Fig. 1 - Fig. 4). This transformation is found to be reversible, since multi-domain contrast reappears inside the grains upon field removal. It should be noted that an intermediate nanodomain state has also been observed during electrical poling of BZT-xBCT. The selected area electron diffraction patterns show neither any reflection splitting nor detectable changes during electrical poling for all compositions studied.
Domain wall movement during the application of electric fields and the absence of any reflection splitting in the electron diffraction patterns for all investigated compositions lead to the conclusion of a large contribution of the extrinsic effect to the piezoelectric response of the Ba(Zr0.2Ti0.8)O3-x(Ba0.7Ca0.3)TiO3.

[1] EU-Directive 2002/96/EC: Waste Electrical and Electronic Equipment (WEEE), Off. J. Eur. Union, 46 [L37] 24–38 (2003).

[2] EU-Directive 2002/95/EC: Restriction of the Use of Certain Hazardous Substances in Electrical and Electronic Equipment (RoHS), Off. J. Eur. Union, 46 [L37] 19–23 (2003).

[3] J. Rödel, W. Jo, K.T.P. Seifert, E.M. Anton, T. Granzow, J. Am. Ceram. Soc. 92 (2009) 1153–1177.

This work was financially supported by the Deutsche Forschungsgemeinschaft under Sonderforschungsbereich 595 and from the state center AdRIA on adaptronics.

Fig. 1: In-situ TEM bright field image of the BZT-0.3BCT along the [1-35]c zone axis at virgin state.

Fig. 2: In-situ TEM bright field image of the BZT-0.3BCT along the [1-35]c zone axis at 2.5 kV mm-1. The direction of the poling field is indicated by the arrow.

Fig. 3: In-situ TEM bright field image of the BZT-0.3BCT along the [1-35]c zone axis at 3.3 kV mm-1. The direction of the poling field is indicated by the arrow.

Fig. 4: In-situ TEM bright field image of the BZT-0.3BCT along the [1-35]c zone axis at 5 kV mm-1. The direction of the poling field is indicated by the arrow.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2216 A TEM study of Bi0.5Na0.5TiO3-based ceramics

Neagu A.1, Tai C. W.1
1Department of Materials and Environmental Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden
alexandra.neagu@mmk.su.se

Ferroelectric materials and especially PZT-based ceramics are frequently used in electronic devices such as actuators, transducers, sensors [1]. Due to recent environmental regulations [2] there is a strong push to replace lead (Pb) in these materials. One such system that is currently considered as a potential replacement for PZT is Bi0.5Na0.5TiO3 (BNT). The challenge in searching for lead-free ceramics is a lack in the fundamental understanding of structure-property relationship in these materials.

In the present work transmission electron microscopy (TEM) has been employed to investigate a series of BNT-based solid solutions. Fig. 1 shows the bright-field image of ferroelectric domains of a ternary system 0.9(Na0.5Bi0.5)TiO3-0.05(Bi0.5K0.5)TiO3-0.05BaTiO3 (BNT-BKT-BT). The domain pattern was recorded close to [001] zone axis at room temperature. The main crystallographic directions are given, according to the corresponding SAED pattern of the same area shown in Fig. 2. A parallel band like configuration of the ferroelectric domains can be observed. In the SAED pattern 1/2 ooe (where “o” and “e” stand for odd and even hkl indexes, respectively) reflections are observed besides the fundamental perovskite reflections, indicating the presence of in-phase oxygen octahedral tilting [3, 4] (a0a0c+, in Glazer notation [5]). The diffuse electron scattering has also been characterized by using rotation electron diffraction (RED) method [6]. This way we can achieve a better understanding of the short-range structural order/disorder of the material using the 3D information in reciprocal space. When tilting the crystal to higher zone axes the diffuse scattering becomes clearly visible as shown in Fig. 3. However, besides the fundamental perovskite reflections we can observe 1/2 ooo superstructure reflections indicating most likely a-a-a- anti-phase octahedral tilting.

[1] J. F. Scott, Science 315, 954 (2007)

[2] P. K. Panda, J. Mater. Sci. 44, 5049–5062 (2009)

[3] C. W. Tai and Y. Lereah, Appl. Phys. Lett. 95, 062901 (2009)

[4] D. I. Woodward and I. M. Reaney, Acta Cryst. B61, 387-399 (2005)

[5] A. M. Glazer, Acta Cryst. B28, 3384 (1972)

[6] W. Wan, J. Sun, J. Su, S. Hovmöller and X. Zou, J. Appl. Crystallogr.46, 1863-1873 (2013)

Acknowledgement: The Kunt and Alice Wallenberg Foundation is acknowledged for supporting the electron microscopy facilities and the project “3D Electron Microscopy for Nanostructure Research”(3DEM-NATUR).

Fig. 1: Bright field transmission electron microscopy image of the ferroelectric domains viewed close to [001]pc

Fig. 2: SAED pattern along a <001>pc zone axis. Pseudo-cubic (PC) system was used to index the pattern

Fig. 3: SAED pattern along a <114>pc zone axis displaying diffuse electron scattering along {04-1}*pc. Pseudo-cubic (PC) system was used to index the pattern
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2264 Structural and electrical properties of hydrothermally synthesized BaTiO3 and BaTiO3/TiO2 nanostructures

Plodinec M.1, Gajović A.1, Šantić A.1, Willinger M. G.2, Čeh M.3, Šipušić J.4
1Ruđer Bošković Institute, Bijenička 54, HR-1002 Zagreb, Croatia, 2Fritz-Haber-Institute der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195 Berlin, Germany, 3Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia, 4Faculty of chemical engineering and technology, University of Zagreb, Zagreb, Croatia
mplodin@irb.hr

BaTiO3 (BTO), is a well-known and widely investigated dielectric material. It is mainly used in capacitors due to its high dielectric constant. The dielectric properties of BTO are controlled by purity and microstructure which are dependent on the methods of preparation. Recent advances in nanotechnology such as MLCC, MEMS, DRAM have resulted in miniaturization of devices [1, 2].

In this work barium titanate nanostructures were synthesized by hydrothermal processing of titanium based precursor in aqueous solution of BaCl2 and NaOH. Several titanium based precursors were used: TiO2 nanopowder, titanate nanotubes (H2Ti3O7), and TiO2 nanotubes array prepared by anodisation of titanium foils. Structural and compositional properties were investigated by XRD, SEM, EDX, TEM, HRTEM, SAED and Raman spectroscopy (ex–situ and in-situ), while electric measurements were studied by impendence spectroscopy.

In the case of synthesis from TiO2 nanopowder precursors, obtained BaTiO3 nanoparticles were found to be about 50 nm in size (Fig. 1a and 1b), while in the case of H2Ti3O7 nanotubes precursor the average size of the particles are somewhat larger. In both cases TiNT, homogenous particles in tetragonal phase were formed. In the case of hydrothermal treatment of TiO2 nanotubes array precursor (Fig. 2a), BaTiO3/TiO2 composites were obtained, where BTO formed the film at the TiO2 nanotubes array (Fig. 2b).

Tetragonal phase of BFO, having 4mm symmetry with c/a axis ratio close to one, is hard or impossible to distinguish from cubic phase by using standard diffraction techniques, so the phase purity and the structure were studied by Raman spectroscopy (RS) as a main technique [3]. In situ low and high temperature RS was used for study of ferroelectric phase transitions from tetragonal to cubic phase. TEM and HRTEM techniques were used to study structure and morphology of all prepared samples. The surface electrical conductivity (Fig. 3a and 3b) and dielectric constant will be discussed in the relation to RS measurements.

References:

[1] N. A. Hill, J. Phys. Chem. B, 2000, 104, 6694–6709.
[2] M. A. Pena and J. L. G. Fierro, Chem. Rev., 2001, 101, 1981–2018.
[3] A. Gajović, J. Vukajlović Pleština, K. Žagar, M. Plodinec, S. Šturm, M. Čeh, J. Raman Spect., 2013, 44, 412-420.

Fig. 1: Hydrothermally prepared BTO nanoparticles from TiO2 ; a) TEM image, b) HRTEM and SAED image

Fig. 2: SEM images of a) TiO2 nanotube arrays prepared by anodization of Ti foil, b) Hydrothermally prepared BTO/TiO2 nanocomposites from TiO2 arrays

Fig. 3: Impedance spectroscopy measurements; a) Shematic view of experiment setup, b) Conductivity measurements dependendance of temperature
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2274 Microstructural Characterization by TEM/STEM and XAFS for Fe85.2Si1B9P4Cu0.8 nanocrystalline soft magnetic alloy

Nishijima M.1, Yasuhara A.2, Matsuura M.1, Takenaka K.1, Makino A.1
1Institute for Materials Research, Tohoku University, Sendai, Japan, 2EM Application Group, EM Business Unit, JEOL Ltd., Tokyo, Japan
m.nishijima@imr.tohoku.ac.jp

The investigation of soft magnetic alloys becomes activated increasingly because of their wide applications, such as transformer, sensors and electric devices. Aiming at the development of the soft magnetic material to achieve lower coecivity (Hc) and higher saturation magnetic flux density (Bs), new Fe85-86Si1-2B8P4Cu1 nanocrystalline soft magnetic alloy (NANOMET) has been developed by Makino et al.[1] which exhibits low Hc of about 5.8 A/m and high Bs of about 1.8 T. According to their studies[1], the magnetic properties are highly related to their heterogeneous structure of the as-quenched state and the nanocrystallization induced by the addition of Cu, P and Si. Therefore, it is quite important to understand the nanocrystallization kinetics of NANOMET. In this study, we focus on the structural change during nanocrystallization by means of transmission electron microscope (TEM) and X-ray absorption fine structure (XAFS). Ribbon samples of Fe85.2Si1B9P4Cu0.8 alloy were prepared by single roller melt-spinning in air atmosphere. As-quenched ribbons were annealed at several temperatures in an infrared furnace. TEM specimens were prepared by Ar ion milling method. TEM characterizations were performed using an atomic resolution analytical electron microscope (JEM-ARM 200F). XAFS measurements of the Fe and Cu K-edge were performed using synchrotron radiation at BL14B2 in SPring-8. Fig. 1 show STEM-HAADF image and their SAED patterns (inset) of Fe85.2Si1B9P4Cu0.8 alloy annealed at (a) 638K for 0s and (b) 693K for 600s, respectively. Due to the higher Fe (85~86 at.%) content, a primary crystalline phase is α-Fe and a few α-Fe nanocrystallites arise in a very limited local area at 638K for 0s even below the onset of the 1st crystallization. The homogeneous structure consisting of fine α-Fe grains with the size of about 12 nm was formed at 693K for 600s. Fig. 2 shows (a) high resolution STEM-HAADF image and (b) STEM-XEDS mapping of an α-Fe nanocrystal precipitated in an Fe85.2Si1B9P4Cu0.8 alloy annealed at 638K for 0s. Fe K-, P L- and Cu K-line are superimposed in Fig. 2 (b), some of fine Cu-rich clusters (1.5 nm or less) are observed inside and/or neighboring the precipitated α-Fe and P is rejected and enriched around the precipitated α-Fe, suggesting that P plays the similar roles to Nb in FINEMET[2]. From our results, heterogeneous precipitation of α-Fe crystallites occurs nearby Cu rich clusters and close relationships between the local structural change around Cu and progress of α-Fe precipitation was confirmed by TEM and XAFS.

[1] A. Makino, IEEE Trans. Magn., vol. 48, pp. 1331-1335, Apr. 2012.

[2] K. Hono, D. H. Ping, M. Ohnuma, and H. Onodera, Acta mater., vol. 47, pp. 997-1006, Mar. 1999.

This work was financially supported by Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan under “Tohoku Innovative Materials Technology Initiatives for Reconstruction” project, “Ultra-low Core Loss Magnetic Material Technology Area”. XAFS measurements are supported by the JASRI/SPring-8 under Proposal Number 2013B1641.

Fig. 1: Fig. 1 STEM-HAADF image and SAED patterns (inset) of Fe85.2Si1B9P4Cu0.8 alloy annealed (a)at 638K for 0s and (b) at 693K for 600s.Brighter contrast grains are α-Fe nanocrystallites and dark contrast is residual amorphous matrix.

Fig. 2: Fig. 2 High resolution STEM-HAADF image (a) and STEM-XEDS mapping of precipitated α-Fe nanocrystal in Fe85.2Si1B9P4Cu0.8 alloy annealed at 638K for 0s. Blue dots in Fig 2(a) indicate the bcc-unit cell of α-Fe (upper left) and white arrow head indicate Cu rich clusters in Fig. 2 (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2523 Atomic resolution electronic state analysis in La2CuO4

Haruta M.1, 2, Nagai T.1, Lugg N. R.3, 4, Neish M. J.3, Nagao M.1, 5, Kurashima K.1, Allen L. J.3, Mizoguchi T.6, Kimoto K.1
1National Institute for Materials Science, Ibaraki, Japan, 2Institute for Chemical Research, Kyoto University, Kyoto, Japan, 3School of Physics, University of Melbourne, Victoria, Australia, 4Institute of Engineering Innovation, University of Tokyo, Tokyo, Japan, 5School of Science and Engineering, Waseda University, Tokyo, Japan, 6Institute of Industrial Science, University of Tokyo, Tokyo, Japan
haruta@eels.kuicr.kyoto-u.ac.jp

  Transition metal (TM) oxides exhibit a variety of physical properties sensitively related to their electronic structures arising from the TM-oxygen polyhedron structure. Today, Cs-corrected STEM-EELS is able to undertake electronic state analysis with atomic resolution. However, experimental spectra include the components from not only the illuminated atomic column but also neighboring columns due to the delocalized nature of the inelastic interactions and the complex elastic and thermal scattering that come from finite specimen thickness. Recently, atomic resolution O K-edge electron energy-loss near-edge structure (ELNES) from distorted metal-oxygen polyhedra was achieved using aberration corrected low-voltage STEM-EELS (FEI Titan operated at 80 keV ) combined with an inversion process to remove the effects of elastic and thermal scattering of the STEM probe from the spectrum imaging data [1].
In the present research, atomic resolution oxygen K-ELNES was studied for La2CuO4 (Fig. 1), which is well known as a parent compound of the high-Tc superconductor La2-xMxCuO4 (M = Sr, Ba), by a combination of first-principles band structure calculations based on the local-density approximation plus on-site Coulomb repulsion (LDA+U) approach and experiment [2]. We will discuss, how the anisotropic chemical bonding of the oxygen 2p state arises not only from hybridization with the Cu 3d state, but also with the Cu 4p and La 5d/4f states. In Fig. 2 we see that the two crystallographically inequivalent O sites show different spectra dependent on the bonding states. In Fig. 3 we show how these different states can be visualized by integrating small energy regions of the EELS data around spectral features associated with the different bonding states. Such an atom-by-atom understanding of the inequivalent oxygen sites in such materials could potentially accelerate efforts to understand the physical properties of complex materials.
[1] N. R. Lugg et al., Appl. Phys. Lett. 101 (2012) 183112.
[2] M. Haruta et al., J.Appl. Phys. 114 (2013) 083712.

This research was partly supported by JSPS Fellowships No. 23-51 and the Discovery projects funding scheme of Australian Research Council (Project Nos. DP110101570 and DP110102228).

Fig. 1: Crystal structure of orthorhombic La2CuO4.

Fig. 2: Experimental O K-edge spectra from crystallographically distinct columns processed by an inversion method[1]

Fig. 3: (a) HAADF-STEM image of the spectrum imaging area. (b) Atomic resolution oxygen mapping. Oxygen mapping discriminated by differences in hybridization states, mainly with (c) Cu 3d, (d) La 5d, (e) La 5d/4f, (f) Cu 4pz and (g) Cu 4pxy using the 2 eV energy window.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2671   TEM AFM study of interface roughness in superconductor/ferromagnet Nb/Co multilayers

Liu L. Y.1, Chacón Hernandez U. D.2, Xing Y. T.3, Suguihiro N. M.2, Haeussler D.4, Baggio-Saitovitch E.2, Jaeger W.4, Solórzano-Naranjo I. G.1
1DEMa, Pontifícia Universidade Católica do Rio de Janeiro, Rio de Janeiro, Brasil, 2Centro Brasileiro de Pesquisas Físicas, Rio de Janeiro 22290-180, Brasil, 3Instituto de Física, Universidade Federal Fluminense, Niterói 24210-346, Brasil, 4Institute for Materials Science, Christian-Albrechts Universität zu Kiel, Germany
lyliu.xing@gmail.com

   

Superconductor (SC)/ferromagnet (FM) Nb/Co multilayers have been produced with magnetron-sputtering with the 100 nm thickness of Nb and 5, 10, 20 nm of Co. The superconducting properties have been investigated by electric transport measurements. The magnetic properties show that with increase of the thickness of Co layers the superconducting transition temperature (Tc) significantly increases. In order to study the unusual behavior, cross-section samples have been prepared to investigate microstructures of the SC/FM interface by means of Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM) and energy-dispersive X-ray spectroscopy line scan analyses in scanning TEM (STEM) mode. Figure 1 (a) are the bright field TEM images showing a Nb – Co multilayer system with uniform thickness of the Nb and the Co layers. The individual Nb layers are polycrystalline and exhibit defects. The Co layers appear uniform in thickness and are interconnected on a larger scale. The dark filed images [figure 1 (b)] shows the size and shape of single crystallites and the texture-like lattice orientation of some of the Nb grains and dislocation contrast is visible in some of the grains. The transmission electron diffraction pattern (inset of figure 1) shows the polycrystalline nature of the Nb-Co multilayer. The surface profiles of two samples show that the sample with thinner Co layers [figure 2 (a)] has a much rough surface than the sample with a thicker Co layers [figure 2 (b)]. From our study we can conclude that the roughness of the SC/FM interfaces plays an important role in the effect of the magnetic layers on Tc: it first increases the area of the interface between the SC/FM layers, which gives stronger proximity effect and, second, enhances the effect of the stray field on Tc based upon nano-scale observations of interfaces topography. It was found that the parameter which determines the effect of magnetic layers on the superconducting layers is not an absolute number of roughness only, but the ratio of the roughness and the thickness of the magnetic layers. On the other hand, these observations invite to elucidate the crystalline defect structure at the Nb/Co interfaces, namely misfit-compensating dislocations density, and the associated strain fields to allow localized coherency at the interfaces.

This research is supported by CNPq, FAPERJ (Brazil).

Fig. 1: (a) Bright-field (BF) TEM micrograph of multilayer cross-section sample Co5; (b) Dark-field (DF) TEM image. Inset shows the transmission electron diffraction pattern. White circle: position and size of aperture chosen for TEM DF imaging. Dark arrow: beam stop blocking the central electron beam.

Fig. 2: AFM surface profile for (a) sample with 5 nm of Co and (b) sample with 10 nm of Co.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2728 EBSD Study of Coincide Site lattice Grain Boundaries in SrTiO3 based Thermoelectric

Azough F.1, Gholinia A.1, Freer R.1
1Materials Science Centre, School of Materials, University of Manchester, Manchester, U.K.
feridoon.azough@manchester.ac.uk

Oxide ceramics are promising Thermoelectric materials owing to high temperature stability. The current focus is to develop improved n type and P type materials for medium to high temperature energy conversion of heat into electricity [1]. La-doped SrTiO3 ceramics are among promising thermoelectric materials for conversion of waste heat to electricity. One of the controlling factors of the thermoelectric efficiency is the resistivity of the bulk ceramic. Grain boundaries play an important role [2] in controlling ion conductivity for example in the case of SrTiO3 the resistance of Sigma 3 grain boundaries is much lower than the general grain boundaries.  Sr0.9La0.1TiO3 thermoelectric ceramics were synthesized by the mixed oxide route. Ceramics were sintered in air atmosphere then annealed in a reducing atmosphere for 8 hours to 32 hours with increments of 8 hours. The population and type of the Coincide Site lattice boundaries of the annealed samples were investigated using EBSD. The observations suggest that resistivity is strongly correlated to the type of grain boundary in reduced ceramics.
The EBSD patterns of the annealed samples were obtained using AZtech EBSD system from Oxford Instruments, with an 20 nA beam current and 3 µm step size at a working distance of 15 mm. Typical EBSD pattern and distribution of the various types of the grain boundaries is presented in Figure 1(a, b).

1.    Fergus J. W, J. Euro. Ceram. Soc. 32(2012) p. 525.
2.    Alftan S. V., et al, Annu. Rev. Mater. Res. 40(2010) P. 577.

We gratefully acknowledge the support of EPSRC through awards EP/1036230 and EP/J000620.

Fig. 1: Figure 1(a,b). EBSD data for sample annealed for 24 hours at 1350°C in hydrogen. a; EBSD pattern, b; content of the Coincide Site lattice Grain Boundaries.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2754 A TEM study of ferromagnetic τ-phase and phase transformation in a Mn-Al alloy

Dhanalakshmi P.1, Chattopadhyay K.1, Madras G.2, Srivastava C.1
1Department of Materials Engineering ,Indian Institute of Science,Bangalore,560012., 2Chemical Engineering Department, Indian Institute of Science,Bangalore,560012.
dhanamaterials@gmail.com

This paper presents microstructural evolution in the binary aluminium manganese Heuslar alloy near equiatomic composition which exhibits attractive magnetic properties. The Heusler phase Mn55Al45 known as τ-phase is not an equilibrium phase in Al-Mn binary system and has a metastable existence. We have successfully synthesize this phase through various processing routes. There exists a debate in the literature about the mechanism of growth of this phase during the formation from the high temperature equilibrium ε phase. By employing various microscopie techniques including structural characterization through transmission electron microscopy, analytical characterization through microprobe, orientation imaging and insitu - transmission electron microscopy. The parameters in the melting process as well as the effect of cooling rate on their microstructure, phase evaluation changes of this metastable ferromagnetic τ-phase ingot have been studied. We are successful in unravelling the pathways for the formation of ferromagnetic tetragonal τ-phase and their decomposition to equilibrium phases. We show that τ-phase forms from high temperature hexagonal ε-phase with little change in composition and containing significant amount of planar defects. It decomposes through an eutectoidal transformation through the formation of cubic-β and rhombohedral-γ2 phase. Along with these results, we shall also present the magnetic properties which can be mapped with the changes in the microstructure.

We would like to acknowledge microscopy facility available at materials department (JEOL 2000FX) and at AFMM centre (FEI F30).

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2788 STEM-EELS Study of SrTiO3 Based Thermoelectric

Azough F.1, Jackson S. S.1, Freer R.1, Kepaptsoglou D.2, Ramasse Q. M.2
1Materials Science Centre, School of Materials, University of Manchester, Manchester, M13 9PL, U.K., 22SuperSTEM Laboratory, STFC Daresbury, Keckwick Lane, Warrington, WA4 4AD, U.K.
feridoon.azough@manchester.ac.uk

Oxide ceramics are promising Thermoelectric materials owing to high temperature stability. Impetus is to develop improved n type materials for medium to high temperature energy conversion of heat into electricity [1]. Ceramics based on SrTiO3-La2/3TiO3 perovskites are among candidate materials for such thermoelectric applications. STEM-EELS has been performed in order to assess for the first time the quantitative effects of composition on the structure and properties of these materials. (1-x)SrTiO3-xLa2/3TiO3 ceramics (x= 0.1, 0.2, and 0.5) were synthesized by the mixed oxide route. Ceramics were sintered in both air and reducing atmosphere. Structures were initially investigated using selected area electron diffraction (SAED) and high-resolution transmission electron microscopy (HRTEM) techniques using a FEI FEGTEM (Tecnai G2) operating at 300 kV. Subsequently, atomic level resolution level structural characterization was carried out using an aberration-corrected Nion (UltrastemTM100). Lattice images were obtained along <100> orientations and sub-grain features to visualize the cation and vacancy distribution in the lattice and structure of precipitates. Electron Energy Loss Spectroscopy was used to determine the distribution of La and Sr in the structure and chemistry of the precipitates. HAADF-EELS study showed that for the compositions with 0.1≤x≤0.5 the distribution of La and vacancies in the structure of cubic SrTiO3 is random as shown in Figure 1. This random distribution is independent of the sintering atmosphere. However for samples sintered in a reducing atmosphere, nanometer size voids and particles are formed (Figure 2). The analysis showed that the precipitates are rich in Ti and the voids contain high content of Ti3+. It has been demonstrated that atomic resolution HAADF-EELS is a powerful technique for determination of lattice site occupancy, valance state of constituent elements and chemistry of the sub-grain features in SrTiO3 based thermoelectric.

[1]  Fergus J. W, J. Euro. Ceram. Soc. 32(2012) p. 525.

We gratefully acknowledge the support of EPSRC through awards EP/1036230 and EP/J000620. SuperSTEM is the UK National Facility for Aberration corrected STEM, funded by the EPSRC.

Fig. 1: [001] HAADF-EELS data for x=0.5.

Fig. 2: a; TEM image showing precipitates and voids, b; HRTEM for  the void, c; HAADF   image for the void, d; Ti L2,3 peaks for two different areas around the void.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2846 Microscopic Investigation on Domain Development in Ferroelectric/Relxaor Composite Oxide

Gi-Yeop K.1, 2, Sung-Dae K.1, Young Mok R.1, Seog-Young Y.2, Si-Young C.1
1Korea Institute of Materials Science, ChangWon, Korea, 2Pusan National University, Pusan, Korea
rlarldug@kims.re.kr

BNT-based piezoelectric composite received intensive attentions as a promising candidate for replacing the environmentally hazardous Pb-based piezoelectric materials such as PZT. Bi0.5(Na(1-x)Kx)0.5TiO3–BiAlO3 (BNKT-BA) ceramic is considered to be one of the replacements for such Bi-based piezoelectric materials due to its large strain value and high depolarization temperature (Td). In spite of its improved piezoelectric properties, it still has low remanent polarization (Pr) and also requires large poling field to make phase transition from non-polar to polar phase. Recently, in order to complement these drawbacks, hard ferroelectric ceramic such as ferroelectric-Bi0.5(Na(1-x)Kx)0.5TiO3 (f-BNKT) was embedded in the BNKT-BA ceramics to decrease the poling field and increase Pr, however, there are still a few reports concerning the microscopic evolution during the phase change in terms of role of the ferroelectric phase on the piezoelectric properties of BNKT-BA ceramics.

In this study, we investigated the microstructure of BNKT-BA + f-BNKT and focused on the low electric field induced phase transitions. We used a modified in-situ electrical biasing transmission electron microscopy (TEM) stage for observing the phase change in real time. Before applying electric field, bright field (BF) images, centered-dark-field (CDF) images and selected area electron diffraction (SAED) patterns of the ceramics were systematically analyzed. From the superlattice spots of 1/2(ooo) and 1/2(ooe) in the SAED patterns (FIG. 1 (b) and (c)), we notice the existence of the R3c phase and the P4bm phase which have anti-phase (a-a-a-) and in-phase (a0a0c+) octahedral tilt symmetry, respectively. The morphology and distributions of these phases are also shown in the CDF images (FIG. 1 (d) and (e)). A notable point is that by adding the f-BNKT particles of 5~20 µm size, most of the BNKT-BA + f-BNKT grains forms in the type of mixture of ferroelectric R3c (R phase) core region and relaxor-like (R+T phase) shell region. On the other hand, the grains of BNKT-BA ceramic without f-BNKT particles are comprised of typical relaxor type (R+T phase). The relaxor phases in the BNKT-BA ceramic never change by applying electric field up to 2kV/mm, whereas the domains are effectively realigned with the assistance of relaxor-like R+T phase even at lower than 1kV/mm, as shown in FIG. 2. Therefore, the inclusion of ferroelectric phases activates the domain propagation and alignment even at the low electric voltage, supposedly due to the formation of local polarized field at the interface between f-BNKT and relaxor BNKT-BA.

This work was supported by the Global Frontier R&D Program (2013-073298) on Center for Hybrid Interface Materials (HIM) funded by the Ministry of Science, ICT & Future Planning. (2013M3A6B1078872)

Fig. 1: TEM micrographs of a core-shell grain in BNKT-BA+f-BNKT. (a) BF image of the grain with zone-axis. (b) and (c) SAED patterns of shell and core regions, respectively. (d) and (e) CDF images using g=1/2(1-32) and g=1/2(-3-1-1), respectively. (f) and (g) BF image and DF image using g=1/2(031) in BNKT-BA grain with [013] zone-axis.

Fig. 2: TEM images of a core-shell grain under electric fields along [013] zone axis. (a) At 0kV/mm. The boundary between R-domain and T-domain is highlighted by yellow dotted lines. (b) At 1kV/mm. (c) At 2kV/mm. (d) After poling. (e), (f) SADE patterns from region R and T, respectively
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2912 Re-constructing the phase relations in Co38Ni33Al29 ferromagnetic shape memory alloy

Kopeček J.1, Jurek K.2, Kopecký V.1, Klimša L.1, Seiner H.3, Sedlák P.3, Landa M.3, Dluhoš J.4, Petrenec M.4, Hladík L.4, Doupal A.4, Heczko O.1
1Department of Functional Materials, Institute of Physics AS CR, Prague, Czech Republic, 2Department of Structure Analysis, Institute of Physics AS CR, Prague, Czech Republic, 3Laboratory of Ultrasounds Methods, Institute of Thermodynamics AS CR, Prague, Czech Republic, 4Tescan Brno, s.r.o., Brno, Czech Republic
kopecek@fzu.cz

Alloys in the Co-Ni-Al system and even their shape memory properties have been studied for decades as the cobalt-based alloys were supposed to have higher transformation stresses and wider intervals of superelasticity than other shape memory alloys (SMA). As these alloys are ferromagnetic the magnetic shape memory effect could be expected like in Ni-Mn-Ga and similar alloys. Additionally, the two-phase structure in the high-temperature austenitic state is an exception within the SMA. It contains a B2 ordered (Co,Ni)Al matrix and A1 fcc solid solution particles. The A1 (interdendritic) particles remain untransformed, whereas the B2 matrix transforms martensitically into the L10 martensite. Only non-modulated L10 martensite was observed in Co-Ni-Al based alloys.

Using the Bridgman method, a set of unidirectional solidified / single crystalline samples was prepared. It was found that martensite observed at room or even higher temperature is not the thermodynamically stable one, but rather stress induced. The equilibrium martensitic transformation is observed close to 200 K. The stress induced martensite existing above its equilibrium temperature has the same structure as the equilibrium one.

There exists a large difference between “as grown” and “annealed and quenched” samples. While the first group shows simple elastic behavior, the second exhibits significant superelasticity with strong orientation dependence. The explanation must be connected with established high temperature equilibrium during annealing and the processes of its relaxation after quenching. The set of various precipitates was already observed in quenched samples. Besides the precipitation in the matrix the annealing causes dissolving of interdendritic particles. Such process enriches the matrix with cobalt atoms and leads to concentration gradients on the particles’ borders.

The recent instrumental development of our group allowed us to use 3D reconstruction techniques with high-performance Xe-FIB. Thus the investigation of interactions transforming matrix and non-transforming particles become possible and the complex geometry of stress induced martensite can be investigated.

Authors would like to acknowledge the financial support from the Czech Science Foundation project P107/11/0391.

Fig. 1: The A1 interdendritic particle entangled by the stress induced L10 martensitic lamellae in sample annealed 1350 °C for 1 h and quenched into the cold water.

Fig. 2: The phase distribution of A1 interdendritic particles (green color) and B2 matrix (red color) observed by EBSD in as-grown Co38Ni33Al29 alloy prepared by Bridgman method.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2951 Study on perpendicular exchange bias in sputter-deposited CoO/CoPt multilayers

SHI J.1, WANG J.2, Sannomiya T.1, Nakamura Y.1
1Department of Metallurgy and Ceramics Science, Tokyo Institute of Technology, Tokyo, Japan, 2National Institute for Materials Science, Tsukuba, Japan
sjtokyotech@gmail.com

Exchange bias (EB) is an interfacial phenomenon associated with anisotropic exchange coupling created at the interface between antiferromagnetic (AF) and ferromagnetic (FM) materials when the system is magnetic field cooled through the Néel temperature (TN) of the AF material.1 Recently, interests in perpendicular exchange biased (PEB) spin valves for spintronics and magnetoelectronics have been dramatically increased due to their high magnetic stability and low device operating current density.2
  In this study, the structural and magnetic properties of room temperature sputter deposited CoO/CoPt multilayer were investigated. As shown in Fig. 1(a), the as deposited multilayer shows strong perpendicular magnetic anisotropy (PMA) about 5x106 erg/cm3 at room temperature. Moreover, after perpendicular magnetic field cooling, the multilayer with AF/FM interfaces exhibits a large shift (PEB) about 1700 Oe in the out of plane hysteresis loop as indicated in Fig. 1(b). Meanwhile, the PMA of the multilayer after field cooling is also enhanced compared with that at room temperature. This is considered due to the strong interfacial exchange coupling between the CoPt and CoO layers, which can be also confirmed from the significantly enhanced perpendicular coercivity. Unlike the Co/ noble-metal structures, the multilayers studied here with relatively thick FM layer (tFM~4nm) surprisingly show strong PMA at as-deposited state (Fig.1 (a)).Therefore, the PMA found in AF/FM multilayer may be partially attributed to the interfacial AF-FM exchange coupling.
  Systematic structural analysis was carried out to clarify the origin of the promising magnetic properties. First, XRD scan indicates a well-defined layer structure and strong (111) texture for CoPt layers (Fig. 2). Then, lattice fringes in TEM image (Fig.3a) support the results of XRD. And one see the local coherent growth of CoPt and CoO crystals penetrates through the multilayer along the growth direction of <111> for both layers(Fig.3b), indicating local epitaxial growth of CoPt on CoO and vice versa (as shown in Fig. 3c). Here, we can conclude that the PMA was attributed mainly to the positive magnetoelastic energy due to the in-plane tensile stress which originates from the local epitaxial growth at the CoO/CoPt interface. It is the first time to prove that the PMA can be also established with metal/oxide interface. Furthermore, with the remarkable PEB, the CoPt/CoO multilayer studied here can be considered as an idea candidate for perpendicular exchange biased (PEB) spin valves for spintronics and magnetoelectronics.

References
1 W. H. Meiklejohn and C. P. Bean, Phys. Rev. 105, 904 (1957).
2 http://www.toshiba.co.jp/about/press/2012_12/pr1001.htm

We want to thank for the collaborative R005 usage of the CREST project "Nanocycle at Nano-in-Macro Interface", supported by Japan Science and Technology Agency (JST).

Fig. 1: Room temperature (a) and low temperature (80K) (b) hysteresis loops of CoO(10nm)/[CoPt(3nm)/CoO(4nm)]8 multilayer. (Field cooling was performed with a 5 kOe external magnetic field perpendicular to the thin film plane.). Insert in (a) shows room temperature hysteresis loops of CoO 10nm/[CoPt 7.6nm/CoO 4nm]3 multilayer.

Fig. 2: Low and high angle XRD profiles of CoO(10nm)/[CoPt(3nm)/CoO(4nm)]8 multilayer using CuKα irradiation.

Fig. 3: (a) STEM-HAADF image of the CoPt/CoO multilayer structure and (b) Zoomed STEM-HAADF image of CoPt/CoO interface indicating the epitaxial relationship between them. (c) Schematic diagram of the interface atom arrangement and epitaxial relationship.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2959 Interparticle interactions for assemblies of magnetic nanoparticles with three-dimensional magnetic vortex

Kim M. K.1, Lee H. Y.1, Jin K.2, Prasanta D.1, Lee J. Y.1, Yoo M. W.1, Lee J. H.1, Chu A.2, Kim M.2, Nam K. T.2, Kim S. K.1
1National Creative Research Initiative Center for Spin Dynamics and Spin-Wave Devices, and Research Institute of Advanced Materials, Department of Materials Science and Engineering, Seoul National University, Seoul 151-744, Republic of Korea, 2Department of Materials Science and Engineering, Seoul National University, Seoul 151-744, Republic of Korea
ngfriend@snu.ac.kr

A very specific spin configuration in magnetic nano-objects, known as a magnetic vortex in different shapes and dimension is promising for bio-applications, information-storage and processing technologies. For example, some literatures report the possibility of artificial structures of magnetic vortices in 2D nanodisks showing exotic physical phenomena. Additionally, magnetic vortex in a cubic or spherical shaped, instead of single-domain magnetic particles, may be exploited for bio applications, therefore, nanoparticles of 3D vortex could be of particular interest for dedicated purposes. Combining these results, self-assembly of isolated or aggregated magnetic nanoparticles is a promising approach to utilize nanostructures with multiple magnetic vortices as an elementary unit. From the perspective of bottom-up approach, the self-assembly of 3D magnetic vortices system can be understood by investigating inter-particle interaction of small clusters of vortex nanoparticles. However a precise knowledge of inter-particle interactions between magnetic-vortex particles is still elusive.
In the present study, we have clarified the magnetic interaction of the permalloy (Py) nanoparticles of 3D vortices as a primary mechanism of forming isolated-single and aggregated-double, triple, and quadruple spheres of different geometrical configurations. The Py nanoparticles of about 100 nm diameters were synthesized by the polyol method (Figure 1). From the transmission electron microscopy (TEM) analysis, preferred geometric configurations for the combination of Py particles were found depending on the number of assembled particles (Figure 2). With the help of micromagnetic siumlations, the interparticle interactions between of the corresponding assemblies were investigated. From the calculations, it is readily shown that magnetic exchange interaction acts as a key factor for forming assemblies of magnetic vortices. The spin configurations of specific assemblies were further investigated by off-axis electron holography (EH). We measured EH from isolated-single and interacting-triple particles of different arrangement. As expected from the micromagnetic simulation, the isolated 100 nm nanoparticles exhibited a vortex state. In the case of interacting particles, the direction of the vortex cores in assemblies is consistent with the calculations. Our results indicate that there is a controllable means of assembling complex nanoparticles of unique 3D vortex spin configuration.

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant No. 2013003460), and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF 201304238).

Fig. 1: (a) TEM image of aggregated Py nanoparticles with diameter of 100 nm. (b) XRD pattern of Py nanoparticles with diameter of 100 nm. (c) SAED pattern of single nanoparticle. (d) Magnetic hysteresis loops of aggregated Py nanoparticles of 100nm recorded at 300 K.

Fig. 2: TEM images of small self-assemblies of 100 nm Py nanoparticles. Observed assemblies are indexed by numerical number and alphabet.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-2981 Effect of SiC doping on improvement of superconductive properties of MgB2 wires studied by TEM

Song M.1, Ye S. J.1, Takeguchi M.1, Matsumoto A.1, Togano K.1, Kumakura H.1
1National Institute for Materials Science, Tsukuba, Japan
minghui.song@nims.go.jp

One of the problems of the promising superconductor materials of MgB2 is that the critical current density (JC) is not high enough for practical use. It has been established that doping with carbon or/and nano-sized inclusions can improve JC of MgB2. SiC is one the most effective dopes for improving the superconductive properties of MgB2. Although many works have been carried out on the doping methods and the changes in physical properties of MgB2, few concerned the effect of SiC doping on microstructure, and especially on the status of other existed impurities in detail. MgO is a kind of impurity in MgB2 which is introduced in the fabrication process of the material and is difficult to be removed. The existence and distribution of MgO inclusions in MgB2 should be also very important to the properties of the material. In the present work, we characterized the microstructure of MgB2 wires, without and with SiC doping, fabricated with internal magnesium diffusion (IMD) process, especially focused on the status of MgO, by means of TEM including tomography, and correlated the microstructure and the distribution of MgO nano-inclusions to the superconductive properties of the wires.

TEM thin film specimens were prepared with a focused ion beam instrument, JEM-9310FIB. JEM-2100F and JEM-3000F were used for the observation and analysis. Two typical areas, called as area A and B respectively hereafter, were observed in both the specimens. Area A was composed of small crystal grains while area B was in amorphous status. The crystal grains were confirmed to be those of MgB2, Mg2Si and MgO in the SiC-doped specimen, and MgB2 and MgO in the specimen without addition of SiC. Area B was confirmed to be amorphous boron with EELS and energy filtering imaging. In HAADF observation, bright contrast particles in size ~ 10 nm were identified which were considered as MgO particles, since MgO had a highest density of the constituents. The MgO nanoparticles distributed dispersively well in the crystalline area in the specimen doped with SiC, while concentrated in a layer-like volume surrounding area B in the specimen without doping of SiC. Figure 1 shows typical images of a MgB2 specimen doped with SiC. TEM tomography revealed that MgO particles in the specimen without doping of SiC distributed mainly on a 3-dementional layer-network which was considered to be the surfaces of starting boron particles. It was considered that doping of SiC seemed to slow down the reaction of Mg with O, and resulted in the dispersion of MgO nanoparticles. Since MgO nanoparticles could act as magnetic pinning centers in superconductor MgB2 wires, the dispersed distribution of MgO particles should be one of the main reasons for the improvement of JC for the MgB2 wires doped with SiC.

Fig. 1: A TEM BF image (a) and a HAADF image (b) of a MgB2 specimen doped with SiC. A and B indicate the crystalline area and amorphous area, respectively. Arrows in (b) show small particles with bright contrast which were considered to be the particles of MgO.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3011 Evidence of a structural and magnetic transition in CaFe5O7 at 360K

Pelloquin D. e.1, Delacotte C. h.1, Hüe F. l.2, Bréard Y. o.1, Hébert S. y.1
1CRISMAT ENSICAEN UMR CNRS 6508, 6 Boulevard du Maréchal Juin, 14050, Caen Cedex 04, France, 2GPM UMR 6634, Avenue de l'Université, BP12 76801 St Etienne du Rouvray. France
denis.pelloquin@ensicaen.fr

Numerous studies are always devoted to mixed valence state iron oxides in materials research due to their complex magneto transport properties like the famous Verwey transition [1]. Among these oxides, a special attention is focused on the orthoferrites LnFeO3 (Ln= rare earth) related to the distorted GdFeO3-type perovskite structure which can exhibit some possible spin reorientation transitions versus temperature and the nature of Ln [2]. Recently, iron based oxides like LnFe2O4 have also focused a large attention due to their ability to exhibit some multiferroic properties [3]. In these systems, both kinds of Fe species (Fe2+ and Fe3+) localize magnetic moments leading to a ferrimagnetic ordering associated to ferroelectric properties. An exciting challenge is to evidence similar properties in other iron based systems. The Ca-Fe-O system offers several interesting candidates like the CaFe5O7 phase in regard to the richness of its phase diagram.

CaFe5O7 oxide exhibits a complex structure which can be described as an intergrowth between one CaFe2O4 unit and n=3 slices of FeO Wustite-type structures [4]. A recent structural study carried out at room temperature combining transmission electron microscopy (TEM) observations and powder X-ray diffraction data has revealed a supercell with a monoclinic symmetry [5]. From the hkl conditions deduced to the electron diffraction study, a structural model considering to this supercell and the centrosymmetric P21/m setting can be proposed. The fine structural analysis combining Rietveld refinements from neutron and X-ray data evidence six independent iron sites and two specific oxygen environments with coordination 6 and 5+1 respectively. According to the chemical formula CaFe5O7, the iron species average state valence is +2.4 and implies the coexistence of Fe+3 and Fe2+, so magnetic interactions could be expected. To base this peculiar feature, the magnetic dependence versus temperature has been studied and susceptibility measurements have revealed discontinuity around 360K (Fig.1). The structural evolution depending on temperature of CaFe5O7 has been also tuned from diffraction techniques. A clear reversible transition (monoclinic to orthorhombic) has been detected in the same temperature range with the disappearing of the supercell (Fig.2) in agreement with the sensivity of the latter under electron beam in Image mode. However a complementary STEM-HAADF study has allowed to image the superstructure (fig.3), with ordered contrasts at the level of iron rows.

[1] E J W Verwey, Nature 144, 327 (1939)

[2] R. Bozorth & al Phys. Rev. Lett., 1, 3, (1958)

[3] M. Hervieu & al, Nature Materiels, 13 (2014)

[4] O. Evrard & al, JSSC 35, 112 (1980)

[5] C. Delacotte & al Key Engineering Materials (in press)

The authors acknowledge the financial support of the french Agence Nationale de la Recherche (ANR), through the program “Investissements d’Avenir” (ANR-10-LABX-09-01) , LabEx EMC3.

Fig. 1: Thermal dependence of magnetic susceptibility

Fig. 2:  electron diffraction patterns of [10-1] zone axis collected at  (a) RT and 450K

Fig. 3: [10-1] HAADF raw image with corresponding FFT and filtered image
Fig. 4:
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3112 Nanoscale investigation of domain-wall pinning and its pinning site

Masseboeuf A.1,2, Nguyen V. D.2, Salvador V.2, Bayle-Guillemaud P.2, Vila L.2, Marty A.2, Attané J. P.2
1CEMES, CNRS, Toulouse, France, 2INAC, CEA, Grenoble, France
aurelien.masseboeuf@cemes.fr

Perpendicular magnetized magnetic materials (or PMA for perpendicular magnetic anisotropy) have been highlighted in the beginning of the 21st century when hard drives manufacturers decided to switch the magnetization perpendicularly to the plane of the disks. On a more fundamental side, PMA materials are promising within the spin-torque research field as their really narrow domain walls (DW) induce a huge magnetization gradient which could favor high mobilities at low current. Unfortunately, these very small walls widths (around 5 nm) is a disadvantage for their characterization as very few techniques offer enough resolution to scrutinize in detail their shape, and moreover their transformation during displacement. Such displacements are by far governed by the presence of structural defects that can sometimes lead to an increase of wall mobility in planar systems [1]. Origin of such defects belong more or less unknown and their effect on DW pinning is still unclear.
This work will present recent results obtained on FePt crystalline layer which exhibit huge crystalline anisotropy (> 10 T) out of the plane of the sample. The magnetization is thus spontaneously divided into well defined Up and Down magnetic domains bounded by very thin domain walls. The high temperature growth implies a relaxation process at the interface with a Pt buffer. Strains are relaxed by the nucleation of micro-twin defects which can be described as nano-volumes of crystal with a c-axis tilted with respect to the growth direction. Such defects are known to pin the domain walls [2,3], but no information on the structure of the wall was experimentally evidenced. Furthermore, even if the micro-twin are well known no clear evidence was reported that the L10 order, which is the origin of the huge crystalline anisotropy, is or not preserved within these defects.

We report in the present work the combination of Holography (Fig 1) and STEM-HAADF (Fig 2) experiments on FePt thin foils revealing at the same time and at the same place the magnetic spin distribution and the atomic chemical ordering which is related to the magnetic domain wall configuration.

References

[1] Lewis, E. R. et al. Fast DW motion in magnetic comb structures. Nat. Mater. 9, 980–983 (2010)
[2] Attane, J. P. et al. DW pinning on defects in FePt thin films. Appl. Phys. Lett. 79, 794–796 (2001)
[3] Jourdan, T. et al. Pinning of magnetic DW to defects (...) Phys. Rev. B 75, 094422 (2007).

This work has been realized thanks to the financial support of Nanosciences Fundation (Grenoble, France).

Fig. 1: Electron Holography experiment exhibiting the Up and Down domain configuration. Top image displays the phase with contour lines representing magnetic flux. Dashed area is the location of STEM HAADF investigation presented in Fig 2. Bottom image is the horizontal gradient thus displaying the vertical magnetic induction component. 

Fig. 2: STEM-HAADF images of the atomic stacking within a microtwin. Top schemes are cristalline structure and the microtwin config. Bottom images show the contrast obtain on such sample where only Platinium atoms can be seen in HAADF signal. The arrangement in the microtwin exhibits a tilted c-axis highlighting a locally tilted anistotropy axis.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3182 Order-disorder phenomena in oxygen deficient Ruddlesden-Popper phases

Ruiz-González L. M.1, González-Merchante D.1, Cortés-Gil R.1,2, Alonso J.3, González-Calbet J. M.1
1Departamento de Química Inorgánica, Facultad de Químicas, Universidad Complutense, 28040-Madrid, Spain, 2CEI Campus Moncloa, UCM-Universidad Politécnica de Madrid, 28040-Madrid, Spain, 3Instituto de Ciencia de Materiales, CSIC, C/ Sor Juana Inés de la Cruz, 3, 28049-Madrid, Spain
luisarg@ucm.es

Manganese related layered compounds belonging to the perovskite related An+1BnO3n+1 Ruddlesden-Popper (RP) family (Fig. 1a) has attracted much attention in the past two decades due to a variety of emerging phenomena such as magnetoresistance. Particular attention has been devoted to the La2-2xSr1+2xMn2O7 system for x around 0.5, where the ground state changes from FM-M to AFM-I associated to a charge ordering state. Nevertheless, compositional variations at the anionic sublattice are scarce compared to other manganese related perovskite systems in which different superlattices have been described as a consequence of the ordering on non-occupied oxygen positions. Due to the influence that oxygen deficiency can play in the above properties, our objective has been to stabilize and characterize new anionic deficient phases in La2-2xAE1+2xMn2O7-δ (AE=Ca, Sr) where Mn in different oxidation states can coexist.

La2-2xAE1+2xMn2O7-δ polycrystalline samples were prepared using a conventional ceramic method. Reduced samples were synthesized in an electrobalance in order to precisely control the oxygen content. According to XRD, SAED and HRTEM studies, the La2-2xAE1+2xMn2O7 topotactic reduction process has led to the stabilization of new La2-2xAE1+2xMn2O7-δ phases with 0<δ<2. The Sr series can be indexed on the basis of a tetragonal (I4/mmm) symmetry while a clear orthorhombic (Bbmm) distortion is found in the Ca system, as shown in the representative SAED patterns (fig. 1b,c) corresponding to LaSrMn2O7-d and La0.5Ca2.5Mn2O7-δ systems along [101]c. Notice the presence of additional reflections in Ca system. Atomically resolved images of these compounds obtained in a JEOL JEMARM200cFEG electron microscope show the presence of order-disorder phenomena in these systems. For instance, a characteristic HAADF image corresponding to La0.5Ca2.5Mn2O6.5 (fig. 2) suggests, according to the atomic number of Ca and La (see the ideal model for LaCa2Mn2O7 at the inset), the major presence of La at the perovskite block whereas Ca is at the rock-salt layers. Nevertheless, the occasional presence of less bright contrast in these blocks suggests the presence of local order-disorder phenomena of La/Ca in this perovskite A site. This situation can be understood because the nominal composition La:Ca=0.5:2.5 deviates from the ideal La:Ca=1:2 ratio. EELS atomically resolved maps confirm the above situation, as shown in fig. 2. Compositional differences at the A site of the perovskite block are evident. Furthermore, EELS studies have allowed identifying Mn in different oxidation states, depending on the oxygen content, in both systems. Magnetization and transport measurements indicate that they are sensitive to the oxygen deficiency.

Fig. 1: Figure 1. (a) Structural models for RP series. Characteristic SAED patterns for (b) LaSr2Mn2O7 and (c) and La0.5Ca2.5Mn2O7

Fig. 2: Figure 2. (a) HAADF image of La0.5Ca2.5Mn2O6.5. Schematic model for the cationic position has been inserted. (b) EELS spectra sum,  over the area marked in (a), showing the Ca-L2,3, Mn-L2,3 and La-M4,5 and signals; (c) HAADF image simultaneously recorded to EELS; (d) mapping for the La-M4,5, (e) Ca-L2,3 (f) Mn-L2,3 signals.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3201 Study of magnetic FeRh nanoalloys : structure and chemical order

Castiella M.1, Tan R.2, Respaud M.2, Gatel C.1, Casanove M. J.1
1CEMES, CNRS UPR 8011 et Université de Toulouse, 29 Rue Jeanne Marvig, 31055 Toulouse Cedex 4, 2Université de Toulouse, INSA-CNRS-UPS, LPCNO, 135 Avenue de Rangueil, 31077 Toulouse, France
marion.castiella@cemes.fr

With the increasing demand for ultra-high density magnetic recording, an important effort was put on the fabrication and control of magnetic nanoalloys. More recently, much attention has been paid to the remarkable magnetic properties of the FeRh alloy, for both fundamental and technological issues. Indeed, the FeRh alloy presents, in a very narrow range of composition (0.48<xRh<0.56), a remarkable magnetic transition from antiferromagnetic (AFM) to ferromagnetic (FM) state (Fig.1). This transition takes place at a temperature close to 370 K in the bulk, i.e. slightly higher than room temperature, which makes this alloy particularly attractive for applications as heat-assisted magnetic recording [1] or microelectronics [2].

From a structural point of view, the equiatomic FeRh alloy crystallizes in a CsCl (or B2) type chemically ordered body-centered cubic (bcc) structure [3-5]. Epitaxial growth of FeRh on MgO substrate requires a suitable lattice match. For α’ phase, it is achieved through an in-plane 45° rotation of the bcc cell with respect to the MgO unit cell since aMgO=0.42 nm= √(2)aα’ (Fig.2).

We will report the influence of the growth parameters on size, morphology and structure of the deposited nanostructures. In particular, we tried to answer the question of the existence of a critical size for chemical order and magnetic properties.

The aim is to optimize growth conditions, thus to obtain the chemically ordered α’ phase epitaxially ultra-thin films.

All the studied films were grown on MgO (001) substrates by dc magnetron co-sputtering from two element targets in ultra-high vacuum chamber with a low pressure. The films were grown at 550 °C with two different thicknesses, 50 and 100 nm. After deposition, some of the films were in-situ annealed at 700 °C for 6 hours.

The evolution of the morphological and structure characteristics was analyzed by high-angle X-ray diffraction (XRD) and transmission electron microscopy (TEM) observations of cross-sectional specimens. TEM experiments were performed in conventional and high-resolution mode (HRTEM) (Fig.3). However, the magnetic properties (studied by VSM) strongly depend on the precise composition, the growth process, the pressure or stress undergone by the alloy and of course the resulting structural phases.

[1] J.U. Thiele, S. Maat, J. Robertson, E. Fullerton, IEEE Transactions on 40 (4) (2004) 2537–2542.

[2] G. Ju, J. Hohlfeld, B. Bergman, R.J.M. van de Veerdonk, O.N. Mryasov, J.-Y. Kim, X. Wu, D.Weller, B. Koopmans, Physical Review

[3] M. Fallot, Annales de Physique (Paris) 10 (1938) 291.

[4] G. Shirane, C.W. Chen, P.A. Flinn, R. Nathans, Physical Review 131 (1) (1963) 183–190.

[5] G. Shirane, R. Nathans, C.W. Chen, Physical Review 134 (6A) (1964) A1547–A1553

Fig. 1: 100-nm-thick Fe100-xRhx thin films deposited at 550 °C. Clear AFM-FM Transition. Ms value close to the bulk in the FM state

Fig. 2: FeRh growth and structure

Fig. 3: Observation of chemical order by Transmission Electron Microscopy, (a) Cross-sectional TEM micrograph of a 100 nm thick FeRh layer, (b) Corresponding Fourier transform of the HRTEM micrograph, (c) Cross-sectional HRTEM micrograph showing the presence of chemically ordered bcc structure
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3214 Hole attractors characterized by aberration corrected microscopy in the La-Ca-Mn-O system

Cortés-Gil R.1,2, González-Merchante D.1, Ruiz-González L.1, Trasobares S.3, Alonso J. M.4, González-Calbet J. M.1
1Dpto. Química Inorgánica, Facultad de Químicas, Universidad Complutense (UCM), 28040-Madrid, Spain., 2CEI Campus Moncloa, UCM-Universidad Politécnica de Madrid, 28040-Madrid, Spain , 3Dpto. Ciencia de los Materiales e Ingeniería Metalúrgica y Química Inorgánica. Facultad de Ciencias, Universidad de Cádiz. Campus Río San Pedro s/n. 11510 Puerto Real (Cádiz) Spain, 4Instituto de Ciencia de Materiales, CSIC, C/ Sor Juana Inés de la Cruz, 3, 28049-Madrid, Spain
rcortes@ucm.es

Among strongly correlated electron systems, La1-xCaxMnO3 is probably one of the most studied due to the peculiar relationship between magnetic and electric properties in addition to the presence of colossal magnetoresistance. The competence between local phenomena as lattice, spin, charge and orbital ordering is on the basis of this complex behaviour. The phase segregation model proposes the coexistence of two types of nanometer-size clusters corresponding to a FM-M phase and a correlated AFM-I one involving a charge ordering state. Actually, hole doping is induced by the substitution of La by Ca. Although this model is in reasonable agreement with numerous experimental results, it has also been proposed that in addition to providing holes to the band, the divalent substituting cation acts as effective attractor for these holes. This effect influences the magnetic response as the Mn4+ location around Ca creates nanoclusters which affects the total magnetic moment. A complete understanding of these phenomena would require an atomically resolved characterization in order to identify the La and Ca positions as well as the Mn3+ and Mn4+ distribution. Nowadays, this is possible as a consequence of the integration of spherical aberration correctors in the TEM. In this context, the aim of this work is to study the Mn4+ location in the La0.9Ca0.10MnO3, according to the hole-attractor model.

La0.9Ca0.10MnO3, prepared by ceramic method, shows an orthorhombic perovskite cell. The average cation ratio was determined by means of EPMA. The oxygen content was determined by thermogravimetric methods. To determine the local cation distribution the aberration corrected JEOL JEMARM200cFEG electron microscope was used. Fig. 1 shows a characteristic HAADF image of La0.9Ca0.10MnO3 along [10-1]c in which apparently ordered perovskite areas are evident. Nevertheless, it should be noticed the different contrast at the dots corresponding to the A sites of the perovskite (ABO3) lattice. According to the HAADF contrast, it could be proposed that the brightest contrast corresponds to La and the less one to Ca. For further compositional information an atomically resolved EELS study was performed. The HAADF image recorded simultaneously to EELS acquisition and the sum spectra obtained over the area marked in figure 1a are depicted on fig. 1a and b, respectively. The chemical maps (fig. 1d-f) suggest a heterogeneous arrangement of the La and Ca cations. A more detailed study of individual atomic positions has led to the detection of Ca free A positions, according to spectra shown in fig. 2a and b. This allows us to localize Ca. Atomically resolved EELS mapping for the Mn oxidation states is in due course.

Fig. 1: (a) HAADF image of La0.9Ca0.1MnO3. Schematic model for the cationic position has been inserted; (b) EELS spectra sum, acquired over the area marked in (a); (c) HAADF image simultaneously recorded to EELS ; (d) mapping obtained for the La-L2,3; (e) Ca-M4,5 and (f) Mn-L2,3 signals.

Fig. 2: Figure 2. HAADF images simultaneously recorded to EELS acquisition and resultant EELS spectra sum corresponding to two different marked cell evidencing the presence of different cation occupation at the A site (a) La and (b) La and Ca.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3223 E-beam induced epitaxial growth at amorphous LAO/STO interface

Kim J. Y.1,2, Moon S. Y.3, Baek S. H.3, Jang H. W.3, Park E. S.2, Chang H. J.1
1Advanced Analysis Center, Korea Institute of Science and Technology, Seoul, Korea, 2Research Institute of Advanced Materials, Seoul National University, Seoul, Korea, 3Electronic Materials Research Center, Korea Institute of Science and Technology, Seoul, Korea
clakiki@snu.ac.kr

Heterostructure oxide LaAlO3(LAO)/SrTiO3(STO) with perovskite structure exhibits excellent high dielectric constant, good magnetic, ferroelectric and insulating properties, high wear resistance, and outstanding resistance against oxidation and are candidates for memory devices and spintronics application.[1] One of the most prominent properties of the material is formation of electrically conducting layer, that is, two-dimensional electron gas (2DEG) at the interface between two insulating oxides LAO and STO. Interestingly, the conductivity was shown in amorphous LAO grown on STO as well as the crystalline LAO [2]. However, the issue of instability of the oxide under electron beam was also raised reporting that amorphous STO, formed by irradiation with 1.0 MeV Au at 400 K, is accelerated to recrystallize epitaxially at the a/c interfaces by electron beam in transmission electron microscopy [1]. For the applications of these oxide materials, it is necessary to study the thermodynamic stability and phase transformation behavior of the heterostructure oxides under electron beam.

In this study, amorphous LAO films were grown on TiO2-terminated STO substrates by pulsed laser deposition (PLD) at room temperature in an oxygen atmosphere. The sample was annealed at 500℃ in vacuum subsequently.[3] The atomic structure change during nucleation and growth at the interface of LAO/STO was studied with HAADF STEM images and EEL spectra collected using a Cs-corrected microscope Titan S 80-300 operated at 300 kV equipped with Gatan Quantum 966 spectrometers. Cross-sectional samples for STEM analysis were prepared by mechanical thinning, precision polishing, and ion milling (PIPS 691).

In HAADF image of annealed a-LAO/STO it was found that epitaxial one atomic layer of La(Al,Ti)O formed on STO. Exposure to electron beam makes this La(Al,Ti)O layer grows from the interface. Figure 1 shows that electron beam positioned even slightly apart from the surface (marked by an arrow in Fig. (b)) for 20 sec can cause epitaxial growth from the interface. Electron irradiation induced nucleation and growth in this material could be explained by a radiation enhanced diffusion, which is resulted from both ionization processes and a strong thermodynamic driving force for crystallization.[4] In this study, the growth behavior of crystalline phase is investigated under systematic microscopic conditions and controlling of size and shape of the crystalline region will be suggested, which can be applied for patterning of crystalline phase in a-LAO.

References

[1] Y. Zhang et al., Phy. Rev. B 72, 094112 (2005)

[2] S.Y. Moon et al. (under submission)

[2] S.Y. Moon et al., App. Phy. Lett. 102, 012903 (2013)

[3] A. Meldrum et al., J. Mater. Res. Vol. 12, No. 7, 1997

The authors thank Y.W. Jeong for important contributions in TEM sampling. This research was sponsored by the KIST Institutional Program (2V03611).

Fig. 1: STEM HAADF image of (a) before and (b) after electron beam positioning for 20 sec at a-LAO annealed at 500℃ in air. La(Al,Ti)O layer grows from top surface (yellow line) in a pyramidal shape
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3263 Study of structural defects in Fe3O4 thin films: atomic scale correlation between imaging and spin calculations

Gilks D.1, Evans R.1, Matsuzaki K.2, McKenna K.1, Lari L.1,3, Susaki T.2, Lazarov V. K.1
1Department of Physics, University of York, Heslington, York, YO10 5DD, UK, 2Secure Materials Center, Materials and Structures Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan, 3York JEOL Nanocentre, University of York, Heslington, York, YO10 5BR, UK
dg522@york.ac.uk

Bulk Fe3O4 exhibits many of the properties required for spintronic device applications. First principle calculations predict Fe3O4 is a halfmetal with only minority spin density states at the Fermi level [1]. Furthermore, Fe3O4 has a high Curie temperature of ~868K. Good epitaxy between Fe3O4 and current oxide barrier materials such as MgO and MgAl2O4 makes realistic spin devices based on halfmetals viable. However, extended growth defects known as anti-phase domain boundaries (APB) frequently found in Fe3O4 films (Figure 1) have a significant impact on the magnetic properties of grown films [2]. To utilize the halfmetallic properties of Fe3O4 we must understand how the presence of various defects affects the magnetic properties and spin dynamics in grown films.

In Fe3O4, O mediates super exchange interactions between the magnetic Fe sites. Such interactions are dependent on both bond length and angle in the various Fe-O-Fe configurations observed in Fe3O4. These short range interactions are dominated by the co-ordination of nearest neighbour Fe sites [3] thus requiring accurate atomistic models to begin to understand APB defects.

In order to simulate the magnetic behaviour of APBs, atomistic models of APB defects have been produced using atomically resolved HAADF STEM images (Figure 2) and image simulations to verify realistic structures.

From this we simulate the magnetisation curves (figure 3a) and the Curie temperature (Figure 3b) of Fe3O4 using super cells containing over 10000 individual spins. This allows us to consider the effective atomistic Heisenberg interactions which act between neighbouring Fe sites, both at and away from a two dimensional defect.

1. Yanase, A. and K. Siratori, Band-Structure in the High-Temperature Phase of Fe3O4. Journal of the Physical Society of Japan, 1984. 53(1): p. 312-317.
2. Margulies, D.T., et al., Origin of the Anomalous Magnetic Behavior in Single Crystal Fe3O4 Films. Physical Review Letters, 1997. 79(25): p. 5162-5165.
3. Wickham, D.G. and J.B. Goodenough, Suggestion Concerning Magnetic Interactions in Spinels. Physical Review, 1959. 115(5): p. 1156-1158.

This research was funded by EPSRC research grants EP/K013114/1 and EP/K032852/1, MEXT Elements Strategy Initiative to Form Core Research Center and Collaborative Research Project of Materials and Structures Laboratory, Tokyo Institute of Technology.

Fig. 1: DF-TEM image of APBs imaged in plan view geometry from a (001) oriented Fe3O4 film

Fig. 2: HAADF-STEM image of an APB running vertically through a (111) oriented Fe3O4 film. This APB can clearly be seen in the centre of the image where the rhombohedral structural motif seen to the left and right of the image is lost.

Fig. 3: Magnetic simulation from bulk Fe3O4 and a region containing an APB. (a) shows the simulated M-H curves for the APB model (red) and the bulk model (black). (b) shows the Curie temperature measurements. 
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3272 Atomic and electronic structure of Fe3O4 oxides heterostructures

Gilks D.1, Lari L.1,2, Matsuzaki K.3, Cai Z.4, Ziemer K.4, Susaki T.3, Lazarov V. K.1
1Department of Physics, University of York, Heslington, York, YO10 5DD, UK, 2York JEOL Nanocentre, University of York, Heslington, York, YO10 5BR, UK, 3Secure Materials Center, Materials and Structures Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan, 4Department of Chemical Engineering, Northeastern University, 360 Huntington Avenue, Boston, MA 02115-5000, USA
dg522@york.ac.uk

Magnetite (Fe3O4) has recently attracted a lot of attention for future spintronic applications due to its 100% spin polarisation at the Fermi level [1]. Incorporating magnetite thin films as an electrode in heterostructures such as spin valves, magnetic tunnelling junctions and other device structures requires growth with atomic control of Fe3O4 thin film interfaces with oxide tunnel barriers and semiconductor layers. Therefore understanding the atomic and electronic structure of Fe3O4 interfaces is crucial for any future development of devices based on halfmetalic Fe3O4.


In this work we study two interfaces of Fe3O4; with MgAl2O4 as a model tunnel barrier and doped SrTiO3(111) as a semiconductor layer important for spin injection and diffusion. Fe3O4 thin films were grown by MBE and PLD and post annealed ex-situ in a CO/CO2 atmosphere in order to improve their stoichiometry and structural ordering. Structural analysis was performed by TEM/STEM and EELS using aberration corrected JEOL 2200FS, Nion Ultrastem 100, and JEOL ARM 005. Electronic calculations have been performed with DFT using full and pseudo potential plane wave codes.


(Fig. 1) shows the interface region of Fe3O4(111)/MgAl2O4(111) in a cross-sectional [1-10] viewing direction. The MgAl2O4 substrate and Fe3O4 layers share the same spinel structure. By following the stacking across the interface we show the FCC O sublattice common to both structures is uninterrupted across the interface. HAADF modelling indicates the sharp interface is defined by the stacking sequence: (.../4O/Mg-Al-Mg/4O/3FeB/4O/…), where Mg is in a tetrahedral site while FeB and Al are in octahedral sites. The DFT calculations confirm this interface is the lowest energy. Similarly to Fe3O4/MgAl2O4 the Fe3O4/SrTiO3(111) interface is atomically sharp as shown in Fig. 2. defined by (...SrO3/Ti/FeA/4O/3FeB...). The clear presence of misfit dislocations in both interfaces is seen. By using GPA analysis we evaluate the strain around the dislocations cores. Finally we performed electronic spin density calculations on the best experimentally matched models as well as other plausible models, and we show that sharp interfaces with bulk like stacking sequence maintain spin polarization.

1. Yanase, A. and K. Siratori, Band-Structure in the High-Temperature Phase of Fe3O4. Journal of the Physical Society of Japan, 1984. 53(1): p. 312-317.

This research was funded by EPSRC research grants EP/K013114/1 and EP/K032852/1, MEXT Elements Strategy Initiative to Form Core Research Center and Collaborative Research Project of Materials and Structures Laboratory, Tokyo Institute of Technology.

Fig. 1: HAADF STEM image of the interfacial region of Fe3O4/MgAl2O4(111) in the cross-sectional [1-10] viewing direction.

Fig. 2: HAADF STEM image of the interfacial region of Fe3O4/SrTiO3(111) in the cross-sectional [1-10] viewing direction
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3406 Characterization of BaTiO3/CoFe2O4 thin films prepared by sol-gel process

Mohallem N. S.1, Andrade H. R.1, Seara L. M.1
1Universidade Federal de Minas Gerais
nelcy@ufmg.br

Nanocomposites formed by ferrimagnetic and ferroelectric materials are a multiferroic material in which magnetoelectric coupling occurs via piezoelectricity and magnetostriction phenomena. These nanocomposites have a variety of applications in tunable microwave devices using electric control of spin wave propagation or new magnetic memories in which the magnetic response is controlled by electric field. Sol-gel process has been an efficient method to produce this kind of thin films due to the good control of the sample morphology, texture, structure, and composition, which can be attained by monitoring in the physical-chemistry parameters of the precursor solution and of the deposition process

In this work, transparent and homogeneous thin films of barium titanate interleaved with cobalt ferrite were prepared by sol–gel method using dip-coating process. The BaTiO3 solution was prepared using tetraisopropyl orthotitanate, alcohol, and barium acetate. The CoFe2O4 solution was obtained by diluting cobalt nitrate and iron (III)-acetyl-acetonat in acidified aqueous solution. The nanocomposite films were dried in air after each dipping and heated at 700 oC for 10 min to convert the amorphous films into crystalline oxides. The samples were characterized by low angle X-ray diffraction (XRD), UV-vis spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and scanning force microscopy (AFM/MFM/PFM).

The nanocomposite films were formed by tetragonal barium titanate particles measuring between 10 and 25 nm, interfacing cubic ferrite cobalt particles measuring between 3 and 10 nm, providing a magnetoelectric coupling. The films showed magnetic and piezoelectric properties measured by magnetic and piezoresponse force microscopy.

CNPq, FAPEMIG, CAPES and Centre of Microscopy of UFMG

Fig. 1: SEM  image of BaTiO3/CoFe2O4 thin films annealed at 700 oC.

Fig. 2: TEM  image of BaTiO3/CoFe2O4 thin films annealed at 700 oC.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3485 Anisotropic nanoscale strain in un-twinned YBa2Cu3O7-x superconducting thin films grown on (110)MgO substrates

Ciancio R.1, Orgiani P.2, Galindo P.3, Arpaia R.4, Bauch T.4, Lombardi F.4, Carlino E.1
1CNR-IOM TASC Area Science Park Basovizza S.S. 14 Km 163.5, 34149 Trieste, Italy, 2CNR-SPIN, UOS Salerno, 84084 Fisciano, SA, Italy, 3CASEM, University of Cadiz, 11510 Puerto Real, Cadiz, Spain, 4Chalmers, Dept Microtechnology and Nanoscience, Quantum Device Phys Lab, MC2, S-41296 Gothenburg, Sweden
ciancio@iom.cnr.it

Very recently, investigation of transport properties of YBCO nano-wires (i.e. lateral width down to 50nm) has revealed a peculiar in-plane anisotropy of the superconducting critical current density Jc when the film is grown on suitable substrates (e.g. [110]-oriented MgO). Such an in-plane anisotropy has been tentatively correlated to the in-plane structural features of the YBa2Cu3O7-d(YBCO) films.

Here we report our results on atomic resolution Transmission Electron Microscopy (TEM) studies and nanodiffraction experiments performed on superconducting (001)YBCO/ (110)MgO thin films prepared for TEM analysis in different geometries.

In Figure 1a a representative bright field plan-view TEM image of the YBCO film is shown. The film has typical domain structure, which is a characteristic fingerprint of the “c-axis” spiral growth mode of YBCO. Domains have an average size of about 50-100 nm, with boundaries along both the in-plane directions. Plan-view nanodiffraction experiments confirmed the "c-axis" growth of the film which is indeed [001]-oriented with the respect to the electron beam only exhibiting the characteristic h00 and the 0k0 diffraction spots. Figure 1b shows a representative nanodiffraction in the [001]zone axis of the film. The diffraction spots of the film are indexed in the presence and the presence of additional spots (pointed by arrows) is also observed whith spacing compatible with the 002 diffraction spots of MgO. By taking the substrate as a reference, we determined that the film and the substrate have a (010)(100)YBCO//(2,-2,0)(0,0,2)MgO orientation relationship. Interestingly, no evidence of twin domains is found within the film.

Cross-sectional HRTEM analyses were also performed across the two-in plane directions of the film. Figure 2a and 2b show bright field HRTEM images taken with the electron beam parallel to the[010] and [100] YBCO crystallographic directions, respectively. Interestingly, a pronounced waving of the YBCO lattice planes along the two in-plane crystallographic directions is observed, with a different wave-periodicity depending on the relative film/substrate crystallographic orientation. We associate the emergence of this feature to a inhomogeneous strain induced by the bared (110)MgO substrate in the two in-plane directions, speculating on the relationship between these structural properties and the high critical current measured in these specimens.

We thank E. Cociancich for the assistance in TEM sample preparation.

The PRIN project 2010 “Oxide - interfacce di ossidi: nuove proprietà emergenti, multifunzionalità e dispositivi per l'elettronica e l'energia” is thankfully acknowledged for the finantial support to this research

Fig. 1: a) Plan view Bright field TEM image of the YBCO film ([001] film zone axis). b) Nanodiffraction taken in the [001] zone axis of the film. In the pattern, diffraction spots of YBCO are indexed and additional spots, compatible with the 002 diffraction spots of MgO, are pointed by arrows. c) Nanodiffraction of the MgO substrate in the [110] zone axis

Fig. 2: HRTEM cross sectional images taken (a) in the [010] and (b) [100] zone axes of the YBCO film. In both the cases a waveness of the YBCO lattice planes is observed.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-3531 Atomic Structure and Properties of Charged Domain Walls in Ferroelectrics

Li L.1, Gao P.1, Nelson C. T.1, Jokisaari J. R.1, Zhang Y.1, Kim S. J.1, Melville A.2, Adamo C.2, Schlom D. G.2, Pan X. Q.1
1Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI 48109, United States, 2Department of Materials Science and Engineering, Cornell University, Ithaca, NY 14853, United States
panx@umich.edu

A ferroelectric domain wall can become electronically active, as a result of charged domain walls (CDWs) with a “head-to-head” or “tail-to-tail” polarization configuration. Such domain walls carrying net bound charge can have distinct properties from uncharged domain walls, such as a metallic conductivity. In this work, we show that CDWs in the rhombohedral-like (R-like, Fig. 1a left) BiFeO3 thin films possess a tetragonal-like (T-like, Fig. 1a right) crystal structure; and the bound charge at the CDW also induces the formation of nano-domains with novel polarization states and unconventional domain walls in nearby regions.

Figure 1b shows a diffraction contrast TEM image taken from a cross-sectional specimen of a 20 nm thick (001)P BiFeO3 film grown epitaxially on an insulating (110)O TbScO3 substrate (P denotes pseudocubic and O denotes orthorhombic), in which triangular 109° (vertical)/180° (inclined) domain wall junctions can be observed. Above the junction near the free surface of the BiFeO3 film, a 71° CDW is observed. The CDW is studied by high angle annular dark field (HAADF) imaging using the TEAM0.5 instrument with a point-to-point resolution of 0.5 Å. The HAADF image is processed to obtain mapping of the lattice parameter and the atomic displacement of Fe cations from the center of four Bi neighbors (DFB). The electric polarization is proportional to -DFB. Figure 2a shows the spatial distribution of -DFB overlaid on the HAADF image. A CDW with “head-to-head” polarization configuration is clearly seen above the triangular junction. Interestingly, the polarization rotates gradually from <111> directions beside the CDW to the out-of-plane orientation at the CDW. The lattice parameter mapping (Fig. 2b) also shows a local increase of the c/a ratio at the CDW. These results suggest the formation of a T-like structure at the CDW, surrounded by the regular R-like phase. The T-like CDW also leads to changes in polarization. As seen in Fig. 2a and b, below the T-like CDW, a nano-domain with a pseudocubic structure, with an in-plane oriented polarization, occurs. As a result, unconventional inclined CDW are observed. For sufficiently thin films, for example, 5 nm thick film as shown in Figure 3a and b, CDWs traverse the full thickness of the film.

In summary, we have found stable charged domain walls (CDWs) in BiFeO3 thin films. These CDWs possess crystal structures, ferroelectric polarization states, and properties different from the bulk film due to local charge compensation and polarization rotation. These CDWs can provide metallic conduction channels in the film, since the accumulation of compensating free charge that screen the bound charge at the CDW can in principle intriguer an insulator-metal transition.

the authors gratefully acknowledge the financial support through DOE grant DoE/BES DE-FG02-07ER46416

Fig. 1: (a) Atomic models of the rhombohedral-like and tetragonal-like structures of BiFeO3. (b) Cross sectional dark field TEM image of domain patterns of 20 nm thick BiFeO3 film on TbScO3 substrate, with the corresponding schematic domain configuration shown below.

Fig. 2: (a) Plot of the -DFB vectors overlaid on HAADF image of a 109°/180° domain wall junction near the free surface of the 20 nm thick BiFeO3 film. (b) The corresponding color map of the c/a ratios. The polarization orientation and bound charge are indicated schematically.

Fig. 3: (a) Plot of -DFB vectors and (b) c/a ratio color map of a 5 nm thick BiFeO3 film grown on TbScO3 substrate. The polarization orientation and bound charge are indicated schematically.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-5849 j k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr

MARTIN J. J.1, ULRICH L.2
1CHU La Timone, Martigues, France, 2Anesthesia, Greek Hospital, Athens, Greece
contact@icnmd2014.org

j k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tio

j k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tioj k kg kgkl hklgkg kg kgkg kgfdi tito hli yyr gil hl juir ujg rug f uruf ufug iuki rig oiopu uitogi utitrur oiutpoyir joi io out uiogig iut i tio

Fig. 1: figure 1: blabla

Fig. 2: figure2: blabla
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-5956 Investigation of the microstructure of Fe67.7B20Cr12Nb0.3 glassy ribbons using high resolution transmission electron microscopy

Whitmore L.1, Ababei G.1, Stoian G.1, Lupu N.1, Chiriac H.1, Budeanu L.1
1National Institute of Research and Development for Technical Physics, 47 Mangeron Boulevard, Iasi, Romania
lwhitmore@phys-iasi.ro

The addition of Cr to Fe-based metallic glasses shifts the ferromagnetic-paramagnetic transition of the amorphous phase due to the magnetic domain structure and magnetic anisotropy distribution, bringing the Curie Temperature to below 40 °C. This makes these materials very useful for temperature sensors and for biomedical applications, and for other applications where they are used as the active elements in magnetic-sensitive sensors. In this investigation we use transmission electron microscopy (TEM) to study the microstructure of Fe67.7B20Cr12Nb0.3 ribbons in relation to a range of thermal treatments. As-cast ribbons are prepared by rapid quenching from melt into the amorphous state. The ribbons are then annealed at temperatures below, close to and above the crystallisation temperature of 510 °C. Magnetic ribbons pose an interesting problem for TEM specimen preparation due to their very small dimensions. Typically 1-2 mm wide and 25 microns thick, traditional methods of preparation are usually avoided in favour of focussed ion beam (FIB) techniques. Using FIB and a Zeiss Libra 200MC microscope, we are able to investigate the evolution of the microstructure, specifically the formation of nano-clusters and nano-crystals, with annealing temperature. This allows an interpretation of the magnetic properties of the ribbons, in particular the magnetization and magnetic susceptibility. Cr and Fe atoms are found to segregate into nano-clusters of a few nanometres size, and the nano-crystalline grain size increases with annealing temperature.

This work was supported financially by the European Commission (FP7-REGPOT-2012-2013-1, Grant Agreement no. 316194, NANOSENS) and by a CNDI–UEFISCDI grant, Project No. 148/2012 (HYPERTHERMIA).

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-5989 Magnetic imaging of ordered arrays of nanowires

Ivanov Y. P.1, Chuvilin A.2, Kosel J.1
1King Abdullah University of Science and Technology, Thuwal, Saudi Arabia, 2CIC nanoGUNE Consolider, San Sebastian, Spain
ivanov.yup@gmail.com

The interest in magnetic nanowires (NW) has considerably increased during the last decade, because of their potential applications in different fields ranging from treatments of diseases [1] to spintronics devices [2]. In particular, NWs fabricated in anodic aluminum oxide (AAO) templates, offer several advantages like high packing density and highly ordered pore distribution. On top of these, they grow inside of an isolating template and perpendicular to the substrate. This feature, could be exploited for high-density data storage by creating 3D storage media. In this context, it is crucial to have a complete understanding of the magnetic behavior of both individual NWs and NWs in an array. Lorenz microscopy and electron holography are appropriate methods to study NWs with diameter less than 100 nm. In particular holography can be used to obtain quantitative information about magnetic and electric fields in materials with a nanometer scale spatial resolution [3].
Here, we report the studies on the magnetic structures of arrays of single crystal hcp Co NWs by a combination of Lorentz microscopy (LTEM) and off-axis electron holography.
Ordered arrays of Co NWs with 45 nm in diameter and 105 nm inter-wire distance have been prepared by electrodeposition into aluminum oxide templates (AOT). The single crystal structure of hcp Co NWs with [100] growth direction has been confirmed by SAED and CBED. For studying individual NWs the AOT has been chemically removed, and the NWs were dispersed on a copper grid. For studying the magnetic structure of the NWs in arrays, cross sections of AOTs with NWs have been prepared (fig. 1a,b).
Fig. 2a shows an LTEM image at under focus (green) and over focus (red) conditions (defocus is 200 µm) of the NW array shown in fig. 1. Most of the NWs are circularly magnetized in the plane of the NW’s diameter. Fig. 2b shows the reconstructed hologram image of the NWs. NWs which have a center spot in the LTEM image contain circular contours, corresponding to the in-plane circularly magnetized vortex shells. The rest of the NWs show the magnetic induction maps corresponding to a complex magnetic state with strong in-plane magnetization components. Using holography data a quantitative analysis of the Co NW’s magnetization as well as of the stray fields between NWs in the array has been performed.

Fig. 1: (a) TEM image of a Co NW array and (b) corresponding tomography image.

Fig. 2: (a) LTEM image at under focus (green) and over focus (red) condition of the NW array presented in fig. 1. Arrows indicate clockwise and anticlockwise rotation of magnetization of vortices. (b) Corresponding reconstructed hologram image (lines correspond to the in-plane component of the magnetic induction, interline distance 0.3 pi).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-12-P-6018 EM of FeTe(Se,S) superconductors

Vasiliev A. L.1,2, Presniakov M. Y.1, Bondarenko V. I.1
1National Research Center “Kurchatov Institute”, Moscow, 123182 Russia 2 –Shubnikov Institute of Crystallography of the Russian Academy of Sciences, Moscow, 119333 , 21 - National Research Center “Kurchatov Institute”, Moscow, 123182 Russia 2 –Shubnikov Institute of Crystallography of the Russian Academy of Sciences, Moscow, 119333
a.vasiliev56@gmail.com

Modern technology of superconducting manufacturing based on Nb3Sn and NbTi for high magnetic field applications is close to the limit of critical current (jc) capabilities and for further improvement the utilization of novel superconducting materials is necessary. The search for new materials pointed to the new copper-free superconductors Fe1+yChx where Ch - S, Se, Te [1]. The transition temperature (Tc) in compounds Fe1+ySexTe1-x at x ≈ 0.5 is close to 15 K [5] at atmospheric pressure Tc=37 K in FeSe0.82Te0.18 at 7 GPa [2]. Overall study of single crystals and thin films of Fe1+yTe1-x(S, Se)x, which includes HR TEM, HR STEM, EDXS was performed to get the correlation between the structure and physical properties. All samples were prepared by FIB (Helios 600, FEI, US) and studied in probe Cs corrected Titan 80-300 TEM/STEM (FEI, USA)

HAADF HR STEM investigations of Fe1.1Te (Fig. 1) demonstrated that Fe1 atoms occupied staggered position and Fe2 (10% occupation) mostly regroup together (Fig.2). HRTEM of FeTe0.9S0.1 crystals indicated to the partial substitution of Te by S with the uprising of the additional reflections in hk0 position (where h and k are odd) on FFT patterns (see insert in Fig.3). The areas where the space doubling were observed were 4-5 nm in size.

To reveal the possible influence of the stress at the interfaces of Fe1.1Te films on MgO and LaAlO3 substrates on Tc the cross sections of these heterostructures were studied (Fig.4). The misfit dislocations, located at the interfaces or the intermediate layers were found and these could release the stress in the Fe1.1Te layers, thus leave the Tc unaffected.

1. Mizuguchi Y., Takano. J. Phys. Soc. Jpn. 2010. V. 79. P. 102001.

2. Margadonna S., et al. Phys. Rev. B 2009. V. 80. P. 064506.

3. Yeh K.W., et al. EPL. 2008. V. 84. P. 37002

Fig. 1: HAADF STEM image of Fe1.1Te single crystal in B=[100] zone axis.

Fig. 2: HADDF STEM image of Fe1.1Te single crystal in B=[100] zone axis after image processing. The groups of Fe2 atoms are shown by arrows.

Fig. 3: HRTEM image of FeTe0.9S0.1 single crystal in B=[100] zone axis. The space doubling are shown by arrows. The FFT is in the insert (note additional reflections in positions 100 and 010).

Fig. 4: HAADF STEN image of Fe1.1Te/MgO interface. Misfit dislocations are shown by arrows.
↑ Go to the Table of content ↑

MS-13. Materials in geology, mineralogy and archeology

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-13-IN-1719 Nanoscale magnetism of magnetite induced by phase transition and oxidation/reduction reactions

Kasama T.1, Almeida T. P.2,1
1Center for Electron Nanoscopy, Technical Univ. of Denmark, Denmark, 2Dept. of Earth Science and Engineering, Imperial College London, U.K.
tk@cen.dtu.dk

Magnetite (Fe3O4) is the dominant carrier of paleomagnetic and paleoclimatic information in rocks and sediments on the Earth and on other planets. It is important to understand the formation and recording mechanisms, as well as fidelity, of the magnetic minerals within natural systems. Below ~125 K, magnetite undergoes a first-order phase transition, known as the Verwey transition, from a cubic structure to a closely-related monoclinic structure. The transition has a profound impact on its magnetic properties. Kasama et al. [1] carried out the first TEM measurements of the magnetic microstructure of magnetite below the Verwey transition, showing strong interactions between magnetic domain walls and twin domain walls. In addition, the ability of magnetite to preserve the remanence of the Earth's and other planets’ magnetic fields is greatly influenced by progressive oxidation or reduction to different magnetic minerals. Here, we use various TEM techniques including off-axis electron holography and environmental TEM (ETEM) to investigate fundamental magnetic properties of magnetite (1) below the Verwey transition and (2) under oxidizing/reducing environments.

(1) The low temperature monoclinic phase has closely-spaced magnetic domains separated by 90˚ or 180˚ magnetic domain walls, which are defined strictly by underlying monoclinic twin domains with a monoclinic [001] easy axis resulting from a large magnetocrystalline anisotropy (Fig. 1). Direct imaging after the application of an external magnetic field during cooling showed that the magnetic field affects the choice of monoclinic c-axis and monoclinic twin formation with some complication due to specimen geometry. Furthermore, magnetic domains in magnetite above or below the Verwey transition can inherit a part of magnetization from the prior phase during zero field cooling or zero field warming through the Verwey transition.

(2) Magnetite nanoparticles were reduced in-situ under 2 mbar H2 atmosphere at 460˚C in a microscope column of the ETEM (Fig. 2). After 5 hours, the particles become rounded and smaller as a result of reduction of magnetite to metallic Fe. The reduced Fe particles have single domain states with dipolar interactions with their neighbors, while the initial magnetite particles have more collective behavior with vortex states. Similar experiments were performed using ~200-nm-sized magnetite particles under oxidizing conditions, showing that the hematitization of the magnetite particles changes their magnetic microstructures dramatically. These results suggest that the magnetic remanence is altered by redox reactions and should be used with an understanding of geological history in a site of interest.

[1] T. Kasama et al., Earth Planet. Sci. Lett. 165 (2011), 229.

We thank R.E. Dunin-Borkowski, J. Jinschek, T.W. Hansen, R.J. Harrison, A.R. Muxworthy, Z.-A. Li and S. Yazdi for discussions.

Fig. 1: (a) Experimental magnetic induction map (1x phase amplification) of monoclinic magnetite obtained using off-axis electron holography. (b) Simulated induction map overlaid onto a Lorentz image. The symbols ‘C0˚’ and ‘C45˚’ refer to c-axes, oriented in-plane and at 45˚ to the plane of the specimen. GB: grain boundary, TWB: twin boundary.

Fig. 2: (a) TEM image of initial cube-shaped magnetite nanoparticles. (b) Electron hologram of the nanoparticles that were reduced under 2 mbar H2 atmosphere at 460˚C in an ETEM column. (c) Magnetic contour map of the reduced nanoparticles shown in (b). The contour spacing is 0.042 radians.
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-13-IN-2076 Imaging and reading micro-to nano-structures of hydrated rocks and mollusc shells: geomineralization versus biomineralization

Baronnet A.1
1Centre Interdisciplinaire de Nanoscience de Marseille (CINaM); UMR 7325-Aix-Marseille Université, Campus Luminy, case 913, 13288-Marseilles, France
baronnet@cinam.univ-mrs.fr

We plan to characterize and interpret the microstructures to nanostructures of abiotic and biogenic minerals with the different imaging, microanalytical and diffraction techniques offered by present-day available nanoscopes. Among them, transmission electron microscopy (TEM) will play a central role.

Serpentine minerals are the product of hydration of the upper mantle rocks by percolating sea water. Their wealth of flat and curved microstructures contrasts with a poor chemical variability. When combined to experimental data these mainly metastable microstructures and their texture may serve to trace back semi-quantitatively temperature paths followed during hydration. In an ophiolite serpentinized at medium-to-low-T, the replacement of anhydrous silicates (olivine and pyroxenes) by lizardite is fully pseudomorphic as the result of an interstitial dissolution/crystallization process in which chemical exchanges in the interfacial fluid are allowed via nanoporosity in the daughter minerals. Interfacial crystallisation from gel as close-packed chrysotile nanotubes also occurs in coeval “crack-seal” veins. Early mineral alteration occurs in independent nanosystems in which local reactions may be written from co-existing solid, relictual and neoformed, nanophases present.

Most shells of molluscs are multiscale composites combining calcite or aragonite crystals and organic matter, from mm scale down to the nm scale as shown here by TEM. Among bivalves and gastropods it will be shown how the carbonate polymorph, the species-specific shell shape and microstructures are mostly, but not fully, controlled by organics. Submicronic granules are ubiquitous bio-units of mineralisation. Organic nodules, fibers and envelopes as extracellular matrix secretions from mantle cells, insure nucleation, crystal propagation and shaping, respectively.

Though belonging to the distinct geo- and bio-worlds, the above two cases share in fact common features: interstitial growth from a gel and tailored shape of the resulting crystals or texture. However biomimetic materials with outstanding functional properties will be within reach only when biogenic crystallisation process(es) will be better understood.

Acknowledgements

We warmly thank Françoise Boudier (Géosciences Montpellier), Olivier Grauby (CINaM) and Julius Nouet ( IDES, Orsay) for their decisive contributions to this work.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-13-O-1424 Visualisation of chemical remanent magnetisation in pseudo-single domain Fe3O4 particles examined by environmental TEM and off-axis electron holography

Almeida T. P.1, Kasama T.2, Muxworthy A. R.1, Williams W.3, Hansen T. W.2, Dunin-Borkowski R. E.4
1Imperial College London, London, United Kingdom, 2Technical University of Denmark, Lyngby, Denmark, 3University of Edinburgh, Edinburgh, United Kingdom, 4Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Research Centre Jülich, Jülich, Germany
t.almeida@imperial.ac.uk

Magnetic minerals in rocks record the direction and intensity of the ambient magnetic field during formation, providing, for example, varied information of the geomagnetic field and past tectonic plate motions. In order to reliably interpret paleomagnetic measurements, the mechanism of chemical remanent magnetisation (CRM) which can induce and subsequently alter magnetic remanence must be fully understood. Currently, models of CRM processes only exist for the smallest, uniformly magnetised grains, termed single domain (SD). However, the magnetic signal from rocks is often dominated by slightly larger grains containing non-uniform magnetisation states and these are termed pseudo-SD (PSD) grains.

Magnetite (Fe3O4) is the most magnetic naturally occurring mineral on Earth, carrying the dominant magnetic signature in rocks and providing a critical tool in paleomagnetism. The oxidation of Fe3O4 to other iron oxides, such as maghemite (γ-Fe2O3) and hematite (α-Fe2O3), is of particular interest as it influences the preservation of remanence of the Earth's magnetic field by Fe3O4. During oxidation, the inverse spinel ferrite Fe3O4 reacts with oxygen to form the Fe2+ cation deficient phase γ-Fe2O3, which can then further oxidise to form hexagonally-close-packed α-Fe2O3.

Environmental transmission electron microscopy (ETEM) enables the detailed investigation of localised chemical reactions under gas atmospheres. Off-axis electron holography permits nanometre-scale imaging of magnetic induction within and around materials as a function of applied field and temperature. The complementary use of these advanced TEM techniques can be used to reveal local changes in magnetisation in minerals as they alter during in situ heating in a controlled atmosphere.

In the present study, synthetic Fe3O4 particles in the PSD size range (< 200 nm) were heated in situ in an ETEM to a temperature of 700 °C in 8 mbar of O2. Oxidation of the Fe3O4 particles was investigated using bright/dark-field imaging and electron energy-loss spectroscopy (EELS). Figs. 1a & 2a show native smooth-surfaced Fe3O4 grains and complementary EELS analysis of the Fe 2p L3 edge (Figs. 1c & 2c) is in good agreement with that of pure Fe3O4. Close examination of Fe3O4 particles after in situ heating revealed surface degradation in the form of nanoparticles (Figs. 1d & 2d) and EEL spectra showed pre-peaks close to the Fe 2p L3 edge (Figs. 1f & 2f) that are indicative of oxidation. The associated effect of CRM was investigated using off-axis electron holography, in the form of reconstructed magnetic induction maps, where the oxidised grains exhibited a loss of overall remanence (Figs. 1b & 1e) and degradation of magnetic vortices (Figs. 2b & 2e).

The authors would like to thank the Natural Environment Research Council (UK) for funding (grant NE/H00534X/1).

Fig. 1: (a) Bright-field TEM image of two Fe3O4 particles, with SAED pattern (inset); and (b) their magnetic induction map. (c) Fe 2p L3 edge of the EEL spectrum acquired from the particles in (a). (d) Dark-field image of the Fe3O4 particles after in situ heating. (e) Magnetic induction map of the particles in (d); and (f) associated EEL spectrum.

Fig. 2: (a) Bright-field TEM image of attached Fe3O4 particles, with SAED pattern (inset); and (b) magnetic induction map. (c) Fe 2p L3 edge of the EEL spectrum acquired from the particles in (a). (d) Dark-field image of the Fe3O4 particles after in situ heating. (e) Magnetic induction map of the particles in (d); and (f) associated EEL spectrum.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-13-O-2615 HRTEM study of recrystallization mechanisms in metamict allanite

Čobić A.1, Klementová M.2, Drábek M.3, Tomašić N.1, Bermanec V.1
1University of Zagreb, Faculty of Science, Division of Mineralogy and Petrology, Zagreb, Croatia, 2Institute of Inorganic Chemistry of the AS CR, v.v.i., Řež, Czech Republic, 3Czech Geological Survey, Prague, Czech Republic
ancobic@geol.pmf.hr

Allanite is a sorosilicate with general formula CaREEAlAlFe2+[SiO4|Si2O7]O(OH) where REE = Ce, La, Nd, Y, but Th and U (in small amounts ~1 wt. %) can substitute for REE [1]. Rather complex crystal structure [2] and chemical composition along with the radioactive decay of, most notably, Th cause the allanite crystal structure to become metamict i.e. amorphous. In order to recrystallize allanite crystal structure, two methods are employed: annealing in air [3] and under hydrothermal conditions [4]. Two mechanisms of recrystallization are possible: epitaxial and nucleation growth. HRTEM investigation is employed in order to compare the recrystallization mechanisms with method of recrystallization.
Two samples (ALF and 1569) of naturally metamict allanite were annealed in air and under hydrothermal conditions at 800°C for 24 h in order to recrystallize the crystal structure. SAED and HRTEM of natural and annealed samples were performed on JEOL JEM 3010 operated at 300kV (LaB6, resolution of 1.8 Å).
Natural ALF sample shows pronounced metamictization (fig. 1a1), although crystalline fragments of original crystal structure are preserved (fig.1a), while natural 1569 sample shows almost complete metamictization (fig. 1b1) with minor preserved crystalline fragments (fig.1b), insufficient to yield observable diffraction pattern (fig. 1b1). After annealing in air at 800°C for 24 h, crystal structure of sample ALF is completely restored (fig. 2a, 2a1) yielding formation of large crystallites (fig. 2a2). For sample 1569, annealing at the same conditions induces recrystallization of small crystallites (fig. 2b1), but with amorphous domains still present (fig. 2b). On the contrary, annealing under hydrothermal conditions at 800°C for 24 h yields complete recovery of crystal structure in both samples (fig. 3), resulting in formation of large crystallites (fig. 3a2, 3b2).
HRTEM investigation of thermally treated metamict allanite showed that in case of incomplete metamictization (fig. 1a), dominant mechanism of recrystallization is epitaxial growth (fig. 2a, 3a), while for completely metamict samples (fig. 1b), both nucleation (fig. 2b) and epitaxial (3b) growth are possible. Which mechanism will occur depends on experimental treatment; if completely metamict samples are annealed in air, dominant mechanism will be nucleation growth (fig. 2b), whilst if annealed under hydrothermal conditions, dominant mechanism is epitaxial growth (fig. 3b). These results prove water as the most important factor in recrystallization of metamict minerals containing crystal water i.e. OH- groups.
[1] Armbruster, T. et al.(2006) Eur J of Miner 18, 551; [2] Dollase, W. A. (1971) Am Miner 56, 447; [3] Berman, J. (1955) Am Miner 40, 805; [4] Čobić, A. et al. (2010) Can Miner 48, 513 (2010)

Fig. 1: Fig. 1. HRTEM, SAED and BF images of natural samples: a) ALF; b) 1569

Fig. 2: Fig. 2. HRTEM, SAED and BF images of samples annealed in air at 800°C: a) ALF; b) 1569

Fig. 3: Fig. 3. HRTEM, SAED and BF images of samples annealed under hydrothermal conditions at 800°C: a) ALF; b) 1569
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-13-O-2759 Use of X-Ray Micro-CT and Scanning Electron Microscopy for Constructing 3D Mineral Maps of Rock Samples

Yakimchuk I. V.1, Varfolomeev I. A.1,2, Korobkov D. A.1
1Schlumberger, Moscow, Russia, 2Moscow Institute of Physics and Technology, Dolgoprudny, Russia
iyakimchuk@slb.com

Continuous development of high-performance computing systems allows researchers to perform more complex and resource consuming calculations. In the past ten years, the number of works devoted to the direct modeling of physical properties (e.g., thermal, hydrodynamic, electrical) of rock samples has significantly increased. The source data for these simulations are 3D digital models of rock structure. In the simplest case, a digital model is represented by a stack of black and white images (black pores and white mineral skeleton) corresponding to the sample’s real pore-space geometry.
One of the most efficient ways for "digitizing" rock specimens is 3D scanning using X-ray absorption micro-computed tomography (micro-CT), which provides volumetric distribution of X-ray attenuation inside the studied object at the beam energy. Most laboratory micro-CT instruments deal with a polychromatic beam using as many photons as possible to obtain statistically reliable images in minimal time. The energy of the beam should be high enough (>60 keV) to penetrate through dense rock samples. These two facts seriously complicate the problem of resolving the spatial distribution of the minerals constituting the sample. Evidently, such information allows constructing more sophisticated digital rock models compared with black and white models, potentially increasing the accuracy of numerical simulations.
There are a number of alternative methods that also provide 3D distributions of chemical elements, such as synchrotron micro-CT with monochromatic beam of the selected energy as well as X-ray fluorescence micro-CT. However, the cost of these solutions is usually quite high.
Another approach for constructing 3D mineral maps combines conventional X-ray absorption micro-CT with X-ray microanalysis via scanning electron microscopy (SEM). The idea is to register the mineral distribution image from SEM with virtual slice of 3D reconstructed image from micro-CT. Having a number of color, textural, and morphological attributes from 3D micro-CT image, we can segment it on a set of clusters. The 2D mineral map (spatially registered with this image) may be used to find correspondence between clusters and minerals and so define characteristic attributes for each mineral. Finally, the clusterized 3D micro-CT image is classified by mineral identification.
We present a short summary of an approach to constructing 3D mineral maps of rock samples using X-ray micro-CT and SEM as well as present first results of the approach’s application.

We are grateful to Sergey Safonov, Dmitry Koroteev, Alexander Nadeev and Mark Andersen for fruitful discussions and support.

Fig. 1: Registration of 3D digital model (from X-ray micro-CT) with 2D mineral map (from X-ray microanalysis via SEM) and further carbonate particle construction in 3D. This specimen is a sandstone sample of 8-mm diameter
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-13-O-3286 Geological Applications of Atom Probe Tomography: New Information from Old Rocks

Valley J. W.1, Cavosie A. J.1,2, Ushikubo T.1, Reinhard D. A.3, Lawrence D. F.3, Larson D. J.3, Clifton P. H.3, Kelly T. F.3, Wilde S. A.4, Moser D. E.5, Spicuzza M. J.1
1WiscSIMS, Dept. Geoscience, Univ. Wisconsin, Madison, WI 53706 USA, 2Univ. Puerto Rico, Mayagues, PR 00681 USA, 3CAMECA Instruments, Inc., Madison, WI 53711 USA, 4Curtin University, Perth, WA Australia, 5Univ. Western Ontario, London, Ont. CAN N6A 5B7
thomas.kelly@ametek.com

Atom probe tomography (APT) makes it possible to study the compositional structure of geological materials at the nanoscale [1]. We have applied APT to three terrestrial zircons of different ages which yields a picture that suggests that the early earth was cool and could have supported life processes as early as 4.3 Ga.
Zrc-1: 4.007 Ga; 01-13b-8-4, Jack Hills, W. Australia [2]: Zrc-2: 2.542 Ga core, 29 Ma rim; ARG-05-28-2, Grouse Creek Mts., Utah [3]: Zrc-3: 4.374 Ga core, 3.4 Ga rim; 01JH36-69, Jack Hills [1]
The 3-D distribution of Pb and Y differ at the atomic-scale in the 3 zircons. Zrc-1 is homogeneous in Pb and Y (Fig. 1). In contrast, incompatible elements, including Pb and Y, are concentrated in sub-equant 5-10nm domains (up to 1 at.% Pb), spaced ~50 nm apart in Zrc-2 (Fig. 2) and Zrc-3 (Fig. 3). U is homogeneously distributed in all three zircons. The average 207Pb/206Pb ratios for these 100-nm-scale specimens, as measured by APT, are 0.17 for the 2.5 Ga Zrc-2, 0.43 for the 4.0 Ga Zrc-1, and 0.52 for the 4.4 Ga Zrc-3. The APT ratios are less precise (±5-10% 2) due to small sample size, but are in excellent agreement with values measured by SIMS, 0.168, 0.427, and 0.548 respectively. The average 207Pb/206Pb ratios within the 5-10 nm Pb-enriched domains are 0.17 in Zrc-2 (Fig. 4a) and 1.2 in Zrc-3. Thus Pb in the Pb-rich domains is radiogenic and unsupported. No Pb is detected outside the Pb-rich domains in Zrc-2 (Fig. 4b), while 207Pb/206Pb = 0.30 outside these domains in Zrc-3. These findings are best explained by diffusion of Pb and other incompatible elements (Y, REEs) into 5-10 nm domains that were damaged by α-recoil and may have been metamict as the result of single U- or Th-decay chains. Diffusion distances of ~20 nm for these elements in crystalline zircon require temperatures above ~700oC for ~106 yr. [4]. This is consistent with the known history of Zrc-2 and -3, which both have younger magmatic overgrowths attesting to reheating at 29 Ma in Zrc-2 and 3.4 Ga in Zrc-3. In contrast, the absence of enriched domains in Zrc-1 suggests that this zircon did not experience similar high-grade metamorphism before or after its deposition within the 3 Ga Jack Hills metaconglomerate. For all 3 zircons, SIMS measurements at 10-20-m scale reintegrate nm-scale features and accurately determine the age of crystallization. Thus APT can provide unique constraints on otherwise cryptic thermal events; on Pb mobility and radiation damage; and for Archean zircons too small to be dated by SIMS, APT can determine 207Pb/206Pb ages.
[1] JW Valley et al. (2014) Nature Geo. 7, 219–223.
[2] AJ Cavosie et al. (2005) Earth Planet. Sci. Lett. 235, 663-681.
[3] A Strickland et al. (2011) Am. J. Sci. 311, 261-314.
[4] DJ Cherniak (2010) Rev. Min. Geochem. 72, 827-869.

Fig. 1: Figure 1. Atom map of Pb isotopes in 4.0 Ga Zrc-1.

Fig. 2: Figure 2. a) Atom map of Y and Pb in 2.5 Ga Zrc-2. b) Isoconcentration surface for Y in Figure 2a.

Fig. 3: Figure 3. a) atom map of Pb and Y in 4.4 Ga Zrc- 3. b) close up atom map of Y clusters. c) atom map of single cluster showing Pb isotopes with Y.

Fig. 4: Figure 4. Partial mass spectra from a) inside the clusters of Figure 2 and b) outside the clusters.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-1420 Identification of twin types in twinned natural quartz crystal by FEGSEM and EBSD analysis

Samardžija Z.1, Lenart A.1
1Jožef Stefan Institute, Ljubljana, Slovenia
zoran.samardzija@ijs.si

Representative sample of the ‘‘v”-shaped twinned quartz crystals from geological location Madan ore, Bulgaria was investigated using a field-emission gun scanning electron microscope (FEGSEM) JEOL JSM-7600F and an electron backscatter diffraction (EBSD) analytical system Oxford Instruments HKL Channel 5 with Nordlys detector. For optimum surface quality quartz sample was finally polished with 0.05-μm colloidal silica on diamond lapping films and coated with a 1.5-nm amorphous carbon layer using precise ion-beam sputtering technique in a Gatan PECS 682 apparatus. This very thin carbon layer was sufficient to prevent charging in the SEM and was adequate also for the acquisition of unblurred, good-quality EBSD (Kikuchi) patterns. SEM operating conditions for the EBSD analyses were set to a voltage of 20 kV, a 10 nA beam current, a working distance of 20 mm and with a specimen tilted to 70o. Quartz crystals are very sensitive to radiation damage which causes rapid amorphization under the intense point-focused electron beam dose. Therefore short pattern acquisition time of 40 ms was applied to avoid structure degradation of quartz consequently allowing us to collect sharp patterns and perform reliable pattern indexing.
The location of the twin boundary was clearly revealed on the SEM micrograph that was recorded using forward scattered electrons (FSE) and on the superimposed EBSD crystal orientation map (COM) where identical crystallographic orientations are displayed in particular colours: green, blue, pink and orange, as shown in Fig. 1. The misorientation angles along the line-profile across the twin boundary were determined relative to starting point in the left part of the crystal (green, A), as shown in Fig. 2. Obtained experimental value for misorientation angle between the inclined c-axes of the unit cells in two twin individuals (A and C) was ≈ 84o and fully agree with theoretical value 84.33o that is exactly attributed to the Japan-law twinned crystal. In the blue region (B) the unit cell is rotated about the c-axis for 60o as compared to the orientation in region A, so confirming the presence of Dauphiné twins which emanate in the vicinity of the Japanese twin boundary. Dauphiné twins in COM are displayed in blue and orange and were also recognized by the distinct change of the FSE contrast in the SEM micrograph.
Using an appropriate experimental FEGSEM-EBSD analytical set-up and optimized sample preparation we have successfully identified the types of twins in the “v”-shaped natural quartz crystal. The evaluation of the EBSD patterns and corresponding crystal orientations directly confirmed Japanese twins type with some Dauphiné twins located near the twin boundary.

This work was supported by the Slovenian Research Agency (ARRS) within project J2-4237.

Fig. 1: The FSE micrograph with superimposed EBSD crystal orientation map of twinned quartz crystal showing the twin boundary location and the Kikuchi patterns acquired from the left (green) and the right (pink) region of the crystal. EBSD analysis confirmed Japanese twin type with some Dauphiné twins (blue, orange) near the twin boundary.

Fig. 2: Misorientation profile along the line shown in Fig. 1 with illustrated unit cell orientations in regions A, B and C. Relative to A region B has no change in c-axis but displays 60o rotation about c-axis which is specific for Dauphiné twinning. Region C clearly showed the rotation of the c-axis by ≈ 84o which corresponds to Japanese twins.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-1819 Earliest life on Earth: new insights from electron and ion beam microscopy

Wacey D.1,2, Saunders M.1, Kong C.3
1Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia, 2Australian Research Council Centre of Excellence for Core to Crust Fluid Systems, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia, 3Electron Microscopy Unit, University of New South Wales, Kingsford, NSW 2052, Australia
David.Wacey@uwa.edu.au

Earth’s geological record holds great potential for decoding the origin and evolution of life on our planet. However, the interpretation of this record is complex and often controversial because Earth’s first life would have been very small, morphologically simple and likely only subtly different from co-occurring non-biological organic material.

Distinguishing between bona fide signs of life and abiotic artefacts requires analytical techniques with excellent spatial resolution in 2 and 3 dimensions, in order to accurately analyse key features of putative cells such as cell-wall ultrastructure, cell contents, and interaction of cells with the minerals that have fossilised them. We here show how TEM of FIB prepared samples and 3D-FIB-SEM reveal new details of fossilised cells at the nanometer to micrometer scale, providing new biosignatures for future studies on Earth or other planets.

Data are presented from three geological formations that play an important role in our understanding of the origin and evolution of life on Earth: 1, The 3,430 million-year-old Strelley Pool Formation of Western Australia, containing Earth’s oldest well preserved cells [1]; 2, The 1900 million-year-old Gunflint Formation of Canada, containing an iconic suite of diverse microfossils used as a benchmark for high quality preservation of early life [2]; 3, The 3,460 million-year-old Apex chert of Western Australia that houses controversial microfossil-like artefacts [3]. Our data include EFTEM maps of the chemistry of the ‘microfossils’ and their surrounding minerals, electron diffraction patterns to identify minerals and crystal orientation, plus serial SEM sections and 3D reconstructions (Figs. 1-2).

We find that the Strelley Pool and the Gunflint materials show many similarities in their style of fossilization and nano-scale morphology, and exhibit multiple features expected of bona fide fossilized organisms. In contrast, the Apex chert microstructures are shown to comprise complex clay mineral aggregates onto which carbon later adsorbed; these show no biological morphology at the nano-scale.

[1] D. Wacey et al., (2011) Nature Geosci. 4, 698–702

[2] D. Wacey et al., (2013) Proc. Nat. Acad. Sci. 110, 8020–8024

[3] M.D. Brasier et al., (2002) Nature 416, 76–81

DW is funded by the Australian Research Council Centre of Excellence for Core to Crust Fluid Systems. We acknowledge the facilities, scientific and technical assistance of the AMMRF at both The University of Western Australia and The University of New South Wales, facilities funded by the Universities, State and Commonwealth Governments.

Fig. 1: 3D reconstruction (A) and orthogonal x-y-z slice view (B) of a cellular microfossil from the 3,430 million-year-old Strelley Pool Formation. Each FIB-SEM slice was approximately 10 μm x 10 μm and the step size between slices was 200 nm. This clearly demonstrates the spherical to elliptical nature of the microfossil.

Fig. 2: 3D reconstruction of carbonaceous (A-B) and pyritic (C) filamentous microfossils from the 1,900 Ma Gunflint Formation. Note that preservation appears better for the pyritic examples with extra details such as putative heterotrophic bacteria (orange spheres) and exopolymers (pale yellow) visible.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-1926 TEM Studies of Clay Mineral Nanotubes: Imogolite and Pseudo-Boehmite

Iijima S.1,2, Zheng L.2
1Meijo University, 2National Institutes of Advanced Industrial Science and Technology / Nanotube Research Center
iijimas@meijo-u.ac.jp

A few tubular structures of nano-meter-scale are found in natural products although carbon nanotubes are known as entirely artificial synthetic products [1]. Some of layer structures of almino-silicate Al2O3-SiO2-H2O clay minerals are known to have a cylindrical form such as chrysotile asbestos. The reason for forming a cylindrical structure is well explained by a mismatch in dimension between the octahedral sheets and tetrahedral sheets. Imogolite is another example of a tubular form of almino-silicate clay minerals [2] but its diameter (2.1nm) is about one tenth of those of chrysotile, and therefore the origin of the curvature might be different. Being in a constant diameter should be explained in terms of crystallography. Another question with imogolite morphology is concerned with its highly anisotropic growth along its tube axis. Here we will propose a growth model for the tubular imogolite. Our model is based on a hypothesis of a chiral structure of imogolite in which the substances could be continuously deposited at the tip end of the tube. Figure 1 shows HRTEM images of imogolite supporting our hypothesis, where linear arrays of periodic dots with about 0.4nm interval are seen at only one side of the tube wall (arrowed). If the imogolite has an orthorhombic symmetry, the dots image should appear equally on both sides of the tube walls. A similar observation has been utilized in determining the chiral structure of a single-walled carbon nanotube [3].

The second example of the tubular structure is concerned with our newly proposed structure model for the aluminum monohydroxide febrile psuedoboehmite AlO(OH) [4]. This mineral is considered as the case where there is no silica in an Al2O3-SiO2-H2O almino-silicate system. Boehmite AlO(OH) is found in a stable crystalline form in nature but pseudoboehmite (PB) is synthesized as sol, a metastable form in a very slow reaction process. In XRD characterization the dried PB sol shows diffused ring patterns corresponding roughly to boehmite. The basic crystal structure of boehmite is a layer of two staggered edge-shared Al-O octahedra held together by hydrogen bonds. Figure 2 shows a low magnification image of the fibrous PB (Fig.2a) and an electron diffraction pattern taken from a bundle of the fibers (Fig.2b) which accords with that of XRD. A HRTEM image of the portion of the PB isolated fibers is shown in Fig.2c, which suggested a tubular structure with a diameter of 2nm. A possible crystal structure of PB will be discussed.

[1] S. Iijima, Nature, 345, 56 (1991).
[2] K. Wada & N. Yoshinaga,　Clay Minerals, 8, 487 (1970).
[3] Zheng Liu, et al., Phys. Rev. Lett., 95, 187406 (2005).
[4] V. Coelho, et al., Revista Materia, 13, 325 (2008).

Fig. 1: The HRTEM images of imogolite showing a regular appearance of the dot feature at the one side walls of the tubes. Such features suggest a chiral structure of the Al-O octahedra around the tube axis.

Fig. 2: (a) The fibrous feature of dried sol of pseudoboehmite (AlO(OH)). (b) An electron diffraction pattern from a bundle of the pseudoboehmite. (c) An enlarged image of a portion of the fiber suggesting a tubular structure with a diameter of 2nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-1972 The interrelation of elemental and phase composition of Norilsk ore type

Mashukov A. V.1, Mashukova A. E.1, Ponomareva S. V.1
1Siberian Federal University
AVMashukov@sfu-kras.ru

Using the methods of X-ray and M?ssbauer spectroscopy, scanning electron microscopy, there were studied the samples of Norilsk ore types in order to identify compounds containing Cu, Ni, Co, Fe, S. Depending on elemental composition there were singled out two sample series.
Maximum concentration in percentage of selected elements for this series is presented below.
1: Ni (0,7); Cu (15,3); Co (2,1); S (17,2); O (20,2); H (0.02); Fe (24,1); Ca (0,1); Mg (0,67);
K (0,54); Al (2,05).
2: Ni (1,94); Cu (23,4); Co (0); S (25,5); O (9,91); H (0,39); Fe (25,9); Ca (0); Mg (0); K (0);
Na (1,12); C (2,51); Si (7,92); Al (1,31).
The research conducted by using the method of scanning electron microscopy and the X-ray microanalysis showed that iron and sulfur are spread uniformly over the scanned area. Sulfur is absent in the inclusions containing Fe and Ni. There are areas, strongly enriched by Fe (Fig. 1). The inclusions of rectangular and rhomboid shapes contain Ni as the content of Fe increases (Fig. 2).
There were identified the inclusions having a high content of Cu, with a maximum concentration of Ni (Fig. 3). The distribution of Co is shown in Fig. 4.
The phases, containing Cu, Ni, Co, have a complex composition.
1: pentlandite (Fe1,63 Ni1,82 Co5,6 S8) - 5,14%; chalcopyrite (CuFeS2) – 44,4%; magnetite (Fe3O4) – 5,77%.
2: pentlandite (FeNiS2) - 3,44; chalcopyrite (CuFeS2) – 66,2%; magnetite (Fe3O4) – 4,68%; bornite (Cu5FeS4) – 0,84%; nickelhexahydrite (NiSO4?[6H2O]) – 3,64%.
The ingrowths of CuFeS2 are characterized by the degree of the structure defectiveness, by various impurities, which are reflected in the studied parameters.
As regards the other sample series the spectra are the superposition of the unsolved doublet, which shows the presence of paramagnetic areas.
The isomer shifts of the samples range from 0,429 до 0,509 mm/s (series 1) and from 0,509 to 1,394 mm/s (series 2). Quadrupole splitting ranges from 0,509 to 2,800 mm/s (series 1) and from 0,509 to 2,688 mm/s (series 2). This indicates that the local electronic structure depends on the genesis of compounds.
Thus, most of the bulk of Cu, Ni is not dissipated in the crystal lattices of the ore, but it is part of the ore sulphides. The presence of the characteristic structures of the solid solutions decomposition shows a wide temperature range of sulphide crystallization.

Fig. 1: The distribution of Fe in the scanned area, x500

Fig. 2: The distribution of Ni in the scanned area, x750

Fig. 3: The distribution of the Cu in the scanned area, x 750 

Fig. 4: The distribution of Co in the scanned area, x750
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-2275 Combining EBSD and TEM to infer the crystallographic and shape orientation relation of acicular TiO2-inclusions with the garnet host lattice

Habler G.1, Proyer A.2, Wirth R.3, Abart R.1
1Department of Lithospheric Research, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria, 2Department of Earth Sciences, University of Graz, Universitaetsplatz 2/II, 8010 Graz, Austria, 3GFZ German Research Centre for Geosciences, Telegrafenberg, 14473 Potsdam, Germany
gerlinde.habler@univie.ac.at

Acicular TiO2 inclusions with preferred shape orientation in natural garnet from a metapelite rock were investigated by EBSD, TEM and light microscopy (U-stage) in order to correlate the shape and crystallographic orientation of inclusions with the garnet host lattice and to characterize the interfaces between inclusions and host. Focused ion beam preparation was applied to selected rutile grains, which had been investigated by EBSD before, in order to obtain exact longitudinal and cross sections of rutile needles of 1-2 micrometer thickness. The data therefore allow the direct comparison and combination of results from EBSD and TEM investigations.
The majority of the rutile grains are acicular, having the needle long axes oriented parallel to Grt <111> or Grt <100> directions. For the majority of Rt-grains the needle long axis does not correspond to the Rt c-axis. TEM data confirmed the notion inferred from EBSD data, that the phase boundary of acicular rutile grains mainly follows Grt {110} and subordinately Grt {100} planes. Isometric rutile grains also have phase boundary segments parallel to Grt {112} planes.
Single point EBSD analyses of 213 rutile grains and the hosting garnet yielded a complex, but strict crystallographic orientation relation between inclusions and the garnet lattice (Proyer et al, 2013). Although there is no unique crystallographic orientation of rutile with respect to garnet, and there are no coincidences of low-indexed garnet and rutile planes, the comprehensive dataset allows inferring systematic orientation relations of rutile and garnet. The majority of Rt-grains are oriented with their c-axes at 12 positions along a cone around Grt <111> directions. In this orientation the Rt lattice is fixed relative to the corresponding Grt <111> direction. Rutile <110> directions corresponding to three different c-axes cones around symmetrically equivalent Grt <111> directions cluster subparallel to the fourth Grt <111> direction. Furthermore, rutile a-axes and Rt <110> directions of this Rt-population seem to avoid Grt <110> directions. Contrastingly, a second subordinate population of Rt needles has c-axes parallel to Grt <111> and one of the a-axes parallel to one of the Grt <110> directions.
TEM data support the interpretations inferred from EBSD analyses, cannot determine the complex orientation relations, which require a larger dataset for detection. Although no coherency of the rutile and garnet lattices was found, the shape and lattice orientation of rutile as well as its boundary geometry are strictly controlled by the garnet lattice.

Proyer et al (2013) Contrib. Min. Pet. 166, 211-234

Contributions by G. Habler and R. Abart were funded by the Austrian Science Fund (FWF): I471-N19 as part of the international DMG-FWF funded Research Network FOR741 D-A-CH. Additional funding was provided by the Austrian Science Fund (FWF): P16194-N06, P22749-N21.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-2452 Study of the oolites Bakchar ore area by scanning electron microscopy.

Kryazhov A.1, Rudmin M.1, Logvinenko O.1, Ilenok S.1
1Tomsk Polytechnic University, Tomsk, Russia
kryazhov@gmail.com

Bakchar ore area was located in the southeastern part of the West Siberian iron-ore basin, 200 km from Tomsk to the northwest. The total area of the cluster is 1200km2, estimated resources of oolitic iron ore constitute28 billion tons, what make this object unique.
Results of research of microinclusions in oolites were received by scanning electronic microscopy. Sampling material was divided in granulometric classes (more than 1 mm; 1-0,5 mm; 0,5-0,2 mm; 0,2-0,1 mm; less than 0,1 mm) by the method of "wet" bout. Samples were pressed and polished then were investigated by SEM Hitachi S-3400N with energy dispersive attachment.
Ore samples can be presented as chlorite hydrogoethite, hydrogoethite types. In all ore oolites inclusions of phosphates of rare-earth elements was observed that show the high content of phosphorus in the Bakchar ores. The size of phosphatic grains is 2-10 microns which were rather evenly distributed in structure of oolites. In rare cases, concentric zones made from entirely rare earth phosphate (monazite group) up to 5 mm (Fig. 1) was observed. Also phosphates were found in "dendrite" form of units (fig. 2) in chlorite- hydrogoethite peas. Besides rare-earth phosphates in oolites were noted calcic (apatite) and ferruterous (vivianite) phosphates. Among other mineral inclusions in ore oolites: quartz, feldspar, glauconite, leptochlorite, galenite, sphalerite, chalcopyrite, ilmenite, zircon, magnetite was also found. It illustrates the high contents of the phosphorus, titan, lead, copper, sulfur and rare-earth elements in the bakcharsky ores
The received results will be used for creation of the technological scheme of oolitic ores of Bakchar ore area.

The work was supported by the State Program: Science №1.1312.2014. Authors would like to thank the head of Department of Geology and Minerals Prospecting of TPU prof. A. Mazurov and prof. V. Voroshilov

Fig. 1: Concentric zones made from entirely rare earth phosphate (monazite group) up to 5 mm

Fig. 2: Mineral incorporating of rare earth phosphate (monazite) as "dentrite" aggregates
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-2457 The mineralogy of Au-bearing skarn zones of the Topolninskoe area (Gorny Altai)

Logvinenko O.1, Kryazhov A.1, Rudmin M.1
1Tomsk Polytechnic University, Tomsk, Russia
kryazhov@gmail.com

This investigation is devoted to the mineralogical data of the Au-skarn zone at Topolninskoe area, Gorny Altai, Russia. Skarn-type metasomatic alteration and mineralization occurs during interactions between Low Silurian carbonates and an Upper Devonian granitoid stock.
Polished thin sections of skarn and metasomatites were studied using scanning electron microscopy (SEM - LEO-1430VP) and wavelength-dispersive X-ray microanalysis (WDX – Oxford instruments, INCA) at V.S. Sobolev Institute of Geology and Mineralogy (IGM), Novosibirsk, Russia.
More than 30 minerals were detected in skarn samples. Main rock forming minerals are clinopyroxene, garnet, calcic amphiboles (ferroactinolite, actinolite and magnesiohornblende), wollastonite, plagioclase, potash feldspar, epidote (clinozoisite, epidote), scapolite, chlorite and calcite. Garnets are grossular–andradite with 0.03 to 4.72 mol. % piralspite, whereas pyroxenes are hedenbergitic to diopsidic in composition.
Fine sulfide mineralization is concentrated in hydrothermally altered rocks. The research showed that ores are formed in three mineral stages: arsenopyrite-molybdenite-pyrite, polymetallic and telluride-sulfotelluride-sulfide. For the first time, such minerals as rucklidgeite (Bi1,97Pb1)3Te4,02, tsumoite Bi1,02Te1, hessite Ag1,81Te1, gersdorffite (Fe0,17Ni0,5Co0,36)As1S0,93, ullmannite Ni1Sb1,35S1,38, cobaltite Co1As1,08S1,19, poubaite Pb1Bi1,71(Se0,56Te0,14S3,85)4, nevskite Bi0,99(Se0,44S0,27) and native bismuth have been detected in this area.
Native gold and electrum prevail in molybdenite, bornite are associated with telluride minerals as fine-grained inclusions; grain size varies from 5 to 20 μm.
As a result of this study the chemical composition and crystallochemical formulas were determined for all silicates and opaque minerals.

Acknowledgement: The work was supported by the State Program: Science
№1.1312.2014. Authors would like to thank the head of Department of Geology and Minerals
Prospecting of TPU prof. A. Mazurov and prof. V. Voroshilov

Fig. 1: Fig.1. Bse images of sulfides: a) gold (Au) and native bismuth (Bi) in chalcopyrite (Cpy); b) native bismuth, nevskite (Nev) and galena (Gn) in arsenopyrite (Ars) grain; c) altaite (Alt) microinclusions in chalcopyrite and bornite (Bn); d) tsumoite (Tsc), hessite (Hes), gersdorffite (Grf) and galena form thin veinlets in chalcopyrite.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-2464 Characterization of sub-micron UO2 inclusions in natural baddeleyite (ZrO2)

O'Connell J. H.1, Lee M. E.1, Janse van Vuuren A.1
1Centre for High Resolution Electron Microscopy, Port Elizabeth, South Africa
jacques.oconnell@gmail.com

Previous work on the chemical composition of inclusions in Phalaborwa baddeleyite (ZrO2) xenocrysts showed that inclusions were a result of the physical alteration of the baddeleyite crystal and the subsequent filling of the cracks by the magma at the end of an emplacement process1. Wavelength dispersive X-ray spectroscopy (WDS) analysis of these secondary inclusions and the baddeleyite matrix did not provide any evidence for the presence of U. Previous work for determining the U-Pb-Th isotope ratios in Phalaborwa baddeleyite for geochronology applications 2,3,4 using SIMS (SHRIMP technique) and LA-ICP-MS analysis (spatial resolution of 25-50 μm), reported on extremely variable concentrations of uranium (51-2124 ppm). However, no evidence for the presence of U inclusions in natural baddeleyite has previously been reported. In this paper we will describe the characterization of small (~1 μm) uranium oxide (UO2) primary inclusions in baddeleyite.

Sections of approximately 2 mm thickness were cut from a Phalaborwa baddeleyite xenocryst using a diamond wire saw and polished to a 0.25 μm finish. FIB lamella were prepared from U containing inclusions which were identified using back-scattered electron (BSE) imaging in an FEI Helios NANOLAB. The FIB lamellae were imaged and analysed using a JEOL ARM 200F TEM with an attached SDD energy dispersive X-ray spectrometer (EDS).

BSE imaging of the baddeleyite matrix revealed small uranium inclusions (~1 μm) intersecting the surface. No U-inclusions were associated with any physical alteration such as crack formation in the zirconia matrix. The HAADF STEM image of a uranium oxide inclusion (bright) in the baddeleyite (dark) is shown in Figure 1 (left) together with the corresponding BF image in Figure 1 (right). Figure 2 shows an SAD acquired over the interface of the inclusion and the surrounding matrix indicating an epitaxial relationship between two crystals. EDS line scans across the boundaries of the inclusion did not provide any evidence for inter-diffusion between U and Zr. These results possibly explain the large variation for U concentrations obtained by previous workers. It is also suggests that UO2 form primary inclusions which were incorporated in the baddeleyite matrix via the initial crystallization process in the earth’s mantle where an immiscible UO2 droplet crystalizes upon cooling of a ZrO2-UO2 melt, followed by the epitaxial growth of ZrO2 on the UO2 nucleation kernels upon further cooling.

References
1. Lee, M.E. et al. (2012) Proc. Microsc. Soc. South. Afr. 42, 57.
2. Heaman, L (2009) Chemical Geology 261, 43.
3. Amelin, Y. and Zaitsev, N. (2002) Geochim. Cosmochim. Acta 66, 2399.
4. Rodionov, N.V. (2012) Gondwana Research 21, 728.

Fig. 1: HAADF STEM (left) micrograph of a UO inclusion (bright) in ZrO2 with the corresponding BF micrograph on the right.

Fig. 2: Partially indexed SAD taken over the UO2-ZrO2 interface.
Fig. 3:
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-2502 Characterization of twin domains in Phalaborwa baddeleyite xenocrysts by EBSD and TEM

Lee M. E.1, Goosen W. E.1, O'Connell J. H.1, Janse van Vuuren A.1
1CHRTEM, NMMU, Port Elizabeth, South Africa
michael.lee@nmmu.ac.za

Baddeleyite is an important mineral which undergoes a martensitic phase transformation from cubic through tetragonal to monoclinic at room temperature for low pressure (<4Gpa)1. A number of high pressure crystalline phases (5-50GPa), including an orthorhombic phase, have been reported for shock recovery experiments of small baddeleyite crystallites (1-30 micron)2. It was also suggested that Phalaborwa baddeleyite xenocrysts are possibly metaminct due to the absence of cathodoluminescence (CL) response3. The presence of twin domains in natural baddeleyite has been reported for twinning in monoclinic baddeleyite (zirconia) occurring along {001} planes and more predominant than along {011} planes and very rarely along {012}4. The volume change (5%) and strain energy (8-14%) during the transformation results in crystal twinning and/or cracking due to catastrophic changes in temperature and pressure as a result of the emplacement process. Twin boundaries in baddeleyite could provide valuable information on the thermal history of the material1 as well as distinguish growth twins from transformation and deformation induced twins. The characterization by transmission electron microscopy (TEM) of twins in baddeleyite from the Mbuji Mayi kimberlite has been previously reported5. However, twin boundaries analysed by electron back scatter diffraction (EBSD) have not been reported for baddeleyite. In this paper we will present results for the analysis of twin domains observed in baddeleyite from the Phalaborwa carbonatite complex by EBSD and TEM.
Polished sections of Phalaborwa baddeleyite were analysed in a JEOL 7001F scanning electron microscope (SEM) and crystal orientation analysis was performed by using am Oxford EBSD system. FIB lamellae using a FEI Helios Nanolab 650 FIBSEM were cut perpendicular to the twin boundaries observed in the polished sections. The FIB lamellae were imaged using the bright field (BF) TEM mode in a JEOL ARM 200F TEM.
The twin bands for baddeleyite are revealed by channelling contrast in the BSE image (Figure.1). Two sets of polysynthetic twins can be identified in the EBSD image shown in Figure 2. Analysis of the EBSD data reveals both 90o and 180o twin boundaries. TEM images showing three distinct twin domains and the corresponding diffraction patterns are shown in Figure 3. The specimen was tilted away from the zone axis shown in the SADs to improve contrast in the BF image.

1, Lumpkin, G.R. and Ewing, R.C. (1992) Am. Mineral. 77, 179.
2. Niihara T. et al. (2009) LPS XL, Abstract #1562.
3. Herd, C.K.D. et al. (2007) LPSC XXXVIII, Abstract #1664
4. Wingate, M.T.D. and Compston, W., 2000. Chemical Geology 168, 75.
5. Kerschhofer, L., Scharer, U., Deutsch, A., (2000) Earth. Planet. Sci. Lett. 179, 219.

Fig. 1: SEM-BSE image showing channeling contrast for the twin domains

Fig. 2: All euler map overlaid on a band contrast image of baddeleyite showing the various twin bands

Fig. 3: BF TEM micrograph of a twin in baddeleyite with superimposed SAD patterns of the three regions.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-2522 3D imaging of chemical distribution from melting in the laser-heated diamond anvil cell

Nabiei F.1, 2, Cantoni M.2, Badro J.1, 3, Dorfman S.1, Gaal R.1, Gillet P.1
1Earth and Planetary Science Laboratory, EPFL, Lausanne, Switzerland, 2Interdisciplinary Centre for Electron Microscopy, Lausanne, Switzerland, 3Institut de Physique du Globe de Paris, Université Paris Diderot, Paris, France
farhang.nabiei@epfl.ch

The laser-heated diamond anvil cell is a unique tool for subjecting materials to pressures over few hundreds of GPa and temperatures of thousands of Kelvins which enables us to experimentally simulate the inaccessible interiors of planets. Meaningful measurements of equilibrium chemical interactions in these experiments depend on stable heating with minimal temperature gradients. However, small sample size, laser profile, thermally conductive diamonds, and uneven absorption and insulation in diamond anvil cell samples cause temperature gradients of 1000s K over a few microns (e.g. Sinmyo et al. 2010). Precious studies have modeled the temperature distribution during laser heating but these models have only included conductive, solid-state transfer (e.g. Dewaele et al. 1998, Kiefer and Duffy 2005, Rainey et al. 2013). During high-pressure melting experiments, convection and diffusion effects are also important to differences in temperature and chemistry (Du et al. 2013).

We have examined samples of San Carlos olivine powder melted in the diamond anvil cell by infrared laser heating from single- and double-side to ~3000 K for 3 minutes at 37 GPa. Recovered samples were analyzed by a combination of focused ion beam (FIB) and scanning electron microscope (SEM) equipped with energy dispersive x-ray detector (EDX). About 300 slices were recorded with 70 μm depth, comprising about 2/3 of the heated zone. Detailed chemical and structural analysis by transmission electron microscopy (TEM) of lamellas prepared from the remaining 1/3 of each sample will also be discussed.

In both single- and double-side heated samples the heated zone included (Mg,Fe)SiO3 perovskite (PV) and two (Mg,Fe)O oxide phases, one Mg-rich, ferropericlase (FP), and one Fe-rich, magnesiowüstite (MW). In the double-side heated sample there is also an Fe-rich melt core. A FP crust was observed around the heated zone in both cases. However, this crust is broken in the upper part of the single-side-heated sample which could be attributed to the lower temperature in this region. MW regions in the single-side-heated sample distributed heterogeneously in the heated zone. These results show the importance of double-sided heating for generating homogeneous temperatures and raise important questions for melting experiments and simulations.

Sinmyo, R., Hirose, K., 2010. . Physics of the Earth and Planetary Interiors 180, 172–178.

Dewaele, A., Fiquet, G., Gillet, P., 1998. . Review of Scientific Instruments 69, 2421–2426.

Kiefer, B., Duffy, T.S., 2005. . Journal of Applied Physics 97, 114902.

Rainey, E.S.G., Hernlund, J.W., Kavner, A., 2013. . Journal of Applied Physics 114, 204905.

Du, Z., Amulele, G., Benedetti, L.R., Lee, K.K.M., 2013. . Review of Scientific Instruments 84, 075111.

Fig. 1: RGB image from one of the central slices from double-side-heated sample. Red, green and blue channels correspond to Fe, Si and Mg respectively. Red arrows in schematic view of diamond anvil cell represent infrared laser direction.

Fig. 2: RGB image from one of the central slices from single-side-heated sample. Red, green and blue channels correspond to Fe, Si and Mg respectively. Red arrow in schematic view of diamond anvil cell represents infrared laser direction.

Fig. 3: Volume rendering from single-side-heated sample. Red, green and gray scales represents Fe, Si and Mg respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-2624 HRTEM investigation of complex modular structures in geo-materials: an important investigation tool to reveal fine nanotextures of titanium-disilicates

Genovese A.1, Camara F.2, 3, Falqui A.1, 4, Sokolova E.5, Hawthorne F. C.5
1Department of Nanochemistry, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova, Italy, 2Dipartimento di Scienze della Terra, Via Valperga Caluso, 35, 10125 Torino, Italy, 3Interdepartmental Center for Crsytallography, Via Pietro Giuria 7, 10126, Torino, Italy , 4King Abdullah University of Science and Technology, Thuwal, Saudi Arabia, 5Department of Geological Sciences, University of Manitoba, Winnipeg, R3T2N2, Canada
alessandro.genovese@iit.it

Lomonosovite, Na10Ti4(Si2O7)2(PO4)2O4, and β-lomonosovite, Na64Ti4(Si2O7)2[PO3(OH)][PO2(OH)2]O2(OF), are typical accessory minerals of nepheline sienites and pegmatites of Lovozero alkaline massif (Kola peninsula, Russia). According to Sokolova [1], they belong to Group-IV of titanium disilicates with the TS block where Ti (+ Mg + Mn) = 4 apfu. Their complex crystal structure (space group P-1) is a combination of TS (Titanium-Silicate) block and I (intermediate) block, interleaved along the c crystal axis and parallel to (001) plane. TS block consists of the central O (octahedral) sheet and two H (heteropolyhedral) sheets containing Si2O7 groups. The TS block is characterized by a planar cell based on t1 and t2 translational vectors forming an angle close to 90°, with t1≈5.5 Å in both phases, t2≈7 Å in lomonosovite (Figure 1a) and ≈14 Å in β-lomonosovite (Figure 2a, after Sokolova et al.[2]). I block is made of a framework of distorted Na polyhedra and P tetrahedra. These titanium silicates exhibit the same structural topology of the TS block characteristic for Group IV. β-lomonosovite is an OH-bearing phase with extensive cation disorder and Na-depletion occurring mainly in the I block and less in the TS block. This charge vacancy is compensated by substitution of OH groups for O atoms, whereas cation disorder results in doubling of the t2 translation.
HRTEM and electron diffraction study of both titanium disilicates exhibited triclinic structures with β angles ≈96°. Lomonosovite [100] sections revealed generally free-defect structure, showing planar cell with translational vectors t2 and t3 forming α angle ≈100° with t2 ≈7 Å and t3 ≈14.2 Å (Figure 1b). On the contrary, β-lomonosovite [010] sections showed ubiquitous lamellar texture with (100) lamellae 20-40 nm thick alternating along [100], generally defective and exhibiting variegated electron contrast (Figure 2b). The (100) lamellae exhibited Na-depletion due to OH substitution to balance charge vacancy and causing greater beam-damage effect with fast crystallinity loss.

[1]. Sokolova, E. The Canadian Mineralogist, 44, (2006), 1273.

[2]. Sokolova et al. The Canadian Mineralogist, 52, (2014), (in press).

We thank Alexander P. Khomyakov (sample 1867/2) of Institute of Mineralogy, Geochemistry and Crystal Chemistry of Rare Elements, Veresaev Street 15, Moscow, 121357, Russia, and K. T. Tait (lomonosovite), Department of Natural History, Royal Ontario Museum, 100 Queens Park, Toronto, ON M5S 2C6, Ontario Canada

Fig. 1: Lomonosovite. a) Atom schema with 2D unit cell (magenta) showing tetrahedra (Si-orange, P-purple), octahedra (Ti-yellow, Na-blue), [4,5]-coordinated Na polyhedra (turquoise). b) [100] projection of free-defect structure; zoon region and its power spectrum consistent with b*c* electron diffraction pattern (insets)

Fig. 2: β-Lomonosovite. a) Atom schema with 2x 2D unit cell (color code as above); blue, pink and yellow spheres are Na, Ca and P sites with occupancy < 50%; red spheres are OH groups. b) [010] projection of Na-depleted and defective lamella; free-defect region and its power spectrum consistent with a*c* electron diffraction pattern (insets).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-2681 Electron microscopy and DFT study of (130) twins in chrysoberyl crystals from Pratinhas, Brazil

Drev S.1, Komelj M.1, Mazaj M.2, Rečnik A.1, Daneu N.1
1Department for Nanostructured Materials, Jožef Stefan Institute, Ljubljana, Slovenia, 2Laboratory for Inorganic Chemistry, National Institute of Chemistry, Ljubljana, Slovenia
nina.daneu@ijs.si

We studied (130) twins of natural chrysoberyl (BeAl2O4) crystals from Pratinhas, Brazil. To determine the local structure of twin boundaries, powder X-ray diffraction analysis (XRD), transmission electron microscopy (TEM) methods and density functional theory (DFT) calculations were used. Chrysoberyl has a slightly distorted hexagonal close-packed (hcp) O-sublattice with Al3+ and Be2+ ions partially occupying octahedral and tetrahedral interstices. The crystal structure was refined by Rietveld analysis (Topas-Academic V4) using our experimental XRD data collected on finely ground bulk material, starting with the atomic coordinates determined by Hazen (1987). The structure of chrysoberyl was refined in the orthorhombic s.g. 62 (Pmnb) with unit cell parameters: a = 5.4825(1), b = 9.4163(2), c = 4.4308(1) and Rwp = 7.8%. The refined structure was then used for interpretation of electron diffraction patterns and lattice images obtained by TEM (JEM2100, Jeol). The specimens were investigated in [001]-projection, where the (130) twin boundaries are viewed edge-on (Fig. 1). EDS measurements show ~1% Fe in bulk chrysoberyl, suggesting an average composition of BeAl1.99Fe0.01O4. The increase of Ti at the twin boundary suggest a transient Ti-exsolution that took place after the twin formation. The twin boundary appears to be coherent, making occasional steps to parallel (130) planes. Following the interface, we observe that the periodicity is attained after every ~1.9 nm, roughly corresponding to 4·[110] interplanar distances. HRTEM images recorded at the defocus value of -60 nm are very sensitive to the positions of Be-atoms, which alternate form one to another side of the O-chains (Fig. 2). Image simulations, based on simple mirror-twin model, match the general contrast features, whereas the interface is not well reproduced and needs some reconstruction. A close look at the interface shows two distinct features, which are a result of different occupation of coordination polyhedra accompanied by some relaxation. Calculations within the framework of the density functional theory (DFT), using pseudo-potential method, suggested that the atomic Cluster-I undergoes a significant relaxation of interfacial Be2+ and O2- positions, whereas in Cluster-II the interfacial Be2+ sites are shifted to neighboring tetrahedral interstices, further away form the boundary. The local charge balance involved with this operations remains unchanged. whereas the contrast of the simulated image based on the DFT relaxed atomic model, shows correct tendency in the interface contrast compared to experimental HRTEM images. Further analysis of (130) twin boundaries will be necessary to verify the possibility of chemically-induced twinning in chrysoberyl (Takeuchi 1997).

References:
[1] Hazen RM: Physics and Chemistry of Minerals 14 (1987) 13-20.
[2] Takeuchi Y: Tropochemical cell-twinning. Terra Scientific Publishing, Tokyo (1997) 319.

Fig. 1: HRTEM image of (130) boundary in natural chrysoberyl crystals (inset) with the corresponding SAED pattern showing the twin operation. EDS analysis indicates the presence of Ti on the twin boundary.

Fig. 2: Refinement of the atomic model, based on HRTEM image simulations and DFT calculations. Major differences occur in atomic clusters I and II, outlined in the HRTEM image.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-2813 Topotaxial transformation of ilmenite (FeTiO3) single-crystals to rutile (TiO2) and hematite (Fe2O3) during thermal treatment

Stanković N.1, Rečnik A.1, Šturm S.1, Daneu N.1
1Department for Nanostructured Materials, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
nadezda.stankovic@ijs.si

Replacement reactions are widespread in nature where minerals are often exposed to significant changes in physical (p, T) and geochemical conditions [1] . During progressive recrystallization, the precursor mineral is transformed into a more stable phase or mineral assemblage. Topotaxial reactions result in the formation of coherent structural intergrowths. During thermally induced oxidation of ilmenite (FeTiO3), for example, the transformation products are structurally coherent rutile (TiO2) and hematite (Fe2O3). Many samples of coherent rutile/hematite intergrowths are found in nature [2] and the transformation is also of high technological importance, for example in the production of titania from ilmenite [3]. In our work we studied atomic-scale mechanisms of ilmenite to rutile/hematite transformation on thermally treated natural single crystal of ilmenite from Zagi Mountain (Pakistan). The crystal was cut into oriented cuboids, which were heated at 600 to 1000°C in air for different periods of time (1, 12, 100 hours). The products were characterized by scanning and transmission electron microscopy (SEM and TEM) in two perpendicular zone axes ([0001]ILM and [10-10]ILM).
After thermal treatment at 800°C and higher we observed progressive exsolution of rutile lamellae in three directions intersecting at 60° within the parent ilmenite. The formation of lamellae starts at the surface and proceeds into the interior of the single crystal. It is accompanied by the formation of cracks as a result of volume change and the formation of a hematite layer on the surface, indicating oxidation and diffusion of Fe ions to the crystal surface (Fig. 1a). The number of rutile lamellae and the thickness of the hematite layer increase with heating time and temperature (Fig. 1b). TEM analyses revealed that rutile and ilmenite are in a coherent orientation relationship: <010>(100)RUT || <0001>{11-20}ILM (Fig. 2a,b). This confirms that the transformation is structurally controlled by ion diffusion though the common hcp oxygen sublattice. The suggested mechanism of transformation includes initial oxidation of Fe2+ in ilmenite to Fe3+ and its diffusion to the surface of the single crystal. The excess Ti ions exsolute within the parent ilmenite structure in the form of rutile lamellae. TEM analyses revealed the presence of an intermediate Ti-O phase prior to the formation of rutile and it is characterised by additional superstructure reflections, indicating that it is a crystallographic shear (CS) phase, possibly containing locally reduced Ti3+ ions (Fig. 2c).


1. Putnis, A., Mineralogical Magazine, 2002. 66(5)
2. Armbruster, T., Neues Jahrbuch für Mineralogy, 1981. 7
3. Zhang, J., et al., Metallurgical and Materials Transactions B, 2013. 44(4)

The financial support of the Slovenian Ministry for Science under the projects PR-04364 and J1-4167 are gratefully acknowledged.

Fig. 1: (a) Progressive recrystallization of ilmenite after 12 hours at 800°C in air results in the formation of rutile lamellae intersecting at 60° and a hematite layer on the crystal surface. (b) At higher temperature, the thickness of rutile lamellae and the width of the surface hematite layer are increased.

Fig. 2: (a) TEM image of ilmenite with rutile lamellae (800°C/12h). (b) Diffraction pattern taken across a typical rutile/ilmenite contact reveals the <010>(100)RUT || <0001>{11-20}ILM orientation relationship. (c) Some rutile lamellae exhibit additional reflections, characteristic for a CS intermediate phase.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-2876 Microscopic methods for the study of biomineralization in ancient sedimentary rocks and modern marine sediments.

Reykhard L.1
1P.P.Shirshov Institute of Oceanology of the Russian Academy of Sciences (IO RAS), Moscow, Russian Federation
mollusc@mail.ru

Biomineralization (the formation or accumulation of minerals by living organisms) can be "biologically induced" (BIM) and "biologically controlled" (BCM) (Lowenstam, 1981; Mann, 1983). To determine the role of biomineralization in the litho- and ore genesis comprehensive studies of Cimmerian oolitic iron ore (N22, Kerch iron pool) and modern silty-clayey marine sediments (QIV, White Sea) were carried out. To study of biomineral formation at the micro- and ultra-micro level comprehensive laboratory tests were performed: a) light microscopy of samples in thin sections; b) Scanning electron microscopy (SEM); b) electron-probe microanalysis (EPMA). BCM in Cimmerian oolitic iron ores may be detected already at the macro level. It is represented by carbonate biominerals made of large (5-7 cm) shells of mollusks and detritus (Fig.1a). Light microscopy showed that calcite-siderite detritus has microfibre structure (Fig.1c). SEM-images show that the shell detritus consists of alternating layers of calcite with different ultramicrostructures (Fig.1c-d). In marine sediments (Fig.2a) BCM is presented by opal. It was discovered as a result of light microscopy of thin sections (Fig.2b,e). It was confirmed by SEM and EPMA. Opal composes fragments of flint shells of diatoms (Fig.2f-g), silicoflagellate skeletons (Fig.2h) and the spicules of siliceous sponges (Fig.2c-d). BIM in oolitic- and pisolitic iron ores resulted in the formation of various oxides and hydroxides of iron. Microscopic analysis of the ores reported presence of such iron biominerals as hematite and goethite, which compose the basic structural elements of ore – oolites, oolite-like formations and cement (Fig.1). Iron biomorphic formations (Fig.1h,j,k) and silicate (Fig.1g,l) biomorphic formations of different shapes found by SEM in the oolitic and pisolitic iron ores, may be an indirect proof of iron and silicate bacteria involvement in iron ore process. In marine sediments BIM is found in the form of framboidal pyrite aggregates. Pyrite framboids are recorded in samples of sediment already in the light microscopy of thin sections (Fig.2b,i). SEM help to visualize framboidstructure and different shape of individual crystallites - globular shape, pentagonal dodecahedron, octahedral and pseudocubic shape (Fig.2j-l). Pyrite framboids often formed on the diatom shells surface residues or inside them (Fig.2i-k). The results of these studies are necessary not only for understanding of biogenic substances role in the formation of sedimentary rocks and ores, but also for the reconstruction of paleoenvironment of sedimentary deposits.
References
Lowenstam H A (1981) Minerals formed by organisms. Science 211:1126-1131
Mann S (1983) Mineralization in biological systems. Struct Bonding 54:125-174

Fig. 1: Biomineralization in Cimmerian oolitic iron ores (Iron Cape Horn, the Black Sea)

Fig. 2: Biomineralization in the bottom sediments (QIV, White Sea)
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-3049 Use of large area phase distribution maps for determination of modal analysis and chemical composition of geological samples

Halodova P.1, Haloda J.1, 2
1Czech Geological Survey, Geologická 6, Prague 5, Czech Republic, 2Oxford Instruments Nanoanalysis, Halifax Road, High Wycombe, HP12 3SE, United Kingdom
patricie.halodova@geology.cz

Quantitative modal analysis of rocks provides an analysis in terms of the distribution and volume percent of the minerals actually present in a rock sample. This type of analysis in many instances cannot be derived from a normal elemental analysis (particulary when polymorphic minerals are present). The combination of field-emission SEM and AztecEnergy AutoPhaseMap software (Oxford Instruments) provides the rapid acquisition of phase distribution and compositional data over the tested large area samples (1 200 mm2) at high-resolution. This technique can be applied to sedimentary, metamorphic and igneous rocks as well as other materials where examination at the micrometre to nanometre scale is required. Also, this method can be successfully applied if only small amount of material is available (rare samples as meteorites, lunar rocks, drill-cores, as well as ceramics, metallographic samples). This large area mapping is fast, non-destructive and accurate for all samples where the analyzed area of the studied material is statistically representative (special care must be taken when analyzing very coarse-grained or heterogeneous rocks). It provides us with the information about identification, distribution and quantitative analysis of phases (for all size ranges including nanoparticles), finds intra-phase variations and can calculate representative composition for each phase and the full-area composition that is comparable to whole-rock geochemical analysis of the specimen (excluding volatile and light elements). This technique is likely to be of use when using the modal analyses of selected domains with defined mineral associations for geochemical modeling (p-T conditions determination, crystallization modelling, etc.).

The authors would like to acknowledge the support of Oxford Instruments NanoAnalysis and the opportunity to use their X-Max 80 SDD EDS detector with AZtecEnergy software.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-3062 THE CONTRIBUTION TO MORPHOLOGICAL AND CHEMICAL CHARACTERISTICS OF THALLIUM MINERALS FROM ALLCHAR

Šoufek M.1, Bermanec V.2, Mikulčić-Pavlaković S.3, Spasevski L.4
1Department of Mineralogy and Petrology, Croatian Natural History Museum, Demetrova 1, 10000 Zagreb, Croatia, 2Department of Mineralogy of Faculty of Science and Mathematics, University of Zagreb, Horvatovac 95, 10000 Zagreb, Croatia, 3Department of Mineralogy and Petrology, Croatian Natural History Museum, Demetrova 1 10000 Zagreb, Croatia, 4Department of Chemistry of Faculty of Science and Mathematics, University of Zagreb, Horvatovac 102A, 10000 Zagreb, Croatia
marin.soufek@hpm.hr

The Allchar deposit, in the Republic of Macedonia, is unique in the world because of the economic concentrations of thallium and the consequent assemblage of thallium minerals. A number of thallium minerals have been found for a first time in Allchar. It is the type locality for ten thallium minerals, many of them found nowhere else: lorandite TlAsS2 (Krenner, 1895), vrbaite S2Tl4Hg3Sb2As8S20 (Ježek, 1912b), raguinite TlFeS2 (Laurent et al., 1969), picotpaulite TlFe2S3 (Johan et al., 1970), parapierrotite Tl(Sb,As)5S8 (Johan et al., 1975), rebulite Tl5Sb5As8S22 (Balić-Žunić et al., 1982), simonite TlHgAs3S6 (Engel at al., 1982), bernardite TlAs5S8 (Pasava et al., 1989), dorallcharite Tl0.8K0.2Fe3(SO4)2(OH)6 (Balić-Žunić et al., 1994), jankovićite Tl5Sb9(As,Sb)4S22 (Libowitzky, pers.com.) and weissbergite TISbS2 (Rieck, 1993). The descriptions of paragenesis were based on only several outcrops and mineral descriptions were made on very small amount of material and are incomplete. They were mostly found in a locality called Crven Dol, at the northern end of the ore body. The Allchar paragenesis is now being better investigated and described, particularly because of great number of SEM and EDS analyses.

References:
BALIĆ-ŽUNIĆ, T. & ENGEL , P. (1982): The crystal structure of rebulite, Tl5Sb5As8S22. Zeitschrift für Krystallographie, 160, 109-125.
BALIĆ-ŽUNIĆ, T., MÖELO, Y., LONČAR, Ž.& MICHELSEN, H. (1994): Dorallcharite, Tl0.8K0.2Fe3(SO4)2(OH)6, a new member of the jarosite-alunite family, Eur. J. Mineral. 6, 255-263.
GOLDSCHMIDT, V. (1904): Lorandit, ein neues Thallium- Mineral von Allchar in Macedonia. Zeitschrift für Krystallographie und Mineralogie, 39, 13-121, Leipzig
JEŽEK, B. (1912): Vrbait, novy thallnaty minerál z Allcharu v Macedonii. Rozpravy České Akademie, v Praze, 21/II čislo 26, 1-11.
ENGEL, P. (1980): Die Krystallstruktur von syntetischem Parapierrotite, TlSb5S8. Zeitschrift für Krystallographie, 151, 203-216.
DICKSON, F.W., & RADKE, AS. (1978): Weissbergite, TISbS2, a new mineral from the Carlin gold deposit, Nevada. American Mineralogist, 63, 720-724.
JOHAN, Z., PIERROT, R., SCHUBNEL, H.-J., & PERMINGEAT, F. (1970): La Picotpaulite TlFe2S3, une nouvelle espèa minérale. Bulletin de la Societe francaise de Mineralogie et Krystalographie, 93, 545-549.
ŠOUFEK, M., BILLSTROEM, K., TIBLJAŠ, D., BERMANEC, V. (1998): Distribution of lead isotopes in wulfenite from Allchar, Macedonia. Neues Jahrbuch für Mineralogie: Monatshefte, 10, 462-468.
BOEV, B., BERMANEC, V., SERAFIMOVSKI, T., LEPITOVKA S., MIKULČIĆ, S., ŠOUFEK, M., JOVANOVSKI G., STAFILOV T., NAJDOSKI M. (2001-2002): Allchar mineral assemblage. Geologica Macedonica, 15-16, 1-23.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-3238 Three dimensional morphometric characterization of rock pore-throats in sedimentary oil and gas reservoirs by scanning electron microscopy

Leyte F.1, Pacheco S. U.2, Garibay V.3, Tovar J. E.4, Palacios E.5, Angulo B. E.1, Esquinca C.1, Martinez E.2, Prado P.3
1Mexican Institute of Petroleum, 2The National Autonomous University of Mexico, 3National Polytechnic Institute
fleyte@imp.mx

It has been established that both, pore-throat size distribution and morphological characterization along with diagenetic fabrics of rocks can be relevant indicators of reservoir flow capacity, storage capacity and potential for horizontal drilling(1,2); Carbonated crystalline rocks may lead to pores network systems ranging from abundant to poor pore systems. These may exhibit polyhedral, tetrahedral, sheet, piping-like or a combination of them. Past investigations have led to the development of techniques to characterize pores properties with techniques such as capillary pressure, pore cast and, lately, SEM dual-beam techniques to digitally reconstruct a 3d pore system object from serial sectioning using ion milling techniques. In this paper we present recent results, developed at IMP, of a novel technique to measure both, pore throats and porosity of carbonated rock specimens, based on systematic studies of pore casting and electron microscopy. Tridimensional determination of shapes, networking pattern and size distribution of pores systems in these rocks are also discussed.

Refererences
1. Chidsey, T. C (2002) “Heterogeneous Shallow-Shelf Carbonate Buildups in the Paradox Basin, Utah and Colorado: Targets for Increased Oil Production and Reserves Using Horizontal Drilling Techniques.” Semi-annual Technical Progress Report. April, 2002-October 5, 2002. Utah Geological Survey.
2. Wardlaw, N. C. (1979) “Geology of Carbonate Porosity”. Short Course: Pore Systems in Carbonate Rocks and Their Influence on Hydrocarbon Recovery Efficiency. April 1st. Huston, Texas.

The authors wish to thank the Mexican Petroleum Institute (IMP) for allowing us to use its facilities to perfrom this resarch. 

Fig. 1: Fig. 1 Examples of carbonated pore systems a) tetrahedral, b) polyhedral-sheet like and c) polyhedral.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-3259 Electron Microscopy of Smectite Clay and the Assembly

Chiou W.1, Kaufhold S.2, Dohrmann R.2, Ishikawa A.3, Kuwamura Y.4, Minoda H.4, Fukushima K.5
1University of Maryland, MD, USA, 2Federal Institute for Geosciences and Natural Resources, Hannover, Germany, 3Nihon University, Tokyo, Japan, 4Tokyo University of Agriculture and Technology, Tokyo, Japan, 5JEOL Ltd., Tokyo, Japan
wachiou@umd.edu

Smectite, the major clay minerals in bentonite, is one of the most interesting and ubiquitous clay minerals in the mineral kingdom. It holds a special place in scientific research because of its great impact on human daily life, e.g., process industries, civil engineering and construction, geology and petroleum, agriculture and food, medicine and pharmaceutical industries, materials science and engineering. The precise characterization of smectitic clays, especially particle size and shape, is crucial in clay research, but is challenging due to particle aggregation (i.e., large surface area to volume ratios and high chemical activity on the clay surface). This paper introduces novel methods of studying smectitic clay particles and their assembly using electron microscopy.

Smectite clay usually occurs only in very small particles. Bentonite clays collected from different localities were purified and dispersed in de-ionized water without any dispersion agent (to avoid possible artifacts). Particle size of < 0.2 μm fraction was collected by the settling (pipet) method. These clay suspensions were very carefully transferred into a special wet environmental cell and then inserted into the TEM column for in situ (WETEM) study [1, 2]. The traditional air-dried, cryo-TEM and SEM methods were also carried out for comparison purpose. Conventional embedding and ultra-microtoming techniques and FIB method were used for clay assembly research.

Conventional electron micrographs of smectite showed broad undulating mosaic sheets, irregular masses of extremely small particles, and irregular flake-shaped aggregates (Fig. 1), though elongated lath-shaped units with flake-, needle-, and rod-like particles were also found. Usually, individual particles can barely be discerned due to particle aggregation in vacuum environment, but the use of WETEM has led to the discovery of different shapes (granular or spherical-like, elongated fiber-like, needle, triangular, polygon and others) of nano-size smectitic clays in addition to those previously reported (Fig. 2). Particle size analysis of ~20 smectite samples showed that almost all smectite particles ranged from 5 to 700 nm in equivalent diameter with a mode between 30 to 125 nm and a mean between 90 to 300 nm regardless of the clay fraction obtained by settling method. Clay aggregates observed in both conventional TEM, WETEM, and SEM revealed many interesting features. With advanced hard and soft wares, surface 3-D images and 3-D tomography can be constructed by images obtained from SEM, TEM and FIB (Fig. 3). The application of 3-D analysis of clays and its assembly promises to increase our understanding and improve the application of clays in many aspects.

[1] A. Fukami et. al., EMSA Proceedings, 45 (1987) 142.

[2] Kuwamura et. al., M&M Proceedings, Suppl. 2. (2013) 1498.

[3] EM work was made the use of NISP Lab (MRSEC, NSF) at UMD. WETEM was done at Nihon U and TUAT.

Fig. 1: Typical conventional TEM micrographs (1a and 1b) show different degrees of aggregation of smectite clay particles. SEM image (1c) depicts undulated flaky mosaic aggregates. Particle size/shape analysis of clay particles in those aggregates is impossible.

Fig. 2: WETEM images reveal dispersed smectite clay particles. A variation of very fine particle size in nm range and shapes such as round, platy, disc- and granular-like (2a), elongated lath-shaped needle- and rod-like particles (2b) are evident.

Fig. 3: SEM micrograph shows clay aggregates (3a). A reconstructed 3D image (3b) using two slightly tilted SEM images of clay aggregate shown in 3a. A 3D TEM tomography image (3c) reconstructed from a series of 140 2D TEM images. Empty areas are pore spaces in the sediment.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-3326 Determination of the 3D texture of hematite aggregates from different structural domains of Quadrilátero Ferrífero (Iron Quadrangle)

Ferreira F. O.1, Lagoeiro L. E.1, Barbosa P. F.2, Rodrigues C. A.1
1Universidade Federal de Ouro Preto, Departamento de Geologia, Ouro Preto, Brazil, 2Centro de Microscopia da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
filippeof@hotmail.com

The polideformed Banded Iron Formations of Quadrilátero Ferrífero (QF) have been extensively studied over the past years, mainly because its ability to preserve the deformation gradient present throughout the region. The EBSD (Electron Backscatter Diffraction) techniques have become a great tool over the past few decades for geologists to study the complex microstructural (and its relation with the regional structures) process that operates at the Quadrilátero Ferrífero.

In this work, we examine samples of hematite aggregates from different metamorphic and deformation domains of Quadrilátero Ferrífero in order to establish a proper correlation between the texture of the aggregates and the deformational process that prevails in different domains.

The QF Banded Iron Formations vary locally from a magnetite rich granular morphology with low preferred orientation at the west, the less deformed region of the QF, to a predominantly homogeneous hematite aggregate with stronger grain shape and crystallographic preferred orientations (CPO), towards the eastern portion. Between these two extremes, samples tend to present both types of microstructures and CPO found in the east and the west. The grain shape preferred orientations as well as the CPO were quantified using EBSD analysis, along with numerical modeling techniques.

First results indicate an increasing in the grain aspect ratio, accompanied by an increase in the grain size as deformation and temperature increase.
ODFs (Orientation Distribution Function) were calculated, employing the M-TEX toolbox for MATLAB™.
Entropy, J and M-index were, then, examined presenting an increase in CPO as we approximate to the eastern portion of QF.

The results confirm previous works indicating a strong relationship between the deformation and temperature conditions with the microstructure generated. The texture quantification of the banded iron formations provides basis work intended to contribute in unveiling the process that acted during deformation events at the Quadrilátero Ferrífero.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-3328 The role of deformation mechanisms related to phase transition in distinct structural domains of the Quadrilátero Ferrífero’s Banded Iron Formations

Ferreira F. O.1, Lagoeiro L. E.1, Barbosa P. F.2, Rodrigues C. A.1, Gonçalves F. L.1
1Universidade Federal de Ouro Preto, Departamento de Geologia, Ouro Preto, Brazil, 2Centro de Microscopia da Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
filippeof@hotmail.com

We investigate the role of phase transformation on the strength of iron formation rocks during the deformation. The rocks come from iron formations in the Iron Quadrangle region, Brazil. Their rocks are deformed in tectonic context of variable intensity. Samples were prepared for optical microscopy and electron backscatter diffraction (EBSD). Three sets of samples were chosen. They correspond to zones of low, intermediate and highly localized deformation. The first correspond to aggregates of granular magnetite grains with variable degree of oxidation to hematite. The aggregates have a random distribution of grain shape and crystallographic planes. The deformation was accomplished mainly by microfracturing leading to a grain size reduction by cracking and a progressive transformation to hematite. In zones more intensively deformed, magnetite grains occurs as isolated clasts surrounded by a matrix of tabular hematite crystals. They are preferred oriented with their longest axis parallel to the foliation and the basal planes of hematite are orientated with their c-axes parallel to the foliation normal. In the highest deformed zones, only tabular hematite grains are present. They have a strong shape and crystallographic preferred orientations. The deformation in this highly deformed domain is accommodated by dislocation creep followed by recrystallization with some grain growth. A grain boundary sliding cannot be ruled out since the crystallographic texture consists of a single maximum around the Z-direction and a spreading of the <a> axis in the foliation plane. Another important aspect of the deformation is the stabilization of grain boundaries, characterized by a crystallography between pairs of neighboring grains in a twinning relationship. All these changes occurring progressively in these shear zones led to a complete modification of the rock fabric, from an initial aggregate of hard load-supporting magnetite phase, in the low deformed shear zones, to weaker and interconnected hematite grains in highly deformed rock.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-3471 A multi-technique approach by SEM/EDS, Optical Microscopy, XRD, FT-IR to characterize limestone from Constantine, North-East of Algeria.

Benguedouar M.1, Bouchear M.1, Benabbas C.2,3
1Materials Sciences and Applications Research Unit, Department of Physics, Faculty of Exact Sciences, Constantine 1 University, Algeria, 2Geology and Environment Laboratory, Department of Earth Sciences, Faculty of Earth Sciences, of geology and Planning, FSTGAT, Constantine 1 University, Algeria, 3Management Institute and Urban Technology, Constantine 3 University, Algeria
mouniabenyamina@gmail.com

The geological context of North East Constantine is very complex. This region is characterized by a superposition of several thrust sheets and a wide variety of sedimentary rocks, such as limestones, sandstones, clays and marls. The establishment and evolution of these rocks took place in a complex geological context marked by paleogeographics, paleoclimatics and paleotectonics changements. These changes have influenced the quality and performance of these materials.
The use of these rocks in development projects, in particular limestone for roads and highways construction, requires a better knowledge and a thorough study of the morphological and chemical characterization of these rocks in the rough.
Calcite is the most important and abundant calcium carbonate containing mineral and it is the major constituent of limestone, as well as others; dolomite, quartz, kaolinite. For that it is necessary to study this element.
In this work, we study two types of limestone from different structures of Kellal Mountain (Constantine, Algeria): white (WKML) and gray (GKML). The characterization of these rocks is made by using an Environmental Scanning Electron (ESEM/EDS) and Optical Microscopy to study the morphological aspect of the existing phases. X- Ray Diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FT-IR) analysis of limestone are required to investigate the structural properties of them. The SEM morphology coupled with EDS results are in good agreement with the previous techniques.
The electron microscopy observations show that the extracted samples (Fig. 1) are made up of calcite as predominant phase with different types.
WKML samples (Fig. 2) is formed by organism's debris (foraminera and equinoderms), have worn bivalve shell (marine fossils), the bivalves fragment are to be in micritic matrix (carbonated mud).
The XRD and FT-IR spectra reveal the present of different phases that composed the rocks. The combination of these advanced techniques, which were not designed with the purpose of answering geological or environmental questions, can generate complementary of geological materials and opening up new approaches in the study of porous geomaterials.

All authors thank, the Process Engineering Laboratory, University of Bejaia and Birine Nuclear Center, Ain Ouassera, Algeria for their collaboration.

Fig. 1: Environmental Scanning Electron Microscopy (ESEM) images and EDS spectrum of WKML; details showing: micro-calcite, equant-calcite grain and dolomite.

Fig. 2: Thin-section micrograph of WKML (Equinoderms and fragment's micritic calcite) under natural light.

Fig. 3: The X-ray diffraction spectrum of WKML samples with high rate of dolomite.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-5721 Refinement of lattice parameter of hematite by Nano-beam electron diffraction

Freitas E. F.2
1Center of Microscopy of UFMG, 2Dep. of Metallurgical and Mining Engineering
freitas.erico@gmail.com

The convergent-beam electron diffraction (CBED) has been used in the lattice parameter determination with good accuracy by using HOLZ reflection lines [1]–[4], and recently a new method to refine the lattice parameters by using the HOLZ reflections observed in the nano-beam electron diffraction pattern (NBDp) has been developed [5]. The HOLZ reflections observed in the NBDp were not used so far because of the displacement in HOLZ spot positions due to the distortion of the projection lens system of the TEM, but such displacements are nevertheless proportional to the distortion coefficients (Crad, Cspi, Cell), and can be taking into account in order to correct the spot positions observed in the NBDp [5]. These microscope parameters are obtained by fitting the spot positions between the experimental and simulated NBD patterns in order to obtain the minimum value of the residual sum of the chi-square [5]. This new method was applied here using a Si standard sample in order to determine the distortion coefficients, and the other required microscope parameters, and then applied in the refinement of the lattice parameters of a sample of hematite containing Al, using the distortion coefficients determined previously. The simulate NBDp were done by using the JEMS software [6] and a standard hematite crystal structure (R-3c, a=0.5035nm and c=1.3732nm) was used in the simulation of the diffraction pattern. The NBDp of both samples (Figures 1 and 2) were performed in the Center of Microscopy of the Universidade Federal de Minas Gerais, using a Tecnai G2-20 (FEI) TEM, with LaB6, operating at 200kV, with camera length of 300mm. The camera length was calibrated by comparison between the experimental and simulated NBDp and the experimental spot positions were measured using ImageJ software by fitting a circle mask in each spot and taking into account its center of mass in pixel units. The distortion coefficients Crad, Cspi, and Cell were estimated in 1E-17, 1E-18, and 1E-2 respectively. The a lattice parameter of hematite sample was measured taking into account the corrected ZOLZ spot positions and it was found a=0.5041nm. The c lattice parameter was kept constant.

References:

[1] J. M. Zuo, Ultramicroscopy, vol. 41, pp. 211–223, 1992.
[2] S. J. Rozeveld and J. M. Howe, Ultramicroscopy, vol. 50, no. 1, pp. 41–56, May 1993.
[3] J. M. Zuo, M. Kim, and R. Holmestad, J. Electron Microsc. (Tokyo)., vol. 47, no. 2, pp. 121–127, Jan. 1998.
[4] P. Paczkowski, M. Gigla, a Kostka, and H. Morawiec, Mater. Chem. Phys., vol. 81, no. 2–3, pp. 233–236, Aug. 2003.
[5] K. Saitoh, H. Nakahara, and N. Tanaka, Microscopy, vol. 62, no. 5, pp. 533–9, Jan. 2013.
[6] P. A. Stadelmann, Ultramicroscopy, vol. 21, no. 2, pp. 131–145, Jan. 1987

Acknowledgements to the Center of Microscopy of UFMG, INCT-Acqua, CAPES. and to FAPEMIG.

Fig. 1: (a) NBD pattern of a Si sample along the <111> zone axis, with some reflections overlaid with the simulated spot positions without distortion correction (a), and with distortion correction (c).

Fig. 2: (a) NBD pattern of a hematite, sample containing Al, along the [001] zone axis, with some reflections overlaid with the simulated spot positions without distortion correction (b), and with distortion correction (c).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-5929 Nanoscale characterisation of meteoritic and geological samples using combined TKD and EDS in the SEM

Trimby P. W.1, Daly L.2, Bland P.2, Jacob D. E.3, Moody S.1, Piazolo S.3, Ringer S. P.1,4
1The Australian Centre for Microscopy & Microanalysis, The University of Sydney, NSW 2006, Australia , 2Department of Applied Geology, Curtin University of Technology, Perth, WA 6845, Australia, 3ARC Centre of Excellence for Core to Crust Fluid Systems and Department of Earth and Planetary Sciences, Macquarie University, NSW 2109, Australia , 4School of Aerospace, Mechanical & Mechatronic Engineering, The University of Sydney, NSW 2006, Australia
patrick.trimby@sydney.edu.au

Electron backscatter diffraction (EBSD) together with energy dispersive X-ray spectroscopy (EDS) in the scanning electron microscope (SEM) has been used to characterise deformation and interphase relationships in the geological sciences for almost 20 years. However, the analysis of sub-micron scale features in bulk rock samples using EBSD-EDS is very challenging due to limitations in the spatial resolution of the two techniques.
The recent emergence of transmission Kikuchi diffraction (TKD) in the SEM enables characterisation of nanostructured materials and materials with high intragranular dislocation densities [1, 2]. To date, most published TKD analyses have been on metallic samples and have not included simultaneous EDS measurements. Here we demonstrate for the first time the application of combined TKD and EDS to characterise sub-micron scale features in two contrasting rock samples.
The first sample is a focused ion beam (FIB) lift-out section from the Allende meteorite. The section was taken from a region containing Pt group element (PGE)-enriched metal grains. The combined TKD-EDS analysis identified 6 phases including 3 Fe-Ni sulphides, olivine, chromite and the PGE-enriched Fe-Ni. The phase distribution, as indexed using TKD, is shown in fig. 1; note how the high spatial resolution of TKD allows identification of grains as small as 100 nm in diameter. Simultaneous EDS spectra were collected, allowing the generation of individual element maps for the whole section. The EDS spatial resolution is in the range of 25-50nm and is far superior to that of EDS on bulk samples. A combined element map of the full section is shown in fig. 2.
A second sample is a FIB section from a polycrystalline diamond aggregate taken from the Orapa Kimberlite in Botswana. This particular section contains 2 inclusions of low-Ni pyrrhotite within a single diamond grain. Combined TKD-EDS analyses were carried out at varying magnifications in order to understand the temporal relationship between the diamond and the inclusions. Fig. 3 shows a combined element map of the whole section, showing an iron oxide rim around the pyrrhotite, as well as small Cu-rich domains within the largest inclusion. A higher magnification TKD analysis of part of the main inclusion was carried out, and this identifies the Fe-oxide as magnetite, and confirms the presence of chalcopyrite within the pyrrhotite.
We will discuss the emergence of the integrated TKD-EDS approach and the implication this has on the characterisation and interpretation of sub-micron scale structures in these and other complex geological and meteoritic samples.

References:
[1] R. Keller and R. Geiss, J. Microsc., 245 (2012), 245-251.
[2] P. Trimby et. al., Acta Mat., 62 (2014), 69-80.

Fig. 1: TKD phase map of a FIB section from the Allende meteorite.

Fig. 2: Combined element EDS map from the same area shown in fig. 1

Fig. 3: TKD-EDS results from a sulphide inclusion in diamond from a kimberlite sample FIB section. The left hand image shows a combined element map of the whole FIB section, with the white box marking an area highlighted in the higher resolution TKD phase map on the right.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-13-P-6005 Understanding mineral weathering on Mars using DualEELS / EDX mapping in STEM

MacLaren I.1, Lee M. R.2, Kovacs A.3, Tomkinson T.1,4, Smith C. L.5
1SUPA School of Physics and Astronomy, University of Glasgow, Glasgow G12 8QQ, UK, 2School of Geographical & Earth Sciences, University of Glasgow, G12 8QQ, UK, 3Ernst-Ruska Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Forschungszentrum Jülich, Leo-Brandt-Straße, D-52425 Jülich, Germany, 4Scottish Universities Environmental Research Center, East Kilbride, UK , 5Natural History Museum, London, SW7 5BD, UK
ian.maclaren@glasgow.ac.uk

Nakhla is a meteorite of Martian origin that is principally composed of olivine (nominally (Fe,Mg)2SiO4), but is criss-crossed by “iddingsite” veins which contain significant amounts of water resulting from the cracking and weathering of the mineral in-situ. The correct interpretation of the weathering sequence provides a valuable snapshot of part of the climate history of Mars. It is already known that on the outer edges of the veins, the olivine has been altered to a carbonate (siderite – FeCO3). It is further known that the inner parts of the veins contain a nanocrystalline or amorphous material with a composition consistent with a smectite clay. A wide range of detailed interpretations of this “clay” have been published.

We have used a JEOL ARM200F cold-FEG Scanning TEM (STEM) operated at 80 or 200 kV equipped with a Gatan GIF Quantum ER and a Bruker XFlash 60 mm2 EDX spectrometer to map the chemistry of a vein in the Nakhla meteorite at resolutions down to 1 nm.

Figure 1 shows a summary of the spectroscopic mapping of this meteorite. At the top left, a high angle annular dark field (HAADF) image is shown of one edge of the vein, with a green box marking the area used for combined EDX and Dual range EELS (DualEELS) mapping. The top right shows a phase map created by using a colour overlay of 4 multiple linear least squares (MLLS) fits to the EELS spectrum in the 5-70 eV range. This shows unaltered olivine as purple, siderite as yellow, a siderite / goethite mix as red, and the “clay” as cyan. Detailed studies using EDX, EELS and electron diffraction investigations (not shown here) confirm the red phase as a siderite / goethite mix. Studies of the oxidation state of iron from the EELS show that the olivine and siderite are both dominated by Fe2+ but the siderite / goethite mix is tending towards Fe3+, as would be expected. This demonstrates that early weathering occurred in a CO2-rich environment and did not oxidise the iron, whereas later weather occurred in a more acidic environment that liberated the CO2 and oxidised the iron.

The “clay” was found to have an inhomogeneous nanostructure, which is shown by the high resolution EELS mapping at the bottom of Figure 1; this does not display a uniform clay composition but is composed of SiO2 globules surrounded by a Fe-, Mg- and O-rich matrix. The Fe in these vein centres is also found to be in a Fe3+ oxidation state. Electron diffraction (not shown here) shows some crystallinity consistent with 2-ring ferrihydrite. The nanostructure of these veins is completely unexpected and requires a totally new explanation for its formation as the final stage in the weathering.

We are indebted to SUPA and the University of Glasgow for the funding of the MagTEM facility used in this work. 

Fig. 1: Analytical STEM studies of olivine weathering on Mars. Top left, survey area for EELS/EDX. Top right: MLLS fitting map olivine - purple, siderite - yellow, siderite/goethite - red, “clay” - cyan. Centre, EDX spectra (same colours). Lower left, chemical maps in the “clay” region. Lower right, overlay map of Fe (red), Mg (green) and Si (light blue).
↑ Go to the Table of content ↑

MS-14. Energy-related materials

↑ Go to the Table of content ↑
Type of presentation: Invited

MS-14-IN-1613 Observation of electrochemical reaction in an all-solid-state Li-ion battery by spatially-resolved TEM-EELS

Yamamoto K.1, Yoshida R.1, Sato T.1, Matsumoto H.2, Kurobe H.1, Hamanaka T.1, Kato T.3, Iriyama Y.3, Hirayama T.1
1Japan Fine Ceramics Center, Nagoya, Japan, 2Hitachi High-Technologies Corporation, Hitachinaka, Japan, 3Nagoya University, Nagoya, Japan
k-yamamoto@jfcc.or.jp

All-solid-state lithium-ion batteries (LIBs) with solid electrolytes have high potential to overcome some present problems of LIB with liquid electrolytes: safety, reliability, lifetime, cost, and energy density. However, the large interfacial resistance of Li-ion transfer at the electrode/solid-electrolyte interfaces prevents their practical use. One effective solution is an in situ formation of electrode materials from the solid electrolytes. Because the electrodes grow from the solid electrolytes with Li insertion reaction, both materials become connected to each other at an atomic scale. Such electrodes were discovered in Li2O-Al2O3-TiO2-P2O5-based solid electrolytes (LATP) [1]. However, the structural growth mechanism and the electronic structure changes due to the Li insertion are still unclear. Here, we used spatially resolved EELS in TEM mode (SR-TEM-EELS) to directly visualize the nano-scale Li profiles and the influence on other elements (Ti and O).

Figure 1(a) illustrates the LIB sample. A Si- and Ge-doped LATP sheet (LASGTP, 90-µm thick) was used as the solid electrolyte. The 800-nm thick film of the LiCoO2 positive electrode was deposited on one side of the sheet by PLD. The Pt current-collector was directly deposited on the other side. Cyclic voltammetry (CV) was carried out for 50 cycles in a vacuum with a sweep rate of 40 mV min-1 (Fig. 1(b)), and the negative electrode was formed in situ near the LASGTP/Pt interface by decomposition with the Li insertion. After the CV, the TEM sample of the negative side region was prepared by FIB.

Figure 2(a) shows the TEM image around the negative side. A slightly uniform contrast layer (about 400 nm) was observed near the Pt. Electron diffraction showed this region was amorphous structure. The SR-TEM-EELS images around the Li-K-edge, Ti-L-edge, and O-K-edge were recorded by the CCD camera (Figs. 2(b) - 2(d)). The Li signals obviously increase in the 400-nm-width region. This is evidence that the negative electrode was formed in this region. In the spectrum image of Ti-L-edge (Fig. 2(c)), we can observe clear chemical shifts of the L2 and L3 edge lines, which shows that the Ti electronic state changed from Ti4+ to Ti3+ due to the Li insertion. The spectrum image of O-K-edge in Fig. 2(d) also shows the spectrum shifts. This indicates that the O also contributed to the electron charge compensation due to the Li insertion.

In conclusions, we succeeded in simultaneous observation of the crystal and electronic changes of the in-situ-formed negative electrode. This technique can potentially be applied not only to LIBs but also to fuel cell batteries, electric double-layer capacitors, and other electrochemical devices.

[1] Y. Iriyama et al., Electrochem. Comm. 8 (2006) 1287-1291.

This work was partially supported by the RISING project of the New Energy and Industrial Technology Development Organization (NEDO) in Japan.

Fig. 1: All-solid-state LIB sample and its cyclic voltammogram. (a) Illustration of the prepared LIB sample. (b) Cyclic voltammogram measured in a vacuum with a sweep rate of 40 mV min-1.

Fig. 2: FIG. 2 TEM image and the SR-TEM-EELS images. (a) TEM image around the negative side. SR-TEM-EELS images, (b) Li-K-edge, (c) Ti-L-edge, and (d) O-K-edge in the region of (a).
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-14-IN-1715 Chemical analysis of energy conversion and storage materials with atomic resolution using aberration-corrected STEM imaging and spectroscopy

Klie R. F.1, Gulec A.1, Paulauskas T.1, Phillips P. J.1, Wang C.1, Nicholls A. W.1
1University of Illinois at Chicago, Chicago, IL, USA
rfklie@uic.edu

While recent advances in energy conversion and storage have revolutionized the consumer electronics market, we still lack a fundamental understanding of the effects that currently limit the devices’ efficiencies and lifetimes. Aberration-corrected scanning transmission electron microscopy (STEM) is becoming one of the most promising characterization tools to study the effects of repeated charging/discharging cycles on electrode aging in Li-ion battery materials or defects in solar-cell devices, due to the wide-range of techniques available on advanced STEM instruments, including the direct imaging of both heavy and light elements, energy-dispersive X-ray and electron energy loss (EEL) spectroscopies and a variety of in-situ methods.

In this presentation, we will present the latest results from the new probe aberration-corrected cold-field emission JEOL JEM-ARM200CF at the University of Illinois at Chicago (UIC), which allows in-situ characterization with 78 pm spatial resolution and an energy resolution of 350 meV in the temperature range between 10 K and 1,300 K using a variety of in-situ heating, cooling, tomography and electrical feedback holders. In particular, we will focus on Li2MnO3-based cathode materials for Li-ion battery applications and CdTe grain boundaries for poly-crystalline solar-cell devices.

Figure 1a is an HAADF image of a [100]-oriented Li2MnO3 particle (corresponding ABF and LAADF images in subfigures (b) and (c)). The Li atoms are directly imaged in the averaged ABF image as confirmed by our multislice simulated ABF (upper) and LAADF (lower) STEM images. EELS results are presented in Figure 2, with the red spectrum highlighting the characteristics of a cycled material, which include a greatly reduced O pre-peak and dramatic decrease in the Mn valence, identified by the Mn L-edge shift to lower energy and increased L3/2 ratio. This part will focus on pertinent structural and electronic differences between pristine and cycled material, with features like atomic ordering of Mn/Li atoms, O vacancy evolution, and Mn valence changes being of particular interest.

Figure 3 shows an atomic resolution STEM-XEDS map of CdCl2 annealed-CdTe [110] using the newly installed Oxford Instruments X-Max 100TLE, a 100 mm2 silicon drift detector on our JEOL ARM200CF. As can be seen in the spectrum image, both the Cd and Te atomic columns are resolved in addition to the Cl signal at the center of a dislocation core. Spectrum images like this will be used to determine the effects of Cl segregation in CdTe solar-cell devices and characterize the structural changes as the result of the CdCl2 post-growth annealing step.

This work is supported by the US Department of Energy (DE-EE-0005956), the National Science Foundation (DMR-0959470, DMR-0846748) and the Joint Center for Energy Storage Research (JCESR) at Argonne National Laboratory.

Fig. 1: (a) HAADF image (scale bar 20 nm) of a Li2MnO3 particle, with high-resolution ABF (b) andLAADF (c) images acquired along the [100] direction (scale bar 0.5 nm). The atomic model of Li2MnO3 is shown towgether with ABF (top) and LAADF (bottom) experimental and multislice simulated STEM images.

Fig. 2: EELS results of the O K- and Mn L L-edges for a cycled material (red) versus a reference (blue).Clear differences include the diminished O pre-peak and the shifted Mn L peak; see text for details.

Fig. 3: Atomic-resolution XEDS map of complex dislocation core in CdTe [110].
↑ Go to the Table of content ↑
Type of presentation: Invited

MS-14-IN-2388 Structure-property relationships in thin-film solar cells on multiple scales by correlative electron microscopy

Abou-Ras D.1, Dietrich J.1,2, Kavalakkatt J.1, Nichterwitz M.1, Schäfer N.1, Schmidt S. S.1, Schaffer B.3, Schaffer M.3, Koch C. T.4, Müller M. M.5, Bertram F.5, Christen J.5, Wilkinson A. J.6, Bauer F.7
1Helmholtz-Zentrum Berlin für Materialien und Energie, Berlin, Germany, 2Technische Universität Berlin, Germany, 3SuperSTEM, Daresbury, UK, 4University of Ulm, Germany, 5University of Magdeburg, Germany, 6University of Oxford, UK, 7Oxford Instruments GmbH, NanoAnalysis , Wiesbaden, Germany
daniel.abou-ras@helmholtz-berlin.de

Thin-film solar cells consist of a stack of polycrystalline layers with thicknesses of few 10 nm to several micrometers, which can be deposited on rigid glass as well as on flexible metal or polymer substrates. A typical p-n setup comprises the use of a p-type absorber layer, an n-type semiconductor layer to form the p-n junction, as well as two electrical contacts, of which one is formed by a transparent conductive oxide. Nowadays, highest power-conversion efficiencies of almost 21 % have been achieved using polycrystalline Cu(In,Ga)Se2 thin films as absorber material [1,2]. The question remains of how such excellent photovoltaic performance is possible, in view of the large densities of extended structural defects present in the absorber layer.

In the recent years, we have addressed this question in various studies, combining different electron microscopy techniques on identical regions of interest on the specimen [3-10]. The present contribution intends to give an overview of this work, showing results from electron backscatter diffraction, electron-beam-induced current, and cathodoluminescence measurements in scanning electron microscopy on scales of up to few hundreds of micrometers, as well as by high-resolution imaging, electron energy-loss spectrometry, and inline electron holography in transmission electron microscopy in the subnanometer range. Challenges in specimen preparation and analysis will be covered, and an outlook on future activities will be presented.

[1] P. Jackson, et al., to be published in phys. stat. sol. (RRL), doi: 10.1002/pssr.201409040.

[2] A. Chirila, et al., to be published in Nature Mater., doi: 10.1038/nmat3789.

[3] D. Abou-Ras, et al., JOM 65 (2013) 1222-1228.

[4] D. Abou-Ras, et al., Phys. Rev. Lett. 108 (2012) 075502-1-5.

[5] D. Abou-Ras, et al., Adv. En. Mater. 2 (2012) 992-998.

[6] S.S. Schmidt, D. Abou-Ras, et al., Phys. Rev. Lett. 109 (2012) 095506-1-5.

[7] J. Kavalakkatt, D. Abou-Ras, et al., J. Appl. Phys. 115 (2014) 014504-1-10.

[8] M. Müller, D. Abou-Ras, et al., J. Appl. Phys. 115 (2014) 023514-1-6.

[9] J. Dietrich, D. Abou-Ras, et al., to be published in J. Appl. Phys. (2014).

[10] D. Abou-Ras, et al., J. Appl. Phys. 107 (2010) 014311-1-8.

The work was supported in part by the BMBF project GRACIS and by the Helmholtz Virtual Institute HVI-520 "Microstructure Control for Thin-Film Solar Cells".

Fig. 1: Scanning electron micrograph of a glass/Mo/CuInS2/CdS/ZnO solar-cell stack acquired in cross-section.

Fig. 2: a) EBSD pattern-quality map of a glass/Mo/CuInS2/CdS/ZnO solar-cell stack with Σ3 grain boundaries highlighted by red lines. b) Monochromatic CL image at 820 nm from the same identical position as in a). The contrast in the CL image was increased substantially in order to make the details close to the Mo back contact visible. Adapted from Ref. [10]
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-1419 Atomic-scale Structure Evolution in Nearly-Equilibrated Electrochemical Process of Rechargeable Battery Electrode Materials using Annular-Bright-Field Scanning Transmission Electron Microscopy

Gu L.1, Ikuhara Y.2
1Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China, 2Institute of Engineering Innovation, The University of Tokyo, Tokyo 113-8654, Japan
l.gu@iphy.ac.cn

Lithium ion batteries have been proved to be one of the ideal candidates in terms of energy density and power density for applications in portable electronics, electric vehicles and smart grid etc. However, limited understanding for the structural evolution of electrode materials at atomic scale amid electrochemical process substantially hinders their further performance optimization. The recent success of annular-bright-field imaging method erected on aberration-corrected transmission electron microscopy has been demonstrated to be a powerful technique to directly visualize individual light atoms, in particular the lithium ions, probing the nearly-equilibrated local structure of lithiated/delithiated electrodes under electrochemical cycling at atomic resolution. This talk presents our recent progress [1-10] to unraveling the atomic-scale structure evolution of electrode materials, extending the current understanding of electrochemical reaction and electrode degradation mechanism.

With the ABF method, we have studied possible electrochemical reaction mechanisms and detailed structure evolution for (partially) lithiated / delithiated 1D-LiFePO4, 2D- LiCoO2, Li2MnO3 and 3D- Li4Ti5O12 and other lithium-based active materials [1-10]. An intriguing staging phenomenon, in which the lithium ions preferably occupy every secondary layer along the c axis in partially delithiated LiFePO4, casts critical insights into two-phase separation mechanism in LiFePO4. Atomically-sharp coherent two phase interface between Li4Ti5O12 and Li7Ti5O12 in partially lithiated Li4Ti5O12 provided solid picture for the two-phase reaction mechanism of zero-strained spinels; whereas a new three-phase sodium-storage mechanism was uncovered despite of the 13% lattice volume change. An unexpected reversible migration of Mn ions Li2MnO3 was observed after shallow delithiation and relithiation, while irreversible cation rearrangement at the surface of electrochemically cycled LiCoO2, leading to the formation of rock-salt phase, was revealed. In addition, we demonstrate the ABF method can be further extended for atomic-scale investigation of oxygen vacancies and other structure evolution of light atoms.

References:

[1] Y. Sun, et al., Nature Commun. 4 (2013) 1870.

[2] R. Wang, et al. Adv. Energy Mater. 3 (2013) 1358.

[3] X. Lu, et al., Adv. Mater. 24 (2012) 3233. (Editor’s Choice in Science 336 (2012) 1621.)

[4] X. Lu, et al., Nano Lett. 12 (2012) 6192.

[5] S. Xin, et al., J. Am Chem. Soc. 134 (2012) 18510.

[6] Y. Q. Wang, et al., J. Am Chem. Soc. 134 (2012) 7874.

[7] X. Lu, et al., Energy Environ. Sci. 4 (2011) 2638.

[8] L. Gu, et al., J. Am Chem. Soc. 134 (2011) 4661.

[9] X. Q. Yu, et al., Adv. Energy Mater., in press.

[10] C. B. Zhu, et al., Adv. Func. Mater., in press.

Fig. 1: Schematic illustration of annular-bright-field imaging geometry. An electron probe with convergent semiangle α is focused to sub-angstrom dimension and scans across the specimen. An ABF detector at the post column subtends a detecting angle ranging from β1 to β2.

Fig. 2: ABF-STEM images of 70nm LiFePO4 viewed at the [010] zone axis. The staging area is marked by the dashed yellow lines.

Fig. 3: Interfacial structure in electrochemically lithiated Li4Ti5O12 sample with about 0.15 mol Li insertion per formula unit along the [110] direction . ABF image near the interface between Li4Ti5O12 phase (region 1) and Li7Ti5O12 phase (region 2). The yellow line indicates the boundary of the interface.

Fig. 4: STEM imaging of a three-phase coexistence region. ABF image in the half electrochemically sodiated Li4Ti5O12 nano-particle showing Li4Ti5O12, Li7Ti5O12 and Na6LiTi5O12 phase boundaries.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-1515 Path-dependence modelling of lithium iron phosphate cathode studied by STEM-EELS spectrum imaging

Honda Y.1, Muto S.2, Tatsumi K.2, Kondo H.3, Horibuchi K.3, Kobayashi T.3
1Graduate school of engineering, Nagoya University, Japan, 2Eco Topia Science Institute, Nagoya University, Japan, 3TOYOTA Central R&D labs., Japan
honda.yoshitake@a.mbox.nagoya-u.ac.jp

LiFePO4 is used as a practical active material for positive electrodes of lithium-ion secondary batteries. It shows several advantages such as low cost, excellent cycle life and safety compared to other candidate materials. Its lithium insertion/extraction proceeds via a two phase mechanism: LiFePO4 ⇔ FePO4 + Li+ + e-. Several models for the charge-discharge mechanism of this material have been proposed, though the Domino-cascade model [1] is now recognized as the most plausible to explain the observed experimental results. However, the path-dependence, the characteristic polarization behavior depending on the preceding charge/discharge history [2] cannot be explained by this model. In this study we re-examine the microstructure of electrochemically half charged Li0.5FePO4 electrodes using scanning transmission electron microscopy (STEM) and electron energy loss spectroscopy (EELS) to clarify the standing problem above.
The lithium content of Li1-xFePO4 electrode was controlled electrochemically using a two-electrode cell. Thin specimens for STEM were prepared by focused ion beam. STEM-EELS spectrum imaging (SI) applied using a Jeol JEM ARM200F equipped with a GIF Quantum EELS, operated at 200 kV. O-K and Fe-L2, 3 spectra were simultaneously measured to distinguish between LiFePO4 (LFP) and FePO4 (FP) phase. The spectral data were collected with a scan step of 3 nm for many particles, from the datacubes of which were well separated into the spectral profiles characteristic of the LFP and FP phases (Fig. 1) and their respective spatial distributions using a multivariate spectral decomposition technique [3].
Particles prepared by lithium extraction from the fully discharged state generally exhibited the structure of LFP shell/FP core, whereas particles by lithium insertion from the fully charged state the FP shell/LFP core structure, contrary to the well accepted Domino-cascade model, as shown in Fig. 2(a) and (b) respectively.
The size dependence of the core/total volume ratios were plotted in Fig. 3 for the many particles. We assumed that the lithium insertion/extraction reactions occurred on the surface of the particles and LFP/FP interface proceeded inward. If the rate of phase transition was proportional to the surface area of each particle, the volume ratios predicted by the model in plotted with the broken line in Figure 3. Assuming the shell layer to act as resistance for ion diffusion, the path-dependence can be roughly explained by the present model.

References:
[1] C. Delmas, et al., Nature Materials 7, 665 (2008).
[2] V. Srinivasan, Electrochem. Solid-State Lett., 9, A110 (2006).
[3] S. Muto, et al., Mater. Trans. 50, 964 (2009).

A part of this work was supported by a Grant-in-Aid on Innovative Areas "Nano Informatics" (Grant number 25106004) from the Japan Society of the Promotion of Science.

Fig. 1: Component spectra extracted with MCR, each corresponding to LFP and FP respectively

Fig. 2: ADF images and spatial phase distributions of (a) Li0.5FePO4 particles prepared by Li extraction and (b) Li0.5FePO4 particles prepared by Li insertion

Fig. 3: Experimental results (symbols) and theoretical prediction (broken line) of size dependence of relative core/total volume ratio, based on a model where lithium insertion/extraction reactions occurred on the surface of the particles with LFP/FP interface proceeding inward, which rate is proportional to the surface area of each particle
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-1764 Atom-by-atom edge chemistry of two-dimensional MoS2 nano-catalysts using STEM-EELS

Ramasse Q. M.1, Seabourne C. R.2, Hansen L. P.3, Zhu Y.3, Moses P. G.3, Brorson M.3, Johnson E.4, Kisielowski C.5, Scott A. J.2, Helveg S.3
1SuperSTEM Laboratory, Daresbury, UK, 2Institute for Materials Research, University of Leeds, UK, 3Haldor Topsøe A/S, Kgs Lyngby, Denmark, 4Nano-Science Center, University of Copenhagen, Denmark, 5National Center for Electron Microscopy, Berkeley, USA
qmramasse@superstem.org

Dwindling resources of fossil fuels and drastically tightened regulations on their purity have put an intense pressure on the oil refining industry to provide more efficient fuel purification, through improved catalysts in particular. The hydrodesulphurisation process makes wide use of planar, single-layer MoS2 nanoparticles as catalysts in removing sulphur from dirty fuels [1]. The chemical reactivity of the MoS2 slabs, whose layered anisotropic structure is related to that of graphene, is associated with their edges [2] and detailed information about the edge structures is thus of the utmost importance to understand and optimise the nature of the catalytic sites.

Scanning tunnelling microscopy and density functional theory (DFT) calculations have in the past provided unprecedented insight on model MoS2 nanoparticles prepared under ultra high-vacuum on planar substrates [3], while recent advances in high-resolution (scanning) transmission electron microscopy have made it possible to resolve directly the individual basal planes viewed in (001) of industrial-style MoS2 particles supported on thin graphite flakes [4]. Atom-by-atom analysis of chemically-sensitive annular-dark-field (ADF) images identified a specific reconstruction at the Mo-terminated edge of the particles [5], with the presence of an additional row of single sulphur atoms offset from the regular S sub-lattice, in good agreement with theoretical predictions: fig. 1.
This pioneering work relied nevertheless on the ability to count each and every atom of the particle and of its substrate. We show here that atomically-resolved electron energy loss spectroscopy (EELS) can instead be used in conjunction with ADF imaging to map the Mo and S sub-lattices directly in monolayer particles: fig. 2. When carefully tailoring the experimental parameters, and the electron dose in particular, the chemistry and detailed structure of the catalytically active particle edges are also unambiguously determined, confirming the presence of single sulphur atoms at the Mo-edge of the particles. Comparisons with ab initio DFT-based simulations of the EELS signal are also used to rationalise site-specific bonding arrangements and the electronic structure of these key edge sites [6].

[1] H. Topsøe et al., Hydrotreating Catalysis, vol. 11, Springer, Berlin (1996).
[2] T. Tenne, Nat. Nanotechnol. 1, 103 (2006).
[3] F. Besenbacher et al., Catal. Today 130, 86 (2008).
[4] C. Kisielowski et al., Angew. Chem. Int. Ed. 49, 2708 (2010).
[5] L. Hansen et al., Angew. Chem. Int. Ed. 50, 10153 (2011).
[6] Q.M. Ramasse et al., In preparation (2014).

This research was supported in part by the the UK Engineering and Physical Sciences Research Council, the Office of Science, Office of Basic Energy Sciences of the US Department of Energy and by the Danish Council for Independent Research (grant HYDECAT) and for Strategic Research (grant CAT-C).

Fig. 1: a) ADF image (deconvolved with a maximum entropy algorithm) of a monolayer MoS2 particle, obtained at 60kV. b) Close-up of the Mo edge revealing a single S termination (as determined from the ADF contrast analysis [5]), offset from the regular S sub-lattice. c) Ball-and-stick model of the edge termination.

Fig. 2: a) Raw ADF image of a monolayer MoS2 particle on graphite, obtained at 60kV. b) Simultaneous ADF and EELS map of the region outlined in red in a), showing the position of the Mo atoms (purple) and the S columns (yellow). c) Simulated S L2,3 EEL spectra for the outer three layers.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-1907 STEM/EELS analysis of SEI layer chemistry on cycled FeOF/C cathode

Sina M.1, Pereira N.1,2, Amatucci G.1,2, Cosandey F.1
1Department of Materials Science & Engineering, Rutgers University, Piscataway, NJ 08854, 2Energy Storage Research Group (ESRG), Rutgers University, North Brunswick, NJ 08902
cosandey@rci.rutgers.edu

For Li-ion batteries, transition metal fluoride/carbon nanocomposites have been under extensive investigation as cathode materials due to their high theoretical capacity between 500 to 800 mAh/g [1,2]. In this study, the structural changes of FeOF/C positive electrode during lithiation/delithiation have been studied as a function of number of cycles (up to 20) under constant cycling current of 50 mA/g and at 60°C. ADF-STEM imaging technique combined with electron energy loss spectroscopy (EELS) were used to track the chemistry of surface layer (SEI) forming on FeOF/C cathode and to determine the bonding characteristics of phases present after cycling. This analysis was done at 200 kV with the JEOL 2010F equipped with Gatan 200 GIF spectrometer having an energy resolution of 0.9 eV.

Upon lithiation-delithiation cycling, the STEM-EELS analysis revealed the formation of a SEI layer as shown in the ADF-STEM image depicted in Fig. 1a. The thickness of this SEI layer is in the range of 20 to 40 nm. The corresponding O-K, F-K and Li-K EELS spectra for the delithiated sample after 20 cycles taken from regions marked 1 and 2 are shown in Fig.1b and 1c respectively. The F-K, O-K, and Li-K vary considerably between these two different regions (1 and 2) suggesting different chemistry in these two different parts of the sample. The edge of the active electrode (region 1) contains only lithium, carbon, fluorine, and oxygen associated with a solid electrolyte interphase (SEI) layer at the cathode. In addition different chemical state is observed for the O-K edge with a pre-peak present in FeOF/C electrode which is missing in the SEI layer (region 1). Quantitative analysis of SEI layer chemistry gives a composition corresponding to Li0.45C0.2O0.05F0.3. Figure 2a and 2b show the Li-K and F-K edge spectra respectively taken from region 1 (edge) and comparison with possible electrolyte (LiPF6) decomposition compounds (LiF, Li2CO3) and with polymer binder (PDVF). The Li-K and F-K EELS spectra from region 1 can be identified as characteristic of LiF [3] from the presence of a post peak in the F-K edge and the separation the separation between the F-K and Li-K peaks of 24.6 eV and 7.2 eV respectively. An additional a small peak marked by an arrow in Fig.2b is also observed in the SEI F-K edge spectrum. This peak with energy of 708.3 eV has been attributed to Fe-L3 line. Furthermore, the energy value of this peak is indicative of ionic Fe with Fe+2 valence state. We have observed that this SEI layer thickness increases with cycle number.

[1] Amatucci, G. G. and N. Pereira (2007), Journal of Fluorine Chemistry 128: 243-262

[2] Sina, M., D. Su, et al. (2013), Journal of Materials Chemistry 1: 11629-11640

[3] Cosandey, F., D. Su, et al. (2012), Micron 43(1): 22-29.

Work supported by NECCES, a DOE-BES-EFRC funded center under Grant DE-SC0001294.

Fig. 1: (a) ADF-STEM image of the fully recharged FeOF/C cathode after 20 cycles, with the corresponding EELS spectra from region 1 and 2, (b) O-K, F-K and Fe-L3,2 edges, and (c) Fe-M and Li-K edges.

Fig. 2: EELS spectra taken from SEI layer (region 1 from Fig. 2a) with (a) Li-K edge, (b) F-K edge and comparison with possible standard phases (LiF, Li2CO3 and PVDF). There is an extra peak on the SEI F-K edge at 708.3 eV which has been attributed to the presence of Fe-L3 peak with Fe2+state.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-1967 Probing the local electronic configurations in tubular cobaltites : impact of the Co2+/Co3+ distribution on the transport and magnetic properties

Bocher L.1, Gloter A.1, Hébert S.2, Stéphan O.1, Pelloquin D.2
1Laboratoire de Physique des Solides, CNRS-UMR 8502, Université Paris-Sud, 91405 Orsay, France, 2Laboratoire CRISMAT, CNRS-UMR6508, ENSICAEN, 6 Boulevard du Maréchal Juin, 14050 Caen, France
laura.bocher@u-psud.fr

Among the numerous different oxide systems, the cobaltites present fascinating and complex physical properties directly correlated with their electronic structure. A strong interplay exists in these Co-based systems between (i) valence and spin states and (ii) low dimensionality and structural anisotropy, all of these yielding the emergence of unexpected magnetic and thermoelectric (TE) functionalities as illustrated in NaxCoO2 [1].

The misfit-layered cobalt oxides built from similar cobalt layers have triggered intensive works regarding their remarkable TE properties at high temperatures. A real driving force of this misfit family is the CoO2 2D hexagonal layers turning out to be at the origin of its metallicity combined with a large thermopower, and more largely of interesting magnetic and electronic phenomena [2].

Another cobalt oxide, Bi4Sr12Co8O28-δ, derived from the 2201-type cuprates reveal an original tubular structure. This complex structure with a 2D structure (24*24*5.4Å3) can be viewed as a stacking of n=2 [Bi2Sr2CoO6] slices separated by single Co-deficient pervoskite [Sr8Co6O16-δ] layers. Hence, squared pillars based on CoOx polyhedra create tubular cavities at the crossing of two [Sr8Co6O16-δ] layers. Besides the complexity of this anisotropic structure, the nature of the CoOx pillars can be fine-tuned by adjusting the oxygen stoichiometry, therefore playing a key role on the local electronic configurations of these systems. As in misfit cobaltites, the Co-tubular family reveals peculiar transport properties and magnetic transitions while their local electronic configurations remain unresolved to date. In these bidimensional cobaltites, one of the pivotal questions is the key role played by each type of Co sites.

Here we attempt to shed light on the [Bi2Sr2CoO6]2[Sr8Co6O16-δ] n=2 member by resolving its atomic structure (Fig. a)) using an aberration-corrected STEM – the NION UltraSTEM200 – coupled to EELS. Relying on high-energy resolution, we probe locally the Co-L2,3 and O-K fine structures, which are highly sensitive to the mixed valence-states and the hybridization geometries. In particular, distinct spectroscopic signatures related to inequivalent oxygen sites are probed in the real-space as illustrated in Fig. b). Hence two different oxygen fine structures (O(3) and O(4)) are identified on the Co sites illustrating the original local bonding environment in these Co-tubular oxides.

[1] I. Terasaki, Y. and K. Uchinokura, Phys. Rev. B 56, R12685 (1997)

[2] S. Hébert, W. Kobayashi, H. Muguerra, Y. Bréard, N. Raghavendra, F. Gascoin, E. Guilmeau, A. Maignan. Phys. Stat. Sol. A 210, 69-81 (2013)

[3] D. Pelloquin, A. C. Masset, A. Maignan, M. Hervieu, C. Michel, B. Raveau, J. Solid State Chem.148, 108-118 (1999)

The authors gratefully acknowledge financial support from the french CNRS (FR3507) and CEA METSA network (www.metsa.fr)

Fig. 1: a) STEM-HAADF image along the [100] direction and the corresponding structural model of the [Bi2Sr2CoO6]2[Sr8Co6O16-δ] phase (inset). b) O-K edge fine structures extracted at four distinct oxygen sites. Each spectrum is integrated over 4 x 4 pixel with a 10ms/pixel acquisition time.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-2078 The atomic structure of outstanding metal oxide catalysts for oxygen evolution reactions

Kisielowski C.1, Garfield D.1, Tilley D.1
1Joint Center for Artificial Photosynthesis, LBNL, 1 Cyclotron Road, CA94720 Berkeley, U.S.A.
CFKisielowski@lbl.gov

A new family of Ce-rich catalysts composed of earth abundant elements was recently discovered in JCAP [1]. These most active catalysts for oxygen evolution are composed of quaternary alloys such as Ni0.3Fe0.07Co0.20Ce0.43Ox. Successive annealing steps trigger phase segregation into CeO2 nanoparticles and a miscible MeOx alloy (Me = Fe, Co, Ni) with size distributions that can be tuned on a scale below 3 nm. It is now understood that atomic resolution imaging of such small particles is challenging because the energy deposited by the electron beam can largely exceed their size dependent total binding energy [2]. Therefore, little is known about the pristine structure of these catalysts.
Our research addresses their atomic structure by either directly investigating the quaternary alloy system, or by studying related single-phase nanoparticles. The specific case of 3-4 nm large Fe2O3 nanoparticles is considered in Figure 1. Electron in-line holography with variable dose-rates and voltages is employed since it allows stimulating and controlling system dynamics at atomic resolution with single atom sensitivity [3]. The method enables imaging with only a few atto Amperes/Å2, which approaches dose-rates that are otherwise used to investigate biological samples at a lower resolution.
We determined a critical dose-rate below 100 e/Å2s from experiments at 80 kV and 300 kV that vary dose-rates. Single images recorded below threshold values are dominated by noise (Figure 1a). Nonetheless, the materials structure is revealed and can be preserved if the phase of the electron exit wave function is reconstructed from focal series of low dose-rate images (Figure 1b-1d). Figure 1b) compares a [111] projection of the crystal structure for images recorded at 80 kV and 300 kV that reveals a somewhat increased image blur. This blur in the 80 kV recording is expected since lens aberrations must be suitably controlled at significantly larger scattering angles to obtain a comparable point-to-point resolution [2]. Fig. 1e) compares an atomic resolution image in [110] projection with two of the expectable crystal structures of FeOx . An agreement of the model with the experiment proves that the catalyst crystallizes as maghemite (Fe2O3) in its tetragonal space group (#96). In the image, the structure can be locally distinguished from its cubic form (#227) because of the different locations of oxygen columns. Thus, low dose-rate in-line holography allows for an identification of pristine crystallographic structures of 3-4 nm large catalysts even if only the location of columns occupied with a few oxygen atoms makes the essential difference.

[1] J.A. Haber et al., Energy Environ. Sci. 7 (2014) 682 - 688
[2] C. Kisielowski et al., Phys. Rev. B 88 (2013) 024305

This work is performed by JCAP supported by the DOE under Award Number DE-SC000499. Electron Microscopy was performed at the NCEM supported by the DOE under Contract No. DE-AC02—05CH11231.

Fig. 1: FIG. 1. Fe2O3 catalysts at 0.9 Å resolution, 80kV and 300 kV. a) A single micrograph acquired in low dose-rate conditions. b-d) Phase images reconstructed from 80 low dose-rate image. b) [111] protections of two particles at 80 and at 300 kV. e) A [110] projection is compared with the Fe2O3 structures in space groups #96 (match) and #227.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-2142 Microstructure analysis of vacuum plasma sprayed electrodes for alkaline electrolysis

Bowen J. R.1, Bentzen J. J.1, Jørgensen P. S.1, Zhang W.1
1Department of Energy Conversion and Storage, Technical University of Denmark, Risø Campus, Frederiksborgvej 399, 4000 Roskilde
jrbo@dtu.dk

The EU FCH-JU RESelyser project seeks to develop high pressure, high efficiency and low cost alkaline water electrolysers that can operate variably and intermittently to meet the demands for integration into energy networks relying on fluctuating renewable energy. The project utilizes NiAlMo alloy electrodes produced at the German Aerospace Center (DLR) by vacuum plasma spraying (VPS). VPS results in a heterogeneous microstructure consisting of a multitude of intermetallic phase subdomains and pores. We present the results of a broad palette of characterization techniques including SEM, TEM and 3D reconstruction by FIB serial sectioning to analyze this complex structure in the initial state and post mortem.
Electrode surfaces and cross sections are analyzed by high resolution SEM and EDS. The analyses of the cross sections reveal a multitude of complex material structures in the activated electrode (See Figure 1) stemming from the vacuum plasma spraying and electrode activation by leaching of Al and some Al containing intermetallic phases. Desert rose like nano flake structures are observed (See Figure 1) on the electrode surface and in the pores on several electrodes. The desert rose structure is confirmed by TEM to consist primarily of NiO and Al2NiO4 like phases (similar lattice parameters). We discuss implications and possible causes of the desert rose structure.
3D reconstructions of the electrodes are made by FIB serial sectioning. The pore space is analyzed in regards to the length, connectivity and tortuosity of the pore transport pathways that allow the KOH electrolyte to infiltrate the porous electrode. Figure 2 shows a reconstructed data cube from an electrode in the initial leached state and the corresponding extracted pore space. The pore space structure is revealed to consist primarily of coarse scale planar like pores parallel to the electrode surface. These thin but coarse pores range in thickness between 2 µm down to some 10s of nanometers at their extremities. In addition, there is a significant pore volume fraction of sub 100 nm wide pores associate with the dissolution of Al from the dentritic morphology of the Raney type NiAl alloy original particles. The combination of the planar pore pathways running parallel to the surface and the fine scale dendritic type pore space significantly complicates the analysis of the 3D pore surface due to the need to reconstruct large volumes at high resolution to both be able to resolve the pores and image the full pathway that connects the pores to the surface of the sample.
The long-term goal is the development of a generic electrode micro/nano structure analysis philosophy for relating electrochemical processes during hydrogen production to highly heterogeneous porous VPS electrodes.

The research leading to these results has received funding from the European Union’s Seventh Framework Programme (FP7/2007-2013) for the Fuel Cells and Hydrogen Joint Technology Initiative under grant agreement n° [278732] 10.

Fig. 1: A FIB cross section of a leached electrode. The surface of the electrode is to the left in the image. The inset shows an SEM surface image of the desert rose like nano flake structures observed on the electrode surface and in the pores on some electrodes.

Fig. 2: A visualization of the 3D reconstruction of an electrode by FIB serial sectioning. (left) A visualization of the reconstructed image data. The bright artefact on the left side is the electrode surface. (right) A surface rendering of the pores in the electrode.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-2233 Strain relaxation of high In-content InGaN epilayers grown by PAMBE

Bazioti C.1, Kehagias T.1, Walther T.2, Papadomanolaki E.3, Iliopoulos E.3, 4, Dimitrakopulos G. P.1
1Physics Department, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece, 2Department of Electronic & Electrical Engineering, University of Sheffield, Sheffield S1 3JD, UK, 3Microelectronics Research Group, Physics Department, University of Crete, P.O. Box 2208, 71003 Heraklion-Crete, Greece, 4IESL, FORTH, P.O. Box 1385, 71110 Heraklion-Crete
kbazio@physics.auth.gr

High indium content InGaN epilayers are particularly interesting for high-efficiency photovoltaic applications. However, defect reduction is crucial in order to increase the internal quantum efficiency of device structures. Such alloys generally exhibit complex microstructural behavior and strain relaxation that are very sensitive to the growth conditions, due to their intrinsic metastable character that leads to the phenomena of indium phase separation and composition pulling.
We have applied a combination of transmission electron microscopy (TEM) characterization techniques, including electron diffraction, HRTEM, geometrical phase analysis (GPA), z-contrast STEM, and EDX, together with high resolution x-ray diffraction (HRXRD), in order to elucidate the influence of strain relaxation on the defect content and indium compositional variations of high alloy concentration InGaN epilayers. Thin films of up to ~500 nm and 10-60% indium content were grown on (0001) GaN templates using plasma-assisted molecular beam epitaxy (PAMBE).
Strain relaxation was found to promote the introduction of a-type threading dislocations, as shown in Fig. 1. The emanation level of TDs was found to differ depending on the growth conditions. At higher growth temperatures or low indium fluxes, phase separation was observed leading to the formation of a strained InGaN interfacial interlayer of lower indium concentration as measured by EDX and GPA, which is shown in Fig. 2(a). Strain relaxation, manifested by the emanation of TDs, took place at and above this strained interlayer, as illustrated in Fig. 2(b). In addition to the discontinuous composition pulling, another characteristic feature of such phase separated epilayers was the appearance of multiple basal stacking faults (SFs) above the internal InGaN interface. Such SFs also acted as TD sources leading to increase of the defect content.
On the other hand, at lower growth temperatures or high indium incident fluxes, TD emanation commenced from the InGaN/GaN interface, showing that strain relaxation took place there (Fig. 3). This was further verified by the observation of regular arrays of misfit dislocations. The epilayer surface morphologies were correlated to the growth modes. Good quality epilayers with a ~40% indium content, showing no mesoscale phase separation, with smooth surfaces, were achieved.

This research has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: THALES.

Fig. 1: Cross sectional TEM (XTEM) images of a 300 nm thick InGaN epilayer with 43% indium content grown under low indium flux, showing emanation of a-type TDs from a strained InGaN interfacial interlayer. (a) Dark field (DF) image with g 1-100. (b) Bright field (BF) image with g 0002.

Fig. 2: (a) Annular DF image of a 200 nm thick InGaN epilayer containing 18% In, grown at high temperature under stoichiometric flux. A self-formed interfacial InGaN layer (s-InGaN) is indicated. (b) HRTEM image showing emanation of TD half loops from the s-InGaN interface (arrows). The GaN/s-InGaN and s-InGaN/GaN interfaces are also indicated.

Fig. 3: XTEM image showing a 450 nm thick InGaN epilayer with 42% indium content showing no phase separation due to the lower growth temperature. (a) BF image with g 1-100. (b) BF image with g 0002.

↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-2539 Polarity-Driven Polytypic Branching in Cu-Based Quaternary Chalcogenide Nanostructures

Zamani R. R.1,2,3, Ibáñez M.3, Luysberg M.4, García-Castelló N.5, Houben L.4, Prades J. D.5, Grillo V.6,7, Dunin-Borkowski R. E.4, Morante J. R.3,5, Cabot A.3,8, Arbiol J.2,8
1IV. Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany, 2Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Bellaterra, Spain, 3Catalonia Institute for Energy Research (IREC), Barcelona, Spain, 4Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute, Forschungszentrum Jülich GmbH, Jülich, Germany, 5Departament d'Electrònica, Universitat de Barcelona, Barcelona, Spain, 6Centro S3, CNR-Istituto di Nanoscienze, Modena, Italy, 7IMEM-CNR, Parma, Italy, 8Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain
reza.r.zamani@gmail.com

Copper-based chalcogenide semiconductor nanocrystals are used in a variety of energy-related applications as a result of their suitable optical, electronic and thermoelectric properties.1-4 In particular, ternary and quaternary copper-based chalcogenides offer a broad range of possibilities for morphological, chemical and structural control through chemical routes, thereby providing an opportunity for further functionality enhancement. For example, controlled combination of branching, polytypism, polarity and cation order can be used to nanoengineer the properties of the materials.

Here, a novel way of realizing property nanoengineering in Cu2CdxSnSey (CCTSe) polypods is presented. The pivotal role of polarity in determining the morphology, growth, and polytypic branching mechanism of the structure is demonstrated. Polarity is considered to be responsible for the formation of an initial seed, which takes the form of a tetrahedron with four cation-polar facets. Size and shape confinement of the intermediate pentatetrahedral seeds is also attributed to polarity, as their external facets are anion-polar. The final polypod extensions also branch out as a result of a cation-polarity-driven mechanism. Aberration-corrected HAADF-STEM is used to identify stannite cation ordering in this material; and linear STEM image simulations.5

Figure 1a shows a STEM micrograph of a CCTSe monopod. The first tetrahedral seed has a stannite structure with tetragonal symmetry (zinc-blende-like, ZB’). Secondary tetrahedra grow on the four facets of the initial one. The nanoparticles then branch out with wurtzite (WZ) structures and form the polypods (polytypic branching). Cation-polarity is maintained along the in the whole structure. However, the surface polarity switches in the secondary tetrahedra in order to retain the cation-polarity along the growth direction.
Aberration-corrected STEM was also used to establish that cation ordering exists in the ZB’ stannite region, while the cations (Cu, Cd, and Sn) are distributed randomly in the WZ branches, as shown in Fig. 2.

References
1 M. Ibáñez et al, Chem. Mater. 24, 562 (2012), 2 M. Ibáñez et al, Cryst. Growth & Des. 12, 1085 (2012), 3 M. Ibáñez et al, J. Am. Chem. Soc. 134, 4060 (2012), 4 M. Ibáñez et al, Chem. Mater. 24, 4615 (2012), 5 R.R. Zamani et al, ACS Nano 8, in press (2014), DOI: 10.1021/nn405747h

The authors acknowledge the European Union FP7 under ‘ESTEEM2’ and ‘nanowiring’, with grant agreement numbers 312483 and 265073, respectively.

Fig. 1: Polarity measurement in a CCTSe monopod: (a) HAADF-STEM image along [201]ZB'=[11-20]WZ zone axis; (b) magnified region indicated on image (a) by colored rectangles after deconvolution of the STEM probe shape; (c) linear simulations of the STEM images shown in (b); (d) intensity profiles measured from single dumbbell units (indicated on images (b)).

Fig. 2: (a) Atomic-resolution HAADF-STEM image acquired along the [111]ZB’ zone axis of tetragonal (ZB') CCTSe (b) deconvolved, and (c) filtered part of the image (a); (d) corresponding linear simulation. (e) 3D atomic model illustrating the ordering effect; (f) intensity profile taken from image (c).
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-2557 Revealing the Origin of “Phonon Glass–Electron Crystal” Behavior in Thermoelectric Layered Cobaltate by Accurate Displacement Measurement

Wu L.1, Meng Q.1, Jooss C.2, Zheng J.3, Inada H.4, Su D.1, Li Q.1, Zhu Y.1
1Brookhven National Laboratory, USA, 2University of Goettingen, Goettingen, Germany, 3Xiamen University, Xiamen, China, 4Hitachi High Technologies Corp., Ibaraki Japan
zhu@bnl.gov

     Measurement of local disorder and lattice vibrations is of great importance for understanding the mechanisms whereby thermoelectric materials efficiently convert heat to electricity. Calcium cobalt oxides (Ca2CoO3)0.62CoO2 is a model system in this regard with a figure of merit ZT above one. The compound has a complex misfit layered structure with significant lattice displacement (both static and dynamic) that is attributed to the reduced thermal conductivity. Its averaged structure consists of two interpenetrating subsystems of a CdI2-type CoO2 layer and a distorted tri-layered rock-salt-type Ca2CoO3 block (Fig.1,2), being incommensurately modulated along the b-axis. It is well known that both static displacement and thermal atomic vibration can effectively scatter phonons to reduce thermal conductivity, however, the exact scattering mechanisms are still unknown, largely because there is no reliable method available for such a measurement that can link the displacement to the phonon scattering.

     Here, we demonstrate that the quantitative acquisition of multiple annular-dark-field images via STEM at different scattering-angles simultaneously (Fig.1) allows us not only to separate but also accurately determine static and thermal atomic displacement in crystals. This is because the intensity characteristics of a STEM image acquired with high angle annular dark field (HAADF) and medium angle annular dark field (MAADF) differ considerably, depending on the nature of the displacement (Fig.2a-b). Unlike diffraction analysis that derives the overall displacement from the intensities of Bragg reflections, we directly measure the atomic displacement in real space, thereby enabling us to refine independently the atomic displacement in the same lattice planes, i.e., in the rigid CoO2 and soft Ca2CoO3 layers (Fig.3), that is crucial to revealing their different nature in phonon scattering.

     Applying our unique method to the layered thermoelectric material (Ca2CoO3)0.62CoO2 disclosed the presence of large incommensurate displacive modulation and enhanced local vibration of atoms, largely confined within its Ca2CoO3 sublayers. Relating the refined disorder to ab-initio calculations of scattering rates is a tremendeous challenge. Based on our approximate calculation of scattering rates, we suggest that this well-defined deterministic disorder engenders static displacement-induced scattering and vibrational induced resonance scattering of phonons as the origin of the phonon glass (Fig.4). Concurrently, the crystalline CoO2 sublayers provide pathways for highly conducting electrons and large thermal voltages [1].

References: [1] Wu, L., Meng, Q., Jooss, Ch., Zheng, J.-C., Inada, H., Su, D., Li, Q., and Zhu, Y., Adv. Funct. Mater. 23, 5728-5736 (2013).

  Work was supported by the U.S. DOE, Office of Basic Energy Science, Material Science and Engineering Division, under Contract No. DE-AC02-98CH10886.

Fig. 1: (a) Simultaneous acquisition of HAADF (114-608 mrad, b, d) and MAADF (46-104 mrad, c, e) images to determine the static displacivemodulation and atomic vibration in the [001] projection (b, c) and the [010] projection (d,e)of (Ca2CoO3)0.62CoO2. The embeddedare simulated images.

Fig. 2: Bottom:Image intensity vs displacement. Calculated intensities (dashed) inthe CoO2 and CoO layers and intensity ratios ICoO/ICoO2(solid) for HAADF (collection angle: 114 - 608 mrad) and MAADF (46-104mrad) as function of (a) thermalmean-square displacement in the CoO layer, and (b) static displacement calculated as two cosine components,A1x & A2x.

Fig. 3: Determining static- & thermal-displacement (a tod) simultaneous STEM images in[010]. Left: HAADF and Right: MAADF. (a)Experimental image; Calculated images with unrelaxed (b), and relaxed model (c)and our refined structure (d). (e) Intensity profiles.Open circles, green-, blue-, and red-lines are from(a), (b), (c), and (d), respectively.

Fig. 4: (a) Thermal conductivity vs temperature:circles are experimental data for (Ca2CoO3)0.62CoO2and MgO. Solid lines are fitting from specific heat, DOS, group velocity, and MFP. Blue and red lines contain the Umklapp and Rayleighterms, while the black line including the displacement and resonance terms. (b) MFP. (c-d) Phonon scattering mechanisms.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-2804 Characterization of nanoscale morphology in photovoltaic polymer blends using energy-filtered scanning electron microscopy

Masters R. C.1, Lidzey D. G.1, Sasam F. C.2, Rodenburg C.1
1University of Sheffield, Sheffield, UK, 2FEI Co., Eindhoven, The Netherlands
rmasters1@sheffield.ac.uk

Modern organic solar cells typically have an active layer consisting of a semiconducting polymer and fullerene blended together in a thin (<100nm) film. In production this blend phase separates on a nanometre scale, producing donor and acceptor phases in the film. The nature of the resulting blend morphology has a huge effect on the efficiency of a solar cell device[1]. As such high-resolution tools for the characterisation of such morphologies are required. We are developing energy-filtered SEM (EFSEM) as a fast, easy-to-implement method to perform this task, building on previous work using the technique to characterise inorganic semiconductor devices[2].

During an SEM exposure, the secondary electrons (SE) emitted by a given material under irradiation by the primary beam have a well-defined energy spectrum[3]. We compare the energy spectra of the SE emitted from different phases in a P3HT:PCBM photovoltaic blend (see fig 1). At energies below 8eV, one phase is brighter than the other; however this contrast is reversed at energies above 8eV. Therefore if all SE are used to image the blend morphology, the contrast in SE emission between the different phases will be relatively small. It is thus very difficult to map such blend morphologies withstandard SEM techniques.

To negate this problem it is possible to place a low-pass energy filter on the SE detected by the through-lens detector in an SEM column. This is done by changing the strength of the electrodes that deflect electrons towards the scintillation detector in the through-lens detector (TLD) assembly of an FEI Sirion SEM[2]. We place our energy filter at 8eV and only image using SE with energies below this threshold. This results in a significantly larger contrast difference between the two blend components than when all electrons are detected, as it excludes the regime in which the contrast between the phases is reversed. Using EFSEM we find we can map the chemical distribution in organic solar cells with sub-nanometre resolution; a feat that is unprecedented with SEM equipment (see fig 2). In our data we see periodicity in our data on two length-scales, at approximately 8nm and 17nm, as displayed in figure 3. We believe that these values correlate to periodicity in the separation between different P3HT and PCBM domains. The ability to image the morphology of a photovoltaic blend with this level of detail could constitute a powerful tool for informing the development of future organic photovoltaic technology.

[1] A.J. Heeger, Adv. Mater. 26 (2014), p. 10
[2] C. Rodenburg et al, J. Phys. Conf. Ser. 241 (2010), p.012074
[3] D. Joy, M.S. Prasad and H.M. Meyer, J. Microsc. 215 (2004), p.77

R. Masters would like to thank Project Sunshine at the University of Sheffield alongside the Faculty of Engineering for his PhD studentship funding.

Fig. 1: Secondary electron spectra of 2 distinct phases seen whilst imaging a P3HT:PCBM blend. Placing a low-pass energy filter at 8eV results in images being taken only from the domain where phase 2 is significantly brighter than phase 1 – this improves the chemical contrast in our images

Fig. 2: EFSEM image of P3HT:PCBM photovoltaic blend, subject to a 5-minute plasma clean to remove surface layers. From previous work we believe the brighter regions to be P3HT-rich and the darker regions to be PCBM-rich. The plot shows a line profile from the image (see yellow arrow), showing the level of detail available.

Fig. 3: Autocorrelation functions applied to our EFSEM images show a two-fold correlation in our data; we postulate that these correspond to periodicity in P3HT-rich and PCBM-rich domains. The equal strength of these peaks leads us to believe that they are two separate correlations, rather than two projections of the same correlation
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-3073 In situ electron microscopy of liquid dispersed 2-dimensional thin-film nanomaterials for electrochemical applications

Doherty E. M.1,2,3, Long E.1,2,3, Downing C.1,2,3, Pettersson H.1,2,3, Nicolosi V.1,2,3
1Trinity College Dublin, Ireland, 2CRANN (Centre for Research on Adaptive Nanostructures and Nanodevices), Dublin, Ireland, 3AMBER (Advanced Materials and BioEngineering Research Centre), Dublin, Ireland
dohertev@tcd.ie

The demands of modern energy consumption present new challenges regarding the storage of electrical and chemical energy. Traditional materials for creating batteries and capacitors can be examined in new ways with a view to increasing their efficiency and lifetime, whilst minimising device size. Research has discovered that the use of 2-dimensional versions of traditional energy storage materials can deliver significant advances in energy-storage technology [1]. However the use of and interactions of these materials with one another is still not well understood. One challenge to date has been how to image liquid-dispersed materials and their chemical interactions in situ. With the advent of a new generation of liquid-cell TEM holders, it is possible to conduct electron microscopy studies on 2D materials dispersed in liquid, thus presenting a new range of experimental conditions for better understanding material interactions and processes.

We conducted electron microscopy including CTEM (conventional transmission electron microscopy) and STEM (scanning transmission electron microscopy) in an FEI Titan 300kV S/TEM using a Liquid Cell TEM holder from Hummingbird Scientific Inc. Imaging can be carried out in both static and dynamic liquid environments and electrical bias can be applied to the materials for electrochemical interaction studies. Examples of the materials we examined include WS2, graphene, MoS2 and MnO2. This data can then be compared with previous, ex situ studies of materials conducted previously in our group [2].

For the first time these 2D layered-nanomaterials have been directly imaged and characterised in a TEM whilst suspended in liquid dispersion. We have demonstrated the ability to measure lattice resolution of these types of materials including graphene. We are also able to investigate and characterise the interactions of these materials in a dynamic environment whilst under electrical bias. It is hoped that using these techniques will provide us with vital information regarding the fundamental science behind the material interactions. This information can then be applied to a range of 2D layered-nanomaterials with the possibility of optimising existing battery and super-capacitor materials and discovering new and inexpensive materials for use in energy-storage technology.

[1] Arico, A.S., et al., Nanostructured materials for advanced energy conversion and storage devices. Nat Mater, 2005. 4(5): p. 366-377.
[2] Mendoza-Sanchez, B., et al., Scaleable ultra-thin and high power density graphene electrochemical capacitor electrodes manufactured by aqueous exfoliation and spray deposition. Carbon, 2013. 52 p. 337-346.


The authors gratefully acknowledge funding from the European Research Council, Science Foundation Ireland and the Advanced Materials and BioEngineering Research Centre.

Fig. 1: Schematic of a Liquid Cell Holder design and use in a Transmission Electron Microscope

Fig. 2: CTEM image and diffraction pattern of a graphene flake in a water/surfactant dispersion. Note the resolution of the lattice spacing of the graphene flake.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-2994 Examining Atomic Scale Radiation Damage in Nuclear Graphite with Transmission Electron Microscopy and Electron Energy Loss Spectroscopy

Freeman H. M.1, Mironov B. E.1, Scott A. J.1, Brydson R. M.1
1University of Leeds
pmhmf@leeds.ac.uk

Graphite moderators within advanced gas cooled or high temperature nuclear reactors are subject to high levels of neutron radiation which results in chemical and physical property changes. As a result, the large blocks can swell and crack which affects neighbouring components and ultimately the lifetime of the reactor, an accurate estimation of which is essential for economic success and plant safety.

 

To better understand the nature of radiation damage in graphite, this paper focuses on the atomic scale using transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS). The former allows us to visualise the atomic structure before and after irradiation and the latter provides information about chemical bonding and specimen density.

 

A methodology has been developed to quantitatively analyse controlled electron radiation damage (due to the TEM’s electron beam) to relate to the damage processes occurring in neutron irradiated graphites. For specimen observation, a non-destructive TEM operating voltage of 80 kV was used, however to induce electron beam damage, a 200 kV operating voltage was required. Experiments were performed at room temperature and reactor environment temperatures (~400 °C) to investigate the effects of thermal annealing (figure 1). All EELS experiments were performed at the magic angle to avoid orientation dependence [1].

 

Collaboration with the University of Bordeaux ‘PyroMan’ research group has led to the use of a computer programme to quantitatively analyse the electron micrographs [2]. Based on 002 fringe analysis, the software provides information on fringe length, spacing, tortuosity and orientation. The EEL spectra were analysed to extract data on sp2 content using the three window method, plasmon electron density and second scattering shell radius (figures 2 and 3).

 

[1] Daniels H, Brown A, Scott A, Nichells T, et al. 2003 Experimental and Theoretical Evidence for the Magic Angle in Transmission Electron Energy Loss Spectroscopy Ultramicroscopy 96 523
[2] Raynal P I, Monthioux M, Da Costa J P, et al. 2010 Multi-Scale Quantitative Analysis of Carbon Structure and Texture: iii. Lattice fringe imaging analysis. Proc. Int. Carbon conf. (USA)

Funding from the National Nuclear Laboratory and EPSRC (grants EP/J502042/1 and EP/I003312/1).

Collaborators in Bordeaux: J P Da Costa, P Weisbecker and J M Leyssale. (University of Bordeaux, Laboratoire des Composites Thermo Structuraux UMR 5801, 33600 Pessac, France).

Fig. 1: TEM images before (a and c) and after (b and d) 200 kV electron beam exposure to a dose of 1 dpa (displacements per atom) at room temperature (a and b) and 400 °C (c and d).

Fig. 2: Evolution of the EELS C K-Edge with electron beam dose at room temperature. The shape of the σ* peak changes significantly with dose and can be integrated along with the π* to extract values for sp2 content. The position of the Multiple Scattering Resonance (MSR) peak is proportional to valence electron density.

Fig. 3: Change in sp2 content with electron irradiation at room temperature for four sets of data. Data is normalised to the sp2 value of the first EEL spectrum in the series.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-3026 In-situ TEM investigation of the electrochemical (de)lithiation of single grain LiFePO4

Basak S.1, Malladi S. K.1, Ganapathy S.2, Wagemaker M.2, Tichelaar F. D.1, Zandbergen H. W.1
1Kavli Institute of Nanoscience, National Centre for High Resolution Electron Microscopy, Delft University of Technology, Lorentzweg 1, 2628 CJ, Delft, Netherlands, 2Department of Radiation Science and Technology, Delft University of Technology, Mekelweg 15, Delft 2629JB, Netherlands
s.basak@tudelft.nl

In understanding the performance of Li-battery materials, a holy grail is the ability to study the effect of particle size, particle boundaries, electrode-electrolyte interfaces, phase transitions, crystal defects, all independently during battery operation [1-2]. In practice however scientists have to rely on macroscopic polycrystalline electrodes with several additives and binders, where the overall performance is determined by a combination of many parameters. Because thorough determination of the effects of all parameters requires very expensive many-parameters studies, in practice researchers restrict to a trial and error approach leaving too much room for interpretation.
In-situ TEM studies on the other hand allow the usage of submicron dimension single particles as electrodes without supplementary additives or binder; enabling easy interpretation of results during the battery operation, even on the atomic scale. These TEM studies can lead to valuable insights on understanding the (de)lithiation mechanism of different electrode materials. However most of the in-situ TEM battery setups are based on STM tip [3-4], where only one edge of the investigated electrode material (generally nanowires) is connected to the electrolyte (either ionic liquid or oxidised lithium). This design forces Li+ ion exchange to take place from that edge alone and blocks the possibility of exchange from all directions. This restriction may lead to partial knowledge about the (de)lithiation mechanism of the electrode material.
We have designed an in-situ nano battery setup based on MEMS based chips, which ensures full coverage of the electrode with the electrolyte, allowing free exchange of Li+ from every part of the electrode. Our in-situ nano battery setup to study the (de)lithiation mechanism of the much debated LiFePO4 [5] is shown in Fig. 1. Here, during charging Li+ ions de-intercalate from LiFePO4 lamella and plate on the opposite gold line. While discharging, these Li+ ions move through LiPON and intercalate back to the lamella.
EELS analysis was carried out during the (dis)charging of the battery. One of our important finding, shown in Fig. 2, is: (de)lithiation always starts from the interface of electrolyte, current collector and electrode irrespective of LiFePO4 crystallite direction, signifying the importance of the interface for faster charging of the battery.

References:
[1] Tarascon et al..; Nature; Vol. 414, Issue 6861, 359-367 (2001).
[2] Goodenough, J.B. et al..; Chemistry of Materials; Vol. 22, Issue 3, 587-603 (2010).
[3] Liu, X.H. et al.; Energy and Environmental Science; Vol. 4, Issue 10, 3844-3860 (2011).
[4] Wang, J.W. et al.; ACS Nano; Vol. 6, Issue 10, 9158-9167 (2012).
[5]C. Delmas et al.; Nature Materials 7, 665 - 671 (2008).

This project is carried out in the framework of NWO-NANO project, No.-11498.

Fig. 1: Nano battery setup: (a) TEM chip (top view) prepared using electron beam lithography; (b) FIB-lamella is prepared from a LiFePO4 crystal; (c) ion beam induced platinum deposition ensures good electrical contact between lamella and current collector (gold pad); (d) chip is placed into electrical TEM holder after sputtering thin LiPON layer.

Fig. 2: a) STEM image of LiFePO4 lamella (highlighted with yellow line)on gold pad. EEL-scan was performed along the red line after charging of battery; b) presence of the edge around 4-7 eV for the position (i), due to presence of Fe3+, indicates the formation of FePO4, while the absence of this edge for position (ii) indicates absence of FePO4.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-3449 Direct Observation of Li2O2 Nucleation/Growth and Electrolyte Degradation by In-Situ Liquid ec-(S)TEM

Mehdi B. L.1, Nasybulin E. N.1, Xu W.1, Thomsen E.1, Engelhard M. H.1, Massé R. C.2, Gu M.1, Bennett W.1, Parent L. R.1, Nie Z.1, Wang C.1, Zhang J. G.1, Evans J. E.1, Abellan P.1, Browning N. D.1
1Pacific Northwest National Laboratory,Richland,WA,USA , 2University of Wisconsin,Madison, WI,USA
layla.mehdi@pnnl.gov

The growing need for high energy density rechargeable batteries used in large-scale energy applications has spawned a wide range of in-situ/operando experimental techniques to provide insights into their operation [1, 2]. The recent development of the in-situ liquid electrochemical stages for (S)TEM (in-situ liquid ec-(S)TEM) has enabled the fabrication of a “nanobattery” to study the details of the electrochemical process by providing real-time information on the dynamic structural changes that occur at the electrode/electrolyte interface during charge/discharge cycles. Here, we demonstrate the application of this cell to study fundamental operational mechanisms such as the formation and decomposition of lithium peroxide (Li2O2) in rechargeable Li-O2 batteries, Li-ion battery electrolyte degradation processes and dendrite formation mechanisms and kinetics at Li and Mg anodes.

Li-O2 batteries are being developed for electric vehicles [3,4] due to their high theoretical energy densities - which are comparable to gasoline. The operation of a Li-O2 battery involves the reversible formation/oxidation of lithium peroxide (Li2O2) at the cathode, the efficiency of which determines the overall battery performance. However, Li-O2 batteries exhibit significant challenges - such as low rate capability, limited charge-discharge cycles resulting from decomposition of both the electrolyte and the electrode material during oxygen reduction and evolution. This leads to accumulation of insulating side products, which causes a high overpotential and fast capacity fading during cycling. Here, we use the in-situ ec-(S)TEM cell to investigate the differences in the growth mechanism of Li2O2 nanoparticles and the decomposition of the side products.

Another example of the in-situ ec-(S)TEM cell is to study electrolyte degradation mechanisms and new electrochemical windows for state-of-the-art Li and Mg batteries. The electrolyte breakdown can be initiated by the localized interaction of the electron beam and provide significant understanding of the reduction/degradation products formed during battery operation, which significantly decreases the time of postmodem analysis. The resulting formation of decomposition products and an example the results obtained for 5 electrolytes commonly used in Li-ion and Li-O2 battery systems is demonstrated in Figure 1 [6].

References:
[1] B. R. Long et al, J Phys Chem C, 115, (2011), 18916
[2] C. A. Bridges et al J Phys Chem C, 116, (2012), 7701
[3] M. Park et al, Adv.Energy Mat., 2, (2012), 780
[4] P. G. Bruce et al, Nat. Mat. 11, (2012), 19
[5] P. Abellan et al, Nano Lett. 14, (2014), 2014

This work was supported by the CII LDRD at PNNL operated by Battelle for the DOE under Contract DE-AC05-76RL01830. The electrolytes were prepared with the support from JCESR, an Energy Innovation Hub funded by DOE-BES. A portion of the research was performed at EMSL, a national scientific user facility sponsored by the DOE-BER and located at PNNL.

Fig. 1: STEM images of e-beam induced degradation mechanisms in LiAsF6 in (a) DOL, (b) DMC, (c) EC/DMC, (d) LiPF6 in EC/DMC, (e) highly stable LiTf in DMSO and (f) EC/DMC for the same electron dose and exposure times. (g) post mortem anlysis LiAsF6 in DMC and the formation of LiF nanocrystals.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-3382 Real-time Observation of Vacancy Dynamics and Phase Transformation in Epitaxial LaCoO3-x Thin Films and Superlattices

Jang J.1, Kim Y.2, He Q.1, Qiao L.1, Biegalski M. D.1, Lupini A. R.1, Pennycook S. J.3, Kalinin S. V.1, Borisevich A. Y.1
1Oak Ridge National Laboratory, Oak Ridge, United State, 2Korea Basic Science Institute, Daejeon, Korea, 3University of Tennessee, Knoxville, United State
jangj@ornl.gov

Transition metal oxides (TMOs) have attracted attention for solid oxide fuel cell, gas sensor and catalytic applications. [1] In many of these cases, a material functionality is dependent on the distribution and transport behavior of oxygen ions. It has recently been demonstrated that, for a static case, oxygen vacancy distribution and vacancy ordering can be characterized at an atomic scale using quantitative aberration-corrected STEM. [2] In this work, we take this approach to the next level by observing the dynamics of vacancy ordering and vacancy injection under the electron beam in LaCoO3/SrTiO3 (LCO/STO) superlattices and LaCoO3-x thin films using high angle annular dark field (HAADF) and annular bright field (ABF) STEM.
We find that while before electron beam exposure films and superlattices do not show any signs of vacancy ordering, they nevertheless contain a substantial amount of vacancies; the ordering is quickly induced by electron beam exposure (Fig.1). We can monitor vacancy ordering by tracking local interatomic spacings, and vacancy injection by tracking global average of the spacings as per Vegard’s law. In (110) projection, multiple lattice distortions can be tracked simultaneously as a function of beam exposure, such as out-of-plane lattice expansion, in-plane Co shift (Fig.2), and octahedral tilt patterns in the surrounding atomic layers.
In the case of 15 u.c. LCO film, beam exposure leads to a sequence of different phases, starting from disordered perovskite LaCoO3-x to a brownmillerite polytype La3Co3O8-x (2 perovskite layers connected 1 tetrahedra layer), to eventually brownmillerite La2Co2O5-x (alternating octahedra and tetrahedra layers in the Fig.3), which is similar to the phase evolution observed in the bulk [3]. Forming oxygen depleted layers couple in complex ways to the existing octahedral tilt system and to each other, giving rise at times to metastable intermediate states that can give us insights into oxygen transport mechanisms in this system. Kinetics of the ordering and vacancy injection, as well as implications for beam-driven material modification at an atomic scale, will be discussed.

[1] J. Maier, Nat. Mater. 4, 805 (2005)
[2] Young-Min et al., Nat Mater. 11, 888 (2012)
[3] Ole H. Hansten et al., J. Mater. Chem. 8 2081 (1998)

The MSE Division, US DOE; through a user project in ORNL’s CNMS, sponsored by the SUF Division, Office of BES, US DOE; The CSGB Division, Office of BES, US DOE.

Fig. 1: HAADF images of LCO/STO superlattice along [100]pc direction (A) as-grown and (B) after electron beam exposure. (C) Atomic spacing map generated from (B) shows that lattice expansion as an indicative of vacancy accumulation develops in the each LCO blocks.

Fig. 2: HAADF images of an LCO block in LCO/STO superlattice along [110]pc direction (A) initial state and (B) after beam exposure. (C) out-of-plane La spacing map generated from (B). (D) in-plane Co-Co spacing map generated from (B).(E) schematic of structural changes in LCO block before/after Vo ordering.

Fig. 3: Sequential HAADF images and models showing the evolution of the structure (A) initial LaCoO3-x along [110]. (B) intermediate 2x1 ordering structure (C) brownmillerite 1x1 ordering structure.
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-3396 Structural Changes of Ta2O5 Based Photocatalyst under Reaction Conditions

Liu Q.1, Crozier P. A.1
1School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ 85287-6106
qliu49@asu.edu

Water splitting using a powdered photocatalyst is a promising clean energy system for converting solar energy into the chemical energy of H2 molecules. Ultraviolet (UV) or visible light is required to generate electron-hole pairs in the catalyst which further reduce/oxidize the water into H2/O2. However, the photocatalyst may undergo a structural transformation under reaction conditions and the structure-activity relationship has not yet been fully understood. In this study, we use Ta2O5 based catalysts as the model material to investigate the structural changes under reaction environments.

The Ta2O5 nanopowders were synthesized using a solvothermal method described elsewhere [1]. The resulting powders were then calcined at 600oC and 800oC for 5h to give a better crystallinity and morphology in the initial photocatalyst. A 450 Watt xenon arc lamp with a mirror selecting the wavelengths from 260 to 320 nm was used to excite the electrons over the bandgap of Ta2O5 which is approximately 3.9 to 4.3 eV wide. The initial Ta2O5 nanopowders were then exposed to UV light for 9h in water vapor. An FEI Tecnai F20 environmental transmission electron microscope (ETEM) was employed to obtain high resolution images of both initial and treated material. The high resolution image (Fig.1) of the initial material shows reasonably well-defined morphology of the nanoparticles where clean and smooth surfaces were observed. Electron and x-ray diffraction patterns revealed an orthorhombic structure of Ta2O5. However after the treatment, the initially clean surfaces were found to be more disordered and amorphous surface layers were present (Fig.2). Also, new lattice spacings from 3.43 Å to 3.58 Å were found in some treated particles that were not previously observed in the initial catalyst.

The functionalization of the catalyst by loading NiO on Ta2O5 was also investigated. The NiO (5 wt%)/Ta2O5 catalysts were synthesized using an impregnation method and were pretreated by H2 reduction at 673K for 1h and subsequent O2 oxidation at 473K for 0.5h. The structure of this reduction-oxidation treated catalyst is shown in Fig. 3a. Photocatalytic reactions of both NiO/Ta2O5 and pure Ta2O5 catalysts were carried out in a glass reactor with a quartz window in a gas-closed system. The evolved H2 gas was detected by a gas chromatography (GC) and Fig.3b shows the H2 peak intensities from the two catalysts. The H2 production was calculated for each catalyst and it was significantly improved from 26 to ~220 μmol/h/g by the NiO loading. Additional in situ experiments will be carried out to understand the fundamental structural evolution of these photocatalysts correlated with their photocatalytic performances.


References:
[1] J. Buha et al, Phys. Chem. Chem. Phys., 12 (2010), 15537.

The support from US Department of Energy (DE-SC0004954) and the use of ETEM at John M. Cowley Center for HR Microscopy at Arizona State University is gratefully acknowledged.

Fig. 1: (a) Initial Ta2O5 photocatalyst showing clean surfaces. (b) Diffraction pattern of the same area revealing an orthorhombic structure of Ta2O5.

Fig. 2: Treated photocatalysts that were exposed to UV light for 9hr in water vapor. A new lattice spacing (3.58Å) and an amorphous surface layer are present.

Fig. 3: (a) The structure of the 5 wt% NiO/Ta2O5 catalyst after reduction-oxidation pretreatment. (b) The H2 peak intensities of both NiO/Ta2O5 and pure Ta2O5 catalysts from the GC. 
Fig. 4:
↑ Go to the Table of content ↑
Type of presentation: Oral

MS-14-O-3447 Synthesis and Characterization of Lead Chalcogenide Nanoparticles and its Application in Photovoltaic Devices.

Garcia-Gutierrez D. I.1,2, Garcia-Gutierrez D. F.1,2, De Leon-Covian L. M.1,2, Hernandez-Casillas L. P.1,2, Arizpe-Zapata J. A.1,2, Gonzalez-Treviño M. T.1,2
1Universidad Autónoma de Nuevo León, UANL, CIIDIT, Apodaca, N.L., México, 2Universidad Autónoma de Nuevo León, UANL, FIME, San Nicolás de los Garza, N. L., México
domingo.garciagt@uanl.edu.mx

The study of lead chalcogenide nanoparticles has gained a lot of attention in recent years, particularly in possible applications in solar cells [1]. Several studies have been made exploring the chemical synthesis of these chalcogenide systems. One of the most popular synthesis methods, the “one-pot method” [2], has been reported to be able to produce a wide variety of different nanostructures. Nevertheless, the nucleation and growth mechanism of the nanoparticles synthesized with this method has not been thoroughly studied. Most of the accepted theories for the nucleation and growth mechanisms have been based on the information obtained in studies of the cadmium chalcogenide systems; fewer studies have been made specifically on the lead chalcogenide systems. In the present work, experimental evidence is presented that supports the viability of a nucleation and growth mechanism that includes the fact that the Pb2+ ions, from the lead precursor (lead oleate), are being reduced to their metallic state, Pb0; as it is evidenced by the presence of Pb nanoparticles in the early reaction stages (Figure 1); and how these lead atoms participate actively in the growth of the lead chalcogenide nanoparticles since the very beginning of the reaction [3]; facts that are not contemplated by currently accepted nucleation and growth mechanisms theories of lead chalcogenide nanoparticles. The experimental evidence presented is based on TEM results, and their related techniques, such as STEM, Aberration Corrected STEM (AC-STEM), EDXS, EELS, SAED and Diffraction-STEM (D-STEM) studies. FTIR and UV-Vis-NIR absorbance studies have been performed to complement the TEM observations, and they were fundamental in understanding the nanoparticles’ capping layer nature (Figure 2), and to correlate the lead oleate nature of the capping layer with the nucleation and growth mechanism proposed. SAED and D-STEM studies allowed us to identify the presence of nanoparticles with two different crystal structures for the case of the ternary system PbSexS1-x (Figure 3), the commonly FCC structure, along with a  body centered tetragonal (BCT) structure, however the observance of this crystalline structure is low. Finally, photovoltaic devices were built based on thin films of PbSe and PbSexS1-x nanoparticles, thin films fabricated by the dip coating technique. These photovoltaic devices showed low efficiencies (η) and short circuit currents density  (Jsc), nevertheless the open circuit voltages (Voc) displayed were among the highest found in the literature (Figure 3).

[1] Choi, J.J. et al. Nano Lett. 9 (11), p. 3749 (2009).

[2] Murphy, J.E. et al. J. Am. Chem. Soc. 128(10) p. 3241-3245 (2006).

[3] D.I. Garcia-Gutierrez et al. J. Nanopart. Res. 15(5), p. 1620, (2013).

The authors aknowledge the financial support recived from CONACYT through grants 154303 and 148691.

Fig. 1: a) HAADF image of Pb nanoparticles with an average size of 2 nm. Inset: SAED pattern corresponding to an hexagonal crystal structure. b) EDXS spectrum showing a clear Pb signal, but no chalcogen signal. Inset: EELS spectrum showing no signal of the chalcogen of interest. 

Fig. 2: a) AC-STEM HAADF image showing one PbTe nanoparticle analyzed. The red line indicates the region where the linescan study was performed. b) EELS-EDXS line profiles for the Pb Ma EDXS signal and the Te M4,5, O K and C K EELS signals. c) FTIR spectrum showing the bands associted to the presence of a lead oleate in the sample.

Fig. 3: a) TEM image showing the synthesized PbSexS1-x nanoparticles. Inset: SAED pattern showing diffraction spots associated to a BCT crystal structure. b) I-V curve of photovoltaic device fabricated with a thin film based on PbSe nanoparticles. c) I-V curve of photovoltaic device fabricated with a thin film based on PbSexS1-x nanoparticles.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1435 Synthesis and Characterization of CdS0.9Se0.1 Nanoparticles Prepared by Chemical Method for Used as Thermoelectric Materials.

Autthawong T.1, Sarakonsri T.1, Thanachayanont C.2
1Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand., 2Thailand Science Park Paholyothin Rd., Klong 1, Klong Luang, Pathumthani 12120 Thailand.
Maman_chem@hotmail.co.th

                    This project studied the appropriated conditions for a synthesis of N-type CdS0.9Se0.1 semiconductor to be used as thermoelectric materials. Cd(CH3COO)2.2H2O, Thiourea and SeO2 as precursors and ethylene glycol as a solvent. Two methods were applied to prepare CdS0.9Se0.1. The first method was a solution method which was applied to prepare selenium powder. NaBH4 was used as a reducing agent and reaction time with 8 hours. The other method was a reflux method, which was used to prepare CdS powder, with 8 hours reaction time and 250 degree Celsius reaction temperature. The product powders were mixed together by with the atomic ratio of Cd: S: Se to 1: 0.9: 0.1. Then it was annealed with different temperatures at 500, 700, and 900 degree Celsius under nitrogen atmosphere and varies annealing time for 5 and 10 hours. Finally, all of the powder products were characterized by X-ray diffraction technique (XRD) while CaRine3.1 for XRD pattern simulation and 3D lattice simulation, Scanning electron microscopy (SEM) with the analysis of Energy dispersive spectroscopy (EDS) for surface composition and Transmission electron microscopy (TEM).
                    From the XRD result of the product powders, powder annealed at 700 degree Celsius under nitrogen atmosphere for 10 hours along with the TEM result of indexing single crystal diffraction pattern can confirm that the powder product is CdS0.9Se0.1 and The SAED of CSS_700_5 and CSS_700_10 in Fig 2 shows the transformation of polycrystalline (CSS_700_5) to single crystalline (CSS_700_10) pattern when increasing annealing time. The SEM images of the CdS0.9Se0.1 are shown in Fig 1. The products were different morphology and random sized, small to large particles with the uncertain-shaped that relate with increasing annealing time and temperature. The image in higher magnification shows accumulated behavior of the particles.

Development and Promotion of Science and Technology Talents Project(DPST), National Metal and Materials Technology Center(MTEC), the Department of Chemistry, Chiang Mai University.

Fig. 1: The SEM images of the CdS0.9Se0.1 product after it was annealed with different temperatures and reaction times. (a) 5000x (b) 10,000x

Fig. 2: The TEM image and the selected area electron diffraction pattern of CdS0.9Se0.1.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1436 Effect of Sn precursor concentration and solution viscosity on Sn particle sizes supported on Graphene; anodes for lithium-ion batteries

Jarulertwathana B.1, Sarakonsri T.1
1Renewable Energy Laboratory, Department of Chemistry, Chiang Mai University, 239 Huay Kaew Road, Muang, Chiang Mai, 50200, Thailand
bjarulertwathana@gmail.com

               Tin/graphene (Sn/graphene) composites are prepared in nano-dimensions for use as anode materials in lithium-ion batteries. The objectives of this research are enhancing both the energy density and mechanical stability of anodic electrodes from high theoretical capacity of Sn and graphene supported material, respectively. The chemical reduction method, which is the simple and low cost method, is used to prepare of Sn on graphene. Sn particles are prepared by using tin(II) chloridedihydrate (SnCl2•2H2O), sodium borohydride (NaBH4) and ethylene glycol as Sn precursor, reducing agent and solvent, respectively. The effect of decreasing SnCl2•2H2O precursor concentration in ethylene glycol solvent and the solvent viscosity on tin particle sizes is studied in this research. The solvent viscosity is varied by adding methanol to ethylene glycol solvent. The result from X-ray diffraction (XRD) technique shows that Sn phase contains in the prepared products. The phase of products will be confirmed by transmission electron microscopy (TEM) technique. The results from scanning electron microscopy (SEM) technique will give the information about size and morphology of Sn particles. The condition that smallest size of Sn is obtained will be used to prepare 10 and 20 percent weight Sn on graphene composites. The charge-discharge voltage and the cycling performance of the products will be investigated. The results of the characterization will be discussed in the 18th International Microscopy Congress.

This work is supported by the Department of Chemisty, Chiang Mai University and the Development and Promotion of Science and Technology Talented Project (DPST).

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1440 Electron Microscopy Investigation of Sb2-xBixTe3 hexagonal crystal structure growth prepared from sol-gel method

Sarakonsri T.1 2, Tongpeng S.2, Thanachayanont C.3, Mitsutaka H.4, Hiroki K.4
1Materials Science Research Center, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand., 2Department of Chemistry, Faculty of Science, Chiang Mai University, 239 Huay Kaew Rd, Suthep, Muang, Chiang Mai 50200, Thailand, 3National Metal and Materials Technology Center, 114 Thailand Science Park, Paholyothin Rd., Klong 1, KlongLuang, Pathumthani, Thailand, 4Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
tsarakonsri@gmail.com

P-type semiconductor Sb2-xBixTe3 has the potential to be used as thermoelectric materials for thermoelectric cooling and power generation because of its high in ZT value at low temperature. In this report, Sb2-xBixTe3 powders with x equals to 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 were prepared by sol-gel method. Bismuth acetate, antimony acetate, and tellurium dioxide were the starting materials which were initially dissolved in 1-propanol, methanol, and ethylene glycol, respectively. Diethanolamine was applied as a stabilizer. The ratio between moles of metals, solvents, and stabilizer was first varied to obtain gel and it was found that 1:60:4 is the most suitable ratio. The gels of Sb2-xBixTe3 were annealed to make powders at 773 K for 2 hours under nitrogen gas atmosphere. X-ray diffraction (XRD) patterns of all samples match with Sb2Te3 phase, but with the trend of peaks shifted to the lower angle when increasing x values suggesting Sb positions were replaced by Bi atoms accordingly. Rod structure composing of a pile up of thin sheets was observed by scanning electron microscopy (SEM) micrographs for all compositions. Transmission electron microscopy (TEM) analysis reviewed the separated sheet of the hexagonal thin films connected to each other in the Z direction. This unique structure resembles the fish bones. STEM images of SbBiTe3 with the corresponding electron diffraction pattern match well with the simulated structure in the 001 zone axis. The growth mechanism this structure will be presented.

National Research University Project under Thailand’s office of the higher education commission (NRU), National Metal and Materials Technology Center (MTEC) MT-B-55-CER-07-295-I

Fig. 1: TEM images of Sb2-xBixTe3 with x = 0.2 (top) and x=0.8 (bottom) and its corresponding SAD patterns
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1462 Optical Microscopy and Wavelet-Based Image Analysis of Experimental Metallurgical Coke

Gornostayev S.1, Heino J.1, Kokkonen T.1, Makkonen H.1, Huttunen S.1, Fabritius T.1
1Laboratory of Process Metallurgy, University of Oulu, P.O. BOX 4300, Oulu, 90014, Finland
Stanislav.Gornostayev@oulu.fi

Metallurgical coke is a key material for blast furnace operation, acting as [Andriopoulos et al., 2003]: an energy source (fuel), a carburisation agent, a reductant, and a structural support. It made of several blends of coal by heating their mix in coke batteries up to 1200oC. Carbonization of coals during the coking process leads to the development of various microscopic textures, which have an influence on chemical and physical properties of the resulting metallurgical coke. The textures of a coke are usually characterized on the basis of their optical behaviour in polarized light. There are three dominating textures [Hideo et al., 1983]: highly oriented banded, mosaic and isotropic.

The effect of addition of high density polyethylene (HDPE) to the textural features of experimental metallurgical coke has been studied using polarized light optical microscopy and wavelet-based image analysis. The samples were made in a laboratory-scale furnace without (100 % coal) and with 2.5, 5.0, 7.5, 10.0 and 12.5 % of HDPE and polished in rounded sections. Optical investigations were done with Olympus BX51 optical microscope equipped with digital camera. Special software [Mattila & Salmi, 2008] developed in MATLAB with its Wavelet Toolbox was used to perform image analysis. According to Makkonen et al. [2009], any given sample of coke can be representatively characterized with wavelet-based image analysis by studying 7-11 points. At every point, 4 images under different positions (20°, -20°, 0°, 90°) of polarizing lenses should be taken. The program divides (Fig. 1B) the images (2048x1536 pix) into 12 sub-areas (512x512 pix) and calculates each sub-area separately. As a result, it gives the amount (%) of isotropic, mosaic and banded textures, as well as pores. In this study, each sample was photographed and analysed in 10 areas (Fig. 1A), which gave 120 sub-areas for the image analysis and 120 datasets per sample for subsequent statistical calculations.

Optical observations of the samples have shown that there are some differences in textural features of cokes prepared with and without HDPE. In the samples containing HDPE, relatively large pores with rounded shapes and smooth outlines (Fig. 2C), which sometimes cover entire area of the image, can be observed more often than in the coke prepared without HDPE. In some cases, the pores in HDPE-containing coke have an almost ideal spherical shape. The calculations have found that the addition of HDPE results in a decrease of mosaic texture and some increase of isotropic texture. Ethylene, formed from the decomposition of HDPE, was considered as a probable gas affecting the texture adjustments. The approach used in this study can be applied to indirect evaluation of reactivity and strength of metallurgical coke.

The research was funded by the Academy of Finland.

Fig. 1: Polished section with locations of the points (A) and a map of sub-areas for the image analysis (B).

Fig. 2: Wavelet-based image analysis of experimental metallurgical coke. A,B - 100% coal, C,D - 87.5% coal and 12.5% HDPE. Scale bar - 50 µm. Textures: isotropic - green, mosaic - red, banded - yellow, pores - blue.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1496 FESEM in the investigations of interaction of molten iron with metallurgical coke

Gornostayev S.1, Heino J.1, Fabritius T.1
1Laboratory of Process Metallurgy, University of Oulu, P.O. BOX 4300, Oulu, 90014, Finland
Stanislav.Gornostayev@oulu.fi

Metallurgical coke is a major energy source for blast furnace (BF) ironmaking. It also acts as a carburisation agent, a reductant, and a structural support [Andriopoulos et al., 2003]. It made of several blends of coal by heating their mix in coke batteries up to 1200oC. The coke reacts with the droplets of molten iron in the bottom of a BF during ironmaking process.

The samples of metallurgical coke were drilled out of working BF. They were cut to slices of 5-7 mm in thickness and 25 mm in diameter, and after that they were polished according to a special procedure [Kekkonen & Gornostayev, 2011]. Investigations with the FESEM Zeiss ULTRA plus have found that the sizes of Fe-Si droplets are within a range of 0.1-3 mm (Fig. 1) in their longest dimention. The droplets vary in shape, contact angle and penetration degree into the BF coke matrix (Fig. 2). There are rounded (Fig. 1A – marked by arrow), elongated (Fig. 1A – larger droplet) and irregular droplets (Fig. 1D).

The shape and penetration degree of Fe-Si droplets may be interrelated, as the penetration depth can be the result of reaction between a certain Fe-Si droplet and the coke matrix. Since the amount of carbon dissolved in molten iron depends on the concentration of Si [Lacaze & Sundman, 1991; Kawanishi, Yoshikawa & Tanaka, 2009], droplets which are under-saturated with silicon may react better and penetrate deeper, forming irregular aggregates (Figs. 1D, 2E and 2F). These under-saturated droplets (Type 3) have small (<90o) contact angle with the surface of coke. Whereas droplets saturated with Si (Type 1) may not react intensively with the matrix and retain more or less the round shape, as shown in Figs. 1A and 2A. These droplets have relatively large contact angle (>90o) with the surface of coke. There is also intermediate type (Type 2) of the droplets, which has contact angle close to 90o. They are partly submersed into the matrix and have semi-spherical shape (Figs. 1B, 1C and 2C). The contact area of Fe-Si droplets with the matrix is also different for all the types listed to above in respect of presence of mineral matter and graphite crystals. Type 1 has limited amount of both (Figs. 2A and 2B), whereas Type 3 has relatively large (above 100 µm) graphite crystals and noticeable amounts of mineral phases (Figs. 2E and 2F). Type 2 (Figs. 2C and 2D) has only single crystals of graphite (which are smaller than in Type 3) and it is somewhere in the middle in respect of these features.

The shape and relationships of Fe-Si droplets with the coke matrix reflect thermal and chemical history of coke-metal interaction under the BF conditions.

The research was funded by the Academy of Finland. Mr. T. Kokkonen is thanked for preparing the samples.

Fig. 1: Appearance of Fe-Si droplets on a surface of BF coke.

Fig. 2: Polished sections of samples of BF coke containing Fe-Si droplets.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1504 Micrograms to Megatonnes: Using Microscopy to Solve Oil Sands Processing Problems

Mikula R. J.1, Munoz V. A.2
1Kalium Research, Edmonton, Alberta T6J 0N1, 2CanmetENERGY, Devon, Alberta T9G 1A8
randy.mikula@oilsandsresearch.com

The development of oil sands as one of the world’s leading oil resources has not come without some challenges. In surface mined oil sands there are significant environmental issues around tailings ponds and management of various waste streams, and overall there are challenges with the carbon dioxide footprint associated with converting the heavy bitumen into transportation fuels. In an industry where two tonnes of ore are needed to produce one barrel of bitumen, on any given day a typical operation will process 500,000 tonnes of oil sands ore using 500,000 tonnes of water. Microscopy is not necessarily the first tool that comes to mind when discussing operational problems on this scale. In fact there are many examples of the applications of microscopy in solving important industry challenges related to tailings handling, separation of water and mineral from the final bitumen products, and generally improving the environmental sustainability of one of Canada’s largest industries.

The micrograph in Figure 1 shows just one of the many examples of the application of microscopy to the production end of the oil sands to crude oil process. The surface mined oil sands bitumen extraction process is quite efficient, with typically more than 95% of the bitumen recovered from the sand and clay minerals. One of the first steps in the bitumen recovery process is the flotation and concentration of the bitumen in a froth. In this froth flotation process, the process temperature is such that bitumen will engulf an air bubble. Figure 1 shows a cryo-SEM image of the interior of such an air bubble where hydrocarbon droplets have collected. With time, the number of hydrocarbon droplets crossing the air-bitumen boundary will increase, suggesting that this behavior might be the first step in the development of a nano-refinery that might help in the upgrading of bitumen to transportation fuels.

Figure 2 shows a micrograph of the mineral particles in a typical oil sands process tailings. Understanding and manipulating the small clay minerals that make up the tailings suspensions is the first step in reclaiming the large tailings ponds associated with surface mined oil sands development. It is an understanding of the processes on the microscopic scale that help define the environmental and production solutions and that have directly led to multi-million dollar changes in important oil sands processes. Some interesting examples will be given where multimillion dollar process changes were made based only on evidence provided by microscopy.

The authors would like to thank Suncor Energy and Syncrude Research for providing oil sands samples, and the technical staff at CanmetENERGY for assistance in sample preparation.

Fig. 1: Cryo-SEM image of the interior of a bitumen froth air bubble at initial attachment (left) and after some minutes (right). The inset x-ray spectra show a high sulphur content associated with the bitumen in the droplets (left), but not in the bulk (right).

Fig. 2: SEM micrograph of the mineral particles making up oil sands tailings. The distinctive plate like nature of the kaolinite clays is evident, as is the presence of many mineral components only a few hundred times larger than the water molecules in which they are suspended.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1609 Degradation analysis of LiCoO2 in an all-solid-state Li-ion battery

Shimoyamada A.1, Yamamoto K.1, Yoshida R.1, Iriyama Y.2, Hirayama T.1
1Nanostructures Research Laboratory, Japan Fine Ceramics Center, Japan, 2Department of Materials, Physics and Energy Engineering, Nagoya University
a_shimoyamada@jfcc.or.jp

All-solid-state Li-ion batteries (LIBs) have been expected as next generation energy storage devices. Research of the battery materials using TEM is increasingly important to analyze the electrochemical reactions in LIBs in nanometer scale [1]. Degradation of electrodes caused by the Li insertion/extraction is one of the serious problems preventing the practical use of all-solid-state LIBs. In this report, we have performed TEM and spatially-resolved (SR)-TEM-EELS to clarify the mechanism of the degradation of LiCoO2 positive electrode in the all-solid-state LIB.

Figure 1 (a) schematically illustrates the LIB model sample. A Li+ conductive glass ceramic sheet of the composition Li1+x+3yAlx(Ti,Ge)2-xSi3yP3-yO12 (LATP) was used as the solid electrolyte. A positive electrode of crystalline LiCoO2 was deposited on one side of the LATP by pulsed laser deposition. The “in-situ formed negative electrode” irreversibly formed by the Li insertion to the negative side LATP was used as the negative electrode [2]. After the bulk LIB sample was charged/discharged 20 times in a vacuum, the region around the LiCoO2/solid-electrolyte interface was lifted out by a micro-sampling method in a FIB system, and the TEM sample was prepared. The TEM image in the positive electrode region (Fig. 1 (b)) shows a different contrast at about 300nm from the interface. Electron diffraction and HRTEM analysis revealed that nanocrystalline compounds of LiCoO2 mixed with CoO existed in the region. Figure 1 (c) shows the spectrum image of Co L2,3-edges detected by SR-TEM-EELS, where the horizontal and vertical axes correspond to the sample position of the Fig. 1 (b) in the horizontal direction and the electron energy loss, respectively. The Co L3-edge spectrum image was clearly shifted to the direction of lower energy loss at the 300nm from the interface, showing Co was reduced to Co2+. The O spectrum change also indicated the existence of CoO in the region. We conclude from the TEM and SR-TEM-EELS analysis that the degradation of the LiCoO2 electrode in this battery is caused by the formation of CoO.


[1] K. Yamamoto et al., Angew. Chem. Int. Ed. 49 (2010) 4414-4417.
[2] Y. Iriyama et al., Electrochem. Commun. 8 (2006) 1287-1291.

This work was supported by the RISING project of the New Energy and Industrial Technology Development Organization (NEDO) in Japan.

Fig. 1: (a) Schematic illustration of the all-solid-state Li-ion battery sample. (b) TEM image around the LiCoO2/LATP interface. (c) SR-TEM-EELS image of Co L2,3-edges. The dotted line shows the interface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1724 Characterization of by-products from the combustion of solid fuels with SEM/EDS and micro-Raman spectroscopy.

Šašek P.1, Viani A.1, Mácová P.1, Peréz Estébanez M.1, Černá M.2
1Centrum Excelence Telč, ÚTAM AV ČR, Batelovská 485-6, 58856 Telč, Czech Republic, 2VŠB- TU Ostrava, HGF, 17. listopadu 15, 708 33 Ostrava, Czech Republic
viani@itam.cas.cz

Coal is still one of the main sources of energy for producing electricity. The environmental impact of the solid products resulting from coal combustion (fly ashes) is mitigated by employing them as secondary raw materials. Their chemical and physical properties are strongly dependent upon the type of coal and the burning plant technology, making their characterization an essential prerequisite for recycling. In recent years, there has been also a concomitant increase in the amount of ashes produced from biomass combustion. Structurally and chemically different, they pose different problems in terms of ecological impact: one of the most relevant is the concentration of heavy metals. In this work, two samples from the combustion of coal and lignite, and two samples from the combustion of biomass, namely straw and hay, have been investigated by means of scanning electron microscopy (SEM) with energy dispersive spectrometry (EDS) and micro-Raman spectroscopy (with laser wavelength 532 nm). Raman spectroscopy, with the aid of the optical microscope, allowed for addressing the laser beam on specific crystals for phase identification at the micrometric scale [1]. X-ray diffraction (XRD) was employed for bulk qualitative analysis of the crystalline fraction. Due to the operating temperatures above 1400 °C, fly ashes from coal combustion showed the presence of partly glassy spherical bodies (around 10 μm in diameter) with alumino-silicate composition. Crystallization of mullite (ideal Al6Si2O13) from the mass was documented. Euhedral to pseudoeuhedral iron oxide crystals were found as a 'coating' on some these particles, suggesting 'condensation' at the grain surface. Their presence in the mass is also an indication of their crystallization from the glass during cooling. These findings are in agreement with XRD results, showing mainly mullite, hematite (Fe2O3) and quartz. Ashes from the combustion of biomass consist mainly of unburned fuel residues, that represent up to 25% in weight. Silica is about 40% in weight and is mainly concentrated in spherical glassy-like particles from nanometric to micrometric in size. Both types of ashes from biomass are high in potassium and phosphates. Typical phases detected by XRD are Arcanite (K2SO4) and Monetite Ca(HPO4). Implications for the use of these by-product as secondary raw materials will be discussed.

[1] Guedes A, Valentim B, Prieto A C, Sanz A, Flores D, Noronha F. Characterization of fly ash from a power plant and surroundings by micro-Raman spectroscopy. Int. J. Coal Geol. (2008) 73, 359-370.

Research supported by the project CZ 1.05/1.1.00/02.0060 from the European Regional Development Fund and the Czech Ministry for Education, Youth and Sports.

Fig. 1: SEM micrograph from the sample of lignite fly ash depicting a spherule with iron oxide crystals emerging from the matrix.

Fig. 2: SEM micrograph from the sample of biomass fly ash depicting an unburnt hay fragment. In the surrounding material, glassy nanometric sized spherules can be recognised.

Fig. 3: Micro-Raman spectrum of a spherical body in fly ash from the combustion of lignite (shown in the inset picture). Hematite (Fe2O3) Raman bands are indicated.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1800 Elucidation of Formation Mechanism of Micro/nano-structures through Competitive Reactions during Initial Hydrogenation in Mg/Cu Super-laminate Composites

Tanaka K.1, Shibata K.2, Ikeuchi S.2, Kikuchi S.3, Kondo R.2, Takeshita H. T.2
1National Institute of Advanced Industrial Science and Technology (AIST), 2Kansai University, 3The University of Shiga Prefecture
koji.tanaka@aist.go.jp

  Mg is one of the promising candidates among hydrogen storage materials because of its abundance, inexpensiveness, light weight, and hydrogen absorption capacity of 7.6 mass% to form MgH2. However, the standard formation enthalpy of MgH2 is -76 kJ(mol H2)-1 and too low to achieve hydrogen desorption under moderate conditions. Another problem is the sluggish reaction of Mg with H2 gas. Thus, various Mg-based alloys and compounds have been investigated to improve the rate and to lower the temperature of hydrogen absorption/desorption.
  Super-laminate composites (SLCs) have been attracting attention since Ueda et al. reported that Mg/Cu SLCs showed reversible hydrogen absorption/desorption at 473K [1]. The improvement of hydrogen absorption/desorption kinetics, its relations with micro/nano-structures, and the effect of initial structures of Mg/Cu SLCs on hydrogen absorption/desorption properties have been reported in previous papers for Mg2Cu-H2 system [2, 3]. However, the structure-property relationship of Mg/Cu SLCs is not fully understood yet. In this paper, we examined the formation mechanism of micro/nano-structures through competitive reactions during initial hydrogenation in Mg/Cu SLCs.
  As shown in fig. 1, three types of MgCu2 forms, (a) an open 3D-network, (b) a sheathing 3D-network and (c) a layer, were observed after hydrogenation of Mg/Cu SLCs at 573K and 3.3MPa of H2 for 86.4ks. It is known that Mg2Cu shows a disproportionation reaction to MgH2 and MgCu2 during hydrogenation like fig. 1(a). We propose that Mg/Cu SLCs could be hydrogenated by other two types of processes [3]. The one is simultaneous hydrogenation of Mg and alloying Mg with Cu to Mg2Cu followed by hydrogenation of Mg2Cu, leading to the formation of sheathing MgCu2 3D-network. The other is hydrogenation of Mg followed by a reaction of MgH2 with Cu, leading to the formation of MgCu2 layer.
  In order to elucidate the formation mechanism, Mg/Cu SLCs, pellets of MgH2 and Mg2Cu, and those of MgH2 and Cu powder as references were prepared, and micro/nano-structures of them were examined with SEM. Mg/Cu SLCs were hydrogenated at 573 K and 3.3 MPa of H2 for 86.4 ks, whereas two kinds of pellets were heated at various temperatures and hydrogen pressures for 86.4 ks.
  The formation of sheathing MgCu2 3D-network and layered MgCu2 is confirmed by SEM observations of a pellet of MgH2 and Mg2Cu powder and those of MgH2 and Cu powder, respectively.

References
[1] T. T. Ueda, M. Tsukahara, Y. Kamiya and S. Kikuchi, Japan Inst. of Metals 2004 Spring Meeting Abstracts, (2004) 170.
[2] K. Tanaka, T. Kiyobayashi, N. Takeichi, H. Miyamura, and S. Kikuchi., J. Mater. Sci. 43 (2008) 3812.
[3] K. Tanaka, H. T. Takeshita, K. Kurumatani, H. Miyamura, and S. Kikuchi, J. Alloy Compd., 580 (2014) S222

 

This work was supported by JSPS KAKENHI Grant Number 23560794.

Fig. 1: Fig. 1 Back Scattered Electron Images of Mg/Cu SLCs after hydrogenation under the conditions of 573 K, 86.4 ks and 3.3 MPa of H2. (a) Hydrogenation of Mg is late after alloying Mg with Cu. (b) Hydrogenation of Mg and alloying Mg with Cu starts at the almost same time. (c) Hydrogenation of Mg is early before alloying Mg with Cu.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1808 In situ TEM observation of Pt/C in reactant gases of the polymer electrolyte fuel cell

Shimizu T.1, Imamura D.1, Yaguchi T.2, Kamino T.1,3
1Japan Automobile Research Institute, Tsukuba, Japan, 2Hitachi High-Technologies Corporation, Hitachinaka, Japan, 3Fuel Cell Nanomaterials Research Center, University of Yamanashi, Kofu, Japan
tshimizu@jari.or.jp

Among the advanced energy sources, the polymer electrolyte fuel cell, PEFC, is one of the most attractive options for automobile application. Two major issues to facilitate widespread dissemination of fuel cell vehicles are the cost and durability of the electrocatalyst. Many efforts are made to develop the electrocatalyst to meet such demands. However, it is necessary to clarify the details of degradation mechanism. In our previous study, in situ TEM observation was carried out to investigate the degradation mechanism of the Pt/C electrocatalyst, focusing on the effect of humidity in the air. We found that the oxidation of carbon support was accelerated at the interface of Pt particle and carbon support in the high humidity condition. In this study, the effect of reactant gases such as hydrogen, nitrogen, oxygen, on the structural change of the Pt/C was investigated. The catalyst sample was Pt supported on high surface area carbon, Pt/C, purchased from TKK. The in situ observation of the catalyst was carried out with a Hitachi H-9500 environmental TEM equipped with an AMT TV camera system. A specimen-heating holder with a gas-injection nozzle was employed for both heating of the specimen and introduction of the reaction gas. The reaction temperature, time, and the total pressure near the specimen was controlled to 220°C, 30 min, and approximately 0.6 Pa, respectively. Although the typical operation temperature of the PEFC ranges from 80°C to 100°C, the reaction temperature of 220°C was chosen to accelerate the in situ reaction with the gases. In the in situ observation, density of electron beam on the specimen was controlled to minimize the radiation damage of the specimen. Figure 1 shows a TEM image (a) and the corresponding selected area diffraction (SAD) pattern (b) of the Pt/C electrocatalyst before reaction. Figure 2 shows a TEM image (a) and the corresponding SAD pattern (b) obtained from the Pt/C electrocatalyst after in situ reaction under oxygen atmosphere. During the in situ reaction, the morphology of the carbon support was gradually changed due to the oxidation (Fig. 2(a)). An analysis of the SAD pattern revealed that the Pt nano particles were agglomerated and crystallized (Fig. 2(b)). We also carried out in situ observation under hydrogen and nitrogen atmosphere. Various interesting phenomena such as a slight shrink of the carbon support and a little increase in the crystallinity of Pt nano particles were observed and characterized.

This work was partly supported by JSPS KAKENHI Grant Number 23510136.

Fig. 1: Pt/C electrocatalyst before reaction. (a) TEM image and (b) selected area diffraction pattern.

Fig. 2: Pt/C electrocatalyst after reaction with O2 (0.6 Pa) at 220°C for 30 min. (a) TEM image and (b) selected area diffraction pattern.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-1988 HRTEM and STEM-EELS study of thin films Pt-CeOx nanocatalysts for on-chip fuel cell technology

Potin V.1, Simon P.1, Lavkova J.1,2, Matolinova I.2, Matolin V.2
1Laboratoire Interdisciplinaire Carnot de Bourgogne, Université de Bourgogne, France, 2Department of Surface and Plasma Science, Charles University, Czech Republic
vpotin@u-bourgogne.fr

In recent years, cerium oxide based materials such as Pt-cerium oxide have received much attention because of their excellent catalytic properties for a variety of reactions. Among others, fuel cells offer an interesting application field of these materials. It was shown that sputtered thin cerium oxide films containing Pt, which had been deposited on the anode side of a fuel cell, exhibited a higher specific power compared to a conventional Pt−Ru catalyst [1-2]. Besides the large scale fuel cells, there is also an increasing interest in miniature fuel cells fabricated on silicon, which could be used as an on-chip power supply for portable electronic devices.

In this study, nanometric Pt-ceria thin films were characterized by TEM after elaboration by physical vapor deposition on various substrates (silicon, carbon foils, carbon nanotubes …). The deposited layers exhibited different morphologies linked to the different substrates [3-4]. More particularly, deposition carried out directly on silicon substrate is linked to flat surface layer whereas elaboration on carbon substrates (nanotubes, carbon foils, intermediate carbon layer grown on silicon substrate) is linked to the presence of porous surfaces. In addition to the substrate type, many effects as the formation of carbides or silicates at the interface, an interaction of ceria with platinum and the presence of the porosity influenced also the structure and the chemistry of the deposited layers.

In all samples, crystallites corresponding mainly to CeO2 and to a less extent to CeC2 crystallographic structures were observed (Fig. 1). STEM-EELS measurements have been carried out on layers grown on silicon with and without intermediate carbon layer. Data analysis of the M4,5 white lines of cerium have pointed out a variation of cerium oxidation state from Ce4+ to a mixture of Ce3+ and Ce4+ depending of the localization of the measurement (Fig. 2).

[1] C. Xu, R. Zeng et al, Electrochimica Acta 51 (2005) p.206

[2] N.V. Skorodumova, S.I. Simak et al, Physical review letters 89 (2002) p.166601

[3] S. Bruyere, A. Cacucci et al, Surface and coating technology. 227, (2013) p.15

[4] M. Dubau, J. Lavkova et al, ACS Applied materials interfaces 6 (2014) p.1213

This research is supported by ANR within IMAGINOXE project (ANR-11-JS10-001) and EU within FP-7-NMP-2012 project chipCAT under Contract No. 310191.The authors acknowledge the support by the Czech Science Foundation under grant No. 13-10396S and J.L. is grateful to the Conseil Regional de Bourgogne (PARI ONOV 2012).

Fig. 1: a-b) HRTEM images of crystallites with corresponding zone axes

Fig. 2: STEM-EELS maps and corresponding schemes showing the oxidation state of cerium on thin layers grown on silicon substrate a-b) with and c-d) without amorphous carbon intermediate layer
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2011 In situ study of Pt-Pd nanoalloys in liquid

De Clercq A.1, Dachraoui W.1, Margeat O.1, Pelzer K.2, Henry C. R.1, Giorgio S.1
1Aix-Marseille Univ., CNRS, CINaM , UMR 7325, 13288 Marseille Cedex 9, France, 2Aix-Marseille Univ., CNRS, MADIREL, UMR 7246, 13397 Marseille Cedex 20, France
suzanne.giorgio@univ-amu.fr

Pt-Pd nanoalloys were prepared in solution from organic precursors and self-assembled in a 2D array on SiO2 substrates, the particles are homogeneous in size and self- organized on the substrate. The size histogram gives an homogeneous average size of 3.2±0.8 nm.

Compared to pure Pt particles, Pt-Pd show a better homogeneity in size and less coalescence. The in situ growth of Pt-Pd particles does not follow the same mechanism as pure Pt particles.

The crystal growth in solution was in situ studied by TEM in a liquid cell.

In situ study by TEM of a solution with Pt and Pd organic compounds in OAm, initiates the nucleation by reduction of the precursors in the electron beam. As soon as the clusters are visible in the liquid, they start to grow without coalescence. The cluster size increases as a function of the time, as t ½, , during the first minute, then the growth rate decreases, which is consistent with a growth mechanism by monomer addition. At the end of the growth, the size of the nanoalloys is between 2 and 2.5 nm.

In the series (fig. 1a-c), the same cluster is imaged in liquid during 540 sec.

The shape starts to be isotropic and facetted after 60 sec, mostly limited by (001) and (111) faces. The particle orientation changes in the liquid, but it is clearly seen along the [110] direction in fig. 1 c.

The same experiment with pure Pt precursors in OAm leads to a fast coalescence of the clusters and to the formation of larger nanoparticles with sizes dispersed between 1 and 5 nm. The nucleation and growth of pure Pt is consistent with a growth mechanism by monomer addition and coalescence events.

The different growth mechanism is probably due to the concentration of Pd atoms on the surface which prevents from the coalescence during the growth.

We thank the Region PACA for the financial support of the PhD Thesis of A. De Clercq

Fig. 1: Nanoalloys imaged in liquid during 540 sec
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2041 HR-STEM and EELS analyses of structural defects in Cu(In,Ga)Se2

Simsek E.1, Ramasse Q. M.2, Abou-Ras D.3, Mainz R.3, Weber A.3, Kleebe H. J.4, van Aken P. A.1
1Max Planck Institute for Intelligent Systems, Heisenbergstr. 3, 70569 Stuttgart, Germany, 2SuperSTEM, STFC Daresbury Laboratories, Keckwick Lane, Warrington, WA4 4AD, United Kingdom, 3Helmholtz Zentrum Berlin, Hahn Meitner Platz 1, 14109 Berlin, Germany, 4Technische Universität Darmstadt, Schnittspahnstr. 9, 64287 Darmstadt, Germany
simsek@is.mpg.de

Cu(In,Ga)Se2 (CIGSe) thin-film solar cells have generated interest with their high power-conversion efficiencies of more than 20% [1,2]. However, in many cases, efficiencies obtained with polycrystalline CIGSe solar cells fall behind this value. This is particular true for solar cells with CIGSe layers produced in multi-stage coevaporation processes without a Cu-rich stage at low temperatures [3]. The reason behind these energy losses and the limitations for the further efficiency increase are not fully understood. In order to gain a better understanding of the compositional properties of the structural defects, which can be relevant for solar cell performance, we analysed the CIGSe absorber layers by high-resolution scanning transmission electron microscopy (HR-STEM) in combination with electron energy-loss spectroscopy (EELS). We obtained Z-contrast images with sub-nanometer resolution and chemical data from the corresponding regions.

Thin foils are prepared by using a focused ion beam (FIB) machine, which produces homogenously thick TEM lamellae along the CIGSe layer. HR-STEM and EELS analyses show striking chemical characteristics for a number of defects present in Cu-poor CIGSe thin films. The elemental distributions at {112} twin planes, which are very frequent in these samples, are the same ones as in the grain interiors, with a homogeneous distribution of all matrix elements (Figure 1). By contrast, within the complex defects, which are closely related to stacking faults, Cu enrichment in combination with In and Se depletion are observed. Cu enrichment and In depletion are also detected at random grain boundaries. However, Se is homogenously distributed in this case. Finally, Cu-Se-rich channels seem to form immediately outside (not within) dislocation cores (Figure 2). The present contribution provides a discussion on the impact of the growth process on the chemical properties of extended structural defects in CIGSe thin films.

References:

[1] P. Jackson, D. Hariskos, E. Lotter et al., Prog. Photovoltaics 19, 894-897 (2011)
[2] Press release, 19.1.2013, EMPA, Switzerland
[3] R. Caballero, C. A. Kaufmann, V. Efimova et al., Prog. Protovolt: Res. Appl. 21, 30-46 (2013)

The work was supported in part by Helmholtz Virtual Institute HVI-520 “Microstructure Control for Thin-Film Solar Cells”. The research leading to these results has received funding from the European Union Seventh Framework Program [FP/2007-2013] under grant agreement no 312483 (ESTEEM2).

Fig. 1: Z-contrast image (a) of the twin boundary along the {112} plane and corresponding elemental map (b) from the EEL spectrum image. Green, blue and red colours show the Cu, In, Se elements respectively.

Fig. 2: Z-contrast image (a) of a dislocation core, corresponding elemental map (b) from the EEL spectrum image. Green, blue and red colours indicate the Cu, In, Se elements respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2074 Cerium reduction at the interface between ceria and yttria-stabilised zirconia and implications for interfacial oxygen non-stoichiometry

Song K.1,2, Schmid H.3, Srot v.1, Gilardi E.4, Gregori D.4, Du K.2, Maier J.4, van Aken P. A.1
1Max Planck Institute for Intelligent Systems, Stuttgart, Germany, 2Shenyang National Laboratory for Materials Science, Chinese Academy of Sciences, Shenyang, China, 3INM-Leibniz Institute for New Materials, Saarbrücken, Germany, 4Max Planck Institute for Solid State Research, Stuttgart, Germany
vanaken@is.mpg.de

CeO2 and Y2O3-stabilized zirconia (YSZ) are two typical candidates for electrolyte materials in solid oxide fuel cells attributed to their high ion conductivities. Theoretical calculations indicate that the oxygen vacancy formation energy is considerably reduced at interfaces and oxygen vacancies expected to segregate to the interfaces might provide highways for rapid ion conduction [1]. The aim of our work is to obtain insights into the structure and chemistry of interfaces between CeO2 and YSZ by scanning transmission electron microscopy (STEM) combined with electron energy-loss spectroscopy (EELS).

Epitaxial CeO2 films were grown on YSZ (111) substrates using pulsed laser deposition. Figure 1 shows an HRTEM micrograph of the CeO2/YSZ (111) interface viewed from the [1-10]YSZ direction. The CeO2 film is approximately 30 nm thick and continuous where the CeO2 film and YSZ substrate have a cubic on cubic orientation relationship ((111) <1-10>CeO2 // (111) <1-10>YSZ). No reaction layers or other phases can be observed at the interface. Periodical misfit dislocations were observed at the interface with extra atomic planes appearing in YSZ.

Figure 2a shows an EELS line scan across the CeO2/YSZ interface, where the extracted EELS spectra (Figure 2b) illustrate the change of Ce-M4,5 edges from the bulk to the interface. It is well known that Ce-M4,5 edges are valence sensitive. Since the presence of Ce3+ is seen as evidence of oxygen vacancy formation, oxidation states of cerium ions near the interface were investigated by EELS for which the intensity ratios of the Ce-M4,5 white lines (Figure 3) were analyzed. Measured spectra were compared with known reference spectra acquired from compounds containing Ce3+ or Ce4+. In addition, quantitative analysis has been performed on the Ce-M4,5 edges to study the ratio of Ce3+ to Ce4+ as a function of the distance from the interface. Thus most of the Ce ions were reduced from Ce4+ to Ce3+ at the interface region with a decay of several nanometers. Possibilities of charge compensations are discussed. Irrespective of the details, such local non-stoichiometries are crucial not only for understanding charge transport in such hetero-structures but also for understanding ceria catalytic properties [2].

References:

[1] M. Fronzi et al., Physical Review B 86 (2012) 085407.

[2] K. Song et al., APL Materials (2014), accepted.

The authors acknowledge funding from the PhD exchange program between the Max Planck Society and the Chinese Academy of Sciences and the Natural Sciences Foundation of China (Grant No. 51221264). The research has received funding from the European Union Seventh Framework Program [FP/2007-2013] under grant agreement no 312483 (ESTEEM2).

Fig. 1: Microstructure of the film shown by the HRTEM micrograph of the CeO2/YSZ (111) interface viewed from the [1‑10]YSZ direction. The interface indicated by the arrow shows periodic misfit dislocations.

Fig. 2: EELS spectrum image from a line scan (top) with Ce-M4,5 spectra (bottom) extracted from the spectrum image, which illustrates the change of Ce-M4,5 edges from the bulk (spectrum 11) to the interface (spectrum 1).

Fig. 3: Ce-M5/M4 white-line intensity ratio vs distance from the CeO2/YSZ interface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2117 Determination of group III atoms gradient throughout CuIn1-xGaxSe2 thin films by plasmon excitation in electron energy loss spectroscopy

Gautron E.1, Arzel B.1, Brohan L.1, Barreau N.1
1 Institut des Matériaux Jean Rouxel (IMN)-UMR 6502, Université de Nantes, CNRS, 2 rue de la Houssinière, BP 32229, 44322 Nantes Cedex 3, France
eric.gautron@cnrs-imn.fr

Lab-scale CuIn1-xGaxSe2 (CIGSe)-based solar cells demonstrate not only the best thin film conversion efficiency (20.8 %) but also overtake the multicrystalline silicon technology (20.4 %). Despite this new record, CIGSe cells efficiency is still far from the theoretical Shockley–Queisser limit. The improvement of the CIGSe technology will not be performed without a better characterization of the material at the nanometer scale. Transmission electron microscopy (TEM) associated with other related techniques like spectroscopy are key tools to reach this objective. However new applications based on “classical” TEM techniques have to be considered to further gain knowledge.

The [Ga]/([Ga]+[In]) ratio (GGI) throughout the CIGSe thin films has been demonstrated for a long time to be of major importance to reach high efficiencies. The present contribution aims at demonstrating the ability of measuring this GGI gradient not by energy dispersive X-ray spectroscopy (EDS) as usually performed but by using electron energy loss spectroscopy (EELS). EELS spectra intensities in the low loss region are high enough to allow very fast acquisitions with low current densities, minimizing irradiation damage and drift during analysis. A systematic study on synthesized powders with different GGI values (figure 1) was performed to establish the dependency of the plasmon energy Ep on GGI ratio. Ep was defined by fitting the volume plasmon peak with the Drude model. This allowed obtaining a calibration curve GGI=f(Ep) which was then applied on CIGSe deposited by a classical 3-stage process (figure 2). GGI gradient obtained by EELS (figure 3) was compared to EDX and secondary ion mass spectroscopy (SIMS) results to demonstrate the reliability of the method. Two CIGSe thin films deposited in the same conditions but on substrates containing different sodium quantities were analyzed. The results are discussed related to photovoltaic performances.

The authors want to acknowledge Marc Souilah, Catherine Guillot-Deudon and Alain Lafond for the supply of CIGSe powders.

Fig. 1: Low loss region EELS spectra acquired on synthesized powders with different GGI values. The calibration curve GGI=f(Ep) was extracted from those data.

Fig. 2: Cross section TEM micrograph of a CuIn1-xGaxSe2 thin film. The film is made of large grains but the GGI ratio varies inside each of them. The CIGSe film is 2 µm thick.

Fig. 3: GGI gradient obtained by EELS on the film shown on figure 2. GGI values were obtained with the calibration curve GGI=f(Ep).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2189 Microstructural evolution of the interaction zone between U–9 wt.% Mo fuel and Zr–1 wt.% Nb cladding alloys

Neogy S.1, Laik A.1, Saify M. T.2, Srivastava D.1, Jha S. K.2, Dey G. K.1
1Materials Science Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India, 2Atomic Fuels Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India
neosuman@barc.gov.in

A few very recent studies have identified the ability of Zr in acting as a diffusion barrier to reduce the deleterious fuel–clad chemical interaction (FCCI), which restricts nuclear fuel designers around the world to successfully utilize the potential of the gamma-phase stabilized U–Mo alloys as reduced enrichment fuels in research and test reactor. Further investigations pertaining to metallurgical interaction between U-Mo and Zr are essential not only to establish Zr as a diffusion barrier in U-Mo fuel but also to envisage Zr-base alloys as cladding as against the currently used Al-alloys.

In this work, metallurgical interaction between U–9 wt.% Mo metallic fuel alloy and Zr–1 wt.% Nb clad material has been assessed through scanning electron microscopy (SEM), electron probe microanalysis (EPMA) and transmission electron microscopy (TEM). Interdiffusion of constituent elements across the fuel–clad interface, together with the phase reactions occurring at high temperature and during subsequent cooling, resulted in development of a layered interaction zone where coexistence of a bcc solid solution phase with varying compositions, along with alpha-U, alpha-Zr and Mo2Zr phases could be noticed (Fig. 1). The instability in the gamma-U(Mo,Zr) matrix leading to phase separation into alpha-U and alpha-Zr phases and the orientation relationships amongst them were established through microdiffraction and composite selected area electron diffraction (SAED) patterns, respectively. The present study is an endeavor to rationalize these observations, which remain unexplained in literature.

Fig. 1: BSE image of the interaction layer (IL) between U–9Mo and Zr–1Nb. Inset shows typical microdiffraction patterns of alpha-Zr and Mo2Zr phases.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2199 Impact of the nanostructure on the electrochemical performance of Li-rich cathode materials

Riekehr L.1, Liu J. L.3, Schwarz B.2, Schmitt L. A.1, Xia Y.3, Ehrenberg H.2
1Technische Universität Darmstadt, Fachbereich Material- und Geowissenschaften, 64287 Darmstadt, Germany, 2Karlsruher Institut für Technologie (KIT), Institut für Angewandte Materialien (IAM), 76131 Karlsruhe, Germany, 3Department of Chemistry, Institute of New Energy, Fudan University, Shanghai 200433, People's Republic of China
lriekehr@st.tu-darmstadt.de

In search for high capacity and high energy cathode materials for the next generation Li-ion batteries (LIB), Li-rich cathode materials are promising candidates. This material class is derived from the classical layered cathode ceramics, such as LiCoO2, commonly used in commercial LIBs. In order to improve stability and capacity, these structures have been blended with Li2MnO3, which can be considered as Li-rich layered material (Li[Li1/3Mn2/3]O2) that is well mixable with classical layered cathode materials [1-3]. The structures of the rhombohedral Li(TM)O2 (TM = Ni, Co, Mn) and the monoclinic Li2MnO3 are depicted in Fig. 1.
It is well known that the synthesis conditions influence the nanostructure and hence the electrochemical performance of Li-rich cathode ceramics [4-6]. In the present study, a molten salt and a coprecipitation synthesis route were chosen in order to prepare two different kinds of samples with the same nominal stoichiometry of 0.5Li2MnO3:0.5Li(Ni1/3Co1/3Mn1/3)O2, hereinafter called MS55 and CP55, respectively. Results of the electrochemical characterization are depicted in Fig. 2. The charge-discharge curves show better characteristics concerning energy density for the MS55 sample in the first cycle and after the 70th cycle. The CP55 shows a lower first discharge capacity and a higher voltage drop after the 70th cycle. High resolution transmission electron microscopy (HRTEM) and electron diffraction has been used to investigate the nanostructure of the pristine material blends. The HRTEM micrograph depicted in Fig. 3 shows the nanostructure of the MS55 sample. The HRTEM pattern can be interpreted as intergrowth of Li(TM)O2 and Li2MnO3 on an atomic scale with only short range monoclinic ordering. Fig. 4 shows the HRTEM pattern obtained from a CP55 crystallite. In contrast to the MS55 sample, the nanostructure consists of blocks which can be described exclusively by monoclinic symmetry and parts which show contrast comparable to the MS55 sample.
This experiment shows the impact of the nanostructure of Li-rich cathode ceramics with the same nominal stoichiometry on the electrochemical performance. It is necessary not only to consider the composition but also to analyze the nanostructure in order to compare the electrochemical performances of nominal the same material, which makes TEM an obligatory tool in the field of research for new Li-rich cathode materials.

[1] Thackeray, M. et al., J. Mater. 17, (2007)
[2] Thackeray, M. et al., Electrochem. Commun. 8, 1531–1538 (2006)
[3] Jarvis, K. et al., J. Mater. Chem. 22, 11550 (2012)
[4] Bréger, J. et al., J. Solid State Chem. 178, 2575–2585 (2005)
[5] Boulineau, A. et al., Solid State Ionics 180, 1652–1659 (2010)
[6] Liu, J.L., et al., J. Electrochem. Soc., 161 (1) A160-A167 (2014)

This work was financially supported by the Sonderforschungsbereich 595 of the Deutsche Forschungsgemeinschaft.

Fig. 1: a) rhombohedral Li(TM)O2 with R-3m symmetry b) monoclinic Li2MnO3 with C2/m symmetry

Fig. 2: Charge-discharge characteristic a) for the first and b) after the 70th cycle

Fig. 3: HRTEM micrograph of the MS55 sample. The pattern can be interpreted as intermixing of Li(TM)O2 and Li2MnO3 on an atomic scale

Fig. 4: HRTEM micrograph of the CP55 sample. The particle contains parts which are monoclinic only and parts that are intermixed on the atomic scale
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2300 Development of Ultra-thin Supercapacitors for Energy Storage

Canavan M.1,3,4, Coelho J.2,3,4, Mendoza-Sanchez B.2,3,4, Pettersson H.1,3,4, Long E.1,3,4, Nicolosi V.1,2,3,4
1School of Physics, Trinity College Dublin, Dublin, Ireland, 2School of Chemistry, Trinity College Dublin, Dublin, Ireland, 3CRANN, Trinity College Dublin, Dublin, Ireland, 4AMBER, Trinity College Dublin, Dublin, Ireland
mcanava@tcd.ie

Supercapacitors currently bridge the gap between batteries and electrostatic capacitors.1 They store energy electrochemically, using reversible adsorption of charges of an electrolyte onto two porous electrodes and the formation of the so-called electric double layer at an electrode/electrolyte interface.2 The aim of this research is to create thin, flexible, high energy density, high power density energy storage devices.

An ultra-thin supercapacitor was produced by exfoliating thin nanoflakes of MnO2 and exhibited good capacitive behaviours. These nanoflakes were produced by dispersion and liquid-phase exfoliation of MnO2 powder in organic solvents such as Benzyl Alcohol.3 The initial stage of this project work showed the flakes can be successfully dispersed in solvents whose surface energy matches that of the MnO2 itself (≈ 37mJ/m2). The presence of MnO2 flakes in the dispersion was confirmed by Transmission Electron Microscopy imaging. Flakes with lateral sizes of 5-20 nm and an estimated thickness of 3-4 layers were observed. The flakes were deposited by ultrasonic spraying onto glass ITO (Indium Tin Oxide) substrates. The capacitive behaviour of the MnO2 electrodes were characterised electrochemically by cyclic voltammetry in O.5M K2SO4 electrolyte. Maximum capacitances of 105 Fg-1/76µFcm-2 were obtained at a scan rate of 5mV/s. It was found that the capacitive behaviour decreased with increased scan rate. It was concluded that exfoliated thin flakes of MnO2 still maintain the same excellent storage capabilities of bulk MnO2, showing promise to be used as a component for hybrid supercapacitors.1,4

References

1.X. Zhao, B. M. Sánchez, P. J. Dobson, and P. S. Grant, “The role of nanomaterials in redox-based supercapacitors for next generation energy storage devices.,” Nanoscale, vol. 3, no. 3, pp. 839–55, Mar. 2011.
2.U. C. Davis and A. Burke, “Ultracapacitors : why , how , and where is the technology,” Journal of Power Sources, vol. 91, pp. 37–50, 2000.
3.Y. Hernandez et al “High-yield production of graphene by liquid-phase exfoliation of graphite,” Nature Nanotechnology, vol. 3, pp. 563–568, 2008.
4.J. N. Coleman et al “Two-Dimensional Nanosheets Produced by Liquid Exfoliation of Layered Materials,” Science, vol. 331, pp. 568–571, 2011.

I would like to thank Joao Coelho, Beatriz Mendoza Sanchez, Henrik Pettersson, Edmund Long, Sean O’Brien and Prof. Nicolosi for all their help and guidance through my project. I am grateful for the advice and encouragement given by all members of the Nicolosi research group.

Fig. 1: TEM image of MnO2 flakes obtained using Benzyl Alcohol under optimum sonication conditions with initial concentration of 10 mg/ml
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2304 High resolution analysis of silicon nano-particle surfaces for lithium-ion batteries

Delpuech N.1, Turner S.2, Lajaunie L.1, Bridel J. S.3, Lestriez B.1, Moreau P.1, Van Tandeloo S.2, Guyomard D.1
1Institut des Matériaux Jean Rouxel (IMN), Nantes, France, 2EMAT, Antwerp, Belgium, 3Umicore Group Research & Development, Olen, Belgium, 4Réseau sur le Stockage Electrochimique de l’Energie (RS2E), France
Philippe.Moreau@cnrs-imn.fr

Silicon based electrodes are much more attractive anode materials for lithium-ion batteries than graphite due to a very high gravimetric energy density (3572 mAh/g vs. 372 mAh/g for carbon) [1]. An important aspect influencing their performance is the initial state of the Si nanoparticle surface [2]. The purpose of this study is to determine at the sub-nanometer scale the exact state of this surface.

In order to complement previous EELS analyses performed on a HF2000, high resolution spectra (both spatially and energy resolution) were performed at the EMAT laboratory in Antwerp (BE) with an FEI Titan “cubed” 50 – 80. By examining multiple particle surfaces, clear changes are observed as a function of the treatment applied to the pristine powder. Thickness and chemical states as observed at the Si L2,3 edge can be determined and interpreted in conjunction with other techniques like FTIR and solid state NMR.

The low loss region can also be characteristic of the chemical state of the surface. Peaks in the 6-10 eV region are often observed but their interpretation is a complex mixture of interface plasmons and interband transitions. Simulations [3] provide a necessary complement and further assessment of the pertinent parameters modified when various silicon surfaces are analyzed.

This study demonstrates the capabilities of sub-nanometer EELS analyses for the monitoring of the interaction of nanoparticles with their environment.

References
[1] D. Mazouzi et al., J.Electrochem. Solid State Lett. 12 (2009) A215.
[2] Y. Oumella et al., J. Mater. Chem. 21 (2011) 6201.
[3] C. A. Walsh, Computer Programs for the Calculation of Electron Energy-Loss Spectra from Interfaces Between Dielectric Media, Cavendish Laboratory, Cambridge, UK, 1992.

Fig. 1: Example of high resolution spectra obtained on a silicon nanoparticle surface after chemical treatment. The 3 successive spectra are spaced by less than 1 nm and the most extreme spectrum displays a signal indicative of Si2+. The HAADF signal is presented in the insert.

Fig. 2: Simulations of low loss EELS spectra considering a 1 or 2.2 nm SiO2 layer on top of silicon for a spherical geometry. The spectra correspond to the aloof beam situation, just like in the experimental spectrum (in red).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2398 Investigating the structure of iron disulfide (pyrite) using high-angle annular dark-field and annular bright-field scanning transmission electron microscopy and electron energy loss spectroscopy for energy applications

Wen C. Y.1, Liou S. C.2
1Department of Materials Science and Engineering, National Taiwan University, Taipei, Taiwan, 2Center for Condensed Matter Sciences, National Taiwan University
cwen@ntu.edu.tw

              Iron disulfide (FeS2) is an earth-abundant and environmental-friendly semiconductor material, which has great potential in photovoltaic and photoelectrochemical applications [1, 2]. It has recently been shown that nanostructured FeS2 can also be used as the cathode in rechargeable batteries with high capacity [3]. On the other hand, synthesised FeS2 nanoparticles may have a sulfur-deficient surface structure, which degrades the efficiency in solar cell applications [1]. In order to find out suitable reaction routes in FeS2 for rechargeable batteries or to improve the surface structure of FeS2 nanoparticels, reliable microanalysis methods to understand the structural characteristics of this material are essential.
              In this work, we first use Cs-corrected scanning transmission electron microscopy (Cs-STEM) to study the structure of natural iron disulfide (pyrite). Pyrite has the space group Pa-3 with the lattice parameter of 5.41Å [4]. Figures 1(a) and (e) show the high-angle annular dark-field (HAADF) images along the [001] and [-211] axes of pyrite, respectively. The intensity in the HAADF images is proportional to z1.7 (where z is the atomic number) [5], so that the columns of iron atoms (z=26), labeled by the green circle in Figs. 1(a) and (e), present higher contrast, whereas those of sulfur atoms (z=16), label by the orange circles, are difficult to display. In order to resolve the sulfur atomic columns, the annular bright-field (ABF) imaging method is used with collection angles between 7 to 18 mrad [5]. In Figs. 1(c) and (g), the iron columns appear to be dark, while the sulfur columns appear to be grey. The stronger contrast difference between iron and sulfur in the ABF images makes the locations of the iron and sulfur atomic columns more distinguishable. These HAADF and ABF images are consistent with those simulated by xHREMTM software [6] (see Figs. 1(b), (d), (f), and (h)). It is therefore useful to combine HAADF and ABF images in Cs-STEM for studying the crystal structure in pyrite.
              The representative electron energy-loss near-edge structure (ELNES) of sulfur L2,3 and iron L2,3 edges of pyrite are shown in Figs. 2 (a) and (b), respectively. The variation of the spectral features in the ELNES of S-L2,3 and Fe-L2,3 edges during the lithiation/delithiation processes of rechargeable batteries will be discussed.

References

[1] D.-Y. Wang, et al., Adv. Mater. 24, 3415 (2012).

[2] D. Richard and G. W. Luther, Chem. Rev. 107, 514 (2007).

[3] L. Li, et al., Nanoscale 6, 2112 (2014).

[4] R. W. G. Wyckoff, Crystal Structures v. 1, (1963).

[5] S. J. Pennycook and P. D. Nellist, eds. Scanning Transmission Electron Microscopy: Imaging and Analysis. Springer (2011).

[6] http://www.hremresearch.com.

This work is supported by the National Science Council of Taiwan (Project no. NSC 100-2112-M-002-019-MY3).

Fig. 1: (a-d) HAADF, simulated HAADF, ABF and, simulated ABF images of pyrite along the [001] axis, respectively. (e-h) HAADF, simulated HAADF, ABF, and simulated ABF images of pyrite along the [-211] axis, respectively. The green and orange circles label respectively the locations of iron and sulfur atomic columns.

Fig. 2: EELS spectra of (a) sulfur L2,3 and (b) iron L2,3 edges of pyrite.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2406 EFFECTS OF HYDROGEN AND RADIATION DAMAGE OF Zr-1% Nb and Zr-2.5% Nb

Vazquez C.1,2, Fortis A. M.1,2, Bozzano P. B.1,2, Versaci R. A.1,2
1Gerencia de Materiales, Centro Atómico Constituyentes-CNEA, Av. Gral. Paz 1499, (B1650KNA), San Martín, Buenos Aires, Argentina , 2Instituto Sabato - UNSAM/CNEA, Av. G. Paz 1499 (B1650KNA) San Martín, Buenos Aires, Argentina.
versaci@cnea.gov.ar

Zirconium alloys are constitutive materials of several components of nuclear fission reactors. Thus, the study of their microstructure and mechanical properties, which are affected by radiation throughout useful life, is essential either for security reasons or eventually for the life extension of the utility.
In the core of a power reactor radiation damage as well as the hydrogen entry occur simultaneously, affecting the mechanical properties. Neutron irradiation that is generated within the reactor forms defects which eventually agglomerate acting as barriers to the movement of dislocations, increasing the mechanical strength, and a loss the ductility.
The aim of this work is to analyze microscopically the interaction between hydrides and crystal defects, in order to interpret how that affects the mechanical properties such as hardness and ductility [1]. The structures of Zr-2.5%Nb and In Zr-1%Nb hydrided were analyzed by transmission electron microscopy.
Zr-2.5%Nb specimens were irradiated at room temperature in the CNEA-RA1 nuclear reactor. They were placed in a capsule located in one of the reactor irradiation channels where the neutron where neutron flux is well known. In this case, neutron fluence was 1.8x1022nm-2 after an irradiation of 500h δ and ε type hydrides parallel to [3 2 1 1] planes. This is consistent with results shown in the literature [2]. In the bright field micrograph “black dots” were observed as in the non irradiated materials. So we can identify them as hydrides and not as a result of the radiation damage. The irradiation dose was not sufficient to see the agglomerate of defects [3].
In Zr-1%Nb, TEM micrographs show hydrides present in the α-Zr matrix located transverse to the traction direction. These hydrides have been characterized as needle-shaped type ﻉ (zeta), with hcp structure, lattice parameters a= 0.33 nm and c=1.029 nm, corresponding to a trigonal crystal with space group P3m1. They are parallel to [0001] planes of α-Zr matrix [4].
At the present, more experiments are on going to clarify the presence and types of hydrides in both alloys.

REFERENCES
[1] A.M. Fortis y C.A. Vazquez, Procedia Materials Science 1 (2012) 520-527.
[2] W. Qin, N.A. P. Kiran Kumar, J.A. Szpunar, J. Kozinski, Acta Materialia 59 (2011), p 7010-7021.
[3] B.V. Cockerram, R.W. Smith, K.J. Leonard, T.S. Byun, L.L. Snead, Journal of Nuclear Material 418 (2011), p 46-61.
[4] Z.Zhao, M. Blat-Yrieix, J-P. Morniroli, A. Legris, L. Thuinet, Y. Kihn, A. Ambarrd, L. Legras, Journal of ASTM International, Vol 5(2008) p 29-50.

Fig. 1: a) Bright field TEM images of ε hydrides in a α-Zr matrix of irradiated Zr-2.5%Nb, b) electron diffraction pattern (zone axis [0 1 0]) of an identified ε hydride, c) dark field image of the hydride shown in b).

Fig. 2: a) TEM bright field images of ﻉ hydrides in α-Zr matrix of Zr-1%Nb, b) enlarged images, c) electron diffraction pattern of the hydrides (zone axis [0001]).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2433 The Nanoscale Mechanisms of Zircaloy Corrosion in Simulated Nuclear Reactor Conditions

Annand K. J.1, MacLaren I.1, Gass M.2
1University of Glasgow, Glasgow, Scotland, 2AMEC Clean Energy Europe, Warrington, England
k.annand.1@research.gla.ac.uk

The nanoscale details of the corrosion of Zircaloy-4 under simulated nuclear reactor conditions in contact with pressurised water or steam have been studied using scanning transmission electron microscopy (STEM). Specifically, we have used dual-range electron energy loss spectroscopy (DualEELS) on a GIF Quantum mounted on our JEOL ARM200F scanning transmission electron microscope to simultaneously study changes in chemical composition and dielectric function of the material at the oxide scale – metal interface with nanometre resolution. This has allowed the correlation of the appearance of different distinct phases with the zirconium-oxygen ratio.
Under all conditions studied, oxygen diffused into the surface of the α-zirconium to levels up to or exceeding 30 atomic %, resulting in measureable plasmon shifts, but a low-loss that still looks similar to that of α-zirconium. This is strong evidence that this layer is therefore metallic α-zirconium containing some interstitial oxygen. Under some conditions, this oxygen-diffused Zirconium is in direct contact with the expected ZrO2 scale. Under other conditions where oxygen supply through the scale to the metal was limited by diffusion kinetics, an intermediate phase is found between the oxygen-diffused zirconium and the ZrO2 scale. This phase was found consistently to contain about 30-50 atomic % oxygen and is clearly a suboxide. Two examples of this are shown in Figures 1 and 2 for samples corroded in pressurised steam and water, respectively; in both cases a well defined layer (green in the false colour scale employed here) exists at the interface of order 100 nm wide. Such suboxide areas are also characterised by a well-defined low-loss spectrum which is not a linear combination of those for ZrO2 and oxygen diffused zirconium, suggesting that this is a distinct crystal phase; this is shown as the green phase in the multicolour phase maps in Figures 1 and 2.
One important difference was observed between corrosion in pressurised water (the normal intended working environment) and corrosion in steam (which should not happen in normal service but can occur in extreme situations). In corrosion in steam the morphology of the metal:oxide interface was observed towards a rougher, saw-tooth form, although the intermediate phases formed were spectroscopically identical to those formed in corrosion in pressurised water. This work shows that nanoresolved electron energy loss spectroscopy can reveal unique nanoscale insights into the precise growth mechanism of oxide scales on zircaloy under different simulated nuclear reactor conditions. This is essential to the understanding of the corrosion mechanisms and thus to the correct prediction of safe operating conditions and lifespan for fuel cladding elements.

We are grateful to AMEC Clean Energy Europe and the EPSRC for supporting this work, especially through the provision of a PhD studentship to KA. We are indebted to the University of Glasgow of SUPA for the provision of the JEOL ARM200F scanning transmission electron microscope. The support of Gatan Inc. and especially Paul Thomas in providing tools for the processing of the data was invaluable.

Fig. 1: EELS analysis of the metal:oxide interface post corrosion in steam. Top left: map of oxygen percentage with position showing an interlayer with around 30% oxygen, as also seen in a line plot across the interface. Lower right: composite of four MLLS fit maps for low loss spectra of distinct phases: red–ZrO2, green–Zr suboxide, blue–Zr, yellow-ZrHx.

Fig. 2: EELS analysis of the metal:oxide interface post corrosion in water. Top left: map of oxygen percentage with position showing an interlayer with around 30% oxygen, as also seen in a line plot across the interface. Lower right: composite of four MLLS fit maps for low loss spectra of distinct phases: red–ZrO2, green–Zr suboxide, blue–Zr, yellow-ZrHx.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2519 Electrochemical investigation and microstructural characterization of Li1.07Mn1.93O4-δ cathode materials

Xu Z.1, Zheng H.1, Gui J.1, Wang J.1
1School of Physics and Technology, Center for Electron Microscopy and MOE Key Laboratory of Artificial Micro- and Nano-structures, Wuhan University, Wuhan 430072, China
wang@whu.edu.cn

Developing advanced cathode materials with superior energy density, power density, and cycle stability has long been an engineering pursuit, since the energy and power density in lithium-ion batteries (LIBs) is largely determined by the cathode materials, which store Li by incorporation into their crystal structure.1 Li-rich and O-deficient Li1+αMn2−αO4-δ is considered as one of the most promising cathode material for secondary lithium batteries with less cost and environmental friendliness. However, it is speculated that oxygen vacancies that induce phase transitions during cycling are harmful for its cycling performance. Thus, a thorough knowledge of the structural characteristics in these materials is of wide technological relevance, which unfortunately remains unclear.

Herein, the serial Li1.07Mn1.93O4-δ samples with different oxygen vacancy δ have been investigated. With the combination of transmission electron microscopy (TEM), X-ray powder diffraction (XRD), and DIFFaX simulation techniques, it is shown that besides the predominant occurrence of cubic spinel LiMn2O4 (SG: Fd-3m), there exist two other phases: (1) the monoclinic Li2MnO3 (SG: C2/m) which exists as nano-scale lamellar variants with 120o rotational twinning stacked randomly along pseudo-three-fold axis [111]c//[103]m, has been identified using selected-area electron diffraction (SAED) and dark field (DF) imaging (Fig. 1).2 XRD simulation (Fig. 2) with DIFFaX considering the stacking disordering shows that the characteristic reflections with 2θ between 20o and 35o in the XRD pattern (CuKα) will become more and more broadened and weakened with the increasing interlayer disordering, indicating the difficulty in the XRD detecting the monoclinic Li2MnO3 phase with stacking disordering.3 Moreover, the extremely disordered stacking sequence may lead to the selective peak asymmetry (Fig. 2c). (2) Secondary tetragonal phases (SG: I41/amd) with different lattice parameters, whereas twinning and defects are commonly observed. The existence of both monoclinic and tetragonal phases will have negative effects on the electrochemical performance of the annealed materials (Fig. 3).4 Our results provide the detailed structural information about the Li1.07Mn1.93O4-δ samples and advance the understanding of corresponding electrochemical properties of this material as well as other layer structured cathode materials for LIBs.

1. Z. Xu, et al. Power Sources 248, 180 (2014).

2. Z. Xu, et al. ACS Appl. Mater. Interfaces 6, 1219 (2013).

3. H. Zheng, et al. in revision, J. Appl. Cryst. (2014).

4. Z. Xu, et al. J. Power Sources 248, 1201 (2014).

This work was supported by the 973 Program (2011CB933300), the National Natural Science Foundation of China (51071110, 51271134, 40972044, J1210061), the China MOE NCET Program (NCET-07-0640), MOE Doctoral Fund (20090141110059), and the Fundamental Research Funds for the Central Universities.

Fig. 1: (a) Experimental SAED patterns of monoclinic Li2MnO3, (b) zoomed-in SAED pattern along the [-1-12]c with pseudo-cubic indexes and (c) the corresponding DF images, which clearly illustrate the presence of three twinning variants.

Fig. 2: (a-c) Simulated XRD patterns with the increasing degree of stacking disordering using DIFFaX program.

Fig. 3: Charge/discharge voltage profiles for the initial (0.1 C) cycles and cycling performance at 1 C rate of discharge (insets) in Li1.07Mn1.93O4-δ with δ values of 0 (a) and 0.299 (b) at room temperature.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2541 Liquid cell electron microscopy of novel two-dimensional energy nanomaterials

Long E. A.1,3,4, Doherty E.2,3,4, Downing C.3,4, Nicolosi V.1,2,3,4
1School of Physics, Trinity College Dublin, Dublin, Ireland, 2School of Chemistry, Trinity College Dublin, Dublin, Ireland, 3CRANN, Trinity College Dublin, Dublin, Ireland, 4AMBER, , Trinity College Dublin, Dublin, Ireland
longed@tcd.ie

Since the discovery of graphene in 2004, two-dimensional layered materials have been the basis for many areas of research[1]. One particular area of interest was their potential use for energy storage and supercapacitors, due in part to their exceptionally large surface-area (indeed, monolayer graphene is nothing but surface and edge).

A wide range of these 2D materials can be produced through liquid exfoliation techniques, one of which involves sonicating in an appropriate solvent (one in which the solvent surface energy closely matches that of the material being exfoliated)[2]. The resulting dispersion can then be centrifuged to aid in the selection of flakes of a particular size, and then used to produce large areas of electrodes via reel-to-reel spraying. Determination of suitable materials for this process involves looking at the viscosity of suitable solvents (for the spraying), characterising the flakes both in-dispersion and as-deposited and measuring the electrochemical properties of the flakes when used as electrodes.

Within our FEI Titan 300kV S/TEM we can carry out STEM (Scanning Transmission Electron Microscopy) or CTEM (Conventional Transmission Electron Microscopy) with a range of imaging modes, in addition to EELS (Electron Energy Loss Spectroscopy) and EDX (Energy-Dispersive X-ray Spectroscopy) to gather information about flake morphology and thickness, and chemical and elemental composition. We can also use a Hummingbird Scientific fluid holder to examine the flakes in both a dispersed state and in standard electrolytes. With the use of a biasing tip for this holder, it is also possible to perform electrochemical measurements whilst simultaneously imaging the electrodes and observe the possible formation of a solid electrolyte interface.

MnO2 is a layered material that has shown particular promise recently as a supercapacitor material, which can be dispersed in a range of solvents including benzyl alcohol (BA). BA is a nice solvent to use, as it is fairly volatile, so easy to remove after spraying and is also non-damaging to the fluid holder components. Figure 1 shows an example of a flake in the fluid cell with BA around it, and can be compared with Figure 2 to see the loss in clarity resulting from the fluid and retaining Si3N4 windows. However, given a thin enough liquid layer (via growth of a bubble), it is possible to achieve lattice (Figure 3) or even potentially atomic resolution[3]. Other promising materials include MoS2, WS2 and Ga2(SO4)3.

References:
[1] K Novoselov et al , Science 306 (2004), pp. 666–9
[2] V Nicolosi et al, Science 340 (2014), pp. 1226419–1226419
[3] G Zhu et al, Chem. Commun. 49 (2013), pp. 10944–6

The authors gratefully acknowledge J Coelho, B Mendoza-Sanchez and C Backes for samples, and funding from the ERC Starting grant 2DNanoCaps, SFI PIYRA, AMBER and ERC Support grants.

Fig. 1: Figure 1: TEM Micrograph of MnO2 dispersed in benzyl alcohol

Fig. 2: Figure 2: TEM Micrograph of MnO2 drop-cast onto a holey carbon grid

Fig. 3: Figure 3: TEM micrograph showing lattice resolution in graphene through a thin layer of water within the fluid cell
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2600 Single-crystallization of LiMn0.4Fe0.6PO4 nanowires via oriented attachments

Kikkawa J.1, Hosono E.2, Okubo M.2, Kagesawa K.2, Zhou H.2, Nagai T.1, Kimoto K.1
1National Institute for Materials Science, 2National Institute of Advanced Industrial Science and Technology
KIKKAWA.Jun@nims.go.jp

Electrospinning, i.e., ejecting a jet of a polymer solution from the tip of a needle using an electric field, is a versatile method for obtaining nanowires (NWs) of various materials [1]. Nevertheless, few attempts to date have successfully fabricated single-crystalline NWs. Recently, advanced single-crystalline NWs of an olivine-structured LiMn0.4Fe0.6PO4 covered with amorphous carbon shells were fabricated using electrospinning [2]. Carbon-coated LiMn0.4Fe0.6PO4 NWs are attractive electrode materials for a high-rate lithium ion battery. However, the single-crystalline NW formation mechanism related to electrospinning remains unclear. For this study, we performed transmission electron microscopy (TEM) to investigate the formation mechanism of single-crystalline NWs from electrospun NWs by heating.

TEM observations were performed using an electron microscope (HF-3000S; Hitachi Ltd.) operated at 300 kV. Results showed that the dried NWs were amorphous (Fig. 1(a)). After heating at 600 °C for 30 min in ambient Ar, crystallized NWs were observed (Fig. 1(b)). NWs were covered with amorphous carbon shells. The selected-area electron diffraction (SAED) pattern (Fig. 1(b)) revealed single-crystalline characteristics of the olivine structure for the NW core, although the outlines of grains are discernible. We also observed polycrystalline NWs composed of crystallographically-orientated and coalesced grains, which indicates the oriented attachment is a key mechanism for single-crystallization [3]. After heating at 800 °C for 10 h in ambient Ar, amorphous gaps left between grains in the NW core (presented in Fig. 1(b)) disappeared, forming a complete crystal core. SAED revealed that single crystallization occurred almost completely beyond the range of approximately 7 μm. We think self-forming amorphous carbon shells play a key role in confining grains and maintaining NW geometry to achieve single-crystalline NW. Details of the single-crystallization mechanism is discussed.

References

[1] E. Hosono, Y. Wang, N. Kida, M. Enomoto, N. Kojima, M. Okubo, H. Matsuda, Y. Saito, T. Kudo, I. Honma, H. Zhou, ACS Appl. Mater. Interfaces, 2, 212 (2010).

[2] K. Kagesawa, E. Hosono, M. Okubo, J. Kikkawa, D. Nishio-Hamane, T. Kudo, and H. Zhou, CrystEngComm, 15, 6638 (2013).

[3] J. Kikkawa, E. Hosono, M. Okubo, K. Kagesawa, H. Zhou, T. Nagai, and K. Kimoto, J. Phys. Chem. C  (in press).

This work was partly supported by "Nanotechnology Platform"(project No. A-13-NM-0060) of MEXT, Japan.

Fig. 1: TEM images and the inset SAED patterns for dried NW after electrospinning, (a) and NW after heating at 600 °C for 30 min, (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2758 The effect of low temperature synthesis (LTS) and Na doping on the structural properties of PbTe thermoelectric materials

Delimitis A.1, Papageorgiou C.2, Kyratsi T.2
1CPERI/CERTH, Thermi, Thessaloniki, Greece, 2University of Cyprus, Nicosia, Cyprus
andel@cperi.certh.gr

PbTe-based thermoelectric (TE) materials lie among the most promising ones for conversion of ‘waste’ heat into electric energy. Their TE efficiency strongly depends on their structure and can be significantly improved by incorporation of nanoscale inclusions, due to enhanced phonon scattering at phase boundaries, interfacial dislocations and/or by band structure engineering. Therefore, bulk nanocrystalline PbTe can combine both improved properties and low cost, making the technology feasible in application level. In the current study, the effect of a novel low temperature synthesis (LTS) and Na doping on the microstructure of PbTe samples is explored using electron microscopy (TEM, HRTEM) and image analysis methods.
The morphology and crystalline quality of the undoped and the 2 at% Na-doped PbTe are depicted in Figs. 1 and 2, respectively. A significant reduction in the particle size is observed, with sizes ranging from 300-500 nm for the undoped PbTe down to 5-80 nm for the 2 at% Na sample. This inevitably leads to a nanocrystalline character for the Na-doped PbTe, as manifested in the Selected Area Diffraction (SAD) pattern inset in Fig 2, too. Na incorporation is envisaged by the formation of elliptical-shape nanoprecipitates –arrowed in Fig.2– with sizes up to 7 nm. They preferentially lie along <110> of PbTe and are located both in the particles’ interior and at grain boundaries. Their Na/Pb ratio, as measured by Energy Dispersive X-ray Spectroscopy (EDS) is up to 8.2 at%, confirming the high Na content.
Fig. 3(a) shows an HRTEM image from a grain boundary at the 2 at% Na-doped sample. PbTe particles usually exhibit (200)-type lattice fringes. A small reduction, up to 2.5%, of their fringe separation distance is measured. This is attributed to slight variations in the Pb/Te ratio, also confirmed by EDS and incorporation of Na atoms inside the host matrix. The extent of residual strain present at the nanograins was measured by the Geometric Phase Analysis (GPA) method and the results are outlined in Fig. 3(b). A uniform strain distribution inside the nanograins is found; however, sharp strain peaks, up to 16%, are commonly observed at grain boundaries.
TE measurements revealed a significant increase in the figure of merit ZT for the Na-doped PbTe (1.38 at 675K, compared to 0.2 for the undoped). This is attributed to a simultaneous increase in carrier concentration and a significant decrease in thermal conductivity due to the in situ nanostructuring which is achieved by LTS and Na incorporation and the enhanced scattering caused by the Na-rich precipitates and grain boundary residual strain present. Consequently, LTS and Na-doping is a highly promising alternative and low cost route for the preparation of PbTe with improved TE performance.

Fig. 1: TEM image and single crystal SAD pattern inset of the undoped sample. The facetted PbTe particle is oriented along [111].

Fig. 2: TEM image and corresponding SAD pattern inset from the 2 at% Na-doped PbTe, revealing the reduction in size and crystallinity of the material. Na nanoprecipitates are depicted by black arrows.

Fig. 3: (a) HRTEM image and corresponding GPA strain map (b) of a grain boundary in 2 at% Na-doped PbTe. A sharp increase of the residual strain, up to 16%, is observed at the boundary, as shown by the strain profile inset in (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2814 Microstructural Analysis of HIPped Steel for Hardfacing Applications in the Nuclear Industry

Tsivoulas D.1, Bowden D.1, Stewart D.2, Preuss M.1, Burke M. G.1
1Materials Performance Centre, The University of Manchester, Manchester, UK, 2Rolls-Royce plc, Derby, UK
dimitrios.tsivoulas@manchester.ac.uk

Hot isostatic pressing (HIP) can be an effective near-net-shape process for the manufacture of nuclear power plant valves from hardfacing alloys. There is strong motivation to replace Co-containing materials such as Stellite alloys that have been used in such applications due to the strong emission of gamma radiation by Co60 which is produced in-service. The microstructure of alloy RR2450, a mixed ferrite-austenite Fe-21Cr-9Ni-8.5Nb-5.8Si-1.8C alloy developed by Rolls-Royce, has been investigated in the present work. The quality of a HIPped component is markedly affected by the microstructure of the powders, particularly for the distribution of coarse hard carbides. Non-homogeneous carbide distributions can be detrimental to the corrosion and/or wear properties of a hardfacing material, hence should be avoided. In this study, field emission gun (FEG) scanning electron microscopy (SEM) coupled with energy dispersive x-ray spectroscopy (EDXS) and electron backscatter diffraction (EBSD) techniques were used to characterise the as-HIPped microstructure. Discrete NbC carbides 100-600 nm in size (arrowed in Figure 1) appeared to be preferentially located at the interfaces of the spherical powder particles that were consolidated during HIPping. In addition, coarse (10-20 μm) NbC carbides were clustered randomly throughout the microstructure. The Cr carbides on the other hand, transformed from a continuous network structure in the powder particles to overall discrete coarse carbides after HIPping exhibiting minor segregation in the matrix. In terms of the imposed strains, there is evidence of considerable straining in the fully consolidated material. Detailed microstructural examination revealed the presence of numerous slip lines and twins in the interior of the austenite grains in the ferritic-austenitic microstructure of the alloy. Also, it is recognized that trace elements can also have a significant impact on the material properties. EDXS analyses both in the FEG-SEM and FEG-scanning transmission electron microscope (STEM) revealed the presence of trace elements (not deliberately added to the alloy). In particular, Al was detected along the boundaries of prior powder particles. Further studies are being directed at identification of experimental parameters that promote such segregation.

We would like to thank the UK Engineering and Physical Sciences Research Council (EPSRC) for sponsoring this project through the Centre for Doctoral Training in Advanced Metallic Systems, as well as Rolls-Royce for additional funding and for providing the materials.

Fig. 1: BSE micrographs and EDXS spectrum images of various features in the RR2450 HIPped steel.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2817 Atomic-resolution STEM study of Cu3Pt nanoparticles used as highly active PEM fuel-cells catalyst

Drazic G.1, Bele M.1, Pavlisic A.1, Jovanovic P.1, Zorko M.1, Hodnik N.1, Jozinovic B.1, Gaberscek M.1
1National Institute of Chemistry, Ljubljana, Slovenia
goran.drazic@ki.si

Various catalysts, such as noble metals, intermetallic alloys, carbon-based supports, metal chalcogenides and carbides, are used to reduce the oxygen reducing reaction temperature and achieve maximum reaction efficiency in proton exchange membrane fuel-cells. The main problem is slow adsorption and reaction kinetics, so searching for more efficient catalysts is one of the main challenges in the field of fuel cells. Among the most promising materials are C-supported Pt-based catalysts. In order to reduce the price of the material, Pt has been alloyed with various transition metal elements. In many cases not only the expected mass activity of the catalyst is improved, but also its specific activity is enhanced due to crystal lattice strains and the ligand effects through the d-band center shift induced by the transition elements. In the case of C-supported CoPt3 particles it has been recently shown that the electrocatalytic activity can be radically increased through core-shell structural ordering of intermetallic nanoparticles. Using a novel, modified sol-gel method the ordered (Pm3-m) Pt-Cu nanoparticles for catalytic oxygen reduction reaction applications were prepared. Tailoring specific parameters like chemical composition, degree of ordering, presence of Pt rich layer at the surface of the nanoparticles and appropriate embedding in carbon matrix the material obtained exhibits a 5-fold improvement of mass activity and a 9-fold improvement of specific activity compared to the Pt/C benchmark. These values exceed markedly the US Department of Energy target for 2017. Detailed analysis of Cu-Pt particles prepared at different conditions showed a core-shell type of alloy. The core consisted of disordered Fm3-m cubic phase where Pt and Cu atoms are statistically distributed inside the spheres. Around this disordered core, an ordered Pm3-m shell could be formed during the annealing procedures. Furthermore, consistently with previous reports on similar alloy systems the existence of a Pt-rich outer layer, 1- 2 nm thick, called skin, can be demonstrated on the surface of particles. It is possible that additional effects besides Pt skin and ordered phase could be present. We observe some twinning and other crystallographic defects like dislocations that could contribute to the strain of the surface platinum and hence to the very high specific activity. The presence of ordered Cu3Pt Pm3-m phase and Pt-rich skin was proved with an atomic-resolution Cs corrected STEM. In Figure 2 cross-sections of Cu-Pt particle are shown. The influence of the synthesis conditions on the formation of ordered Pm3-m structure and Pt-rich skin will be explained in detail and the impact of those parameters on the final properties will be discussed.

The work was financed through the P2-0148 research program by Slovenian Research Agency.

Fig. 1: STEM images (a. BF-STEM, b. ADF-STEM) of Cu3Pt nanoparticles.

Fig. 2: HAADF-STEM images of a. - Cu3Pt nanoparticle with ordered Pm3-m structure. At the surface of the particle 2nm thick Pt rich skin is present. b.- atomic resolution image of the Pt rich skin layer at the surface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2875 Degradation Mechanisms of Platinum Nanoparticle Catalysts in Proton Exchange Membrane Fuel Cells: The Role of Particle Size

Yu K.1, Groom D. J.1, Wang X.4, Yang Z.2, Gummalla M.2, Ball S. C.3, Myers D. J.4, Ferreira P. J.1
1Materials Science and Engineering Program, The University of Texas at Austin, TX, 78712, USA, 2United Technology Research Center, CT, USA, 3Johnson Matthey Technology Center, Blount’s Court, Sonning Common, Reading, RG4 9NH, UK, 4Argonne National Laboratory, Argonne, IL, 60439, USA
kangyu@utexas.edu

Proton exchange membrane fuel cells (PEMFCs) are promising energy conversion devices due to their high efficiency, high energy density and low operation conditions. Pt nanoparticles are widely used as the catalysts in cathode and anode for the half cell reactions. However, the durability of Pt nanoparticles still remains the most significant obstacle for large scale application of PEMFCs, especially in the cathode. In general, a significant decrease in electrochemical surface area (ECA) is observed.

In this work, five membrane electrode assemblies (MEAs) with platinum (Pt) nanoparticles of different average sizes (2.2, 3.5, 5.0, 6.7, and 11.3 nm) in the cathode were analyzed before and after potential cycling (0.6 to 1.0 V, 50 mV/s). MEAs with 2.2nm and 3.5nm show significant growth in mean particle sizes after 10,000 potential cycles, while the other samples do not (Fig.1a).

To understand the aforementioned particle growth, we need to consider the following possible mechanisms: (i) modified electrochemical Ostwald ripening (MEOR), (ii) platinum dissolution and re-precipitation inside the membrane and (iii) particle migration and coalescence [1]. As MEOR is an isotropic process, a comparison of the particle size distributions (PSDs) of spherical particles and PSDs of all the particles indicates that this mechanism plays a significant role in the degradation of 2.2nm and 3.5nm samples, but not in the other samples (Fig.1b). Re-precipitated particles in the membrane are found among almost all the samples (Figure 2a-e), but their amount is minor comparing to the particles in the cathode (Figure 2f), which reveals that re-precipitation plays an insignificant role in the degradation of PEMFCs. In terms of coalescence there are three plausible mechanisms: (i) particles migrate and coalescence, (ii) particles in proximity grow in size due to MEOR and make contact and (iii) soluble Pt species re-precipitate to bridge two particles followed by MEOR (Figure 3a). In any case, coalesced particles occur among all samples, although the 2.2nm sample shows the highest extent of coalescence (Fig.3b). However, as the carbon support exhibits a convoluted 3D structure, as shown by in-situ tomography (Fig. 3c,d,e), it is difficult for particles to coalesce through a migration mechanism.

In summary, Pt dissolution seems to be the controlling mechanism for degradation, as it assists the MEOR process and two plausible mechanisms of coalescence. Thus, reducing Pt dissolution is essential to prevent ECA loss and catalyst performance degradation.

References:

[1] Z. Yang, S. Ball, D. Condit, M.Gummalla, Journal of The Electrochemical Society, 158, 2011, B1439-B144

This work was supported by the Fuel Cell Technologies Office of the U.S. Department of Energy’s (DOE) Office of Energy Efficiency and Renewable Energy.

Fig. 1: Figure 1. (a) Average particle size after 10,000 cycles for various initial particle sizes. (b) Average spherical particle size after 10,000 cycles for different initial particle sizes.

Fig. 2: Figure 2. Cathode-membrane interface of MEAs of initial sizes of (a) 2.2nm, (b)3.5nm, (c)5.0nm (d) 6.7nm, (e)11.3nm and (f) low magnification image of cathode-membrane interface of MEAs of initial sizes of 2.2nm.

Fig. 3: Figure 3. (a) Coalescence of particle by three plausible mechanisms. (b) Coalesced particles in 2.2nm cycled MEA. (c), (d) and (e) In situ tomography images of the carbon support after -60°, 0°and +60° tilts, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2911 Li K-Edge mapping study for surface modified LiCoO2 using STEM-EELS measurement

Taguchi N.1, Akita T.1, Sakaebe H.1, Kuniaki T.1, Ogumi Z.2
1National Institute of Advanced Industrial Science and Technology (AIST), Osaka, Japan, 2Kyoto University, Kyoto, Japan
n-taguchi@aist.go.jp

For improvement of the properties of Li ion battery materials, a lot of modification processes such as surface modification have been examined [1]. To study the mechanism of these modification effects on the materials, it is very important to understand atomic structures and chemical states of modified materials. Analytical TEM measurement such as STEM-EELS method is a powerful tool for investigating local information about the sample [2].

In the present study, we prepared a cathode material (LiCoO2) with small particle size by Pechini method in order to avoid damage from sample preparation such as ion beam milling and focused ion beam [3]. Surface modification was performed using the sol-gel method. Al oxide, Mg oxide, and Si oxide coating sample were prepared. And the samples dispersed on Cu mesh with carbon micro-grid were directly observed without thinning process. A TITAN3 G2 60-300 electron microscope (FEI), equipped with EDS(Super-X : Bruker) and EELS(Quantum : Gatan) was used for analytical TEM measurement. For EELS measurements, a monochromator was used to achieve higher energy resolution. ZLP was less than 0.3 eV with 0.05 eV/ch.

The EELS measurement around the Li K-edge is carried out for surface modified cathode materials. Although the Co M-edge completely overlaps Li K-edge, it becomes possible to obtain a sharp Li K-edge peak by using a monochromator. Using this benefit, we constructed a elemental map using the Li-K edge in nm-order spatial steps, and discussed the relationship between electrochemical properties and Li concentration distribution in the particle

A schematic image of the EELS analysis of the Li K-edge is shown in Figure 1. Using the Co-M edge intensity, we constructed a Li-K/Co-M intensity ratio map [4]. The Li distribution in the LiCoO2 particle after charge discharge cycle was drastically changed by the coating species, and the homogeneity of Li ion distribution as shown in figure 2 corresponded well with the trend of capacity retention after the cycling test.

Reference

[1] J. Cho, Y. J. Kim, and B. Park, Chem. Mater. 12, 3788, (2000).
[2] J. Kikkawa, T. Akita, M. Tabuchi, M. Shikano, K. Tatsumi, and M. Kohyama, Appl. Phys. Lett. 91, 054103, (2007)
[3] Z.S. Peng, C.R. Wan, and C.Y. Jiang, J. Power Sources, 72, 215-220 (1998).
[4] N. Taguchi, T. Akita, H. Sakaebe, K. Tatsumi, and Z. Ogumi, Electrochem. Soc. 160, 11, A2293 (2013)

This work was supported by the “Research and Development Initiative for Scientific Innovation of New Generation Battery (RISING project)” of the New Energy and Industrial Technology Development Organization (NEDO, Japan).

Fig. 1: (a) Schematic image for reconstruction of the map of Li-K/Co-M edge intensity map of bare LiCoO2. (b) EELS spectra around Li-K edge for both the initial LiCoO2 and the charged state (4.2V ~ Li0.5CoO2) are shown at bottom left. Difference spectrum of (initial LiCoO2) – (4.2 V charged LiCoO2 ) is shown at bottom right.

Fig. 2: Li-K/Co-M intensity map for 100th discharged (3.0-4.5V /Li metal) (a) bare LiCoO2, (b) coated LiCoO2 (MgO 1 wt%). The contrast of the image is normalized as the Li-K/Co-M intensity ratio. The x in the intensity scale corresponds to the x in LixCoO2.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2919 Exploring the connection between thermal displacements and chemistry in Bi2Te3-xSex

Dycus J. H.1, Oni A.1, Sang X.1, Chan T.1, Koch C.1, LeBeau J. M.1
1Department of Materials Science and Engineering/ North Carolina State University, Raleigh, NC 27695, USA
jhdycus@ncsu.edu

Bismuth telluride-selenide alloys are commonly employed thermoelectrics at room temperature. In these materials, phonon scattering plays a pivotal role in controlling the thermoelectric properties where decoupling phonons and charge carriers induces a net heat flux that results in an electric potential. Understanding the heat flow requires knowledge of phonon scattering as a function of alloying, at defects, and within the microstructure. In the case of Bi2Te3-xSex alloys, the structure consists of stacked ‘quintuple’ layers in the sequence Te(1)-Bi-Te(2)-Bi-Te(1) [1]. Se can substitute into either the Te(1) or Te(2) sites. The position of alloying impurities in the crystal lattice greatly influences thermoelectric properties, as the local bonding environment changes at different crystal orientations. Additionally, impurities will change the bond strength in the structure, further affecting phonon transport. Determining impurity site preference and influence on bonding thus provides a vital component needed to understand the relationship between alloy content and properties.

In this talk, we will present an atomic scale structural and chemical study of Bi2Te3-xSex alloys using aberration corrected scanning transmission electron microscopy (STEM). Further, we employ RevSTEM, a new technique that corrects drift and distortion in scanning microscopy, providing precise and accurate intensity measurements [2]. From HAADF, see Figure 1 and Figure 2(a,c), we will show that Se appears randomly distributed across the Te sites as the intensity of Te(1) and Te(2) columns are equivalent. By employing state-of-the-art atomic resolution energy dispersive x-ray spectroscopy (EDS), Se primarily resides in Te(2) crystal sites, as also shown in Figure 1. Using this information, we will show that a typical atomic number HAADF interpretation suggests that Te(2) atom columns should appear darker (Se=34, Te =52), but this is not observed.

Through a combination of imaging at different ADF collection angles and results from EDS, we will demonstrate that the observed intensities are due to phonon scattering as a result of Se incorporation. As shown in Figure 2(c-f), LAADF STEM shows a significant drop in Te(2) intensity for the alloy, which is not observed in pure Bi2Te3 or in HAADF images. We will show, through a comparison of experiment and simulations, the results can be explained by reduction of the average thermal displacements in the Te(2) layers. These results also explain prior literature suggesting that bond strength of the Te(2) layer changes upon incorporation of Se [1].

References

[1] J. R. Wiese and L. Muldawer, Journal of Physics and Chemistry of Solids 15 (1960), p. 13

[2] X. Sang and J. M. LeBeau, Ultramicroscopy 138 (2014), p. 28

The authors acknowledge the Analytical Instrumentation Facility (AIF) at North Carolina State University, which is supported by the State of North Carolina and the National Science Foundation.

Fig. 1: Atomically resolved Bi2Te2.7Se0.3 imaged down the [100] zone axis in a RevSTEM series. Top left: Atomic positions of Bi2Te3 along [100]. Top right: atomic resolution EDS map overlayed with individual Te, Se, and Bi signals. Note the Se signal only resides in the Te(2) position.

Fig. 2: Bi2Te2.7Se0.3 and Bi2Te3 ADF images (a-d). The Te(2) intensity decreases relative to Te(1) exclusively in (b). Line profiles from Bi2Te2.7Se0.3 (e) and Bi2Te3 (f) extracted from (a-d) with corresponding atom positions, Bi (blue), Te (red), and Se (green). Arrows indicate the drop in Te(2) intensity for Bi2Te2.7Se0.3 but not Bi2Te3.
Fig. 3:
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2978 Microstructural study of diffusion in a CeFe4Sb12/Cu thermoelectric couple

Boulat L.1, Fréty N.1, Ravot D.1, Viennois R.1, Dadras M.2
1Institut Charles Gerhardt, Université Montpellier 2, UMR 5253 CNRS-UM2-ENSCM-UM1, cc 1504, Place E. Bataillon, 34095 Montpellier Cedex 5, France, 2Centre Suisse d'Electronique et de Microtechnique SA, Jaquet-Droz 1, Case Postal, CH-2002 Neuchâtel, Switzerland
laetitia.boulat@univ-montp2.fr

The performance of thermoelectric devices is highly dependent on the figure of merit Z (Z = α².σ / κ where α is the Seebeck coefficient, σ the electrical conductivity and κ the thermal conductivity) but is also greatly related to the interface properties between the thermoelectric material and the electrode metal. In this study the thermal stability of a CeFe4Sb12/Cu thermoelectric couple and the role of transition metal nitride interlayer on the interfacial reactions were investigated to 673 K during 6 h. Tantalum nitride thin films have been selected as diffusion barriers due to the unique properties of this material such as low electrical resistivity and good thermal stability. Microstructural evolution and interfacial reactions were performed by Scanning Electron Microscopy (SEM) coupled with Energy Dispersive X-ray Spectrometry (EDS) and Transmission Electron Microscopy (TEM). These analyses showed that the CeCu2 and Cu2Sb phases, which are detrimental to the thermoelectric device efficiency, are formed in the annealed CeFe4Sb12/Cu couple. However it appeared that the TaN interlayer can interestingly inhibit the formation of these phases. Indeed, results showed that the tantalum nitride-based films, embedded in an oxynitride amorphous phase and with a size of about 5 nm, can inhibit the interdiffusion of Sb, Ce and Cu elements. Consequently tantalum nitride-based films appear as promising diffusion barriers for thermoelectric energy systems made of CeFe4Sb12/Cu couples. 

Fig. 1: SEM observation of cross-sectional of as-prepared CeFe4Sb12/TaN/Cu
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2982 In-situ TEM Observation on Hydrogen Storage Materials

Matsuda J.1, Yoshida K.2, Uchiyama N.3, Akiba E.1,4
1International Institute for Carbon-Neutral Energy Research, Kyushu University, Fukuoka, Japan, 2Institute for Advanced Research, Nagoya University, Nagoya, Japan, 3ATSUMITEC CO., LTD., Hamamatsu-shi, Shizuoka, Japan, 4School of Engineering, Kyushu University, Fukuoka, Japan
junko.matsuda@i2cner.kyushu-u.ac.jp

While global warming and fossile fuel drain pose a problem, hydrogen could generate electricity without discharging CO2 and also serve as a secondary energy carrier for electric power. Technology to store and transport hydrogen compactly and safely is indispensable in order to realize hydrogen energy society. Hydrogen storage materials are able to make hydrogen gas 1/1000 or less volume and carry it.
Metal hydrides which interstitial sites are occupied by hydrogen atoms are one of the hydrogen storage materials; they are promising candidates to be applied to hydrogen storage tanks in fuel cell vehicles and stationary storage systems. When the metal hydrides are formed, lattice defects such as vacancy, dislocation and stacking fault are introduced due to large volume expansion. In LaNi5-based intermetallics, misfit dislocations along a-planes and c-planes in the hexagonal unit cell are observed after hydrogenation [1,2]. Regarding Ti-V based BCC alloys, twin boundaries and stacking faults are formed parallel to (111) in FCC hydrides to accommodate anisotropic expansion along c-axis [3]. These lattice defects have a great effect on hydrogention/dehydrogenation properties such as absorption pressure, kinetics and cycle ability. Formation of lattice defects are suggested to relate to microstructure evolution during hydrogention/dehydrogenation. This study aims to elucidate the hydrogenation mechanism for storage materials with various crystal structures from the microscopic point of view, in order to improve the hydrogenation/dehydrogenation properties.
First, a new sample holder with an ex-situ cell for transmission electron microscope (TEM) has been developed. Commercially available Pd powder was observed by TEM after exposed to hydrogen in the ex-situ cell. Moreover, we have succeeded in high-resolution TEM observation on hydrogenation of Mg6Ni films using environmental TEM with aberration corrected. As shown in Figs. 2, it is found that MgH2 crystallization occurs following Mg2NiH4. This suggests that Mg2Ni has a catalytic effect on hydrogenation of Mg. In the future, relation between the hydrogenation mechanism and Mg/Ni ratio of the Mg-Ni films will be revealed.
References
[1] T. Yamamoto, H. Inui, M. Yamaguchi, Intermetallics, 9 (2001) 987.
[2] J. Matsuda, Y. Nakamura, E. Akiba, J. Alloys Compd., 509 (2011) 7498.
[3] J. Matsuda, Y. Nakamura, E. Akiba, J. Alloys Compd., 509 (2011) 4352.

Development of TEM holder with ex-situ cell was supported by The New Energy and Industrial Technology Development Organization (NEDO), Japan. This study was partially supported by a grant-in-aid for Scientific Research (24560853) from the Japan Society for the Promotion of Science, Japan.

Fig. 1: TEM imgaes of Pd powder: (a) before hydrogenation (as-received) ; (b) after exposed to hydrogen of 0.01 MPa for 60 minutes. It is noted that crystals with the diameter more than 100 nm are observed.

Fig. 2: High-resolution TEM imgaes of Mg6Ni films taken in the hydrogen atmosphere of 80 Pa: (a) 40 seconds; (b) 100 seconds passed after electron irradiation.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-2992 In situ Raman microscopic study of lithium metal surface

Hyono A.1, Shibuya S.1, Ueda M.1, Ohtsuka T.1
1Hokkaido University, Sapporo, Japan
hyono@eng.hokudai.ac.jp

Background: When one uses metallic lithium for anodes of lithium batteries, it is expected to be the higher capacity than that of the carbon. However, there is a serious problem, such as dendritic growth during charging, for using metallic lithium. This dendritic lithium brings about a short circuit of electrodes and produce dead-lithium. It is assumed that the dendrite formation is affected by the surface compounds on the lithium electrode including the Solid Electrolyte Interphase. In this study, to investigate the surface change on the lithium electrode in organic solvent, we used the technique of in situ Raman spectroscopy.
Experimental: The apparatus for in situ Raman spectroscopy of the electrodes under in-situ condition is shown in Fig. 1. Lithium foil was fixed at the center of the cell, facing an optical window. In front of the incidence slit, confocal optical system for collection of Raman scattering light and cut-edge optical filter were equipped. The detection of Raman scattering light was done by highly sensitive CCD camera. Excitation of Raman process was performed by a laser light at 532.0 nm wavelength. In the cell, Luggin capillary connected to a reference electrode Ag/AgCl/saturated KCl (SSC) was inserted and the counter platinum electrode was fixed around the working electrode. The cell was assembled in a glove box filled with Ar gas, fixed to the apparatus and filled with electrolyte solution. The solutions used were 1 M LiClO4 /PC (propylenecarbonate), 1 M LiClO4/PC+DMC (dimethylcarbonate) and 1 M LiBF4/PC.
The electrochemical treatment and measurement were performed by using a potentiostat with a function generator. The potential was stepwise changed from OCP to anodic or cathodic direction with 0.1 V step. The potential was kept at each potential for 15 min. and then Raman spectra were measured.
Results: The photographs of the Lithium surface at -2.9 V (OCP) and -2.2 V vs. SSC in 1 M LiClO4 solution are shown in FIg. 2. Before polarization, the surface exhibited a bright color with burnish. Dark areas began to appear at -2.6 V and the area spread with the increase of potentials. At -2.2 V the almost whole surface turned to dark. In other solvents, the same changes were observed. Raman spectra of the lithium surface at the dark areas in organic solutions under in-situ condition are shown in Figure 3. In 1 M LiClO4/PC and 1 M LiClO4/PC+DMC, two broad peaks around 530 cm-1 and 930 cm-1,and the other peak around 970cm-1 appeared in 1 M LiBF4/PC.
Discussion: Comparing the spectra of the lithium surface in 1 M LiClO4 /PC and 1 M LiBF4/PC, the peaks belong to compounds generated from electrolyte molecules, i. e. the peaks of 530 cm-1 and 930 cm-1 are LiCl and LiClO4 in LiClO4 solution, and the one of 970 cm-1 is LiF in LiBF4 solution.

Fig. 1: Schematic illustration for in situ Raman spectroscopy

Fig. 2: Photographs of lithium surface at -2.9 V and -2.2 V

Fig. 3: Raman spectra of Li electrodes surfaces
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3002 Microstructural changes of the Sanicro25 steel for coal fired power plants caused by thermal exposure and fire-side corrosion

Rutkowski B.1, Lipińska-Chwałek M.1,2, Cempura G.1, Gil A.1, Cieszyński K.3, Czyrska-Filemonowicz A.1
1AGH University of Science and Technology, Al. A. Mickiewicza 30, 30-059 Kraków, Poland, 2Forschungszentrum Jülich GmbH, 52425 Jülich, Germany , 3RAFAKO S.A, ul. Łąkowa 33, 47-400 Racibórz, Poland
rutkowsk@agh.edu.pl

Due to its high creep strength, high corrosion resistance and good weldability Sanicro25 (22Cr25NiWCoCu), high-chromium austenitic steel developed by Sandvik Materials Technology, is a promising material for superheater and reheater tubes of coal fired power plants operating at Advanced Ultra Super Critical (A-USC) steam conditions of 700 °C and 30 MPa [1].
The aim of the present study was to describe microstructural changes of Sanicro25 steel caused by the two-steps thermal exposure: 30 h in a real combustion environment of the 0.5 MW test rig (pulverized fuel combustion rig), followed by 970 h in the laboratory corrosion test set at 650 °C under a gas mixture simulating USC boiler environment (University of Stuttgart, [2]).
The microstructural investigation was carried out using SEM and TEM (Merlin Gemini II of Zeiss and a probe Cs corrected Titan3 G2 60-300 of FEI equipped with a ChemiSTEM system, respectively). The TEM analysis was conducted using the lamellae prepared by FIB and extraction double-replicas. Phase identification was performed by STEM-EDS and electron diffraction (SAED) methods. The SAED patterns were interpreted with the help of a JEMS.
The microstructure of as-received Sanicro25 steel was consisted of the austenitic matrix and primary Nb-rich M(C,N) precipitates. During exposure at 650˚C for 1000 h, the intensive precipitation of M23C6 carbides (where M= Cr, W, Si) from the supersaturated matrix occurred. The numerous M23C6 carbides as well as some Cu-rich precipitates at the grain- and twin boundaries were observed (Fig.1). STEM-EDS images of the microstructure of the scale and the steel observed on the FIB lamella taken from the outer surface of Sanicro25 tube are shown in Fig. 2. During exposure at 650 ˚C, a protective, 200 nm thick Cr-based scale with Fe-rich outermost film was developed. Beneath the scale (up to a depth of 1000 nm), the Cr-depletion zone with unstable (Cr,W,Si)23C6 carbides was formed. Following carbides dissolution, the W-rich precipitates were formed in the Cr-depletion zone. Additionally, Si diffused through the grain boundaries to the scale-metal interface, where it internally oxidized forming large Si-containing crystallites. Beneath the scale, the internal oxidation zone with Si- and Mn- rich precipitates was formed (Fig.3).

References
[1] R. Rautio, S. Bruce, Advanced Materials and Processing, April 2008, 35-37.
[2] M. Lipińska-Chwałek, M. Stein-Brzozowska, B. Rutkowski, A. Gil, J. Maier, A. Czyrska-Filemonowicz, submitted to 10th Liège Conference, 2014

The financial support by EIT KIC InnoEnergy (“NewMat” project) and AGH-UST (statutory project, 2014) is kindly acknowledged.

Fig. 1: Sanicro 25 after thermal exposure at 650 °C for 1000 h; a) STEM-DF image: M23C6 and Cu-rich precipitates in the austenitic matrix, b) STEM-EDS elemental map of Cr and Cu

Fig. 2: The changes of the Sanicro 25 microstructure beneath Cr-based scale; a) STEM-HAADF-image: W-rich and Cu-rich precipitates in Cr-depletion zone; b) STEM-EDS elemental map

Fig. 3: The internal oxidation zone (beneath the Cr-based scale) composed of Si- and Mn- rich precipitates; a) STEM-HAADF image, b and c) STEM-EDS elemental maps
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3111 TEM Study of Li-ion battery cells

Persson J. M.1, Mayer J.1, 2
1Central Facility for Electron Microscopy, RWTH Aachen University, Germany, 2Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, Jülich Research Centre, Germany
persson@gfe.rwth-aachen.de

The most popular rechargeable storage devices today are lithium ion batteries, with uses in consumer electronics and as the primary source of power for modern electric vehicles [1]. For both of these applications a high energy density is needed and, especially for the automobile industry, a significant drawback of the current generation of lithium ion batteries is their limited lifetime [2]. A solid understanding of the lithium ion batteries is required in order to improve their performance. Using modern high resolution electron microscopy the atomic structure of the lithium containing structures can be probed and compared with in situ electron microscopy methods. Comparing results from cells that have been cycled only a few times and cells that have been cycled many times may help us understand the reasons of ageing.

Here we have been studying commercially available lithium ion batteries for the effect of continuous cycling. The anodes of these batteries were made of grains of intercalated graphite with conductors of copper. Thin solid electrolyte interphase (SEI) layers were formed on the surface of the graphite grains by cycling the battery a few times. The SEI needs to be present as it protects the material from cointercalation by the solvents used. Unfortunately one of the ageing mechanisms in these batteries is the eventual buildup of thicker SEI layers, preventing the flow of lithium.[3]

Figure 1 shows an SEM micrograph of the cross-section of an anode from a many times cycled lithium ion battery. The layering of the graphite inside the grains is clearly visible, as are the areas between the layers. Using a focused ion beam liftout method a sample was prepared for transmission electron microscopy. The result can be seen in Figure 2.

While there is still need for further investigation on the subject, the preliminary data suggest that this method is usable for measuring and comparing the thickness of the SEI layer as well as tracking contaminations stemming from the electrolytes, in the material. The latter is one possible explanation to the bright areas in figure 1, shown by energy dispersive X-ray spectroscopy to be rich in fluorine, copper, phosphorous and sulphur.

References:
[1] B. Scrosati et al., Energy Environ. Sci. 4 (2011), 3287
[2] Y-K. Sun et al., Nature Materials 8 (2009), 320
[3] P. Verma et al., Electrochemica Acta 55 (2010), 6332

The authors would like to thank ECC Repenning GmbH for providing materials and to the BMBF via the MEET Hi-EnD project for funding. Instrumentation was in part provided by the ER-C, FZ Jülich.

Fig. 1: SEM micrographs of a lithium-intercalated, several times cycled, graphite structure from a lithium ion battery anode. (b) Shows a magnified view of an area in (a). The layering of the graphite can be seen clearly, as well as several inhomogeneities (brighter spots).

Fig. 2: TEM micrographs showing the same graphite structure as in figure 1. The sample was made from the same source using a focused ion beam liftout method. (b) Shows a magnified view of on area in (a).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3122 New Insights into SCC Initiation in Alloy 600 using Advanced Analytical Electron Microscopy

Bertali G.1, Burke M. G.1, Scenini F.1, Zhong X. L.1
1Materials Performance Centre, University of Manchester, Machester UK 1
m.g.burke@manchester.ac.uk

Stress corrosion cracking (SCC) is an important failure phenomenon in a variety of alloys used in power generation applications. In light water reactor (LWR) plants, materials such as austenitic stainless steels and Ni-Cr-Fe alloys (Alloy 600) can be susceptible to Primary Water SCC (PWSCC). Although there is an extensive amount of data on PWSCC crack growth rates and fracture, initiation of SCC continues to be a topic of research, particularly in terms of localized intergranular oxidation.  In this study, Alloy 600 (Ni-16Cr-9Fe-0.045C) was exposed for 66 and 120 h to a hydrogenated steam environment at atmospheric pressure and 480°C (O2 partial pressure = 9.88x10-26 atm which is lower than the dissociation pressure of Ni/NiO at 480°C). This reducing environment was used to avoid protective Ni-rich surface oxide formation and to accelerate the intergranular oxidation that is generally occurs at lower temperatures in a LWR environment [1]. This oxidation system successfully simulated PWR oxide morphologies [2-4]. Field emission gun (FEG) scanning electron microscopy (SEM), focused ion beam (FIB) and analytical electron microscopy techniques were used to characterize the type and extent of preferential oxidation associated with SCC initiation.

The exposed solution-annealed and water-quenched samples were evaluated in an FEI Quanta 650 SEM equipped with Oxford Instruments SDD and EBSD systems. A variety of grain boundary (GB) oxide morphologies were observed, reflecting the type of grain boundary, Fig. 1.   Surface oxide morphology from FIB cross-sections was correlated with intergranular oxidation susceptibility.  More detailed analyses were performed using the FEI Titan G2 80-200 aberration-corrected S/TEM with Super EDX.  STEM-EDX microanalysis confirmed the presence of interconnected subsurface Cr-rich oxides and intergranular Cr-rich oxide (Fig.2), with a Ti/Al-rich oxide delineating the original grain boundary location. Analyses showed strong correlation between the surface GB oxide morphology and its susceptibility to intergranular oxides. Microstructural evidence of grain boundary migration and the associated depletions and enrichments that develop also appear to be notable factors in the preferential GB oxidation/SCC initiation.

References

1. Scott PM, Le Calvar M., in Proc. 6th Intl Symp. on Environ. Deg. of Matls in Nuclear Power Systems- Water Reactors; 1993, 657.

2. Scenini F, et al., Proc.12th Intl Symp. on Environ. Deg. of Matls in Nuclear Power Systems- Water Reactors 2005, 891.

3. Bertali G, et al., Proc.16th Intl Symp. on Environ. Deg. of Matls in Nuclear Power Systems- Water Reactors 2013, in press.

4. Lindsay J, et al., Proc.16th Intl Symp. on Environ. Deg. of Matls in Nuclear Power Systems- Water Reactors, 2013, in press.

Fig. 1: Secondary electron images showing (a) the surface morphology of the GB oxide, Ni surface nodules, and nodule-free zone (NFZ); and (b) a FIB cross-section through the oxidized GB.

Fig. 2: HAADF STEM image and corresponding Titan EDX spectrum images for Cr, O and Ni showing the oxidation and a Ni-rich nodule at the surface.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3130 EELS-Compositional analysis of SiNWs anodes for Lithium microbatteries

FLOREA I.1, LEVEAU L.1,2, GOHIER A.2, LAIK B.3, MOREAU P.4, COJOCARU C S.1
1LPICM, École Polytechnique, Route de Saclay, 91128, Palaiseau Cedex, France,, 2Renault SAS, DREAM/DETA/SEE, 1, Avenue du Golf, 78288 Guyancourt, France , 3ICMPE/GESMAT, UMR 7182 CNRS-UPEC, 2 à 8 rue Henry, Dunant, 94320 Thiais, France, 4IMN, Université de Nantes - CNRS, UMR6502, 2 rue de la Houssinière, 44322 Nantes Cedex, France
lenuta-ileana.florea@polytechnique.edu

Nowadays Li batteries became one of the most efficient systems for energy storage[Tarascon M.-Nature2001].Intensive efforts are needed to improve their energy density and to extend their life cycle as for example by using new negative electrode materials that insert Li at low potentials.Among all the studied anode materials,silicon nanowires(SiNWs) have proved to be very interesting candidate[Laïk B.-El.Acta 2008].Besides their high theoretical capacity (3579 mAhg-1) they exhibit good performances in terms of resistance to fracture during the volume expansion and their 1D character facilitates axial charge transport and radial Li ion diffusion. Nevertheless, the main drawback remains their progressive degradation due to the large volume changes during the electrochemical cycling which will affect later the anode performances.Improving the battery design remains a challenge and probing the reaction kinetics and microstructural evolution during battery operation may provide extremely useful information for this.The key-issue addressed by this work is the assessment of an accurate correlation between the electrodes performances, their morphological, structural and chemical characteristics.From the experimental point of view, the only techniques that allow accessing chemical and morphological characteristics together with structural information of the studied object are the TEM characterization based techniques. In addition, the continuous technological development made possible the design of specific sample holders such as vacuum TEM holders that enable the analysis of sensitive materials like cycled SiNWs without air exposure and without severe beam damaging.Thus the approach proposed herein aims at combining the conventional TEM using a special TEM holder and the EELS technique, for the assessment of morphological and chemical composition modifications of SiNWs during the lithiation/delithiation cycling. From a morphological point of view Fig.1 illustrates, as expected, the volume expansion of SiNWs during the cycling.As for the chemical composition, EELS spectra recorded for various positions of the electron probe covering energy ranges below 20eV (plasmons region), allowed the identification of LixSi alloys within the cycled NWs.Firstly for the lithiated SiNWs a detailed analysis of the EELS spectra allowed identifying one main peak at 13eV revealing the signature of Li13Si4 alloy.Peaks at 9eV and 20eV were also identified, assigned to LixSiOy alloy and electrolyte degradation products on the surface.For the delithiated NWs, a main peak at 16eV marking the Si signature was found.These findings highlight the utility of the EELS technique and a special sample holder for probing the compositional changes of SiNWs during the electrochemical cycle.

Fig. 1: TEM image of the SiNWs before A) and after B) lithiation, showing silicon volume expansion. The electrolyte used in the electrochemical reaction was a conventional organic mixture of ethylene carbonate (EC) and dimethyl carbonate (DMC) with 1M LiPF6 salt.

Fig. 2: A) TEM image of SiNWs which suffered a complete lithiation; B) Experimental EELS plasmon spectra taken along the green line illustrating the presence of the main peak at 13eV. The peak at 9eV indicating the reaction of surfacic SiO2 with Li, and a large peak at 20eV assigned to the presence of the Solid Electrolyte Interphase (SEI).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3190 Local Imaging and Measuring of Distortions in the Oxygen Sub-Lattice of Complex Mixed Oxides

Lunkenbein T.1, Girgsdies F.1, Noack J.1, Trunschke A.1, Schlögl R.1, Willinger M. G.1
1Fritz-Haber-Institute of the Max-Planck-Society, Berlin, Germany
lunkenbein@fhi-berlin.mpg.de

“It would be very easy to make an analysis of any complicated chemical substance; all one would have to do would be to look at it and see where the atoms are. The only trouble is that the electron microscope is one hundred times too poor.” With this statement in his famous speech “There is plenty of room at the bottom” R. Feynman has already highlighted the future importance of transmission electron microscopes (TEMs) in 1959.

Nowadays modern Cs corrected TEMs are powerful enough to obtain point resolution below 50 pm.[1] However, direct imaging of light elements next to heavy elements remains complex. In probe corrected scanning transmission electron microscopy (STEM) recent developments tackle this challenge, resulting in the revival of the annular bright field (ABF) detector. In contrary to the contrast detected by the high angle annular dark field (HAADF) method, which is due to Rutherford scattering and proportional to Z2, the ABF detector is also sensitive to light elements.[2]

Using the ABF detector, we investigated orthorhombic (Mo,V) oxides crystallized in a structure analog to the M1 structure (ICSD no. 55097) of MoVTeNb oxide. The obtained micrographs were compared with Rietveld refined X-ray diffraction (XRD) data. Fig.1 shows an ABF image where the oxygen atoms brighten up. Furthermore we directly measured metal-oxygen bond angles and discussed the oxidation states of the metal centers.

Our results prove Feynman´s prediction. Seeing where the atoms are, generates in particular in heterogeneous catalysis a deeper understanding of the functionality of materials on the way towards tailor-made catalysts.

Refernces:

[1] a) R. Erni, M. D. Rossell, C. Kisielowski, U. Dahmen, Phys. Rev. Lett. 2009, 102, 096101; b) K. Takayanagi, S. Kim, S. Lee, Y. Oshima, T. Tanaka, Y. Tanishiro, H. Sawada, F. Hosokawa, T. Tomita, T. Kaneyama, Y. Kondo, J. Electron Microsc. 2011, 60, S239-S244.

[2] a) P. E. Batson, Nat Mater 2011, 10, 270-271; b) S. D. Findlay, N. Shibata, H. Sawada, E. Okunishi, Y. Kondo, Y. Ikuhara, Ultramicroscopy 2010, 110, 903-923.

The authors acknowledge Jeol and the Max-Planck-Society for help and financial support.

Fig. 1: (A) Atomic resolution ABF-STEM image of (Mo,V)Ox. The white rectangle displays the orthorhombic unit cell, which is in good agreement with the ABF image. Metal sites are partially highlighted with blue circles and oxygen sites are labeled with red circles. (B) Corresponding HAADF image of the same region.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3194 Probing the microscopic grading mechanism of sodium-containing layered lithium rich cathode materials in lithium ion batteries by aberration-corrected STEM

Huang W.1, Wu C. Y.1, Wang G. Q.1, Xie J.1, Zeng Y. W.1, Jin C. H.1, Zhang Z.1
1State Key Laboratory of Silicon Materials, Key Laboratory of Advanced Materials and Applications for Batteries of Zhejiang Province, Department of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, P. R. China.
tmacweiwei@126.com

Lithium ion battery (LIB) plays an important role in portable devices and with promising application in automobile industry and sustainable technology. So far, many works have been done to improve the energy density of LIBs. Lithium-rich layered cathodes can display a reversible capacity more than 300 mAhg-1, nearly twice the capacity of the traditional cathode materials. Unfortunately, this kind of material suffers from a poor rate capacity and cycling performance. Sodium and cobalt doping is confirmed as a useful way to improve the electrochemical performance of lithium-rich material, however, a detailed understanding on the associated mechanisms is still lacking.
In this work, by using the Cs-corrected STEM combined with EDS and EELS, we carried out systematic characterizations on the sodium-cobalt containing lithium-rich cathodes and found sodium ions have two different aggregations behaviors inside: either to distribute homogeneously in lithium layer, while the others gather with cobalt to form a sodium-cobalt enriched layer, as evidenced by the HAADF-STEM and STEM-EDS mapping. More importantly, we found that the presence of the latter structure is actually response for the capacity fading upon cycling, and eventually lead to the cracking of electrode materials and failure of the whole battery. This finding is of particular importance to understand the fading mechanism of such lithium-rich cathodes, and will be quite suggestive for the design and modification of cathode materials.

The work on microscopy was done in the EM Center of ZJU. This work is financially supported by the NSFC (51222202), the 973 program (2014CB932500), the Program for Innovative Research Team in University of Ministry of Education of China (IRT13037) and the Fundamental Research Funds for the Central Universities (2014XZZX003-07).

Fig. 1: HAADF image of a sodium-containing Li-rich particle, the brightlines are the Na-Co enriched layers

Fig. 2: EDS map of Co element in the same particle, the bright yellow lines are the Co enrichment areas which are also accompanied with Na enrichment

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3196 TEM investigation of He bubbles in 9%Cr ferritic steel after neutron irradiation

Klimenkov M.1, Materna-Morris E.1, Möslang A.1
1Karlsruhe Institute of Technology (KIT), Institute for Applied Materials - Applied Materials Physics (IAM-AWP)
michael.klimenkov@kit.edu

To provide quantitative microstructural information on helium bubble development under the influence of irradiation, 3 heats of EUROFER 97 composition with the addition of natural boron as well as pure 10B isotope were fabricated. He concentrations after irradiation were ~81, ~415, and ~5800 appm for these three specimens (alloy 1, alloy 2 and alloy 3), respectively. Neutron irradiation with a dose of up to 16 dpa was performed in the temperature range from 250°C to 450°C. This allowed for a detailed study of the influences of radiation parameters on the size, morphology, spatial distribution, and density of He bubbles. These investigations are necessary to correlate radiation-induced changes of the microstructure to the mechanical properties.

The cavities were not detected in alloys 1 and 2 irradiated at 250°C. Irradiation at this temperature leads to the formation of point defects and dislocation loops of 5-10 nm in size (Figs. 1a, 2a). However, their spatial density that exceeds bubble density by one order of magnitude, increase further strength and hardness which are dominated below 350°C by irradiation induced interstitial type loops. Irradiation at 400°C produces clear differences in the microstructures of alloy 3 due to the 5800 appm He content. The randomly distributed cavities and bubbles were detected in the specimen irradiated at 350°C (Fig. 1b,2b). The cavities exhibit a mainly faceted shape, which indicates the formation of empty voids rather than filled bubbles. In the alloys 1 and 2 irradiated at 450°C, no formation of faceted voids was detected, the He-filled bubbles were mainly located in dislocations, precipitates, or grain boundaries. The bubbles are smaller than 10 nm and show a sharp size distribution with a maximum at 5 nm. In some cases, bubbles are located on dislocations forming a 2-dimensional net (Fig. 2c). The density of bubbles in the specimens irradiated at this temperature depends on the He concentration: 2.4*1021 m-3 for alloy 1 with 82 appm He and 8.1*1021 m-3 for alloy 2 with 415 appm He. The presented TEM investigations of irradiated material clearly show the strong influence of transmutation-produced He on the formation of He bubbles or cavities. It can be supposed that faceted cavities >10 nm, similar to the imaged individual bubble in Fig. 1b-2b, are empty voids.

The HRTEM investigations of an individual cavity are shown in Fig. 3. The image (a) and the Fast Fourier Transformation (FFT) pattern (b) exhibit the orientation of the cavity’s facets. The cavities often show a hexagonal shape, the facets are oriented in <110> directions, if the matrix is oriented close to the [111] zone axis.

Fig. 1: TEM images of alloy 1 irradiated at 250°C 350°C, and 450°C are shown in parts a, b, and c, respectively.

Fig. 2: TEM images of alloy 2 irradiated at 250°C 350°C, and 450°C are shown in parts a, b, and c, respectively.

Fig. 3: HRTEM image of a cavity in alloy 2 irradiated at 350°C (a) and fast Fourier transformation image showing crystallographic orientation (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3224 Influence of cooling speed on crystallization and final electrochemical properties of cathode material

Kazda T.1, Čudek P.1, Vondrák J.1, Sedlaříková M.1
1Brno University of Technology, Brno, Czech Republic
xkazda02@stud.feec.vutbr.cz

The lithium - ion electrochemical power sources are rechargeable systems used in many electrical devices. In standard lithium - ion power sources there is lithiumcobaltoxide (LiCoO2) used as cathode material which can provide the working voltage of 3.7 V against lithium [1], [2]. In order to get more effective power it is necessary to change cathode or anode material. For this purpose there was a new cathode material lithiumnickelmanganeseoxide (LiNi0.5Mn1.5O4) prepared and tested, which can provide 4.7 V against lithium [2], [3]. This means that the new electrochemical power system can provide almost 20 % more power than a standard battery with a similar capacity [4].
The method of solid state reaction was used for the preparation of the LiNi0.5Mn1.5O4 cathode material. The three-step annealing process was used for reaction. The conditions of the first and second annealing steps were the same, while during the third step the cooling speed of the material after annealing was changed. It was experimentally proved that reducing the cooling speed improved the electrochemical properties such as stability during cyclic voltammetry and stable characteristics during discharging (Fig. 1). The improvement was reached by getting better crystallinity of the prepared material which was also proved by scanning electron microscopy diagnostic method.
Prepared materials were observed by scanning electron microscope (SEM) Vega 3 XMU with LaB6 cathode by Everhart - Thornley scintillation secondary electron detector which can provide a very good topography exploration. Observations of the samples were realized in vacuum conditions using 30 kV accelerating voltage for getting a good resolution. Studying of the topography of prepared materials in SEM showed the difference in the crystal structure (Fig. 2, Fig. 3) of differently prepared cathode materials for lithium - ion power source. The experiments will be discussed in more detail in poster presentation.

References

[1] Ohzuku T, Brodd RJ (2007) An overview of positive-electrode materials for advanced lithium-ion batteries. Journal of Power Sources 174 (2):449-456
[2] Xiao J, Chen X, Sushko PV, Sushko ML, Kovarik L, Feng J, Deng Z, Zheng J, Graff GL, Nie Z, Choi D, Liu J, Zhang J-G, Whittingham MS High-Performance LiNi0.5Mn1.5O4 Spinel Controlled by Mn3+ Concentration and Site Disorder. Advanced Materials 24 (16):2109-2116.
[3] Hu M, Pang X, Zhou Z Recent progress in high-voltage lithium ion batteries. Journal of Power Sources 237 (0):229-242.
[4] Chen X, Xu W, Xiao J, Engelhard MH, Ding F, Mei D, Hu D, Zhang J, Zhang J-G (2012) Effects of cell positive cans and separators on the performance of high-voltage Li-ion batteries. Journal of Power Sources 213 (0):160-168

This work was financially supported by FEI Company, Centre for Research and Utilization of Renewable Energy under project No. LO1210 – „Energy for Sustainable Development (EN-PUR)” and project FEKT-S-14-2293.

Fig. 1: The comparison of the capacities during the 10 cycles by 0.5 C for LiNi0.5Mn1.5O4 cooling 0.5°C/min and LiNi0.5Mn1.5O4 cooling 0.2°C/min

Fig. 2: Crystal structure of LiNi0.5Mn1.5O4 cooled 0.5°C/min

Fig. 3: Crystal structure of LiNi0.5Mn1.5O4 cooled 0.2°C/min
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3334 Understanding the Surface Structure of LiNi0.45Mn1.55O4 Spinel Cathodes with Aberration-Corrected HAADF STEM

Amos C. D.1, Song J.1, Goodenough J. B.1, Ferreira P. J.1
1The University of Texas at Austin
charles.amos@utexas.edu

In order for Li-ion batteries to mature to a level useful for integration into the current or future energy infrastructure, basic problems such as cyclability, cost and rate capability must be overcome. LiNi0.5Mn1.5O4 (LNM), a spinel cathode material, has the advantage of being both cost-effective and a high-rate capable material, but it is plagued with cyclability problems. In the LNM system the main contributor to cycling degradation is the high operating voltage which leads to solid-electrolyte interphase (SEI) formation. We find that excess-Mn doping of this material (LiNi0.5-XMn1.5+XO4 where x=0.05) leads to increased cyclability through natural passivation [1]. To understand the exact role that excess Mn plays in the passivation of this cathode material, it is crucial to determine the surface’s atomic structure. This is because the surface structure determines how reactive the cathode will be with the electrolyte during oxidation and reduction cycles.

In order to understand how excess-Mn LNM reacts with the electrolyte, it is critical to understand the different phases that form in this system. In this regard, aberration-corrected HAADF STEM was used to identify the surface and bulk structures in the excess-LNM system. HAADF STEM confirms the spinel structure (Fig. 1) and shows good agreement with STEM simulations in the bulk. Near the surface however, other phases are observed. These include a rock-salt structure which is expected from x-ray diffraction (XRD) results and a new phase, defined here as “ring-type structure”, because of the characteristic rings that are formed within the first few atomic surface layers. All three phases are observed near the surface, however only the spinel is found within the bulk of the particles. Also, the rock-salt phase and ring phase do not necessarily have to exist in close proximity to one another even though they are found near each other in Fig. 1. This is evidenced in Fig. 2 where only the spinel and ring phases are present. HAADF STEM enables a detailed characterization of these phases and has led to an important understanding of the cycling degradation mechanisms in the excess-Mn LNM system. In turn, this work enables us to develop a well-suited cathode material for future energy storage that will potentially spur the evolution of the future sustainable energy landscape.

This work was supported by a NASA Office of the Chief Technologist’s Space Technology Research Fellowship, the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under award number DE-SC0005397, and a grant from the National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health.

Fig. 1: HAADF STEM image of LiNi0.45Mn1.55O4 along the [110] zone axis. Shown in the figure are the normal spinel phase (blue), the rock-salt phase (green) and the ring phase (red). The image has been deconvoluted for clarity.

Fig. 2: HAADF STEM image of LiNi0.45Mn1.55O4 along the [110] zone axis. Observed are the normal spinel phase in the bulk (blue) and the ring phase at the surface (red). No rock-salt phase is observed in this image.

Fig. 3: Structural models of the ordered spinel (left), rock-salt phase (center) and ring phase (right) oriented along the [110] zone axis. The yellow region indicated in the right figure is the characteristic ring observed in this phase. Blue atoms: Ni, purple atoms: Mn, and green atoms: Li. Multi-colored atoms indicate a fractional occupancy.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3348 Imaging and Spectroscopy of Filler-Matrix Interaction in a Ceramic Nanocomposite: First Evidence

GULGUN M. A.1, SHAWUTI S.1, CEH M.1,2, STURM S.1,2
1SABANCI UNIVERSITY FENS AND SUNUM, 2JOSEF STEFAN INSTITUTE
m-gulgun@sabanciuniv.edu

An inorganic salt (Na2CO3)-oxide nanoparticles (samarium doped ceria, SDC, (Sm0.2 Ce0.8O1.9)) composite showed an unexpected sinergy in electrical behavior [1-3]. The ionic conductivity of the composite shows a marked increase as the average oxide particle size decreases and when the oxide particle to matrix salt ratio is tailored to an optimum value (Figure 1) [4]. It was suggested that the interfacial interaction of oxide nanoparticles with the amorphous carbonate salt matrix would enhance the conductivity by generating new pathways for ionic transport.
High resolution transmission electron microscopy and energy filtered imaging was utilized to investigate the extend and type of this interfacial phenomenon. A TEM bright field image of the nanocomposite is shown in Figure 2a. Energy filtered imaging provided the first evidence for the influence of oxide surface on the structure of solid amorphous salts in the interfacial region. The interaction may not only create a new pathway fort he conduction but also increase the mobility of the conducting ion complexes. By altering the surface properties of the oxide nanoparticles it is possible to control the extend of this interaction.
An JEOL ARM 200 CFEG STEM and GATAN Quantum 965 ER Spectrometer were utilized to investigate the interaction between the oxide surface and the amorphous carbinate matrix phase. Energy filtered imaging of the composite using C_K, Na_ K, Sm_ L and Ce_L edges with a three window method proved to be problematic since Na-K (1074 eV) line and Sm_L (1075 eV) line are only 1 eV away from each other. However, the Ce_L and C_K line images are useful to visualize the carbonate shell around the ceria particles (Figure 2 b and c).

[1] B.Zhu, J.Power sources, 93 (2001) 82.
[2] B.Zhu, J. Power sources, 114 (2003) 1.
[3] B. Zhu, X. Liu, M. Sun, S.J, J. Solid State Sci., 8 (2003) 1127.
[4] S. Shawuti and M. A. Gulgun, 'Solid Oxide-Molten Carbonate Nano-composite Fuel Cells: Particle Size Effect', in review for J. Power Sources, 2014 Jan.

Fig. 1: Figure 1. The Nyquist plot for nanocomposites with different average particle sizes (PS) taken at 350°C, showing that the impedance of the composites increase with increasing PS.

Fig. 2: Figure 2 a. TEM bright field image of the SDC – Na2CO3 nanocomposite electrolyte. The light grey areas between the SDC oxide particles are the amorphous carbonate matrix.

Fig. 3: Figure 2 b. Energy filtered image taken Ce_M line showing the oxide particle locations clearly.

Fig. 4: Figure 2 c. Energy filtered image taken C_K line showing the concentration of carbonte ion clearly.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3351 Electron microscopy study of 2-dimensional lithium cobalt oxide

McGuire E. K.1,2, Mendoza-Sánchez B.1,3, Coehlo J.1,3, Downing C.1,4, Nicolosi V.1,2
1AMBER (Advanced Materials and Bioengineering Research Centre), Trinity College Dublin, Ireland, 2School of Physics, Trinity College Dublin, Ireland, 3School of Chemistry, Trinity College Dublin, Ireland, 4Advanced Microscopy Laboratory, Trinity College Dublin, Ireland
emcguir@tcd.ie

Liquid exfoliation of bulk material is an efficient and scalable synthesis method to produce single- and few-layers 2-dimensional (2D) flakes of greatly improved surface area as compared to the raw material [1]. Lithium cobalt oxide (LiCoO2) is the most popular material currently utilized for commercial rechargeable batteries [2]. The charge storage capacity of battery materials is proportional to the surface area of electroactive materials. Here, we investigate the possibility of improving the charge storage capability of commercially available LiCoO2 (particle size in the micrometer range) by producing 2D flakes (lateral dimension of approx 400 nm) of enhanced surface area.

Commercially available LiCoO2 and CoO2 were processed by liquid phase-exfoliation in various solvents to obtain 2D flakes. A fundamental question however is whether the exfoliation procedure affects the material’s electronic properties, crystal structure and stability.

The low atomic weight of lithium makes its detection and analysis by electron microscopy or spectroscopy challenging [3,4]. In order to overcome this difficulty we employ a combination of electron microscopy techniques, including high-resolution transmission electron microscopy (HR-TEM), electron diffraction, scanning transmission electron microscopy (STEM), energy dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS), to elucidate the structure and stability of exfoliated LiCoO2 and compare this with exfoliated CoO2. The combination of imaging and spectroscopy techniques allows us not only to assess the crystal structure and stability of these materials but also to evaluate their chemical structure. A detailed electron microscopy study of these materials will answer key questions relating to their structure, stability and potential as battery materials, as well as further our understanding of how liquid exfoliation affects materials at the atomic level.

[1] V. Nicolosi et al, Science 340 (2013), p. 1420.

[2] MS Whittingham Chem Rev 104 (2004), p. 4271.

[3] F Wang et al, ACS Nano 5 (2011), p. 1190.

[4] Y Shao-Horn et al, Nature Materials 2 (2003), p. 464.

The authors gratefully acknowledge the support of SFI and AMBER.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3377 TEM characterization of In-free transparent conductive oxides: the case of Al:ZnSnO

Hessler-Wyser A.1,2, Jeangros Q.1,2, Morales Masis M.1, Dauzou F.1, Ding L.1, Nicolay S.1, Ballif C.1
1Laboratory of photovotaics and thin-film electronics, Ecole Polytechique Fédérale de Lausanne, 2000 Neuchâtel, Switzerland, 2Interdisciplinary Centre for Electron Microscopy, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland, 3PVCenter, Centre Suisse d'Electronique et de Microtechnique, 2000 Neuchâtel, Switzerland
aicha.hessler@epfl.ch

Novel optoelectronic technologies, such as organic light emitting devices or flexible solar cells, require electrodes that are flexible, transparent and conductive. Materials such as In-Sn-O (ITO) and In-Ga-Zn-O (IGZO) both gather those three required properties and have been extensively studied in the last years. However the downside is that they are made out of scarce and expensive indium. Therefore there is a need from the market to find alternative solutions. Amorphous transparent oxide semiconductors are a relatively new class of materials, which could fulfil the requirements for replacing In in transparent conductive oxides (TCO), and in particular Zn-Sn-O (ZTO) is an excellent candidates as it is inexpensive, abundant and non-toxic.

This work presents the development and characterization of amorphous Al-doped ZTO grown by co-sputtering ZnO:Al and SnO2 with varying Sn/Zn composition ratio. 150 nm layers were simultaneously deposited on glass substrates and TEM Cu grids with thin C film for top-view characterization by STEM (on FEI Technai Osiris, Fig. 1). Same conditions were used for deposition of 300 nm thick layers for optical and electrical characterization. Hall mobility and free carrier concentration were determined by Hall-effect measurements using the Van der Pauw configuration. Optical transmission and absorptance spectra in the range from 320 to 2000 nm were determined using a UV-Vis-NIR spectrophotometer equipped with an integrating sphere. TEM lamellae were extracted by FIB for x-section.

A clear variation of the structural, electrical and optical properties was observed, indicating an ideal Sn/Zn ratio of 4.3 (measured by EDX), which was then used to deposit uniform layers by tuning the sputtering power of each target and rotating the substrate. Furthermore, although amorphous, the layer presented a columnar structure, which could explain a lower conductivity compared to crystalline layers (Fig.2). Hydrogen is known to act as a shallow electron donor in several TCOs and thus to improves their electrical properties. H2 plasma treatments were applied to the AZTO films under temperature and time conditions ranging from 50 to 200 °C and from 1 to 5 minutes, respectively. Electrical measurements show an improved behaviour after treatment, whereas optical transmittance degrades. SEM observations reveal that reduction of tin oxide is the responsible for the degraded optical properties. SIMS was also performed to assess a possible H penetration into the layer, but only revealed the oxygen depletion in the top 100 nm of the layer due to oxide reduction. This shows that EM studies play a key role in linking film morphology and physical processes occurring during H2 plasma treatment as well as electrical and optical properties.

The author thank EU FP7 for financial support (Flex-O-Fab project)

Fig. 1: Top view BF (top) and HAADF (middle) STEM micrographs and diffraction patterns (bottom) of 5 different Sn/Zn ratio ZTOs. Layers with Sn/Zn≤4.3 are amorphous.

Fig. 2: Top view (top) and cross-section (bottom) STEM micrographs of ZTO with Sn/Zn = 4.3 while rotating the substrate.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3391 Intermetallic phase formation in SmartWires: novel concept of interconnection technology for solar cells

Hessler-Wyser A.1, 3, Faes A.2, Cattin J.1,3, Baumgartner Y.1,3, Levrat J.2, Escarré J.2, Champliaud J.2, Despeisse M.2, Gattaneo G.1, Ufheil J.4, Papet P.5, Yao Y.6, Söderström T.6, Ballif C.1,2
1Laboratory of Photovoltaics and Thin-Film Electronics, Ecole Polytechnique Fédérale de Lausanne, 2000 Neuchâtel, Switzerland, 2PV-Center, Centre Suisse d'Electronique et de Microtechnique, 2000 Neuchâtel, Switzerland, 3Centre Interdisciplinaire de Microscopie Electronique, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, 4Somont, 79224 Umkirch, Germany, 5Roth&Rau Research AG, 2068 Hauterive, Switzerland, 6Division Module, Meyer Burger AG, Gwatt, Switzerland
aicha.hessler@epfl.ch

Standard busbars and ribbons are the commonly used contacting technology for crystalline silicon solar cells. For costs and efficiency constraints, alternative contacting solutions with less shadowing effects are developed. The innovative SmartWire Contacting Technology (SWCT) offers excellent perspectives as it combines several advantages: (1) reduction of production steps, (2) efficiency improvement by lowering ohmic losses in the existing metallization, (3) a reduced consumption of the costly raw materials by 85%, (4) enhancement of the module reliability and (5) improved aesthetics.

This work presents material characterization of those SmartWires when contacting c-Si heterojunction solar cells by scanning and/or transmission electron microscopy (SEM/(S)TEM), combined with analytical X-ray dispersive sepctroscopy (EDX), for as-fabricated and degraded modules. SWCT consists of polymer foils supporting copper wires coated with InSn alloy that has a low melting point (117 °C) and melts during the module lamination process (T = 160 °C). This leads to solder contacts to the solar cell silver metallization thanks to interdiffusion of metallic species and intermetallic phase formation occurring both at front and back contact of the cell. Degradation tests like thermo-cycling and damp heat IEC tests show a strong effect on electrical properties of the contacts. Therefore a detailed investigation of the microstructure evolution of the contacts and the formed phase is needed.

SEM-EDX was performed on an FEI xlf30 equipped with a Si-drift detector (Oxford), whereas TEM observations were carried out on FEI Technai Osiris with the dedicated ChemiSTEM technology. TEM lamellae were extracted by focussed ion beam (Zeiss, Nvision) from embedded samples. Simultaneously, diffusion tests were performed on In-Sn coated wires alone covered by silver paste, then analysed by SEM. In all cases, three ternary phases of Cu-In-Sn were found (Fig.1). Surprisingly, no silver was found in the tin phase that is directly in contact with silver paste, but significant silver amounts (up to 12 at%) were measured in the In-rich intermetallics (Cu2In3Sn). Degraded cell (80°C in dry air during 1500 h) shows that silver back contact disappears when in contact with the SmartWire, and that silver content of the In-rich phase raises up to 20 at%. Furthermore, silver grains segregate at the In-rich/Cu-rich phase interface (Fig. 2). Those results explain the disappearance of silver at the cell back surface.

The authors thank F. Bobard and D. Laub for sample preparation.

Fig. 1: SEM BSE micrograph (left) of a wire outer layer with the three phases differentiation (right). The wire was coated with silver paste (visible in the upper part of the image) and annealed at 160°C.

Fig. 2: SEM micrograph of the extracted TEM lamella (left), EDS mapping of the region (middle) and silver segregation at the inferface between Cu2(In,Sn) and Cu2In3Sn.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3395 Study of structure and defects in Li-rich layered oxide material for Li-ion batteries.

Shukla A. K.1, Ramasse Q.2, Darbal A.7, Das P.5, Mendoza J.6, Estrade S.6, Ophus C.4, Duncan H.3, Chen G.1
1Environmental Energy Technologies Division, Lawrence Berkeley National Laboratory, 2SuperSTEM, 3Kinestral Technologies, Inc, 4National Center of Electron Microscopy, Lawrence Berkeley National Laboratory, 5NanoMegas, 6University of Barcelona, 7AppFive
akshukla@lbl.gov


Transformations in Li-rich layered oxides have been extensively studied recently for their potential application in Li-ion batteries. These materials have attracted a lot of interest due to the high capacity offered by them. However, the structure of these materials in their pristine state is not clearly understood. Several reports have assigned their structure to be trigonal (R-3m), monoclinic (c2/m), or a combination of both (composite). The present study discusses the structure of Li1.2(Ni0.13Mn0.54Co0.13) O2 prepared with two different morphologies: plates and needles, using the results obtained from aberration corrected (scanning) transmission electron microscopy, electron energy loss spectroscopy (EELS), convergent beam electron diffraction and precession electron diffraction tomography and question the validity of the the claims of them being “composite”. It was found that these materials consist of domains which correspond to variants of monoclinic structure. It will be shown how diffraction-based experiments on such materials can often lead to misleading conclusions, since analysis of diffraction-based techniques inevitably assign them as trigonal, although the present study shows that the three-fold symmetry observed in electron diffraction patterns result from the combination of the variants having monoclinic structure.

Furthermore, results from STEM and EELS experiments showed that the pristine materials have several defects. The plates exhibited a differently ordered structure on their surface, and the needles exhibited several cobalt-rich line defects. These results prescribe that extreme care should be taken while interpreting the electron microscopy results obtained from cycled samples.

This work was supported by the Assistant Secretary for Energy Ef- ficiency and Renewable Energy, Office of Vehicle Technologies of the U.S. Department of Energy under the Batteries for Advanced Transportation Tech- nologies (BATT) Program. The authors also acknowledge support of the National Center for Electron Microscopy, Lawrence Berkeley Laboratory.

Fig. 1: STEM HAADF image showing the structure of monoclinic Li1.2(Ni0.13Mn0.54Co0.13) O2

Fig. 2: Inverted and color-coded HAADF STEM image showing the three variants (in projection) of the monoclinic structure.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3439 Electron Microscopy Characterization of the Nanostructure of ZnO-based hybrid solar cells

Wisnivesky Rocca Rivarola F.1, Divitini G.1, Saberi-Moghaddam R.2, Friend R. H.2, Ducati C.1
1Department of Materials Science and Metallurgy, University of Cambridge, Cambridge, UK, 2Cavendish Laboratory, University of Cambridge, Cambridge, UK
fw299@cam.ac.uk

Organic-inorganic hybrid materials have been widely studied for being a promising technology to obtain highly efficient and low cost devices for solar energy harvesting. One of the most extensively studied semiconductor materials for hybrid solar cells is zinc oxide (ZnO). Its wide band gap and high exciton biding energy (60 meV) are attractive features for application in optoelectronics, ultraviolet light emitters, piezoelectric devices, chemical sensors and photovoltaic devices [1] .
The main photovoltaic processes in hybrid solar cells occur in the light absorbing photoactive layer (bulk-heterojunction,BHJ), namely: exciton generation, dissociation and charge transport recombination. The relationship between the device efficiency and the morphology of the active layer makes thorough morphology characterization a key element to enable optimization of photovoltaic devices. A combination of microscopy techniques has to be used in order to characterize the chemical and morphological composition of the active layer with high spatial resolution.
In this work, we present a study of the morphology of the BHJ in hybrid solar cells made with ZnO and P3HT functionalized with a carboxylic acid group. ZnO was deposited over an annealed P3HT matrix through atomic layer deposition (ALD), and the resulting composite was probed using scanning transmission electron microscopy (STEM), energy–dispersive x-ray (EDX) spectroscopy and high resolution transmission electron microscopy (HRTEM). The microscopy images and spectra obtained were analyzed to quantitatively examine the geometry of the resulting structure and correlate the nanostructure with charge transport efficiency. Cross-sectional imaging of a lamella of the BHJ incorporated into a device was also performed to complement plan-view images obtained, allowing analysis of the features with a 3D perspective. A correlation between the measured solar cell performance parameters and the morphological features characterized was obtained by modelling the system using a simulation tool (TiberCAD).
Results obtained showed that the recrystallized P3HT film formed an organized structure such that the deposition of ZnO particles occupied the spaces between the P3HT chain stacks. This resulted in a film composed by arrays of ZnO particles, separated by 10 nm wide polymer sections. EDX mapping of the samples were obtained for compositional analysis (Figure 1) and High resolution TEM was employed to complement STEM characterization (Figure 2), by probing the crystallography of the film’s nanostructure.

[1] E. Guziewicz et al, J. Appl. Phys., volume 105, (2009) p. 122413.
[2] J. Bouclé et al, J. Mater. Chem., volume 17 (2007) p. 3141.

F.W.R.R. gratefully acknowledges funding from CNPq under grant number 246050/2012-8.

Fig. 1: STEM image of the recrystallized P3HT/ZnO film and EDX mapping images of sulphur (S,blue), zinc (Zn, yellow), carbon (C, red) and oxygen (O, orange).

Fig. 2: HRTEM image of the ZnO/P3HT composite. The FFT is displayed on the top right along with the equivalent crystal orientation on the [0 0 0 1] zone axis.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3454 Initial Observations of the Lithiation of Tin Nanoneedles

Janish M. T.1, Mackay D. T.2, Jungjohann K. L.3, Liu Y.3, Carter C. B.1,3, Norton M. G.2
1U. of Connecticut, Storrs, CT, USA, 2Washington State University, Pullman WA, USA, 3Center for Integrated Nanotechnologies (CINT), Sandia National Laboratories, Albuquerque, NM, USA
matthew.janish@uconn.edu

Metallic Sn has generated considerable interest as a candidate anode material for lithium-ion batteries (LIBs) due to its high theoretical specific capacity of 994 mAh/g. However, during cycling a large volume change occurs; the high associated stresses cause mechanical failure of the electrode [1], and this prevents bulk Sn from being used in LIBs. Nanostructured Sn offers a possible route to circumvent this problem [2]. A smaller, open structure accommodates the volume changes, which reduces the local stresses and prevents mechanical failure [3]. Such Sn nanostructures have been prepared using a template-free, low-temperature, industry-scalable process [2]. This paper will report initial TEM observations of the microstructural changes that occur in this material during the lithiation and delithiation processes.

The method described in [2] was adapted for in-situ TEM experiments by electroplating the Sn needles onto a Cu TEM grid. A Nanofactory TEM-STM holder and FEI Tecnai F30 TEM operated at 300 kV were used for the study. The grid was cut in half and affixed to a piece of Al wire with a conductive epoxy for mounting in the holder. Li metal was used as the counter electrode, and was mounted in the Nanofactory holder on a piece of tungsten wire in a glove box with a dry-He atmosphere. A layer of Li2O formed on the surface of the Li metal during the transfer to the TEM; this oxide acted as the solid electrolyte during the experiment.

The Li2O was brought into contact with a needle as shown in Figure 1, and a voltage was applied. The volume change in the needle after lithiation is obvious, though only the needle making contact with the electrolyte reacts. Other needles are just visible at the top corners of both Fig. 1a and 1b, and appear unchanged between the two. Some materials undergo an irreversible transformation during the first cycle; Si, for example, forms an amorphous material after delithiation. The diffraction patterns shown in Figure 2 demonstrate that this is not the case for Sn. Fig. 2a and 2c are consistent with β-Sn (tetragonal) while the pattern in Fig. 2b is that of a polycrystalline Li-Sn alloy—i.e. the needle is crystalline before, during and after lithiation. This result is consistent with measurements made using this material in button cells where reproducible behavior over many cycles is seen [2].

References:

1. Park, CM et al., Chem Soc Rev, 2010. 39(8): 3115-41

2. Mackay, DT et al., J of Mat Sci, 2013. 49(4): 1476-1483

3. Huang, JY et al., Science, 2010. 330: 1515-1520

4. Wei, Z et al., J Power Sources, 2013. 223: 50-55

5. MTJ acknowledges a GAANN Fellowship from the Dept of Education

This work was performed at Sandia National Laboratories at CINT, a DOE-BES supported national user facility, and in the Materials Characterization Department. Sandia National Laboratories is a multiprogram laboratory managed & operated by Sandia Corporation, a Lockheed Martin Corporation, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DEAC04-94AL85000.

Fig. 1: An Sn needle a) before and b) during lithiation. In ‘a’ the the tip of the needle is about to touch the Li source which is located at the bottom of the image. In ‘b’ the Li source has been moved to contact the needle, and a voltage has been applied. The dimensions of needle have increased and the needle is still crystalline.

Fig. 2: Diffraction patterns from key timepoints during the experiment: a) before lithiation, consistent with β-Sn but off-axis; b) after lithiation, consistent with a polycrystalline Li-Sn alloy; c) after delithiation, consistent with β-Sn and nearly on the [001] zone axis; and d) partial re-lithiation after separating the electrodes, caused by the beam.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3501 TEM of High Dose Neutron Irradiated Austenitic Stainless Steel with Use of Precise 1 mm Disc Punching Methodology from Minimized Tensile Specimens

Michalička J.1, Bublíková P.1, Namburi H. K.1, Rosnecký V.1, Keilová E.2, Kočík J.2, Ernestová M.2
1Research Center Rez, Rez, Czech Republic, 2UJV Rez, Rez, Czech Republic
mja@cvrez.cz

In today’s world, energy consumption is increasing drastically to meet the economy needs. Nuclear energy is considered as one of the means for energy source. Nuclear industries are aspiring for building modern Nuclear Power Plants (NPP’s) with enhanced safe operation and energy through-put as main concerns. Modern NPP’s require structural materials with high performance that can resist the effects of harmful irradiation conditions. This keep great challenging tasks for researchers to understand existing failure mechanisms due to the irradiation, assess current and develop new materials that are more appropriate for structural stability of NNP’s. Structural and System Diagnostics department at Research Center Rez focuses to study the irradiated and non-irradiated materials behavior and their degradation aspects. This includes also microanalysis of structural materials of nuclear reactors.

 

The presented study is a part of post irradiation examination of CW 316 austenitic stainless steel used for baffle bolts in Reactor Vessel Internals (RVI) of Pressurized Water Reactors (PWR). The material was exposed to neutron dose 15 dpa (displacements per atom) under temperature 300°C. A combination of effect of complex irradiation-induced damage formed in a nano-metric scale together with applied mechanical load and corrosive PWR primary water environment may turn out the RVI component to be sensitive to intergranular Irradiation Assisted Stress Corrosion Cracking (IASCC).

Our main concern is to be able to perform TEM study of localized area adjacent to a crack of broken mechanical testing specimen of the highly radioactive RVI materials and to study cracking mechanisms. To reduce the radioactivity, minimized tensile specimens must be used generally. In particular, we have tested tensile specimens with shank diameter 2 mm, see Fig. 1. Therefore, a standard 3 mm TEM discs could not be punched-out, but a tailored methodology specialized in 1 mm TEM thin foil preparation had to be developed from the first cut up to the final electron transparent TEM foil and applied on radioactive material.

The first part of the study describes whole process from bulk sample handling, including e.g. remote-controlled material cutting in shielded hot-cells and TEM disk polishing in glow-boxes, up to the main final procedure of electrolytic-polishing of 1 mm TEM foils.

The second part shows results of TEM analysis of the studied material including radiation-induced defects observed by advanced techniques using different diffraction or phase contrast conditions and as well as deformation microstructure relevant to the cracking of the neutron irradiated stainless steel.

This work has been supported by the SUSEN Project CZ.1.05/2.1.00/03.0108 realized in the framework of the European Regional Development Fund (ERDF).

Fig. 1: Fig. 1: A schematic drawing of miniaturized Slow Strain Rate Test (SSRT) tensile specimens used for IASCC studies on neutron irradiated RVI materials. Area Of Interest (AOI) is indicated.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-3527 TEM visualisation of Sodium-Vacancy Ordering in olivine NaxFePO4 (x≈0.7)

Roddatis V. V.1,2, Arenal R.3,4, Galceran M.1, Acebedo B.1, Peral I.5, Rojo T.1,6, Casas-Cabanas M.1
1CIC Energigune, Albert Einstein 48, 0150 Miñano, Spain, 2Georg-August-Universität Göttingen, Institut für Materialphysik, Friedrich-Hund-Platz 1, 37077 Göttingen, Germany, 3Laboratorio Microscopias Avanzadas (LMA), Inst Nanociencia Aragon (INA), Univ Zaragoza, 50018 Zaragoza, Spain, 4Fundacion ARAID, Zaragoza, 50004, Spain, 5CELLS – ALBA, Campus Universidad Autonoma de Barcelona, 08193 Bellaterra (Barcelona), SPAIN, 6eDepartamento de Química Inorgánica, Facultad de Ciencia y Tecnología, Universidad del País Vasco, 48080 Bilbao, Spain
vroddatis@material.physik.uni-goettingen.de

       The microstructural changes and phase transitions taking place during the charge/discharge cycles critically influence battery performance and lifetime. Recently, NaFePO4 has attracted a great attention as the one of promising materials for sodium ion batteries. The formation of superstructures in NaxFePO4 (x≈0.7) during FePO4 to NaFePO4 transformation was reported by several groups, however no direct imaging of sodium/vacancy ordering has been provided yet. Here we report the observation of Na atoms/vacancies ordering in NaxFePO4 (x≈0.7) nanoparticles as revealed by electron diffraction (ED), high resolution (scanning) transmission electron microscopy (HR(S)TEM), atomic modeling and HRSTEM image simulations.

       An intermediate NaxFePO4 (x≈0.7) phase, as a powder, was prepared both chemically and electrochemically. The superstructure formation was confirmed for particles obtained for both synthesis methods. Electron diffraction patterns (Figure 1 a-c) collected from main zone axes and accompanied with local Energy Dispersive X-ray (EDX) analysis revealed the presence of several phases with the same elemental composition but different Na/vacancy ordering. Different superstructures were found in different particles as well as coexistence of differently oriented domains with superstructure was demonstrated within one nanoparticle (Figure 1 d-f). It was also observed that electron beam strongly influenced on the structure of NaxFePO4 phase causing redistribution of Na atoms. This process was found to be extremely fast, however the use of cooling specimen holder allowed reducing significantly the beam influence and tracing the structural transformations. Thus, it has been discovered that FePO4 to NaFePO4 transformation proceeds through the formation of different commensurate and incommensurate phases.

This work was supported by Etortek project energiGUNE’10 and funding from the European Union FP7 under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative – I3).

Fig. 1: (a)-(c) ED pattern along [100], [010] and [001] directions, respectively; (d) low magnification HAADF image of NaxFePO4 particle; (e) and (f) HRSTEM images from area 1 and area 2 (Fig.1d), respectively. Atomic models are shown in insets.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-5708 TEM analysis of irradiated and non irradiated MgH2 thin films

Raskovic – Lovre Z.1, Kurko S.1, Ivanovic N.1, Fernández J. F.2, Fernández J. R.2, Sturm S.3, Mogstad T.4, Novakovic J. G.1, Novakovic N.1
1Vinča Institute of Nuclear Sciences, Belgrade, Serbia, 2Universidad Autónoma de Madrid, Madrid, Spain, 3Jožef Stefan Institute, Ljubljana, Slovenia, 4Institute for Energy Technology, Kjeller, Norway
saso.sturm@ijs.si

Among the diverse Mg-nanostructures, adequate as H storage materials the Mg thin films draw interest since of its relatively easy synthesis and low reactivity. MgH2 thin films were obtained in inline sputtering system using high purity Mg target, annealed in vacuum to increase the stability and reduce the oxidation. Thin films irradiation was done using 60keV Ar+ ions with fluence 1015 ion/cm2. TEM analyses were performed by using JEM-2010F TEM/STEM microscope operated at 200 keV, equipped with EDXS-ISIS300 and EELS system. TEM specimens were prepared by applying conventional cross-section technique. TEM image (Fig. 1a) shows that non-irradiated film is homogeneous. HRTEM image acquired from the non irradiated film central region indicates that film consists of randomly orientated crystallites. To measure the average crystallites size, first the corresponding fast Fourier transform (FFT) was performed (inset in Fig.1b). The Bragg-mask filter was applied in the FFT allowing the contribution of only Bragg reflections to the formation of the inverse FFT image (Fig. 1c). The crystallite size is ranging between 6-7 nm. The irradiated film shows notably diverse microstructure i.e. large crystal grains are imbedded in a crystallites matrix (Fig. 2a) and can reach the size of 80 nm (dark-field TEM image, Fig.2b). Although the severe microstructural changes can be expected only in the 80 nm surface region, the film is completely recrystallized throughout all thickness which is attributed to the thermal effects of the irradiation. Fig. 3a show composed SAED pattern acquired from the several region of the non-irradiated film. The bottom region SAED pattern is characterized by smaller number of diffraction rings and contains mainly the MgO phase, while the rest of the film is composed of the MgH2 phase. SAED analysis shows that the e-beam irradiation of those MgH2 films didn’t promote hydrogen desorption, as it is earlier observed. The SAED patterns acquired in different regions of the irradiated film did not show large differences, which suggests that the distribution of crystal phases is uniform all over the film, but there is different phase distribution comparing to non-irradiated film (Fig. 3b). The diffraction rings corresponds to MgH2 and MgO phases. MgO crystallites are uniformly present thorough all film thickness, while the MgH2 phase is characterized only by a small amount of discrete crystals. The most intense diffraction rings correspond to large Mg crystallites.

This work was financially supported by the Slovenina Research Agency and European Union as part of the Framework 7 program by [FP7/2007-2013] under grant agreement n°312483 (ESTEEM2). Authors also thank Spanish MINECO and CAM by financial support under contracts MAT2011-22780 and CCG10-UAM/ENE-5245 and MEST Serbia under grant III 45012.

Fig. 1: Fig.1.a) TEM image of non-irradiated film. b) The HRTEM image of the central film region with the corresponding FFT pattern. c) Bragg-masked HRTEM image with indicated crystallites.

Fig. 2: Fig.2.a) Bright-field and b) corresponding dark-field TEM image of the irradiated film.

Fig. 3: Fig.3.a) SAED patterns acquired from the surface, central and bottom region of the non-irradiated film. Calculated SAED patterns of MgO (full line) and MgH2 (short dashed line) phases are superimposed. b)SAED pattern acquired from the irradiated film shows the presence of MgH2, MgO and Mg (long dashed line) phases.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-5718 TEM study of the nanosized Li2MnO3

Burlaka L.1, Grinblat J.1, Markovsky B.2, Kovacheva D.3, Aurbach D.1,2
1Bar Ilan Institute for Nanotechnology and Advanced materials, Ramat Gan 52900, Israel, 2Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel, 3Institute of General and Inorganic Chemistry, Academy of Sciences, Sofia 1113, Bulgaria
tapuzinka@gmail.com

     Lithium manganese oxides, such as layered trigonal LiMnO2, cubic spinel-type LiMn2O4 and monoclinic Li2MnO3 etc. are electroactive intercalation host materials in lithium cells. Among them Li2MnO3 is one of the most interesting compounds from the point of view of its electrochemical behavior. Usually, in the layered compounds, during the charge/discharge cycles, Li‏ ions leave/fill their sites, and charge neutrality of the unit cell is preserved by oxidation/reduction of Mn atoms. By contrast, the Li2MnO3 compound is electrochemically inactive within the voltage range 2.0 V- 4.4 V because lithium can be extracted without oxidation of the Mn ion due to the fact that all manganese ions in the Li2MnO3 structure are tetravalent and cannot be oxidized further. On the other hand, the Li2MnO3 electrode can be activated at >4.4 V when Li ions are extracted together with oxygen, and in this condition the expected theoretical reversible capacity of the material can reach about 460 mAh/g for complete Li extraction. Moreover, when synthesized in a nanocrystalline form, Li2MnO3 becomes electroactive, probably due to the factors associated with the material’s defect chemistry and significantly larger area of working surface possessing more active sites for the electrochemical reactions.
     Despite the dramatic effect of the nano-size morphology (<20 nm) on the electrochemical behavior of the material, little work has been done on understanding the mechanism of activation processes and their role in accessing higher levels of energy storage, as well as understanding the details of structural reorganization during lithium intercalation/deintercalation cycles. A clue to solving these issues could be found in systematical studies of the structural changes occurring in the material in the course of charge /discharge cycles.
     In this study, we present our results of TEM investigations of the structural evolution caused by cycling the nano-sized Li2MnO3 material. The material was synthesized by a self-combustion reaction (SCR). To characterize the particles on the nanometer scale, convergent electron-diffraction technique was applied along with the conventional methods of TEM microscopy and powder XRD analysis. It was shown that in the course of electrochemical cycling the Li/Mn cation ordering in the transition metal layers, which is characteristic of the Li2MnO3 monoclinic structure, gradually disappears. The main structural change accompanying the cycling process is a transition to spinel-like structure (more thermodynamically stable than the layered one), which apparently involves migration of Mn ions from their octahedral sites in the Mn layers into octahedral sites in the Li layers, with concomitant displacing of Li ions to tetrahedral sites.

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-5858 Microstructural characterization of Na0.44MnO2 nanorods as a cathode material for Na-ion batteries

KAYA P.1, DEMIREL S.2, OZ E.2, ALTIN E.3, ALTIN S.2, BAYRI A.2, AVCI S.4, TURAN S.1
1Department of Materials Science and Engineering, Anadolu University, Eskisehir, Turkey, 2Physic Department, Inonu University, Malatya, Turkey, 3Scientific and Technological Research Center, Inonu University, Malatya, Turkey, 4Department of Materials Science and Engineering, Afyon Kocatepe University, Afyon, Turkey
pkayamse@gmail.com

Rechargeable batteries are essential components for powering ever-more demanding portable electronic devices such as laptops and smart phones [1]. Over the last years and decades, the intercalation-based lithium ion battery (LIB) technology has been at the center of this research, due to its high power density and capacity [2]. However; due to high cost and limited Li sources; research has recently also focused on alternative energy storage technologies and sodium ion batteries are one of the promising candidate instead of lithium ion batteries [2, 3].
NaMn2O4 nanorods were synthesized by using conventional solid state method. Na2O2 and Mn2O4 were mixed in agate mortar and then sintered at 750oC for 24 h under oxygen atmosphere. After sintering, the powders were pressed into pellets and the pellets were heat treated at 250-900oC for 24 h under oxygen atmosphere. The samples were characterized by employing XRD, SEM and TEM techniques. XRD patterns of the samples were recorded by automated Rigaku RadB Dmax x-ray difractometer with CuKa radiation was used for the XRD analysis. Sintering the sample at 250oC and 450oC is not enough to from the Na0.44MnO2, since it still contains Na2O2 and Mn2O4 (MnO2) phases as the main phases. During the next step of sintering at 650oC for 24 h, the Na0.44MnO2 phase starts forming (Figure 1). Scanning electron microscopy (SEM) investigations were carried out by using a Schottky emitter field emission gun (FEG) SEM (Zeiss SUPRA 50 VP) equipped with in-lens, back scattered (BS), electron back scattered diffraction (EBSD) and energy dispersive x-ray (EDX) detectors. For transmission electron microscopy (TEM) analysis, powder sintered at 750°C for 24 h was chosen and dispersed in 2 - propanol and then suspension was dropped on the copper grid. TEM studies were conducted by using 200 kV field emission TEM (JEOL™ JEM-2100F) equipped with STEM high angle annular dark field (STEM-HAADF) detector (Model 3000, Fischione), parallel electron energy loss spectrometer (PEELS) and energy filter (Gatan™ GIF Tridiem), and energy dispersive spectrometer (EDS) (JEOL™ JED-2300T). According to microstructural characterization results, the length of the rods reach to ~500µm, while their widths range between ~100 nm – ~2 μm (Figure 2).

References
[1] M. Armand, J.-M. Tarascon, Nature, 451, 652 (2008)
[2] N. Bucher, S. Hartung, A. Nagasubramanian, Y. L.Cheah, H. E. Hoster, and S.Madhavi, ACS Appl. Mater. Interfaces, 6 (11), 8059–806, (2014)
[3] Y. Lu, S. Zhang, Y. Li, L. Xue, G. Xu, X. Zhang, Journal of Power Sources, 247, 770–777 (2014)



Fig. 1: Bottom panel: XRD patterns showing the evolution of Na0.44MnO2 phase after various heat treatments. Top panel: XRD pattern for the nanorods scratched from the surface of the main matrix.

Fig. 2: SEM image of the samples sintered at 700 for 24 h taken by using INLENS dedector.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-5860 The enhancement of capacity by B substituted LiMn2O4 cathode materials and their cycling performance

Demirel S.1, Oz E.1, Altin E.2, Altin S.1, Ozdemir M.3, Oner Y.4, Boyraz C.5, Avci S.6
1Physics Department, Inonu University, Malatya, Turkey, 2ScientificandTechnological Research Center, Inonu University, Malatya, Turkey, 3Department of MechanicalEngineering, Marmara University, Istanbul, Turkey, 4Physics Department, Marmara University, Istanbul, Turkey, 5Department of Physics Engineering, Istanbul Technical University, Istanbul, Turkey, 6Department of Materials Science and Engineering, AfyonKocatepe University, Afyon, Turkey
demirel.srkn@gmail.com

Energy production and consumption that rely on the combustion of fossil fuels have had severe impacts on world economics and ecology [1]. The rechargeable li-ion batteries have became the part of energy cycles.Li-ion batteries have found very broad and promisingapplications in modern technology [2, 3].In this research, LiBxMn2-xO4 cathode materials were synthesized via using conventional solid state reaction method.The XRD, SEM, temperature dependence resistivity (R-T) and micro strain of samples were analyzed and the results were compared by the cycle life and battery performance.The lattice parameters were calculated by Jade 5.0 using Rietveld refinement technique. The samples for x≤0.5 have no impurity phases. The xrd pattern of LiMn2O4 samples for as seen in Figure 1. Electrochemical measurements were carried out at room temperature (24°C). To assess thequasi open-circuit voltage profiles, galvanostatic charge-discharge cycles wererecorded at a slow scan mode using a MTI-BST8 system as seen Figure 2.We report that the B substitution increases the capacity of the coin cell which is a promising result for energy storage technologies.

[1]M. Armand,J.-M. Tarascon,Building better batteries, Nature, 451, 652 (2008)
[2]Timothy J. Kucharski ,Yancong Tian , Sergey Akbulatov and Roman Boulatov, Chemical solutions for the closed-cycle storage of solar energy,Energy Environ. Sci.,2011, 4, 4449-4472
[3]Th. Dumont, T. Lippert, M. Döbeli, H. Grimmer, J. Ufheil, P. Novák, A. Würsig, U. Vogt, A. Wokaun, Influence of experimental parameter on the Li-content of LiMn2O4 electrodes produced by pulsed laser deposition, Applied Surface Science, 252, 13, 2006, 4902–4906.

This study was supported by TUBITAK (The scientific and Technological Research council of Turkey) under grant no TUBITAK 112M487 and Inonu University Research found under grant no BAP 2013-154 and BAP 2014-02

Fig. 1: XRD pattern of LiMn2O4.

Fig. 2: Cyclic voltammetry of LiBxMn2-xO4.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-5873 Battery Performance and Structural Properties of Boron Substituted Li-ion Batteries

Oz E.1, Demirel S.1, Altin E.2, Altin S.1, Ozdemir M.3, Oner Y.4, Boyraz C.5, Avci S.6
1Physics Department, Inonu University, Malatya, Turkey, 2Scientific and Technological Research Center, Inonu University, Malatya, Turkey, 3Physic Department, Marmara University, Istanbul, Turkey, 4Department of Physics Engineering, Istanbul Technical University, Istanbul, Turkey, 5Department of Mechanical Engineering, Marmara University, Istanbul, Turkey, 6Department of Materials Science and Engineering,AfyonKocatepe University, Afyon, Turkey
erdinc_oz_86@hotmail.com

   LiCoO2 is one of the important cathode materials for rechargeable battery technology and it has been studied extensively in the literature[1]. Up-to-date the studies based on the improvement of the performance of the battery have been focused on cathode materials’ structural and electrochemical properties[2]. However, lithium rechargeable batteriessuffer from capacity fade and slow recharging. Capacity fade is linked to the changes in materials’ properties due to repeated charge/discharge cycles [3]. Therefore, it is necessary to optimize the cathode material and interfaceproperties to allow faster recharging and prevent capacity fade of the battery. Boron substitution in cathode materials have been reported to increase the battery performance [4, 5].
   Here, we report the synthesis, structure and electrochemical properties of LiCo1-xBxO2 (x=0-1). The compounds were fabricated using conventional solid state reaction technique. The lattice parameters were calculated by Jade 5.0 using Rietveld refinement technique.The samples for x≤0.25 have no impurity phases which indicates that the B3+ions substitute perfectly for Co3+ ions in the LiCoO2. The LiBO2 and Li6B4O9 impurity phases start forming on the samples for x>0.25 as seen in Figure 1.Increasing boron content in the samples, the grains show a well-defined round shaped structure.Electrochemical measurements were carried out at room temperature (24°C). To assess thequasi open-circuit voltage profiles, galvanostatic charge-discharge cycles wererecorded at a slow scan mode using a MTI-BST8 system as seen Figure 3.

References.
[1]T. Nagaura, K. Tozawa, Progress in Batteries & Solar Cells 9, 209 (1990).
[2]R.J. Gummow, D.C. Liles, M.M. Thackeray A Reinvestigation of the Sructures of Lithium-Cobalt-Oxides Whith Neutron-Diffaction Data. Materials Resaearch and Bulletin. 28, 1177-1184(1993).
[3] J. N. Reimers., J. R. Dahn Electrochemical and In Situ X‐Ray Diffraction Studies of Lithium Intercalation in LixCoO2. J. Electrochem. Soc, 139, 8, 2091-2097 (1992).
[4] A. Mauger, X. Zhang, H. Groult, C.M. Julien, LiCo1-yByO2 as cathode materials for rechargeable lithium batteries”, ECS Transactions, 35 (34), 141-148 (2011)
[5] R. Alcantara, P. Lavela, and J. L. Tirado, Structure and Electrochemical Properties of Boron-Doped LiCoO2, J. Solid State Chem.134, 265-273 (1997).

This study was supported by TUBITAK (The scientific and Technological Research council of Turkey) under grant no TUBITAK 112M487 and Inonu University Research found under grant no BAP 2013-154 and BAP 2014-02.

Fig. 1:  XRD patterns of LiCo1-xBxO2 for x=0, 0.125,0.25,0.375, 0.5, 0.75, and 1. The Blue (top) and red (middle) and orange (bottom) vertical bars are the Bragg angle positions for LiCoO2 (PDF#50-0653), LiBO2 (PDF#51-0517) and Li6B4O9 (PDF#47-0170) phases, respectively.

Fig. 2: Charge-discharge profiles of LiCo1-xBxO2cathodematerials (x=0 and x=0.125).
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-5900 NanoSIMS and TEM analysis of deuterated ZIRLO and Zr-1%Nb nuclear fuel cladding alloys

Aarholt T. M.1, Moore K. L.1, Frankel P.2, Preuss M.2, Lozano-Perez S.1, Grovenor C. R.1
1Department of Materials, Oxford University, Parks Road, Oxford, UK, 2Materials Performance Centre, School of Materials, University of Manchester, Manchester, UK
thomas.aarholt@materials.ox.ac.uk

NanoSIMS (Secondary Ion Mass Spectrometry), SEM (Secondary Electron Microscopy) and TEM (Transmission Electron Microscopy) have been used to study the hydrogen pickup of ZIRLO, a commercial zirconium alloy currently employed in the cladding material in nuclear reactors and Zr-1%Nb, a binary research alloy with particularly strong corrosion resistance. Samples were oxidised in autoclave in water for 80 and 180 and 540 days at 360°C to produce different levels of oxidation, two samples of each at pre- and post-transition. Further oxidation in 5% deuterium-rich water for 45 days at 350°C introduced deuterium to the metal and oxide, simulating the transit of hydrogen through oxidised samples in wet corrosion at the given moment of transition. The alloys were analysed in cross sections using NanoSIMS. Deuterium was found to distribute differently across the oxide in samples that experienced different oxidation times, with an interesting correlation to the breakaway behaviour of these oxides. In particular, peaks of deuterium concentration where found in the first micron of oxide. Large zirconium hydrides (deuterides) were found in the metal. SEM with Energy-Dispersive X-ray Spectroscopy (EDS) was used to relate the effect of second-phase particles to the distribution of deuterium in the areas analysed by NanoSIMS. Second phase particles containing Fe and Nb were found. TEM foils made by DualBeam Focussed Ion Beam allowed for microstructural comparison of the two alloys at pre and post-transition.

I would like to thank Westinghouse for funding this research.

Fig. 1: Cross section of post-transition Zr-1%Nb after 540 days oxidation in water and an additional 45 days in deuterated water, showing a slice through the oxide into the metal. NanoSIMS analysis shows large accumulation of deuterium in the oxide as well as a large deuteride (ZrH) in the metal.

Fig. 2: Line profile comparing the deuterium signal (red) with the oxygen signal (blue) from a cross-section of ZIRLO (deuterium image shown on the lower right) after 180 days oxidation in water and an additional 45 days in deuterated water. A peak in deuterium within the first micron of oxide is found to be consistent throughout measurements.
↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-6035 Focused Ion Beam serial sectioning tomography of Li-ion battery electrodes

Song B.1, Ying S.1, Sui T.1, Liu L.2, Kim T. H.1, Lu L.2, Korsunsky A. M.1
1MBLEM, Department of Engineering Science, University of Oxford, Oxford OX1 3PJ, UK, 2Department of Mechanical Engineering, National University of Singapore, Block EA#07-08, 9 Engineering Drive 1, Singapore 117575
alexander.korsunsky@eng.ox.ac.uk

The structural features of the lithium ion battery cathodes at the micro- and nano-scale were studied using serial material removal (sectioning) using the combination of Focused Ion Beam milling and SEM imaging using the Tescan LYRA3 XM instrument. The serial sectioning of as-fabricated and cycled cathodes allowed revealing the changes that take place in the internal arrangement of the particles of the active material. In the case reported here, the active material for the battery cathode consisted of the mixed layered transition metal oxides of Mn, Ni and Co that were bonded by the polymer matrix loaded with carbon black to provide electron conductivity. To understand the evolution of the structural arrangements within the battery electrode upon cycling, charge-discharge cycling was applied to one of the two nominally identical samples, and then their 3D structure was analysed by repeated layer removal by FIB. Comparing the raw results and tomographic reconstructions reveals that charge cycling leads to visible changes in the morphology of the layered oxides. The observations were consistent with the appearance of internal flaws (cracks and voids) within the mixed oxide particles. 3D reconstruction of allowed the shape and connectivity of these defects to be observed and analysed. The complex meandering morphology of the cracks observed suggests the presence of weak internal interfaces at the nanoscale. The possibilities of further studies opened up by these results are also discussed.

Colleagues at Tescan UK and Tescan Brno (CZ) are gratefully acknowledged

↑ Go to the Table of content ↑
Type of presentation: Poster

MS-14-P-6046 Formation of desert rose structures in vacuum plasma sprayed electrodes for alkaline electrolysis

Bentzen J. J., Zhang W., Jørgensen P. S., Bowen J. R.
Department of Energy Conversion and Storage, Technical University of Denmark, Risø Campus, Roskilde, Denmark
jabe@dtu.dk

The EU FCH-JU RESelyser project is concerned with the development of high pressure, high efficiency and low cost alkaline water electrolysers that can be operated variably and intermittently to meet the demands for integration into energy networks relying on fluctuating renewable energy. The project utilizes NiAlMo alloy electrodes produced at the German Aerospace Center (DLR) by vacuum plasma spraying (VPS). VPS results in a heterogeneous microstructure consisting of a multitude of intermetallic phase sub domains and pores. Prior to electrolysis operation the electrodes are activated by leaching of Al and some Al containing intermetallic phases leaving micrometer pores and nanometer dendritic pores increasing the surface area available for the electrolysis reactions.
The vacuum plasma sprayed electrodes were analyzed by high resolution SEM and TEM before and after electrolysis operation and after storage in water. Analyses of cross sections and electrode surfaces revealed desert rose like nano flake structures on the surface and in the pores on several electrodes. The formation of the desert rose structure appeared to be related to the electrolysis operation as well as the duration of storage in distilled water. The size of the faceted flakes varied from tens of nm to a couple of µm where the thickness varied from a few nm to ~50 nm. The desert rose structure was confirmed by TEM to consist primarily of NiO and Al2NiO4 like phases (similar lattice parameters). The possible implications for the application and performance of the electrodes are discussed.

This work is funded by the European Union’s Seventh Framework Programme for the Fuel Cells and Hydrogen Joint Technology Initiative under grant agreement n° [278732] 10.

Fig. 1: Formation of desert rose nano flakes on the electrode surface and in pores. SEM images of surface a) as sprayed; b) leached, washed and dried; c) leached, washed and stored in water 3 d; d) leached, washed, stored 120 d in water, and operated as electrode for ~30 min.; e) leached, washed, stored 90 d in water, and operated as electrode for 28 d.
↑ Go to the Table of content ↑

LS-1. Live imaging of cells, tissues and organs

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-1-IN-2399 Regulated exocytosis and diabetes

Lim C.1, Bi X.1, Wu D.2, Kim J.3, Gunning P. W.4, Hong W.5, Han W.1
1Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR), Singapore, 2The Key Laboratory of Regenerative Biology and The Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China, 3Seoul National University, Seoul, Korea, 4Oncology Research Unit, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia, 5Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Singapore
weiping_han@sbic.a-star.edu.sg

Diabetes is the result of insufficient insulin secretion from pancreatic beta-cells and impaired insulin signaling in the muscle and adipose tissue. At the cellular level, insulin signaling stimulates glucose transporter 4 (GLUT4) to translocate from intracellular stores to the cell surface in muscle and adipose cells to promote glucose clearance from the circulation. Insulin-stimulated GLUT4-storage vesicle (GSV) translocation and fusion with the plasma membrane is mediated by Akt and its downstream effectors. To discover novel Akt substrates in the regulation of GSV exocytosis, we performed GST pulldown experiments and mass spectrometry. We identify actin-capping protein Tropomodulin 3 as a novel Akt2-interacting partner in 3T3-L1 adipocytes. We demonstrate that Tropomodulin 3 is phosphorylated at Ser71 upon insulin-stimulated Akt2 activation, and Ser71 phosphorylation is required for insulin-stimulated GLUT4 insertion into the plasma membrane and glucose uptake. Phosphorylated Tropomodulin 3 regulates insulin-induced actin remodeling, an essential step for GSV fusion with the plasma membrane. Furthermore, interaction of Tropomodulin 3 with its cognate tropomyosin partner, Tm5NM1 is necessary for GSV exocytosis and glucose uptake. Together, these results establish Tropomodulin 3 as a novel Akt2 effector that mediates insulin-induced cortical actin remodeling and subsequent GLUT4 plasma membrane insertion. Our findings suggest that defects in cytoskeletal remodeling may contribute to impaired GLUT4 exocytosis and glucose uptake.

This work was supported by A*STAR Biomedical Research Council (WH) and Australian National Health and Medical Research Council (RM321705 to PG). We thank Clement Khaw and Ron Ng of SBIC-Nikon Imaging Centre, Singapore for assistance in TIRFM imaging. DW wishes to acknowledge the financial supports and funds from the National Basic Research Program of China (2011CB504004 and 2010CB945500).

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-1-IN-6089 Imaging developmental dynamics using light sheet microscopy

Tomancak P.1
1Max Planck Institute of Molecular Cell Biology and Genetics
tomancak@mpi-cbg.de

Selective Plane Illumination Microscopy (SPIM) has the potential to fundamentally change the way we study early development by enabling live, in toto imaging of embryos over long periods of time while monitoring fluorescent gene activity reporters. In order to make this emerging technology more accessible to the scientific community we have developed an open access platform that describes in great detail how to build and operate a simple, affordable and modular SPIM microscope[1-3]. We show that the OpenSPIM is capable of recording cellular level morphogenesis and pattern formation during embryogenesis of Drosophila as well as in other developmental and model organism contexts. OpenSPIM is suitable for parallelization, allowing increase in imaging throughput by building OpenSPIM farms, it is an excellent teaching tool [4] and we hope that it will nucleate an interdisciplinary community where technology developers interact with biologists to create customized light-sheet microscopy solutions. We also provide a comprehensive software solution under Fiji [5] for processing of SPIM image data that scales even to the largest datasets. We hope that OpenSPIM in its radical openness will demonstrate that the benefits brought to science by the Open Source approach apply equally well to hardware. We will present an overview of how the research community can benefit from the work we have invested in SPIM hardware and software and particularly how the microscopy community can and should contribute to its further development.

[1] Pitrone P. Schindelin J. et al. "OpenSPIM: an open access light sheet microscopy platform" Nature Methods, 10(7), pp. 598-599 (2013) [2] Pitrone P. Schindelin J. et al. "OpenSPIM: an open access light sheet microscopy platform" extended pre-print http://arxiv.org/abs/1302.1987 [3] http://openspim.org [4] http://openspim.org/2013-04-07_-_Open_SPIM_in_South_Africa [5] http://fiji.sc/SPIM_Registration

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-1-O-1407 Imaging of oxygenation in 3D tissue models by means of cell-penetrating phosphorescent nanosensors

Papkovsky D. B.1, Dmitriev R. I.1
1University College Cork, Cork, Ireland
d.papkovsky@ucc.ie

Recently we have developed several cell-penetrating phosphorescent probes (small molecule and nanoparticle based structures [1,2]) which allow real-time, high-resolution imaging of O2 concentration in respiring cells and tissues and detailed metabolic and physiological studies with 3D tissue models. The probes can be used on standard imaging platforms and in different detection modalities, with preference to confocal microsecond FLIM/PLIM (i.e. phosphorescence lifetime based O2 sensing) or ratiometric intensity imaging, under one and two-photon excitation [1]. The utility and performance of these probes and O2 imaging method have been demonstrated with 2D cell cultures, multi-cellular spheroids (neurospheres from primary neurons and cancer cell spheroids), slices of brain and colon tissue and live animals [2-4].

In the talk we will describe different probe structures, their use and O2 sensing principles, mechanisms of cellular uptake, toxicity and safety aspects. This will be illustrated with examples of imaging experiments performed with different cell and tissue models, including multi-parametric and functional imaging of live tissue, effects of hypoxic environment, drug action and metabolic stimulation. These new chemistries and imaging methodologies provide powerful tools for life science and biomedical research with long-ranging applications.

References:

1. Kondrashina AV et al, A phosphorescent nanoparticle based probe for sensing and imaging of (intra)cellular oxygen in multiple detection modalities, Adv Funct Mater, 2012, 22: 4931.

2. Dmitriev RI et al, Imaging of neurosphere oxygenation with phosphorescent probes, Biomaterials, 2013, 34: 9307.

3. Dmitriev RI et al, Small molecule phosphorescent probes for O2 imaging in 3D tissue models, Biomater. Sci., DOI:10.1039/C3BM60272A.

4. Tsytsarev V et al, In vivo imaging of brain metabolic activity using a phosphorescent oxygen-sensitive probe, J. Neurosci Meth., 2013, 216(2): 146-51.

Science Foundation Ireland, Grants 12/RC/2276, 12/TIDA/B2413, and EC FP7 Program, grant FP7-HEALTH-2012-INNOVATION-304842-2.

Fig. 1: Fig. 1. 3D FLIM image of a respiring neurosphere showing heterogeneous distribution of probe lifetime and O2 (left), and phosphorescence decays for ROIs with high (green) and low (orange) O2 concentration (right).

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-1-O-2128 Label free optical imaging of engineered neural tissue formation by second harmonic signals from collagen type I

Sanen K.1, Paesen R.1, Martens W.2, Lambrichts I.2, Phillips J. B.3, Ameloot M.1
1Biophysics, BIOMED Hasselt University, Hasselt, Belgium, 2Functional Morphology, BIOMED Hasselt University, Hasselt, Belgium, 3Department of Biomaterials & Tissue Engineering, University College London, Eastman Dental Institute, London, UK
kathleen.sanen@uhasselt.be

A variety of optical microscopy techniques can visualise individual cells in their extracellular matrix (ECM), most of them requiring exogenous dyes. Many labels have been subject of discussion because of phototoxic effects and perturbation of native cellular behavior. Interestingly, some biological molecules and structures can generate intrinsic optical signals, thereby making the use of exogenous dyes redundant. Cellular autofluorescence can be observed by one- or two-photon excitation (TPE) of for example NADH and flavins. Another intrinsic optical effect is Second Harmonic Generation (SHG), where laser light interacting with non-centrosymmetric molecules such as collagen type I generates frequency-doubled light. The resulting images with high contrast and submicron resolution can be further analyzed to obtain specific quantitative information.

Despite the advantages of these nonlinear optical microscopy methods, their use in the biomedical field is not widespread. In areas of tissue engineering, these techniques could be of great value for non-invasive characterization of biomaterials. Collagen type I hydrogels have been proposed for many regenerative applications due to their native-like ECM properties, inherent biocompatibility and suitability as carriers for different cell types. When a collagen type I hydrogel solution seeded with dental pulp stem cells (DPSCs) is casted into a mould with tethering bars positioned at each end, the contractile forces generated by DPSCs create a uniaxial tension along the tethered hydrogel (Fig 1a), resulting in longitudinal cell alignment within this 3D matrix (Fig 1b).[1] Although this engineered neural tissue (EngNT) containing DPSCs represents the desired end result for neuroregenerative applications [1], time-lapse experiments monitoring changes of hydrogel architecture are lacking.

In order to truly understand ECM remodeling by enclosed cells, it is essential to monitor the interaction of these cells with the 3D construct in time without the use of fluorescent labels. To this end, we performed TPE and second harmonic imaging of live EngNT, which revealed a marked change in collagen type I organization before and after cell alignment (Fig 2). Furthermore, we applied our in house developed image correlation spectroscopy approach [2] to characterize the spatial organization and structural characteristics of collagen type I fibers in time. This research demonstrates the application of nonlinear label free optical techniques for high resolution biomedical imaging.

[1] Martens W, Sanen K, et al. FASEB J. 2013 Dec 18 [Epub ahead of print]
[2] Paesen R, Sanen K, et al. Acta Biomaterialia. 2014 Jan 18 [Epub ahead of print]

This research was supported by the FWO (Fonds voor Wetenschappelijk Onderzoek Vlaanderen, grants 11N0914N and GO29112FWO).

Fig. 1: Construction of EngNT. A collagen type I hydrogel containing DPSCs was casted into a rectangular mould and tethered at each end. Following overnight incubation, the DPSCs contracted the hydrogel (a) and formed chains of along the longitudinal axis of the gel (b) (www.jamesphillips.org).

Fig. 2: EngNT formation visualized by label free optical imaging techniques. EngNT exhibited autofluorescence of DPSCs (red) and SHG of collagen type I (green) (λex = 900 nm). (a) Shortly after casting, cells appeared round and collagen fibrils were randomly oriented. (b) After contraction, alignment of cells and collagen fibrils was observed.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-1-O-3353 Sensitive imaging of cellular processes using two-photon polarization microscopy (2PPM)

Lazar J.1,2, Timr S.3, Bondar A.1,2
1Institute of Nanobiology and Structural Biology, Nove Hrady, Czech Republic, 2University of South Bohemia, Budweis, Czech Republic, 3Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic
lazar@nh.cas.cz

Membrane proteins are difficult to study, due to their requirement of a lipid membrane for function. We have taken advantage of the cell membrane requirement for exceptionally sensitive functional imaging of membrane proteins, using two-photon polarization microscopy (2PPM). In 2PPM, differences in fluorescence intensity observed with distinct linear polarizations of the excitation light are used to infer information on molecular orientation of a fluorescent label attached to a membrane protein. 2PPM can be used for imaging of ligand interactions with G protein coupled receptors, activation of G-proteins, changes in intracellular calcium concentration, and many other cellular processes, in living cells and organisms, with sensitivity comparable to, or exceeding that of current FRET probes. Crucially, in contrast to FRET, polarization fluorescence microscopy only requires presence of a single fluorescent protein. Reliance on a single fluorescent label allows using many of the existing fluorescently labeled constructs as optical probes of molecular processes involving membrane proteins. Furthermore, the need for a single label allows facile observations of multiple processes simultaneously. Importantly, polarization microscopy offers a clear path towards development of new genetically encoded optical probes of membrane protein function, including a usable genetically encoded optical sensor of cell membrane voltage. Apart from simply observing a molecular process to occur, 2PPM also allows making insights into molecular mechanisms of the processes being observed. Our results indicate that in many biological applications, 2PPM will complement or even replace other imaging modalities.

Supported by FP7 Marie Curie International Reintegration grant PIRG-GA-2007-209789 ‘MemSensors’ (J.L.); Czech Science Foundation grant P205/13-10799S (J.L.); University of South Bohemia Grant Agency fellowship 141/2013/P (A.B.)

Fig. 1: A mammalian cell expressing a fluorescently labeled membrane protein, imaged by 2PPM. Images acquired using excitation light polarized vertically (left, colored green) and horizontally (middle, colored red) show clear differences (right, showing the other two images overlaid). The differences can be used for sensitive imaging of molecular events.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-1-O-3492 Intravital imaging of osteoclast dynamics by using multiphoton microscopy

Kikuta J.1,2, Furuya M.1,2, Kowada T.3, Kikuchi K.3,4, Ishii M.1,2
1Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan, 2Japan Science and Technology Agency, CREST, Tokyo, Japan, 3Laboratory of Chemical Imaging Techniques, WPI–Immunology Frontier Research Center, Osaka University, Osaka, Japan, 4Department of Material and Life Sciences, Graduate School of Engineering, Osaka University, Osaka, Japan
jkikuta@icb.med.osaka-u.ac.jp

Osteoclasts are bone-resorbing giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. Upon the stimulation of essential factors such as M-CSF and RANKL, osteoclast precursor monocytes attach to the bone surface, fuse with each other to form giant cells and mediate bone resorption. To reveal the regulatory mechanism of osteoclast ‘function’ and ‘differentiation’, we generated various kinds of fluorescently-labeled mice.
First, we generated the mice expressing GFP under the promoter of vacuolar type H+-ATPase a3 subunit, that was abundantly expressed in differentiated osteoclasts. We succeeded in visualizing fluorescently-labeled mature osteoclasts in intact bone tissues, and identified two different populations of living mature osteoclasts, ‘static - bone-resorptive’ and ‘moving - non-resorptive’. We also developed a pH-sensing fluorescent chemical probe to detect the acidification by bone-resorbing osteoclasts in vivo. By means of this probe, we could visualize ‘bone-resorption’ by osteoclasts and found that the pH value in the resorption pit created by osteoclasts should be within the range of 4–6.
We next generated the double-fluorescently-labeled mice where Green and Red fluorescent proteins were expressed under the promoter of cell differentiation markers in different stages. We crossed CSF1R-EGFP mice where EGFP was expressed in CSF1R+ osteoclast precursor monocytes, with TRAP-tdTomato mice where tdTomato was expressed in TRAP+ mature osteoclasts. By intravital multiphoton imaging, we could chase the state of osteoclast ‘differentiation’ as a color change, in addition to 4-dimentional information such as x/y/z and temporal information. We named this strategy 5D imaging technology.
Finally, we crossed TRAP-tdTomato mice with Col1a1-ECFP mice where ECFP was expressed in Col1a1+ osteoblasts. By intravital bone imaging of the mice, we succeeded in visualizing the interaction of bone-resorbing osteoclasts with bone-forming osteoblasts (‘coupling’) in living bones.
These approaches would be quite beneficial for studying the osteoclast dynamics in vivo and thus useful for evaluating novel anti-bone resorptive drugs currently developed in the world.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-1402 Thrombus Development Processes are dependent on Endothelial Injuries: Visualyzed by In vivo Two-photon Molecular Imaging

Nishimura S.1
1The Univ of Tokyo, Jichi Medical Univ 1
snishi-tky@umin.ac.jp

Aim: The thrombotic cellular mechanisms associated with cardiovascular events remains unclear, largely because of an inability to visualize thrombus formation. Thus, we developed in vivo imaging technique based on single- and multi-photon microscopy to revealed the multicellular processes during thrombus development.

Methods: We visualized the cell dynamics including single platelet behavior, and assessed dynamic cellular interplay in two thrombosis models using two photon microscopy to CAG-eGFP mice in which GFP was expressed ubiquitously. Hoechst was injected to visualize nucleus, and dextran to blood flow. Mice with anestesia is set on to inverted microscopy, and visualization was performed through small incision. (Figure a, b).

Results: First, we visualized that rapidly developing thrombi composed of discoid platelets without EC disruption was triggered by ROS photochemically induced by moderate power laser irradiation (Figure c). In this model, thrombus consisted by discoid platelet aggregations without leukocyte recruitment. The second model is, thrombus with EC disruption. High power laser induced EC erosion and extravasations of circulating leukocytes with thrombus development. Inflammatory cytokine, adhesion molecules dynamically control these two processes. (Figure d)

As for the thrombus formation with EC disruption, chemokine expressions in endothelium and leukocyte (especially neutrophils) recruitment played a significant role in these processes. Leukocyte was immediately recruited into the subendothelial layers with bleeding and hemostatic reactions. TLR4 signaling also contributed to these steps, and pretreatmet of LPS markedly enhanced these steps. Thrombus included calcium activated cores and deformed platelets. Immigrated leukocyte also showed the increase of intracellular calcium.

Summary: These results indicated that endothelial function, especially inflammatory status, determined the thrombotic reaction. Leukocyte also contributed with TLR4 signaling. In sum, using our imaging system can be a powerful tool to analyze thrombus formation and evaluate the therapeutic strategies.

Fig. 1: In vivo imaging visualize cell kinetics in thrombus
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-1403 Artery Cell Contraction via ROS and NO Balance Examined by In Vivo Multi-photon Imaging Technique

Nishimura S.1
1the Univ of Tokyo, Jichi Medical Univ 1
snishi-tky@umin.ac.jp

Aim The blood pressure is regulated dynamically by arterial wall tensions in vivo, but the previous methods did not clarify their detailed mechanisms in living animals due to the lack of visualization technique. Thus we aimed to visualize cell kinetics during artery contractions processes by improving previous photochemical reaction technique.

Methods We recently developed the in vivo fluorescent imaging technique based on two photon microscopy, which enabled us to directly visualize the femoral artery in living animals. In addition, by combining with the visualization technique with laser induced injury models, we could visualize transient artery contraction in response to ROS (reactive oxygen species) (Figure a). Using this novel animal model, we elucidated the functional contribution of smooth muscle cell of artery to hypertensive diseases. We also directly measured the ROS and NO (nitric oxide) production in smooth muscle cells, which was analyzed and quantified by novel tracking software.

Results: We observed that ROS and NO production was counter-balanced in transient artery contractions (Figure b and c). As for the molecular mechanisms, we confirmed that this contraction is via PKC and NADPH signaling. The contraction was inhibited by Ca blocker, isosorbide, sildenafile, and angiotension II blockers administrations.

In addition, by treating the artery with CaCl solution, the aneurysm formation was also visualized. In the early phase, the hyper-responsiveness of artery wall, and transient thrombus formation was observed. In the later, the wall was stiffened, collagen contents were increased, and aneurysm was observed in last.

Conclusions These results indicated the ROS and NO counter balances dynamically regulate artery contractions. eNOS also contributed to these contractions, and imaging can visualize various dynamic signaling. .In sum, our imaging system can be a powerful tool to analyze the molecular mechanisms of hypertension and artery aneurysm.

Fig. 1: In vivo imaging of artery contractions, femoral artery in living mice
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-1404 Protective effect of Derris reticulata extract against alloxan-induced cell death in pancreatic RINm5F cells

Chudapongse N.1, Kumkrai P.1
1School of Pharmacology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, THAILAND
nuannoi@sut.ac.th

Derris reticulata Craib. (Family: Leguminosae) is a climbing plant distributed in the tropical regions of Asia and East Africa which is traditionally used as anti-coughing and expectorant. Several pharmacological activities of this plant, such as anti-inflammatory effect and antiviral action against herpes simplex virus type 1, have been documented. It was also employed as alternative diabetes treatment by local medicinal plant practitioners in some parts of Thailand. The aims of this study were to evaluate antioxidant and protective effects of the aqueous extract of Derris reticulata (ADR). Antioxidant activities were determined using FRAP, ABTS and DPPH scavenging methods in vitro. The scavenging activities of ADR against DPPH and ABTS free radicals were found at the IC50 of 239.85 ± 0.13 and 515.05 ± 0.13 µg/ml, respectively, whereas the FRAP value of ADR was 0.23 ± 0.05 µmol of Fe2+/mg dried extract. Morphological changes and density of pancreatic RINm5F cells were observed under inverted microscope. The protective effect of ADR against alloxan-induced cell death was studied by MTT assay. It was found that alloxan, a free radical producing agent, decreased the number of RINm5F cells and altered their morphology as well as caused cell detachment from plate. In accordance with microscopic examination, the result from MTT assay showed that pretreatment of cells with ADR increased cell viability after exposure to alloxan. These findings suggest that antioxidant activities of ADR may play a key role in the protective effect against alloxan-induced toxicity in vitro.

This study was financially supported by SUT Research and Development Fund. We thank Dr. Paul J. Grote for plant verification.

Fig. 1: Photomicrograph of pancreatic RINm5F cells (200×). Panel A: control, Panel B: cells treated with alloxan (9 mM), Panel C-H: cells pretreated with Derris reticulata extract (50, 100, 150, 200, 250 and 500 µg/ml, respectively) before exposure to alloxan.

Fig. 2: Cytoprotective effect of Derris reticulata extract. Cell viability was determined by the MTT assay. RINm5F cells were pretreated with the extract for 12 hours before exposure to alloxan (9 mM). Results are mean ± S.E.M. (n=3). a, b and c (p < 0.05) significant difference from control, alloxan and the lower doses, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-1485 Systemic Sclerosis: a pathological model linking ROS overproduction to DNA damage and fibrosis.

Spadoni T.1, Svegliati S.1, Gabrielli A.1
1Università Politecnica delle Marche, Ancona, Italy
t.spadoni@univpm.it

NADPH oxidase (NOX) is a multi-protein complex producing reactive oxygen species (ROS) in response to growth factors. Among the seven members of the NOX family there are key differences in their activation, subunit composition, localization and expression. NOX overactivity or overexpression are often associated with chronic diseases, characterized by tissue damage and fibrosis. Such changes are consistent with the involvement of ROS in causing damage to biomolecules including protein, DNA, and lipid membranes. Scleroderma (SSc) is a chronic fibrotic disease which can affect skin and internal organs, resulting in significant morbidity. SSc fibroblasts isolated from lesional areas of patients overproduce ROS and overexpress type I collagen and a-smooth muscle actin (a-SMA), and show chromosomal aberrations. Although the evidence that oxidative stress contribute to the establishment of fibrosis, the role of the single NADPH oxidase members has not been previously investigated in scleroderma.
In this study we focused on the role of NOX enzymes in SSc fibroblast activation and on the possible link between ROS induced-DNA damage and fibrosis.
Scleroderma skin fibroblasts show enhanced expression of NOX 2 and 4 mRNA and protein compared to normal fibroblasts. Incubation with DPI or transfection with siRNAs against specific NOX mRNA downregulate cell activation, DNA damage, and type I collagen expression in scleroderma cells. SSc fibroblasts show high levels of phosphorylated ATM (ataxia telangiectasia mutated, the major regulator kinase of the cellular response to DNA damage) compared to normal cells. Incubation with KU55933, a specific inhibitor of ATM, leads to downregulation of ROS in SSc fibroblasts as well as in normal cells stimulated by bleomycin, an inducing agent of fibrosis. Moreover inhibition of ATM causes a significant reduction of type I collagen expression, as confirmed in ATM-/- cells.
In the present study, we identified NOX 2 and 4 as critical components of NADPH oxidase complex in SSc fibroblasts and we provide evidence that ROS produced by NOX may play a important role in the pathological activation of dermal fibroblasts. These data strongly demonstrate the pathological link among ROS, DNA damage and collagen production, and suggest new targeting strategies in the treatment of fibrotic diseases, such as Scleroderma.

This work was supported by Fondazione di Medicina Molecolare e Terapia Cellulare, Università Politecnica delle Marche, Ancona, Italy.

Fig. 1: ROS production. SSc fibroblasts were transfected with a control siRNA (CTRL) or siRNA against NADPH oxidase subunits (NOX2 or NOX4). In the upper pannel cells were incubated with DHE and the fluorescence were analyzed using a confocal microscopy. In the lower pannel, after treatment with DCF ROS production was measured using a microplate reader.

Fig. 2: Activation state. SSc fibroblasts were transfected with a control siRNA (CTRL) or siRNA against NADPH oxidase subunits (NOX2 or NOX4). In the upper pannel a-SMA expression was detected using a confocal microscopy. In the lower pannel, collagen and fibronectin mRNA levels were measured in real-time PCR.

Fig. 3: DNA damage. SSc fibroblasts were transfected with a control siRNA (CTRL) or siRNA against NADPH oxidase subunits (NOX2 or NOX4) or treated with DPI. In the upper pannel phosphorylation of H2A.X was analyzed using a confocal microscopy. In the lower pannel, the same marker was detected in western blot.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-1502 PHOTOTOXICITY OF CRAJIRU EXTRACT (Arrabidaea chica) INCORPORATED IN POLYMERIC NANOEMULSION AGAINST MURINE MAMMARY ADENOCARCINOMA CELLS (4T1)

Silva R. C.1, Muehlmann L. A.2, Longo J. F.2, de Azevedo R. B.2, Degterev I. A.3, Lucci C. M.2
1Institute of Metrology, Quality and Technology, Rio de Janeiro, Brazil., 2Institute of Biological Sciences, University of Brasilia, DF, Brazil., 3Center of Biological & Natural Sciences, Federal University of Acre, Rio Branco, Brazil.
renata.veterinaria@gmail.com

Photodynamic therapy (PDT) is an alternative therapy to cure a lot of diseases, including cancer. A new set of photosensitizing (PS) drugs arising from natural plants, as vegetable oils and extracts have been tested in cancer therapy. However, most of these PS drugs present some difficulties for clinical use and in this context, polymeric nanoemulsions, kinetically stable systems, have several potential as drug carrier systems. The aim of this study was to evaluate the possible photodamage of the chloroform extract of aerial parts of the Amazon plant - crajiru (Arrabidaea chica) - incorporated in polymeric nanoemulsion (NanoECr) in PDT against murine mammary adenocarcinoma cells (4T1) in vitro. By dynamic light scattering measurements, NanoECr had an average hydrodynamic diameter of 370.5 ± 264.31 nm, and presented characteristics of a stable formulation. After incubation of 4T1 cells with different NanoECr concentrations and times, in the absence of irradiation (in the dark), non-toxic concentration (54 µg/mL) was determined by MTT and microscopy techniques and the maximum time of interaction between NanoECr and 4T1 cells (15 minutes) was determined by confocal microscopy (Figure 1). Associating 54 µg/mL of NanoECr for 15 minutes of incubation and irradiation using a laser of 670 nm wavelength at different energy doses, cell death occurred when the cells were irradiated with energy ranging from 8.57 J/cm2 to 85.7 J/cm2 and only at the maximum energy dose of 85.7 J/cm2 used in this experiment occurred 100% of cell death. Evaluating the type of cell death, cells irradiated at an energy dose of 25.7 J/cm2 had death by apoptosis, visualized by intense cytoplasmic staining of the apoptotic bodies with acridine orange in confocal microscopy (Figure 2) and the presence of blebs and mitochondria and endoplasmic reticulum damage observed in Transmission Electron Microscopy (TEM - (Figure 3). At an energy dose of 85.7 J/cm2, necrosis was the type of cellular death occurred noted by intense staining of cells cytoplasm in red (ethidium bromide) in confocal microscopy and cell membrane damage with leakage of cellular contents and presence of vacuoles inside the cells observed in TEM (Figure 3). At Scanning electron microscopy (SEM), in both energy doses (25.7 J/cm2 and 85.7 J/cm2), disruption/disintegration of the plasma membrane with the presence of numerous perforations in its structure and detachment of the cells from the substrate were observed (Figure 4). Damage was not observed when cells were incubated with NanoECr in the absence of irradiation or irradiated without NanoECr incubation. We can conclude that the chloroform extract of the aerial parts of crajiru incorporated in polymeric nanoemulsion is a potential PS formulation for use in PDT.

The authors thank the financial funding from MCT-INCT, FAP-DF, CAPES and CNPq.

Fig. 1: Confocal micrographs of 4T1 cells exposure in vitro to NanoECr for 15 minutes in the dark. A) Phase contrast, B) Nucleus cell stained with DAPI (blue), C) NanoECr fluorescence in cell cytoplasm (red), D) Overlay of A, B and C.

Fig. 2: Confocal micrographs of 4T1 cells stained with acridine orange and ethidium bromide after PDT. A) Control - Bright field, B) Control – Fluorescence, C) PDT 25.7 J/cm2. Blebs in plasma membrane represented by intense orange staining (arrows) indicating apoptosis, D) PDT 85.7 J/cm2. Cells stained in red indicating cell death by necrosis.

Fig. 3: TEM Electron micrographs of 4T1 cells. A) Control, B) Cell incubated with 54μg/mL of NanoECr in the dark. White arrows indicate amorphous vesicles, C) PDT 25.7 J/cm2. Black arrows indicate blebs in plasma membrane, D) PDT 85.7 J/cm2. Note the release of cytoplasmic contents and vacuoles (star) inside cells. Nu = cell nucleus.

Fig. 4: SEM micrographs of 4T1 cells. A) Control, B) Cell incubated with 54μg/mL of NanoECr in the dark. Note the presence of diffuse nanostructures in the membrane (arrow), C) PDT 25.7 J/cm2, D) PDT 85.7 J/cm2. Note the presence of perforations in plasma membrane (arrows). In D, total disintegration of the cell.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-1729  α1-Adrenergic receptors regulate Ca2+ modulation of acinar cells in rat lacrimal grand

Kurosawa C.1, Saino T.2, Kurosaka D.1, Satoh Y.2
1Departments of Ophthalmology, Iwate Medical University, Morioka, Japan, 2Department of Anatomy, Iwate Medical University, Yahaba, Japan
kurosawa.chika@gmail.com

The lacrimal gland is the primary source for aqueous portion of the tear film. This portion contains water, electrolytes and proteins, which are necessary for the health and the maintenance of the cells of the ocular surface. Noradrenaline (NA), released from sympathetic nerves, is a major stimulus of lacrimal gland secretion. Here, lacrimal gland acinar cells response to adrenergic receptors activation were examined, with special reference to intracellular Ca2+ concentration ( [Ca2+]i ) dynamics.
In the present study, detection of mRNA of acinar cells specific to adrenergic receptor subtypes was determined by RT-PCR. All kinds of adrenergic receptors were detected except α2c and β1 in acinar cells of lacrimal glands.
NA (30 μM) induced an increase in [Ca2+]i in acinar cells. NA-induced [Ca2+]i changes showed a biphasic behavior; the first step involved a steep phase of rapidly increasing [Ca2+]i, followed by the second plateau phase step. The removal of extracellular Ca2+ and the use of Ca2+ channel blockers did not completely inhibit the NA-induced [Ca2+]i increases. This reaction did not persist for long and the second plateau phase disappeared. Furthermore, suramin (a G protein antagonist) inhibited these increases. Phenylephrine (an α1 adrenoceptor agonist) induced a strong increase in [Ca2+]i. However, clonidine (an α2 adrenoceptor agonist) and isoproterenol (a β adrenoceptor agonist) failed to induce a [Ca2+]i increase.
These findings indicated that NA activation resulted primarily in Ca2+ mobilization from intracellular Ca2+ stores and that NA activates α1 adrenoceptors which cause an increase in [Ca2+]i by production of IP3. Our results suggested that α1 adrenoceptors were key receptors in calcium-related cell homeostasis and exoclines in lacrimal glands.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-1763 3D mapping of tissue oxygen in chronically inflamed mouse bladder

Zhdanov A. V.1, Golubeva A. V.1, Cryan J. F.1, Papkovsky D. B.1
1University College Cork, Cork, Ireland
a.zhdanov@ucc.ie

Painful bladder syndrome (PBS) is a chronic disorder characterized by severe clinical manifestations and unmet medical need in terms of effective diagnostics and treatment. Systemic administration of cyclophosphamide (CYP) in mice has been proposed as a relevant preclinical model of chronic bladder malfunction. We demonstrated recently that chronic CYP treatment induces bladder inflammation associated with activation of proliferation and apoptosis [Physiological Reports (2014), In Press]. Both processes are energy demanding, and therefore elevated respiration and local deoxygenation are expected in the inflamed bladder tissue. Since molecular oxygen is a master regulator of cellular function, the knowledge on O2 availability and consumption rate is critically important for understanding of the molecular mechanisms underlying PBS and successful treatment of the disease.
To build the O2 map of normal and inflamed bladder, we applied newly developed cell-penetrating phosphorescent O2 sensitive probes and imaging technique based on microsecond PIM-TCSPC confocal microscopy [1-3], which demonstrated good performance in the experiments with 2D cell cultures, multicellular 3D models (spheroids and scaffolds), brain slices, rodent intestine segments and human colon biopsies (Fig. 1). Here we correlate high-resolution imaging data on O2 levels in whole bladder with the results of functional, morphological and immunological tests, aiming to analyse diagnostic and prognostic value of O2 imaging platform. A potential of the platform for wide range biomedical applications and further development will be discussed in the presentation.
References:
1. A.V. Kondrashina, R.I. Dmitriev, S.M. Borisov, I. Klimant, I. O'Brien, Y.M. Nolan, A.V. Zhdanov, D.B. Papkovsky, A phosphorescent nanoparticle‐based probe for sensing and imaging of (intra) cellular oxygen in multiple detection modalities, Advanced Functional Materials, 22 (2012) 4931-4939.
2. R.I. Dmitriev, A.V. Zhdanov, Y.M. Nolan, D.B. Papkovsky, Imaging of neurosphere oxygenation with phosphorescent probes, Biomaterials, 34 (2013) 9307-9317.
3. R.I. Dmitriev, A.V. Kondrashina, K. Koren, I. Klimant, A.V. Zhdanov, J.M. Pakan, K.W. McDermott, D.B. Papkovsky, Small molecule phosphorescent probes for O2 imaging in 3D tissue models, Biomaterials Science, (2014).
4. A.V. Golubeva, A.V. Zhdanov, G. Mallel, T.G. Dinan, J.F. Cryan, The mouse cyclophosphamide model of bladder pain syndrome: tissue characterization, immune profiling and relationship to metabotropic glutamate receptors, Physiological Reports, (2014). 

This work was supported by Enterprise Ireland grant CF/2012/2346.

Fig. 1: Confocal PLIM image of rat colon produced using Pt-Glc O2 sensitive probe. Phosphorescence intensity image of mucosal layer (left) is converted into LT image using SPCImage program. Tissue O2 levels (μM) are determined from LT using the conversion function. Single colon crypt is highlighted by quadrangle.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-2273 Human myometrial telocytes: in vitro low-level laser stimulation

Campeanu R. A.1, Radu B. M.1, 2, Cretoiu S. M.3, 4, Banciu D. D.1, Banciu A.1, Cretoiu D.3, 4, Popescu L. M.3, 4
1University of Bucharest, Bucharest, Romania, 2University of Verona, Verona, Italy, 3Carol Davila University of Medicine and Pharmacy, Bucharest, Romania, 4Victor Babeş National Institute of Pathology, Bucharest, Romania
sanda@cretoiu.ro

Background. Telocytes (TCs) are a brand-new cell type frequently observed in the interstitial space of many organs (see www.telocytes.com). TCs are defined by very long (tens of μm) and thin prolongations named telopodes. Dilations, called podoms (~300 nm) alternate with podomers (80-100 nm). The mechanisms of telopodes’ elongation and ramification is not known.
Methods. TCs were identified in a myometrial interstitial cell culture based on morphological criteria and by CD34 and PDGFRα immunopositivity. We report here the low-level laser stimulation (LLLS) using a 1064 nm Nd:YAG laser, with an output power of 60 mW, of the telopode growth in cell culture. The laser beam was focused through the 100x/1.3 Oil objective.
Results. LLLS of TCs determines a higher growth rate of telopodes in pregnant myometrium primary cultures (10.3 ± 1.0 μm/min) compared to non-pregnant ones (6.6 ± 0.9 μm/min). Acute exposure (30 min) of TCs from pregnant myometrium to 1 μM mibefradil, a selective inhibitor of T-type calcium channels, determines a significant reduction in the LLLS growth rate (5.7 ± 0.8 μm/min) compared to LLLS per se in same type of samples. Meanwhile chronic exposure (24 h) completely abolishes the LLLS telopodes growth in both non-pregnant and pregnant myometrium. The initial direction of telopode growth was modified by LLLS, the angle of deviation being more accentuated in TCs from human pregnant myometrium than in TCs from non-pregnant myometrium.
Conclusion. TCs from pregnant myometrium are more susceptible of reacting to LLLS than those from non-pregnant myometrium. Therefore, some implications are emerging for low level laser therapy in uterine regenerative medicine.

This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number 82/2012 (PN-II-PT-PCCA-2011-3.1-0553).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-2282 Atomic force microscopy reveals differences in cell membrane properties in nuclear myosin I mutant

Venit T.1, Petr M.1, Hozák P.1
1Institute of Molecular Genetics, Prague, Czech Republic
tomas.venit@img.cas.cz

Nuclear myosin I is a nuclear isoform of the well-known “cytoplasmic” Myosin 1c protein. Located on the 11th chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus [1]. Previous studies revealed its roles in nuclear processes such as RNA Pol I and RNA Pol II transcription [2, 3], chromatin remodeling [4], and chromosomal movements [5]. In contrary, Myo1c was shown to act in different plasma membrane and cytoplasm related processes.

In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen and strikingly, no obvious phenotype related to previously described functions has been observed. Surprisingly, we found that NM1 KO skin fibroblasts are more resistant to hypotonic stress in comparison to WT cells suggesting the role of NM1 in plasma membrane dynamics.

To explore the mechanical properties of NM1 KO cells in detail, we used Atomic Force Microscopy (AFM). We show that plasma membrane of NM1 KO cells has higher elasticity in comparison to WT cells, and therefore it is more resistant to cell swelling processes occurring during hypotonic treatment. This suggests that NM1 might acts as a dynamic link between plasma membrane and cytoskeleton, influencing the fluidity of the cortical layer of the cell.

1. Pestic-Dragovich et al. (2000) Science. 290, 337-41.

2. Percipalle et al. (2003) PNAS, 6475-80.

3. Philimonenko et al. (2004) Nature cell biology. 6, 1165-72.

4. Percipalle et al. (2006) EMBO reports. 7, 525-30.

5. Chuang et al. (2006) Current biology : CB. 16, 825-31.

OP EC CZ 1.07/2.3.00/30.0050 "Founding the expert platform for phenotyping and imaging technologies" Operational Program Education for Competitiveness by MEYS, CR + European Social Fund, by the GACR (P305/11/2232), by the TACR (TE01010118), by the MEYS CR (LH12143, LD12063, OP EC CZ 1.07/2.3.00/30.0050 and TE01020118), MIT CR (MIT FR-TI3/588 and MIT FR-TI4/660) and by the GAUK (Reg. No. 633012) .

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-2402 Smooth muscle cells of testicular venules show different responses to various transmitters, when compared with smooth muscle cells of arterioles; with special reference to intracellular calcium dynamics

Sasaki K.1, Hirakawa M.1, Saino T.1, Sato Y.1, 2
1Department of Anatomy (Cell Biology), 2Department of Medical Education
yisatoh@iwate-med.ac.jp

[Background/Aim] It has been well known that shape and distributions of smooth muscle cells (SMC) are different between artery/arterioles and vein/venules. The morphological differences of SMC can indicate different role in vivo, however, very few data has been available on physiololical characters of SMC of vein/venule. Intracellular Ca2+ concentration ([Ca2+]i) plays an essential role in stimulus-response coupling in a great variety of tissue/cells, including vascular SMC. We observed Ca2+]i dynamics of SMC of intact venules to clarify which transmitters can elicit any response of venules, and compered with those of SMC of arterioles. [Materials and Methods] Real-time confocal microscopy (Nikon RCM/Ab) was employed to study the alteration in [Ca2+]i. Venules and arterioles (external diameters <100 μm) were isolated from rats (Wistar, male), and loaded with a fluorescent Ca2+-indicator, Indo-1. Ringer' solution containing various transmitters/modulators was perfused around the specimens. [Results] SMC of arterioles are fusiform in shape, while those of venules are polygonal. 5-Hydroxytryptamine (5-HT), ATP, and angiotensin II elicited an increase in [Ca2+]i in most SMC of arterioles. The [Ca2+]i increase and the following contraction were persistent. When noradrenaline (NorAd) was used as a stimulant, [Ca2+]i increase was observed only in a portion of the SMC, and the response was relative transient. On the contrary, the[Ca2+]i increase in SMC of venules during 5-HT or ATP stimulation was faint, but NorAd can induce an evident increase in SMC. Response to angiotensin II was also significant. The increase of SMC of venules was not inhibited, when extracellular Ca2+ was removed. Thapsigargin, inducing depletion of intracellular Ca2+ store, suppressed the response of SMC of venules to the transmitters. [Discussion] 5-HT and ATP has not been considered as “pressor substances”, even though both induced strong vasoconstriction of arterioles. This may mean that not only vasoconstriction of arterioles is a unique factor for increase of systemic pressure. However substances known as “pressor substances” induce SMC response of venules as well as arterioles. About 65% of whole blood was contained in vein/venules, therefore the reduction of vascular volume of venous system can play a pivotal role in increase of systemic blood pressure.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-2478 Diagnosing Pathological Conditions of the Connective Tissue Using Atomic Force Microscopy Study of the Extracellular Matrix

Timashev P. S.1, Kotova S. L.2
1Institute of Laser and Information Technologies, Moscow, Russia, 2N.N.Semenov Institute of Chemical Physics, Moscow, Russia
timashev.peter@gmail.com

Extracellular matrix (EM) forms a basis of human connective tissue, providing its specific mechanical properties. The EM structure, and, particularly, the packing of collagen, the main protein EM component, depends on the functional activity of cells and may significantly change in the presence of a pathological process.
Here we have applied atomic force microscopy (AFM) to diagnose morphological changes in the EM of connective tissue caused by two different pathological processes – connective tissue dysplasia leading to pelvic organ prolapse (POP) and a neoplastic process (tumors of bone). AFM imaging was performed on air on deparaffinized tissue sections.
Our AFM studies showed marked deviations from the normal EM morphology of human skin and pelvic ligament for patients with POP. The deviations were observed at all the levels of the EM texture, including microtexture (packing of collagen fibers), nanotexture (arrangement of collagen fibrils) and structure of individual collagen fibrils. In particular, we observed visible separation, thinning and fragmentation of collagen fibrils and fibers, disintegration and disordering of collagen structures up to the complete destruction of the specific tissue architecture (Fig.1). The nanoindentation study revealed significant deterioration of the mechanical properties of the collagen fibrils bundles in the skin of POP patients, as compared to the skin of healthy subjects.
In the AFM study of bone tumor tissue, we compared the morphology of chondrosarcoma of histologically malignant grade I, II and III. A benign chondroma tumor was used as a control. The AFM imaging showed a clear correlation between the content of the fibrous collagenous elements in the EM of a bone tumor and the degree of its malignancy (Fig.2). While the EM of chondroma and grade I chondrosarcoma were represented mostly by the network of collagen fibrils, the grade II chondrosarcoma contained a substantial fraction of non-fibrous elements, and AFM images of the EM of the grade III chondrosarcoma showed only the non-fibrous amorphous material.
The AFM data on the EM structure of normal and pathologically altered connective tissue were found being in a good agreement with the data of the standard morphological methods (including histological and electron microscopy analysis) on the same clinical specimens. Thus, AFM and related techniques may serve as either an independent, or a complementary diagnostic tool for tracking pathological changes in the connective tissue.

This study was financially supported by the Russian Foundation for Basic Research (grant No. 12-02-00633-а)

Fig. 1: AFM-diagnostics of pelvic organ prolapse (POP). The loss of order in the collagen fibrils packing in the extracellular matrix of the skin of POP patients (A-C) as compared to normal skin (D). The scan sizes are 3x3 µm.

Fig. 2: AFM-diagnostics of the bone tumors’ degree of malignancy. The fraction of fibrous collagenous elements of the extracellular matrix decreases with the malignancy grade. C – chondroma (benign tumor); CS1, CS2, CS2 – I, II and III grade chondrosarcoma, respectively. The scan sizes are 3x3 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-2775 Accurate and Fast Correlative Light and Electron Microscopy of Cells under Vacuum and Near-Native Conditions

Sueters J.1, Liv N.1, Haring M. T.1, Zonnevylle A. C.1, Kruit P.1, Hoogenboom J. P.1
1Delft University of Technology, Faculty of Applied sciences, Department of Imaging Physics, Delft, The Netherlands
J.Sueters@tudelft.nl

Biological investigation of samples, both in their native state as well as under dried, fixated conditions, is predominantly achieved by light microscopy (LM). LM allows biomolecule identification, but has a diffraction-limited resolution and is incapable of reveiling the ultrastructure. On the other hand, electron microscopy (EM) offers sub-nm resolution, but only allows grey scale images of the cellular ultrastructure of dehydrated and/or frozen samples without providing the functional biomolecular information. By combining both techniques in CLEM, biomolecule identification within the ultrastructure is possible at high resolution and sensitivity.

Nowadays, the number of researchers using CLEM is still relatively limited, compared to the amount of LM and/or EM users. This can be related to transfer steps between separate EM and FM setups, crucial differences in sample preparation protocols, the need for fiducial markers visible in both LM and EM to retrieve regions of interest, and limitations in correlation accuracy. Additional challenges are to increase spatial resolution in LM and incorporate temporal resolution in EM.

We have recently developed a setup that enables LM and EM imaging simultaneously on the same area of a sample [1]. This eliminates the transfer and ROI retrieval steps [2]. Thus allowing for easy, quantitative investigation of large areas and datasets with a drastic reduction of inspection time and reduced risk of sample contamination2. With this microscope, high registration accuracy down to five nanometer can be obtained without the need for fiducial markers.

However, comprehensive investigation of biological activities of cells requires high resolution imaging in the native state of the cell. Therefore, a microchip was designed as part of this setup, with a thin electron transparent window as well as a light transparent window [3]. This facilitates correlative microscopy of cells in liquid. We will present the microscope design, show application examples and discuss the possibilities for near-native state correlative imaging. We expect that with new developments like our setup, CLEM can become a powerful tool for fundamental biological research, applied industrial research and medical diagnostics.

[1] A.C. Zonnevylle et al., Integration of a high-NA light microscope in a scanning microscope, Journal of Microscopy (2013), volume 252-1, p. 58-70.

[2] N. Liv et al, Simultaneous Correlative Scanning Electron and High-NA Fluorescence microscopy, PLoS ONE (2013), volume 8-2.

[3] N. Liv. Et al., Scanning electron microscopy of individual nanoparticle bio-markers in liquid, Ultramicroscopy (2013), doi: 10.1016/j.ultramic.2013.09.002.

This work is in collaboration with Delmic BV. We want to thank Ruud van Tol, and Carel Heerkens for their assistance and STW for financial support.

Fig. 1: Optical components onthe outside of the vacuum door (A),integrated microscope in SEM with final electron lens above and light objectivelens below the sample (B) and vacuum parts of the CLEM platform [1].

Fig. 2: Schematicillustration of the simultaneous observation with fluorescence (from below) andscanning electron microscopy (from above) of a sample in liquid, shielded fromthe vacuum by a thin, electron-transparent membrane [3].

Fig. 3: (A) Fluorescenceimage of CV1 cells, fixed during uptake of EGF-conjugated QDs (B), the enlargedimaged of the boxed area in a), (C) SEM image of the same area (reversedcontrast) and (D) Similar high-magnification SEM image of part of a CV1 celltaken in liquid. Arrows point to the internalized EGF-conjugated QDs.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-2776 Interaction of Escherichia coli bacteria with silica nanoparticles studied by Atomic Force Microscopy

Gammoudi I.1, Mathelié-Guinlet M.1, Moroté F.1, Grauby-Heywang C.1, Moynet D.2, Cohen-Bouhacina T.1
1Laboratoire Ondes et Matière d'Aquitaine, Université de Bordeaux, Talence, France, 2Biothérapie des Maladies Génétiques et Cancers, Université de Bordeaux, Bordeaux, France
ch.heywang@loma.u-bordeaux1.fr

Understanding the mechanisms of interaction between nanoparticles (NPs) and  bacteria is important for many reasons: some toxic NPs can be used for their bactericide power, whereas others can be used in the design of sensors for the selective detection of bacteria. Toxic effects of NPs on bacteria can be due to the formation of defects or holes in the bacteria membrane, to the generation of reactive oxygen species or to the dissolution of NPs in ions interacting with the metabolism.
Atomic force microscopy (AFM) is a powerful method to study the interaction of NPs with bacteria: the tapping mode enables not to damage biological samples, which can be also imaged in contact in liquid. Moreover the AFM tip is able to discriminate between “hard” (NPs) and “soft” (bacteria) materials, leading to a high contrast in phase images. In this work, we applied AFM to the study of silica NPs interacting with Escherichia coli (E. coli) bacteria. These bacteria were incubated with NPs (previously dispersed in ultra pure water at a concentration low enough to avoid the formation of aggregates), and then immobilized either on mica or on a multilayered cushion of polyelectrolytes (formed by the Layer by Layer method). Silica NPs with diameters ranging from 4 nm to 200 nm were used. AFM images were performed in air after drying the samples and in liquid.
In the absence of NPs, AFM first confirms the formation of stable E. Coli biofilms, even after several rinsing steps, and enables to determine their shape and dimensions (mean length width and height at 1.8 µm, 1.2 µm and 0,15 µm respectively, in agreement with previous results obtained in the case of healthy bacteria). The morphology of bacteria does not change after their incubation in the presence of NPs of 100 nm or 200 nm of diameter, suggesting that bacteria are still healthy (Fig. 1 and 2). Moreover, AFM images suggest that some NPs are aggregated and wedged between the bacteria and the surface of the substrate. In the case of NPs of 4 nm and 10 nm of diameter, the morphology of bacteria is modified: bacteria adopt more unusual spherical shapes. Moreover, the height of some of them is clearly lower than in the case of healthy bacteria, suggesting that these bacteria are emptied of their content (Fig. 3 and 4). In some cases, it is possible to observe a partial collapse of the membrane on itself.
These results show that the size of NPs is a crucial parameter in their toxicity mechanism. NPs of 100 nm seem to be too voluminous to cross by diffusion the bacteria membranes, which remain intact, even if some NPs are aggregated at the surface of bacteria. On the contrary, in the case of smaller NPs, the empty appearance of bacteria suggests that membranes are disrupted, leading to the release of cellular compounds.

Authors thank NSI platform of LOMA (CPER COLA2) and JPK Instruments for AFM equipment and technical support.

Fig. 1: AFM amplitude image (5x5 µm) of a bacterium interacting with silica NPs (diameter 100 nm) showing a still healthy bacterium with unchanged morphology.

Fig. 2: AFM phase image (5x5 µm) of a bacteria interacting with silica NPs (diameter 200 nm): the contrast is different for one NP as compared to other ones, suggesting its endocytosis.

Fig. 3: AFM topography image (5x5 µm) of bacteria interacting with silica NPs (diameter 10 nm).

Fig. 4: AFM phase image (5x5 µm) corresponding to the Fig. 3. On the left, we observe the print of a bacterium which is not observed in the topography image.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-2859 Microspectroscopy monitoring of singlet oxygen in single mammalian cells

Scholz M.1, Dědic R.1, Valenta J.1, Breitenbach T.2, Hála J.1
1Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University in Prague, Czech Republic, 2Center for Oxygen Microscopy and Imaging, Department of Chemistry, University of Aarhus, Denmark
Marek.Scholz@mff.cuni.cz

Singlet oxygen (1O2), the lowest excited state of molecular oxygen, is up to several orders of magnitude more reactive than the ground state oxygen and readily oxidizes a wide variety of biomolecules, such as lipids, nucleic acids and proteins. 1O2 is involved in a number of biochemical processes, including cellular signaling, immune response, or polymer degradation. The most important way of 1O2 generation is the energy transfer from light-excited triplet states of organic dyes - photosensitizers - to ground state oxygen. Larger amounts of 1O2 are cytotoxic which can be utilized in photodynamic therapy of various diseases, namely cancer. The photosensitizer administrated to the patient accumulates selectively into the target tissue which is then irradiated by light of an appropriate wavelength and the production of 1O2 triggers processes leading to cell death. It is highly desirable to investigate and understand how 1O2 acts on cellular and subcellular level. A very weak near-infrared phosphorescence of 1O2 allows for its direct imaging by means of luminescence microscopy, but this is still an experimental challenge. The recent introduction of improved near-infrared 2D-array detectors brings new possibilities into the field.
The contribution presents our new setup for 1O2-based luminescence microscopy using a novel near-infrared sensitive 2D-array detector NIRvana. The setup allows for simultaneous observation of VIS fluorescence of the photosensitizer together with near-infrared phosphorescence of 1O2 and the photosensitizer. The ability of the system to detect 1O2 emission is evaluated and 1O2-based images of single mouse fibroblast cells loaded with photosensitizer TMPyP are demonstrated. Our work expands the earlier pioneering work by Ogilby's group [1,2]. However, its greatest novelty is the insertion of a spectrograph in the detection path, which allows us to acquire spectral images of individuals cells, where one dimension is spatial and the other is spectral. These spectra provide important implications for the further development of the technique. Finally, present limitations and future directions are discussed.

[1] I. Zebger et al: Direct Optical Detection of Singlet Oxygen from a Single Cell, Photochemistry and Photobiology, 2004, 79, 319–322.
[2] J. Snyder et al: Optical detection of singlet oxygen from single cells, Physical Chemistry Chemical Physics, 2006, 8, 4280–93.

The work was supported by project number 848413 from the Grant Agency of Charles University and project P501/12/G055 from the Czech Science Foundation.

Fig. 1: NIR luminescence based images and spectra together with bright-field and VIS fluorescence-based images of 3T3 mouse fibroblasts. The cells were incubated for 5h with 100 µM TMPyP in D2O-based saline solution.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-2871 3D reconstruction of live cell organelles from information entropy point of view

Romanova K.1
1University of South Bohemia, Faculty of Fisheries and Protection of Waters, Institute of Complex Systems, Zámek 136, 373 33 Nové Hrady, Czech Republic
romanova@frov.jcu.cz

A major challenge of live cell imaging is keeping cells alive and functioning as naturally as possible for the duration of the experiment [1]. A single 2D image only provides a small part of the complete context of the research and the relationship between structures in the sample cannot be fully determined in 2D. 3D imaging gives researchers more information about cellular structure and function. Our goal is to investigate images of observed objects' positions that as possible credibly copy their positions and determine the states of elementary units which determine the state of the organism: in our case, organelles. A microscopic image thus must be understood as information about the sample, and the microscope must be seen as an information channel. If we want to reconstruct an organelle in three-dimensional space we have to have a method for automatic features extraction. Computational image analysis allows us to provide rapid and automated processing of large datasets.

In order to understand the development of the image at various levels of de-focus, we introduce the point divergence gain PDG. We examine the change in the information which occurs when the particular point is replaced by the point from the next image. This may result in both an increase and decrease in the information. The change in an image series due to a z-scan is the result of either an object's appearance or disappearance from the image or changes in the point spread function of the object with the distance from its ideal focal plane. To determine the borders of the determinable point spread function, we calculate image points which carry the same information in consequent images captured upon moving the object along the lens' optical axis (z-scan). In this way, we may precisely identify the border of the point spread function for different identifiable objects [2, 3].

References:

1. Melanie M. Frigault, Judith Lacoste, Jody L. Swift, Claire M. Brown2009 Live-cell microscopy – tips and tools, J Cell Sci 122, 753-767. doi: 10.1242/jcs.033837.

2. Stys D, Urban J, Vanek J, Cisar P (2011) Analysis of biological time-lapse microscopic experiment from the point of view of the information theory. Micron 42:360365. Epub 2010 Feb 12. doi: 10.1016/j.micron.2010.01.012.

3. Stys D, Jizba P, Papacek S, Nahlik T, Cisar P (2012) On Measurement of Internal Variables of Complex Self-Organized Systems and Their Relation to Multifractal Spectra, IWSOS 2012, LCNS 7166:3647, Kuipers and Heegaard eds. Springer: Heidelberg Dordrecht London New York.

Results of the LO1205 project was obtained during financial support of MEYS CR in the frame of NPU I programme. This work was also financed by grants Postdok JU and GA JU 134/2013/Z.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-3095 Cell wall formation in root hairs and the influence of heavy metals

Lichtscheidl I.1, Rumé-Strobl J.1, Adlassnig W.1
1Core Facility of Cell Imaging and Ultrastructure Research, University of Vienna, Vienna, Austria
irene.lichtscheidl@univie.ac.at

Root hairs are long tubular cells with unidirectional growth; cell wall formation is restricted to their tips and depends on the coordination of numerous molecular and cellular processes including exocytosis of pectins, endocytosis of excess plasma membrane, cellulose synthesis and the turgor pressure of the vacuole. They increase the root surface tremendously and serve as contact between plant and soil in order to improve nutrient uptake.

Exo-/endocytotic vesicles are too small to be resolved by conventional light microscopy, wherefore the tip region of root hairs is usually called “clear zone”; they become visible only in electron micrographs. Visualizing their dynamic behaviour and their coordination with the cytoskeleton is however needed in order to understand the underlying principles of signaling and growth regulation. We therefore applied video-enhanced contrast light microscopy and ultraviolet microscopy and thus were able to visualize these vesicles in their living state and to analyze their movements. Staining of the plasma membrane with fluorescent dyes yielded information about the dynamics of exo- and endocytotic processes during cell wall secretion and membrane recycling.

We investigated the influence of heavy metals, especially of zinc, on structure and growth of root hairs of Triticum aestivum, spring wheat. In addition, we analysed the absorption and transport of zinc into roots and root hairs by using fluorescent tracer dyes specific for heavy metals. We compared the results from fluorescence with data from the scanning electron microscope where we used energy-dispersive X-ray spectroscopy (EDX) in order to localize heavy metals.

We wish to express our gratitude to Dr. Miroslav Ovecka, University of Olomouc, for constant help and discussion. This work was supported by the Austrian OEAD project appear 43, BIOREM.

Fig. 1: Tip of growing root hair after zinc treatment, showing abnormal cell wall thickening. a: vesicles in the clear zone in the bright-field after contrast-enhancement by video techniques. b: plasma membrane and endosomes in FM4-64.

Fig. 2: Root hairs after staining with Newport Green, a fluorescence tracer dye specific for heavy metals such as zinc.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-3129 SHG Imaging as a Method for Precision Characterization of Collagen Structures in Combination with Nanoindentation

Sepitka J.1, Burdikova Z.2, Filova E.3, Lukes J.1
1Czech Technical University of Prague, Hysitron Nanomechanical Applications Lab, Prague, Czech Republic, 2Department of Biomathematics, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 3Department of Tissue Engineering, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
jaroslav.lukes@fs.cvut.cz

Studying the mechanical properties of native biological tissues is a great challenge in the field of biomechanics. Studying hardly accessible structures that play a very important role within a locomotive system, such as cartilaginous endplates (CEP), is especially challenging. Experimental techniques in biomechanics have undergone a hlarge evolution recently. CEP is approximately a 0.6mm thin layer of hyaline cartilage located between an intervertebral disc (IVD) and a vertebral body (VB). Calcification or any mechanical damage of a CEP can cause nutritional and metabolic waste flow restrictions both inward and outward from the IVD, respectively. Degenerative processes influence the mechanical properties of the tissue and therefore, this paper will aim towards understanding the material properties of CEPs. Information about local mechanical properties could help identify CEP malfunction and provide possible designs for IVD replacement.
Due to the very small thickness of CEPs, instrumented nanoindentation is a suitable method for determining mechanical properties. This is especially the case when using nanoscale Dynamic Mechanical Analysis (nanoDMA), which was developed to test viscoelastic materials. The aim of this work is to determine CEP localization using optical imaging methods and Second Harmonic Generation imaging (SHG) in combination with nanoindentation. The local mechanical properties of native CEP were measured and related to the inner microstructure. It is evident that the large scattering of CEP mechanical properties is due to the inhomogeneity of the microstructure.

The presented study was supported by the Technology Agency of the Czech Republic, grant no. TA01010185.

Fig. 1: Anisotropic structure of CEP. SHG imaging with two photon femtosecond laser (exc. 860 nm) linear-polarised beam: upper raw images: vertebral body - VB, cartilaginous endplate - CEP, nucleus pulposus – NP. Polarisation at various angles (red 0°, blue 60°, green 120°).

Fig. 2: Isotropic structure of CEP. SHG imaging with two photon femtosecond laser (exc. 860 nm) linear-polarised beam: upper raw images: vertebral body - VB, cartilaginous endplate - CEP. Polarisation at various angles (red 0°, blue 60°, green 120°).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-3248 Metal Treatment on Physcomitrella patens Compared to two Bryophyte Species Naturally Occurring on Metal Contaminated Sites

Sassmann S.1, Weidinger M.1, Bock B.1, Antreich S.1, Adlassnig W.1, Lang I.1
1University of Vienna, Cell Imaging and Ultrastructure Research, Althanstrasse 14, A-1090
stefan.sassmann@univie.ac.at

Bryophytes inhabit extremely different habitats, ranging from dry fallen river banks (e.g. Physcomitrella patens) to metal contaminated sites (e.g. Pohlia drummondii and Mielichhoferia elongata). Therefore, some bryophyte species are considered stress tolerant, and even the supposedly metal sensitive moss P. patens showed increased tolerance to Cu-EDTA in earlier studies.

For the present experiments, the bryophytes were cultivated on sterile agar plates and tested for zinc (as Zn-EDTA, ZnCl2 and ZnSO4) and copper (as Cu-EDTA, CuCl2 and CuSO4) over a period of five weeks (Fig. 1).

Despite of the high tolerance towards Cu-EDTA of P. patens, we measured changes in growth and metal uptake analyzed by X-ray microanalysis in a scanning electron microscope (Fig.2) if the metal is offered with different anions. Here, especially the uptake of EDTA chelated metals was significantly lower compared to metal offered as ionic salt. Modelling of ion availability explained most of the differences in toxicity.

Changes in the cellular content of reactive oxygen species (ROS) after staining with 2,7-dichlorofluorescein diacetate (H2DCFDA) were analyzed in a confocal scanning microscope (Fig.3) and the three different bryophyte species compared. P. patens showed only low H2DCFDA fluorescence in control cells, in contrast to metal treated cells were increased ROS could be detected for chloroplast associated mitochondria, the nuclear region and the cell wall region.

Further investigation of cellular localization of metal deposition was performed using FluoZin-3 and is ongoing in transmission electron microscopy studies.

This research was supported by the Vienna Anniversary Foundation for Higher Education (grant H-2486/2012 to S.S.) and the ŒAD (Appear/43/Biorem). Many thanks are due to Irene Lichtscheidl, University of Vienna.

Fig. 1: P. patens plants grown on control and zinc spiked media over a period of 5 weeks.

Fig. 2: Scanning electron microscope micrograph of P. patens leafy gametophyte (scale bar = 1 mm).

Fig. 3: P. patens leaf cells stained with H2DCFDA for H2O2 detection. a, Control cells; b, Plants grown on 1 mM ZnCl2 and c, plants grown on 0.1 mM CuSO4 show increased fluorescence in close vicinity of the chloroplast, in the nuclear region (*), mitochondria (arrowhead) and the cell wall (scale bar = 10 µm).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-3346 VISUALIZATION OF LIVING BLOOD CELLS - NEW OPPORTUNITIES FOR CELL DIAGNOSTICS

Vasilenko I.2, Beljakov V.3, Nasirov M.3, Topuzoov A.3
1Maimonides State Classical Academy, 2Russian Medical Academy of Postgraduate Education, 3N.I.Pirogov Russian National Research Medical University
vasilenko0604@gmail.com

Studying structural features and functional full-value of blood cells are extremely significant in solving pathogenesis processes, diagnosis of diseases, and assessment of the treatment efficiency. Conventional microscopic methods do not always meet the growing requirements for sensitivity, low invasiveness, fast activity, and spatial resolution. Therefore, new technologies of cellular visualization are quite topical because they allow performing in the real time the efficient quantitative analysis of the structural parameters and cellular function without image fixation and contrast. Phase-interference methods investigate so called functional images of the cells. The refractivity index studied enables assessment of the inner cellular structure which reflects its function, directly or indirectly.
The informative biotecnology was worked out, based on the divice-program complex (DPC) “Biony” for clinical and laboratoty diagnostics with digital image processing and new type biocensors (Westtrade LTD, Russia). The chief DPC module is a computerized phase-interference microscope (CPM) which is a modified Linnick’s interferometer with phase modulation of the bearing wave. Helium-neon laser (λ=633 nm) is a source of light. DPC enables measurement of integral parameters of the phase micro-objects with high sensitivity, and а method of automatic reading of interferograms allows achieving measurement resolution of λ/150, where λ is a radiation wave length if the time of measurement is 15-30 sec. Complex algorithm of the live cells analysis includes topogram, 3D-image, set of profiles, histogram of the phase heights distribution, calculation of morphometric parameters of single cells, integral analysis of the cellular population by signs of measuring, and creation of cytograms.
The authors identified and characterized the vital phase pictures of all blood cells: erythrocytes, neutrophiles, lymphocytes, and thrombocytes. Within the frames of one method, the morphologic features of these cells were estimated as well as their size and functional activity. The regularities of morphologic changes in the blood cells condition in norm and pathology were revealed and quantitatively assessed (with regard for their age-caused peculiarities).
Acquirement of important quantitative information concerning cellular condition using technically available and not very expensive modalities of interference microscopy presents new opportunities for practical application of live functioning cells as prospective biosensors for diagnostic purposes.

Fig. 1: Phase-interference image of living peripheral blood erythrocyte

Fig. 2: Phase-interference image of living peripheral blood platelet

Fig. 3: Phase-interference image of living peripheral blood lymphocyte

Fig. 4: Phase-interference image of living peripheral blood neutrophil
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-3493 Visualization of hepatocytes and immune cells in the liver by intravital multiphoton microscopy

Matsumoto S.1,2, Maeda S.1,2, Kikuta J.1,2, Ishii M.1,2
1Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka, Japan, 2Japan Science and Technology Agency, CREST, Tokyo,Japan
j.kikuta1981@gmail.com

Background: The liver is a vital organ in our body. It has various kinds of functions, such as detoxification, protein synthesis, bile production, and metabolism. Hepatocytes are the main cells of liver and organized into plates separated by vascular channels (sinusoids). Recently, the development of biological imaging techniques with intravital multiphoton microscopy enables us to visualize dynamic cell migration and cell-cell interactions in vivo. Observation of living cells in the liver would be very useful for understanding physiology and pathology of liver function. However, there are few studies examined by intravital liver imaging due to its difficulty.
Objective: This study aimed to establish the new imaging system for visualizing living liver with intravital multiphoton microscopy.
Methods: A mouse was anesthetized with isoflurane. The liver was exposed, and then the mouse was immobilized in a custom-made stereotactic holder. Before imaging, nuclei of hepatocytes or sinusoids were visualized by intravenous injection of hoechst33342 or eFluor 650NC, respectively. The liver tissues were observed by using inverted multiphoton microscopy.
Results: We succeeded in visualizing 3D structure of liver, hepatocytes, and blood flow in real-time. By using the mice expressing EGFP under the promoter of LysM, we also observed the movement of immune cells around hepatocytes for several hours.
Conclusion: We have developed the new technique for visualizing living liver with intravital multiphoton microscopy. This imaging technique would be quite beneficial for studying physiology and pathology of liver function in vivo.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-3518 Recovery of the plant Golgi apparatus during Brefeldin A treatment

Sedlakova Z.1, Hawes C.1
1Oxford Brookes University, UK
zone.cz@gmail.com

This research used live cell imaging to report on the disassembly and subsequent recovery of the Golgi apparatus in tobacco leaf epidermal cells and Arabidopsis thaliana during Brefeldin A (BFA) treatment. BFA, a macrocyclic macrotone, is a fungal drug commonly used in vesicle-mediated protein trafficking research for its ability to disturb the guanine-nucleotide exchange factors (GEFs) function resulting in a blockage of protein trafficking. To observe the effect of the drug on the plant Golgi apparatus and to reveal more about the related protein trafficking mechanism, different concentrations of the BFA were used and tested on tobacco leaf epidermal cells and multiple tissues including leaf, hypocotyl and root tissue of A. thaliana. The Golgi related proteins of interest included a small GTPase ADP ribosylation factor (ARF), trans specific sialyltransferase (ST), a cis/medial specific part of a β1,2-N-acetylglucosaminyl-transferase-I (GnTI), and a post-Golgi compartment protein (PS1). Those proteins were fluorescently tagged using green fluorescent protein (GFP) and monomeric red fluorescent protein (RFP) and underwent 3 hr or overnight BFA incubation.
The results have confirmed previously described disruptive effect of the BFA on the Golgi apparatus and protein trafficking in general to be time and dose dependent and even tissue specific in A. thaliana. For the ARF-GFP activity in tobacco, the protein retreat from the Golgi into the cytosol was seen after just 60 min incubation with 25µg/mL BFA and indicated the Golgi recovery in longer overnight samples only. In the case of PS1-GFP in tobacco, the Golgi disruption was recorded after 60 min BFA incubation with the protein locating to vacuoles. The reappearance of Golgi bodies occurred after 210 min drug treatment. For ST-GFP, the results from tobacco showed slower disassembly of the Golgi apparatus and the protein retreat into the endoplasmic reticulum (ER) after 90 min of 25µg/mL BFA treatment but much faster progression of Golgi depletion was recorded in ST-GFP expressing roots of A. thaliana. On contrary, the Golgi disassembly in the leaves and hypocotyl of A. thaliana were recorded after 120 min drug incubation and the Golgi recovery was the fastest in hypocotyl with just 210 min after the BFA incubation initiation, showing the diverse effect of BFA across Arabidopsis tissues.
To assess the manner in which Golgi recovery occurs further, A. thaliana expressing both cis/medial located GnTI-mRFP and trans located ST-GFP markers was used in additional BFA treatment experiment. The results of this experiment suggested that the reassembly of the Golgi apparatus happens in cis to cis/medial and trans manner.

The authors would like to thank Dr Anne Osterrieder and Dr John Runions, both from Oxford Brookes University, for their help during the research.

Fig. 1: Wild-type tobacco epidermal cells expressing ST-GFP. Micrographs show three BFA [25 µg/mL] treatment times: 0, 120 min treatment and overnight (20 hrs). The ER and the nuclear envelope are clearly visible at 120min of BFA treatment but no ERis seen in before and overnight samples - showing the recovery ability of ST proteins. Scale bars = 20 µm.

Fig. 2: A. thaliana leaves coexpressing cis located GnTI-mRFP (red) and trans located ST-GFP (green) treated with BFA [25 µg/mL] for 150min. The merged micrograph shows that the ER proteins come from different parts of the Golgi apparatus. The reassembly of the Golgi apparatus happens in cis to cis/medial and trans manner. Scale bars = 20 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-3523 Synthesis and application of new fluorescent dyes

Kozák J.1, Kužmová E.1, Reyes-Gutiérrez P. E.1, Devadig P.1, Joshi V.1, Sonawane M.1, Talele H.1, Severa L.1, Jirásek M.1, Hubálková P.1, Hájek M.1, Teplý F.1, Cebecauer M.2
1Institute of Organic Chemistry and Biochemistry, v.v.i., AS CR, 2J. Heyrovský Institute of Physical Chemistry, v.v.i., AS CR
jardakozak1@gmail.com

We have generated a growing family of cationic dyes with interesting fluorescent properties that are markedly environment sensitive (e.g., DNA, protein, heparin or artificial membranes). Our library of 1500 dyes is based on 19 skeletal structures which are easily modified by standard commercially available chemicals to achieve desired properties. In contrast to a majority of commercial organic dyes our dyes are nonplanar, a feature which may bring new opportunities for detection and visualization of important biological structures. Altogether, this indicates their potential use in various applications such as flow cytometry and microscopy.

A set of 150 selected dyes have been tested for their ability to penetrate and label specific structures in human cell lines (HeLa, CCRF-CEM, HGC-27, Hep G2 and U2-OS). Highly selective labelling of mitochondria was prevalent for our novel dyes but other localizations such as plasma membrane, endoplasmic reticulum or nucleolus can be detected. A good example of specificity of our novel fluorescent probes are dyes Cellmem8 and Mito19 which were shown to highly colocalise with commercial dyes CellMaskTM (PCC = 0.73) and MitoTracker® (PCC = 0.81), respectively, see Figures 1 and 2. Conversely, DNA sensitive dye Celldead3 cannot penetrate cytoplasmatic membrane and therefore is able to discriminate dead cells, see Figure 3.

Our dyes show good biocompatibility and are promising candidates for live-cell imaging. The simple one step synthesis and easy purification process make them even more attractive.

We gratefully acknowledge the support of IOCB and the Academy of Sciences of the Czech Republic (RVO: 61388963).

Fig. 1: Colocalization of Cellmem8 (A) with commercial dye CellMaskTM Deep Red (B) shown in HeLa cells. C shows merge of the images A and B. Zeiss LSM 780, Plan-Apo 63x / 1.4 NA oil immersion objective.

Fig. 2: Colocalization of Mito19 (A) with commercial dye MitoTracker® Red (B) shown in HeLa cells. C shows merge of the images A and B. Zeiss LSM 780, Plan-Apo 63x / 1.4 NA oil immersion objective.

Fig. 3: Staining of dead cells (HGC-27) with our dye – Celldead3 and GelRed. A shows the merge of bright field and cells stained with Celldead3 and GelRed. B shows the colocalization (PCC = 0.83) of Celldead3 (C) and GelRed (D). Zeiss AxioObserver.A1, 20x objective.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5723 Quantification of aortic and cutaneous elastin and collagen morphology in a mouse model of Marfan syndrome by multiphoton microscopy

Cui J. Z.1, Tehrani A. Y.1, van Breemen C.1, Esfandiarei M.1, 2
1Child & Family Research Institute, Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, BC, Canada, 2Department of Biomedical Sciences, College of Health Sciences, Midwestern University, Glendale, AZ, USA
mesfan@midwestern.edu

Marfan syndrome (MFS) is an autosomal dominant connective-tissue disorder caused by heterozygous mutations in fibrillin-1 gene (FBN1). In a mouse model of Marfan syndrome, conventional Verhoeff-Van Gieson staining displays severe fragmentation, disorganization and loss of the aortic elastic fiber integrity. However, this method involves chemical fixatives and staining, which may alter the native morphology of elastin and collagen. Thus far, quantitative analysis of fiber damage in aorta and skin in Marfan syndrome has not yet been explored. In this study, we have used an advanced noninvasive and label-free imaging technique, multiphoton microscopy to quantify fiber fragmentation, disorganization, and total volumetric density of aortic and cutaneous elastin and collagen in a mouse model of Marfan syndrome.

Thoracic aorta and dorsal skin samples were harvested from Marfan and control mice aged 3-, 6- and 9-month. Elastin and collagen were identified based on two-photon excitation fluorescence and second-harmonic-generation signals, respectively, without exogenous label. Measurement of fiber length indicated significant fragmentation in Marfan vs. control. Fast Fourier transform algorithm analysis demonstrated markedly lower fiber organization in Marfan mice as compared with control. Significantly reduced volumetric density of elastin and collagen and thinner skin dermis were observed in Marfan mice. Cutaneous content of elastic fibers and thickness of dermis in 3-month Marfan resembled those in the oldest control mice. Our findings of early signs of fiber degradation and thinning of skin dermis support the potential development of a novel non-invasive approach for early diagnosis of Marfan syndrome.

This work was supported by grants from the Canadian Institutes of Health Research (MOP-111266 to C. V. B. & M.E.). We thank Drs. Thomas Abraham, Furquan Shaheen, Damian Kayra, and Kevin Hodgson for their technical assistance with the multiphoton microscopy.

Fig. 1: Measurements of aortic elastin & collagen volume and fiber organization using multiphoton microscopy and Fast Fourier analysis. A) 3-D reconstruction of aortic segments from 6-month old control and MFS mice (Elastin, green; collagen, purple). B) Analysis of the elastic fiber orientation using two-dimensional fast Fourier transform algorithm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5840 LEUKOCYTE RECRUITMENT TO ADIPOSE TISSUE INDUCED BY EXPERIMENTAL FOOD ALLERGY IS REGULATED BY CHRONIC ALLERGEN INGESTION BY SENSITIZED MICE

BATISTA N. V.1, PEREIRA R. V.2, FONSECA R. C.2, FERREIRA A. V.3, CARA D. C.2
1Departamento de Bioquímica e Imunologia - Universidade Federal de Minas Gerais, Brasil, 2Departamento de Morfologia - Universidade Federal de Minas Gerais, Brasil, 3Escola de Enfermagem - Universidade Federal de Minas Gerais, Brasil
nathaliavb@gmail.com

Introduction and objective: Leukocyte recruitment to adipose tissue is involved in several pathological conditions such as obesity [1]. We have shown that in the context of food allergy there is an increase in the monocyte recruitment to adipose tissue [2]. The aim of this study was to investigate whether prolonged ingestion of antigen by previously sensitized mice would revert the adipose tissue inflammation caused by experimental food allergy. Leukocyte recruitment is a hallmark event of inflammation and happens as a cascade of steps. The leukocytes first roll in the vessels, then adhere to them and it results in the transmigration of leukocytes to the tissue [3]. Methods: Male BALB/c mice were sensitized with Ova in Al(OH)3 on day 0 and received a booster on day 14. The control group (Ova-) wasn’t sensitized with Ova. After day 21 all animals received an Ova diet for 7 (Ova+ 7 days) or 14 days (Ova+ 14 days) (n=8). Results: The allergic process, that happened after 7 days of Ova ingestion by sensitized mice, was evidenced by a significant increase in the anti-Ova IgE level measured by ELISA. This process resulted in a significant body weight and adipose tissue loss that had the peak 7 days after the challenge with progressive recovery after this day. This loss was followed by a decrease in the area of adipocytes and an increase in the level of cytokines involved in the cellular recruitment such as IL-6 (Fig. 1A) and TNF-alfa (Fig. 1B) in the adipose tissue. Also in this tissue there was an increase in the leukocyte recruitment, rolling cells (Fig. 2A) and adhesion events (Fig. 2B) in the microvasculature of the adipose tissue, visualized by intravital microscopy. After 14 days of oral challenge, sensitized mice showed an anti-Ova IgE level similar to the mice that were only sensitized. With this developed desensitization to Ova all parameters analyzed were significantly improved although they did not reach the basal levels. Conclusion: Our data suggest that the continued ingestion of Ova by sensitized mice leads to a desensitization to this antigen with a reduction in the leukocyte recruitment to adipose tissue caused by experimental food allergy.

References:
[1] Nishimura S et al., In vivo imaging in mice reveals local cell dynamics and inflammation in obese adipose tissue, J Clin Invest, 118 (2008) 710-721.
[2] Dourado LP et al., Experimental food allergy leads to adipose tissue inflammation, systemic metabolic alterations and weight loss in mice. Cell Immunol, 270 (2011)198-206.
[3] Klaus Ley, Integration of inflammatory signals by rolling neutrophils, Immunol Rev, 186 (2002) 8-18.

Acknowledgements: Financially supported by CAPES, CNPq and FAPEMIG.

Fig. 1: Figure 1 - Kinetics of IL-6 (A) and TNF-α (B) production in epididymal adipose tissue in non-sensitized or sensitized after Ova challenge. Data are reported as means ± SEM for six mice in each group. * P < 0.05 compared to Ova - group and # P < 0.05 compared to Ova + after 7 days of Ova consumption.

Fig. 2: Figure 2 – Intravital microscopy was used to assess the rolling (A) and adhesion (B) of leukocytes in microvasculature of epididymal adipose tissue in vivo. Data are reported as means ± SEM for six mice in each group. * P < 0.05 compared to Ova - group and # P < 0.05 compared to Ova + after 7 days of Ova consumption.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5847 Time-lapsed videomicroscopy in cancer biology

Kripnerová M.1, Tuková J.1, Pešta M.1, Červinka M.2, Rudolf E.2, Hatina J.1
11) Department of Biology, Faculty of Medicine in Pilsen, Charles University in Prague, Czech Republic, 22) Department of Medical Biology and Genetics, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Czech Republic
michaela.kripnerova@lfp.cuni.cz

The evolution of cancer cell population is a very dynamic process, involving numerous cell-cell and cell-matrix interactions constantly shaping the cancer cell population. Time-lapse videomicroscopy of living cell cultures can be very instrumental in following and dissecting these interactions and the resulting evolution within the cancer cell population.

We demonstrate this approach by two examples. First, we have followed the fate of putative cancer stem cells over prolonged time scale. A derivative daughter of the bladder cancer cell line BFTC-905 has been transfected with an expression vector coding for the GFP reporter under the control of a doxorubicin-responsive promoter and cultured in doxorubicin containing medium; cancer stem cells, by virtue of their constitutive expression of multidrug resistance efflux pumps, constantly eliminate doxorubicin out of the cell and therefore cannot switch on the doxorubicin-regulated GFP, in progenitor cells is this capacity limited, leading marginal GFP expression level, whereas in differentiated cancer cells strong GFP expression could be recorded. The time lapse videomicroscopy is thus able to unravel the intrinsic hierarchy of carcinoma cell lines. In the second experiment, we cocultured subperitoneal fibroblasts with the A2780 ovarian carcinoma cells. Peritoneal spreading is the preferred way of ovarian cancer metastasis and the chemotaxis of ovarian cancer towards subperitoneal fibroblasts is supposed to represent one of the underlying mechanisms. By long-term coculture recording, we could really observe a strong chemotaxis of ovarian carcinoma cells towards sparsely grown subperitoneal fibroblasts. These examples illustrate the power of time-lapsed videomicroscopy in analysing complex dynamic processes in cancer biology.

The work was supported by the specific student research grant of the Charles University SVV 260 050.

Fig. 1: Cocultivation of subperitoneal fibroblasts with the A2780 ovarian carcinoma cells, magnification 100x
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5852 Live imaging of early oral epithelium reveals epithelial migration of tooth progenitor cells

Prochazka J.1, Prochazkova M.1,2, Spoutil F.3, Hoch R.4, Shimogori T.5, Wittmann T.6, Klein O. D.1,7
1Department of Orofacial Sciences and Program in Craniofacial and Mesenchymal Biology, University of California, San Francisco, CA, USA, 2Department of Anthropology and Human Genetics, Faculty of Science, Charles University in Prague, Czech Republic, 3Institute of Experimental Medicine AS CR, Prague, Czech Republic, 4Department of Psychiatry, Nina Ireland Laboratory of Developmental Neurobiology, University of California, San Francisco, CA, USA , 5RIKEN Brain Science Institute, Laboratory for Molecular Mechanisms of Thalamus Development, 2-1 Hirosawa Wako, Saitama 351-0198, Japan, 6Department of Cell and Tissue Biology, University of California, San Francisco, CA, USA , 7Department of Pediatrics and Institute for Human Genetics, University of California, San Francisco, San Francisco, CA
jan.prochazka@ucsf.edu

Live-imaging of embryonic tissues provides unique tool to underpin cellular and molecular mechanisms of morphogenesis and organ formation. In our system we used early embryonic mandibles explanted at embryonic day 11 into organ culture. This approach enabled us to perform long time-lapse confocal microscopy imaging of early oral epithelium for up to 48 hours. The speed of scanning and length and intensity of tissue exposure to excitation lasers are critical parameters to avoid photo-bleaching of fluorescence reporters, as well as to keep the tissue healthy, and to ensure that assessed developmental processes are comparable with development in vivo. To accomplish that, we established imaging setup for organ culture with use of spinning disc confocal microscope. Spinning disk imaging technology provides unique combination of sufficient speed and resolution for 48h long time-lapse imaging with minimal photo-toxicity for tissue. The use of described live-imaging microscopy setup allowed us to examine the cellular mechanisms of tooth development initiation. We identified a migratory population of Fgf8-expressing epithelial cells in the embryonic mandible which provides most of the epithelial cells required for development of future molar tooth. This population migrates anteriorly towards Shh-expressing epithelial cells at the site where tooth placode will initiate. Furthermore, inhibition of Fgf and Shh signaling disrupted the oriented migration of cells, leading to failure of tooth development. The time-lapse data allowed us to perform statistic analysis of cell movement in time and evaluate the differences in cell behavior under Fgf and Shh signaling compromising conditions compared to normal development. Our results demonstrate the importance of collective epithelial cell migration in the initiation of tooth development and provide the first example of such cell behavior during mammalian organ formation, suggesting that epithelial migration might be more frequent mechanism of morphogenesis also in other organ systems and proper live-imaging is only direct way to analyze this phenomenon.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5865 Experiences of building and adapting the OpenSPIM

Harris J.1, Stramer B.2, Thackery J.2, Schwertner M.3, Kamp V.3, Fleck R. A.4
1Nikon, Imaging Centre, Kings College London, London, SE1 1UL, 2Randall Division of Cell and Molecular Biophysics, Kings College London, London, SE1 1UL, 3Linkam Scientific Instruments, Tadworth KT20 5LR, 4Centre for Ultrastructural Imaging, Kings College London, London, SE1 1UL
roland.fleck@kcl.ac.uk

Selective Plane Illumination Microscopy (SPIM) is an emerging technique with potential in the life sciences. SPIM promises fast, minimally-invasive, optical-sectioning (3D acquisition) of fluorescent specimens over time. Its principal advantage over more established fluorescence techniques (e.g, wide field epi-fluorescence microscopy, confocal and multiphoton microscopy) is the generation of larger volume (x-y-z) data sets with potentially lower levels of light induced damage to the tissues. SPIM is particularly suited to live cell studies of whole organisms (e.g., Drosophila embryos, Zebra fish embryos etc).

SPIM generates 3D information by focusing a thin laser light-sheet into the specimen using a first objective lens and taking 2D images of the illuminated slice with a second detection lens placed perpendicularly to the light sheet. .. Sequential, 3D stacks are acquired by moving the specimen orthogonally to the light-sheet between consecutive images. By mounting the sample in a rigid medium, e.g. low temperature gelling agar, and mounting it in the sample chamber in front of the detection lens, it is possible to rotate the sample and collect 3D stacks from multiple angles. These multiple 2D images can then be used to reconstruct the fluorescence distribution in the 3D volume.

Presently there is one commercial SPIM platform available (Zeiss Lightsheet Z.1), however, it is possible to build your own SPIM based on the designs featured on the OpenSPIM website: (http://openspim.org/Welcome_to_the_OpenSPIM_Wiki). This allows groups to gain initial experience of SPIM and/ or optimise a system for a specific task. In a collaboration between Kings College London and Linkam Scientific, we have built a SPIM based around the OpenSPIM platform and we will discuss our experience of building and operating the OpenSPIM along with the parallel development of a temperature regulated stage for use with the system.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5869 Enhancement of FRET AB by time resolved spectral detection

Pala J.1,2, Stixová L.3, Čmiel V.4,5, Provazník I.4,5
1Third Medical Faculty, Charles University Prague, CZ, 2Comipa s.r.o., Klecany, CZ, 3Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, CZ, 4Technical University Brno, CZ, 5International Clinical Research Center - Center of Biomedical Engineering, St. Anne’s University Hospital Brno, CZ
jan.pala@lf3.cuni.cz

Fluorescence resonant energy transfer (FRET) is a very popular method used both in wide-field and confocal laser scanning microscopy nowadays. This is because FRET can easily provide information about the short distances between molecules in nanometer range. FRET Acceptor Photobleaching (FRET AB) is quite simple method based on bleaching the acceptor, but the drawback is that there are needed a lot of tests and controls to confirm FRET. Our proposal is to use so-called TimeGate function of confocal laser scanning microscope Leica TCS SP8 X. This function is available for the hardware combination of excitation by pulsed picosecond White Light Laser (WLL) and spectral hybrid HyD detectors based on GaAsP. WLL is a laser freely tunable in the spectral range 470-670 nm, so that the wavelength and intensity can be tuned for ideal excitation. Spectral detection by HyDs can be expanded by time resolved detection by TimeGate function with arbitrary time windows in the range 0-12 ns. It is based on time delay between the excitation pulse and detection of fluorescence. As the minimum opening of the TimeGate window is 3.5 ns with the stepsize 0.1 ns or lower, the TimeGate function can be applied to distinguish between the donor and FRET channels.

We performed FRET for well-known interacting partners p53 and 53BP1 in mouse mbryonic fibroblasts (MEFs). The fact that p53 and 53BP1 can interact was well described previously (Iwabuchi K, et al. Proc Natl Acad Sci U S A. 1994). Although the significance of the interaction between 53BP1 and p53 is not completely elucidated, it is evident that 53BP1 plays an important role in chromatin-based DNA damage response signaling (Panier S, et al. Nat Rev Mol Cell Biol. 2014). The 53BP1-p53 interaction is mediated by the tandem-BRCT repeats of 53BP1 (residues 1702–1972) and the DNA-binding domain of p53 (residues 94–292) (Joo WS, et al. Genes Dev. 2002). In our experiments, we have used laser lines 488 nm and 591 nm for analysis by FRET AB with two HyDs with spectral bandwidth set adequately to excitation laser lines. Different time windows were tested. Here, we present results with the time window set in the range 0.3-3.8 ns. Format of the images was 512 x 512 pxls (30.75x30.75 microns). Preferentially, we investigated the interactions between p53-GFP and 53BP1-Alexa 594. In classic settings, FRET efficiency was approximately 25 % (Fig 1), while the FRET resolution was increased by the setting of the TimeGate function (efficiency 45.5±6.1%, Fig 2). Therefore, TimeGate function seems to be very useful tool for advanced FRET application.

The work was supported by by the Education for Competitiveness Operational Programme (ECOP) CZ.1.07/2.3.00/30.0030.

Fig. 1: FRET analysis for the interactions between well-known interacting partners p53 and 53BP1 without TimeGate function in MEFs.

Fig. 2: FRET analysis for the interactions between well-known interacting partners p53 and 53BP1 with TimeGate function in MEFs.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5881 Simplified time resolved observation of cardiomyocytes by TimeGate function of hybrid detectors and pulsed white light laser

Baiazitova L.1, Cmiel V.1,2, Pala J.3,4, Provaznik I.1,2
1Department of Biomedical Engineering, Faculty of Electrical Engineering and Communication, Brno University of Technology, CZ, 2International Clinical Research Center - Center of Biomedical Engineering, St. Anne’s University Hospital Brno, CZ, 3Third Medical Faculty, Charles University Prague, CZ, 4Comipa s.r.o. Klecany, CZ
xbaiaz00@stud.feec.vutbr.cz

Enzymatically isolated cardiac cells are widely used in cardiovascular research due to their electromechanical abilities. Adult cardiomyocytes can be used as single unit models in short-term experiments or in primary cultures prepared for long-term experiments. Freshly isolated myocytes do not remain viable for the time needed for long-time experiments. There is often a need to prolong their life and functionality; continual electrical stimulation is often used for such purpose.
In any case, cardiomyocytes viability and functionality is necessary to be monitored. LIVE / DEAD Cell Imaging Kit (Life Technologies) using confocal microscopy supported by lifetime imaging technique described below were used in our research.

Experiments were performed on the confocal laser scanning microscope Leica TCS SP8 X equipped with gateable hybrid (HyD) detectors as internal spectral detectors. The picosecond White Light Laser (WLL) Leica Microsystems freely tunable in the spectral range 470-670 nm with 80 MHz repetition rate was used for excitation. Combination of gateable HyD detectors and WLL enables both spectrally and time resolved study of cardiomyocytes. Time resolved settings of HyD detectors in Photon Counting Mode was adapted by TimeGate function that allows arbitrary setting of time window /band in the range of 0-12 ns with the minimum opening of the time window 3.5 ns.

Nine TimeGate bands with the 1 ns step size and minimum opening of 3.5 ns were measured. Images acquired in photon counting mode were loaded in Matlab and developed image processing algorithm was applied to stack of images. Proposed simplified time resolved observation is not the standard Fluorescence Lifetime Imaging (FLIM) as the time resolution and photon counting is not comparable with the standard FLIM systems. Cardiomyocyte viability may be evaluated in numerous criteria such as fluorescence intensity loss of calcein with related ethidium homodimer fluorescence intensity increase or cell shape changes. Finally, the calculated pseudo coloured time resolved fluorescence images were compared to basic intensity fluorescence images of calcein to find new possibilities in cardiomyocyte viability evaluation

The work was supported by European Regional Development Fund Project FNUSA-ICRC (No. CZ.1.05/1.1.00/02.0123)

Fig. 1: Comparison of flurescence intensity image with resulted pseudo-coloured time resolved image from 0 ns to 11.5 ns. Images are in resolution of 512x512 pixels and 133x133 um in real size.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5882 DNA repair studies in living cells

Bartova E.1
1Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, 612 65, Brno, Czech Republic
bartova@ibp.cz

The maintenance of genome integrity is fundamental for proper cellular functions. Cells are continuously exposed to genotoxic factors, including UV irradiation or oxidative stress induced by pollutants. Therefore, faultless DNA repair is more than demanding for genome stability. Genotoxic stress generally leads to induction of DNA lesions that must be repaired in order to avoid deleterious chromosomal translocations. Therefore, in irradiated chromatin of living cells we analyze kinetics and appearance of proteins involved in DNA repair pathways or proteins recognizing the changes in radiation-caused chromatin conformation. For induction of DNA lesions we are using various sources of radiation, including UVA lasers or gamma-rays. From the view of various types of DNA lesions, we study the cell cycle dependent recruitment of selected proteins at radiation-damaged chromatin. An example represents PCNA protein according to which it is possible to recognize cells in the non-S-phase (Fig. 1A) and the S-phase (Fig. 1B) of the cell cycle. To distinguish G1 and G2 phases of the cell cycle, we are using HeLa-Fucci cellular system expressing RFP-cdt1 in the G1 phase, and GFP-geminin in the G2 phase of the cell cycle. For example, by this experimental approach we showed that coilin, a protein of Cajal bodies, has ability to recognize DNA lesions appearing in both G1 and G2 cell cycle phases. Our aim is also to study protein-protein or protein-DNA interactions and kinetics in locally induced DNA lesions of living cells after intervention to epi-genome.

Work was supported by Grant Agency of the Czech Republic, project Nos: 13-07822S.

Fig. 1: Figure 1. Recruitment of RFP-PCNA (red) at UVA-induced DNA lesions in HeLa cells stably expressing histone H2B tagged by GFP (green). (A) Nuclear pattern of PCNA in non-S-phase of the cell cycle and (B) nuclear pattern of PCNA in S-phase. DNA lesions were induced at selected regions of interest (white frames) by UVA-laser.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5884 Multi-modal superduper chemiluminescent proteins enabling multicolor functional imaging and manipulation

Suzuki K.1, Arai Y.2, Nagai T.2
1Department of Biotechnology, Division of Advanced Science and Biotechnology, Graduate school of Engineering, Osaka University, 2The Institute of Scientific and Industrial Research, Osaka University
ksuzuki40@sanken.osaka-u.ac.jp

Chemiluminescence imaging had shed light on not only bioscience but also medical and pharmaceutical research fields in situations when fluorescence cannot be used, for example non-invasive deep tissue imaging of whole animals in the anesthetized condition. In spite of their potential utility, the universal applications had been precluded by its low brightness. To overcome this problem, we previously developed a bright chemiluminescent protein, Nano-lantern (Saito K. et al. Nat. Commun. 3, 1262, 2012), which enabled us real-time imaging of tumor tissue in freely locomoting mouse. However, the brightness of Nano-lantern is not enough to trace faster biological phenomena such as protein dynamics without compromising the spatial resolution. Moreover, the luminescent color of Nano-lantern has been limited to green. Here we report further development of a superduper chemiluminescent protein, the enhanced Nano-lantern (eNano-lantern) and its color variant (red-eNano-lantern). The eNano-lantern emits approximately 8 times brighter signal than that of Nano-lantern mainly due to the fast substrate turn-over rate. The emission peak of red-eNano-lantern is 585 nm which is relatively transparent in living tissue than green color. With the use of eNano-lantern, we could perform sub-video rate imaging of representative intracellular structures in living cells with high spatial resolution. Furthermore, by engineering the eNano-lantern, we could make a novel Ca2+ indicator which gets brighter up to 170% upon Ca2+ binding. To make maximal use of newly developed palette of these superduper chemiluminescent probes, we would like to demonstrate in the conference not only multiple malignant tumors imaging in an awake mouse but also multifunctional video rate imaging of protein dynamics in conjunction with optogenetic manipulations at single cell level.

This work was supported by a grant for “Development of Systems and Technology for Advanced Measurement and Analysis” from JST.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5955 Termination pattern of nanocrystalline diamond controls cell behavior

Brož A.1, 5, Ukraintsev E.2, Babchenko O.2, Kromka A.2, Rezek B.2, Verdánová M.1, 3, Ostrovská L.1, 4, Sauerová P.4, 5, Hubálek Kalbáčová M.1, 4
1Institute of Inherited Metabolic Disorders, 1st Faculty of Medicine, Charles University in Prague, Ke Karlovu 2, 12853 Prague 2, Czech Republic , 2Institute of Physics, Academy of Sciences of the Czech Republic, v. v. i., Cukrovarnická 10, 16253 Prague 6, Czech Republic , 3Department of Genetics and Microbiology, Faculty of Science, Albertov 6, 128 43, Prague 2, Czech Republic, 4Biomedical Centre, Faculty of Medicine in Pilsen, Charles University in Prague, 134 00 Pilsen, Czech Republic, 5Institute of Physiology AS CR, Vídeňská 1083, 142 20, Prague 4, Czech Republic
abroz@lf1.cuni.cz

Introduction & Materials and Methods
The aim of the study was to investigate the behavior of human osteoblastic cells (SAOS-2) on nanocrystalline diamond (NCD) film with altered wettability during the cultivation period of 48 h with a focus on primary adhesion. The NCD was already proven to be biocompatible material that can be utilized in implantology as a surface coating1 or for biosensor development2. Surface termination and thus wettability of the NCD was achieved by exposition to oxygen (NCD-O) or hydrogen (NCD-H) microwave plasma. Two different surface termination patterns of the NCD were prepared: 1) half of the surface terminated by O and the other half by H, 2) surface with 200 um wide O/H alternating stripes. Differences in cell behavior caused by these various termination patterns were studied by live cell imaging phase contrast microscopy and subsequent image analysis based on image thresholding and cell tracking in NIS Elements software. Using these methods we determined the number of adherent and non-adherent cells and parameters of each cell migration track on the NCD samples with O/H halves and O/H stripes.
Results & Conclusion
The results show that the cell behavior, especially cell adhesion is strongly influenced by termination pattern of the NCD. Cell adhesion takes place earlier on the NCD-O than on the NCD-H. Cells also proliferate more on NCD-O than on NCD-H if they are cultivated on O/H stripes. However, this trend was not observed on NCD with O/H halves. Generally, cells move more on NCD-H compared to NCD-O. These results indicate that the different plasma treatment of NCD which resulted in patterned wettability can be used to control cell behavior.

References:
1 Jaatinen JJ, Korhonen RK, Pelttari A, Helminen HJ, Korhonen H, Lappalainen R, Kröger H. Early bone growth on the surface of titanium implants in rat femur is enhanced by an amorphous diamond coating. Acta Orthop. 2011 Aug; 82(4): 499-503.
2 Izak T, Novotna K, Kopova I, Bacakova L, Rezek B, Kromka A. H-terminated diamond as optically transparent impedance sensor for real-time monitoring of cell growth. Phys Status Solidi B. 2013 Dec; 250(12): 2741-2746.

Acknowledgements:
This work was supported by: Czech Science Foundation - GACR P108/12/0996 and 14-04790S. Charles University in Prague, First Faculty of Medicine - project PRVOUK-P24/LF1/3 and Faculty of Science - project GAUK no.: 501212 and grant SVV-2014-260081 and by the project ED2.1.00/03.0076 from European Regional Development Fund.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-5985 ELECTRON MICROSCOPY AS A TOOL FOR DISSEMINATION, STUDY AND CONSERVATION OF BOTANICAL GARDEN “Xíitbal neek’” FROM CENTRO DE INVESTIGACION CIENTÍFICA DE YUCATÁN A.C. (CICY)

Andrade S.1, Campos G.1, Moreno O.1
1Centro de Investigación Científica de Yucatán, A.C. 130 No.43, Chuburná de Hidalgo, 97200, Mérida, Yucatán, México. sbac@cicy.mx
silvana74@gmail.com

The Centro de Investigación Científica de Yucatán, A. C. (CICY) is a public center of scientific and technological research, which forms human resources and generates knowledge in the various fields of science, from the biological sciences to the area of materials and sustainable energy development. Among the most important aspects of the center is the Regional Xíitbal neek’ Botanical Garden, which was founded in 1983. Its regional aspect refers to the flora of the Yucatan Peninsula as an object of study, and its Mayan name means: the place where the seeds sprout, referring to the ex situ cultivation of wild species from seed. Its collections contribute to the conservation of regional plant resources, either conducting or supporting research on the regional flora, letting know people its importance and value or supporting specific conservation activities. It was declared a Living Museum of Plants by the Secretariat of Environment and Natural Resources and registered as a Management Unit for Wildlife Conservation (key MA-SEMARNAT-0169-JB-YUC-09). At the same time, Laboratory Scanning Electron Microscopy (SEM) has been working since 2004 to support research both within the research center and research centers in the region. The scanning electron microscope has been particularly important and has contributed to the imaging and EDS microanalyses for comparative studies of plant tissues; systematic and phylogenetic studies in plants (pollen, trichomes, waxes, seed); morphology of tissues infested by fungi, morphological characterization of the development of many plant species and taxonomic studies and microorganisms (phycoflora, yeast); anatomy of wood; metal content in plant tissues, microanalysis from archaeo-paleontological samples. Besides the valuable information for scientific study carried out both at the Centre for Research and for the country, the microscope is a strong diffusion tool, because through image, species of tiny dimensions imperceptible to the human eye can be observed. Throughout Multidisciplinary we can converge and disclose to children, youth and adults the importance of scientific progress. Gathering this tool (the microscope) with today’s natural wealth within the botanical gardens, in order to preserve our natural resources. The pictures from the summary belongs to the representative vegetation of the Regional Botanic Gardens. The image was obtained using a Scanning Electron Microscope JEOL 6360LV, Laboratory of Scanning Electron Microscopy CICY.

Fig. 1: Yucatan´s Scorpions wich are members of the class Arachnida and are closely related to spiders, mites, and ticks.

Fig. 2: Morphology  details of Acalypha Hispida (Euphorbiaceae) flower in Yucatán, México.

Fig. 3: Morphology of Artemisia (Astaraceae)  pollen grain in Yucatán, México.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-1-P-6076 The importance of Rapamycine usage on CD 44 and RHAMM expressions on breast cancer cell lines

Inan S.1, Temel M.1, Onal T.1, Oztatlici M.2, Cam F. S.2, Turkoz Uluer E.1, Ozbilgin K.1
1Celal Bayar University, Faculty of Medicine, Dept. of Histology&Embryology, Manisa, Turkey, 2Celal Bayar University, Faculty of Medicine, Dept. of Medical Genetıcs, Manisa, Turkey
sevincinan@yahoo.com

CD44 is a member of superfamily hyaluronan (HA) binding proteins (HABPs) that play a role in cell adhesion, migration, invasion and survival. CD44 and Receptor for HA-mediated motility (RHAMM) are increased during tissue repair and carcinogenesis. Mammalian target of Rapamycin (mTOR) is a serine/threonine protein kinase which belongs to the phosphatidylinositol 3-kinase (PI3K) family. Rapamycine is a macrocyclic antibiotic, has been known to inhibit mTOR by destabilizing the mTOR-Raptor complex. The aim of this study was to examine the effects of Rapamycine on the distributions of CD44 and RHAMM expressions, using indirect immunohistochemistry and RT-PCR methods on non-invasive MCF-7 and invasive MDA-MB 231 breast cancer cell lines.

MCF-7 and MDA-MB231 cells were cultured in RPMI-1640 medium; containing 10% fetal bovine serum, 1% L-glutamine and 1% antibiotic solution in a humidity incubator at 37°C, containing 5% CO2. Cells were grown on cover slips and incubated with Rapamycine for 24 and 48 hours. Cells were immunostained with anti CD44 and anti-RHAMM primary antibodies using avidin-biotin-peroxidase method. Staining intensities were measured by using semi-quantitative method and ANOVA statistical test, to compare the results. The total RNA was extracted using EruX Universal RNA Purification Kit according to manufacturer’s instructions from MCF-7 and MDA-MB-231 cell lines. The cDNA synthesis from total RNA was performed using EurX dART RT-PCR Kit and RT-PCR process from obtained cDNAs was caried out using the Solis BioDyne qPCR Mix Plus.

It was observed that MCF-7 and MDA-MB 231 cells had strong CD44 and RHAMM immunostainings on their cell surfaces and in the cytoplasms, especially in mitotic cells (Figure 1-4). Decreased RHAMM immunoreactivity was detected on MCF-7 and MDA-MB 231 cells in Rapamycine treated groups while CD44 immunoreactivty was detected as decreased on only MDA-MB 231 cells. Compared the gene expression profile of CD44 and RHAMM genes, gene expressions were detected as relatively increased in MCF-7 cells than MDA-MB 231 cells. Decreased RHAMM expression was detected both MCF-7 and MDA-MB 231 cells, while CD44 expression was decreased only in MDA-MB 231 cells in treatment with Rapamycine in RT-PCR method.

CD44 and RHAMM suggested novel prognostic markers for breast cancers and hyaladherins could be used as a target for cancer therapy. Rapamycin is the most well studied mTOR inhibitor and this might be effective on invasive breast cancer treatments in addition to chemotherapy and/or radiotherapy.

References :

1 Zhao Y, Zhang T, Duan S, Davies NM, Forrest ML. Nanomedicine: Nanotechnology, Biology and Medicine. 2014

2. Nyga A, Cheema U, Loizidou M. Journal of Cell Communication and Signaling. 2011;5:3,239

The material used in this project was funded by Scientific Research Committe of Celal Bayar University (Project Number: 2010-098)

Fig. 1: Immunoreactivity of CD 44 in MCF-7 cells

Fig. 2: Immunoreactivity of RHAMM in MCF-7 cells

Fig. 3: Immunoreactivity of CD 44 in MDA-MB-231 cells.

Fig. 4: Immunoreactivity of RHAMM in MDA-MB-231 cells.
↑ Go to the Table of content ↑

LS-2. Structure and function of cells and organelles

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-2-IN-2259 Visualizizing gene expression in living cells

Tantale K.1, Müller F.2, Zimmer C.2, Basyuk E.1, Bertrand E.1
1IGMM-CNRS UMR 5535; 1919, route de Mende; 34293 Montpellier Cedex 5; France, 2Institut Pasteur; 28, rue du Docteur Roux; 75015 Paris; France.
edouard.bertrand@igmm.cnrs.fr

Gene expression is a fundamental process in all organisms, and we have a great deal of information about this process at the biochemical and structural level. However, we still have a poor understanding of transcription at the cellular level. A recent and fascinating discovery about gene expression relates to stochastic noise : transcription is not constant but fluctuates randomly over time, even for genes at steady-state. Different cells of a clonal population will therefore express different amount of any given mRNA, and this is very important as it creates phenotypic variability. However, because of the lack of appropriate technologies, these phenomena are not well understood.

Measuring transcription at the level of single cells requires tools able to detect and quantify single molecules of mRNAs, both in live and fixed cells. Single molecule FISH and MS2-tagging of RNAs provide such powerful tools. Building on this, we have developped microscopy approaches meeting these single molecule requirements. Thus, it is now possible to label specific mRNAs in live or fixed cells and to detect every molecule with a high spatial and temporal resolution. By measuring in real-time the amount of nascent RNAs at the transcription site of the gene of interest, it is possible to follow the stochastic variations of the corresponding promoter and to measure activity of RNA polymerase II in real-time. This data provide important insights into the mechanisms of transcription and gene expression.

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-2-IN-2643 The junctional epithelium keratinocyte, a unique undifferentiated epithelial cell

Nanci A.1, 2, Wazen R. M.1, Moffatt P.3
1Faculty of Dentistry, Université de Montréal, Montreal, QC, Canada, 2Faculty of medicine, Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, QC, Canada, 3Shriners Hospital for Children, McGill University, Human Genetics, Montreal, QC, Canada
antonio.nanci@umontreal.ca

The tooth is the only structure in the body that breaches its integrity at the level of the oral mucosa. To palliate to this weak link in the body, the oral mucosa has adapted by developing a specialized structure called the junctional epithelium (JE) which forms an adhesive ‘collar’ around the tooth that essentially seals off periodontal tissues from the oral environment. Integrity of the JE is thus essential for maintaining a healthy periodontium and preventing the spread of bacterial infection. Disease sets in when the structure of the JE starts to fail. The JE is considered as an incompletely differentiated epithelium that produces components for tooth attachment instead of progressing along a keratinization pathway. It is a stratified squamous non-keratinizing epithelium whose cells derive from basal cells situated away from the tooth surface. The basal cells rest on a typical basal lamina (BL), which interfaces with dermal connective tissue. Suprabasal cells all have similar appearance, and quite remarkably maintain the ability to undergo cell division. The JE turns over quite rapidly, at least in some species. The most superficial cell layer of the JE provides the actual attachment of the gingiva to the tooth surface by means of a structural complex called the epithelial attachment. This complex consists of an atypical BL formed and maintained by the flattened superficial cells. This BL adheres to the mineralized tooth surface rather than connective tissue in a yet unknown manner and the JE cells attach to it by hemidesmosomes. Characterization of the structural components of the atypical BL of the JE has been particularly challenging. However, it is now widely accepted that it contains laminin-332 but not γ1 chain laminins, type IV and VII collagens, setting it apart functionally and compositionally from typical BLs binding to connective tissues. In this invited presentation, I will review the structure of the JE, present novel constituents of the BL that may mediate its adhesion to mineral, and discuss regeneration of the JE. In particular, I will focus on a protein called odontogenic ameloblast-associated (ODAM). This unique protein with no known homology is normally expressed only by the JE but is highly upregulated during neoplastic transformation of epithelial cells. We have thus hypothesized that ODAM is a matricellular protein that participates both in structuring of the atypical BL and in modulating the cell status of JE keratinocytes and transformed epithelial cells.

Canadian Institute of Health Research, Natural Sciences and Engineering Research Council of Canada, Network for Oral and Bone Health Research, Shriners of North America.

Fig. 1: (A) Scanning electron micrograph of a section showing the various components of the junctional epithelium (JE). (B) Electron micrograph of a region similar to the boxed area. Immunoperoxidase (C) and immunogold (D) labeling illutrating the expression of ODAM by the JE and its localization among cells of the JE and in the basal lamina.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-2-O-1549 Simultaneous Imaging of Cryo-Bright Field, Dark Field STEM and SEM Using Unroofed Living Cells .

Usukura J.1, Minakata S.1
1 EcoTopia Science Institute, Nagoya University, Nagoya, Japan 1
usukuraj@esi.nagoya-u.ac.jp

Cryo-electron microscopy has been used so far for single particle analysis of purified proteins. Recently, technical improvements on rapid freezing and cryo-microtome permitted structural analysis of organelle or macromolecules in cells using vitreous cryo-sections. However, cryo-sections show quite low contrast due to few electron scattering. They complicate a finding and a focusing of objects. In order to overcome those difficulties, we are improving unroofing techniques for preparation of cryo-electron microscopy of cells together with development of SEM based cryo-STEM. We succeeded simultaneous imaging of cryo-dark field and bright field STEM and secondary electron with extremely high contrast and accuracy.

Materials and Methods: HeLa or ECV cells were cultured on the molybdenum mesh grid covered with carbon coated polyvinyl Formvar. Cultured cells were washed once with HEPE Ringer’s solution and placed in KHMgE buffer for unroofing by weak sonication. Plunging them into liquid ethane using Leica EM GP quickly froze unroofed cells. Frozen samples were then brought into SU9000 SEM (Hitachi High-Technologies Co.) by using newly developed cryo-transfer holder.

Results and Discussion: Simultaneous imaging of cryo-dark field and bright field STEM and secondary electron was very useful for evaluating sample condition and judging the irradiation damages. Sometimes mesh grids became frosted during observation because of inadequate absorbing water vapor by anti-contamination trap. Comparison the STEM with SEM images elucidated obviously that such frosting occurred always underside of the grid.　SEM also was capable of visualizing how much the sample received irradiation damages while observing. The unroofing technique was developed originally for describing membrane undercoat in freeze-etching replica; it was so useful that we could apply it to atomic force microscopic (AFM) imaging. We improved and extended this technique further for cryo-electron microscopy. Soluble proteins in cytoplasm flew out on unroofing, though cytoskeleton and structural proteins are remained in situ. Expectedly, actin filaments and microtubules were found on the cytoplasmic surface of cell membrane with extremely high contrast. In the cortical area, the filaments often extended in parallel, as shown in Fig.1. In other regions, however, fine filaments extended in various directions on the membrane, while aggregating and dispersing at several points, which eventually divided the membrane surface into several domains (Fig. 2). The images obtained from samples prepared in this way must therefore be comparable between different observation methods. Confirmation of results by two different observation methods is very important for determining real structures.

This study was supported by the grant, “Development of Systems and Technologies for Advanced Measurement and Analysis” Program from the Japan Science and Technology Agency (JST).

Fig. 1: Simultaneous imaging of cryo-bright field (A), dark field (B) and SE (C). This sample received slightly irradiation damage, because SE image is not flat.

Fig. 2: Cryo-bright field STEM image showing actin filaments network of membrane undercoat.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-2-O-1870 Interplay between endoplasmic reticulum sheets and dynamic actin filament arrays revealed by multi-scale microscopy

Joensuu M.1, Belevich I.1, Vihinen H.1, Jokitalo E.1
1Institute of Biotechnology, University of Helsinki, Finland
eija.jokitalo@helsinki.fi

The endoplasmic reticulum (ER) comprises an elaborate 3D network of diverse structural subdomains where functions are distributed according to their specific requirements. The tubule-associated ER functions include interactions with several other cell organelles 1,2,3. Based on the distribution of ER sheet-bound ribosomes4 and the direct correlation between RER abundance and secretion activity of the cell5, protein synthesis, folding and quality control of proteins are functions assigned especially for ER sheets. Proper ER operation requires an intricate balance within and in-between dynamics, morphology, and functions, but how these processes are coupled in cells has remained unclear.
Using multi-scale microscopy comprising of live cell imaging, thin section TEM and two 3D-EM methods, electron tomography and serial block face imaging, we characterized the interplay between ER and the actin cytoskeleton in mammalian cells6. First, we showed that an array of dynamic actin filaments localize in the polygons defined by the surrounding ER structures. Depolymerization of these actin filament arrays led to increased sheet fluctuation and transformations resulting in small and less abundant sheet remnants and a defective ER network distribution. We propose that these filaments have a role in maintaining the sheet-tubule balance and sheet structure by regulating ER sheet remodeling events. Second, we performed a screen where over 200 known functional actin-binding proteins were depleted one-by-one and analyzed by LM to identify those that would have a role on ER morphology and/or dynamics. Based on the screen, we identified the unconventional motor protein myosin 1c (myo1c) localizing to the ER associated actin filament arrays, and revealed a novel role for myo1c in regulating these actin structures. Myo1c depletion and dominant-negative expression of mutated form with abolished actin-binding domain led to loss of the actin filament arrays and to subsequent loss of ER sheets similarly as observed after actin depolymerization using drugs. We propose that ER -associated actin filaments have a role in ER sheet persistence regulation, and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network. In addition to tubular ER associations with microtubules, interactions with the actin cytoskeleton are essential to create and maintain the ER sheet persistence and the characteristic architecture of the ER network in mammalian cells.
1Friedman and Voeltz, Trends Cell Biol. 21 (2011), 709-
2Ylä-Anttila et al., Autophagy 5 (2009),1180-
3Kornmann, Curr.Opin.Cell Biol. 25 (2013), 443-
4Puhka et al., Mol.Biol.Cell 23 (2012), 2424-
5Wiest et al., J.Cell Biol. 110 (1990), 1501-
6Joensuu et al., Mol.Biol.Cell (2014), Epub ahead of print

We thank Mervi Lindman and Antti Salminen for technical assistance. This work was supported by the Academy of Finland (project 131650 to E.J.) and Biocenter Finland. M.J. is a student of the Integrative Life Science doctoral program, University of Helsinki.

Fig. 1: Microtubules and actin filaments can be found in close proximity to ER. High-pressure-frozen, freeze-substituted Huh-7 cells were subjected to electron tomography. From the resulting tomogram, microtubules (blue), actin filaments (red), and ER(yellow) comprising tubules and highly fenestrated sheets were modelled.

Fig. 2: Time-lapse confocal frames (A) show that the relocation and disappearance of actin arrays (magenta) and ER sheet (green) dynamics are interdependent. ER models (yellow) from control (B) and myo1c-depleted cell (C) reveal consumption of abundant, large and fenestrated sheets (B) into unevenly distributed network of tubules and sheet remnants (C).

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-2-O-3517 Autophagic hallmarks and gene expression accompany programmed cell death in stress-induced pollen embryogenesis and pollen development

Solís M. T.1, Bárány I.1, Cano V.1, Risueño M. C.1, Testillano P. S.1
1Pollen Biotechnology of Crop Plants group. Centro de Investigaciones Biológicas (CIB) CSIC. Ramiro de Maeztu 9, 28040 Madrid, Spain.
pilartestillano@gmail.com

The microspore or immature pollen grain follows in vivo the gametophytic program and differentiates to form the mature pollen. At defined stages, the tapetum, anther tissue with a key nursing role during microspore development, undergoes programmed cell death (PCD). In vitro, upon the application of a stress treatment the microspore can be deviated towards embryogenesis leading to plant regeneration, the so-called microspore embryogenesis, a process which represents an important tool in plant breeding to obtain double-haploid plants; <span> the efficiency of the process is affected by cell death of microspores after the stress. Plant programmed cell death (PCD) seems to share some features with both apoptosis and autophagy in animals; nevertheless, recent data on PCD in plants indicate that classification is not clear yet. Increasing evidences indicate that autophagy plays critical roles in both PCD processes, during development and stress responses.

In this work we studied the existence and dynamics of autophagy compartments, machinery and genes, specifically Beclin1/ATG6 and ATG8, in relation to ROS/RNS production, caspase-like activity and ultrastructural rearrangements during two PCD processes of the pollen pathways,: the PCD of microspores in culture after the stress treatment inducing embryogenesis, and the PCD of tapetum during pollen development, in Brassica napus and Hordeum vulgare, by a multidisciplinary approach.

Early after microspore embryogenesis induction by stress treatments, cell death increased together with ROS and NO production, caspase 3-like activity and Beclin1/ATG6 gene expression induction. During pollen development, in tapetal cells at PCD initiation, apoptotic-like features as nuclear condensation, cytochrome C release and high caspase 3-like activity were detected concomitantly with an increase of vesicles, vacuoles and different autophagic-like structures in the cytoplasms, some of them were labelled by ATG8.

Results will be discussed on the light of the participation of autophagy in the PCD during the two pollen developmental pathways.

Rodríguez-Serrano M, Bárány I, Prem D, Coronado MJ, Risueño MC, Testillano PS (2012) NO, ROS and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley. J. Exp. Botany, 63, 2007-2024.

Solís MT, Chakrabarti N, Corredor E, Cortés-Eslava J, Rodríguez-Serrano M, Biggiogera M, Risueño MC, Testillano PS (2014) Epigenetic changes accompany developmental programmed cell death in tapetum cells. Plant Cell Physiol. 55, 16-29.

Work supported by projects funded by Spanish MINECO (BFU2011-23752) and CSIC (PIE 201020E038).

Fig. 1: PCD and autophagic features in microspores and tapetum. A: Dead microspores (arrows) stained by Evans blue after stress to induce embryogenesis. B: Anther section with tapetum (Tap) in PCD. C: Electron micrograph with autophagic structures (arrow) in tapetum during PCD. Bars A, B: 20µm, C: 100nm.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-2-O-3522 Phosphoinositide islets in the cell nucleus: a novel nuclear compartment?

Sobol M.1, Filimonenko V.1, Šmíd O.1, Yildirim S.1, Kalendová A.1, Hozák P.1
1Institute of Molecular Genetics, Prague, Czech Republic
sobol@img.cas.cz

The eukaryotic nucleus is recognized as a highly organized and well-orchestrated organelle, where nuclear architecture plays a role in gene regulatory networks, epigenetic regulation of gene expression, and RNA processing events. So far, mostly protein complexes have been considered as important in the three-dimensional nuclear organization. We introduce novel structures containing phosphatidylinositol 4,5-bisphosphate (PIP2) which seem to be of importance as well. It was previously described that PIP2 is present in interchromatin granule clusters (IGC). Recently, we published data on PIP2 involvement in nucleolar organization and transcription of genes coding rRNA. Based on this, we mapped PIP2-containing structures using pre-embedding immunolabeling and 3D electron tomography also in nucleoplasm. In addition to PIP2 detected in IGC and in the nucleolus (Figure 1a), PIP2-positive structures are present in nucleoplasm as 70-100 nm roundish “phosphoinositide islets” (Figure 1a, b). Using electron energy-loss microscopy and super-resolution light microscopy (SIM, STED), we demonstrated that they are composed mostly of phosphoinositides, and they are surrounded by chromatin. To explore the possible functions of the PIP2-containing islets, we mapped relative localization of PIP2 with a wide range of nuclear proteins involved in transcription, splicing, and chromatin organization using advanced immunogold electron microscopy and SIM. We demonstrate that at the periphery of the islets, PIP2 co-localizes or is located in close proximity to nascent transcripts as well as to the proteins engaged in Pol II transcription, mRNA splicing, and organization of chromatin. Direct binding, gradient fractionation, and mobility assays also showed nucleoplasmic interactions between PIP2 and proteins engaged in the chromatin remodelling and trancsription by Pol I and Pol II. Further, we revealed recruitment of some nuclear skeletal proteins into complexes with PIP2 using pull-down and 2D-electrophoresis assays. Taken together, our data allow us to suggest that newly observed PIP2-containing islets might play an important role in chromatin organization and nuclear architecture, thus participating in regulating gene transcription.

This work was supported by the GACR (P305/11/2232); the MSMT CR (LD12063); the TACR (TE01020118); the project „BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University“ (CZ.1.05/1.1.00/02.0109) from the European Regional Development Fund; the IMG (RVO68378050).

Fig. 1: PIP2-containing structures in the cell nucleus. a, statistical mapping of PIP2 distribution in the nucleus of HeLa cell (N – nucleoplasm, NL – nucleolus, ICG – interchromatin granules clusters, Cyt - cytoplasm); b, PIP2-islets in the nucleoplasm of a HeLa cell visualized by immunogold labeling using anti-PIP2 antibody.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1399 Immnue-LCM analysis of M1/M2 macrophages in engineered dental pulp tissues

Kaneko T.1, Yamanaka Y.1, Ito T.1, Okiji T.1
1Division of Cariology, Operative Dentistry and Endodontics, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
tomoendo@dent.niigata-u.ac.jp

We have recently reported that macrophages are generated in dental pulp-like tissues engineered by culturing stem cells from human exfoliated deciduous teeth (SHED) in a scaffold placed in the canal space of human tooth slice. We hypothesized that these macrophages are activated to M1 and/or M2 directions, and participate in absorbing the scaffold by their phagocytic ability. Thus, to examine the types of activated macrophages in the engineered dental pulp-like tissue, we performed immune-laser capture microdissection (LCM) of CD68 (anti-human macrophages)-immunoreactive cells. The CD68-immunoreactive cells in the pulp-like tissues that had been fixed with formaldehyde, demineralized with 10% formic acid, and embedded in paraffin were retrieved by LCM and analyzed for the mRNA expression of M1 and M2 macrophage markers with real-time PCR. Results demonstrated that the expression level of CCL18, an M2 macrophage marker, was significantly higher in CD68-immunoreactive cells retrieved from the area where most of the scaffold was absorbed, as compared with CD68-immunoreactive cells in the scaffold-remaining area. On the other hand, the mRNA expression level of inducible nitric oxide synthase (iNOS), an M1 macrophage marker, was significantly decreased in the scaffold-remaining area. These results suggested that macrophages activated to the M2 direction are preferentially distributed in the scaffold absorption area of the engineered dental pulp-like tissues. The immune-LCM method presented here allows for the quantitative analysis of gene expression in paraffin-embedded tissue sections from specimens that have been demineralized. This immune-LCM method constitutes a new approach for histochemistry of mineralized tissues such as bone and teeth, opens the door for the acquisition of new data from archived specimens, and is suited for the analysis of relatively rare cell types within a tissue.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1393 Scanning electron microscopic studies on sertoli cell of dog (Canis lupus familiaris)

Choudhary O. P.1, Dhote B. S.2, Singh I.3, Bharti S. K.4, Sathapathy S.5, Mrigesh M.6, Singh G. K.7
1Department of Veterinary Anatomy, College of Veterinary and Animal Science, G.B. Pant University of Agriculture and Technology, Pantnagar-263 145, India
dr.om.choudhary@gmail.com

The Sertoli cells, which nurture maturing germ cells, play an important role in the process of spermatogenesis. Scanning electron microscopic configuration was never accurately described, probably because of its complicated morphological features as well as its close attachment to germ cells.
The study was carried out in 10 dog’s testes samples collected from Small Animal Operation Theater, Department of Veterinary Surgery, College of Veterinary and Animal Sciences, G.B.P.U.A. & T., Pantnagar. The testes from sexually mature dogs were fixed in 2.5% glutaraldehyde with 0.1 M phosphate buffer, after perfusing with the same fixative through the testicular artery. They were cut into smaller pieces to be fixed in 2.5% glutaraldehyde overnight. The materials were than washed in 0.1 M phosphate buffer for 10 minutes at 4°C and rinsed in 8N hydrochloric acid (HCL) at 60°C for 15-20 minutes. The specimens, washed repeatedly in Hanks' balanced salt solution (HBSS) for about 10 minutes. They were postfixed in 1% osmium tetroxide, dehydrated with graded ethanol (30%, 50 %, 70%, 90% and 100%) and dried with liquid CO2 at the critical point for 60-90 minutes by using EMITECH K850. Finally, they were coated with gold by using JEOL JFC-1600 auto fine coater and observed under a JEOL JSM-6610LV scanning electron microscope.
In present study the basal portion of the seminiferous epithelium, spermatogonia and/or spermatocytes were located in compartments enclosed by adjacent Sertoli cells. From the basal aspect, they were situated in successive recesses. In the middle portion, early round spermatids halfway embedded in the Sertoli cell were recognized. The exposed surfaces of these spermatids were wrapped with ramifying processes which were derived from the Sertoli cell. In the apical portion, only the heads of the maturing spermatids invaded the Sertoli cell. As the spermatid matured, the apical Sertoli process varied in range to finally release the spermatid head. It is probably that the maturing spermatids gradually leave the apical Sertoli process and ultimately segregate themselves from the seminiferous epithelium. The average diameter of seminiferous tubule and sertoli cell was 70.893±0.4106μm and 6.1566±0.0509μm respectively.
Keywords: Sertoli cell, dog, spermatogonia, seminiferous tubule, SEM.

The authors are grateful to the Incharge and Technician, Electron Microscopic Laboratory,Pantnagar for providing facilities and support for carrying out this research.

Fig. 1: Showing Basal portion of the seminiferous epithelium from the basal aspect. Some spermatogonia are situated in successive recesses. These recesses consist of continuous Sertoli cells. BM basement membrane, (Sp) Spermatids in lumen

Fig. 2: Showing mature spermatozoa (Sp) produced from sertoli cell (Sc) of dog
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1412 PIP2: A new key player in the transcription of ribosomal genes

Yildirim S.1,2, Castano E.1,3, Dzijak R.1, Philimonenko V. V.1, Sobol M.1, Venit T.1, Hozák P.1
1Institute of Molecular Genetics ASCR v.v.i. Department of Biology of the Cell Nucleus, Vídenská 1083, 142 20, Prague 4, Czech Republic., 2The Hospital for Sick Children, Peter Gilgan Centre for Research and Learning, 686 Bay Street, M5G0A4 Toronto, Ontario, Canada., 3Biochemistry and Molecular Plant Biology Department, CICY. Calle 43, No.130, Colonia Chuburná de Hidalgo C.P. 97200, Mérida, Yucatán, México
sukriye.yildirim@sickkids.ca

Nucleolus is the most prominent structure within the cell nucleus. It is formed around the nucleolar organizing regions (NORs) which contain the 5.8S, 18S, and 28S rRNA genes in tandem repeats. Nucleolus has three well defined subcompartments: fibrillar centers (FCs), dense fibrillar component (DFC), and granular component (GC). While transcription of pre-ribosomal genes by RNA polymerase I (Pol I) takes place at the FC/DFC border, assembly of the ribosomal subunits into ribosomes takes place in the GC.

We showed that phosphatidylinositol 4,5-bisphosphate (PIP2) is localized at the transcriptionally active sites of nucleoli, namely FC and DFC. PIP2 directly interacts with transcription initiation factor UBF which facilitates the open chromatin structure by displacing the histone H1 and makes a complex with Pol I in the nucleolus. We found that PIP2 is synthesized and present on the active ribosomal promoter during the transcription, and PIP2 depletion reduces Pol I transcription which can be rescued by the addition of exogenous PIP2, but not by its precursor (phosphatidylinositol 4-phosphate) and hydrolysis products (inositol 1,4,5-trisphosphate and diacylglycerol). In addition, PIP2 also binds directly to the pre-rRNA processing factor fibrillarin, in a transcription-dependent manner. PIP2 binding to UBF and fibrillarin causes conformational changes in these proteins that are important for their binding to nucleic acids.

Based on these findings, we suggest a model, in which PIP2 may act as a bridge between Pol I, UBF and fibrillarin to connect transcription initiation and early maturation steps. The link between RNA synthesis and maturation may dictate nucleolar structures in the FC and DFC regions, where PIP2 may form a framework which allows gathering of proteins to work in concert for efficient transcription of ribosomal RNA genes.

This work was supported by the GACR [P305/11/2232], MSMT [LC545, LC06063], the institutional grant [RVO68378050] and the student program of the GACR [204/09/H084 to S.Y.].

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1444 Membrane surface negative charge characteristic of leukemic cells

Marikovsky Y.1
1Dept. Biological Chemistry Weizmann Institute of Science Rehovot, Israel
yehuda.marikovsky@weizmann.ac.il

During cell transformation, changes occur in the macromolecular architecture on cell membrane surfaces, mainly involving carbohydrate-containing components of the peripheral cell coat. One of the cell surface changes responsible for the different behavior of transformed cells appeared to be the surface anionic sites. The surface topography of negatively charged membrane sites is visualized and evaluated by electron microscopic (EM) observation with cationic ferritin (CF) particles. CF, a polyvalent ligand induces a regrouping-relocation of CF-specific anionic sites and induces a formation of clusters and patches of CF particle binding sites on the surface of leukemic cells and not on surfaces of normal lymphocytes. This probably is due to a more fluid lipid layer in the surface of leukemic cells than on surfaces of normal lymphocytes. The difference in the CF particle pattern as observed in EM between membrane cell surfaces of leukemic cells and those of normal lymphocytes appears to be a characteristic exhibiting an alteration in structural composition and membrane behavior of transformed cells different of that of normal cells. This characteristic has been shown also in a variety of leukemic cell lines and various transformed malignant cell lines. This findings were fully reported at an earlier publication.                                                                                                                                                                                                                                                                                                                             

 

Thanks to Prof.Yechiel Shai for his continuous support of my research activity.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1446 The ultrastructure and biochemical composition of the notochordal sheath in dogfish Scyliorhinus canicula L.

Dujmović I.1, Vukojević K.2, Saraga Babić M.2, Bočina I.3
11 Faculty of Philosophy, University of Split, Teslina 12, 21 000 Split, 22 School of Medicine, University of Split, Šoltanska 2, 21 000 Split, 33 Faculty of Science, University of Split, Teslina 12, 21 000 Split
idujmovic@ffst.hr

The peculiar structure of the notochordal sheath and its biochemical composition in dogfish Scyliorinhus canicula L. were studied using transmission electron microscopy, histochemical and immunohistochemical techniques. The notochordal sheath surrounds notochord and it is the product of the notochordal cells. The notochord defines all Chordates, and plays an important role in the vertebrate development. It is the main skeletal element of an embryo and it induces the formation of the surrounding tissue during early embryogenesis. In dogfish Scyliorhinus canicula L., the notochordal sheath separates the cartilaginous vertrebra from the underlying notochordal cells (Fig.1A) and it is a layer made of collagen fibrils (Fig. 1B). The part of the sheath close to the marginal notochordal cells, consists of electron dense material (Fig. 1A and 1B) presumed to be the elastic fibers which was then confirmed by using Verhoeff's staining (Fig. 2A). This structure is known as elastic membrane in notochordal sheath of some other vertebrates. Some membrane pores could be seen all along the elastic membrane (Fig. 1A and 1B). The elastic membrane is more resistant to degenerating processes than notochordal sheath itself (Fig. 1C). The outer part of the sheath, close to the cartilaginous matrix, contains collagen type 1, which was confirmed by using antibody to collagen type 1 (Fig. 2B). Contrary, the hyaline cartilage in the vertebra wasn't labelled by antibody to collagen type 1. The inner part of the notochordal sheath was also positive to antibody to intermediate filament vimentin when using the immunofluorescence technique (Fig. 2C).

We thank Mrs. Asja Miletić for her skilful technical assistance and Department of Pathology at Clinical Hospital Dubrava, Zagreb for using their transmission electron microscope.

Fig. 1: (A) Notochordal sheath (ns), elastic membrane (arrow), pores (arrowheads), notochordal cells (nc), vacuoles (v). Scale bar: 5 µm. (B) Collagen fibrils (arrow), elastic membrane (em), notochordal cells (nc), vacuoles (v). Scale bar: 1 µm. (C) Degenerating sheath (arrow) with conserved elastic membrane (arrowhead). Scale bar: 10 µm.

Fig. 2: (A) Collagen fibrils (arrowhead) and elastic membrane (arrow). Verhoeff staining. Scale bar: 25 µm. (B) Collagen type 1 (arrowhead), centrum of the vertebra(cv), elastic membrane (arrow). DAB. Scale bar: 50 µm. (C) Positive staining to vimentin (arrow), notochord (n), centrum of the vertebra (cv). Alexa fluor. Scale bar: 25 µm.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1471 Localization and Distribution of Ghrelin in the Gastrointestinal Tract of the Golden Apple Snail, Pomacea canaliculata

Ngernsoungnern P.1, Ngernsoungnern A.1
1School of Anatomy, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
npiyada@sut.ac.th

Ghrelin, a 28 amino acid peptide, is an endogenous ligand for the growth hormone secretagogue receptor. Ghrelin was firstly isolated from the rat stomach. However, ghrelin has later been identified in the gastrointestinal tract of many species. In the present study, the localization and distribution of ghrelin were investigated in the gastrointestinal tract of the golden apple snail, Pomacea canaliculata, by immunohistochemistry technique using antibody that raised against the N-terminus of ghrelin of human origin. In addition, levels of ghrelin in segments of the gastrointestinal tract were measured using enzyme-linked immunosorbent assay (ELISA). Immunoreactivity of ghrelin was identified in the entire segments of the gastrointestinal tract. The immunoreactive cells were localized in the mucosal layer of the esophagus, stomach, intestine, and rectum. In the esophagus, the immunoreactivity was observed in the apical region of the epithelial cells. However, the immunoreactivity was found at the basal region of the epithelial cells lining the stomach, intestine, and rectum. It was found that ghrelin level was highest in the esophagus, but was in the lowest level in the rectum. The levels were 74.14 ± 3.56, 59.21 ± 4.22, 26.13 ± 2.76, and 10.87 ± 3.62 ng/ml in the esophagus, intestine, stomach, and rectum, respectively. These results suggest that ghrelin may participate in the digestive function of this species. Moreover, it seems that ghrelin has a high degree of preservation during evolution from invertebrates to vertebrates.

This work was financially supported by the Thailand Research Fund, the Commission on Higher Education, Ministry of Education, and Suranaree University of Technology.

Fig. 1: Localization of ghrelin in the epithelial cells lining the intestine of the golden apple snail.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1474 Observations on the ultrastructure of endocrine cells in axolotl testis

Keyhani E.1
1Laboratory for Life Sciences, 19979 Tehran, Iran
keyhanius2002@yahoo.com

Axolotls have become interesting tools for various studies such as limb regeneration where programmed cell death triggers regenerative responses in many different organisms [1, 2]. Cardiac mutant axolotls with heartbeat failure due to heart cells’ inability to form organized myofibrils are invaluable for heart disease research [3]. More recently, axolotl eggs were found instrumental in the fight against cancer, and, furthermore, axolotls accepting brain transplant restore function of that brain part. These and other recent investigations show the potential value of axolotl for human health research and thus warrant further investigations on structure-function relationship in various axolotl cells, tissues, and organs. Here we report the ultrastructure of endocrine cells in testes of the axolotl Ambystoma mexicanum.

Five male adult axolotls (~ 2 years old) were killed after anesthesia in tricaine methanesulphonate. Testes were removed, diced, doubly fixed with glutaraldehyde-osmium and embedded in epon. Sections double stained with uranyl acetate and lead citrate, were examined in Philips EM 300 electron microscope, at accelerating voltage of 60 kV with a 25 μm objective aperture.

Fig. 1 shows, at low magnification, the general structure of an endocrine testis cell cut longitudinally. The nucleus exhibited a highly irregular and elongated shape, and well visible perinuclear chromatin. In the cytoplasm, numerous mitochondria (average diameter 0.4 to 1.2 μm) and lipophilic granules (liposomes) were seen. Liposomes varied in aspect with respect to lipophilic content: there were empty and translucent liposomes of average diameter 0.35 to 0.7 μm (Fig. 1), liposomes with a central lipophilic granule, translucent periphery, and of average diameter 0.45 to 1 μm (Fig. 1), and lipid-filled liposomes of average diameter 0.3 to 0.9 μm, occasionally fused together (Fig. 2A). Still in the cytoplasm, smooth endoplasmic reticulum (SER), either sparse or abundant (Fig. 2B), sometimes formed a three-dimensional mesh of 30-90 nm thick anastomosing tubules (Fig. 2C).

Mitochondria exhibited vesicles (average diameter 70-100 nm) and tubules that were projected by the inner membrane into the matrix. Tubules were about 15-20 nm in inner diameter, with discrete enlargements where the diameter reached up to 35 nm (Fig. 3A and insets). Fig. 3B (and inset) showed mitochondria cross-sections with tubular structures 20-35 nm in diameter. Such mitochondria with different types of tubules, together with abundant SER, are characteristic of cells producing steroid hormones.

References

[1] King, R.S. & Newmark, P.A. (2012) J Cell Biol. 196:553-562.

[2] Mc Cusker, C. & Gardiner, D.M. (2011) Gerontology 57:565-571.

[3] Lemanski, L. et al. (1995) Cell Mol Biol Res. 41:293-305.

Fig. 1: Fig. 1 – Low magnification of axolotl testis endocrine cell. N: nucleus; m: mitochondria (arrows); L: liposomes (arrows point to liposomes with central lipophilic granule; arrow heads point to empty liposomes).

Fig. 2: Fig. 2 – A) Lipid-filled liposomes (L); N: nucleus. B) Smooth endoplasmic reticulum (SER); m: mitochondrion. C) Aggregate of smooth endoplasmic reticulum (SER); m: mitochondrion.

Fig. 3: Fig. 3 – Mitochondria and organization of cristae and tubules inside them; v: vesicles. A) Insets a) and b) show further magnifications of mitochondrial internal structure (x 86,500 and x 144,000, respectively). B) Inset shows a higher magnification (x 50,000) of tubular-like structure inside mitochondria (please see text).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1475 Artistic compositions by surfactant in axolotl lung alveoli

Keyhani E.1
1Laboratory for Life Sciences, 19979 Tehran, Iran
keyhanius2002@yahoo.com

Lung surfactant is a mixture of dipalmitoylphosphatidylcholine and specific proteins (SP-A, -B, -C, and -D) that covers the interior lung alveoli epithelium; it is synthesized by type II alveolar cells and packaged in membrane-bound lamellar inclusion bodies. By lowering interfacial tension, lung surfactant insures a negligible work of breathing and uniform lung inflation [1]. This paper reports the fine structure of surfactant in the lung of axolotlt Ambystoma mexicanum, revealing amazing pictures that evoke artistic compositions but that also show elaborate organizational patterns.

8 male and female adult axolotls (~ 2 years old) were killed after anesthesia in tricaine methanesulphonate. Lung portions were removed, immediately diced into 1-mm pieces, doubly fixed with glutaraldehyde (2.5%)-osmium (1%) at pH 7.4, dehydrated in ethanol and embedded in Epon. Thin sections were collected on 200-mesh grids, doubly stained with uranyl acetate and lead citrate, and examined in Philips EM300, at 60 KV accelerating voltage and 50 μm objective aperture.

Three predominant morphological types were observed.

Type I inclusions or tubular myelin (average size 3.7 x 1.7 μm) showed square to rectangular grid patterns, strikingly resembling a fishing net organization (Fig. 1). The lattice appeared to be formed by membranous elements of ~ 6 nm diameter, crossing in two directions perpendicular to each other. Each unit (square or rectangle) shared its walls (membranous elements) with four neighbors, had an internal perimeter of ~ 17 x 27 nm, and measured ~ 27 x 36 nm for center to center spacing of its walls. A centrally located dense line of 2 nm diameter with a fuzzy contour crossed the middle section of each unit longitudinally. High resolution revealed a globular structure of the membranous elements (Fig. 1, inset, arrows).

Type II inclusions consisted of arrays of osmiophilic lamellar bodies displaying concentric whorls of lamellae reminding of spider webs. However, the overall appearance of such inclusions resembled representations of an owl’s head (Fig. 2 A, B) ranging from 1 x 0.8 to 3.1 x 2.9 μm.

Type III consisted of osmiophile-dense granules of average size 4.7 x 1.8 μm (Fig. 3).

Recent research indicated that lung surfactants have micro-nano scale organizations or domains that play a critical role in functioning of membrane proteins, phase segregation and liquid ordering of soft material in two dimensions [2]. The structures reported here revealed micro scale as well as micro-nano scale organization (Fig. 1, inset) in axolotl lung surfactant.

References

[1] Braun, A., et al. (2007) Biophys J. 93:123-139.

[2] Nag, K. et al. (2007) Modern Research and Educational Topics in Microscopy (Mendez-Vilas A. and Diaz, J., eds), Badajoz: Formatex; 483-490.

Fig. 1: Fig. 1 – Type I inclusion – Note the fishing net organization of surfactant. Inset: globular structure of membranous elements (arrows).

Fig. 2: Fig. 2 – Type II inclusions: arrays of osmiophilic lamellar bodies displaying concentric whorls of lamellae reminding of spider webs, but with overall appearance resembling representations of owl’s heads.

Fig. 3: Fig. 3 – Type III inclusions: osmiophile dense granule. Note membrane formation (arrows).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1478 Micro-morphology of the liver of the juvenile Nile crocodile, Crocodylus niloticus

Van Wilpe E.1, Groenewald H. B.1
1University of Pretoria, Pretoria, South Africa
erna.vanwilpe@up.ac.za

There is a lack of knowledge regarding hepatic metabolism and the pathogenesis of hepatic disease in the reptilian liver (1). The Nile crocodile is an important keystone reptile for aquatic biodiversity in Africa (2) and is one of the top commercially utilized species of crocodiles in the world (3). The morphology of the liver of mammals, birds and reptiles have been investigated comprehensively, but studies of the Nile crocodile liver are deficient. This presentation explores the histology and ultrastructure of the liver of the juvenile Nile crocodile, Crocodylus niloticus. Livers from five juvenile Nile crocodiles, obtained from Izintaba Crocodile Farm in South Africa, were perfusion-fixed and prepared using standard techniques for light and transmission electron microscopy. Several of the microscopical features are comparable to that described in other reptiles, most notably the absence of the classic lobulation pattern usually found in vertebrates and the presence of collagen trabeculae in the liver parenchyma of some crocodilian species. However, a few distinctive findings differentiate the juvenile Nile crocodile from the reptiles studied. For instance, the presence of a basal lamina between hepatocyte groups and Glisson’s capsule, the variable location of the Kupffer cells, the presence of conspicuous tubular structures in Kupffer cells and the coexistence of stellate and myofibroblasts (Fig. 1) in the space of Disse. The establishment of baseline data for the liver of the Nile crocodile is essential for comparative studies with other crocodilians and for the assessment of the pathology of liver disease.


References
1. DIVERS, S.J. & COOPER, J.E. 2000. Reptile hepatic lipidosis. Seminars in Avian and Exotic Pet Medicine, 9:153-164.
2. ASHTON, P.J. 2010. The demise of the Nile crocodile (Crocodylus niloticus) as a keystone species for aquatic ecosystem conservation in South Africa: The case of the Olifants River. Aquatic Conservation: Marine and Freshwater Ecosystems, 20: 489–493.
3. ROSS, J.P. 1998. Status Survey and Conservation Action Plan, edited by J.P. Ross. Switzerland: IUCN-SSG/CSG. http://www.iucncsg.org/ph1/modules/Publications/action_plan1998/plan1998a.htm#Contents

 

The authors are grateful to the University of Pretoria for sponsoring attendance of ICM2014 and the Izintaba crocodile farm for donating the juvenile crocodiles.

Fig. 1: Figure 1. Myofibroblastic cells (M) and stellate cell (SC), containing a lipid droplet (L), occupying the same area in the space of Disse between two sinusoids (stars). Note endothelial cell cytoplasmic extensions (arrows). H, Hepatocytes.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1481 Plants related to early evolutionary events (Bryophytes) contain Lacandonia granules previously discovered in flowering plants

Alonso-Murillo C. D.1, Jimènez-Garcìa L. F.1
1National Autonomus University of Mexico
yiribom@hotmail.com

The early evolution of the plant kingdom is controversial, and bryophytes are argued to be the earliest divergent plants. Indeed, the origin and diversification of land plants marks an interval of unparalleled innovation in the history of plant life. For example, from a simple plant body consisting of only a few cells, land plants (non-vascular plants) evolved an extraordinary array of complex organs and tissue systems (vascular plants). Previous ultrastructural analysis of the Lacandonia schismatica nucleus revealed the presence of Lacandonia granules (32 nm in diameter). Cytochemical, immunocytochemical and in situ hybridization studies suggested that Lacandonia granules are involved in mRNA storage. In addition, Lacandonia granules have been located in other flowering plants of the order Triuridales (such as Triuris alata) and also in non-flowering plants (such as the gymnosperm Ginkgo biloba). Therefore, in order to understand the evolution of RNA processing, we demonstrated the presence of Lacandonia granules in three species of non-vascular plants (Bryophytes). In this study, we analyzed three species of Bryophytes (Marchantia polymorpha, Polytrichum junniperum and Anthoceros punctatus) using transmission electron microscopy and found granules in sporophyte cell nuclei of all species. In addition, the granules are about twice the size of ribosomes; therefore, we showed ribosomes (as internal comparative structures) in the cytoplasm of cells of all three studied species. Moreover, the presence of few granules in the interchromatin and perichromatin space was a constant feature of all nuclei of Bryophytes. Thus, non-abundant particles of around 32 nm in diameter were observed in the perichromatin and interchromatin space. Perichromatin fibers are usually present in continuity with granules, forming a fibrogranular environment. Finally, we demonstrated that these particles are positive after the EDTA regressive technique preferential for ribonucleoproteins. In summary, this study suggests that Lacandonia granules contribute to the understanding of the spatiotemporal organization of several mRNA processing factors in the nuclear subcompartments and verifies the conservation of the event throughout the evolutionary process in the Plant Kingdom.

We thank the Biological Sciences Doctorate program at National Autonomus University of Mexico(UNAM) and financially supported by CONACYT.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1507 Presence and Distribution of Ghrelin Receptor in the Nervous System of Pomacea canaliculata

Ngernsoungnern A.1, Ngernsoungnern P.1
1School of Anatomy, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
apichart@sut.ac.th

Growth hormone secretagogue-receptor 1a (GHS-R1a), a member of the G-protein couple receptor family, is a natural receptor for gastrointestinal hormones, especially ghrelin peptide. The ghrelin-GHS-R1a complex has an ability to stimulate the releasing of growth hormone from the pituitary gland. In vertebrates, GHS-R1a has been identified in the central nervous system, mainly the hypothalamus. In the present study, the immunohistochemistry technique was used to examine the localization and distribution of GSH-R1a in the nervous system of the golden apple snail, Pomacea canaliculata. Pairs of the snail ganglia were investigated, including cerebral, buccal, and pleuropedal ganglia. Histologically, the snail neuronal cells had been classified into four types, according to their sizes into NR1-4 (small-, medium-, large-sized neurons and giant neuron, respectively). From the immunoperoxidase technique, the immunoreactivity of GHS-R1a was identified in the neurons housed in the pleuropedal ganglion. In contrast, no immunoreactive neurons were observed in the cerebral and buccal ganglia. The immunoreactivity showed a strongly staining only in the NR1 and NR3. The immunoreactive neurons was scattered throughout the pleuropedal ganglion. However, the highest number of the immunoreactive neurons was observed at the peripheral of the neuropil. This result was confirmed by the immunofluorescence technique. In summary, we have demonstrated that GHS-R1a was localized and distributed in the ganglion of this gastropod mollusk. We suggest that ghrelin peptide which is a ligand for this receptor could play a physiological function in this species.

This work was supported by Suranaree University of Technology.

Fig. 1: Localization of ghrelin receptor in neurons of the snail pleuropedal ganglion. Scale bar = 100 um.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1544 Fluorescent sesquiterpene lactone trilobolide: focus on endoplasmic reticulum and mitochondria

Rimpelová S.1, Jurášek M.2, Peterková L.3, Kmoníčková E.4, Drašar P.5, Ruml T.6
1Institute of Chemical Technology in Prague, Prague, Czech Republic, 2Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
silvie.rimpelova@vscht.cz

Sesquiterpene lactones are bioactive natural compounds with cytotoxic, antibacterial, antifungal, antiviral, antimalaric and immunomodulative effects. One of these compounds is trilobolide, which is a structurally related analog of the well-known thapsigargin (recently in the second stage of clinical trials for prostate cancer treatment). We isolated trilobolide from Laser trilobum and revealed that it is a potent immunostimulatory agent strongly inducing IFN-γ secretion and nitric oxide production in primary macrophages. Trilobolide, similarly to thapsigargin, is a potent inhibitor of sarco-/endo-plasmic Ca2+ ATPase (SERCA), which maintains concentration gradient of calcium ions between sarco-/endo-plasmic reticulum and cytosol. To study biological relevance and to track sesquiterpene lactones in live cells, fluorescently labeled derivatives are inevitable approach. We designed, synthesized and properly characterized a series of novel fluorescent conjugates of this natural compound. The live-cell imaging experiments proved that trilobolide derivatives localize in endoplasmic reticulum of various cancer cell lines (PC-3, LNCap, both derived from prostate carcinoma, U-2 OS from osteosarcoma and HeLa cells from cervix carcinoma). Moreover, we found that trilobolide disrupted calcium homeostasis, which resulted in fragmentation of mitochondrial network. Cytotoxicity, metabolic activity and nitric oxide production of cell treated with trilobolide derivatives were also tested. Recently, we focus on targeted delivery of trilobolide and its potent derivatives.

This work was supported by MŠMT ČR MSM6046137305, GA ČR 304/10/1951, GA CR, 305/07/0061 and P503/11/0616.

Fig. 1: Live-cell fluorescent image of trilobolide localization (50nM, 30 min) in HeLa cells. A. trilobolide-BODIPY, B. pentamethinium salt staining mitochondria, C. merge of images A and B.

Fig. 2: Live-cell fluorescent image of trilobolide localization (100nM, 30 min) in HeLa cells. A. trilobolide-BODIPY, B. pentamethinium salt staining mitochondria, C. merge of images A and B.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1657 Three-dimensional reconstruction of Angomonas deanei reveals association of the protozoa organelles and a symbiotic bacterium

Catta-Preta C.1 2, De Souza W.1 2 3, Motta C.1 2
1Laboratório de Ultraestrutura Celular Hertha Meyer, IBCCF, UFRJ , 2Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, UFRJ , 3Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO)
carol.cattapreta@gmail.com

Co-evolution between primitive organisms associated by symbiotic relationship can offer valuable information about the origin of organelles and the evolution of of the eukaryotic cell. Some monexenic protozoa from the Trypanosomatid family, as Angomonas deanei, maintain a mutualistic relationship with a bacterium that divides in synchrony with other host cell structures. In this work we performed structural analysis using high-pressure freezing and freeze substitution associated to the electron tomography technique in order to study the symbiont ultrastructure and its association with protozoan organelles. Such analyses were combined with genomic data obtained from the bacterium and its host Trypanosomatid. Our results showed a close association of the symbiont with the host nucleus, reinforcing the idea that the protozoan controls the bacterium division and that during this process the nucleus serves as a topological reference to the symbiont segregation. During the protozoan cytokinesis, the bacterium maintains its position close to the nucleus ensuring its inheritance to each daughter cell. Interestingly, our genomic data showed that the symbiont presents a reduced content (±830 kb), indicating a massive gene loss as those from the dcw (division and cell wall) cluster. These sequences code proteins of the bacterial division ring, known as Z-ring and those involved in peptidoglycan layer synthesis and septum formation. Such structures are not detected in the symbiont envelope after using classical optical and electron microscopy techniques. In this work, the use of electron tomography membrane contrast revealed a detailed ultrastructure of the symbiont envelope that presents a reduced cell wall and lacks the Z ring. Another interesting aspect of this symbiotic relationship is the intense metabolic exchange between the associated partners, as the symbiont obtainment of phosphatidylcholine from the host Trypanosomatid. According to this idea the high-resolution microscopy showed that the endoplasmic reticulum presents an intimate relationship with the bacterium, with some contact points observed between these structures. Taken together, our resuls assume that an ultrastructural and metabolic association between the symbiont and host cell structures is essential to maintain the endosymbiosis in trypanosomatids. 

Financial support: CNPq, FAPERJ.

Fig. 1: (A-C) FIB-SEM. Symbiont (green); Protozoa nucleus (blue); Glicossomes (yellow); ER (red) (D-E) Electron Microscopy. Symbiont (S); ER (arrow); Nucleus (N); Glicossome (G). (F) Kinetoplast (K) in pink. (G-H) Cryo-techniques were used to verify membrane fusion events. Mithocondrion (M) in yellow; ER (red); Symbiont (green). Scale bar: 0,5 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1728 Influence of surface topography on osteoblast-like cell behavior to zirconia

Yoshinari M.1
1Tokyo Dental College, Tokyo, Japan
yosinari@tdc.ac.jp

Zirconia, particularly, tetragonal zirconia polycrystals (TZP) have been used in medical and dental fields with their outstanding mechanical properties, biocompatible and esthetic performance. The importance of 3-dimensional (3-D) surface characterization in evaluating cellular response has been noted in addition to conventional 2-D surface characterization. Moreover, the synergetic effect with micro- and nano-topography was reported in enhancing the cell response on the biomaterial surfaces. This study aimed to clarify the influence of surface topography on osteoblast-like cell behavior to TZP. Mirror-polished; blasted with 50- or 150-µm alumina (SB50 and SB150); and SB150 acid-etched with hydrofluoric acid (SB150E) were prepared on TZP. Titanium specimen with alumina-blasted and acid-etched was also prepared as a control. The Sa (average roughness) and Sdr (developed interfacial area ratio) values were evaluated using an electron beam 3-D surface roughness analyzer (ERA-8900FE; Elionix, Tokyo, Japan).　Initial attachment and proliferation assay of mouse osteoblast-like cells MC3T3-E1 were performed with WST-1-based colorimetry. In addition, alkaline phosphatase (ALP) activity as well as the gene expressions of type 1 collagen and osteocalcin was determined as a differentiation of the cells. Significantly higher Sdr values were obtained in the SB150E than in the other specimens despite no apparent difference in Sa values was observed between SB150 and SB150E, indicating that both micro- and nano-topographies produced on the SB150E surfaces. Although no clear differences were observed in initial cell attachment among specimens, the proliferation rate and expression of ALP activity on the SB150E specimens was significantly higher than those on the other specimens. These results indicate that the creation of micro- and nano-topographies on TZP by surface treatment offers a promising method for enhancing the proliferation and differentiation of MC3T3-E1 cells.

This research was supported by the Foundation of the Japan Medical Association, by Oral Health Science Center Grant hrc7 from Tokyo Dental College, and by a “High-Tech Research Center” Project for Private Universities: matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology) of Japan, 2006-2011.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1930 Keratocyte interaction with stromal microfibril bundles in murine cornea

Behzad A. R.1, Brown E. S.2, Burns A. R.2, Hanlon S. D.2
1Imaging and Characterization Core Lab. King Abdullah University of Science and Technology, Thuwal, KSA, 2College of Optometry, University of Houston, Houston, United States of America
ali.behzad@kaust.edu.sa

 Corneal transparency is made possible by uniform size and spacing of stromal collagen fibrils which are organized into sheets of parallel fibrils laid down in criss-crossed fashion. Keratocytes, interspersed between collagen lamellae, make extensive lateral connections and form layers parallel with the corneal surface. Understanding the architecture of ECM is limited when using conventional 2D ultrastructural information from thin sections. Serial block-face imaging with a scanning electron microscope (SB-SEM) provides a z-stack of images that can be reconstructed to show 3D details of spatial distribution and organization of ECM ultrastructural components.

Our initial TEM observations of the mouse cornea revealed what appeared to be small random electron dense patches within the corneal stroma. Computer-aided 3D reconstructions of images generated from SB-SEM showed these patches were in fact fibers, distinctly different than stromal collagen lamellae. In this study, we used SB-SEM imaging and 3D reconstruction to characterize these stromal fibers and their relationship to the ECM and keratocytes in the mouse cornea.

Corneas from adult mice were fixed and embedded in resin blocks for histological sectioning using a custom protocol for enhanced contrast. SB-SEM images (100 nm intervals between images) were obtained using a Gatan 3 View system mounted in Quanta 200FEG SEM. Z-stacks were obtained from the stroma in the limbus, paralimbus, and central cornea. Keratocytes and stromal fibers were segmented and reconstructed in 3D using Amira 5.2 software.

Reconstruction of segmented z-stacks provided evidence the corneal stroma contains a layered network of ~100nm diameter fibers running parallel with the corneal surface and lying between and within the collagen lamellae. High magnification TEM images of the fibers show that each fiber consists of numerous microfibrils ~10nm in diameter (Fig 1). Some fibers appear to attach posteriorly to Descemet’s membrane and anteriorly to peripheral lymphatic vessels (Fig. 2). The fibers were frequently observed in juxtaposition with keratocytes and the surface of keratocytes contained complementary invaginations or “grooves” which encompassed these fibers.

3D reconstruction of SB-SEM images revealed a network of fibers composed of microfibrils within the murine cornea (Figs. 3 and 4). The association of these fibers with Descemet’s membrane and lymphatics suggests they may serve as mechanical force transducers capable of opening lymphatic vessels for drainage when an injured cornea becomes edematous. The physical association of fibers with stromal keratocytes suggests the fibers are secreted by and/or assembled by keratocytes. Additionally, the fibers may serve as a scaffold for the keratocyte network.

 

The authors gratefully acknowledge funding from the National Institutes of Health under grant numbers EY17120, and P30EY007551.­

Fig. 1: Electronmicorgraph of corneal stroma showing a fibril bundle made of numerous microfibrils that are 10 nm in diameter (arrows).

Fig. 2: TEM image showing the cable (arrows) associated with lymphatic endothelial cell.

Fig. 3: Serial Block-face image reconstruction of cables from the stack of electronmicrographs from central cornea. The reconstructed cables form an intricate network in layers parallel with the corneal surface.

Fig. 4: Stromal elements segmented and reconstructed from the stack of electronmicrographs from paralimbus area. Keratocytes are in yellow, orange, light and dark blue, light and dark green, and purple. Microfibril bundles are in red.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1932 The Expression of Chondroitin Sulfate in Human Amniotic Fluid Cells during Short Term Culture

Aungsuchawan S.1, Keawdee J.2, Ongchai S.3, Kongtawelert P.4, Pothacharoen P.5
1Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 2Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 3Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 4Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, 5Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
sirinda001@hotmail.com

A biological molecule, chondroitin sulfate (CS) is one type of the glycosaminoglycan (GAG) that has a wide variety of biological processes. The human amniotic fluid cells (hAFCs) have the potential to differentiate into multiple cells lineages. The objective of this study is to investigate the pattern expression of chondroitin sufate epitope (WF6) in hAFCs associate with the series of the day culture from days 0 to day 30. The hAFCs were obtained from the amniotic fluid of the pregnant women at 18 weeks of gestation (n= 5). All protocols approved by the Medical Ethical Review Board of Faculty of Medicine of Chiang Mai University.They were cultured in RPMI 1640 medium containing 20% FCS, AmnioMAX-C100 16 % and antibiotics. The expression of chondroitin sufate epitope (WF6) were continuously detected by immunocytochemistry analysis. The level of the chondroitin sulfate WF6 epitope in hAFCs were correlated together with both parameters. The first cycle, it gradually increased from day 0 to day12 and continually subsided from day 12 to day 18. The second cycle, it increased from day 18 to day 27 and decreased after day27 to day30. It might be conclude that the expression of chondroitin sulphate WF6 epitope in hAFCs gradualy increased and released in a cycle pattern that might be correlated with biological activities such as proliferation or differentiation. It is the benefit basic data for further studies.

References

Bossolasco, P., Montemurro, T., Cova, L., 2006. Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential. Cell Res. 16, 329-336.

Cipriani, S., Bonini, D., Marchina, E., Balgkouranidou, I., Caimi, L., Zucconi, G.G., Barlati, S., 2007. Mesenchymal cell from human amniotic fluid survive and migrate after transplantation into adult rat brain. Cell Biol. 31, 845-850.

Fthenou, E., Zafiropoulos, A., Katonis, P., Tsatsakis, A., Karamanos, N.K., Tzanakakis, G.N., 2008. Chondroitin sulfate prevents platelet derived growth factor-mediated phosphorylation of PDGF-Rb in normal human fibroblasts severely impairing mitogenic responses. J. of Cellular Biochemistry. 103, 1866-1876.

Sugahara, K., Mikami T., Uyama, T., Mizuguchi, S., Nomura, K., Kitagawa. H., 2003. Recent advances in the structural biology of chondroitin sulfate and dermatan sulfafe.Current Opinion in Structure Biology. 13,612-620.

Pothacharoen, P., Teekachunhatean, S., Louthrenoo, W., Yingsung, W., Ong-Chai, S.,Hardingham, T., Kongtawelert, P., 2006. Raised chondroitin sulphate epitopes and hyaluronan in serum from rheumatoid arthritis and osteoarthritis patiens. Osteoarthritis and Cartilage. 14, 299-301.

The Faculty of Medicine Research Fund, Chiang Mai University, Thailand

Fig. 1: Immonofluorescent localization of WF6 epitope on the hAFCs at series days of the culture Day0(a), Day6(b), Day12(c), Day15(d), Day24(e) and Day27(f).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1938 Altered structural analysis of the brain in a Drosophila of Alzheimer’s disease model: A FIB-SEM microscopy study

Park S. J.1, Schertel A.2, Han S. S.1
1School of Life Sciences and Biotechnology, Korea University, Seoul, Korea, 2Carl Zeiss NTS GmbH, Oberkochen, Germany
fufujini@korea.ac.kr

Alzheimer’s disease (AD), one of the most progress neurodegenerative brain diseases that leading cause of dementia, has been extensively researched for years. However, our knowledge of its synaptic structure, which is a basis to understanding neurodegenerative disorders, is still unclear. Defining the structures of neurons and their synaptic connections are significant goals of brain research. To study synaptic connectivity, three-dimensional (3D) reconstructions of the nervous system are very helpful. In this study, the 3D structure of brain synapses in the Drosophila was analyzed using focused ion beam scanning electron microscopy (FIB/SEM). This technique is one of the most useful for 3D reconstruction, as the process of obtaining serial images is fully automated and thus avoids the problems inherent in hand-operated ultrathin serial sectioning. In this study, we visualized and quantitatively analyzed the ultrastructural characteristics of the calyx region of a Drosophila mushroom body, which is a neuropil organ that plays an important role for learning and memory in insects. We used transgenic line of Drosophila melanogaster Swedish mutant APP (Swe-APP), which is characterized by early onset AD and increased Aβ production. The 3D images of normal and AD brains reported in this study reveal characteristic features of AD such as appearance of autophagy, abnormal axon formation, and increased mitochondrial size. This 3D analysis reveal structural change as a basis for understanding neurodegenerative disorder.

Fig. 1: Ultrastructure of axon terminals. Comparison of axon terminal ultrastructure between normal (A, B) and AD models (C–E). Note that the AD model occasionally demonstrates aberrant large (C) or normally sized (D, E) axon terminals. The active zone, which is the site of neurotransmitter release (arrowhead), can also be observed. Scale bar, 1 μm

Fig. 2: 3D reconstruction of nine individual presynaptic axons each from the normal (A) and AD models (B). Axons pass in many different trajectories other than the parallel direction. In the normal model (A), most axons appear long and straight. However, in the AD model (B), axons are relatively short and of various sizes.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-1957 The secretory pathway in electron microscopy: Sec16 and ER exit sites

Schmidt K.1,2, Sealey M.2, Fedorenko I.1,2,3, Tögel S.4, Mallinger B.1, Wegscheider R.1, Ellinger A.1, Warren G.2
1Department of Cell Biology and Ultrastructure Research, Medical University of Vienna, Vienna, Austria, 2Max F. Perutz Laboratories, University of Vienna and Medical University of Vienna, Vienna, Austria, 3University of Applied Sciences Wiener Neustadt for Business and Engineering Ltd, Wiener Neustadt, Austria, 4Karl Chiari Lab for Orthopaedic Biology, Department for Orthopaedics, Medical University of Vienna, Vienna, Austria
katy.schmidt@meduniwien.ac.at

Generating the cellular surface, and in many cases also the extracellular environment is fundamental to all cells from bacteria to eukaryotes and essential for the multicellular organization of tissues and organisms. Consequently, disruptions of the underlying process, secretion, can cause malformations and diseases. The secretory pathway starts with newly synthesized proteins and lipids leaving the ER at distinct structures termed ER exit sites (ERES), defined by the presence of Sec16. Sec16 is a large membrane-associated protein supposed to create a platform for the generation of COPII-ensheathed vesicles at ERES. The COPII vesicle coat consists of Sec23/Sec24 forming the inner shell and Sec13/Sec31 comprising the outer layer. COPII-coated vesicles transport the cargos to the Golgi where they are processed before being routed to their final destinations. To study the morphological relationship of involved organelles in the early secretory pathway we are have investigated Trypanosoma brucei, the causative agent of African sleeping sickness. T. brucei is good model since it contains only a single ERES and Golgi at a defined location in a specific state of its live cycle. Phenotypic analyses of trypanosome cells following Sec16 knockout, depletion or overexpression revealed an intriguing link between ERES and Golgi size. In particular, increased levels of Sec16 resulted in larger ERES, and concomitantly a larger Golgi whereas decreased levels had the opposite effect. We are now exploring the changes in size and shape of these organelles in 3D using electron tomography. In a second approach, and to link secretion with medically relevant conditions, we are investigating the early secretory pathway and the structure of the extracellular matrix during cartilage degeneration (osteoarthritis).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2030 Morphology of leaf starch granules of Arabidopsis thaliana wild type and mutants (sex1 & nex1)

Annunziata M. G.1, Maximova E.1, Stitt M.1, Hartmann J.2, Lunn J. E.1
1Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam–Golm, Germany, 2Max Planck Institute of Colloids and Interfaces, 14476 Potsdam–Golm, Germany
maximova@mpimp-golm.mpg.de

Starch, together with sucrose, is the main end product of photosynthesis in many plants. The main pathways of starch synthesis and degradation are well known but our understanding of their regulation is still fragmentary. The aim of this work was to identify novel starch excess mutants to provide new insights into the pathway and regulation of starch metabolism. We identified a T-DNA mutant with insertion in a gene encoding a protein not previously linked to starch metabolism which we called NEX1. To characterize the nex1 mutant in detail we are comparing its phenotype with already known starch excess mutants via: 1) metabolite profiling through a 24-h diurnal cycle; 2) analysis of starch granule morphology; 3) transcript profiling (qRT-PCR) of ~80 starch related genes; 4) growth analysis. To investigate the morphology of the leaf starch granules from Arabidopsis thaliana (L.) Heynh. wild type (WT) and mutants (sex1 & nex1) we performed transmission electron microscopy (TEM). Pieces (2 mm2) of mature leaves were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer and post-fixed in 1% OsO4. The tissues were embedded in Spurr’s epoxy resin and examined with an EFTEM (Zeiss, Germany). Some ultrastructural alterations were observed in mesophyll cells in WT plants and mutants (sex1 and nex1) during day and night. Differences were found in plastids, especially, in the size and shape of starch granules, as well as in the organization of thylakoid membranes, predominantly in granal stack, size and number. Electron micrographs showed that starch grains occupy most of the plastid volume in mutants at the end of the day. Starch granules of WT plants are smaller, flat and a little elongated [Fig. 1]. In contrast, in the sex1 mutant, which accumulates up to three times more starch than WT, the granules are flattened-elongated, running parallel to the long axis of the plastid with transverse crystalline regions (dark part in starch granules) and amorphous (lighter part - less crystalline material) [Fig. 2]. The starch granules in nex1 are highly variable in size, but are generally larger than WT granules, and more rounded than both WT and sex1 granules [Fig. 3]. The nex1 mutant contains only slightly more starch than WT plants at the end of the day, but degrades little or none of its starch at night. The biochemical reason for this defect is unknown. The genetic lesion in the nex1 mutant might lead to a deficiency in one or more of the enzymes that are required for starch degradation. Another possibility is that the nex1 lesion causes synthesis of abnormal starch granules that are resistant to hydrolysis by β-amylase or the action of other starch degrading enzymes.

Fig. 1: Transmission electron micrographs of mesophyll cells in Arabidopsis thaliana WT Col0 leaves at the end of the day - fragment of the cell. Notice a few starch grains in stroma of the plastids. Starch granules of WT plants are smaller, flat and a little elongated compared with mutants (sex1 & nex1). Scale bar: 2000 nm

Fig. 2: Transmission electron micrographs of mesophyll cells in Arabidopsis thaliana sex1 mutant leaves at the end of the day - fragment of the cell. Notice a strong starch accumulation. The starch granules are flattened-elongated with transverse crystalline regions appearing as dark bands, and occupy most of the plastid volume. Scale bar: 2000 nm

Fig. 3: Transmission electron micrographs of mesophyll cells in Arabidopsis thaliana nex1 mutant leaves at the end of the day - fragment of the cell. Starch granules in plastids are rounded and very variable in size ranging from very small to big compared with wild type and sex1 mutant granules. Scale bar: 2000 nm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2064 Nuclear phosphatidylinositol-4,5-bisphosphate in a complex with histone demethylases.

Uličná L.1, Kalasová I.1, Vacík T.1, Hozák P.1
1Institute of Molecular Genetics ASCR v.v.i. Department of Biology of the Cell Nucleus, Vídeňská 1083, 142 20, Prague 4, Czech Republic
ulicna@img.cas.cz

Phosphorylated derivatives of phosphatidylinositol (phosphoinositides - PIs) are essential regulators of cytoskeletal dynamics, membrane trafficking, and are the basis of a ubiquitous membrane signalling system. In addition to their cytoplasmic roles, they are also involved in important nuclear processes as DNA transcription, pre-mRNA splicing, or mRNA export out of the nucleus. To widen our knowledge about phosphoinositides roles in the nucleus, we study the interacting partners of phosphoinositides in various nuclear processes.
We decided to study lysine-specific demethylase 7B (KDM7B; PHF8) and lysine-specific demethylase 2A (KDM2A; FBXL11) as possible PIs - interacting proteins. They contain PHD conserved domain and they were identified as components of nuclear PtdIns(4,5)P2 complexes by neomycine extraction. FBXL11 is a histone demethylase required to sustain centromeric integrity and genomic stability, particularly during mitosis. PHF8 acts as a coactivator of rDNA transcription, by activating polymerase I mediated transcription of rRNA genes.
Our experimental data confirm that PHF8 and FBXL11 are indeed in a complex with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Using confocal flourescence microscopy, we show that both FBXL11 and PHF8 partially colocalize with PtdIns(4,5)P2 in the nucleoplasm and in nuclear speckles, dynamic intranuclear compartmens, where many pre-mRNA splicing factors are stored. These data suggest that PtdIns(4,5)P2 may have a role in regulation of gene expression and maintenance of genomic stability through the interaction with PHF8 and FBXL11.

This study was supported by the project BIOCEV (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund – and by the Grant Agency of the Charles University (No. 606112), Grant Agency of the Czech Republic (P305/11/2232) and by the institutional grant RVO: 68378050.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2069 Actin filaments in the nucleus

Kalendová A.1, Yamazaki S.2, Kalasová I.1, Harata M.2, Hozák P.1
1Institute of Molecular Genetics, Department of Biology of the Cell Nucleus, Academy of Sciences of the Czech Republic, Videnska 1083, Prague, Czech Republic, 2Laboratory of Molecular Biology, Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi 1-1, Aoba-ku, Sendai, Japan
kalendova@img.cas.cz

Actin is in the cytoplasm present either as a monomer or in form of filaments, which is the major component of cytoskeleton. It is required for maintenance of cell shape, motility, vesicle movement, cytokinesis, and signalling. In last decades it has been well documented by a multiple studies that actin localizes also to the cell nucleus1, where it participates in transcription2 and chromatin remodeling3. However, the state of nuclear actin is not fully understood yet. It is anticipated that besides monomers actin exists also in oligomeric or polymeric form4. Recent study showed formation of nuclear actin filaments after overexpression of beta-actin fused to a NLS and flag tag5. Here we confirmed a presence of such actin filaments in the nucleus upon overexpression of EYFP-actin fused to NLS (EYFP-NLS-actin) in human U2OS cell line. These filaments seem to be emanating from below nuclear envelope, ranging variably through the nucleus. They can be visualized by phalloidin and they do not bind any of actin-binding proteins (spectrin, vinculin, paxillin, nuclear myosin 1) as tested by immunolocalization, which suggests their differential behaviour from the cytoplasmic ones. In addition, nuclear actin filaments do not seem to enter heterochromatin regions, active chromatin or interchromatin granules. Surprisingly, cells possesing nuclear actin filaments seem to exhibit higher transcriptional activity in comparison to the controls. The question still remains upon which stimulus the actin filament formation occurs and how does it change the cellular processes in general.

1. Pederson T. and Aebi U., JSB 140:3–9, review, 2003.

2. Philimonenko, V.V., Zhao, J., Iben, S., Dingova, H., Kysela, K., Kahle, M., Zentgraf, H., Hofmann, W.A., de Lanerolle, P., Hozak, P. and Grummt, I. , Nat. Cell Biol. 6: 1165-1172, 2004.

3. Zhao, K., W. Wang, O.J. Rando, Y. Xue, K. Swiderek, A. Kuo, and G.R. Crabtree, Cell. 95:625–636, 1998.

4. McDonald , D. , G. Carrero , C. Andrin , G. de Vries , and M.J. Hendzel , JCB 172: 541 – 552, 2006.

5. Kokai E1, Beck H, Weissbach J, Arnold F, Sinske D, Sebert U, Gaiselmann G, Schmidt V, Walther P, Münch J, Posern G, Knöll B.: Histochem. Cell Biol. 141:123-35, 2014.

Grant Agency of the Czech Republic (P305/11/2232), by the Technology Agency of the CR (TE01010118), by the Ministry of Education, Youth and Sports of the CR (LH12143, LD12063, OP EC CZ 1.07/2.3.00/30.0050 and TE01020118), Ministry of Industry and Trade of the CR (MIT FR-TI3/588 and MIT FR-TI4/660), by the Human Frontier Science Program (RGP0017/2013) and by the institutional grant RVO: 68378050.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2121 Amyloid-β affected mitochondrial membrane as potential source for the autophagosome biogenesis

Kim M. J.1, Je A. R.1,2, Kim H. J.1,2, Kim H. G.2, Huh Y. H.1, Kweon H. S.1
1Division of Electron Microscopic Research, Korea Basic Science Institute, Daejeon 305-806, Korea, 2Department of Pharmacology, College of Medicine, Dankook University, Chungnam 330-714, Korea
hskweon@kbsi.re.kr

Autophagy plays a crucial role in maintaining cellular homeostasis by carrying out continual degradation and recycling of cellular components [1]. However, overactive autophagy may promote neuronal cell death in neurodegenerative disorders including Alzheimer's disease (AD). Recent evidence has demonstrated that amyloid-β (Aβ) induces the formation of doubled-membrane-bound vacuole, known as the autophagosome [2]. In spite of various studies on the ultrastructure of autophagy, the membrane formation mechanism of autophagosome is still poorly understood. The purpose of this study is to identify the organelles that provide the source of membrane in the early autophagosome biogenesis. For this, we have performed immunocytochemical analysis, cryo-immunogold labeling and ultrastructural analysis, and three-dimensional electron tomography [3] in animal- and cell-model for AD. In the APP/PSEN1 transgenic (TG) mice that express human amyloid precursor protein, the deposition of Aβ plaques and distrophic neurite (DN) were detected in the hippocampus and cortex regions. We also identified the loss of peroxiredoxin, an endogenous cytoprotective antioxidant enzyme, and the accumulation of Aβ in the hippocampal mitochondria of transgenic mice with severe disruption of mitochondrial cristae. In the Aβ-stimulated murine microglial cell line, BV-2, we found that mitochondrial membranes were disrupted by the depletion of peroxiredoxin. In addition, the number of LC3, autophagosome marker, positive autophagosome was increased significantly within the cytoplasm. We also found that cytochrome c oxidase subunit IV (COX IV) and Beclin-1, a pre-autophagosome marker, were co-localized in tiny tubule-like structure and on some autophagosome membrane around the Aβ-affected mitochondria. These results suggest that the fragments of mitochondrial membrane are involved in the generation of pre-autophagosomal membranes during AD development. Our in vivo and in vitro studies strongly support the hypothesis that the mitochondria may be a potential source for the membrane biogenesis in the autophagosome formation.

References

[1] Nixon RA, Trends Neurosci. 29: 528-535 (2006).

[2] Kim MJ et al., Appl Microsc. 42: 179-185 (2012).

[3] Choi KJet al., J Biosci. 39: 97-105 (2014).

This research was supported by the Korea Basic Science Institute grant (E34700) and the Bio & Medical Technology Development Program of the National Research Foundation funded by the Ministry of Science, ICT & Future Planning (2013M3A9A9050076).

Fig. 1: Immunofluorescence and cryo-immunogold electron microscopy of LC3 in Ab-treated BV-2 cells. (A) LC3 positive signals were detected in cytoplasm and (B) LC3 were localized in autophagosome (AU) of Ab-treated BV-2 cells. Immunogold EM experiments were performed using anti-LC3 (15 nm) and anti-Aβ antibodies (10 nm).

Fig. 2: Sequential double cryo-immunogold electron microscopy of Beclin-1 and CoxIV in Ab fibril-treated BV-2 Cells. In Ab-treated cells, both CoxIV (15 nm) and Beclin-1 (10 nm) were co-expressed in damaged mitochondria (A), fragmented mitochondria (B) and autophagosome membrane (C).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2127 Golgi-endoplasmic intermediates in herpes virus infected cells

Wild P.1, Schraner E. M.1,2, Käch A.3, Rohrer J.4, deOliveira A. P.2, Ackermann M.2, Tobler K.2
1Institute of Veterinary Anatomy, University of Zürich, Switzerland, 2Institute of Virology, University of Zürich, Switzerland, 3Center for Microscopy and Image Analysis, University of Zürich, Switzerland , 4Biotechnology, Zurich University of Applied Sciences, Wädenswil, Switzerland
elisabeth.schraner@uzh.ch

The Golgi complex plays a crucial role in herpes virus morphogenesis and intracellular transport. Herpes viruses comprise the capsid, tegument and envelope with embedded glycoproteins. Capsids assembled in the host cell nucleus are transported to the Golgi complex. There, they bud at Golgi membranes acquiring tegument and envelope. Concomitantly, a transport vacuole is formed enclosing the virion. This process is referred to as wrapping (1). Capsids also bud into large vacuoles or Golgi cisternae, which may enlarge to engulf multiple virions (2). Another yet hypothetical route would be intraluminal transportation of virions from the perinuclear space (PNS) via ER into Golgi cisternae (3, 4). Virions, which derive by budding of capsids at the inner nuclear membrane into the PNS, are intraluminally transported out of the PNS into the ER (5). Transportation of virions from the ER into Golgi cisternae would be possible provided ER cisternae are connected to Golgi cisternae. To detect possible ER-Golgi intermediates, we infected cells with herpes simplex virus 1 or bovine herpes virus 1 infected cells. Confocal microscopy of cells stably expressing β 1,4-galactosyltransferase 1-green fluorescent protein revealed that the Golgi complex is located close to the nucleus. Cryo-filed emission scanning electron microscopy demonstrated that the Golgi complex was covered to a large extent by an intact membrane so that the Golgi complex appeared as an entity. Transmission electron microscopy of rapidly frozen and freeze substituted cells (Müller, 1992) clearly revealed PNS-ER-Golgi connectivity with enclosed virions supporting the hypothesis of a direct intraluminal transportation route from the PNS into Golgi cisternae. ER-Golgi intermediates were reported in many other cells suggesting that other substances might be intraluminally transported.

 

References

1. Roizman B, Knipe DM, & Whiley RJ (2007) Herpes simplex viruses. Fields Virology, ed D M K, P M, Howly (Lipincott-Raven Publishers, Philadelphia), 5th Ed Vol 2, pp 2501-2601.

2. Stannard LM, Himmelhoch S, & Wynchank S (1996) Intra-nuclear localization of two envelope proteins, gB and gD, of herpes simplex virus. Arch. Virol. 141(3-4):505-524.

3. Leuzinger H, et al. (2005) Herpes simplex virus 1 envelopment follows two diverse pathways. J. Virol. 79(20):13047-13059.

4. Wild P, et al. (2002) The significance of the Golgi complex in envelopment of bovine herpesvirus 1 (BHV-1) as revealed by cryobased electron microscopy. Micron 33(4):327-337.

5. Schwartz J & Roizman B (1969) Concerning the egress of herpes simplex virus from infected cells: electron and light microscope observations. Virology 38(1):42-49.

The authors thank Bernard Roizman, University of Chicago, for providing the deletion mutant R7041(∆Us3)

Fig. 1: The ER runs from the perinuclear region into the outermost lamella of a Golgi field

Fig. 2: The ER transists via one Golgi field to the next

Fig. 3: The outer nuclear membrane continues via ER into Golgi membrane. The ER cisterna contains virions indicationg intraluminal transportation.

Fig. 4: Virions in vacuoles and Golgi cisternae contradicting wrapping. Two capsids are in the process of wrapping.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2137 Phosphatidylinositol 4,5-bisphosphate interacts with lysine-specific histone demethylase 1 in the nucleus.

Kalasova I.1, Ulicna L.1, Vacík T.1, Hozák P.1
1Institute of Molecular Genetics ASCR v.v.i. Department of Biology of the Cell Nucleus, Vídenská 1083, 142 20, Prague 4, Czech Republic.
kalasova@img.cas.cz

The eukaryotic nucleus is a highly structured cellular compartment composed mainly of proteins and nucleic acids. In addition to these abundant molecules, the nuclear interior also contains minor components such as phosphoinositides. Phosphoinositides are phosphorylated forms of phosphatidylinositol - a negatively charged glycerol-based phospholipid. In the cytoplasm, phosphoinositides are well known signalling molecules involved in regulation of membrane dynamics, cell architecture and motility, modulation of ion channels and transporters, or generation of second messengers. Moreover, it has been suggested that phosphoinositide signalling occurs also in the cell nucleus. Phosphatidylinositol 4,5-bisphosphate (PIP2) localizes to nucleoli, nuclear speckles, and small foci in the nucleoplasm. It was shown that nuclear PIP2 is required for DNA transcription, pre-mRNA processing, and export of mRNA to the cytoplasm. Here we present a novel PIP2 binding protein - lysine-specific histone demethylase 1 (LSD1). LSD1 was previously shown as a potential PIP2 interacting protein using neomycin extraction. We confirmed that LSD1 forms a complex with PIP2 within the nucleus and that the LSD1-PIP2 interaction is direct. As shown by structured illumination microscopy, LSD1 localizes to the edges of nuclear speckles and co-localizes with PIP2 in small PIP2 foci in the nucleoplasm. These data indicate that PIP2 through LSD1 may regulate histone demethylation and therefore may play a role in regulation of gene expression.

This project is supported by the project BIOCEV (CZ.1.05/1.1.00/02.0109) from the European Regional Development Fund, by the Grant Agency of the Czech Republic (GA P305/11/2232), by the Ministry of Education, Youth and Sports of the CR (LD12063), by the Charles University Grant Agency (606112), and by the institutional grant (RVO: 68378050).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2146 Three-Dimensional Ultrastructural Analysis of Neuronal Cells in Brain and Pharmacological Effects of Bromocriptine on Motor Behaviors of hLRRK2 (G2019S) Transgenic Mice

Je A. R.1,2, Huh Y. H.1, Choi J.2, Lee Y.2, Lee J. K.2, Kim H. J.1,2, Kim M. J.1, Kim H. R.2, Kim H. G.2, Kweon H. S.1
1Division of Electron Microscopic Research, Korea Basic Science Institute, Daejeon 305-806, Korea, 2Department of Pharmacology, College of Medicine, Dankook University, Chungnam 330-714, Korea
hskweon@kbsi.re.kr

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic causes of late-onset, autosomal dominant Parkinson’s disease (PD).1-2 Recently, our group has produced transgenic mice by the insertion of human LRRK2 mutant (G2019S) gene in mouse, showing motor impairments similar with PD.3 In this study, we first investigated the three-dimensional ultrastructural alteration of subcellular organelles in neuronal cell of LRRK2 transgenic mouse using high voltage electron microscope and electron tomography. Most of the mitochondria in neuronal cells were swollen and the mitochondrial membrane and cristae were severely disrupted in the striatum and substantia nigra of transgenic mice. Redundant loops of myelin sheath were shown in both striatum and substantia nigra region of LRRK2 mice (Figures 1 and 2). After we confirmed the structural deterioration of subcellular organelles of neuronal cells, we further investigated pharmacological effects of bromocriptine, a dopamine D2 receptor agonist, on the motor behavior of transgenic mice. LRRK2 (G2019S) transgenic mice (9~20 months old) have motor deficiency in rota-rod test, and also the mice showed impairments in total moving distance, rearing frequency and moving duration in open field test. Treatment with bromocriptine (10mg/kg, for 7days) to LRRK2 (G2019S) transgenic mice had a positive effect on the retention time on rota-rod when compared with control group. In addition, transgenic mice showed increased moving distance, frequency rearing and moving duration that were comparable to control group. These results indicate that administration with bromocriptine can decreased motor deficiency in transgenic mice. These data suggest that LRRK2 (G2019S) transgenic mice model is of value in the screening of drugs against a dopamine D2 receptor agonist for neurodegenerative disease like PD. In future studies, we will analyze whether the bromocriptine treatment can also recover ultrastructural integrity of neuronal cells in striatum and substantia nigra of LRRK2 (G2019S) transgenic mice.

References

[1] Hardy J et al., Curr. Opin. Genet. Dev. 19: 254-265 (2009).

[2] Lesage S and Brice A, Hum. Mol. Genet. 18: 48-59 (2009).

[3] Lee KS et al., Korean J. Physiol. Pharmacol. 17: 89-97 (2013).

This research was supported by the Korea Basic Science Institute grant (E34700) and the Bio & Medical Technology Development Program of the National Research Foundation funded by the Ministry of Science, ICT & Future Planning (2013M3A9A9050076).

Fig. 1: Ultrastructural alterations of mitochondria and myelin sheath in neuron. Most of the mitochondria (m) in neuronal cell were swollen and the mitochondrial membrane and cristae were severely disrupted in LRRK2 transgenic mice. ds, dendritic spines; m, mitochondria; my, myelin sheath; ps, presynaptic axon.

Fig. 2: Three-Dimensional reconstructions of the mitochondria in substantia nigra from wild-type and LRRK2 (G2019S) TG mouse, with section thickness ranging from 300 to 500 nm.The tilt series containing 121 images were recorded using HVEM, over a tilt range from -60° to 60°, with an interval of 1°.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2187 Structural analysis of mouse primary cilia and centrosome

Kuwahara R.1, Shigeta M.2
1Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan, 2Kyoto Prefectural University of Medicine, Kyoto, Japan
kuwahara@uhvem.osaka-u.ac.jp

A Cilium is one of the organelles found on the surface of eukaryotic cells, and centrosome is the organelle which acts as microtubule organizing center (MTOC) of animal cells and regulates the cell cycle. The formation of cilia and centrosome is organized by centrioles, which are important for cell division, polarity and motility.
Cilia are projections from cell surface and have two types: one kind is motile cilia, which exert mechano-signaling found in airway, ovary, ventricle and sperm. The other is primary cilia, non-motile cilia, which exert sensory function found in kidney, bone, inner-ear, and eye. Several studies have reported that dysfunction of primary cilia lead to many diseases such as cystic kidney disease, and disease caused by dysfunction of cilia is called Primary Ciliary Dyskinesia(PCD). Similarly, deregulation of centrosome is associated with several disease including cancer and developmental diseases. Despite the important roles of primary cilia and centrosome, little is known about their structures.
This study was performed to determine the structure of primary cilia and centrosome. To this end, we established several mutant cells which were loss of PCD related genes such as Mks1, Cep290, Ift88 and verified the structural change of mutant cells. We focused on the observation of basal body and trandition zone in primary cilia, which are particularly important for protein transport into cilia. And to clarify the centrosome structure during cell division, we characterized the centrosome structure of mitosis.

This work was supported by "Nanotechnology Platform" (project No. 12024046) of the Ministry of Education, Culture, Sports,Science and Technology(MEXT), Japan.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2208 DEB-1 localization and its dynamics in the gonads of C.elegans during meiosis

Fukalová J.1, Rohožková J.1, Hozák P.1
1Institute of Molecular Genetics, Dept. of Biology of the Cell Nucleus, Academy of Sciences of the Czech Republic, Prague, Czech Republic
fukalova@img.cas.cz

C. elegans is a well-established model organism for the study of meiosis. In the adult C. elegans hermaphrodite, germline nuclei progress more proximally, away from the distal tip of the gonad arm, they exit the mitotic cell cycle and enter meiotic prophase I. Cytologically recognizable nuclei in the pachytene stage of meiotic prophase I are visible before the “loop”.
Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Recently, it was found that vinculin can be localized to nuclei, binding especially to regions around nuclear pores and under nuclear lamina. In addition we localized vinculin for the first time in meiosis-specific structures – the synaptonemal complexes. Regarding our previous observation we suggest vinculin as a possible new actor in the mammal meiosis. To support this possibility we found ortholog protein of vinculin in C. elegans called DEB-1. DEB-1 is a muscle attachment protein found in dense bodies, and is required for attaching actin filaments to the basal sarcolemma.
First of all we localized DEB-1 in isolated gonad of hermaphrodite with specific monoclonal antibody. We detected specific diffused signal in the entire gonad and within rachis (the central part of gonad), where distal tip cell and sheet cell covering the gonad were marked more intensive. To determine the consequence of decreased presence of DEB-1 on meiosis in hermaphrodite gonads, we transformed worms with DEB-1 RNAi. Lack of DEB-1 (deb1-/-) disrupts the regular arrangement of germline nuclei. In detail, in comparison to mock transformed control, the anterior part of gonad contained a lot of apoptotic cells, also was missing the transition zone where chromosomes should form chromosomal bouquet. In closer view chromosomes we found, that chromosomes are not paired in the whole gonad.
We used immunolocalization for electron microscopy to confirm and characterize the appearance of DEB-1 in gonad of hermafrodite worm. TEM analysis can reveal changes in the morphology of germline nuclei within WT gonads and deb1-/-. We compared the DEB-1 localization changes via immunogold labeling on ultrathin sections of C.elegans and evaluated grouping of gold particles by spatial statistics with previously developed plugins to the Ellipse image processiong program.

This study was supported by the Technology Agency of the Czech Republic (TAČR, FEI) , Reg. No MEYS TE01020118, 253306.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2215 Gadolinium in the lactating mammary gland tissue. Ultrastructural and microanalytical study

Ayadi A.1, Maghraoui S.1, Mhamdi M.1, Badri N.1, Zaroui A.2, Ben Jemaa N.3, Tekaya L.1
1Laboratoire de physiologie Faculté de Médecine de Tunis (Université Tunis El Manar), 2Service de cardiologie Hôpital la Rabta (Université Tunis El Manar), 3Service d’Embryologie Centre de maternité, la Rabta (Université Tunis El Manar)
tekaya.leila@gmail.com

Gadolinium (Gd), a rare earth, is frequently used in the medical and industrial domains. In the medical field Gd is essentially employed as a contrast agent in the Magnetic Resonance Imaging (MRI). In the industrial field it is used in manufacture televisions, car batteries, computer components, CD Roms, reason for which previous works have attempted to study its behavior in many organs such as liver, kidney etc. However, the precise intracellular localization of this element in the lactating mammary gland cells remains poorly understood.The aim of this work was to study the intracellular localization of Gd in the rat lactating mammary gland and the chemical form of the intracellular deposits. The ultrastructural localization  of Gd was studied using the conventional transmission electron microscopy(CTEM).The chemical determination of the intracellular deposit was performed using electron probe microanalysis (EPMA). Ultrastructural observations showed the presence of many dense granules in the lysosomes of mammary glandular epithelial cells of the Gd treated rats. The EPMA showed that the intralysosomal deposits were composed of Gd and phosphorus. These results showed that after its intraperitoneal administration, Gd was concentrated in the lysosomes of the glandular epithelial cells as an insoluble Gd phosphate salt. The present observations remind those previously published showing that after its parenteral administration Gd was observed in the liver, the kidney, the spleen and the bone marrow. More sophisticated methods have to been carried out to study the impact of the presence of Gd or other mineral elements on the organism.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2218 Highly toxic aberrant astrocytes have abundant and diverse secretory granules

Jiménez-Riani M.1, 2, Díaz-Amarilla P.1, Trías E.1, Casanova G.2, Barbeito L.3, Olivera-Bravo S.1
1Departamento de Neurobiología Celular y Molecular, IIBCE, Uruguay, 22Unidad de Microscopía Electrónica, Facultad de Ciencias, UdelaR, Uruguay, 33Institut Pasteur Montevideo, Uruguay
mjimenez510@gmail.com

Astrocytes are known to secrete a number of signaling molecules that participate in the communication of both neurons and glial cells. These signaling molecules, named gliotransmitters, are stored in two types of secretory vesicles: translucent small synaptic-like vesicles and large dense-core vesicles. It also has been described that hippocampal astrocytes have a 1,4,5-triphosphate(IP3)-induced Ca2+ dependent regulatory pathway that is characterized by the expression and stimulated exocytosis of Secretogranin II (SgII) [1].

Recently our group isolated a new population of highly toxic anomalous astrocytes that were named AbAs (Aberrant Astrocytes) [2]. Since AbA toxicity is mostly due to released soluble factors, we hypothesized that AbAs are enriched in regulated secretory vesicles and prototypic markers such as SgII and Chromogranin A (Chr-A). Confluent cultures of AbAs in basal conditions showed a difuse perinuclear expression of both markers. Western blotting and semiquantitative analysis of the bands positive to SgII and Chr-A related to β-actin bands indicate that AbAs expressed more SgII and Chr-A than neonatal astrocytes.

TEM analysis allowed us to identify an enormous heterogeneity in cytoplasmic granules, with differences in size, morphology, electron-density and membranous content. They are frequently found clustered together in areas of the cytoplasm. This abundance of cytoplasmic granules and the fact that AbA cells release soluble factors and are SgII and Chr-A positive suggests that in some of these secretory granules there might be the toxic component found in the extremely toxic AbA conditioned media. TEM immunogold labeling is needed to confirm.

References:

[1] Yong Suk Hur, et al (2010). PlosOne. Vol 5, issue 8, e11973

[2] Díaz-Amarilla P, et al. (2011). Proc Natl Acad Sci U S A 108: 18126–18131.

Uruguayan Program for the Development of Basic Sciences (PEDECIBA), National Agency for Research and Innovation (ANII).

Fig. 1: See below

Fig. 2: TEM of AbA cells showing the heterogeneity of dark cytoplasmic granules. Note the differences in size, morphology and shape.

Fig. 3: Western blotting evidencing the presence of SgII and Chr-A in AbA cells and neonatal astrocyte culture. The quantification of the relative areas to β-actin show a significant increase of SgII in AbA cell culture compared to the astrocytes. * indicates p<0.05.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2244 Structural study of centriole duplication process by using mice over-expressing centrosomal proteins

Gogendeau d.1,2, Trepout s.1,2, Messaoudi c.1,2, Tassin a. m.3, Marco s.1,2, Basto r.4
1Institut Curie, Centre de Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France, 2INSERM U759, Campus Universitaire d'Orsay, Bât. 112, 91405 Orsay cedex France, 3CNRS, Centre de Génétique Moléculaire, Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France, 4CNRS UMR 144, 12 rue Lhomond, 75005, Paris, France
delphine.gogendeau@curie.fr

Centrosomes are the major microtubules organizing centers of animal cells. Centrosomes are composed of two centrioles surrounded by a mesh of proteins called the pericentriolar material. Centrioles consist of a cylinder surrounded by nine sets of microtubules triplets and their structure is remarkably conserved throughout evolution. Centriole duplication is tightly regulated during the cell cycle. In KE37 human cells, it has been recently shown that mother and daughter centrioles are transiently linked by fibers (connecting stalk) at the initiation of duplication (Guichard et al. 2010). Presently, we are interested to identify the molecular players contributing to the formation of the connecting stalks. To this purpose we are using mice strains that over-express centriole-duplication proteins to further characterize, by cryo-electron-tomography, the initial steps of centriolar duplication in biological conditions closer to the native state.

As a proof of concept and with the aim of characterizing mouse centrioles, we have purified centrosomes from the TTA1.6 lymphocyte cell line. Cryo-electron tomograms of mouse centrosomes show that they contain two centrioles with the canonical structure: centriole appendages and nine microtubule triplets where the cap on the A-microtubule can be identified depending on the maturation stage (figure 1). This validates the use of mice centrosomes to study centriole duplication by cryo-electron tomograms.

References:

- Guichard et al, EMBO J. 2010 May 5;29(9):1565-72.

This work is funded by French Government “Agence Nationale de la Recherche” ANR- 11-BSV8-0016

Fig. 1: Section of cryo-tomograms from wild type mice TTA1.6 lymphocytes isolated centrosomes. A) Orthogonal projection of a centriole, showing the nine microtubule triplets B) Centriole where distal appendage is visible (arrow). Scale bars=100nm

Fig. 2: Longitudinal section of a mature centriole and its pro-centriole surrounded by pericentriolar material. The cartwheel is visible inside the procentriole (arrowhead). Other sections from the same tomogram reveal the presence of capped microtubules in the pro-centriole and uncapped microtubules in the mature centriole (insets). Scale bar=100nm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2262 Telocytes: new players in the interstitium of human uterus

Cretoiu D.1, 2, Cretoiu S. M.1, 2
1Carol Davila University of Medicine and Pharmacy, Bucharest, Romania, 2Victor Babeş National Institute of Pathology, Bucharest, Romania
dragos@cretoiu.ro

Background. Although the role of interstitial space is frequently neglected there are some new cellular elements – the telocytes (TCs)– that seem to be vital (along with other cells). Our goal is to bring further evidence of TCs involvement in a lot of processes such as: tissue patterning during development and tissue renewal after injury. These processes are probably mediated by the vesicular transport carriers which can create an adequate microenvironment for precursor cells.
Methods. We used transmission electron microscopy, immunohistochemistry (IHC), immunofluorescence (IF), on human myometrial tissue and in cell cultures.
Results. TCs have a tiny cell body with distinctive extensions named telopodes (Tps). A TC can have 1-5 Tps with alternating podoms (dilated segments) and podomers (thin segments). Podoms provide accommodation for mitochondria, ER and caveolae, a trio involved in calcium homeostasis. Tps have a dichotomous branching pattern, building a 3D network due to homo-and heterocellular junctions. In human uterus the heterocellular contacts were seen to be established between TCs and myocytes. Moreover, TCs establish cell-cell nanocontacts with immune cells. TCs release shed vesicle and/or exosomes, thus sending macromolecular signals to surrounding cells probably modifying their transcriptional activity.
Conclusion. Telocytes are supposed to be involved: (1) in intercellular signaling; (2) as stem cell adjutants involved in tissue renewal; (3) as sensors for steroid hormones; (4) in the guidance of immune cells; (5) as stretch sensors; and (6) as contractility modulators.

This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number 82/2012 PN-II-PT-PCCA-2011-3.1-0553.

Fig. 1: A.Two cellular bodies (TC1, TC2) can be easily seen in the interstitial space between smooth myocytes. One telocyte has long, convoluting telopodes (TC2). B.Details at higher magnification from area marked in A with a dotted square. Note the heterochromatin mostly confined to the periphery of the nucleus but also dispersed throughout.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2285 Deletion of CD133 C-terminal cytoplasmic domain affects the architecture and dynamics of plasma membrane protrusions and the release of small extracellular membrane vesicles in human colon cancer cells.

Lucchetti D.1, Palmieri V.2, Fanali C.1, Farina M.1, Svelto M.1, Cufino V.1, De Spirito M.2, Sgambato A.1
1General Pathology Insitute, 2Physics Insistute
dona.lu87@libero.it

CD133, a transmembrane glycoprotein considered a marker of colon cancer stem cells, is confined to lipid microdomains enriched in cholesterol. The cytoplasmic tail of CD133 contains a PDZ-binding domain, which could potentially interact with the cytoskeleton changing the dynamics of plasma membrane protusions. Aims of this study was to evaluate the functional effects of the deletion of CD133 C-terminal domain on the architecture and dynamics of plasma membrane protusions in HCT116 human colon cancer cells. Effects of this deletion have been also evaluated on the levels of released Extracellular Vesicles (EVs), membrane-bound sacs that are shed from the surfaces of cells into the extracellular environment. Indeed a link between the release of CD133 contained in membrane vesicles, cell protrusions and the cellular differentiation has been suggested.
The cDNAs encoding the full length CD133 molecule and its variant deleted of the C-terminal (C-term) domain were cloned into the expression vector pCDNA3 and transfected into HCT116 cells. CD133/1-PE antibody was used for immunofluorescence. Conditioned culture medium of colon cancer cells was collected and subjected to differential centrifugation for EVs isolation. EVs were morphologically characterized by Electron Microscopy (TEM) and their quantities were determined using Bradford assay. The number and length of plasma membrane protusions were studied with Scanning Electron Microscopy (SEM). Cells were stained with Laurdan and the multiphoton microscopy for image analysis was used to measure membrane fluidity.
Derivatives of the HCT116 cells (HCT-CD133) overexpressing full length CD133 displayed long protrusions on cell surface while cells (HCT-CtermCD) overexpressing the C-term variant expressed abnormal membrane structures (Figure1). Immunofluorescence analysis showed that CD133 is uniformly distributed in HCT-CD133 cells, while it forms platforms of aggregated proteins in HCT-CtermCD cells (Figure2). CD133 overexpression increased cell fluidity both in its full length and, more markedly, in its C-term variant (Figure3). A reduction in EV quantities was evident in the HCT-CtermCD cells, when compared with HCT-CD133 cells (Figure4). In conclusion an intimate relation between CD133 expression, membrane fluidity regulation and protrusions in colon cancer cells was showed. These findings suggest that changes in membrane microdomain organization associated with the expression of the C-term variant of the CD133 protein could be involved in impaired extracellular membrane traffic. Given the importance of membrane microdomains in signal transduction and in various membrane trafficking events, the evaluation of new players, such as CD133, in the architecture of plasma membrane protrusions is crucial.

Fig. 1: Fig.1 Representative images of HCT-V (control vector), HCT-CD133 and HCT-CtermCD obtained with SEM. The expression of full-length CD133 causes the appearance of long protrusions (D) and in increased the number on cell surface while HCT-CtermCD cells abnormal membrane structures are evident.

Fig. 2: Fig.2 CD133 distribution in HCT-CD133 (A) and HCT-CtermCD (B) cells.

Fig. 3: Fig.3 Mean Whole Cell GP. Increased fluidity in HCT-CD133 and HCT-CtermCD with a more pronounced increase in the latter (A). The most affected compartment in the HCT-CtermCD cells is the plasma membrane (B). Indeed while GP of HCT-CD133 has a small increase compared to HCT-V, the outer membranes of CD133-CtermCD display the highest GP (C-D).

Fig. 4: Fig.4 Identification and characterization of EV: exosome (Exo) and microvesicles (MV). (A) TEM for morphological study of exosomes (Exo) and microvesicles (MV); (B) isolated EV were lysated and protein concentration was determined using the Bradford method.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2318 ULTRASTRUCTURE OF THE SPERMATOGENESIS OF Spondylus calcifer (Carpenter, 1857) AND S. princeps (Gray, 1825) (MOLLUSCA: PELECIPODA: SPONDYLIDAE)

Villalejo-Fuerte M.1, Camacho M. A.1, Arellano M.1, Cuellar N.2, Lopez E. O.3, Esther U.3
11 Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas, Paz, Baja California Sur, México. , 22 Departamento de Biología. Facultad de Ciencias Naturales. Universidad de Oriente, Santiago de Cuba., 33 Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas
mvillale@ipn.mx

Marcial Villalejo-Fuerte1, Marian Alejandra Camacho Mondragón1, Marcial Arellano Martínez1, Nilia Cuélllar Araújo2, Esther Uría Galicia3, Edgar Óliver López Villegas3

1 Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas-IPN, Av., S/N, Col. Playa Palo de Santa Rita. CP 23096. La Paz, Baja California Sur, México.

2 Departamento de Biología. Facultad de Ciencias Naturales. Universidad de Oriente, Santiago de Cuba.

3 Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Unidad Profesional Lázaro Cárdenas, Prolongación de Carpio y Plan de Ayala s/n, Col. Santo Tomas C.P. 11340 Delegación Miguel Hidalgo México, D.F.

*corresponding author: mvillale@ipn.mx

ABSTRACT

Spondylus calcifer (Carpeter, 1857) and S. princeps (Gray, 1825) are bivalve gonochoric and free spawning with fertilization occurring in seawater, both species are distributed on coastal rock of American Pacific Ocean. We describe for the first time the ultrastructural development of the cells during spermatogenesis, and the morphology of the mature spermatozoa were characterized by transmission electronic microscopy. The testes in both species are tubular. The development of the spermatogonia and spermatocites of 1st and 2nd order are concentrically arranged inside the tubules with the less developed cells closer to the wall, and the spermatids and spermatozoa closer to the lumen. The tubules are surrounded by a matrix of connective tissue with accessory cells which function is equivalent to the Sertoli cells in mammals. Four stages were distinguished in gametes development of both species: Spermatogonia (4-8 μm), Spermatocites (3-4 μm) (primary and secondary), Spermatids (4-8 μm) and Spermatozoa (~2 μm head length). In both species, the spermatozoid shows similar morphology, which supports the close phylogenetical relationship between them. Both species also have four mitochondria arranged circularly in the midpiece, typical of external fertilization species and ancestral spermatozoids. The evolutionary implications and relevance of these results for the taxonomy of the Spondylus species and sustainable use of their populations are discussed.

EDI, COFAA, IPN, CONACyT

Fig. 1: Figure 1. Transmission electron micrographs of spermatogenesis in Spondylus princeps. Spermatogonia (spg); nucleus (n); mitochondria (m); primary spermatocyte (spt1); secondary spermatocyte (spt2); spermatid (spm); support cells (sc).

Fig. 2: Figure 2. Transmission electron micrographs of mature spermatozoa of Spondylus calcifer. Nucleus (n); flagella (fl); pseudopodia- like projections (pp).

Fig. 3: Figure 3. Transmission electron micrographs of mature spermatozoa of Spondylus princeps. mitochondria (m); acrosome (ac).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2321 Staining in non-isotope metals to cell structure in serial sections from animal tissues

Muranaka Y.1, Nishida T.1, Park S. M.2, Park P.3
1Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Japan, 2Research Center for Environmental Genomics, Kobe University, Japan, 3Emeritus Professor of Kobe University, Japan
muranaka@uhvem.osaka-u.ac.jp

Uranium has been used widely as the pre-stain of electron double stain for electron microscopy. However, the use and purchase have become more difficult in recent years. Therefore, the study on developing alternative uranium is underway. Some non-isotopic heavy metals such as hafnium chloride (HF), samarium chloride (SC), platinum blue (PB) were recommended in other studies as alternative stains of uranyl compounds to TEM sections from aldehyde-and osmium-fixed biological tissues. The cell contrast in tissues stained with the metals and lead was compared in the nearly same spatiality sites of serial sections with that in tissues with uranyl acetate (UA) and lead (Pb). The stains used were in the six followings: 2% UA aqueous solution and 50% EtOH, 4% HF in 50% and 100% EtOH, 4% SC solution, Pb solution while the test tissues were liver, kidney, small intestine, nerve, skeletal muscle of mice. Seven serial sections with 60 nm in thickness were cut from each of the blocks. Each of sever sections were stained with different staining methods used here. The contrast of cell images due to different stains was examined with penetration rates of stains in sections. The staining mechanisms of stained in nucleic acid and proteins will be discussed from the result obtained. In addition, the non-isotope staining solution are explored to replace the uranium in field of the ultra-high voltage electron microscope such as the method of three-dimensional reconstruction. Information of penetration of the stain and the contrast of the image is very useful.

Fig. 1: Hepatocytes TEM image stained with 2% uranium acetate in aqueous solution. Ultrathin sections of the three following the ultra thin section in Figure 1, is used 2, 3 and 4.

Fig. 2: Hepatocytes TEM image stained with 4% hafnium chloride in 50% alcohol solution. Staining of the cell substrate is comparable to uranium acetate.

Fig. 3: Hepatocytes were stained with TI blue stock solution was diluted 5-fold with 50% alcohol. in comparison with other stains, Ti blue is difficult to obtain the contrast such as staining of chromatin is low.

Fig. 4: Hepatocytes were stained with 4% aqueous solution of chloride Samaniumu. Staining closest to uranium is obtained. Necessary to study the permeability of the resin, the staining of the different organizations.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2344 Structural changes of cherry tomatoes (Lycopersicon esculentum) packed in bags based on thermoplastic corn starch/talc nanocomposites

Lopez O. V.1,2, Castillo L. A.2, Garcia M. A.1, Barbosa S. E.2, Villar M. A.2
1CIDCA (CONICET-UNLP), La Plata, Argentina, 2PLAPIQUI (CONICET-UNS), Bahía Blanca, Argentina
mvillar@plapiqui.edu.ar

Fruits are dehydrated to enhance storage stability, minimize packaging requirement and reduce transport weight [1]. The aim of this work is to evaluate the feasibility of using packages based on thermoplastic starch/talc nanocomposites to dehydrate cherry tomatoes (Lycopersicon esculentum), evaluating their cells structural changes. Tomatoes were packed within bags of thermoplastic corn starch (TPS) with 0 and 5% w/w talc. Tomatoes were sorted for uniform size, color, and without physical damage. To decrease fruits initial microbial charge, half of them were dipped in chlorinated water (250 ppm Cl2) before being packaged. Packaged samples were stored (50% RH-25ºC) for 2 months. Samples were removed from bags, weighted and conditioned. Cubic samples were immersed in glutaraldehyde to preserve their cellular structure. Samples were rinsed with phosphate buffer before post-fixation step by immersing them in osmium tetroxide; then they were washed with bidistilled water, dehydrated using acetone, and dried until the critical point. Finally, samples were examined in a JEOL JSM-35 CF electron microscope and mean cell diameters (Dc) were determined by considering around 100 cells using AnalySis Pro 3.0 software program. Samples suffered alterations in their color and volume due to natural dehydration (Fig. 1). Macrostructural changes were induced by microstructural modifications. Fresh tomatoes tissue has a well-organized structure of rounded cells and intercellular spaces (Fig. 2a). Dehydration caused reduction in tomatoes volume due to large moisture gradients that induced microstructural stresses, collapsing capillaries and decreasing intercellular contact [2]. Both visual observation and SEM demonstrated the lack of microbial activity in all dehydrated samples. Lost weight for tomatoes packed in TPS and TPS-talc bags was 92 and 69%, respectively. Dc for samples within TPS was lower than those corresponding to tomatoes packed in TPS-talc bags (Fig. 2b and 2c). Lost weight is in good agreement with Dc and it is attributed to the higher capacity to retain water of TPS-talc bags compared to TPS ones. Talc enhances films barrier properties due to it hinders vapor diffusion. Chlorine treatment caused a higher damage in cell tissues (Fig. 2c and 2d). Despite chlorine affected tomatoes structure, lost weight was similar than those corresponding to unchlorinated ones. Dc corresponding to chlorinated samples could not be determined due to tissue destruction and cells collapse. In conclusion, bags based on TPS/talc nanocomposites were suitable to dehydrate naturally tomatoes cherries without affecting fruits microbial quality.

[1] Sagar V. & Sureh K. J Food Sci Technol 47(1), 15-26, 2010.
[2] Zotarelli M., Almeida P. & Laurindo J. J Food Eng 108, 523-531, 2012.

Authors gratefully acknowledge to Viviana Sorrivas and Cecilia Gutierrez Ayesta (CCT-CONICET, Bahía Blanca, Argentina) for their technical support.

Fig. 1: Photographs of: a) fresh tomatoes, b) and c) dehydrated tomatoes packed in TPS and TPS with 5 % w/w talc, respectively, d) and e) dehydrated chlorinated tomatoes packed in TPS and TPS with 5 % w/w talc, respectively.

Fig. 2: SEM micrographs (500x) on tissues of: a) fresh tomatoes, b) and c) dehydrated tomatoes packed in TPS and TPS with 5 % w/w talc, respectively, d) and e) dehydrated chlorinated tomatoes packed in TPS and TPS with 5 % w/w talc, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2357 Human neutrophil activation by Kojic Acid

Frade P. R.1,2, Costa J. P.1,2, Rodrigues A. D.2,4, Farias L. S.1,2, Do Nascimento J. M.3, Silva E. O.1,2
1Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Pará, Brazil, 2Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Brazil, 3Laboratório de Neuroquímica, Instituto de Ciências Biológicas,Universidade Federal do Pará, Belém, Pará, Brazil, 4Laboratório de Microscopia Eletrônica, Instituto Evandro Chagas, Secretaria de Vigilância em Saúde do Ministério da Saúde, Belém, Pará, Brazil
edileneoli@gmail.com

Neutrophils has a crucial role on cell physiology and act as professional phagocytes to eliminate pathogens. During the cell activation, morphological and physiological changes results in various antimicrobial effectors mechanisms and production of superoxide anion and other reactive oxygen species can be upregulated. The Kojic Acid (KA) is a secondary metabolite synthesized by some species of fungi that has several applications, largely used as a food additive, tyrosinase inhibitor and macrophage activator. The aim of this study was to perform an in vitro analysis of human neutrophils activation by KA. Human neutrophils were isolated from buffy coats of healthy human donors by density sedimentation (Histopaque® 1077-density-gradient). Neutrophils were treated for 1 hour with 50 μg/mL of KA and positive control cells were treated with 100 nM of phorbol 12-myristate 13-acetate (PMA) and compared with control without treatment. The morphological analyses were performed by optical microscopy (Figure 1), scanning electron microscopy and transmission electron microscopy (Figure 2). Before the treatment cells exhibited rounded shape that changed to a polarized morphology with many cells exhibiting an increase of the cell volume, membrane projections and high spreading ability (Figure 1 and 2). Treated cells analyzed by immunofluorescence microscopy showed enhanced spreading, associated with a rearrangement of actin filaments (Figure 3). For superoxide detection, treated neutrophils were analyzed with a cytochemical assay using nitroblue tetrazolium salt (NBT). Treated cells showed formazan deposits distributed in the cellular cytoplasm, showing intense superoxide production (Figure 4C), in comparison with the untreated cells (Figure 4A). The similar results were observed to positive control treated with PMA (Figure 4B). The reaction was observed in approximately in 75% of the KA-treated cells. Increase ROS production was detected by labeling with CellROX® green. The number of CellROX labeled cells was superior to untreated cells (Figure 4E), revealing that KA stimulated a large production of ROS. In conclusion, this study demonstrates that KA significantly induced neutrophils activation.

CAPES, CNPq, UFPa , Instituto Nacional de Biologia estrutural e Bioimagem, PRONEX/FAPESPA/CNPq,Ministério da Saúde-MS

Fig. 1: The morphological analysis by optical microscopy (A-B) and Morphometric analysis (C). (A) Control cells with typical morphology (B) Neutrophils treated with 50 µg/mL of KA. (C) Morphometric analysis showed a significant increased in the cell area when neutrophils were treated with 50 µg/mL KA. Test T was used. (*) P <0.05. Bar: 10 μm.

Fig. 2: The morphological analysis by scanning electron microscopy (A-B) and transmission electron microscopy (C-D). (A,C) Control cells with typical morphology (B,D) Neutrophils treated with 50 µg/mL of KA. Treated cells with extensive cytoplasmic projections and increase of cell volume. Bar: 2,5 μm.

Fig. 3: Actin filaments detected by fluorescence in neutrophils exposed to 50 mg/mL KA for 1 h (A–C) Fluorescence labelling of actin filaments with phalloidin and DAPI in untreated cells (B-D), KA-treated cells (B-D) with enhanced filopodium establishment. Bars: 10 μm.

Fig. 4: Detection of Superoxide production by NBT assay (A-D) and ROS Production by CellROX® (E). (A) Non-treated neutrophils;(B) Cells treated with 100 nM PMA; (C) Neutrophils treated with 50 µg/mL KA. Bars: 10 μm (D)Number of neutrophils that presented formazan deposits (E)The number of CellROX labeled cells. ANOVA was used. (*) P <0.05.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2358 Differentiation in vitro of bone marrow cells into macrophages induced by Physalis angulata

SILVA B. M.1,3, RODRIGUES A. D.3,4, FARIAS L. S.1,3, NASCIMENTO J. M.2, SILVA E. O.1,3
1Laboratório de Biologia Estrutural/Laboratório de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Brazil. , 2Laboratório de Neuroquímica, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Pará, Brazil. , 3Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Brazil , 4Laboratório de Microscopia, Instituto Evandro Chagas, secretaria de vigilância em saúde, ministério da saúde, Belém, Pará, Brazil.
edileneoli@gmail.com

The bone marrow is a hematopoietic tissue that in the presence of cytokines and growth factors generates all circulating blood cells. These cells are important to protect the organism against pathogens and for establishing an effective immune response. Some studies in literature have been showing action of immunomodulatory effects of different products obtained of plant extracts. Thus, the aim of this study is to evaluate the immunomodulatory properties aqueous extract of the root plant Physalis angulata (AEPa). Bone marrow cells (BMC) were obtained by flushing femurs, and maintained in cultures treated with AEPa at a concentration of 100 µg/mL. It was observed by optical microscopy (Figure 1) increase of cellular area, high spreading ability and number of cytoplasmic projections. Furthermore, AEPa did not promote the proliferation of lymphocytes and polymorphonuclear leukocytes, however promotes increased the number of macrophages in the culture. The ultrastructural analysis by MET (Figure 2) of BMC treated showed spreading ability, high number of cytoplasmic projections and increase of autophagic vacuoles. Flow cytometry showed increased labeling LC3b (Figure 2e) in treated BMC, indicating autophagic process. Immunophenotyping was performed by flow cytometry. F4/80 labeling (Figure 3), a specific marker for mononuclear phagocytes revealed that AEPa seems to stimulate differentiation of BMC into macrophages and did not stimulate differentiation into dendritic cells (Figure 4). Thus, these results demonstrate that AEPa can promote the differentiation of BMC into macrophages in just 96 hours of treatment and AEPa could be used as an immunomodulator agent.

CNPq, CAPES, Ministério da Saúde-MS, Instituto Nacional de Biologia Estrutural e Bioimagem (INBEB), FAPERJ, PRONEX/CNPQ/FAPESPA.

Fig. 1: Morphological analysis of BMC treated with AEPa by 96 hours. a) Untreated control. b) BMC treated with AEPa, observing the spreading increased cell (arrow). c) BMC treated with M-CSF, observe the process of cell fusion (arrow). Bar 10μm. d) Morphometric analysis. ANOVA was used, Student t. (*) P <0.05.

Fig. 2: Ultraestrutural analysis in BMC treated with AEPa by 96 hours. a) Untreated control. b) BMC treated with M-CSF. c-e) BMC treated with AEPa, observe cytoplasmic projection (arrows) and presence of autophagic vacuoles (*). Bar (a-d) 5μm, (e) 1 μm. f) Fluorescence intensity of BMC labeled with LC3b. ANOVA was used. (*) P <0.05.

Fig. 3: Detection of the surface markers F4/80 by flow cytometry in BMC treated with AEPa by 96 hours. a) Peritoneal macrophages. b) Untreated control. c) BMC treated with AEPa. d) BMC treated with M-CSF. BMC treated with AEPa expressed more F4/80 than untreated BMC. e) Fluorescence intensity of BMC labeled with F4/80. ANOVA was used. (*) P<0.05.

Fig. 4: Detection of the surface labelled CD11c (dendritic cells) in BMC treated with AEPa 96 hours. Analysis by indirect immunofluorescence. a) Untreated control. b) BMC treated with AEPa. Analysis by flow cytrometry. c) Fluorescence intensity of BMC labeled with F4/80. ANOVA was used. (*) P <0.05.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2430 Architecture of the cuticle and epidermal cells: imaging and analyses of mineralized matrix assembly and disassemby in isopod crustaceans by TEM, FEG-SEM and EDXS

Žnidaršič N.1, Mrak P.1, Žagar K.2, Čeh M.2, Štrus J.1
1Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia, 2Jožef Stefan Institute, Department for Nanostructured Materials, Jamova 39, 1000 Ljubljana, Slovenia
nada.znidarsic@bf.uni-lj.si

Assembly and disassembly of extracellular matrices can be considered from the cell biology perspective, including tissue/cell ultrastructure and matrix dynamics, and from the physico-chemical standpoint of mechanisms lacking direct cellular control. In general, chitin-based and collagen-based networks are the two alternative organic scaffolds for biomineralized structures. Crustacean cuticle is an epidermal apical matrix composed of chitin-protein fibers and mineralized by calcite, amorphous calcium carbonate and calcium phosphate. Cuticle de novo formation during embryonic development and during molting in adults involves secretion of cuticular constituents, elaboration of an ordered hierarchical structure, calcification, establishment of mechanical connections to muscles and formation of surface cuticular structures. Simultaneously, the old cuticle disassembly, resorption and shedding is performed.

In our study a cuticular matrix in different forming / degradation phases and associated tissues were imaged by a combination of scanning and transmission electron microscopic techniques (SEM, TEM). Imaging was performed on samples prepared primarely to preserve the mineral component (methanol fixation) and on samples prepared to determine the cell ultrastructure. Cuticular matrix displayed different ultrastructures with respect to its renewal phase, homogenous matrix or helicoidal pattern and layering were recorded (Fig. 1a). A very intense connection of the forming matrix and underlying epithelial cells was evidenced by the presence of numerous protrusions of the apical cytoplasm into the chitinous scaffold (Fig. 1b). Epidermal cells contained electron dense vesicles at the apical plasmalemma, abundant rough endoplasmic reticulum, various amounts of glycogen, Golgi complexes with surrounding vesicles, mitochondria and microtubules beneath the cell surface. Ecdysal space is an unique extracellular compartment between the old degrading cuticle and the newly assembling cuticle, in which we observed a variety of structures related to cuticle renewal, the most outstanding were several types of spherules (Fig. 2a). A network of cytoskeletal and junctional elements constitutes the cuticle-muscle attachment complexes. In premolt specimens the newly produced cuticle is already extensively connected to underlying tendon cells by massive arrays of fibers, expanding from the tendon cells, traversing the entire new cuticle and ecdysal space and protruding into the distal layers of the detached cuticle (Fig. 2b). Tendon cells are characterized by extensive parallel arrays of microtubules and their basal membrane is involved in prominent anchoring junctions with muscle cells. Our results suggest that the cuticle of the newly hatched youngs is already calcified.

Fig. 1: Figure 1a: Ultrastructural pattern of chitin-protein fibers in methanol fixed cuticle in early larva manca of Porcellio scaber. Figure 1b: Intense interconnections of the epidermal cell (ec) and its apical cuticular matrix in assembly phase in adult P. scaber as evidenced by prominent cytoplasmic protrusions (→) into the chitinous scaffold.

Fig. 2: Figure 2a: Spherules in the ecdysal space of premolt Ligia italica. Figure 2b: Newly forming chitinous matrix (nc) in premolt L. italica was anchored to the apical parts of tendon cells (tc) by numerous bundles of filaments (⌂), that traversed the new cuticle and the ecdysal space (es) and extended deeply into the old cuticle (oc).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2455 Detailed membrane association between mitochondria and surrounding endoplasmic reticulum in mammal cells revealed by three-dimensional reconstruction using focused ion beam scanning electron microscope

Ohta K.1,2, Okayama S.1, Togo A.1, Nakamura K.1
1Dep. Anatomy, Kurume University School of Medicine, Fukuoka, Japan, 2Riken Quantitative Biology Center, Osaka, Japan
kohta@med.kurume-u.ac.jp

Physical contacts between mitochondria and the endoplasmic reticulum (ER) and the mitochondria-associated ER membrane (MAM) around these contact points have important roles for several cellular functions, including phospholipid transport, Ca2+ signaling, and autophagy. It has recently been found that these contact sites are closely related to mitochondrial division and contribute to mitochondrial quality control. Ultrastructural relationships of contact sites have been analyzed in yeast cells using cryo-electron tomography methods, but not so far in the mammalian cell. The mammalian cell MAM is larger than the yeast MAM in size, so detailed 3D understanding of its organization through either transmission electron microscopy observation of ultrathin serial sections (low spatial resolution) or electron tomography (the structure is too large) is difficult. We used a 3D reconstruction method using a focused ion beam/scanning electron microscope (FIB/SEM), which reconstructs the specimen through ion-beam serial ablation and SEM block face imaging—a potential method to observe mesoscopic scale objects with a 10-nm spatial resolution. Using this method we attempted to reveal the entire 3D organization of the MAM in the mammalian cell. A resin embedded HeLa cell and rat hepatocytes were used as specimens and the entire membrane organization of the cells was reconstructed by FIB/SEM (Quanta 3D FEG, FEI Co.). The reconstructed volumes clearly depicted the ER contact sites on the outer mitochondrial membrane, showing two types of membrane interaction. One is a tether structure between the ER and mitochondria that has been reported in mammalian cells, which is a similar structure to that referred to as ERMES (ER-mitochondria encounter structure, identified in yeast). The other membrane interaction is direct adhesion between the ER and the mitochondrial membrane. These two types of contacts were present on a mitochondrion simultaneously. At the portion of mitochondrial constriction that implies mitochondria division, ER contacts were also observed around the constriction. Although the structure of the MAM in mammalian cells was slightly different from those in yeast, in which a ring-like ER tube surrounds the constriction, in the mammal cell a large part of the constriction site was also wrapped by small ERs. These observations suggest that the MAM in mammalian cells is also related to mitochondrial fission and its quality control. We need to further research whether MAM-related proteins are localizing here.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2500 Does adipokinetic hormone play a role in neuronal communication within the insect brain?

Weyda F.1, Kodrík D.1,2, Stašková T.2, Pflegerová J.2
1Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic, 2Institute of Entomology, Biology Centre, ASCR, Branišovská 31, 370 05 České Budějovice, Czech Republic
weydafk@seznam.cz

Adipokinetic hormones (AKHs) are a group of well-known insect metabolic neurohormones. Those hormones are responsible for activation of insect organism under stress conditions and for keeping the body homeostasis. AKHs are synthesized and released from an endocrine retrocerebral gland - the corpus cardiacum, however, small amounts of the hormone have been identified also in the brain. Interestingly, a role of AKH in the brain is not satisfactory elucidated. To contribute to solving of this problem we studied a subcellular localization of AKHs in the brain of the firebug Pyrrhocoris apterus (Heteroptera, Insecta), which possesses two AKHs: octapeptides Pyrap-AKH and Peram-CAH-II. Confocal and immunoelectron microscopy revealed that in brain neurones the hormones are synthesised in specialized secretory granules that are localized predominantly in neuronal bodies, from them they are probably transported into the axons, where the hormones might play a role in neuronal signalling. This was supported by recording of the positive AKH immunoreaction in axons unequivocally outside the granules. The neurotransmittor function of AKH in insect brain is assumed for a long time, nevertheless, the situation is far from clear and the direct proof is still missing.

For immunoelectron microscopy we have used 4% formaldehyde (EM Grade) fixation, embedding into Epon-araldite resin or into LR White resin, polyclonal rabbit anti-Pyrap-AKH as primary and anti-rabbit as secondary antibody conjugated with colloidal gold. Sections were stained with lead citrate, carbon coated and examined under the Jeol 1010 transmission electron microscope.

Acknowledgement. This study was supported by grant GACR 14-07172S (DK) from the Czech Science Foundation, and by project No. Z50070508 of the Institute of Entomology, funded by the Academy of Sciences of the Czech Republic. The authors thank Miss J. Zralá and Mrs. D. Rienesslová for their technical assistance, and Dr. D. Doležel for a kind providing of the RP49 gene primers.

Fig. 1: Immunoreaction in secretory vacuoles of corpora cardiaca (CC).

Fig. 2: Immunoreactive secretory vacuoles in some neurone cell body inside of brain.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2507 Evaluation of morphological differences of breast cancer cells using various biological markers

Simsone Z.1, Freivalds T.1, Harju L.1, Gudrā D.2, Kudaba I.3,4, Liepniece-Karele I.4,5, Bērziņš J.1, Buiķis I.1
1Institute of Experimental and Clinical Medicine, University of Latvia, Riga, Latvia., 2University of Luxembourg, Faculty of Science, Communication and Technology, Luxembourg, Luxembourg, 3Oncology Centre of Latvia, Riga East University Hospital, Riga, Latvia, 4Riga Stradins University, Riga, Latvia, 5Centre of Pathology, Riga East University Hospital, Riga, Latvia
z.simsone@gmail.com

Cancer cells more or less differ from cells of tumor origin. Cancer cells nucleus has undergone chromatin structure condensation and shape and also there are exchanged nucleolus volume and staining properties. Nevertheless it is suggested that cancer stem cells are responsible for resistance to anticancer treatment and tumour recurrence. There are various cell surface and nucleus antigens to identify cancer stem cells. For example CD44, CD24 and ADLH are known as breast cancer stem cell biological markers.
THE AIM OF THE STUDY is to determine cancer cell resistance, cell population heterogeneity and amount of DNA in luminal and triple negative breast cancer cell population using paraffin sections. It is necessary to find new criteria to estimate the efficiency of anticancer therapy.
MATERIAL AND METHODS
In two types of breast cancer- triple negative (n=11) and luminal (n=32) the CD44, CD24, and ALDH antigen expression were studied to evaluate cancer stem cell amount in population. Antigen expression was estimated immunohistochemicaly in paraffin sections by semi-quantitative method. In addition, both types of cancer histological samples were stained by Feulgen method to measure DNA amount in the cancer cell nuclei. HeLa cell culture was transfected with plasmid pGFP-N3 containing GFP gene.
RESULTS
The CD44 and ALDH expression was higher in triple negative breast cancer than in luminal breast cancer. We observed CD24 antigen expression in two cell types- in large- polyploid cells (with size 10-20µm) and in small cells- microcells (with size ~9µm). In both types of breast cancer we observed polyploid cells and microcells with phenotypes CD44+/CD24-/ALDH+ and CD44+/CD24-/ALDH+. According to these studies we conclude, that the luminal breast cancer small cell population contain cells with low and high DNA concentration, but large cells exhibit only low DNA concentration. In triple negative breast cancer cell population small and large cells contain high and low concentration of DNA.
In transfection experiments with HeLa cells we observed the GFP expression in microcells and little bit later in large cells after UV irradiation.
CONCLUSION
Based on this study, it seems that development and behaviour of microcells and polyploid cells are similar, but polyploid cells are in later developing phase after anticancer treatment. Consequently, our study suggested that both small and large cells with high concentration of DNA could be responsible for resistance but small cells could be resistant cell population progenitor.

Our research was accomplished with support from the University of Latvia, Faculty of Biology Student Governing. We wish to thank Riga East University Hospital Oncology Centre of Latvia and Pathology Centre for cooperation.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2529 Planar chromatin in interphase nuclei of human cells

Chicano A.1, Daban J. R.1
1Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain.
andrea.chicano@uab.cat

It was observed previously in our laboratory that the chromatin of human and chicken metaphase chromosomes at metaphase ionic conditions is organized as multilaminar platelike structures (1,2), instead of forming fibrillar structures (3). Furthermore, we found that chromatin fragments obtained from metaphase chromosomes digested with micrococcal nuclease associate spontaneously to form chromatin plates (4). These observations and results obtained in other laboratories led us to the proposal of a physical model for the structure of condensed chromosomes (5). Now we are interested in the study of chromatin structure in interphase cells. We have developed procedures for the preparation of nuclei from interphase HeLa cells, and we have assayed different treatments and ionic conditions to produce the extrusion of chromatin from partially denatured nuclei. The structure of the extruded material was visualized using transmission electron microscopy. We have used ultrasonication, rapid passage through a syringe needle, and vortexing in the presence of glass beads to induce emanation of chromatin from nuclei. The best yield was obtained with samples homogenized in the presence of glass beads. We have also obtained nuclei at different stages (G1, S, and G2) of the interphase using a fluorescence activated cell sorter, and we have denatured them using glass beads. Finally, we have digested interphase nuclei with micrococcal nuclease to obtain chromatin fragments (Fig. 1) and to study the structure of the aggregates produced at a high concentration of chromatin in the presence of different concentrations of Mg2+. Electron microscopy analysis of the preparations obtained in these experiments indicate that interphase chromatin from HeLa cells can form amorphous aggregates, short chromatin fibers, and planar chromatin. The micrographs corresponding to chromatin fragments (obtained by nuclease digestion of G1, S, and G2 nuclei) extensively dialyzed against solutions containing 2.5 mM Mg2+ (Figs. 2-4) and 5 mM Mg2+ show many self-assembled chromatin plates.
(1) J. M. Caravaca, S. Caño, I. Gállego and J. R. Daban (2005) Chromosome Res., 13:725-743.
(2) I. Gállego, P. Castro-Hartmann, J. M. Caravaca, S. Caño and J. R. Daban (2009) Eur. Biophys. J., 38: 503-522.
(3) J. R. Daban (2011) Micron, 42: 733-750.
(4) M. Milla and J. R. Daban (2012) Biophys. J., 103: 567-575.
(5) J. R. Daban (2014) J. R. Soc. Interface, 11: 20131043.

Work supported by MINECO research grant BFU2010-18939, and by a predoctoral fellowship (PIF-UAB) to A.C.

Fig. 1: (a) Isolated nuclei from HeLa cells were digested with micrococcal nuclease. (b-e) Chromatin fragments in 1 mM Pipes (pH 7.2) and 1 mM EDTA. Scale bars: (a) 1 µm, (b-e) 200 nm.

Fig. 2: Chromatin fragments from G1 nuclei associated by dialysis against an aqueous solution containing 2.5 mM Mg2+. Scale bar 200 nm.

Fig. 3: Chromatin fragments from S nuclei associated by dialysis against an aqueous solution containing 2.5 mM Mg2+. Scale bar 200 nm.

Fig. 4: Chromatin fragments from G2 nuclei associated by dialysis against an aqueous solution containing 2.5 mM Mg2+. Scale bar 200 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2676 The 3D-structure of a mouse seminiferous epithelium observed with serial block face-SEM

Hasebe Y.1, Haruta T.1, Searle S.2, Galloway S.2, Yarwood A.3, Nishioka H.1, Suzuki T.1
1JEOL Ltd. 3-1-2 Musashino, Akisima, Tokyo 196-8558 Japan, 2Gatan　UK. 25 Nuffield Way, Abingdon Oxon, OX14 1RL, UK, 3JEOL UK Ltd. Silver Court Watchmead, Welwyn Garden City AL7 1LT, UK
yhasebe@jeol.co.jp

 Cells in biological tissues contact other cells three-dimensionally and interact with each other. And cellular differentiation is considered to be induced by the interaction of cells. On the seminiferous epithelium, spermatogenesis takes place from spermatogonia to spermatids. It is important to sturdy interaction between cells which revealing arrangement and fine structure cell groups and Sertoli cell. Sertoli cells hold and feed these cell groups. Recently, the cellular network is frequently observed with confocal laser scanning microscopy(CLSM), Because it is easy to observe the connections between cells with CLSM. However, the observation with CLSM needs to label fluorescence markers on molecules of the specimen. Therefore if molecules in the tissue are unknown, CLSM is not enabled to be used for the observation of the specimen. Also, as the spatial resolution of CLSM is 200 nm, it is not enough to observe objects such as organelles, intercellular bridges and so on. On the other hand, the spatial resolution of scanning electron microscopy(SEM) is far better to be a few nanometers. Besides, SEM can be used to observe in a wide range of magnification. Therefore SEM is useful for the observation the cellular network such as organelles.

 Recently serial block face-SEM(SBF-SEM)[1]was developed. In this method an ultramicrotome is set in the specimen chamber of a SEM, the surface of a resin embedded specimen is cut with a diamond knife of the ultramicrotome, and the backscattered electron image (BEI) of an exposing specimen surface is taken. Then the old surface whose image has already been taken is cut out in a predetermined thickness to expose a new specimen surface underneath the older one to be taken another BEI. This process is repeated a number of times until the whole specimen is covered. All the BEIs taken are stored in a PC and used to reconstruct the three dimensional image of the specimen. The advantage of this method is that a wider area prepared with an ultramicrotome can be observed as a high resolution image with SEM.

 In this presentation we report the three dimendional structure of a seminiferous epithelium observed with the SBF-SEM. An SEM used in this study was an FE-SEM, JSM-7100F(JEOL, Japan), equipped with the Gatan 3View®(Figure 1). The specimen was a mouse seminiferius epithelium stained with the NCMIR method. This method had been developed to enhance BEI contrast together with eliminating the effect of charging. A part of the result is shown in Figure 2.

Reference

[1] Denk. W, Horstmann. H. Serial block-face scanning electronmicroscopy to reconstruct three-dimensional tissue nanostructure. Plos Biol. 2 p 1900-1909. (2004) 

Fig. 1: An Fe-SEM, JSM-7100F(JEOL, Japan),  combined with the Gatan 3View®.

Fig. 2: A mouse testis semifoerous epitithelium.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2602 Is coenzyme-Q has a protective effect against atorvastatin induced myopathy? A histopathological & immunohistochemical study in albino rates

Ahmed M. S.1
1Department of Medical Education, College of Medicine, King Saud University
msalah28@hotmail.com

Is coenzyme-Q has a protective effect against atorvastatin induced myopathy? A histopathological & immunohistochemical study in albino rates

Khalil MS1, Nehal Khamis2, Abdulmajeed Aldrees3, Abdulghani HM4.

1 Department of Histology, Faculty of Medicine, Suez Canal University, Egypt; College of Medicine, Department of Medical Education, King Saud University, Riyadh, KSA.

2Department of Pathology, Faculty of Medicine, Suez Canal University, Egypt; Department of Medical Education, College of Medicine, King Saud University, Riyadh, KSA.

3Department of Physiology, College of Medicine, King Saud University, Riyadh, KSA.

4Department of Medical Education, College of Medicine, King Saud University, Riyadh, KSA.

Introduction

In addition to its hypercholesterolemia reducing effect, statin has pleiotropic effects that may extend their use to the treatment and prevention of various diseases such as cancer, osteoporosis, multiple sclerosis, rheumatoid arthritis, type II diabetes, and Alzheimer’s disease. Therefore, the number of patients taking statin is expected to increase. The statin induced myopathy, which may result from reduced muscular coenzyme Q10 levels, limits their use. The current study investigates if the supplementing with Co Q10 could ameliorate the statin induced myopathy.

Materials and Methods

Forty adult male albino rats were randomized into 4 groups, 10 rats each and the following was administered using nasogastric tubes: Group 1: 2 ml of 0.5% carboxymethyl cellulose once daily. Group 2: 100mg/kg/ day Coenzyme Q10 dissolved in 2ml of cotton seed oil. Group 3: 10 mg/kg once daily atorvastatin dissolved in 0.5% carboxymethyl cellulose. Group 4: concomitantly received Co Q 10 and atorvastatin similar to groups II and III respectively. Plasma creatine kinase levels were measured by using spectrophotometer. The right extensor digitorum longus muscle sections were stained for histological (Haematoxylin & Eosin, Masson trichrome and Phosphotungstic acid haematoxylin ) and immunohistochemical (cytochrome C and Bax) examinations. Quantitative measures of cytochrome C and Bax were carried out using image analyzer.

Results

Atorvastatin induced increased total cytokeratin kinase, skeletal muscle variations in the sizes and shapes, necrosis, disorganization, nuclear pyknosis, karyorrhexis, karyolysis, dismantled plasma membrane, excess collagen fibers and lipid deposition in addition to loss of cross striation. Atorvastatin increased the intensity of the immune-positive reactions of Cytochrome C and Bax. These changes were ameliorated by concomitantly giving Coenzyme Q10.

Conclusion:

Co Q10 may ameliorate the atorvastatin induced skeletal muscle injury.

This work was funded by the College of Medicine Research Centre, Deanship of Scientific

Research, King Saud University, Riyadh, Saudi Arabia

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2609 Chloroplast ultrastructure of Fagus sylvatica leaves – 3D visualization using dual-axis electron tomography and serial sectioning

Radochová B.1, Michálek J.1, Janáček J.1, Čapek M.1, Lhotáková Z.2, Bílý T.3, Nebesářová J.3, Kubínová L.1, Albrechtová J.2
1Institute of Physiology ASCR, Prague, Czech Republic, 2Charles University in Prague, Czech Republic, 3Biology Centre ASCR - Institute of Parasitology, České Budějovice, Czech Republic
b.radochova@seznam.cz

Chloroplast ultrastructure is usually examined using transmission electron microscopy (TEM). However, due to the size of chloroplasts (diameter of about 3 to 8 µm), there is only limited extent of three-dimensional (3D) structural information when ultrathin sections (in average 70-80 nm) are used for examination. Therefore, several microscopy techniques for 3D reconstruction and visualization of chloroplast ultrastructure are used, including both serial sectioning and electron tomography. Each of them has advantages, disadvantages and pitfalls. Electron tomography is analogous to the tomographic techniques used nowadays; however the angular range allowed by the conventional TEM specimen holders is more restricted (usually to ± 60°or 70°) resulting in a wedge of missing information and subsequently in anisotropic resolution of tomograms. Dual-axis tomography, where images from two orthogonal axes are processed, is therefore used to improve isotropy in resolution. For electron tomography, thickness of the sections around 200 nm is usually recommended (depends on the voltage of an electron microscope). Serial sectioning technique is based on acquiring of TEM images from standard ultrathin sections that are subsequently aligned and visualized in 3D. It is both less time consuming and less technically demanding, but the resolution in the z axis is limited (to about twice the section thickness). Therefore, this method is useful mainly for rough estimation of chloroplast spatial organization.
In the present study, we used both dual-axis electron tomography and serial sectioning for visualization of the spatial arrangement of chloroplast ultrastructure in beech (Fagus sylvatica) leaves sampled at the experimental site Bílý Kříž (Beskids Mts., Czech Republic). Samples were chemically fixed, processed using microwave tissue processor and embedded into Spurr´s epoxy resin. Electron tomography projections of two orthogonal axes were acquired from 200 nm thick sections using JEOL JEM-2100F microscope. Tomograms were 3D reconstructed by the IMOD software package (Boulder Laboratory, Colorado). Serial sections (at least five consecutive sections) were viewed in JEOL JEM-1010 microscope and acquired image data were aligned using special software (Link MRC). Chloroplast components (e.g. thylakoids, starch grains and plastoglobuli) were traced in reconstructed series of images by interactive segmentation and then visualized using 3D plug-in in Ellipse software (ViDiTo, Košice, Slovakia). The most interesting part of beech chloroplast visualized in 3D was the occurrence of protrusions. These protrusions of chloroplast envelopes sometimes formed open pockets which included cell organelles (predominantly mitochondria).

This work was supported by Czech Science Foundation (P501/10/0340) and by the Academy of Sciences of the Czech Republic (RVO: 67985823) and by Technology Agency of the Czech Republic (TE01020118).

Fig. 1: Fig. 1: 3D visualization of beech chloroplast acquired via dual-axis electron tomography. Thylakoids in green, starch grains in yellow and plastoglobuli in brown. Note a cytoplasm-filled pocket with mitochondria in blue.

Fig. 2: Fig. 2: 3D visualization of beech chloroplast acquired by serial sectioning of ultrathin sections. Thylakoids in green, starch grain in yellow and plastoglobuli in brown. Note a stroma filled protrusion on the right side of the chloroplast.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2626 Colocalization of Amelotin, Odontogenic-Ameloblast Associated and Secretory Calcium-Binding Phosphoprotein-Proline-Glutamine-rich 1 in the basal lamina at the interface of ameloblasts and maturing enamel

Wazen R. M.1, Maia L.1, Dos Santos Neves J.1, Moffatt P.3, Nanci A.1, 2
1Faculty of Dentistry, Université de Montréal, Montreal, QC, Canada, 2Faculty of medicine, Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, QC, Canada, 3Shriners Hospital for Children / McGill University, Human Genetics, Montreal, QC, Canada
rima.wazen@umontreal.ca

An atypical basal lamina (BL) is present between the apical plasma membrane of post-secretory ameloblasts and the surface of maturing enamel. Unlike elsewhere in the body, this BL binds to mineral rather than connective tissue, and likely has had to adapt by integrating specialized molecules. It is rich in glycoconjugates and contains laminin-332 but not γ1 chain laminins, type IV and VII collagens. However, its precise composition still remains elusive. Screening of the secretome of ameloblasts has led our lab to identify genes encoding for three novel proteins called odontogenic ameloblast-associated (ODAM), amelotin (AMTN) and secretory calcium-binding phosphoprotein-Proline-Glutamine-rich 1 (SCPPPQ1). These genes reside in the secretory calcium-binding phosphoprotein (SCPP) gene cluster on human chromosome 4 (5 in rat and mouse) that encodes for proteins involved in the regulation of mineral deposition. We have previously demonstrated that AMTN and ODAM localize at the cell-tooth interface but still little is known about the distribution of SCPPPQ1 and the relationship between the three proteins. Objective: To compare the localization of AMTN, ODAM and SCPPPQ1 during amelogenesis. Methods: Mice and rats were perfused, hemimandibles were decalcified and processed for embedding in paraffin or LR White resin. Immunohistochemistry with rabbit antibodies against rat AMTN, ODAM and SCPPPQ1 was performed on paraffin sections. Single or double postembedding colloidal gold immunolabeling was carried out on ultrathin resin sections. Results: Immunolabeling confirmed that ODAM and AMTN were found in the BL at the cell-enamel interface throughout maturation. Labeling for ODAM seems to appear slightly earlier than that for AMTN. SCPPPQ1 was also detected at the cell-tooth interface but only from the later part of mid-maturation, and reactivity intensified into late maturation. Co-localization of the three proteins in the BL was confirmed by dual immunogold labeling. Both ODAM and SCPPPQ1 exhibited intense Golgi reactions in ameloblasts while cellular labeling for AMTN was extremely weak and seen only at the very beginning of maturation. Conclusions: The ultrastructural localization of AMTN, ODAM and SCPPPQ1 indicates that they are novel components of the BL associated with maturation stage ameloblasts. The sustained Golgi labeling for ODAM and SCPPPQ1 and the very restricted AMTN cellular reaction suggest that ODAM and SCPPPQ1 undergo renewal throughout maturation, while AMTN seems to be produced during a narrow time-frame and to remain stable. While the function of these proteins remains to be determined, their temporospatial distribution suggests that they may contribute to the mechanism binding ameloblasts to enamel, and/or to enamel maturation events.

CIHR, NSERC, RSBO, Shriners of North America.

Fig. 1: Dual immunogold labeling shows that ODAM, AMTN, SCPPPQ1 co-distribute and represent novel constituents of the basal lamina at the cell-tooth interface during maturation stage of amelogenesis. (A) SCPPPQ1 (large gold particles) and AMTN (small gold particles) and (B) SCPPPQ1 (large gold particles) and ODAM (small gold particles).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2627 Light Microscopy , Electron Microscopy and Co-relative light electron Microscopy (CLEM) to study RTN4B (Reticulon 4B) / NogoB and Endoplasmic Reticulum.

Kumar D.1, Rämö O.1, Vihinen H.1, Belevich I.1, Viita T.1, Joensuu M.1, Vartiainen M.1, Jokitalo E.1
1Institute of Biotechnology, University of Helsinki
darshan.kumar@helsinki.fi

The endoplasmic reticulum (ER) is the largest membrane-bound organelle spreading throughout the cytoplasm as a continuous membrane-enclosed network in mammalian cells. It comprises of the nuclear envelope along with a dynamic network of peripheral interconnected tubules and membrane sheets [1].Sheets are predominant over tubules in the central area of the cell and long interconnected tubules close to the plasma membrane(PM) at the periphery [2]. The ER in mitotic cells has been observed to undergo both spatial reorganization and structural transformation of sheets towards a more fenestrated and tubular form [2,3]. ER subdomains are believed to play a crucial role in a variety of functions ranging from Ca2+ handling to protein translocation [4].The Reticulons(Rtns) are membrane bound proteins found in all eukaryotic forms.In addition to the ER, the Rtns are known to insert to other cellular membranes including the PM and the Golgi apparatus.Rtn4B’s close neighbour Rtn4A is known to give rise to ER tubule structures in association with DP1 [5].
The aim of this project is to screen interacting partners for Rtn4B.We first narrowed down our search for interacting partners within the proteins of the Rtn family,and then broadened our scope towards other potential candidates.The screening was performed using the BiFC (Bimolecular fluorescence complementation) method [6] (Fig 2).These interacting partners will then be studied for their effects on ER structure maintenance and dynamics using a variety of imaging techniques such as immuno EM,electron tomography and serial block face scanning EM[3].First, we showed that Rtn4B was forming dimers and/or homo-oligomers as biochemical data has suggested [7], as BiFC signal was detected between two Rtn4B-constructs (Fig.1A).The BiFC signal (Green) in Fig.1B shows to co-localize with the signal from immunolabelled endogenous Rtn4b(Red) suggesting that the oligomerization was not restricted to certain subdomain of the rtn4B-positive ER or rtn4B molecules. In addition, using CLEM, we narrowed down the BiFC-GFP positive signal to pin-point the morphological changes on ER upon the overexpression of locked Rtn4b dimers/oligomers at an EM level (Fig.3).Electron tomography revealed the induction of tight network of narrow tubules that were connected to ER sheets and tubules in cells over-expressing BiFC-Rtn4b dimers/oligomers.
[1]Bobinnec Y,et al., Cell Motil. Cytoskeleton, 54 (2003)217–225.
[2]Puhka M, et al,. J. Cell Biol.,179 (2007)895-909.
[3]Puhka M, et al., MBoC,23(2012)2424-2432.
[4]Baumann O, et al., Int. rev. cytol.,205 (2001)149-214.
[5]Voeltz GK, et al., Cell,124(2005)573–586.
[6]Kaddoum L, etal. , BioTechniques,49(2010)727-736.
[7]Shibata.Y, et al., J. Biol. Chem.,283(2008)18892-18904.

This work was supported by the Academy of Finland along with VGSB and ILS doctoral programmes.

Fig. 1: Huh-7 cells expressing constructs with positive BiFC signal post 24hr incubation; A. 20X image of GFP signal from dimerization of RTN4b. B. 63X image of cells expressing GFP from RTN4b dimerization & antibodies against RTN4B(in red). C&D. 63X images of GFP from RTN4b dimerization. GFP(green),Nucleus(Blue) & anti-RTN4b(Red)

Fig. 2: Overview of BiFC technique

Fig. 3: A. Images from the same region of interest at different levels of microscopy of the positively expressing BiFC cells for Rtn4b dimers. B. Micrograph showing ER morphology from overexpression of Rtn4b dimers/oligomers. C. Serial tomography generated model from overexpression of Rtn4b dimers
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2907 Serial block face SEM for modeling cells of the pancreatic islet of Langerhans

Shomorony A.1, Pfeifer C. R.1, Zhang G.1, Xu H.2, Cai T.2, Kim Y. C.3, Aronova M. A.1, Notkins A. L.2, Leapman R. D.1
1National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, MD, USA, 2National Institute of Dental and Craniofacial Research, NIH, Bethesda MD, USA, 3U.S. Naval Research Laboratory, Washington DC, USA
leapmanr@mail.nih.gov

Serial block-face scanning electron microscopy (SBF-SEM) enables the reconstruction of large biological tissue volumes (105-106 µm3), i.e., extending over 100-μm in three dimensions [1]. The technique is complementary to electron tomography (ET), which provides higher resolution 3D ultrastructure from smaller volumes (e.g., 10-102 µm3). The SBF-SEM incorporates an automated ultramicrotome, which cuts plastic-embedded specimen blocks that are stained with heavy metals and imaged with backscattered electrons [1,2], resulting in a spatial resolution <10-nm in the plane of the block face and 25-nm in the perpendicular direction, as determined by the minimum slice thickness.

Here, we have applied SBF-SEM to quantify the architecture of mouse pancreatic islets of Langerhans, which are microscopic endocrine organs of diameter ~100 µm containing insulin-secreting beta cells, glucagon-secreting alpha cells, somatostatin-secreting delta cells, and polypeptide cells, as well as a network of capillaries lined by fenestrated endothelial cells. Using SBF-SEM, we have shown that structural parameters (number of secretory granules, total cell volume, nuclear volume, mitochondrial volume, and blood vessel geometry, etc.) can be derived with much higher accuracy than is possible using stereological measurement on randomly selected TEM images.

Fig. 1A shows x-y, y-z, and x-z slices through a portion of an islet acquired by SBF-SEM at a beam energy of 1.5 keV with 24-nm pixels in the plane of the block face and 50-nm thick sections. The visualization of a single beta cell in Fig. 1B, recorded with 5-nm pixels in the x-y plane and 25-nm slices along the z-axis, reveals a compact spherical nucleus surrounded by a branching network of mitochondria. Cross sections through an alpha and beta cell are shown in Fig. 2A and 2B, respectively. Sub-volumes of the beta cell (Fig. 2C) were used to determine the numbers of secretory granules per unit volume, from which it was possible to estimate the mean number of granules within a beta cell based on the average cell volume minus the average volume of the nucleus and mitochondria. Similar measurements were performed for alpha cells. It was found that the volume of alpha cells was 60 ± 5% that of the beta cells. Nuclear volumes of the two cell types were approximately equal but beta cell nuclei were round compared to a more irregular shape for the alpha cell nuclei (Fig. 3A and 3B).

The SBF-SEM is a promising tool for quantifying differences in ultrastructure between pancreatic islets in normal and disease models and can yield quantitative data that would be difficult to obtain by conventional TEM.

[1] W. Denk, H. Horstmann, PLoS Biology 2 (2004) 1900.

[2] Deerinck, T.J. et al. Microsc Microanal 16 (suppl 2) (2010) 1138.

Research supported by the intramural programs of NIBIB and NIDCR of the National Institutes of Health.

Fig. 1: (A) Orthoslices through region of mouse pancreatic islet of Langerhans obtained by SBF-SEM, showing distribution of secretory cells and capillaries; (B) visualization of beta cell showing plasma membrane (purple), nucleus (green) and network of branching mitochondria (red).

Fig. 2: (A) Slice through alpha cell, showing glucagon-secretory granules (GSG), mitochondria (M), endoplasmic reticulum (ER); (B) slice through beta cell, showing insulin-secretory granules (ISG) with dense cores and halos, mitochondria (M), and Golgi (G); (C) volume of beta cell from which number of ISGs per unit volume can be determined.

Fig. 3: Visualization of plasma membranes (purple) and nuclear membranes (green) for two beta cells and two alpha cells, showing larger beta cells with round nuclei and smaller alpha cells with irregularly shaped nuclei.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-2926 Nucleus Size and Shape Mystery

Ganguly A.1, Bhattacharyya D.1
1Advanced Centre for Treatment, Research & Education in Cancer, Navi Mumbai, MH 410210 India
asgc.ganguly@gmail.com

Regulation of intracellular organelle architecture is a fundamental cell biological problem. “Karyoplasmic ratio” [KR] (the ratio of the nuclear volume to cell volume) remains roughly constant in cells with different cellular conditions, widely different DNA contents, and ranging from single-cell eukaryote to mammalian cells [1, 2]. However, the strong association of aberrant nuclear size and shape with tumor grade [3] tempts to speculate that nuclear size and shape is tightly regulated in normal cells which is disrupted in case of cancer cells. In this study, mammalian cell lines and budding yeast were used to develop assay systems to monitor nuclear size and shape under live cell condition with the nucleus and the cell membrane labeled with two different colored fluorescent proteins. In one approach, the variations of KR in different immortalized and transformed cell lines of same tissue origin, were determined. A significant increase was observed in KR of transformed cell lines compare to immortalized cell lines. Present concepts rely on nuclear size is determined by a limiting soluble component that originates in the cytoplasm and is transported to the nucleus. Importin subunit beta 1-334 does not allow import [4]. RanT24N & RanQ69L are very well documented for nuclear transport perturbation [5, 6]. We observed a significant increase in KR upon overexpression of Importin subunit beta 1-334(P<0.001), RanT24N (P<0.01) and RanQ69L (P<0.05) in immortalized cell line HaCaT. This increased KR phenotype was rescued when wild type Importin subunit beta and Ran were overexpressed along with respective dominant negative proteins. Interestingly, Nucleolus volume was also found to increase significantly and numbers of nucleolus decreased upon Importin subunit beta 1-334(P<0.01) and RanQ69L (P<0.01) overexpression. Our initial results suggest that the nuclear transport indeed an important factor for regulating nuclear size and also have a correlation with nucleolar volume in mammalian cells. In another approach, >200 genes, responsible for strange nuclear shape were identified from ‘mitocheck’ database [7].Mutational effect of 27 genes (with reported yeast homolog) were studied in Saccharomyces cerevisiae. Out of 27 genes only 4 genes showed altered nuclear shape and increased nuclear surface area. For example, KAR3 deletion showed increased nuclear surface area compare to wild type cells although KR is not changed significantly. Using Auxin induced degron system, immediate depletion of KAR3 protein in wild type strain is analyzed presently under live cell condition. Functional implications of all four genes will be studied.

[1]. PMID:15596464
[2]. PMID:17998401
[3]. PMID:15343274
[4]. PMCID: PMC1169714
[5]. PMID: 7988569
[6]. PMID:9739075
[7]. PMID:20360735

Bhattacharyya Lab Member,
ACTREC Imaging facility and Genomics facility
ACTREC for project funding and doctoral fellowship

Fig. 1: Effect of Importin Subunit Beta 1-331 overexpression on Nucleus in HaCaT cells: Significant increase(P<0.001, N=100) in KR was observed when transfected with Importin subunit beta 1-334 along with nuclear marker Lamin-GFP and cell membrane marker F-mcherry; confocal images were taken including z stack, 3D rendering was done for volume measurement. 

Fig. 2: Effect of Importin Subunit Beta 1-331 overexpression on Nucleolus in HaCaT cells:Significant increase (P<0.01, N=50) in nucleolar volume and decrease in numbers were observed when transfected with Importin subunit beta 1-334 and Fibrillarin-GFP; confocal images were taken including z stack, 3D rendering was done for volume measurement.

Fig. 3: Effect of KAR3 deletion on Nucleus Shape in S. cerevisiae: Nucleus shape is drastically changed with increased nuclear surface area in KAR3 deletion condition compared to Wild type cells in which nucleus membrane and cell membrane are tagged with Nup116-GFP and Ras2-mcherry respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3001 Ultrastructure of Cytoplasmic and Nuclear Inosine-5’-monophosphate Dehydrogenase 2 Inclusions

Jůda P.1, Šmigová J.1, Raška I.1
1Charles University in Prague, First Faculty of Medicine, Institute of Cellular Biology and Pathology, Czech Republic
fpaj.work@gmail.com

Inosine-5´-monophosphate dehydrogenase (IMPDH) is a key enzyme in the de novo biosynthesis of guanosine nucleotides. IMPDH catalyzes NAD-dependent oxidation of inosine 5´-monophosphate (IMP) to xanthosine 5´-monophosphate, the first and rate-limiting step towards the synthesis of guanosine triphosphate (GTP) from IMP. This reaction can be inhibited by specific inhibitors like ribavirin or mycophenolic acid that are widely used in clinical treatment when required to inhibit the proliferation of viruses or cells. However, it was recently found that such an inhibition affects the cells, leading to redistribution of IMPDH2 and appearance of IMPDH2 inclusions in cytoplasm. According to their shape, these cytoplasmic inclusions have been termed “Rings and Rods” (R&R). In this work we focused on subcellular localization of IMPDH2 protein and ultrastructure of R&R inclusions. Using microscopy and Western blot analysis, we showed the presence of nuclear IMPDH2 in human cells. We found that the nuclear pool represents approximately 22% of cellular IMPDH2 and it has an ability to form Rod structures after inhibition by ribavirin. Concerning the ultrastructure, we observed that R&R inclusions in cellulo correspond to accumulation of fibrous material that is not surrounded by a biological membrane. The individual fibers were made up of regularly repeated subunits with length about 11 nm. Together, we showed the localization of IMPDH2 also inside the nucleus of human cells and described the ultrastructure of R&R inclusions.

This work was funded by the Czech Science Foundation [P302/12/G157], the Charles University in Prague [UNCE 204022] and [Prvouk/1LF/1], OPPK [CZ.2.16/3.1.00/24010] and OPVK [CZ.1.07/2.3.00/30.0030]. PJ was partially supported by grant [GA UK 610512] from the Charles University in Prague.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3109 Two strategies for zinc sequestration in the moss Pohlia drummondii and Physcomitrella patens

Sassmann S.1, Weidinger M.1, Wernitznig S.1, Lang I.1
1Cell Imaging and Ultrastructure Research, The University of Vienna, Austria Althanstrasse 14, A-1090 Vienna, Austria
ingeborg.lang@univie.ac.at

High resolution light and electron microscopy combined with specific detection techniques for metal ions is the key for novel findings on the cellular and subcellular level. All these methods, including various cryo-fixation techniques, are applied in the Core Facility at the University of Vienna, Austria, to address tricky questions and allow for its examination from different points of view.

One of our current research topics is focussed on growth analyses on zinc spiked media in the moss species Pohlia drummondii and Physcomitrella patens. X-ray microanalyses in the scanning electron microscope showed a clear uptake of the metal by the plantlets but the cellular distribution remained unclear. Fluorescence labelling with the zinc-specific dye FluoZin-3 shows the retention of the metal in the cell wall of P. drummondii which might enable this species to inhabit former mining sites. In P. patens, normally living at non-contaminated sites, the zinc-specific dye enters the cell and is apparently scavenged in the cytoplasm. Recent X-ray microanalyses confirm this phenomenon: mosses from metal habitats show less uptake than P. patens. To detect the influence of zinc on the photosynthetic pathway, the production of autochthone starch was measured in polarized light microscopy and by staining with Lugol's iodine. Transmission electron micrographs indicate the abundance of starch grains in control cells whereas zinc-treated cells of P. patens contain less autochthone starch.

Taken together, we propose an extracellular barrier to enable the colonization of metal contaminated sites (as for P. drummondii) and intracellular uptake in combination with detoxification mechanisms in P. patens.

Many thanks to Ursula Lütz-Meindl, the University of Salzburg, Austria, for fruitful discussions and technical support in high pressure freezing and freeze substitution. This work was supported by the Vienna Anniversary Foundation for Higher Education (grant H-1939/2008 to IL and H-2486/2012 to SS)

Fig. 1: Scanning electron micrograph of a young moss plantlet (Physcomitrella patens), grown on ZnCl2 1mM medium.

Fig. 2: The zinc specific dye FluoZin-3 is retained at the cell wall of Pohlia drummondii (a); it is taken up into the cytoplasm of Phycomitrella patens (b). Autochthone starch in a chloroplast of P. patens (c).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3120 Single Cell Compression Testing

Lukes J.1, Grzanova P.2, Fuzik T.2, Sepitka J.1
1Czech Technical University of Prague, Hysitron Nanomechanical Applications Lab, Prague, Czech Republic, 2Institute of Chemical Technology, Prague, Czech Republic
jaroslav.lukes@fs.cvut.cz

Mechanical properties of a cytoskeleton or a cell itself are considered to be a quantitative parameter for cell diversification or disease. A nanoindenter or an atomic force microscope is usually used for the assessment of the mechanical properties of a single cell. Appropriate testing probes as well as mechanical models must be chosen, in order to correctly interpret the mechanical loading and derive the intrinsic material characteristic of a cell. In this case, the compression tests of a single cell were performed by a Hysitron TI 950 TriboIndenter® [Hysitron, Inc., Minneapolis, USA] nanomechanical test instrument with a 100 µm diamond flat end probe (90° fluid cell conical).
However, a clear visualization of the living cell needs to be established in order to precisely position the probe with the X and Y coordinates of the cell. There are two microscopy regimes available for the TriboIndneter – bright field and fluorescence, both top-down. Based on previous experiences, COS-1 cells [ATCC code: CRL-1650] were used due to their long viability and good adhesion properties. The cell line was derived from an African green monkey kidney; the cells grow attached to the base (adherent) and have the same morphology as fibroblasts [ATCC, USA].
The practical use of green fluorescence of EGFP modified cells exposed to a blue light applied by the TriboIndnter microscope will be discussed and compared to standard bright field microscopy also available for cell localization. Compressive load-displacement data demonstrating a critical bursting force of a cell membrane will be also presented.

This research was supported by Grant Agency of the Czech Technical University in Prague, grant No. SGS13/176/OHK2/3T/12.

Fig. 1: Fluorescence image demonstrates an adhered cell to a cover slip. Beneficially, the top most location, where a cell nucleus is located, can be targeted for a compression test.

Fig. 2: Image shows the less detailed bright field image of single cell inside of PBS. Rounded dead cells can be also identified.

Fig. 3: Displacement controlled compression data. The bursting of a cell membrane can be identified as a drop in the force signal during the test.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3151 Piecemeal degranulation is the main secretory process of eosinophils during the experimental acute Schistosoma mansoni infection

Dias F. F.1, Amaral K. B.1, Malta K. K.1, Silva T. P.1, Chiarini-Garcia H.2, Melo R. C.1
1Federal University of Juiz de Fora, Juiz de Fora, Brazil, 2Federal University of Minas Gerais, Belo Horizonte, Brazil
ffdias@gmail.com

Schistosomiasis is a chronic disease caused by trematoda Schistosoma mansoni that triggers granuloma formation [1]. Eosinophils, cells of the innate immune system, are recruited and migrate to sites of granulomatous response playing effectors functions against parasite eggs through the release of secretory granule-derived proteins [2]. These granules exhibit an ultrastructurally unique morphology, with a crystalline core, and store a significant number of cytokines and cationic proteins in preformed pools within them [3]. Since little is known about the processes of secretion of these cells in response to helminthic infections, this work aimed to investigate the secretory processes involved in the eosinophil secretion during the experimental infection with Schistosoma mansoni. Female Swiss mice (n=6) were infected with 100 cercariae of S. mansoni percutaneously. Animals were euthanized (animal ethical approval CEUA/FIOCRUZ # LW-32/2012) after 55 days post-infection (acute phase) and liver fragments were processed for light microscopy (LM), conventional transmission electron microscopy (TEM), and immunogold electron microscopy using a pre-embedding approach for detection of Major Basic Protein (MBP), the major cationic protein stored within eosinophil secretory granules. LM revealed the presence of high number of infiltrating eosinophils surrounding the S. mansoni eggs into hepatic granulomas of infected mice, some of them in close contact with the S. mansoni eggs surface (Fig. 1). TEM revealed distinct eosinophil degranulation processes in the S. mansoni-infected livers, such as classical granule exocytosis and, mainly piecemeal degranulation (PMD). PMD (Fig. 2) was characterized by morphological changes of specific granules (enlargement, reduced electron-density, core disarrangement and coarse granule matrix) and presence of a high number of cytoplasmic vesicles, indicative of a vesicle-mediated transport of granule-stored products. Granules undergoing PMD were immunolabeled for MBP (Fig. 3). This means that the secretion of eosinophil products during the acute infection is occurring through mobilization and release of specific molecules. Altogether, our findings confirm that eosinophils are key cells in the S. mansoni acute infection and identify PMD as a major process of secretion in response to the infection. Moreover, the understanding of all events and mechanisms governing differential sorting, packing and secretion of granule-stored mediators may be also fundamental to the goal of specifically blocking eosinophil secretion as a therapeutic strategy.
References:
[1] Gryssels et al. (2012). Infect Dis Clin N Am, 26: 383–397.
[2] Shamri et al. (2011). Cell Tissue Res, 343: 57-83.
[3] Melo & Weller (2010). Histol Histopathol, 25: 1341-1354.

Supported by CAPES, CNPq and FAPEMIG.

Fig. 1: A hepatic granuloma shows infiltrating eosinophils (*) surrounding and in close contact with the S. mansoni egg (arrows). Livers from S. mansoni-infected mice were processed for light microscopy and 5μm-thickness sections were stained with hematoxylin-eosin (HE). Bar: 30µm.

Fig. 2: Electron micrograph of an eosinophil in process of piecemeal degranulation (PMD). Secretory granules show morphological changes (*) – enlargement and core disarrangement - indicative of a vesicle-mediated transport of granule-stored products. Vesicles (arrows). Livers from S. mansoni-infected mice were processed for TEM. N: nucleus. Bar: 1.3μm.

Fig. 3: (A, Ai) Eosinophil secretory granules undergoing PMD are immunolabeled for Major Basic Protein - MBP (arrowheads) (A). Liver fragments were immunolabeledd for MBP, using pre-embedding immunogold electron microscopy. N: nucleus. Bar: (A) 1.3μm; (Ai) 0.65μm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3154 The extension and contraction mechanism of the proboscis of a ciliate, Lacrymaria olor

Yanase R.1, Sonobe S.1
1University of Hyogo, Hyogo, Japan
ryuji.yanase@gmail.com

A single cell microorganism, Lacrymaria olor has an elastic neck called “proboscis” and captures foods by using oral part associated with its distal end. The proboscis can be extended to be ten times longer than the original cell length quickly and repeatedly. The aim of this research is focused on its mechanism. In the previous researches, it is thought that the extension is elicited by the movement of the oral cilia and sliding in the microtubule bundles located under the cell membrane including the undercoat structure called “pellicle”. However, molecules concerned with such motility have been unknown. Accordingly, at first, we analyzed contribution to the extension and contraction of the movement of oral cilia by using a high-speed camera. And also, we observed the movement of the oral part and proboscis separated from the body of Lacrymaria olor. From these observations, it is concluded that the oral part highly contributes to the directional changes and extension power of the proboscis. Moreover, we stained the microtubules with fluorescence antibody and observed them by a confocal microscope. And I observed the ultrathin section of Lacrymaria olor by an electron microscope to see the microtubules located. From these observations, it is revealed that the microtubules are oriented in a spiral mode on the whole body of Lacrymaria olor. But, in spite of total coverage of microtubules on the body, the proboscis can move freely. It may indicate that the microtubules may also move or change the shapes to make the movement of the proboscis possible.

We thank A. Miyazawa and Y. Nishino for assistance with electron microscopy, K. Hatta and M. Ito for the use of high-speed camera, and M. Hayakawa and I. Hayakawa for discussion and valuable comments.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3161 A CLEM approach to investigate ultrastructural changes associated with the overexpression of lamin B1 in a mammalian cell line.

Marotta R.1, Ruffilli R.1, Giacomini C.2, Gasparini L.2, Falqui A.1
1Electron Microscopy Lab, Nanochemistry Department, Istituto Italiano di Tecnologia, IIT, Genoa, Italy, 2Neuroscience and Brain Technologies/Neuro Technology, Istituto Italiano di Tecnologia, IIT, Genoa, Italy
roberto.marotta@iit.it

Lamins are intermediate filament proteins constituting the major structural component of the nuclear lamina, a fibrillar meshwork that lines the inner nuclear membrane in eukaryotic cells. The nuclear lamina plays various cellular functions, providing skeletal support for the nuclear envelope, mediating the attachment of the nuclear envelope to interphase chromatin, and allowing the proper organization and anchoring of the nuclear pore complexes. Vertebrates synthesize a variety of lamins, namely lamin A, B and C, which are encoded by different genes or generated by differential RNA splicing. Recently, gene duplication and protein overexpression of lamin B1 (LMNB1) have been reported in pedigrees with autosomal dominant leukodystrophy (ADLD). However, how the overexpression of LMNB1 affects nuclear ultrastructure remains unexplored. To investigate the morphological changes associated with the overexpression of LMNB1 we transiently transfected mammalian cells with bicistronic expression vectors containing cDNA for both the LMNB1 protein and the enhanced green fluorescent protein (EGFP) reporter or the EGFP reporter alone. Coupling in vivo confocal fluorescent microscopy with transmission electron microscopy (TEM) and electron tomography (ET), we were able to selectively focus our ultrastructural investigation only on EGFP-positive transfected cells. The over-expressed LMNB1 was located to the nuclear lamina, as revealed by confocal fluorescent microscopy. TEM and ET observations on the LMNB1 over-expressing cells revealed the presence of membrane structures forming extensive arrays of stacked cisternae aligned with the nuclear envelope, or laying inside the nucleoplasm. These cisternae clearly expressed LMNB1 within their stacks, as revealed by immuno EM. Moreover, in the nuclear envelope of those cells overexpressing LMNB1 the nuclear pores complexes were clustered together. To minimize the artifacts due to chemical fixation and room temperature dehydration, the transfected cells, once analyzed by confocal fluorescent microscopy, were processed by high pressure freezing and freeze substitution. Respect to conventional EM preparation, nuclear membranes appeared smoother; and the stacked cisternae were separated by a larger nucleoplasmic space. These morphological data clearly demonstrate that LMNB1 overexpression deeply alters the structural organization of the cell nucleus.

We are grateful to Mattia Pesce for technical assistance during confocal fluorescent analysis.

Fig. 1: Fluorescence images of nuclei from LMNB1-EGFP transfected HEK293 cells growing on a grid carbon-printed on an ACLAR disk. The nuclei were counterstained with Hoechst-33342.

Fig. 2: Fluorescence images of nuclei from LMNB1-EGFP transfected HEK293 cells. Higher magnification of a region of interest from Fig.1 showing the over-expressed LMNB1 located at the nuclear lamina (double arrowhead), in the nucleoplasm (arrow), but also in the cytoplasm (arrowhead). The nuclei were counterstained with Hoechst-33342.

Fig. 3: Representative TEM image of HEK293 cells overexpressing LMNB1 showing an array of stacked membranes aligned with the nuclear envelope (arrowheads). Abbreviations: cyt, cytoplasm; n, nucleus.

Fig. 4: Representative TEM images of HEK293 cells overexpressing LMNB1 showing isolated cisternae present inside the cytoplasm close to the membranes stack (arrowheads). Abbreviations: cyt, cytoplasm; n, nucleus.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3192 Actin dependent gliding motion of a diatom, Bacillaria paradoxa.

YAMAOKA N.1, SUETOMO Y.2, SONOBE S.1
1University of Hyogo, Hyogo, Japan, 2Iwakuni City Microlife Museum, Yamaguchi, Japan
bacillaria_paradox@yahoo.co.jp

Diatom is a major group of algae, and the most popular type of phytoplanktons. Some of pennate diatoms can adhere to the substratum and glide over it. This motion is called “gliding”. Bacillaria paradoxa belongs to pennate diatoms and forms a colony consisting of 2-30 cells. Neighboring cells show active gliding each other, but its mechanism and physiological meanings are not understood. We established a culture system of B. paradoxa with artificial sea water (ASW) in our laboratory. From the observation of transmission and scanning electron microscope, the neighboring cells are connected with mucilage secreted from the raphe, the elongated slit in the frustules. Alexa 488-phalloidin staining revealed that two actin filament bundles are present along with the raphe.
Both latrunculin B (LB), an actin polymerization inhibitor, and 2, 3-butanedione monoxime, a myosin inhibitor, completely inhibited gliding motion. After removal of the LB, gliding motion restored. These results suggest an important role of actomyosin system in gliding motion.
Electron microscopy revealed the detailed structures of frustules. Arch-shaped structures called fibula are present across the raphe in the interior of the frustules. Each fibula passes through the cytoplasm. We could observe some filamentous structures between the fibula and the outer wall. These filamentous structures are located a similar place to that observed in fluorescence microscope. We suppose that these fibers are actin bundles. We found a high-electron dense structure on the plasma membrane between actin bundles and the raphe. In addition, we could observe a low-electron dense material filling the space on the raphe and excrete to the extracellular space, probably mucilage. Actin bundles are associated with high electron dense structures on the plasma membrane and it is associated with the low electron dense materials on the extracellular surface. The high-electron dense structures were only observed in particular places, and the mucilage was always observed with this high density structures. This high-electron dense structure might be a complex of trans-membrane proteins and myosin that drives movement of the mucilage. These structures should play a key role in the gliding motion.
We found that a single cell separated from a colony shows a motion in back and forth at a point attaching to the substratum, and succeeded in revealing a motion of the mucilage extending from rapha using micro beads. We presented a curtain model for the gliding motion of B. paradoxa.

We would like to thank A. Miyazawa and Y. Nishino for technical support.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3219 Three-dimensional analysis of phagophore biogenesis at ultra-structural level

Vihinen H.1, Biazik J.2, Eskelinen E. L.2, Jokitalo E.1
1Institute of Biotechnology, University of Helsinki, Helsinki, Finland, 2Department of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland
helena.vihinen@helsinki.fi

Autophagy is the major degradative process in eukaryotic cells that involves degradation of unnecessary or dysfunctional cellular components and even whole cell organelles. The autophagosomal mechanism is crucial for the cells since the inability to degrade defective organelles, clear microbes or remove harmful protein aggregates will result in the onset of diseases. Autophagy begins with the formation of the phagophore, a flat membrane cistern that enwraps portion of cell cytoplasm with/without cell organelles. When phagophore membrane achieves complete closure around the cargo, a double membrane vesicle, termed the autophagosome, is formed. The autophagosomes are then further maturated by fusing with lysosomes forming autolysosomes. The autolysosome degrades the sequestered cargo by the lysosomal hydrolases and degradation products are then recycled back to the cytoplasm. A consensus is emerging that the phagophore is interconnected with the endoplasmic reticulum (ER) and nucleates from a subdomain of the ER termed the omegasome [1]. However, several other organelles such as the mitochondria, Golgi complex, plasma membrane and lysosomes have also been linked with phagophore formation.

Here we will report on the findings that serial block face scanning electron microscopy (SB-EM) and electron tomography (ET) are offering in the field of phagophore biogenesis. SB-EM allows the generation of a three dimensional ultrastructural overview of cells revealing the occurrence and distribution of autophagosomes in large number of cells, as well as the organelles in close proximity to the autophagosomes (Figure 1). The actual membrane contact sites are below the resolution limit of SB-EM method but they can be revealed by utilizing ET (Figure 2) [2]. As SB-EM allows analysis of proximity in large volumes and ET specific contact sites of membrane bound organelles, these two methods nicely complement each other. As a result, this investigation has identified membrane contact sites between the phagophore and membranes originating from the mitochondria, ER exit site, Golgi complex and late endosomes/lysosomes. Identification of direct membrane contact between the phagophore membrane and adjoining organelles has potential to direct future research in membrane flux experiments to help determine whether membrane contacts also signify lipid translocation between the phagophore and the aforementioned organelles.

[1] Q. Lu et al., Dev Cell 21 (2011) 343-57.
[2] P. Ylä-Anttila et al., Autophagy 5 (2009) 1180-5.

The authors gratefully acknowledge funding from the Academy of Finland and Biocenter Finland. Ms. Mervi Lindman is thanked for excellent technical assistance with ET and SB-EM specimen preparation.

Fig. 1: SB-EM indicates the presence of ER in close vicinity of the forming autophagosomes. NRK-52E cells were starved for 1 h and prepared for EM using chemical fixation. The block face was imaged at nominal magnification of 3500x with a voxel size of 11 x 11 x 40 nm3.

Fig. 2: The phagophore and ER contact sites as revealed by ET. Tilt series were acquired at 14,500x or 19,000x nominal magnification and the membrane contact sites were primarily found between the forming phagophore membrane and the ER membrane located inside of forming autophagosome.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3262 CHARACTERIZATION OF ELASTICITY OF WILD-TYPE AND MODIFIED CANCER CELLS USING ATOMIC FORCE MICROSCOPY-BASED METHODS

Crawford K.1, Duan B.2, Sevim S.3, Iyison N. B.2, Torun H.1
1Department of Electrical and Electronics Engineering, Boğaziçi University, Bebek/Istanbul, Turkey, 2Department of Molecular Biology and Genetics, Boğaziçi University, Bebek/Istanbul, Turkey, 3Department of Mechanical Engineering, Boğaziçi University, Bebek/Istanbul, Turkey
kcrawford@g.hmc.edu

Viscoelastic properties of diseased cells are often drastically altered from their healthy counterparts, most noticeably in cancer cells (1). However, as there is no unifying experimental method across studies, the reported values for Young’s modulus and viscosity obtained using atomic force microscopy (AFM)-based methods vary widely. This study attempts to provide a unifying methodology for AFM of cell material properties and reports on the effects of various experimental and data analysis methods on the reported cell stiffness. As cancer cells are often used as model systems with transfected genes for other biological experiments, the effect of transfection on the viscoelastic properties of the cells is presented. All experiments were performed with Huh7 hepatic cancer cells (donated by Dr. Mehmet Öztürk, Bilkent University, TR). With these methodological goals, the various groups probed were: living non-transfected cells, fixated non-transfected cells, living cells with the transfection reagent only (ThermoScientific, USA), living cells with an empty plasmid, living cells with a cytoplasmically-expressed protein (GFP, Clontech Laboratories, USA), and living cells with a membrane-expressed protein (AST, Genbiotek, TR). The basic method of probing mechanical properties of cells by AFM is shown in Figure 1. Force spectroscopy and AFM force-clamp methods were employed using a sharp-tip probe and colloidal probes with diameters of 10μm and 45μm (NovaScan, USA). However, because the 45μm bead was comparable to the cell diameter, the current Hertzian model must be revised. With the current model, the 45μm bead data yields Young’s moduli that are two orders of magnitude greater than the 10μm bead. Data analysis methods were: basic Hertzian contact model and a finite-thickness Hertzian corrected model (2). Figure 2 summarizes the differences in fit between the Hertzian contact model and the finite-thickness corrected model. Because of the better fit for the finite-thickness model, it was used in the remainder of the analysis. Figure 3 compares results across different types of AFM probes using a Hertzian model; this shows for both experimental groups tested, the sharp tip probe showed a 5x greater Young’s modulus than the 10μm probe. Figure 4 compares all the steps of transfection using the thin-film model; these results show that, from the empty plasmid transfection state to GFP, a 5x difference in cell stiffness was found.

References

1. Cross, S E, et al. "AFM-based Analysis of Human Metastatic Cancer Cells." Nano. 19.38 (2008): 384003.

2. Dimitriadis, E "Determination of Elastic Moduli of Thin Layers of Soft Material Using the Atomic Force Microscope." Biophys Jour. 82.5 (2002): 2798-810.

The authors would like to acknowledge funding from the Whitaker Fellowship and TUBITAK grant no: 212TO11.

Fig. 1: Example set-up of an AFM cell stiffness experiment.

Fig. 2: A typical force curve from an AFM cell stiffness experiment with the Hertzian and thin-film corrected fits. The larger figure is the entire force curve, with the Hertzian fit in green and the thin-film correction fit in red. The inset figure is zoomed around the contact point (in black) to show the goodness of fit.

Fig. 3: The difference in Young’s moduli when using different types of cantilevers, as shown with two different transfection groups. The light grey bars are the 10μm colloidal probe, while the dark grey is the sharp-tipped probe.

Fig. 4: Comparison of Young’s moduli among different transfection groups. NT stands for ‘no transfection.’ TR stands for ‘transfection reagent only.’ EP stands for ‘empty plasmid.’ GFP stands for the cytoplasmically-expressed green fluorescent protein. AST stands for the membrane-expressed allatostatin protein.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3300 Molecular, structural and ultrastructural analyses of eosinophils in the jejunal mucosa of irritable bowel syndrome patients

Salvo-Romero E.1, Fortea M.1, Sánchez-Chardi A.2, Lobo B.1, González-Castro A.1, Cardoso F.2, Azpiroz F.1, Santos J.1, Vicario M.1
1Neuro-immuno-gastroenterology Laboratory, Digestive Diseases Research Unit, Department of Gastroenterology, Institut de Recerca Vall d’Hebron & Hospital Universitari Vall d’Hebron, CIBEREHD, 08035 Barcelona, Spain, 2Servei de Microscòpia. Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
Alejandro.Sanchez.Chardi@uab.cat

Introduction: The eosinophil is a multifunctional resident of the gastrointestinal tract that displays extensive regulatory ability to promote and balance mucosal inflammation.  In irritable bowel syndrome (IBS), the prototype of gastrointestinal functional disorders, mucosal immune activation associated with altered barrier function and psychological stress has been identified. However, the role of specific leukocytes such as the eosinophil remains unknown. Here we used qualitative and quantitative approaches combining gene expression with light and electron microscopy techniques (structure, ultrastructure and immunolocalization) in order to study the role of eosinophil in IBS etiology.
Methods: Healthy (H) subjects (n=12) and age-matched, naïve participants fulfilling diarrhea-prone IBS (IBS-D) Rome III criteria (n=17) were included. Jejunal biopsies were obtained by Watson's capsule in all participants. RNA was isolated for the study of gene expression by microarray and identification of differential expression associated with biological functions by Ingenuity Pathway Analysis. Mucosal eosinophil counts were evaluated by immunohistochemistry for major basic protein (MBP) and its ultrastructure was analyzed by transmission electron microscopy as well as the quantification of corticotropin-releasing factor (CRF) by immunolocalization.
Results: The analysis of gene expression revealed functions related to eosinophil activity (P<0.0001) and stimulation (P<0.001) as differentially expressed between IBS-D and H. The number of MBP+cells was similar in both groups (H:44±7; IBS-D:83±19 cells/mm2), however the ultrastructure showed fragmentation of cytoplasmic granules and the presence of tubular structures and sombrero-like vesicles, indicative of secretory activity in the IBS-D group (Figure 1). In the intestinal mucosa, labelling of CRF was only present in the granules of eosinophils, and its content was higher in the group of patients (IBS-D:5.8±2.4, H:2.6±0.9 particles/granule; P<0.05).
Conclusions: Mucosal eosinophil activation in IBS-D was identified by gene expression and further confirmed by ultrastructure analysis. Additionally, although there are no differences in eosinophils density, the increased CRF granular content suggest the existence of alternative pathways of eosinophil activation involving the stress response.

Fig. 1: Representative images of degranulation of eosinophils (black arrows) from healthy (H) and diarrhea-prone IBS (IBS-D) subjects. Labelling of CRF localized in eosinophil granules (white arrows).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3303 Fibrocyte - Leishmania (L.) amazonensis interaction: Toll “like” Receptor-2 participation

Côrte-Real S.1, Guerra C.1, Macedo-Silva R.1, de Lima C.1, Diniz V.1, Carvalho J.2
1Structural Biology Laboratory, Oswaldo Cruz Institute / Fiocruz, Rio de Janeiro, Brazil, 2Biology and Tissue Ultrastructure Laboratory, Rio de Janeiro State University, Rio de Janeiro, Brazil
scrf@ioc.fiocruz.br

Introduction: Fibrocytes emerge as important part to understand the progression of many diseases because they have been identified in areas related lesions generated in these processes. However, the fibrocytes morphology and their behavior when interacting with parasites is poorly understood [Macedo-Silva et al, 2014]. Fibrocytes has hematopoietic origin and are characterized by simultaneous expression of molecules CD45 or CD34 and extracellular matrix proteins production [Bucala et al, 1994]. Fibrocytes may have an important role in the innate immune response and development of acquired response, once they have Toll "like" (receptors TLRs) [Balmelli et al, 2007], stimulate T limphocytes and produce cytokines [Chesney et al, 1997]. In this study, we evaluate the presence of toll-2 in infected fibrocytes with Leishmania (L.) amazonensis. Methods and Results: We use primary culture of peripheral blood fibrocytes from mice and promastigotes of Leishmania (L.) amazonensis. Fibrocytes were characterized by Epifluorescence using a rat anti-CD45 and a rabbit anti-Hsp47 primary antibody. Following were incubated with anti-rat IgG FITC-conjugated anti-rabbit IgG and TRITC-conjugated secondary antibodies. Fibrocyte culture infected were fixed with 2% paraformaldehyde, blocked solution of FcRs, incubated with anti-TLR-2 and pos-incubated with secondary antibody conjugate immuno-gold at 1h/37ºC. After were fixed in 2,5% glutaraldehyde, pos-fixed with OsO4, dehydrated in ketonic series, included in PolyBed 812 resin and observed in transmission electron microscope- Jeol 1011 of Platform-IOC/Fiocruz. Ultrastructural analysis showed TLR-2 at the plasmatic membrane and in parasitophorous vacuole containing the parasite. We also analyzed the fibrocytes morphology and their ability to internalize Leishmania promastigotes. Conclusion: Since TLRs signaling after the invasion of microorganisms confers specificity to the immune cells in response to different pathogens and are present in the Leishmania phagosome in fibrocytes, peharps these may be involved in the development of leishmaniasis, and are target cell studies for the production of therapies most effective and least toxic to the control and treatment of the disease.

Supported: Fiocruz/IOC, CNPq, Faperj

Fig. 1: Fig. (A) Scanning electron micrograph showing the adhesion of promastigotes at the fibrocyte; (B) Transmission electron micrographs showing fibrocyte with promastigote in narrow vacuole; (C) TLR-2 in the plasmatic membrane of fibrocyte (arrow) (D) in parasitophorous vacuole containing Leishmania (arrowhead).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3306 The development of silica phytoliths in plants is morphologically pre-determined even under the absence of silica

Martinka M.1,2, Soukup M.1, Ravaszová F.1, Švancárová M.1, Lux A.1,3
1Department of Plant Physiology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska dolina B2, 842 15, 2Institute of Botany, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 845 23, Slovak Republic, 3Institute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 845 38, Slovak Republic
martinkambio@yahoo.com

Many plant species are able to accumulate high amounts of silicon and form biogenic opal – silica phytoliths [1]. There is a potential to use the silica phytoliths, because of their physical and chemical features, in electro-technical and building industry [2]. The goal of this study was to characterise the silicon- and cadmium-dependent silica phytoliths formation in endodermal cells and to test the possibility to change the size and morphology of silica phytoliths with the aim to produce particles of requested features for industry.

Tested plants of Sorghum bicolor were cultivated in hydroponics containing wide concentration range of Na2SiO3, as silicon source, and of cadmium. After three-day treatment the seminal roots of plants were investigated by a combination of negative phase contrast and differential interference contrast, fluorescent, transmission and scanning electron microscopy (equipped with EDXA) to characterise the development, exact localisation, shape and morphology of silica phytoliths related to the composition of cultivation media and to the endodermal cell wall modifications.

At the concentrations of silicon above 0.8 µmol/L the silica phytoliths form uniformly in endodermal cells of S. bicolor seminal roots with a positional effect consistently with a gradual development of suberin lamellae. The low silicon concentrations in the hydroponic media affect negatively the size of silica phytoliths and the continuous size-dependent gradient shifts towards the base of the root. Plants form, even under silicon deficient conditions, in the responsible cells morphologically pre-determined areas for silica phytoliths formation. The cadmium affects not only the size, but also the exact localisation of phytoliths. At still relatively low concentrations of cadmium the phytoliths stop to form and the silicon is spread ectopically in the tissues of root.

Based on the results the exact localisation of silica phytoliths is pre-determined and silica phytoliths are formed uniformly under natural conditions. However, their development, size and morphology can be changed by silicon starvation or by use of compounds affecting the silicon metabolism and/or mechanisms of phytoliths formation in plants. The further investigation, understanding, and modification of processes involved in the phytoliths development can help to produce particles of requested features for industry.

1. M.J. Hodson et al., An Bot 96 (2005), p. 1027-1046.

2. S. Neethirajan et al., Trends Biotech 27 (2009), p. 461-467.

This work was supported by the Slovak Grant Agency [VEGA 1/0817/12]; and by the Slovak Research and Development Agency, under the contract No. APVV-0140-10.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3379 Protective effects of curcumin in liver damage induced by high-fat diet

Ozdas Beyhan S.1, Tanrıkulu-Kucuk S.2, Yapislar H.3, Seyithanoglu M.4, Oner-Iyidogan Y.4, Akin D.5, Koçak H.2, Dogru-Abbasoglu S.4, Kocak-Toker N.4
1Department of Medical Biology and Genetic, Faculty of Medicine, Istanbul Bilim University, Istanbul, Turkey , 2Department of Biochemistry, Faculty of Medicine, Istanbul Bilim University, Istanbul, Turkey , 3Department of Physiology, Faculty of Medicine, Istanbul Bilim University, Istanbul, Turkey , 4Department of Biochemistry, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey , 5Department of Pharmacology, Faculty of Medicine, Istanbul Bilim University, Istanbul, Turkey
sule.ozdas@istanbulbilim.edu.tr

This study investigated the hepatoprotective effects of curcumin against liver damage induced by a high-fat diet. Curcumin is a diarylheptanoid and it is the principal curcuminoid of the popular South Asian spice turmeric (1). In vitro, curcumin modulates the inflammatory response by down-regulating the activity of cyclooxygenase-2, lipoxygenase, and inducible nitric oxide synthase enzymes; and inhibits several other enzymes involved in inflammation mechanisms(2).Thus, this study aimed to elucidate the impact of curcumin on modulation of these molecular mediators on liver damage, steatohepatosis and inflammation in high fat diet-induced rat. We also examined several potential underlying mechanisms of curcumin including antioxidant activity and lipid metabolism. In our study, Sprague-Dawley male rats were divided into four groups (n=8/group). One group of rats were fed diet containing 10 % fat by weight and designated as the control group. Other group (HF) was fed high fat diet containing 60% fat and third group (HF+Cur) was fed high fat diet supplemented with curcumin (1000 mg/kg diet) for 16 wks. Last group of rats were fed with normal diet supplemented curcumin (1000 mg/kg diet). Total free fatty acids, triglycerides were measured in plasma, Dien Conjugate (DC) content and antioxidants levels were measured.Hem Oxygenase (HO-1) expression was demonstrated by western blotting (data not shown). In addition, the presence of fat droplets, peri-portal fibrosis and glycogen was examined histologically (Figure.1-2). In recent studies curcumin through a series of complex mechanisms, alleviated the adverse effects of high fat diet on weight gain, fatty liver development, dyslipidemia, expression of inflammatory cytokine in rats(4). According to our results, lipid droplets within hepatocytes, infiltration of inflammatory cells, and necrotic foci in the liver were morphologically alleviated by curcumine (HF+Cur) in a compared with the HF group. These findings indicate that curcumin suppressed development of steatohepatosis, reduced fibrotic tissue, and preserved glycogen levels in liver and it may protect against liver damage caused by a high-fat diet partly by modulating the antioxidant activity and the lipid metabolism. Therefore, curcumin may provide a promising natural nutri-therapeutic strategy against liver disease.
1- Kolev, Tsonko M. et al. International Journal of Quantum Chemistry 2005,102(6):1069–79.
2- Abe Y, Hashimoto S, Horie T. Pharmacol Res 1999, 39 (1): 41–7.
4- Hasan ST et al. Atherosclerosis. 2014 Jan;232(1):40-51.

Fig. 1: Hematoxylin and eosin stained liver tissues all groups. A- Control group, normal liver tissue structure. In control group, normal liver tissue structure. B/D- HF group, inflammation area in the liver tissue. C- Focal degeneration area and cytoplasmic vacuolization in hepatocytes. E/F- HFCur group, liver tissue is similar to control group.

Fig. 2: Reticulin stained liver tissues of all groups. A-B-C Control group, normal liver tissue architecture. D-E, HF group, dark black staining of the reticulin fibers with reticulin stain. The other white circular structures near the reticulin fibers are fat globules. F- HF-Cur group, liver tissue is similar to control group.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3405 Effects of Electrical Stimulation at Different Frequencies on the Cytoskeleton of Frog Nerve Terminals

Mukhitov A. R.1, Nikolsky E. E.1
1Kazan Institute of Biochemistry and Biophysics of RAS
alexmukhitov@mail.ru

The cytoskeleton plays a crucial role in a variety of important cellular processes including the functioning of the neuromuscular endings. Two main classes of the cytoskeletal structures: microtubules and microfilament involved in all main stages of synapse maturation and function. At the mouse model it was shown that the NMJ are normally able to switch between modes of synaptic transmission that require different biochemical pathways, depending on the frequency of stimulation [1]. At the same time response of a cytoskeleton on electrical stimulation at various frequencies remains insufficiently studied. The present study aimed to investigate changes of tubuline and actin cytoskeleton in the motor nerve terminals of frogs in response to electrical nerve stimulation at different stimulus frequencies. The experiments were done on the muscle m. cutaneus pectoris of Rana ridibunda. The nerve of the dissected muscle was stimulated with a frequency of 10 Hz or 100 Hz during 5 min or 10 min. Then the muscle was fixed by 4% PFA. Synaptic sites were identified by staining acetylcholine receptors with a-bungarotoxin conjugated with TRITC. Primary antibodies included rabbit polyclonal antibodies against actin, mouse monoclonal antibodies against beta-tubuline. Secondary antibodies were conjugated with ATTO647N and ATTO488 dyes. Visualization of cytoskeletal elements was carried out by means of laser confocal microscope Leica SP5. The obtained data processed statistically and analyzed by means of the LAS AF 4.0 and ImageJ 1.42 programs. The morphology of microtubules and microfilaments bundles and the average mean of fluorescence in the nerve terminals were estimated. We found that electric stimulation of a nerve induces the changes of the neuronal endings cytoskeleton depending on both stimulus frequency and stimulation duration. The increase dyes-labeled MTs was observed in nerve terminals after stimulation. In addition MT bundles became more branched (Fig. 1). The actin cytoskeleton density was decreased in nerve terminals depending on nerve stimulation frequency (Fig. 2). It was shown that the F-actin-based network may participate in creating a scaffold for SV clustering, and in supporting ordered vesicle mobility [2]. Ours results are consistent with a role of the cytoskeleton in the synaptic vesicle recycling pathway and the transport of cargo by molecular motors.

 

[1] Maeno-Hikichi Y. et al. Frequency-Dependent Modes of Synaptic Vesicle Endocytosis and Exocytosis at Adult Mouse Neuromuscular Junctions // The Journal of Neuroscience, 2011, 31(3):1093–1105.

[2] Hirokawa N. et al. The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1 // J Cell Biol, 1989, 108: 111–126.

The reported study was partially supported by RFBR, research project No. 13-04-01595 a.

Fig. 1: Effects of low- and high-frequency electrical stimulation on the tubuline cytoskeleton of frog nerve terminals. A – Mean fluorescence intensity of dyes-labeled MTs in terminals, B – morphology of MT cytoskeleton: green - MTs (ATTO 488), yellow - acetylcholine receptors (TRITC-conjugated alpha-bungarotoxin). 

Fig. 2: Effects of low- and high-frequency electrical stimulation on the actin cytoskeleton of frog nerve terminals. A – Mean fluorescence intensity of dyes-labeled F-actin in terminals, B – morphology of actin cytoskeleton: red - actin bundles (ATTO 647N), yellow - acetylcholine receptors (TRITC-conjugated alpha-bungarotoxin). 
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3409 In vitro clot structure of metabolic syndrome induced transient ischemic attack patients using scanning electron and atomic force microscopy

van Rooy M. J.1, Buys A. V.2, Pretorius E.1, Duim W.3
1Department of Physiology, School of Medicine, Faculty of Health Sciences, University of Pretoria, 2Unit of Microscopy and Microanalysis, University of Pretoria, 3Department of Neurology, School of Medicine, Faculty of Health Sciences, University of Pretoria
antoinette.buys@up.ac.za

The metabolic syndrome is a common, very complex set of heterogeneous risk factors that are interlinked and increases the prevalence of atherosclerosis and type II diabetes mellitus and chronic inflammation. The subsequent atherosclerosis and consequent inflammation in turn increases the risk for thrombo-embolic stroke and myocardial infarction. Both thrombo-embolic stroke and MI are caused by the formation of pathological thrombi or where an atherosclerotic plaque is ruptured resulting in an occlusion of a blood vessel. Transient ischemic attack (TIA) in contrast to thromboembolic stroke is caused by the temporary occlusion of a cerebral artery resulting in neurological symptoms typically lasting less than one hour. Several studies have been conducted on the relation of metabolic syndrome and TIA, but very few have focused on the alterations resulting in TIA. In this study the morphology of the formed thrombus is examined to determine what changes take place in the coagulation characteristics of metabolic syndrome affected individuals and how this alters the structure of the clot.

Participants in this study included a control and experimental (TIA) group. After written consent was obtained 5 ml of blood was drawn. Venipuncture of TIA patients was done within 48 hours after symptoms developing. Whole blood and platelet rich plasma (with and without thrombin) was prepared following standard preparation protocols for SEM and AFM. SEM was used to investigate red blood cell (RBC) and platelet morphology, platelet activation and fibrin fiber arrangement. AFM was employed to examine RBC morphology, roughness and mechanical properties. Furthermore platelet and fibrin fiber mechanical properties were also studied.

Preliminary results indicate increased platelet activation and aggregation with decreased elastic modulus. The fibrin networks from TIA patients appear more disorganized and curved when compared to controls. No RBC shape changes can be seen when using SEM, however, when whole blood was artificially activated by the addition of thrombin, the RBCs of the control group show dramatic alterations in shape due to the presence of fibrin fibers exerting a force on the RBCs. These findings are corroborated by AFM results indicating increased Young’s modulus and significant changes in the 1st and 3rd order membrane roughness indicating changes in the membrane macromolecular composition.

Dentali F, Romualdi E, Ageno W. The metabolic syndrome and the risk of thrombosis. Hemat J. 2007;92(3):297-299.
Palomo I, Alarcól M, Moore-Carrasco R, Argilés JM. Hemostatis alterations in metabolic syndrome (Review). Int J Molec Med. 2006;18:969-974.
Sunshine JL. Transient Ischemic Attacks: Added Specificity from Modern MR Imaging. Am J Neuroradiol. 2002;23:4-5.

Fig. 1: Figure 1: RBCs of experimental group. A: SEM, scale 1µm. B: AFM, scale 1µm. C: Surface Roughness Image, scale 200nm. D: Surface profile original and 1st-3rd order.

Fig. 2: Figure 2: Platelet morphology. A: SEM, scale 1µm. B: AFM, scale 1µm.

Fig. 3: Figure 3: Fibrin network arrangement and morphology. A: SEM, scale 1µm. B: AFM, scale 1µm.

Fig. 4: Figure 4: Whole blood clot morphology in vitro, scale 1µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3441 The Effect of Electrical Nerve Stimulation on the Dynamics of Posttranslational Modified Tubulin in Frog Motor Nerve Terminals

Mukhitov A. R.1
1Kazan Institute of Biochemistry and Biophysics of RAS
alexmukhitov@mail.ru

The cytoskeleton plays an important role in most processes necessary for cell life. In neurons, MTs actively participate in the initial steps of neuronal polarization, the organization of intracellular compartments, the remodeling of dendritic spines and the trafficking of cargo molecules to pre-, post- or extrasynaptic domains [1]. Post-translational modifications of tubuline are highly dynamic processes where tubuline’s functional properties are altered by addition of a chemical group or another protein to its amino acid residues. Posttranslational modifications of tubulin can be used to monitor the dinamics of microtubules. It was shown that long-lived stable microtubules are enriched for acetylated tubulin, whereas newly polymerized microtubules are enriched for tyrosinated tubulin [2, 3]. The present study aimed to investigate changes in both level and distribution of the posttranslational modified form of tubuline in the motor nerve terminals of frogs in response to electrical nerve stimulation at different stimulus frequencies. Our studies were done on the muscle m. cutaneus pectoris of a frog of Rana ridibunda. The nerve of the allocated muscle was stimulated with a frequency of 10 or 100 Hz during 5 min or 10 min. Then the muscle was fixed by 4% PFA. Synaptic sites were identified by staining acetylcholine receptors with a-bungarotoxin conjugated with TRITC. Primary antibodies included mouse monoclonal anti-acetylated tubulin antibody, mouse monoclonal antibodies against tyrosinated tubuline. Secondary antibodies were conjugated with ATTO647N and ATTO488 dyes. Visualization of cytoskeletal elements was carried out by means of laser confocal microscope Leica SP5. The obtained data processed statistically and analyzed by means of the LAS AF and ImageJ 1.42 programs. The morphology of microtubules bundles and the average mean of fluorescence in the nerve terminals were estimated.  We found that electric stimulation of a nerve induces the changes of the neuronal endings cytoskeleton depending on stimulus frequency. The increase dyes-labeled tyrosinated MT  was observed in nerve terminals after stimulation. The alteration of level and distribution tyrosinated MTs were depends on both the stimulus frequency and the stimulation duration. At the same time significant changes of the acetylated MTs of the cytoskeleton was not observed.

[1] Janke C. and Kneussel M. Tubulin post-translational modifications: encoding functions on the neuronal microtubule cytoskeleton // Trends in Neurosciences, 2010, Vol.33, No.8, p. 362–372.

[2] Palazzo A. et al. Cell biology: tubulin acetylation and cell motility // Nature 2003, 421:230.

[3] Fukushima N. et al. Posttranslational modifications of tubulin in the nervous system // J Neurochem, 2009, 109:683– 693.

The reported study was partially supported by RFBR, research project No. 13-04-01595 a.

Fig. 1: Effects of low- and high-frequency electrical stimulation on the level of acetylated forms of MT cytoskeleton in nerve terminals. A – Mean fluorescence intensity of dyes-labeled acetylated MTs in terminals, B – morphology of MT cytoskeleton: green - acetylated MTs (ATTO 488), yellow - acetylcholine receptors (TRITC-conjugated alpha-bungarotoxin).

Fig. 2: Effects of low- and high-frequency electrical stimulation on the level of tyrosinated form MT cytoskeleton in nerve terminals. A – Mean fluorescence intensity of dyes-labeled tyrosinated MTs in terminals, B – morphology of MT cytoskeleton: green tyrosinated MTs (ATTO 488), yellow - acetylcholine receptors (TRITC-conjugated alpha-bungarotoxin). 

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3450 Effect of the Obesity Induced by High-Sugar Diet on Inflammatory Response During Experimental Infection by Mycobacterium bovis BCG

Assis A. A.1, Toledo D. A.2, Rodrigues G. S.1, Moreira D. A.1, Melo R. C.1, Gameiro A.1, Andreazzi A.1, * D' Avila H.1
1Federal University of Juiz de Fora, Juiz de Fora, Brazil *davila.bizarro@ufjf.edu.br, 2Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
danielkbssa1@hotmail.com

Tuberculosis (Tb) is a public health problem with around 1.4 million deaths per year [1]. In the lungs is observed an intense influx of cells to the site of infection where they can form structures called granulomas [2]. It has been observed the differentiation of macrophages (MØ) in “foamy cells" in granulomas [3]. The foam aspect of MØ is a reflex of intracellular lipid accumulation [3,4,5]. Lipid body structural features, including lipid and protein composition may vary according to the cell type, activation state and inflammatory environment and thus may determine different cellular functions for lipid bodies [6]. Obesity is another health problem worldwide, causing the deaths of almost 3 million of people [1]. It is associated with chronic inflammatory response of white adipose tissue due to infiltration of MØ, responsible for overexpression of TNF-α and IL-6 [7]. Our objective was to evaluate the involvement of obesity in influx and activation of cells in experimental infection with M. bovis BCG in mice, aiming to clarify the physiopathology of Tb and the role of metabolic disorder in bacterium replication. C57BL6 mice were divided in 2 groups fed with high-sugar diet or common chow. After 90 days, the mice were intrapleurally infected by BCG and the control received saline. (Animal euthanasy ethical approval: #109/2012 CEUA/UFJF). The leukocyte influx and lipid body enumeration was performed at 24h after infection. It was observed an increase in the mass of fat in the high-sugar diet (Mean ± SEM: from: 0,112 ± 0,017 in control to 0,219 ± 0,002 in high-sugar group; n=10) and a significant reduction in influx of leukocytes into the pleural cavity (2,05 ± 0,366 in control to 20,100 ± 5,460 in infected on common chow group; from: 4,480 ± 0,615 in control to 8,080 ± 2,168 in infected on high-sugar diet group; n=5) as well as the neutrophils and eosinophils migration in obese mice. Also, there was less lipid body formation in obese compared with normal animals (from 1,200 ± 0,060 in control to 3,920 ± 0,738 in infected group on common chow; from1,713 ± 0,081 in control to 1,460 ± 0,102 in infected on high-sugar diet group). Our data suggest that the largest quantity of adipose tissue disadvantage the BCG. The fact the reduction in the lipid body formation and leukocyte migration in obese individuals indicated a correlation of obesity under the progression of experimental infection with M. Bovis.

[1] World Health Organization, Geneva, Switzerland, 2013
[2] PloS one (12): 581, 2012
[3] J Immunol (176): 3087-3097, 2006
[4] Int Immunopharmacol (8): 1308-1315, 2008
[5] Immunology (106): 257–266, 2002
[6] Journal of Histochemistry & Cytochemistry (59): 540-556, 2011
[7] Exp Biol Med Maywood (235):1412, 2012

Research Support Foundation of Minas Gerais (FAPEMIG); National Council for Scientific and Technological Development (CNPq) and Federal University of Juiz de Fora

Fig. 1: Leukocyte migration in control individuals, performed at 24h after infection, showed mononuclear cells (arrow). Bar = 50 μm

Fig. 2: Leukocyte influx in infected individuals, performed at 24h after infection, showed neutrophils (black arrow indicated) and eosinophils (white arrow indicated) migration. Bar = 50 μm

Fig. 3: Lipid bodies (black arrow) observed on mononuclear cells from the pleural cavity of the infected group. Bar = 50 μm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3509 Analysis of nucleolar structure by atomic force microscopy

Segura-Valdez M. L.1, Zamora-Cura A.1, Chávez-Rosasles R.1, Jiménez-García L. F.1
1Department of Cell Biology, Faculty of Sciences, UNAM
lourdes_segura@ciencias.unam.mx

The nucleolus is the site for ribosome biogenesis and other several functions in eukaryotes. It is composed by rDNA, rRNA and its precursors, proteins and other RNAs as UsnRNAS. The ultrastructure of the nucleolus includes three main compartments known as fibrillar centers, dense fibrillar component and granular component. Instersticies are also areas in the nucleolus. Here we show recent advances in the analysis of the structure of the nucleolus by atomic force microscopy, to test the putative nanoscale approach of this organelle. Samples were prepared as for standard transmission electron microscope, as described previously (1-5). Briefly, samples of plant (Lacandonia schismatica, Gingko biloba, Allium cepa) and animal (HeLa, cells, Hep2 cells) cells were fixed with 2.5% or 6% glutaraldehyde buffered with PBS. They were then postfixed with osmium tetroxide, dehydrated with a series of graded ethanol and embedded with epoxy resin. Semithin sections were mounted on coverslips and observed by an atomic force microscope operating in contact mode. Results show that at least two different phases are observed in the nucleolus (Figure 1), displaying also different height parameters. We conclude that nucleolus may be studied by atomic force microscope to revealed nanoscale parameters of every subcompartment.
References
1.- Jiménez-García, L.F., Segura-Valdez, M. de L. (2004). Visualizing nuclear structure in situ by atomic force microscopy. Pp 191-199. En: Atomic Force Microscopy: Methods and Protocols in Biomedical Applications [Braga, P.C. & Ricci, D. (Eds.)]. Methods in Molecular Medicine. Humana Press, New Jersey, USA.
2.- Segura-Valdez, M.L. et al. (2010). Visualization of cell structure in situ by atomic force microscopy. En: Méndez-Vilas, A. & Díaz, J. (eds) Microscopy: science, technology, applications and education (Microscopy Book Series), Formatex, Badajoz, Spain.
3.- Segura-Valdez, M.L. et al. (2012). Cell Nanobiology. En: Oxidative Stress and Chronic Degenerative Diseases - a Role for Antioxidants (J.A. Morales-González, ed.).
4.- Jiménez-García, L.F. and Fragoso-Soriano, R. (2000). Atomic Force Microscopy of the Cell Nucleus. J. Struct. Biol. 129: 218-222.
5.- Fragoso-Soriano, R.J. et al. (2009). Atomic Force Microscopy Imaging of Thin Sections of Lacandonia Granules. J. Scann. Probe Microsc. 4: 1-5.

We thank funding from CONACyT 180835.

Fig. 1: Atomic force micrograph of the nucleolus (n) of Ginkgo biloba meristematic cells. Two phases are observed within the nucleolus (arrows).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3510 Lacandonia granules are present in Welwitschia mirabilis

Agredano-Moreno L. T.1, Segura-Valdez M. L.1, Muñiz Díaz de León M. E.2, Jiménez-Ramírez J.2, Jiménez-García L. F.1
1Departamento de Biología Celular, Facultad de Ciencias, UNAM, , 2Departamento de Biología Comparada, Facultad de Ciencias, UNAM.
agredano-moreno@ciencias.unam.mx

Lacandonia granules are extranucleolar ribonucleoprotein (RNPs) particles, 32 nanometers in diameter intermixed with fibrils that were first described in the nucleoplasm of the plant Lacandonia schismatica (Jiménez-García, et al. 1992). Cytochemical and imunocytochemical studies suggest that these particles are equivalent to perichromatin and Balbiani ring granules described in mammals and salivary glands cells of the insect Chironomus tentans, respectively (Agredano-Moreno & Jiménez-García, 2000). In addition, Lacandonia granules have been reported in other close groups as Triuris brevystilis (Jiménez-García, et al. 1992), and even in the tree Ginkgo biloba (Jiménez-Ramírez et al. 2002). In the present work we analyzed the presence of Lacandonia granules in Welwitschia mirabilis. Samples of plant leaves grown in greenhouse were processed for electron microscopy. Light, electron and atomic force microscopy revealed that nuclei are reticulated according to compact chromatin distribution. Granules of 32 nm in diameter, positive to the EDTA regressive technique for RNP, distributed in the perichromatin and interchromatin space were observed among strands of reticulated chromatin (Figure 1). Stereo pair images from ultrathin sections showed fibers and granules intermingled. Our results show that Lacandonia granules are also present in the order Gnetales, associated to reticulated chromatin.

References
Jiménez-García, L.F., Agredano-Moreno, L.T., Segura-Valdez, M. de L. Echeverría, O., Martínez, E., Ramos, C.H., Vázquez-Nin, G.H. 1992. The ultrastructural study of the interphase cell nucleus of Lacandonia schismatica (Lacandoniaceae:Triuridales) reveals a non-typical extranucleolar particle. Biol. Cell. 75:101-110

Agredano-Moreno, L.T., Jiménez-García, L.F. 2000. New evidence that Lacandonia granules are ultrastructurally related to perichromatin and Balbiani ring granules. Biol. Cell. 92:71-78

Jiménez-Ramírez, J., Agredano-Moreno, L.T., Segura-Valdez, M de L., Jiménez-García, L.F. 2002. Lacandonia granules are present in Ginkgo biloba cell nuclei. Biol. Cell. 94:511-518

DGAPA-PAPIME PE211412

Fig. 1: Electron micrograph showing Lacandonia granules (arrows) in the cell nucleus of meristematic cell of Welwitschia mirabilis. EDTA regressive staining for RNP. Chromatin (C). Bar is 80 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-3532 Mitochondria need superresolution microscopy: hypoxic cristae inflation is reflected distinctly by biplane FPALM with intermembrane space vs. matrix marker

Špaček T.1, Alán L.1, Engstová H.1, Plecitá - Hlavatá L.1, Ježek P.1
1Institute of Physiology, Czech Academy of Sciences, Prague, Czech Republic
spacek@biomed.cas.cz

While cryo-electron microscopy indicated the cristae width (intracristal space sections) increase from 18±5 nm to 24±10 nm upon mild 5% O2 hypoxia in hepatocellular carcinoma HepG2 cells, 3D super-resolution photo-activated localization microscopy of a biplane detection scheme (Biplane FPALM)[1], indicated thickening or thinning of mitochondrial (mt) network tubular contour, depending on the marker. Biplane FPALM with the inter-membrane space (IMS) marker, a truncated lactamase-beta (LactB) conjugated with Eos, indicated hypoxic IMS inflation by increasing diameter of Eos-LactB contour for mt network. In turn, the matrix-addressed Eos (mtEos) indicated hypoxic decrease in diameter for mtEos- visualized tubular network. The outer mitochondrial membrane (OMM) marker, Eos-FIS1tr, visualizing hollow tubules, indicated no changes. Thus the real mt tubule diameter did not altered upon hypoxia. The apparent hypoxic thinning of the mt matrix contour, observed previously by 4Pi and conventional confocal microscopy, was in fact reflecting the altered cristae morphology. Our case provides warning to interpretations of conventional confocal data for mt network, since their resolution merely equals to the real diameter of mt tubules. Since mitochondria likely change cristae conformation with physiological state, e.g. at state-4 to state-3 transitions (the former is resembling the low substrate state, the latter state with cell maximum ATP production), these morphology changes must be reflected in images collected by superresolution microscopy with the intracristal space marker (a part of IMS protruding beyond the OMM contact sites), matrix space marker or inner membrane marker. Mitochondrial cristae in vivo morphology represents a great challenge for superresolution microscopy.

[1] M.J. Mlodzianoski, J.M. Schreiner, S.P. Callahan, K. Smolková, A. Dlasková, J. Šantorová, P. Ježek, J. Bewersdorf. "Sample drift correction in 3D fluorescence photoactivation localization microscopy". Opt. Express 19, 15009-15019 (2011).

Supported by grant 13-02033 (GACR)

Fig. 1: Mitochondrial tubules as imaged by Eos-LactB at normoxia

Fig. 2: Mitochondrial tubules as imaged by Eos-LactB at hypoxia
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-5753 Ultrastructural changes in vascular beds during retinal vasculogenesis and angiogenesis of newborn mouse.

Eun Kyung Choi 1,4, Jeong Hun Kim 2, Young Suk Yu 2, Man Gil Yang 1 , Nam Hyun Jung 3, Sung Sik,Han 4
1 Laboratory of Electron microscopy/pathology Biomedical Research institute, Seoul National University Hospital, Korea 2 Department of Ophthalmology, College of Medicine, Seoul National University & Seoul Artificial Eye Center Clinical Research Institute, Seoul National University Hospital, Seoul, Korea 3 College of Life Sciences and Biotechnology, Korea University, Korea. 4 Department of Life Sciences , korea University ,Seoul, Republic of korea
cek1002@naver.com

Purpose: To analyze the cellular events involved in the construction of the organized vascular architecture and to characterize the tight junction in developing vessels of retina.

Methods: On post-natal days (P)4, P8, P12, P16, and P56, electron microscopy for retinal vessels and immunohistochemistry for CD31, ZO-1, glial fibrillary acidic protein, and α-smooth muscle actin were performed. ZO-1 expression was measured with progression of retinal vessel development in whole retina. Leakage was assessed by immunohistochemistry for CD31 on retinal vessels perfused with fluorescein conjugated dextran as well.

Results: The recruitment of pericytes and astrocytes to vascular tube of endothelial cells is closely associated with the formation of tight junction in developing retinal vessels. At P4, endothelial cells of retinal vessels behind the invading front directly contact to pericytes, but not to foot processes of astrocytes yet, where ZO-1 was already weakly immunoreactive along retinal endothelial cells. With the progression of retinal development, foot processes of astrocytes are gathered around retinal vessels and the maturation of tight junction in endothelial cells is clearly defined, which was temporally and spatially accordant to the expression of a tight junction protein, ZO-1. In addition tight junction could be formed with contact of pericytes to endothelial cells without the prominent ensheathment of astrocytic foot processes which was coincided with the appearance of a tight junction protein, ZO-1.

Conclusion: Our data suggests that the tight junction of endothelial cells in blood-retinal barrier could be developed by cellular interactions between pericytes, asctrocytes and endothelial cells. Moreover, ZO-1 as well as occluding or claudin might demonstrate the tightness of blood-retinal barrier in developing retina.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-5795 In Situ Visualizing T-Tubule/SR Junction Reveals Novel Arrangement of Ryanodine Receptor-1 Using Electron Tomography

Yang Y.1, Song X.2, Tang Y.3, Lei C.4, Cao M.5, Shen Y.6, Zhu J.7
1Department of Biophysics, Second Military Medical University, Shanghai 200433, China, 2School of Life Science and Technology, Tongji University, 200092 Shanghai, China
yjyang22@163.com

Abstract:

Background, Membrane-integral proteins are critical executants of life activities. It is still lack of efficient technologies to determine the structures and arrangement of membrane-integral proteins in their native phospholipid-bilayer environment. Here, we confirmed the possibility of using Cryo-Electron Tomography to visualize ryanodine receptor-1 (RyR1) in its close-to-life state. Results, We found that RyR1 was distributed in subregion of sarcoplasmic reticulum (SR) membrane, where T-tubule was most closely neighboring. Interestingly, we visualized the relationship between RyR1 and SR membrane for the first time, showing that most mass of RyR1, in umbrella-shape, was exposed to SR, whereas only a small portion, in stalk-shape, was exposed to the cytoplasic environment. Based on this observation, we established a new modulating model of RyR1, in which, we postulated that dihydropyridine receptor (DHPR) directly interacted with the stalk-shaped part of RyR1, while FK-506 binding protein 12 kDa (FKBP12) directly interacted with the umbrella-shape of RyR1 to regulate the process of calcium releasing from SR during excitation-contraction coupling. Conclusions,Electron Tomography could be used as an efficient method to determine the structure and arrangement of single membrane-integral protein within intact cells in its native context of membranes, SR, vesicles and other molecular complexes.

Keywords: membrane-integral proteins; RyR1; macromolecular architecture; Electron Tomography; DHPR

Fig. 1: Figure 1. In situ visualizing the T-Tubule/Sarcoplasmic Reticulum junction in 2 dimensional picture using Electron Microscopy. The T-Tubule membrane and Sarcoplasmic Reticulum membrane was closely neighboring with each other.

Fig. 2: Figure 2. Surface-rendered views of the final 3D map of the T-Tubule/SR junction from different direction using IMOD software construction. The density fitting usage of computational methods combined with Electron Tomography showed whole structure of T-Tubule/SR junction.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-5808 Ultrastructural alterations in the liver and kidney of Nile telapia (Oreochromis niloticus Linn.) induced orally by ultra-low doses of endosulfan

Polsan Y.1, Sakaew W.1, Hipkaeo W.1
1Department of Anatomy, Faculty of Medicine, KhonKaen University, KhonKaen, Thailand
yadpol@kku.ac.th

Endosulfan, an organochlorine pesticide, is widely used in agriculture and hence found its contamination in natural water. Thus it’s highly toxic to aquatic organisms, including fish. The purpose of this study was to elucidate the ultrastructural changes in the liver and the kidney of Nile telapia (Oreochromis niloticus Linn.) treated with endosulfan. In a laboratory experiment, 20 Nile tilapias were exposed to 0.5 µg kg-1 of endosulfan food dry weight for 28 days and another 20 were used as the control group without any treatment. The liver and the kidney were then collected and fixed in Karnovsky’s fixative for 24 hr and processed to investigate under the transmission electron microscope (TEM). Observation on liver alterations, the hepatocytes showed enlargement of nucleoli, increase in number and size of both Golgi fields and rough endoplsmic reticulum (rER) lamellae, as well as proliferation of peroxisomes and lysosomes. These alterations represent the morphological equivalent of a general stimulation of hepatic metabolism. In addition, proliferation of the rER is indicated the onset of biotransformation processes under the influence of food-borne endosulfan. In the kidney, the ultrastructural alterations were observed in the proximal tubular epithelial cells (PT) which showed the nuclear condensations, rER fragmentations and massive swelling of the mitochondria. Disorganized brush borders and increased numbers of large vacuoles and lysosomes were also observed in PT. The pathological alteration in organ subsequently in contact with toxicants appeared as a useful biomarker of pollutant exposures and effects. Endosulfan, as a mixture of the toxic substances, may well have contributed to the overall toxicity of the chemical released during the agricultural used and the subsequent fish killed.

This research was funded by Division of Research Administration KKU. We would also like to thank Asst.Prof. Chanarong Aranyanat for his guidance in TEM technique.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-5874 Sticky business: How imaging tools reveal molecules involved in malaria pathogenesis

Rug M.1, Cyrklaff M.2, Lemgruber L.2, Frischknecht F.2, Prescott S.3, Hanssen E.4, Maier A. G.5, Cowman A. F.6
1Centre for Advanced Microscopy, The Australian National University, Canberra, Australia, 2Department of Infectious Diseases, University of Heidelberg Medical School, Heidelberg, Germany, 3Department of Chemistry, Melbourne University, Melbourne, Australia, 4Bio 21, Melbourne University, Melbourne, Australia, 5Research School of Biology, The Australian National University, Canberra, Australia, 6The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia
melanie.rug@anu.edu.au

Once the malaria parasite Plasmodium falciparum has invaded the human host erythrocyte it starts remodelling its new home immensely. A new trafficking apparatus is set up within the red blood cell, which is devoid of any organelles itsself. Nutrients are delivered from the outside of the cell to the parasite and proteins from within the parasite to the cytoplasm and surface of the host cell via this route. Both events are essential for the survival of the parasite. The latter event provides the parasite with the opportunity to escape the human immune response by transporting a number of proteins underneath and onto the red blood cell membrane. The major virulence factor Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has to be displayed on elevated structures on the surface of the erythrocyte, so called knobs, in order to confer cytoadherence to various ligands on endothelia of different organs in the human host. This leads to occlusion of blood vessels and to the most severe symptoms in malaria patients (e.g. cerebral malaria). We have studied a number of exported proteins and will report on microscopical techniques we used in order to characterise these proteins including immunofluorescence assay, scanning electron microscopy, various transmission electron microscopy approaches (including cryo tomography) and atomic force microscopy. The deletion of one of the proteins presented  results in dramatic alterations in morphology of organelles essential for protein transport to the surface of the infected red blood cell (so called Maurer’s clefts) and in actin cytoskeleton linkage. Other known Maurer’s clefts resident proteins localise to vesicular structures that appear to represent deformed Maurer’s clefts. We will report on possible functions of these proteins in PfEMP1 transport to and display on the surface of the infected red blood cell and Maurer’s cleft architecture.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-5906 THE EFFECTS OF CYCLOOXYGENASE 1 and 2 INHIBITION ON RENOMEDULLARY INTERSTITIAL CELLS IN RATS

Demirci S.1, Seckin İ.1, Sönmez H.2, Ekmekci H.2, Erozenci L. A.3, Popovici M.4
1Department of Histology and Embryology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey, 2Department of Biochemistry, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey, 3Department of Molecular Biology and Genetics, Faculty of Science and Letters, Istanbul Technical University, Istanbul, Turkey, 4Iuliu Hațieganu University of Medicine and Pharmacy, Cluj-Napoca
sibell.demirci@gmail.com

Renomedullary interstitial cells (RMICs) are the dominant cell type in inner renal medulla[1]. Their most distinct characteristic is multiple lipid droplets which believed to be storage units for precursors of prostaglandins in their cytoplasm[2,3]. Especially prostaglandin E2 (PGE2) is synthesized by RMICs in the kidney and produced by three steps[4-7]: 1)Arachidonic acid release from membrane phospholidips; 2)Formation of prostaglandin H2 from arachidonic acid by the action of cyclooxygenases; 3)Specific prostaglandin synthesis. In this study, we examined the effects of PGE2 inhibition on RMIC function. We formed four groups: First group was control. Second group in which we inhibited arachidonic acid release was injected with dexamethasone; third group was treated with ip indomethasine to inhibit non-specific cyclooxygenase and fourth group was injected with ip celecoxib to examine selective cyclooxygenase-2 inhibition. We dissected renal medulla of sacrificed animals after 10 days and counted lipid droplets in 50 random RMICs for each animal in electron microscopy. Our morphometric analysis showed that the number of lipid droplets was significantly decreased in dexamethasone group and was significantly increased in indomethasine and celecoxib groups when compared to control. Medullary hyaluronan, CD44 immunoreactivity and RMIC apoptosis were significantly increased in all groups when compared to control. 24-hours urine values collected on the 10th day were significantly increased in dexamethasone and indomethasine groups; in celecoxib group was similar to control. These results indicate that lipid droplets may be storage units of arachidonic acid, PGE2 inhibition may lead functional changes and apoptosis in RMICs.

This work was supported by Scientific Research Project Coordination Unit of Istanbul University; Project Number: 8402. The authors thank Azize Gumusyazici and Ercument Boztas, the techniciancs of the electron microscopy laboratory in Istanbul University, Cerrahpasa Medical Faculty.

Fig. 1: The graphics of urine volume, urine pH, serum PGE2 levels and number of lipid droplets.

Fig. 2: A) The Alcian blue staining in renal papilla. B) The Alcian blue staining in renal inner medulla. C) The semi-thin sections dyed with toluidin blue.

Fig. 3: Immunohistochemical determination of Caspase-3 and CD44

Fig. 4: 1)Electron micrographs of RMICs in control group.2) Different RMICs in DEX group.3) Different RMICs in IND group.4) Different RMICs in CXB group.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-5923 Sperm ODF2: analysis using ODF2-EGFP transgenic mice

Toshimori K.1
1Department of Reproductive Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba, 260-8670, Japan
ktoshi@faculty.chiba-u.jp

Outer dense fiber 2 (ODF2) is a main structural protein of the sperm tail outer dense fibers as well as a cell-cycle dependent scaffold component of centrosome in somatic cells. ODF2 is located in the main part of the sperm tail from the connecting piece to the distal part of the principal piece by analysis with anti-ODF2 antibody. ODF2 has many isoforms encoded by transcript variants, but few have been well studied since there are no specific antibodies against each isoform.

In this study we cloned five transcript variants of Odf2 gene (Odf2-1a ,-2a ,-3a ,-3b, -4) from mouse testes. Odf2-2a was identified with the transcript variant 3, encoding isoform c (ODF2c). We established an ODF2c-EGFP transgenic (Tg) mouse line and analyzed the expression and localization of ODF2c during the spermatogenesis and the fate of ODF2c in the fertilization process using the Tg mice.

ODF2c-EGFP was first detected in the proximal part of the tail of step 4-5 spermatids and then found in the midpiece to principal piece as the tail developed. In mature sperm ODF2c-EGFP was strongly expressed from the distal end of the connecting piece to the proximal part of the principle piece, but it was detected neither in the proximal and main part of the connecting piece nor in the main and distal part of the principal piece nor in the end-piece. In the fertilization process, the signal of ODF2c-EGFP could be chased until the formation of male pronucleus.

This is the first report of ODF2c Tg mice. The result of the study suggested that ODF2c is associated with the outer dense fibers in the distal part of connecting piece, midpiece and proximal part of the principal piece, and that other isoforms participate with the rest of the structure of outer dense fibers. Since ODF2c-EGFP signal is detected clearly not only during sperm formation but also in the fertilization process, ODF2c Tg mouse sperm will be a useful tool for live-imaging to monitor the events from spermiogenesis throughout to the pronuclear formation.

The authors would like to thank Mr. T Mutoh for his technical assistance. This work was partially supported by a grant from the Japan Society for the Promotion of Science to KT (22112504, 22390033, 24112706) and in part to CI (24592441) and to KY (23791809).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-5958 Three-dimensional distribution of the mitochondrial DNA in the mammalian cell revealed by FIB/SEM tomography.

Okayama S.1, Ohta K.1, Higashi R.2, Nakamura K.1
1Department of Anatomy, Kurume University School of Medicine, kurume, Japan, 2EM lab, Kurume University School of Medicine, kurume, Japan
okayama_satoko@med.kurume-u.ac.jp

[Introduction]

Mitochondrial fission and fusion events are fundamental mechanisms for quality control of the mitochondrial functions. It has been known that the mitochondrial DNA (mtDNA) frequently divide into offspring mitochondria after the fission that has been observed as nucleoid by fluorescent microscopy and the mtDNA dynamics is considered to be coordinated with the mitochondrial turnover. Candidates of molecular mechanisms of the relationships between mtDNA division and the mitochondrial fission have been suggested recently, but their ultrastructural aspect are still unclear. Visualization of the mtDNA at electron microscopic level is a quite important step to understand how they involved in this mechanism, but it is quit difficult to observe by a conventional electron microscopic method. In the present study, we tried to visualize the localization of the mtDNA / nucleoid at EM level by immuno-electron microscopy. We also attempted to visualize 3D distribution of the nucleoid within the mitochondria using focused ion beam scanning electron microscopy (FIB/SEM).

[Materials & Methods]

After chemical fixation in mixture of 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1M phosphate buffer, HeLa cells were immunohistochemically labeled with anti-TFAM IgG antibody(Abnova, USA)and anti-DNA IgM antibody(Progen, Germany). For pre-embedding method, HRP conjugated secondary antibody and DAB reaction was preformed. For immunogold method, nanogold conjugated secondary antibody was used. Immunologically labeled specimens were then strained by OTO method, embedded in resin and applied for the FIB/SEM tomography(Quanta 3D FEG, FEI) and 3D reconstruction was done on computer software(Amira, USA).

[Results & Discussion]

We could not identify any nucleoid-like structure within the mitochondria even in a complete 3D reconstruction by FIB/SEM tomography of the HeLa cells prepared by conventional specimen preparation. In pre-embedding immuno-electron microscopy, DAB immunoreaction products (IR)were observed in the matrix of some mitochondria. Interestingly, DAB-IR depicted in the globular region of the mitochondrial matrix(0.4µm diameter)frequently localizing in the peripheral end of the mitochondrial matrix just adjacent to the inner membrane. In post-embedding immunogold method, gold labels were also observed in a part of the matrix adjacent to the mitochondrial inner membrane. These immunocytochemical results were well coincide with the fluorescent microscopic observation.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-5963 The development of apoplasmic barriers in the cells of primary cortex affects phytoremediation characteristics of plants

Martinka M, Demko V, Lux A
Comenius University in Bratislava Science Park, Comenius University in Bratislava, Mlynska dolina, 842 15 Bratislava, Slovak Republic
martinkambio@yahoo.com

Environmental pollution affects the quality of pedosphere, hydrosphere, atmosphere, lithosphere and biosphere. Phytoremediation, using the plants for remediation and being more cost-effective and fewer side effects than physical and chemical approaches, has gained increasing popularity in both academic and practical circles [1]. The most important plant features affecting their decontamination (phytoremediation) characteristics are the ability to take up and transport the pollutants [2]. These processes may be influenced by changes of pollutant transfer via apoplasmic space of roots [3]. The goal of this study was to find out the relationship between the cell wall modification and the translocation ability of pollutants in the plants.
Tested plants of Allium cepa, Arabidopsis thaliana, and Sorghum bicolor were cultivated in cadmium nitrate (50 µM) containing substrate. After seven-day treatment the roots of plants were investigated by fluorescent microscopy (visualization of apoplasmic barriers with FY088 under Axioskope 2 plus, Carl Zeiss) and transmission electron microscopy (visualization of apoplasmic barriers after contrasting with uranyl acetate, potassium permanganate, lead citrate under JEM 2000FX, JEOL). The aim was to characterise the development and thickness of suberin lamellae in primary cortical tissues related to the accumulation of cadmium in aboveground parts of plants. The cadmium concentration was measured by AAS (Perkin Elmer 1100, USA).
The suberin lamellae in endodermal cells of investigated species are developing with prominent positional effect (starting in endodermal cells against the phloem poles and finishing in the cells against the xylem poles of vascular tissues). The thickness of suberin lamellae in fully matured endodermal cells differs between the tested species: A. thaliana plants develop the thinnest suberin deposits (in the range 45 – 68 nm), whereas A. cepa plants the thickest suberin deposits (several hundreds nm). The accumulation of cadmium in the aboveground parts of plants is the lowest in A. cepa and the highest in A. thaliana.
Based on the results the thicker the apoplasmic barriers are developed the lower is the uptake and translocation of cadmium via apoplasmic space, and thus the less efficient is the decontamination of the environment by phytoremediation methods. The further investigation and modification of processes involved in the apoplasmic barrier development may help to produce plants with more effective phytoremediation characteristics.

1. Lone M. I., He Z.-L., Stoffella P. J., Yang X 2008. Zhejiang Univ Sci B. 9 (3): 210–220.
2. Neil W. 2007. Phytoremediation. Humana Press, Totowa, New Jersey, 480 pp.
3. Lux A., Martinka M., Vaculík M., White P. J. 2011. J Exp Bot 62 (1): 21–37.

This contribution is the result of the project implementation: Comenius University in Bratislava Science Park supported by the Research and Development Operational Programme funded by the ERDF Grant number: ITMS 26240220086.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-5996 Mesoscale organization of insect virus-polyhedra crystals revealed by rapid-freeze, freeze-fracture EM

Morone N.1, Mori H.2, Heuser J. E.3
1Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan
morone@icems.kyoto-u.ac.jp

Cytoplasmic polyhedrosis viruses (CPVs or 'Cypoviruses') are icosahedral ds-RNA viruses that assemble in infected cells of lepidopteran (insect) larvae. They quickly become embedded in elaborate protein crystals called 'polyhedra'. A great deal is known from X-ray diffraction about the internal crystal structure of these highly-ordered polyhedra, but the molecular mechanism(s) by which they assemble and incorporate virions in the cytoplasm of infected insect cells is much less well understood. Here, we have analyzed their molecular mechanisms of assembly, both in their natural environment inside intestinal cells of larval silkworms, and in Sf21 cells in tissue culture. Avoiding the artifacts and difficulties of previous EM-approaches, all samples were here prepared by "quick-freezing" directly from life, followed by freeze-fracture to gain entry into the cells, and then "deep-etching" and rotary Pt-replication to achieve true-to-life, topologically accurate 3-D images of the polyhedra and their associated virions. This approach yielded novel and definitive images of polyhedra in situ, and thereby revealed unique differentiations within cellular environs wherein polyhedra form, and the exact mechanism of inclusion of nascent virions into the polyhedra. Specifically, it allowed us to focus directly on the edges of polyhedra, right where the nascent viruses are being captured and engulfed during the growth of the polyhedra. This allowed us to compare the success of virion-capture and polyhedron-assembly in a variety of different experimental conditions, including the substitution of mutant viruses and incomplete virions lacking critical recognition-factors. Additionally, it allowed us to determine exactly how the architecture of the infected cells' cytoskeletons is altered, to permit this elaborate crystal-formation and virus-incorporation to occur. In this presentation, these observations and results will be described and illustrated by means of realistic three-dimensional images presented in "anaglyph" stereo form. References: 1) Coulibaly F, Chiu E, Ikeda K, Gutmann S, Haebel PW, Schulze-Briese C, Mori H & Metcalf P. The molecular organization of cypovirus polyhedra. Nature 446, 97-101 (2007). 2) Chiu E, Coulibaly F and Metcalf P. Insect virus polyhedra, infectious protein crystals that contain virus particles. Curr Opin Struct Biol 22, 234-240 (2012).

We are most grateful for support from Satoshi Abe and Takafumi Ueno (Tokyo Institute of Technology). This work was supported by WPI-iCeMS and Grants-in-Aid for Scientific Research, JSPS-MEXT in Japan (to NM and JEH).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-6059 STRUCTURE OF NUCLEOLI IN OOCYTES FROM BIDDER´S ORGAN OF CUBAN ENDEMIC TOADS (ANURA: BUFONIDAE, PELTOPHRYNE)

Sanz-Ochotorena A.1, Segura-Valdez M. L.2, Rodríguez-Gómez Y.1, Lara-Martínez R.2, Jiménez-García L. F.2
1Human and Animal Biology Department. Faculty of Biology. University of Havana, 2Cellular Nanobiology Laboratory. Faculty of Sciences. National Autonomous University of Mexico
luisfelipe_jimenez@ciencias.unam.mx

The nucleolus is the nuclear subdomain that assembles ribosomal subunits in eukaryotic cells and it is the largest structure in the cell nucleus. The nucleolar organizer regions of chromosomes, which contain the genes for pre‐ribosomal ribonucleic acid, serves as the foundation for nucleolar structure. The nucleolus disassembles at the beginning of mitosis, its components disperse in various parts of the cell and reassembly occurs during telophase and early G1 phase. In amphibian´s oocytes there are a lot of nucleoli during previtellogenic stages which synthesize part of the necessary machinery that brings the maternal cytoplasm to start protein synthesis in the early embryo. On the other hand, Bidder´s organ (OB) is a vestigial organ in any member of the Bufonidae family located in cranial tip of the male and maybe female gonad. Normally it is inactive and contains little previtellogenic follicles or previtellogenic oocytes. The main goal of this work is to show the presence and structure of nucleoli in nuclei from oocytes of Bidder´s organ in Cuban toads as well as trying to explain why their presence and development in apparently vestigial and possibly nonfunctional structures.
Three male’s toads of Peltophryne fustiger, P. peltocephala, P. florentinoi, P longinasus, P. gundalchi, P. taladai and P. cataulaciceps were collected in their habitats through Cuban archipelago and ethically euthanized. Gonad fragments were fixed in Bouin's fluid, Paraformaldehyde and Glutaraldehyde to be processed for light microscopy and TEM respectively.
The results show that the gonad of the male from all studied species has one pair of Bidder’s organs just above the testes on each cranialis part. Previtellogenic oocytes were seen and as in other members of the Bufonidae Family it was found that ovarian and Bidderian oocytes were morphologically identical. Two or three nucleoli were observed in each nucleus whose structure resemble normal morphology, with ribosomes presents.
Although the presence of previtellogenic follicles is referred to OB as a rudimentary ovary, the structure and dimensions of the nucleoli in those nuclei of Cuban toads indicates that they are active cells capable of producing ribosomes, to ensure protein synthesis. No vitellogenic follicles that may suggest a real function of body Bidder as a producer of viable female cells were observed, however we do not know the reason for the maintenance of the BO. So, it´s possible to suggest the possibility that Bidder´s organ may be an active endocrine organ rather than a simple, rudimentary ovary.

Dirección General de Cooperación e Internalización of the National Autonomous University of Mexico (DGECI-UNAM) for the partial financial support.

Fig. 1: Two nucleoli in Bidder´s oocyte from Peltophryne fustiger

Fig. 2: Nucleolus in Bidder´s oocyte from P. peltocephala

Fig. 3: Peltoprhyne. sp. Bidder´s organ oocytes where nucleoli are observed. On top, cyst of spermatozoa in testicular zone. Mallory technique 400X
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-2-P-6060 STAGES OF SPERMATOGENESIS REVEALED BY TEM IN ELEUTHERODACTYLUS FROGS (ANURA: ELEUTHERODACTYLIDAE).

Rodríguez-Gomez Y.1, Sanz-Ochotorena A.1, Segura-Valdez M. L.2, Lara-Marrtínez R.1, Jiménez-García L. F.2
1Faculty of Biology, University of Havana, Cuba, 2Faculty of Sciences, Universidad Nacional Autónoma de México, Mexico
luisfelipe_jimenez@ciencias.unam.mx

Spermatogenesis includes a sequence of events where from a spermatogonia differentiate other cell types during maturation: primary spermatocyte, secondary spermatocytes, spermatid and sperm. In vertebrates there are two models of spermatogenesis: the longitudinal or cysts in anamniotes and radial in amniotes. Spermatogenesis in amphibian is cystic, in which a Sertoli cell associated with a primary spermatogonia enclosing and forming the cyst wall. Observations in sex cells of amphibians have led to important generalizations, such as the suggestion of a correlation between the structural morphology and the conditions in which fertilization occurs. In the latter, the transmission electron microscopy (TEM) has opened a whole new and expanding field in morphology. It's has been used in many groups of frogs, in which was helpful suggesting evolutionary trends and taxonomic relationships. That's why our goal is to describe the stages of spermatogenesis in five species of the genus Eleutherodactylus using TEM.
Three mature male of five species of Eleutherodactylus (E. blairhedgesi, E. planirostris, E. riparius, E. thomasi, E. varleyi) were collected from nature in occidental Cuba. Frogs were sacrificed and testes were removed and fixed in 2.5% glutaraldehyde. We process samples with TEM techniques and were viewed using a JEOL JEM 1010 TEM.
TEM shows spermatogonia, spermatocytes I and II, different kinds of spermatids and bundles of spermatozoa arrangement in their respective cysts. Spermatogonia begin the spermatogenic process. These cells are the largest in the spermatogenetic lineage. Many mitochondria, a prominent Golgi apparatus, and lipid inclusions in their cytoplasm can be observed. Spermatocytes I are spherical cells, somewhat smaller than the spermatogonia. The chromatin is slightly condensed in their nuclei. Spermatocytes II are scarce and observed with the nuclear material showing different degrees of coiling. Early spermatids are round cells having a smaller size than spermatocyte II and with more compacted chromatin. Late spermatids are easy to identify because their nuclei even more compact.
Spermatozoa arise in clusters inside the cysts. They have an extraordinary nuclear coiling and a cytoplasmic reduction. An elongated acrosome is located at the top of the slender head, showing a substance of moderate electronic density. Between the acrosome and the nucleus appears a subacrosomal space. We distinguished an undulating membrane related to the tail. It’s consists of a fiber yuxtaxonemal part associated with the axoneme, an axial sheath and an axial fiber. The dynein arms in the axoneme are very short or absent. When the spermatozoa reach maturity are released from the bundle and reach the locular lumen and the ductuli efferentes.

Dirección General de Cooperación e Internacionalización UNAM, Mexico, for partial financial support.

Fig. 1: Spermatogonia of  E. planirostris where the nucleus with a nucleolus (nu) therein detailed.

Fig. 2: Primary spermatocytes of E. riparius.

Fig. 3: Spermiogenesis in E. planirostris upon tail formation. Note the mitochondria (m) around the future axial sheath (as).

Fig. 4: E. thomasi sperm head where the acrosome (ac) and the nucleus (n) is observed.
↑ Go to the Table of content ↑

LS-3. High-resolution localization of molecular targets and macromolecular complexes

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-3-IN-2308 Engineering of bacterial phytochromes for in vivo imaging

Shcherbakova D. M.1, Piatkevich K. D.1, Filonov G. S.1, Verkhusha V. V.1
1Albert Einstein College of Medicine, New York, USA
vladislav.verkhusha@einstein.yu.edu

Fluorescence imaging is a powerful and widely used approach for biological research. However, lack of genetically-encoded probes for in vivo imaging is the major limitation in this field. Mammalian tissues are relatively transparent in a near-infrared optical window of 650-900 nm due to significant decrease in hemoglobin and melanin absorbance and still low water absorbance. Therefore, probes with fluorescence spectra within the near-infrared range are preferable for imaging in mammalian tissues and whole animals. On the basis of bacterial phytochromes we have engineered three types of near-infrared fluorescence probes, which utilize present in mammalian tissues heme-derived biliverdin as a chromophore. These probes include several spectrally distinct permanently fluorescent proteins (iRFP670, iRFP682, iRFP702, iRFP713 and iRFP720), fluorescent proteins that are photoactivatable from low to high brightness (PAiRFP1 and PAiRFP2) and bimolecular fluorescence complementation probe that reports on protein-protein interactions (iSplit). The designed near-infrared probes were imaged in tumor models in living animals using fluorescence and photoacoustic techniques. The multicolor whole-body imaging aided by the developed probes should become common approaches in cell and developmental biology, in studies of cancer and pathogen invasion and in biomedicine. The near-infrared fluorescent probes extend the methods developed for cell microscopy into deep-tissue imaging, including multicolor labeling, cell photoactivation and tracking, and detection of protein interactions and enzymatic activities.

This work was supported in part by grants GM073913, CA164468 and EB013571 from the US National Institutes of Health.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-3-O-2334 Single Molecule Localisation Microscopy of the distribution of chromatin nanostructures using standard DNA dyes

Szczurek A. T.1, Prakash K.1,2, Lee H. K.1, Żurek-Biesiada D. J.3, Best G.4,5, Hagmann M.4,5, Dobrucki J. W.3, Cremer C.1,2,4, Birk U.1,4
1Institute of Molecular Biology, Mainz, Germany, , 22Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany,, 33Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland, 4Kirchhoff Institute for Physics, Heidelberg University, Heidelberg, Germany, 55University Hospital Heidelberg, Heidelberg University, Heidelberg, Germany
aleksander.szczurek@gmail.com

In order to investigate DNA/chromatin structure, one may employ optical microscopy as a method of choice, due to feasibility and availability. However, conventional light optical imaging approaches suffer from diffraction of the visible wavelengths used, preventing the resolution of structures that are spaced closer than about 200 nm laterally and 600 nm axially. On the other hand, many other approaches, in particular Electron Microscopy and Electron Spectroscopy Imaging, provided a great amount of knowledge on chromatin nanostructures, down to the few nanometer resolution range. However, these important advantages are balanced by a number of drawbacks, such as the time-consuming sample preparation and the harsh preparation conditions which are likely to alter the structure of interest. To overcome this dilemma, recently novel visible light ‘superresolution’/’nanoscopy’ imaging approaches have emerged. Presently especially structured illumination microscopy and Single Molecule Localisation Microscopy (SMLM) have been established as useful approaches [1]. Using fluorescence-based SMLM methods, it was shown that one may use DNA basepair analogs labelled with special fluorophores, ubiquitous H2B histone targeting, or cyanine derivated DNA dyes in order to better resolve chromatin in the eukaryotic cell nucleus or DNA fibers and bacteria. However, no SMLM studies were performed so far on single cell nuclei with directly stained DNA. Here we present novel application of specific DNA dyes in order to obtain nuclear DNA density maps with high optical and structural resolution. For this, we applied a special SMLM variant, Spectral Precision Distance Microscopy (SPDM) [2]. In mammalian cell nuclei, this SPDM technique yielded a single molecule localisation precision in the order of 15 - 30 nm, corresponding to an optical (two-point) resolution of roughly 40 - 70 nm. We investigated various DNA structures and obtained data with a single DNA fluorophore density as high as 5000/µm2. This constitutes a significant improvement in comparison to previously obtained SPDM data for H2B-GFP histones (100 - 400/µm2). We anticipate that in the near future, this approach may contribute to obtain a three dimensional DNA mapping of genome nanostructures of various cell types, such as stem cells and differentiated cells, or normal and cancer cells. Such precise density maps may also serve as a basis for numerical modeling of the nuclear genome.

[1] C. Cremer, B.R. Masters, Resolution enhancement techniques in microscopy, Eur. Phys. J. H. 38 (2013) 281–344.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-3-O-2434 Single-molecule correlative microscopy using optical excitations in low energy-loss spectra of quantum dots

Pfannmöller M.1,2, Röder I. V.2, Béché A.1, Wacker I.2, Verbeeck J.1, Bals S.1, Schröder R. R.2
1EMAT, University of Antwerp, Antwerp, Belgium, 2CellNetworks, Universitätsklinikum Heidelberg, Heidelberg, Germany
martin.pfannmoeller@uantwerpen.be

Correlative light and electron microscopy allows the combination of dynamic imaging in the light microscope (LM) with visualization of cellular ultrastructures in the electron microscope (EM). The use of fluorescent, semiconductor nanocrystallites (quantum dots, QDs) offers a way to apply only one marker for both modalities. Since they are more electron opaque than biological materials, QDs can be identified in the EM (Fig. 1).
Multi-colour labelling is feasible with QDs in LM but separation in EM is hindered since conventional contrast of different QD particles is similar. Use of different sizes or shapes would allow for segmentation. To prevent varying labelling efficiencies and penetration depths into plastic sections [1], we use two different commercially available QDs: QD-655 and QD-705 (Molecular Probes, USA). The only difference of these core-shell particles is a minimal variation in core composition to alter optical absorption and emission characteristics.
We prepared ultrathin sections of resin (HM20) embedded muscle tissue containing neuromuscular junctions. Acetylcholine receptors (AChRs) of the synapse were labelled by antibody-conjugated QDs-655 and QDs-705. As shown in the light micrographs in Fig. 2A,B, the fluorescent signal of both types is concentrated at synapses. This is verified by the signal of an Alexa Fluor 555 dye conjugated to bungarotoxin, which was used as control to label AChRs (Fig. 2C). Synapses can be relocated in the EM with single QDs at the postsynaptic membrane (Fig. 2D). To separate the randomly distributed QDs we applied scanning transmission electron microscopy (STEM) in combination with electron energy-loss spectroscopy (EELS) at both high spatial and energy resolution. This allows acquisition of EEL signals in the low-loss regime including optical excitations. To identify the spectral features we used samples that were labelled with either only QD-655 or only QD-705 and acquired EELS data sets for single particles. Multivariate statistical analysis and machine learning was applied to learn on the known data sets followed by classification of the unknown data sets from the sample with combined labelling. An ensemble of particles at a synapse is shown in Fig. 3A,B. Selected QDs were classified and highlighted. EEL spectra from the learning and classified sets (Fig. 3C) indicate the separating features, which are attributed to low-energy interband excitations.
Our results show that minor differences of spectral properties of correlative markers on biological samples can be detected in the analytical STEM. This opens the door to multi-colour EM imaging at the single molecule level.
[1] Giepmans, B.N.G. et al.: Nature methods (2005), 2, 743.
[2] Microscopes: FEI Titan G2, Libra 200 (Carl Zeiss Microscopy GmbH).

We acknowledge financial support of the European Soft Matter Infrastructure (ESMI) and the German Federal Ministry for Education and Research, project NanoCombine, grant no. FKZ: 13N11401.

Fig. 1: Figure 1: Conventional electron micrographs of QD-655 and QD-705. A,B: AChRs of neuromuscular junctions were labelled with QDs by immunostaining. Contrast for both particles is very similar in conventional TEM bright field images. C,D: Dark field STEM images of single particles reveal the nearly identical core-shell structure.

Fig. 2: Figure 2: Correlative microscopy of neuromuscular junctions in LM and EM. A,B,C: Labelled ultrathin sections show a correlative signal of QDs-655 (A), QDs-705 (B), and of an Alexa Fluor 555 (AF555) control staining (C). D: Bright field TEM image of the relocated synapse (marked in A-C) at lower resolution to show the ultrastructure of the synapse.

Fig. 3: Figure 3: Optical excitation signals allow separation of QD-655 and QD-705. A: Dark field STEM image of a synapse labelled with both QDs, with selected, classified particles in the white boxed region in false colour. B: Enlargement of box in A. C: Spectra of QD-655 and QD-705 learning sets and average spectra from the classified particles in A-B.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-3-O-2560 Intracellular distribution of phosphatidylinositol-3,5-bisphosphate revealed by quick-freezing and freeze-fracture replica labeling

Takatori S.1, Tatematsu T.1, Akano T.1, Matsumoto J.1, Cheng J.1, Fujimoto T.1
1Nagoya University Graduate School of Medicine, Nagoya, Japan
takatori@med.nagoya-u.ac.jp

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-3-O-2791 Tracking drug pathway in living cells by superresolution microscopy

Hagmann M.1, Celik N.2, Rossberger S.1,2, Licha K.3, Dithmar S.2, Birk U.1,4, Cremer C.1,4,5
1Kirchhoff Institute for Physics, University of Heidelberg, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany, 2Department of Ophthalmology, University Hospital of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany, 3mivenion GmbH, Robert-Koch-Platz 4, 10115 Berlin, Germany, 4Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany, 5Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Heidelberg, Germany
martin.hagmann@kip.uni-heidelberg.de

We present the application of superresolution microscopy to analyze the uptake behavior of pharmaceuticals by adherent living cells.
By a combination of Structured Illumination Microscopy (SIM) and Spectral Position Determination Microscopy (SPDM)[1] in one setup[2] we have obtained the density distribution of labelled pharmaceuticals within the cell at a substantially enhanced resolution.

High light intensities in the order of 10 kW/cm² as necessary for SPDM is hazardous to living cells which is why we imaged the cells using SIM for which about a 100 fold lower light intensity suffices. By that approach we were able to image the cell and the drug density distribution inside and outside of the cell at an approximately two-fold resolution improvement when compared to the conventional Abbé limit.
As the drug distribution can be studied by both SIM and SPDM, accepting the cell apoptosis, we ultimately determined the location of the individual drug molecules inside the cell from an SPDM snapshot with accuracy about 10 fold better than the conventional diffraction limit.

As an application example, we used transiently transfected human retinal pigment epithelium cells where GFP (green fluorescent protein) was targeted against the nucleus and RFP (red fluorescent protein) against the plasma membrane. As an antibody we used Bevacizumab labeled with 6s-IDCC[3] which is used to treat age related macular degeneration[4].

References

[1] C. Cremer, "Optics far beyond the diffraction limit", in Springer Handbook of Lasers and Optics (F. Träger, ed.), pp. 1359{1397, Springer Berlin Heidelberg, 2012.
[2] S. Rossberger, G. Best, D. Baddeley, R. Heintzmann, U. Birk, S. Dithmar, and C. Cremer, "Combination of structured illumination and single molecule localization microscopy in one setup", Journal of Optics, vol. 15, p. 094003, Sept. 2013.
[3] J. Pauli, K. Licha, J. Berkemeyer, M. Grabolle, M. Spieles, N. Wegner, P. Welker, and U. Resch-Genger, "Fluorescent labels with tunable hydrophilicity for the rational design of bright optical probes for molecular imaging", Bioconjugate Chemistry, vol. 24, pp. 1174-1185, July 2013.
[4] S. Rossberger, T. Ach, G. Best, C. Cremer, R. Heintzmann, and S. Dithmar, "High-resolution imaging of auto fluorescent particles within drusen using structured illumination microscopy", British Journal of Ophthalmology, vol. 97, no. 4, pp. 518-523, 2013.

The authors would like to thank Rainer Heintzmann for support with the reconstruction of the images. Further thanks is also extended to Gerrit Best (KIP) and Florian Schock (KIP) for fruitful and inspiring discussions and support throughout the project.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-3-P-1626 Dynamics and organization of homomeric alpha3 glycine receptors in the plasma membrane using single particle detection

Notelaers K.1,2, Rocha S.2, Paesen R.1, Smisdom N.1, Jochen M. C.3, Rigo J.1, Hofkens J.2, Ameloot M.1
1Biomedical Research Institute, Hasselt University and School of Life Sciences, transnational University Limburg, Agoralaan building C, 3590 Diepenbeek, Belgium, 2Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Heverlee, Belgium, 3RNA Editing and Hyperexcitability Disorders Helmholtz Group, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany
kristof.notelaers@uhasselt.be

The mobility and spatial organization of membrane proteins are important factors in their interaction capabilities and physiological roles. In the case of neurotransmitter receptors, lateral diffusion and local trapping are essential in the regulation of their synaptic localization and functionality. One of these receptors is the strychnine-sensitive glycine receptor (GlyR), one of the main mediators of synaptic inhibition. Currently this research project is focusing on homomeric GlyRs consisting of alpha3 subunits. This subunit, encoded by the GLRA3 gene, exhibits two splice variants (GlyR alpha3 L and K) forming receptors with different clustering and desensitization properties. Single particle techniques are now being used to gather more information on nanoscale properties of these different receptors in the plasma membrane. This includes studying the diffusion in living cells using single particle tracking (SPT) and investigating the spatial distribution in the membrane of fixed cells using direct stochastic optical reconstruction microscopy (dSTORM) [1]. GlyR trajectories measured by SPT are analyzed for the detection of anomalous diffusion. Both confined and directed motion are revealed in the receptor populations, offering new perspectives on the regulation of GlyR alpha3 membrane trafficking [2]. The super-resolution images generated by dSTORM are analyzed using pair-correlation analysis [3]. Hereby complementary quantitative information is retrieved concerning the clustering properties of the GlyRs.

[1] K. Notelaers, N. Smisdom, S. Rocha, D. Janssen, J.C. Meier, J.M. Rigo, J. Hofkens, M. Ameloot, Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor alpha3 isoforms in the cell plasma membrane, Biochimica et biophysica acta, 1818 (2012) 3131-3140.
[2] K. Notelaers, S. Rocha, R. Paesen, N. Smisdom, B. De Clercq, J.C. Meier, J.-M. Rigo, J. Hofkens, M. Ameloot, Analysis of α3 GlyR single particle tracking in the cell membrane, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1843 (2014) 544-553.
[3] K. Notelaers, S. Rocha, R. Paesen, N. Swinnen, J. Vangindertael, J. Meier, J.-M. Rigo, M. Ameloot, J. Hofkens, Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy, Histochem. Cell. Biol., (2014) 1-12.

Fig. 1: Representation of the experimental approach used for studying homomeric alpha3 glycine receptors (GlyR) in the plasma membrane of HEK 293 cells. Imaging is sub-divided in live-cell diffusion measurements and fixed-cell aggregation measurements.

Fig. 2: Identification of anomalous diffusion sections in GlyR alpha3 trajectories obtained by live-cell imaging. A. Detection of confined motion (circle), based on confinement probability level (Lc). B. Detection of directed motion (arrow), based on directed motion probability level (Ld).

Fig. 3: Co-expression aggregation study of the GlyR alpha3K (A, C: red) and alpha3L (B, C: green) in fixed HEK 293 cells, showing micrographs and pair crosscorrelation analysis. The splice variants exhibit co-clustering (C, magenta inset). The crosscorrelation is fitted in order to determine the co-cluster radius (ξ^c).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-3-P-2212 Ultrastructure of collagen fibrils after acupuncture treatment during rat tendon healing

Almeida M. S.1, Freitas K. M.1, Oliveira L. P.1, Vieira C. P.1, Guerra F. D.2, Dolder M. H.1, Pimentel E. R.1
1Institute of Biology, Campinas, Brazil, 2Institute of Biomedical Science, Alfenas, Brazil
marcosfisiobr@gmail.com

ABSTRACT
Previous study showed that electroacupuncture (EA) increases the concentration and reorganization of collagen molecules in rat tendon healing (ALMEIDA et al., 2012). However, the analysis of the ultrastructure of collagen fibrils after acupuncture (AC) treatment is unknown. Objectives: To assess the effect of AC protocols on ultrastructure of collagen fibrils during tendon healing. Methods: The Wistar rats were divided into: not tenotomized (normal group), tenotomized (teno group) as well as tenotomized and submitted to manual AC at points Zusanli (ST36 group), Chengshan (BL57 group), Zusanli associated with Chengshan (SB group) and electrical stimulation at points mentioned (EA group). The Mass-average diameter (MAD) (EDWARDS et al., 2005; FLINT et al., 1984) and the reorganization of collagen fibrils diameter were determined at days 7, 14 and 21 after tendon injury. Results: The MAD increased at days 14 and 21 of the healing process in BL and SB groups. In the EA group, the MAD initially increased and at day 21 it decreased. The reorganization of collagen fibrils diameter in EA group at day 14 and SB group at day 21 was similar to the normal (N) group according to Kolmogorov and Smirnov test for two samples. Thick fibrils were not found at EA group in day 21. Conclusion: These results indicate that the use of EA up to day 14 and manual AC at Zusanli and Chengshan points up to day 21, improves the ultrastructure of the tendon, indicating the strengthening of the tendon structure. These data suggest a potential use of AC in rehabilitation protocols. Future studies need to investigate the mechanisms activated by AC during the tendon healing.

This work was funded by CNPq and CAPES

Fig. 1: Electron micrographs of collagen fibrils at days 7, 14 and 21. Note the mixture of thick and thin fibrils in group N. The diameter of collagen fibrils decreases in groups T and E-36. In groups BL57 and SB at days 14 and 21 there are thick fibrils, as well as in the EA group at day 14. At day 21, thick fibrils are not seen in EA group. Bar: 500 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-3-P-2234 Tunable close-to-nature cellulose for enzymatic degradation studies via dynamic high resolution AFM

Ganner T.1, Roŝker S.2, Eibinger M.3, Chernev B.2, Kraxner J.2, Mayrhofer C.2, Aschl T.1, Zahel T.3, Nidetzky B.3, Plank H.1
1Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Graz, Austria, 2Center for Electron Microscopy, Graz, Austria, 3Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria
thomas.ganner@felmi-zfe.at

The introduction of second generation fuels, based on enzymatic cellulose breakdown, might be one of the favorite prospects to encounter our ever growing need for new energy resources. The abundant bio-availability and accessibility of cellulose in our biosphere explains the extensive effort in today’s research to find applicable economic ways for the efficient degradation of cellulose. While the process of enzymatic degradation is known almost for a century the diverse structures of cellulose, its recalcitrant nature and the complex interplay of different enzymes during synergistic degradation elucidate the efficiency problems still present.

In recent years versatile in-situ studies using Atomic Force Microscopy (AFM) revealed strongly varying degradation mechanisms on different cellulose phases (crystalline/amorphous). This becomes highly relevant if they are side by side as found in nature. In order to emulate natural circumstances but still being able to control phase-related composition and its according dimensions, it is necessary to develop an artificial substrate which furthermore provides nanoflat surfaces to allow high-resolution AFM imaging.

In this study we present two new types of substrates to study the degradation of cellulose dynamically in liquid environments via high-resolution AFM. For the first approach, we introduce preparation protocols, entirely based on highly crystalline AVICEL cellulose, to fabricate a substrate with tunable content of amorphous and crystalline cellulose and a surface roughness below 10 nm. We used Raman spectroscopy, X-ray diffraction (Figure 2), scanning and transmission electron microscopy to demonstrate the tunable multiphasic nature of the cellulose substrates further denoted as “mixed amorphous-crystalline cellulosic model substrate” or shortly MACS.

Although well suited, there remains the issue of size control with respect to the crystalline areas in MACS. Therefore, a second concept is introduced, based again on a mixture of amorphous and nanocrystalline cellulose whiskers with typical dimensions of 300 nm to 30 nm. The combination of both phases in a tunable manner results in a nanoflat substrate further denoted as nanocomposite cellulose (TFNC).

Complementary in-situ AFM degradation studies with different enzyme types on MACS and TFNC confirm the expected behavior regarding affinity and activity of different cellulases (Figure 1) and demonstrate the close-to-nature character of both substrates. Hence, both approaches allow conclusions regarding the degradation of natural cellulosic materials, thus helping to understand the complex nature of the process.

The authors thank the Austrian Science Fund for project funding (grant P 24156-B21) and Prof. F. Hofer, Prof. W. Grogger, Ing. H. Schröttner, Dr. Stefan Mitsche, Dr. H. Rattenberger and S. Rauch for support.

Fig. 1: Dynamic AFM measurements of enzymatic degradation of MACS cellulose using Trichoderma reesei cellulases. Due to the different degradation resistivity, highly crystalline particles (top left corner) are much less affected than the surrounding amorphous matrix.

Fig. 2: X-ray diffraction characterization of MACS substrates revealing the tuneability of the crystal content indicated by the evolution of the 101, 10-1, 021 and 002 related peaks of cellulose Ia together with the amorphous peak.

Fig. 3: Dynamic AFM measurements of TFNC substrates during enzymatic degradation revealing the excavation of crystalline parts (a --> b) followed by their full decomposition (b --> d).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-3-P-2479 Living cells toward electron microscopy? Let’s do CLEM, it can be easy!

de Marco A.1, Hekking L.2, Bittermann A. G.3, Guenthert M. M.3, Wepf R. A.3, Langhorst M.1
1FEI Company, Munich, Germany, 2FEI Company, Eindhoven, Nederlands, 3ScopeM ETH, Zurich, Switzerland
alex.demarco@fei.com

Correlative light and electron microscopy (CLEM) aims at combining the large field of view and chemical specificity of fluorescence microscopy with the high resolution ultra-structural details revealed by electron microscopy. CLEM can be extremely powerful in extending electron microscopy analysis to rare events that are impossible to target based on EM morphology alone. Furthermore, efficient workflow solution can speed up complicated experiments by extended automation of different steps along the workflow.

Here, we present different ways of using fluorescence to efficiently target areas for 3D analysis using AutoSlice&View. In the first experiment we started from an already resin embedded tissue and imaged with both modalities. This was possible thanks to a protocol that has both the fluorescence signal and contrasting agent in the embedded block. Due to purely image-based correlation, it was straightforward to directly target a specific area identified by the fluorescence signal without the use of fiducial markers of any kind. The second experiment covers imaging from living cells in the light microscope, to resin embedded cells in the electron microscope. Here we could identify a living cell with an endocytic event occurring through light microscopy and follow that very structure all the way through the sample preparation until the final high resolution 3D electron microscopy. In this second workflow we also introduced automation of the sample preparation steps including measures for efficient relocation procedures.

These experiments clearly highlight the strong potential of using correlative approaches to target small sub-volumes in larger volumes for efficient AutoSlice&View acquisition of 3D electron microscopy data. The use of a flexible software framework is needed to accommodate different workflows, while automation of sample preparation steps will help to cut down the complete experiment time tremendously.

Fig. 1: Images through the complete experiment: from living cells in the light microscope, to fixed embedded cells first in the light microscope and then in the FIB/SEM. The yellow arrowhead indicates the cell of interest, which has been tracked throughout the workflow

Fig. 2: Images through the complete experiment. From top to bottom images collected in the light microscope first and in the FIB/SEM then. In the middle panel are displayed the overlays between the LM and EM images used to correlate. The yellow arrowhead point at the cell followed throughout the workflow
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-3-P-2756 The "blinking" FISH: Localization microscopy of chromatin nanostructure using combinatorial oligonucleotide fluorescence in situ hybridization (COMBO-FISH)

Stuhlmueller M.1, Schwarz-Finsterle J.1, Huellen K.2, Mueller P.1, Spachholz E.2, Lux J.1, Hildenbrand G.1, Bach M.1,4, Hinderhofer K.2, Cremer C.1,3,4, Hausmann M.1
1Kirchhoff-Institute for Physics (KIP), University Heidelberg, D-69120 Heidelberg/Germany, 2Institute of Human Genetics, University Hospital Heidelberg, D-69120 Heidelberg/Germany, 3Institute for Pharmacy and Molecular Biotechnology (IPMB), University Heidelberg, D-69120 Heidelberg/Germany, 4Institute of Molecular Biology (IMB), D-55128 Mainz/Germany
c.cremer@imb-mainz.de

Expansions of DNA repeats are responsible for many human hereditary disorders. The fragile X syndrome (FXS), a neuro-developmental disorder, is the most prominent cause of heritable mental retardation. In the last years it became evident that unusual structural features of the chromatin and the nuclear dynamics in these expandable repeats causes instability of the gene region and lead to their further expansion. In this study the quantification of the repeat expansion and the resulting nanostructural changes were examined by a novel interdisciplinary approach that is based on combinatorial oligonucleotide fluorescence in situ hybridization (COMBO-FISH) and spectrally assigned localization microscopy. For this, fluorescence labelled oligo-nucleotides were used to label the repeats uniquely as well as the FMR1 gene regionin 3D intact cell nuclei. The localization of the hybridized and labelled oligo-positions with nanometer precision was performed using Spectral Precision Distance/Position Determination Microscopy (SPDM) in the ‘blinking’ mode. The correlation of the obtained data on the single cell level with standard human genetic diagnostic methods with bulk DNA may contribute to understand the impact of chromatin nano-architecture on repeat instability and expansion. Furthermore, a new diagnostic approach based on optical sequencing is envisaged to count repeats in cell nuclei especially in cases of premutated alleles that are very unstable with a strong tendency to expand to the full mutation upon transmission to the next generation.

C. Cremer et al. (2011)Superresolution Imaging of Biological Nanostructures by Spectral Precision Distance Microscopy (SPDM), Biotechnology Journal 6: 1037 – 1051

Figure legend

Left: Dual colour COMBO-FISH experiment on human fibroblast cells. (1) conventional wide-field fluorescence image of the fragile X gene region labelled with Alexa488 and Alexa568. (2) SPDM image λex: 488nm. (4) SPDM image λex: 568nm. (3) Overlay of (2) + (4). Right: (A) Reconstruction of the SPDM data, the intensity values of the individual signals are proportional to the number of adjacent signals. (B) The positions of the signals are shown in green and the determined area of the clusters in red. (C) Visualization of the detected structure

This work was funded by the FRONTIER innovation fund of the University of Heidelberg within the excellence initiative of the DFG.

Fig. 1: see Text
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-3-P-2778 Behavior of oxidized lipids in monolayers and Langmuir-Blodgett films studied by surface pressure measurements and Atomic Force Microscopy

Grauby-Heywang C.1, Mathelié-Guinlet M.1, Faye N. R.1, Moroté F.1, Cohen-Bouhacina T.1
1Laboratoire Ondes et Matière d'Aquitaine, Université de Bordeaux, Talence, France
ch.heywang@loma.u-bordeaux1.fr

Some lipids are particularly sensitive to oxidation, such as unsaturated ones. The presence of oxidized lipids in cellular membranes or simplified membrane models promotes important changes in the lipid packing or in phase separation processes. Here, we study by Atomic Force Microscopy (AFM) the behavior of oxidized lipids in Langmuir-Blodgett (LB) films. LB films were transferred on hydrophilic mica surfaces, polar head groups of lipids being separated from mica by a thin water layer of 1 nm thick. We used two strategies : either following the evolution of LB films of an unsaturated phospholipid (palmitoyl-oleoyl-phosphatidylcholine, POPC) naturally ageing in contact of atmospheric oxygen, or incorporating a known amount of a defined oxidized derivative of POPC in POPC monolayers before their LB transfer (“mixed” monolayers and LB films). Two oxidized lipids were studied, PoxnoPC and PazePC, both characterized by a shortened oxidized chain ending with a polar group (aldehyde or carboxylic acid group, respectively).
First, homogenous AFM images of freshly prepared LB films of POPC confirm that this lipid is a homogenous liquid expanded phase. On the contrary, AFM images of naturally ageing POPC LB films show the appearance of small circular domains after 2 days of exposure to air. These domains (not observed if the samples are kept under vacuum) are characterized by a higher thickness (+0.8 nm) as compared to the intact POPC areas, likely due to a reversal of the more polar oxidized chain to be in contact with the water layer between the film and the mica surface.
In the case of mixed monolayers, surface pressure measurements show different results according to the polarity of the oxidized shorter chain. PazePC/POPC monolayers behave ideally (additivity of mean molecular areas taking into account the ratio of both molecules into the monolayer), whereas PoxnoPC induces an expansion of mixed monolayers. In all cases, AFM images are on the whole homogenous. However, they also show the presence of some higher circular spots (2-8 nm higher than the surrounding phase) in PazePC/POPC LB films suggesting the formation of aggregates of PazePC, ejected during the monolayer compression.
Finally, these results suggest a different behavior of oxidized lipids using the two strategies previously described. In particular, they support the hypothesis of a natural oxidation occurring in areas of POPC LB films presenting likely a looser packing or a defect.

Authors thank NSI platform of LOMA (CPER COLA2) for AFM equipment and technical support.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-3-P-2780 Novel multiple immunolabeling method of ultrathin resin sections for high resolution SEM

Wandrol P.1, Vancova M.2,3, Nebesarova J.2,4
1FEI Czech Republic, Brno, Czech Republic, 2Institute of Parasitology, Biological Centre of ASCR, v.v.i, Ceske Budejovice, Czech Republic, 3. Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic , 4Faculty of Science, Charles University in Prague, Czech Republic
nebe@paru.cas.cz

At present the multiple immunolabeling is limited by the number of suitable labels, which is possible to distinguish in the electron microscope. The most often ccolloidal gold nanoparticles (AuNPs) conjugated with an antibody localize a bond between the molecule of interest (antigen) and the antibody within the cell ultrastructure. For simultaneous labeling of more antigens AuNPs with different diameters are used. However they must be small enough to ensure good labeling efficiency together with good visualization of particles in cell structures, typically in the range 6 – 15 nm. Therefore the detection of more than two molecules is difficult because of a narrow diameter range. Recently several approaches of immunodetection of more than three proteins by the electron microscope were used, such as using nanoparticles of different shapes [1] or composition [2].

This abstract proposes method that doubles number of localized proteins compared to commonly used methods. Maximum number of simultaneously detected antigens using nanoparticles of various shapes in TEM is now 5 [3]. This method increases this number up to ten. Moreover, it allows using two antibodies conjugated with AuNPs of the same diameter as well as two different primary antibodies produced in the same animal. Since the diameter of NPs influences the labeling density, the possibility of using NPs with the same diameter allows simultaneous comparison of two antigen concentrations in the studied specimen area. In contrary to current methods of immunolocalisation, high resolution scanning electron microscope (HRSEM) working in BSE and transmission mode is used for the evaluation of immunolabeling results.

The general concept is to label both sides of the ultrathin section with different antibodies conjugated to same electron-dense nanoparticles and to distinguish on which side the label is by advanced imaging method. The scheme in Figure 1 illustrates the principle of the method involving sequential recording of two images at different energies (E1 around 1 keV, E2 in the range 15-30kV) and by means of different detectors (BSE for E1, STEM for E2). Markers on the top side of the ultrathin section are visualized in image I1, markers on both sides in image I2. Labels on the bottom side in the image I3 is result of the subtraction of I1and I2 images. Final image is then correlation of the I1, original I2 and I3 images and localizes both types of molecules within the ultrastructure.

References:

[1] M Slouf et al, Colloids and Surfaces B-Biointerfaces, 2012, 100, p.205-208

[2] J Nebesarova et al, EMC 2012 Conference Proceedings (2012) Manchester, LS 2.3

[3] VV Philimonenko et al, Histochem Cell Biol 2014, Jan 22 (Epub ahead of print)

The authors acknowledge funding from the Technology Agency of the Czech Republic, project TE01020118

Fig. 1: Ultrathin section is labeled for double immuno-detection at both sides. The diagram shows the sequential acquisition of images 1 and 2 at different energies using different detectors and their following processing.

Fig. 2: Figure 2. Images of the ultrathin section of salivary glands (Ixodes ricinus) labelled at the upper side against BSA and at the bottom side against hemoglobin (10nm AuNPs). A. BSE image shows top side particles; B. STEM image visualizes particles on both sides C. Final processed image showing particles on the top side (red) and bottom (yellow)

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-3-P-3193 UV-activated conversion of Hoechst and DAPI to their green-emitting protonated forms and their application in super-resolution microscopy

Żurek-Biesiada D.1, Szczurek A.2, Waligórski P.3, Prakash K.2, Cremer C.2,4, Dobrucki J.1
1Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland, 2Institute of Molecular Biology (IMB), Mainz, Germany, 3The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków, Poland, 4Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Heidelberg, Germany
dominika.zurek@uj.edu.pl

KEY-WORDS: DNA-binding dyes, Hoechst, DAPI, photoconversion, protonation, super-resolution microscopy

BACKGROUND. In our recent paper we have reported that, upon excitation with UV, DNA-binding dyes Hoechst 33258 and DAPI undergo photoconversion [1]. Although a typical loss of fluorescence (photobleaching) is observed, apparently a fraction of the population of the blue-emitting form of the dye do not lose the ability to fluoresce but is are converted into a blue-excited, green-emitting form. Mass spectrometry data suggest that the observed spectral changes are associated with UV-induced protonation of the Hoechst molecule [2].

Goal. The purpose of this work is to shed more light on the photophysics of the process of UV-activated conversion of the DNA-binding dyes and to apply those dyes in super-resolution microscopy.

Methods. Spectrofluorimetry, mass spectrometry, spectrally-resolved confocal microscopy, SPDM

RESULTS and CONCLUSION. We demonstrate that the UV-generated form of Hoechst 33258 presents different spectral characteristics, with its absorption and emission spectrum shifted towards longer wavelengths. The photoproduct is stable in time, UV-dose dependent and the process of photoconversion does not require the presence of the DNA. Moreover, Hoechst 33258 exhibits the same spectral properties as the forms of Hoechst that can be obtained by subjecting the dye to a highly acidic environment (pH 0.5-3.0). By using mass spectrometry and spectrofluorimetry we demonstrate that exposing Hoechst to UV leads to generation of three protonated forms of the dye. The spectral properties and affinity to nucleic acids of these protonated forms differ from the original blue-emitting form. Our recent findings suggest that Hoechst may be used as a DNA probe in super-resolution microscopy (Szczurek et al., submitted). The key to a successful exploitation of the phenomenon of photoconversion is understanding of the photophysics of this process.

Reference
[1] Zurek-Biesiada D, Kędracka-Krok S, Dobrucki JW. UV-activated conversion of Hoechst 33258, DAPI, and Vybrant DyeCycle fluorescent dyes into blue-excited, green-emitting protonated forms. Cytometry A 83(5):441-51 (2013).
[2] Żurek-Biesiada D, Waligórski P, Dobrucki J. Mass spectrometry and fluorimetry analysis of blue-excited green-emitting protonated forms of Hoechst 33258 generated by UV illumination (in preparation).
[3] Szczurek A, Prakash K; Lee HK, Żurek-Biesiada D; Best G, Hagmann M, Dobrucki J, Cremer C, Birk U. Single molecule localisation microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes (manuscript submitted).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-3-P-3243 Structural analysis of inhibitor-bound Arp2/3 Complexes

Chemeris A.1, Ydenberg C.2, David V.3, Gautreau A.3, Goode B.2, Sokolova O.1
1Lomonosov Moscow State University, Moscow, Russia, 2Brandeis University, Boston, MA, 3CNRS UPR3082, Gif-sur-Yvette, France
Angelina1707@mail.ru

Dynamic reorganization of actin cytoskeleton networks is vital to the proper formation of organ systems during embryogenesis, and to such processes as wound healing, neuronal activity, and inflammatory responses. These dynamic rearrangements of actin filamentous networks are controlled by the activities of numerous actin-binding proteins (ABPs). One of the important ABP, Arp2/3 complex directly binds to the side of existing actin filament to nucleate formation of a daughter filament. This ‘branched nucleation’ activity of the Arp2/3 complex is essential for cell motility, endocyosis, and intracellular transport of specific organelles and vesicles in virtually all eukaryotic cell types. On the other hand, it is unclear how the densely branched networks of filaments are subsequently ‘pruned’ or debranched during phases of rapid actin network turnover, which is equally essential in vivo. Further, still not well understood is what conformational changes in Arp2/3 complex are associated with inactivation state.

Here we use single particle EM to determine the 3D structures of Arp2/3 complex bound to two different inhibitors: Gmf1 and Arpin. Arp2/3 complex bound to each of these ligands was isolated by affinity chromatography, applied to EM grids negative stained, and analyzed on a JEOL 2100 electron microscope at 200 kV. Projections of Arp2/3 complexes were captured by CCD camera (Gatan) at 8 Mpix resolution and 3D reconstructions were generated using random Conical Tilt method implemented in EMAN2.1, with subsequent refinement in IMAGIC-4D.

Our results show that GMF-bound Arp2/3 complex exists in two inactive conformations, suggestive of two separate Gmf binding sites on the complex (Fig. 1), as was proposed earlier (1). 90% of Arp2/3 complexes bound to Arpin adopted an inactive conformation (Fig. 2). The binding site for Arpin is overlapping with the binding site for VCA domains (3), suggesting a mechanism for Arpin inhibition of Arp2/3 complex (Fig. 3). Additionally, we have demonstrated that different cellular factors, working in concert, like Crn1 and Gmf1, have an increasing inhibitory effect, implicated in more Arp2/3 complexes in inactive conformation (Fig. 2B). Thus our results provided an improved level of mechanistic understanding of Arp2/3 complex regulation by determining the conformations (Fig. 4) of an inactive complex.

This work has been in part supported by CRDF-global (to OS).

Fig. 1: Reconstruction of the Gmf1-bound Arp2/3. (A) Class-sum images of the active Arp2/3 with Gmf1-GFP. (B) Schematic of the possible binding sites of Gmf1. (C) Imposed open states for Gmf-bound I and II. (D),(E)-3D reconstructions of Gmf-bound I and II Arp2/3 states with a docked crystal structure of the Arp2/3.

Fig. 2: (A) Analysis of relative frequencies of Arp2/3 complex conformations with and without Arpin. (B) Cooperate inhibition of theArp2/3 complex by Gmf1 and Crn1.

Fig. 3: Arp2/3 complex bound to Arpin. (A) Single-particle EM class-sum images of the Arp2/3 complex with Arpin in the open conformation. (B) Schematic, demonstrating the possible binding site of Arpin (open circle), based on positions of Arpin blobs (filled circles).

Fig. 4: Conformation changes in the Arp2/3 complex. (A) Three basic conformations of the Arp2/3 complex. (B) Inactive conformations of theArp2/3. Arrows show the conformational changes upon (A) activation and (B) inactivation.
↑ Go to the Table of content ↑

LS-4. Structure of macromolecules and macromolecular assemblies

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-4-IN-2313 Cryo-electron microscopic and genetics studies of flagellar dynein regulation.

Kikkawa M.1
1Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo
mkikkawa@m.u-tokyo.ac.jp

Cilia/flagella are cell organelles conserved among eukaryotes and generate fluid flow. The bending motion of flagella is driven by axonemal dyneins and requires concerted activity of several hundreds of flagellar proteins.

To elucidate the mechanisms of dynein motor regulations in cilia/flagella, we have developed a new method to label specific proteins using biotin-streptavidin system. This labeling technique attaches the BCCP (Biotin Carboxyl Carrier Protein) tag to the target protein, which are subsequently enhanced by adding streptavidin and biotinylated cytochrome c. The 3D structure of labeled-flagellar were visualized by combining with cryo-electron tomography and sub-tomographic averaging. This enables us to locate flagellar proteins in 3D structure.

Using this system, we identified nonspecific intermolecular collision between central pair and radial spoke as one of the regulatory mechanisms for flagellar motility. By combining cryo-electron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to radial spoke head paralyzed flagella. Moreover, the motility defect in a central pair projection mutant could be rescued by the addition of exogenous protein tags on radial spoke heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of central pair- and radial spoke-mediated regulation of flagellar motility. These results suggest that mechano-signaling between central pair and radial spoke regulates dynein activity in eukaryotic flagella.

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-4-IN-2446 Dissecting Limitations in high-resolution single particle cryo-EM

Fischer N.1, Haselbach D.1, Kirves J.1, Rodnina M. V.1, Chari A.1, Stark H.1
1Max-Planck-Institute for biophysical Chemistry
hstark1@gwdg.de

Recently numerous high-resolution structures were obtained for macromolecular complexes by single particle cryo-EM techniques. The importance of the development of new generation pixel detectors and the possibility to correct for motion by the alignment of image frames has been particularly stressed and is considered to be one of the main important recent hardware developments. These detectors are also used within the context of modern high-end microscopes, and in parallel, considerably improved computational image processing tools were developed over the years. It is thus difficult to determine the exact contribution of the new detectors to the latest success in high-resolution cryo-EM. We wanted to understand the influence of the detector and other hardware components in more detail and systematically recorded large image datasets of a biochemically well-defined macromolecular complex (70S ribosome-EF-Tu-kirromycin complex) varying only one imaging parameter at the time. We studied data recorded on CCD and the Falcon pixel detector (DDD) using either a normal FEI Schottky field emitter (SFEG) or the high-brightness gun (XFEG) in a Cs corrected Titan Krios.

Using the DDD/XFEG setup, we obtained the structure of the ribosome at 3.1 Å resolution which is identical to the resolution obtained by X-ray crystallography for the same complex from a different organism. Surprisingly, using images recorded on CCD camera still results in a 3.9 Å reconstruction which shows the possibility of high-resolution structure determination on CCD cameras. A more detailed analysis revealed however, that a ~3 times higher image statistics is required for CCD images to obtain the same resolution as for DDD images.

One conclusion from this analysis is that most structural studies are not necessarily limited by the detector but by the lack of biochemical control during purification and handling of the macromolecular complex. Macromolecules may easily become damaged during the purification procedure and further destabilized being in a non-optimum buffer environment. We have therefore developed a screening method to find maximum stabilization conditions by systematically screening the chemical space. Having analyzed >80 different macromolecular complexes, we found a very broad pH distribution in buffers that were most stabilizing. This is in severe conflict with an EMDB database analysis where the current entries reveal a narrow pH distribution around pH 7.5. The correct pH is one of the most important factors for stabilization of macromolecules and we speculate that screening optimum buffer conditions will provide a substantial boost in the functional understanding and structure determination of macromolecular complexes.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-4-O-1658 Phase-plate Imaging for Cryo-TEM: Types, Benefits and Applications

Marko M.1
1Wadsworth Center, NY State Dept. of Heath, Empire State Plaza, Albany NY 12201-0509 USA
mike.marko.em@gmail.com

Vitreously frozen specimens for cryo-TEM retain a near-native state, free of artifacts due to chemical fixation or stains that limit resolution, but their low-Z composition yields weak amplitude contrast. Imaging by phase contrast, at low electron dose, is required. Thus, SNR maximization is important; it is improved by zero-loss energy filtering [1] and noise-free “counting” imaging detectors [2], which, along with use of an FEG and 300 kV, bring us closer to “all that physics will allow”. However, the oscillating CTF of traditional defocus phase-contrast imaging remains a spatial-frequency-dependent hindrance to good SNR.  Recording “in-focus” images with a phase plate can be the next step in optimizing imaging [3].

Phase plates increase the SNR--especially at lower spatial frequencies--for cryo-electron tomography as well as for particle-picking and alignment of sub-100 kDa molecules in single-particle cryo-EM. They have been used to reveal structures in 3D not seen before [4].

While there are many types are in use, under development or proposed [review: 5], a typical phase plate creates a phase shift between the unscattered beam and electrons scattered by the specimen. The most-common type uses a thin film to shift the phase [3,6]. While scattering in the film slightly reduces contrast and may degrade the envelope function at the highest spatial frequencies, this type is easy to make and operate, and is the only one in routine use.

In principle, phase plates without films should be preferred. Those using a voltage applied to an electrode in or near the path of the unscattered beam [e.g. 7,8] have adjustable phase shift (either positive or negative), which can be optimized for accelerating voltage and intrinsic phase shift. Phase shift can also be created by a magnetic field [9] or by a laser [10]. Also, blocking half of the back-focal-plane diffraction pattern over a range of low spatial frequencies boosts phase contrast over that range [11].

We will discuss applications and operating considerations of various types of phase plate, and present our recent work on construction and use of Zernike phase plates [12].

[1] R. Grimm et al., J. Microsc. 183(1996)60-68.
[2] X. Li et al, Nat. Meth. 10(2013)584-590.
[3] R. Danev et al., Ultramicroscopy 109(2009)312-325.
[4] R. Rochat et al., J. Virol. 85(2011)1871-1874.
[5] R. Glaeser, Rev. Sci. Instr. 84(2013)11101
[6] M. Malac et al., Ultramicroscopy 118(2012)77-89.
[7] R. Cambie et al., Ultramicroscopy 107(2007) 329-339.
[8] S. Hettler et al., Microsc. Microanal. 18(2012)1010-1015.
[9] C. Edgecomb et al., Ultramicroscopy 120(2012)78-85.
[10] H. Müller et al., New J. Phys. 12 (2010)073011.
[11] B. Buijisse et al., Ultramicroscopy 111(2011)1696-1705.
[12] M. Marko et al., J. Struct. Biol. 184(2013)237-244.

Supported by NIH grant 8R01GM103555.

Fig. 1: 1.  Examples of film-type phase plates in the objective-lens back focal plane (not to scale).  A. Zernike; unscattered electrons go through central hole, others shifted by π/2 [3,12].  B. Hilbert; half of diffraction plane shifted by π [3].  Hole-free; unscattered electrons phase-shifted by induced, charged-up spot [6]. 

Fig. 2: 2.  Some film-free phase-plate types (in back focal plane; not to scale).  A. Central electrode or ring-magnet shifts phase of unscattered beam [5,7,9].  B. Coaxial cable: inner-conductor potential forms electrostatic field at unscattered beam [8].  C. Single-sideband blocking of low frequencies gives uniform transfer at 0.5 over that range [11].
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-4-O-2305 Revealing drug-induced conformational states of macromolecular complexes by high-resolution cryo-electron tomography

Maletta M.1, Marsalek L.3, Sani M.4, Simonetti A.5, Fabretti A.6, Slusallek P.2, Peters P. J.7
1Cell Biology II, NKI-AVL, Amsterdam, The Netherlands, 2German Research Center for Artificial Intelligence, Saarbrücken, Germany, 3Eyen SE, Prague, Czech Republic, 4Vironova AB, Stockholm, Sweden, 5Architecture et Réactivité de l´ARN, Université de Strasbourg, CNRS, France, 6Laboratory of Genetics, Dept. of Biosciences and Biotechnology, University of Camerino, Italy, 7Department of Health Medicine and Life Science, Maastricht University, The Netherlands
lukas.marsalek@eyen.se

Drug-induced conformational changes of macromolecular complexes provide indispensable insight into the function of cellular nanomachines and aid, among other things, to rationally design drugs targeting them. Unfortunately, only a few techniques exist, that allow observation of those changes in close-to-native environment.

Cryo-electron tomography (CET) is emerging technique that allows determining three-dimensional structure of macromolecular complexes in quasi-native environment by studying frozen-hydrated samples. However, despite providing tremendous insights into structure and organization of cellular organelles, flagella, microtubules and other large and/or symmetric objects, its ability to observe smaller asymmetric macromolecular complexes in resolution sufficient to reveal drug-induced conformational states has been disputed so far.

We present a high-resolution cryo-electron tomography pipeline (HRCET), which builds on open software (IMOD and PyTom) and combines state-of-the-art computational and imaging techniques like cryo-electron tomography (CET) with direct electron detector (DED), sub-volume averaging (SVA), robust contrast transfer function (CTF) correction, template-bias prevention or X-ray fitting to allow for unambiguous detection of moderate-sized conformational changes on even asymmetric molecular nanomachines.

Using the proposed pipeline, we detect antibiotic-induced change of 70S E. Coli ribosome. We show that drug-stabilized, drug-free and partially-assembled states can be computationally separated with sufficient resolution to allow for docking of high-resolution X-ray structures, facilitating biological interpretation of the observed changes. We also show that a semi-quantitative estimation of the drug affinity is possible using our technique.

The presented pipeline for high-resolution cryo-electron tomography (HRCET) represents another step towards user-friendly, highly reproducible workflow necessary for widespread application of cryo-electron tomography, ultimately allowing HRCET to become a reliable tool for answering some of the key questions that arise in practical drug design process.

We wish to thank the PyTom development team, Sascha De Carlo, Rishi Matadeen, Stefano Marzi, David Mastronarde, Raimond Ravelli and Miloš Vulović for their enthusiasthic support, suggestions and feedback on this work. The authors have been supported financially and with equipment by Dutch national e-infrastructure, NeCEN, SURF Foundation and EU FP7 grant agreement nº 267038.

Fig. 1: Workflow for high-resolution cryo-electron tomography.Tilt series are collected using Titan KRIOS with Falcon I DED. IMOD (http://bio3d.colorado.edu/imod/) package combined with our own code is used to align and reconstruct the tomograms. Using PyTom (http://pytom.org/), particles are localized and averaged to arrive at final structures.

Fig. 2: Analysis of the found conformational states of 70S ribosome.A) 70S ribosome with EF-G. B) 70S only. Density relative to EF-G is overlapped as a green mesh. C) Density corresponding to the EF-G alone. D) Map of the 50S only, which was also identified in the datasets. Atomic ribbons are fitted X-ray structures (2WRI, 2WRJ).
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-4-O-3051 Neutralizing antibodies can initiate genome release from human enterovirus 71

Pavel P.1,2, Perera R.1, Cardosa J.3, Suksatu A.1, Kuhn R. J.1, Rossmann M. G.1
1Purdue University, West Lafayette, United States of America, 2CEITEC, Brno, Czech Republic, 3Sentinext Therapeutics, Penang, Malaysia
pavel.plevka@ceitec.muni.cz

Antibodies were prepared by immunizing mice with empty, immature particles of human enterovirus 71 (EV71). EV71 is a picornavirus that causes hand, foot, and mouth disease. In infants and small children the infection may proceed to encephalitis that can be fatal or result in permanent brain damage. The capsid structure of EV71 empty particles is different from that of the mature virus and is similar to “A” particles encountered when picornaviruses recognize a potential host cell prior to genome release. The "A" particles are expanded relative to the stable form of the virions and are prone to release their genomes. The monoclonal antibody E18, generated by this immunization, induced a conformational change when incubated with mature virus, transforming infectious virions into A particles. Thus, binding of E18 to the virus might correspond to receptor interaction. The resultant loss of genome that was observed by cryo electron microscopy and a verified by flouresecent Sybr-GREEN dye assay. The release of the genome inactivated the virus. As the mechanism for virus inactivation has now been established the E18 has the potential to be developed for anti-EV71 therapy. Antibodies recognizing epitopes similar to that of EV71 could be prepared in mice. The antibody-mediated virus neutralization by the induction of genome release has not been previously demonstrated. Furthermore, the present results indicate that antibodies with genome-release activity could also be produced by immunization with immature particles for other picornaviruses.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-4-O-3255 Observation of intracellular complexes of filopodia at molecular resolution with cryo-ET

Aramaki S.1, Mayanagi K.2, Aoyama K.3,4, Yasunaga T.1
1Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Fukuoka, Japan, 2Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan, 3Application Laboratory, FEI Company Japan Ltd., Tokyo Japan, 4Graduate School of Frontier Biosciences, Osaka University
shinji@yasunaga-lab.bio.kyutech.ac.jp

Introduction: Cryo-electron tomography (cryo-ET) give us nanometer resolution structure of biological samples. Samples which observed by cryo-ET are fixed at liquid nitrogen temperature so it can keep biological condition structures. This technique have developed dramatically during last decade. In our research we have tried to unveil the intracellular structure of filopodia with this technique. Filopodia are very important structure for cells. They have very thin and finger-like (needle-like) structure and have an important role like antennae for cells. This structure associate with a lot of cell processes such as cell migration, neurite outgrowth and so on. However mechanisms of filopodia are still unknown. Therefore we would like to reveal the formation mechanisms of filopodia with cryo-ET.
Methods: We use NG108-15 (neuroblastoma/glioma) as a model nerve cell. These cells are cultured on the QUANTIFOIL (Au 0.6/1) directly and manually plunge frozen by second cryogen such as liquid ethane. In this method the samples are rapidly frozen, so it is able to avoid the artifact from any chemical fixations. Hence we can reveal the detail 3D structure of biological condition samples with transmission electron microscope. We use Tecnai G2 Polara, FEI Company operated at 200kV with the GIF 10eV energy filter.
However hydrated biological samples are very week for electron beam, and so we have to minimize radiation of electron beam and EM images must be with low dose system.Thus it cause low signal-to-noise ratio (S/N ratio) of image, so we need to process the acquired images with computers.
Results: We succeeded to observe the ultrastructure of filopodia. In filopodia, actin filaments are bundled by fascin in 36 nm period and this is equal to the half pitch of actin filaments. Moreover we using helical averaging technique to intracellular acin filaments and get atomic resolution actin filament model, and the bundling region of actin filament by fascin are observed. However we could not observe any motor proteins such as myosin because of crowd environment in the cell.
Conclusion: At the first, we are going to reveal the actin bundling mechanisms by fasin. Next, we would like to unveil the mechanisms of motor protein like myosin. It is very important to understand the mechanisms of formation of filopodia. It will give us new insight to the mechanisms of intracellular transportation.

Fig. 1: (Upper Left) XY-plane, (Upper Right) YZ-plane, Segmented image of the reconstructed volume of filolpodia.

Fig. 2: (Left) Interaction region beteen actin filaments and fascin of the reconstructed image. (Right) Helical averaged image of an actin filament.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1453 Atomic force microscopy investigation of amyloid fibril formation of Escherichia coli RNA polymerase σ(70) subunit

Dubrovin E. V.1, Koroleva O. N.1, Khodak Y. A.1, Kuzmina N. V.1, Yaminsky I. V.1, Drutsa V. L.1
1M.V.Lomonosov Moscow State University
dubrovin@polly.phys.msu.ru

σ70 subunit is a part of Escherichia coli RNA polymerase holoenzyme and plays a key role in transcription initiation. Using atomic force microscopy (AFM) imaging in different conditions (both in air and in liquid), we have found that this protein forms amyloid fibrils under a wide range of cationic concentrations including physiological ones. These fibrils have straight cylindrical shape based on a helical structure with diameter 5.4 nm and length from several tens nanometers up to several microns (figure 1). By the utilization of ultrafilters, which either allowed only σ70 monomers passing through or, oppositely, retaining them, we have proved that σ70 subunit aggregation is a spontaneous process and does not require any additional catalyst. To understand the mechanism of aggregation we have studied three mutant variants of σ70 subunit devoid the whole 1.1 region (N-terminus) or its part. All studied mutant proteins showed either the same or better ability to aggregate compared to the wild type of σ70 subunit. The obtained data allowed us proposing a model of σ70 subunit aggregation, which is based on the domain swapping mechanism accompanied by partial rearrangement of protein structure with subsequent intermolecular β-sheets formation due to the exposure of amyloidogenic regions. σ70 subunit of Escherichia coli RNA polymerase may serve as a good model object for studying amyloid fibril formation and searching factors influencing this process. Directed fibril formation can be also utilized in molecular architecture and other nanobiotechnology applications.

The President grant program for young researchers (grant MK-312.2013.2) is acknowledged

Fig. 1: AFM height image of a σ70 linear aggregate.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1454 AFM study of DNA self-organization on molecular nanopatterns on graphite

Dubrovin E. V.1, Kuzmina N. V.1, Yaminsky I. V.1
1M.V.Lomonosov Moscow State University
dubrovin@polly.phys.msu.ru

DNA is one of the most important molecules for utilization in biological nanotechnologies, since it has unique recognition capabilities, high mechanical rigidity, physicochemical stability and possibility for repeated denaturation-hybridization cycles. Directed DNA immobilization on solid substrate through a "bottom-up" approach is one of the challenging tasks for bionanotechnology. In this work, peculiarities of DNA adsorption on modified highly oriented pyrolytic graphite (HOPG) were studied using atomic force microscopy (AFM). Three alkane derivatives with the same chain length (C18) but different functional groups (-COOH, -OH and -NH2) were used for HOPG modification: stearic acid (CH3(CH2)16COOH), stearylamine (CH3(CH2)16CH2NH2, octadecylamine) and stearyl alcohol (CH3(CH2)16CH2OH). All three modifiers form regular nanopatterns on its surface due to particular orientation of carbon chains along the crystallographic axes of graphite. We have shown that these molecular nanopatterns allowed immobilization of DNA in extended form. Moreover, DNA molecules self-organize on octadecylamine and stearyl alcohol nanopatterns along three directions, reflecting crystallographic axes of HOPG. The influence of the type of functional group in the nanopattern and its concentration on the shape of adsorbed DNA molecules was studied and analyzed using statistical analysis of fluctuations of the contours of biopolymers. Conformation of adsorbed DNA molecules were defined using the analysis of the scaling exponent ν, which relates mean squared end-to-end distance and contour length of the polymer. The persistence length of DNA adsorbed on molecular nanotemplates was determined from AFM images and compared to that of native DNA.

The President Grant program for young researchers (grant MK-312.2013.2) is acknowledged.

Fig. 1: AFM height image of λ-DNA molecules adsorbed on octadecylamine nanopattern on highly oriented pyrolytic graphite.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1465 Single Particle Cryo-EM of the Archaellum (Archaeal Flagellum)

D'Imprima E.1, Neiner T.2, Banerjee A.2, Tripp P.2, Mills D. J.1, Albers S. V.2, Vonck J.1
1Max Planck Institute of Biophysics, Frankfurt am Main, Germany, 2Max Planck Institute for terrestrial Microbiology, Marburg, Germany
eddimpri@biophys.mpg.de

The methods of X-ray crystallography and NMR spectroscopy can provide detailed information on molecular structure and dynamics. At the cellular level, optical microscopy reveals the spatial distribution and dynamics of molecules tagged with fluorophores. Electron microscopy (EM) overlaps with these approaches, covering a broad range from atomic to cellular structures. In this field single-particle electron cryo-microscopy (cryo-EM) is used to resolve the three-dimensional structure of large macromolecular complexes. The specimen to be analyzed is prepared by rapid cooling of a thin layer of an aqueous solution of macromolecules on an EM grid, a thin amorphous layer of ice is formed, in which objects are visible without any staining agent. Because of recent progress in the EM and image processing methods, cryo-EM has become a major tool for structural biology in the molecular to cellular size range.

We decided to use single particle cryo-EM to investigate the structural interactions in the archaellum. Both bacteria and archaea use their flagellum to swim towards favorable conditions. The bacterial flagella assembly system is related to the type III secretion system, however archaella (Jarrell & Albers, Trends Microbiol. 20, 307-12, 2012) contain proteins that are homologous to type IV pilus components. It is therefore interesting to understand the assembly mechanics of archaella and their evolutionary connection to the known surface appendages, e.g., flagella and type IV pili in gram-negative bacteria. We are focusing on understanding the archaella assembly via characterizing homologously and heterologously expressed Fla genes and building an interaction map of the different Fla proteins.

Our first targets are interactions between three proteins involved in archaellum assembly and rotation: FlaX, a 28-kDa monotopic membrane protein, essential for crenarchaeal archaella assembly, that shares a distant similarity with methyl accepting chemotaxis proteins; FlaH, a predicted soluble ATP-binding protein and FlaI, an ATPase that hexamerizes upon binding ATP (Reindl et al., Mol. Cell 49, 1–14, 2013). FlaX forms high molecular weight complexes. To study the oligomeric state of FlaX directly, we performed cryo-EM and found that in vitro FlaX forms rings of various sizes with a diameter of 26–38 nm (Banerjee et al., JBC 287, 43322–30, 2012). Class averages display a rotational symmetry of 15–23, 18 and 19 being most common (Fig. 1). Biochemical experiments show that FlaX and FlaH interact. Preliminary results suggest that FlaH binds to the inside of the FlaX ring (Fig. 2).

We thank Prof. Dr. Werner Kühlbrandt for his support.

Fig. 1: Fig. 1. The upper row shows class averages after multireference alignment in order of increasing size. The lower row shows the same averages with rotational symmetry applied from 15 (left) to 23 (right). Adapted from Banerjee et al., 2012. Fig. 2. Cryo-EM micrograph of FlaXH complex shows densities inside the FlaX ring. Scale bars: 25 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1489 Single particle electron microscopy of prokaryotic ATP synthases

Allegretti M.1, Mills D. J.1, Mayer F.2, Cossio P.1, Peng G.1, Hummer G.1, Mueller V.2, Vonck J.1
1Max Planck Institute of Biophysics, Frankfurt am Main, 2Goethe University, Frankfurt am Main
maallegr@biophys.mpg.de

Single particle EM (SP) is emerging as an invaluable tool for the study of biological structures and protein complexes in their native environment from nanometer to sub-nanometer resolution. With the emerging new direct electron detection cameras SP is becoming the most suitable technique to get three-dimensional models and conformational variability at intermediate to high resolution of macromolecular complexes (> 150 kDa in size) when crystals are not available or as a complementary technique to X-ray crystallography.
ATP synthases are ubiquitous rotary machines conserved in all three domains of life, which convert a transmembrane electrochemical gradient into chemical energy by a rotary catalysis mechanism. They consist of a soluble F1 or A1 part and a membrane-bound Fo or Ao part. X-ray structures are known for many subunits and subcomplexes of the ATP synthases, but the structure of the transmembrane domain is still not completely understood.
At the moment SP is the most suitable technique to get a three dimensional reconstruction of this kind of asymmetric protein complexes. Progress has been made in determining structures of the archaeal and mitochondrial ATP synthases, but there is still a lack of structural knowledge about the small bacterial F1Fo ATP synthases and nothing is known about the variability of the ring stoichiometry in the Ao of A-type ATP synthases.
This work focuses on two stable ATP-synthases from two thermophiles, the bacterium Aquifex aeolicus (Peng G. et al., FEBS Letters 2006, 580, 25, 5934-5940) and the archaeon Pyrococcus furiosus (Vonck J. et al., J Biol Chem 2009, 285, 84, 10110-10119). 2D image classes and 3D reconstructions of negatively stained protein from Aquifex aeolicus show two different conformations, one with a bent central stalk and an unusual round conformation of the F1 head, the other with a triangular shape head. The involvement of the bent central stalk in blocking the rotation of the rotary machine in hydrolysis is under investigation.
A cryo-model of the archaeal ATP-synthase at 13 Å (data collected on film negatives) suggests a very small ring in the membrane side compared to other archaeal species, interesting feature from a bioenergetic point of view for the ATP production of the anaerobic Pyrococcus furiosus. Data collection with the FEI-Falcon-II direct electron detector is in progress and initial results show that better maps can be obtained from a much lower number of particles than on film. In addition, the consistency of the hypothesis of a very small ring (eight subunits) is being further investigated calculating the Bayesian probability with which the raw particles fit four ATP-synthase models owing different ring stoichiometries (Cossio P. & Hummer G., J Struct Biol 2013, 184, 3, 427-437).

we thank Werner Kühlbrandt for the possibility to have the state-of-the-art microscopes in the Structural Biology Department of the Max Planck Institute of Biophysics.

Fig. 1: Aquifex aeolicus F1Fo. a) Electron micrographs of A. aeolicus F1Fo stained with uranyl acetate 1%; class averages at the bottom right showing two different conformations; scale bar:50nm. b) 3D reconstructions (UCSF-Chimera). c) The same 3D reconstructions (UCSF-Chimera solid viewer) show the different conformations of the head subunits.

Fig. 2: Pyrococcus furiosus A1Ao. a) Electron micrograph of A1Ao embedded in vitreous ice; scale bar:50nm. b) Map of the A1Ao complex with (right) and without (left) fitted available atomic models. c) A slice through the Ao domain with the c-ring crystal structure from E. hirae superimposed; the crystal has a larger diameter than the EM density.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1595 Assembly and molecular architecture of PI4K IIα complexes

Peterek M.1,2, Bouřa E.3, Němeček D.1,2
1Central European Institute of Technology, Masaryk University, Brno, CZ, 2Department of Biochemistry, Max Planck Institute, Martinsried, DE, 3Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, CZ
peterek@ceitec.muni.cz

The phosphatidylinositol 4-kinases (PI4K) catalyze the production of phosphatidyl inositol phosphates (PIPs) that regulate membrane associated signal transduction and protein trafficking in eukaryotic cells [1]. The PI4Ks are also hijacked by several RNA viruses to generate membranes enriched in phosphatidylinositide 4-phosphate lipids that are used as viral replication platforms [2]. Two types of PI4K's have been identified in mammalian cells: the type II kinases (α and β isoforms) that are stably associated with membranes and the type III kinases (α and β isoforms) that are soluble and remain in cytosol [3]. While type III kinases share homology to structurally well characterized PI3 kinases the structure of type II kinases is unknown.

We expressed and purified a recombinant PI4K IIα kinase and discovered that it can assemble into large multimeric complexes. Here, we present the initial structural analysis of these complexes that were imaged by negative stain and cryo electron microscopy. Electron micrographs showed heterogeneous particles of globular shape ranging from 110 to 310 Å in diameter. The particles were separated into three groups based on their overall diameter, aligned and classified in 2D using EMAN package. Most particles (~70%) appeared as small compact spheres of ~110 Å diameter. About 17% particles had an oval shape with dimensions ~250 x 200 Å. Interestingly, about 13% of the particles formed large rings with ~310 Å outer diameter, ~140 Å thickness and ~90 Å inner hole. While the small particles likely represent a tight assembly of several PI4K IIα molecules (54 kDa), the two larger particles exhibit similar architecture as some other membrane binding proteins – respectively, the intermediate particles appear as compact assemblies of the human caveolin-3 protein [4] and the large rings are not dissimilar to assembled ESCRT proteins at the necks of budding viruses from the cell membrane [5]. 

[1] Greaham, T.R. and Burd, C.G. (2011) Trends Cell Biol. 21:113-121.
[2] Altan-Bonnet, N. and Balla, T. (2012) Trends Biochem. Sci. 37:293-302.
[3] Balla, A. and Balla, T. (2006) Trends Cell Biol. 16:351-361.
[4] Whiteley, G., Collins, R.F. and Kitmitto, A. (2012) J. Biol. Chem. 287:40302-16.
[5] Votteler, J. and Sundquist, W.I. (2013) Cell Host Microbe. 14:232-241.

This work was conducted in the Central European Institute of Technology supported by the project CZ.1.05/1.1.00/02.0068 financed from European Regional Development Fund.

Fig. 1: Electron micrograph of negatively stained PI4K IIα shows heterogeneous complexes of assembled PI4K subunits – besides small round particles (arrowheads) the micrograph also shows large complexes of disk-like appearance in both top and side views (arrows).

Fig. 2: Class averages (first column) and representative particles of three major groups of PI4K IIα complexes: (1) Round particles of ~110 Å diameter, (2) oval particles of ~250 x 200 Å diameter and (3) large disk-shaped particles with ~310 Å diameter and ~90 Å inner hole.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1597 Assembly and Molecular Architecture of the TSC1-TSC2 Complex

Guha D.1, Nellist M.2, Plitzko J. M.3, Nemecek D.1,3
1Central European Institute of Technology, Masaryk University, Brno, CZ, 2Department of Clinical Genetics, Erasmus Medical Center, Rotterdam, NL, 3Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, DE
guha.dipanjan@rediffmail.com

The TSC1-TSC2 complex plays an important role in the mechanistic target of rapamycin (mTOR) signaling pathway that integrates signals from extracellular growth factors, nutrients, energy deficit or inflammation and downstream controls cell metabolism and growth [1]. TSC1 is a ~130 kDa hydrophilic protein that shows no sequence homology to other known vertebrate proteins and likely guides and stabilizes the proper assembly of the TSC1-TSC2 complex [2]. TSC2 is a ~200 kDa protein that contains a conserved 163 amino acid region close to the C-terminus that is homologous to GTPase activating proteins (GAPs). The TSC1-TSC2 complex possesses RHEB GAP activity [3], and conversion of active GTP-bound RHEB into the inactive GDP-bound form by the complex downregulates the activity of mTOR complex 1 (TORC1) [1]. Pathogenic mutations in either TSC1 or TSC2 genes lead to tuberous sclerosis complex (TSC), an autosomal dominant disorder characterized by neurological symptoms, skin and renal abnormalities [4]. Recently, it has been shown that TSC1 and TSC2 assemble into high molecular weight complexes (>1 MDa) [5, 6]. However, the molecular architecture of this complex is unknown. We co-expressed epitope-tagged TSC1 and TSC2 in HEK 293T cells and imaged the affinity purified TSC1-TSC2 complexes by negative stain electron microscopy. Micrographs showed small rings of the complex that had a disk-like appearance. Initial alignments and classification of ~1000 particles using EMAN [7] revealed two kinds of rings: a smaller ring with ~90-Å diameter and 20-Å inner hole that seems to be composed of 5 subunits (Figure 2a) and a larger ring-like structure (~120 Å diameter and ~30 Å inner hole) that may correspond to a hetero-octamer of four TSC1 and four TSC2 subunits, according to the estimated average molecular mass of the complex. Ongoing data analysis aims to identify the different subunits in the two respective complexes and provide the structural basis for their function.


[1] Laplante M, Sabatini DM (2012) Cell, 149, 274–293.
[2] Sun, W., et al. (2013) Nat.Commun., 4, 2135.
[3] Maheshwar, M. M., et al. (1997) Hum.Mol.Genet., 6, 1991–1996.
[4] Gomez, M., et al. (2013) The tuberous sclerosis complex, Oxford University Press.
[5] Hoogeveen-Westerveld, M., et al. (2012) BMC Biochem., 13, 18.
[6] Menon S., et al. (2014) Cell 156, 771–785.
[7] Tang, G. et al. (2007) J. Struct. Biol., 157, 38-46.

This work was conducted in the Central European Institute of Technology supported by the project CZ.1.05/1.1.00/02.0068 financed from European Regional Development Fund.

Fig. 1: Scheme of the mechanistic target of rapamycin (mTOR) pathway. Growth factor signals and energy status regulate the activity of the TSC1-TSC2 complex. The RHEB GAP activity of the TSC1-TSC2 complex inhibits the activity of mTOR complex 1 that controls cell metabolism and growth.

Fig. 2: Epitope-tagged TSC1-TSC2 complexes were affinity purified from HEK 293T cells (inset shows Coomassie-stained SDS-PAGE gel of the purified complex). Electron micrograph of negatively stained complexes shows individual particles that appear as disk-like structures with an overall diameter ~100 Å (arrows)

Fig. 3: Classification of TSC1-TSC2 particles revealed two kinds of ring-like structures. Class average of the larger ring (top left) has ~120-Å diameter and ~30-Å hole, whereas the average of the smaller ring (bottom left) has ~90-Å diameter with ~20-Å hole. Representative particles from each class are shown in the right columns.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1671 Electron-cryomicroscopy of macromolecular complexes using a direct electron detector

Mills D. J.1, Allegretti M.1, Vonck J.1
1Max Planck Institute of Biophysics, Frankfurt am Main, Germany
deryck.mills@biophys.mpg.de

The introduction of direct electron detectors with higher detective quantum efficiency and fast read-out marks the beginning of a new era in electron cryo-microscopy. Data can be collected with a much higher signal-to-noise ratio than was possible before (Figure 1) and the effects of beam-induced specimen motion can be corrected by subframe alignment. Near-atomic resolution reconstructions can be obtained when all conditions are met: 1) pure and homogeneous protein sample; 2) optimal ice thickness and sample concentration in the cryo specimen; 3) optimal coma-free microscope alignment.

Using the FEI Falcon II direct electron detector in video mode on an FEI Polara electron microscope, we have reconstructed a map of the 1.2 MDa F420-reducing hydrogenase (Frh) from methanogenic archaea from a data set of 27,000 particles1 (Figure 2). Video frames were aligned by a combination of image2 and particle3 alignment procedures to overcome the effects of beam-induced motion. The reconstructed density map at 3.4 Å resolution showed a significant improvement over a recent reconstruction from a much larger data set recorded on film4. All secondary structure, densities for the cofactors (four FeS clusters, an FAD, a NiFe cluster and two other ions, most likely Fe and Zn) as well as clear side chain densities for most residues were clearly resolved (Figure 3) and allowed determination of an atomic model1 (Figure 4).

1 Allegretti, M., Mills, D. J., McMullan, G., Kühlbrandt, W. & Vonck, J. eLife 3, e01963, 2014.

2 Li, X., Mooney, P., Zheng, S., Booth, C. R., Braunfeld, M. B., Gubbens, S., Agard, D. A. & Cheng, Y. Nature Methods 10, 584-590, 2013.

3 Bai, X.-c., Fernandez, I. S., McMullan, G. & Scheres, S. H. W. eLife 2, e00461, 2013.

4 Mills, D. J., Vitt, S., Strauss, M., Shima, S. & Vonck, J. eLife 2, e00218, 2013.

We thank Greg McMullan for his help in setting up the Falcon II detector in video mode, Özkan Yildiz and Juan Castillo for computer setup and Werner Kühlbrandt for his support.

Fig. 1: Part of a cryo-electron micrograph of the 1.2 MDa Frh complex at 900 nm defocus. The image was collected using an FEI Polara at 300 kV on the Falcon II direct electron detector. The complexes are easily recognisable. Scale bar: 25 nm.

Fig. 2: Reconstruction of the tetrahedral Frh complex at 3.4 Å resolution. Each of the twelve FrhABG heterotrimers is shown in a different colour.

Fig. 3: A slice of the map density for one heterotrimer without and with the fitted atomic model. Each of the three subunits FrhA, FrhG and FrhB is shown in a different color as in Figure 4.

Fig. 4: The hydrogenase Frh regenerates the reduced form of F420, a coenzyme of several enzymes in the methanogenesis cycle, using molecular hydrogen. Frh consists of 3 subunits, FrhA (43 kDa), FrhG (26 kDa) and FrhB (31 kDa) with several cofactors as shown. The electron transfer chain is clearly visible in the atomic model built in the EM density.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1746 Electron Microscopy Studies on the Pore-Forming Toxin

GANASH M. A.1
1king Abdulaziz university, Bology department, Kingdom of Saudi Arabia - Jeddah
, P.O. Box : 80200, 
Zip Code : 21589
mganash@hotmail.com

Electron microscopy is an important tool, which can provide essential low- and medium-resolution information on the size and quaternary structure of membrane proteins stabilized with detergent or in a lipid membrane. This abstract describes the results from the electron microscopy studies on APEC ClyA protein.

Escherichiacoli cytolysin A also known as hemolysin E (ClyA, also known as hemolysin E, HlyE) is a 34 kDa cytolytic α-helical pore-forming toxin. The crystal structure of soluble monomeric E. coli K-12 ClyA was previously solved at high resolution and this showed that ClyA had a novel structure that had not previously been seen in the data bank of proteins. Avian pathogenic E. coli (APEC), strain JM4660ClyA is 75% sequence identical to E. coli K-12 ClyA and has many significant similarities.

To investigate Avian pathogenic E. coli ClyA pore formation, purified Avian pathogenic E. coli ClyA was incubated with detergent 1% ß-octylglucoside (ß-OG). The result shows the majority of the pores were circular from the top view and bind together in complex but uniformly sized clusters. The pores can be seen as spikes from the side view. The clusters may well be spherical assemblies of pore, not unlike virus particles. Seen from above some appear to have a central pore surrounded by 6 others. This may be consistent with 12- fold symmetry seen for K12 ClyA pores in the crystal structure. Future work will focus on further experiments on electron microscopy of the pores forming toxins to enable greater understanding of mechanisms of pore formation.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1927 Electron microscopy and single-particle 3D reconstruction of negatively stained 60-160 kDa proteins provides valuable structural information at moderate resolution.

Ryazantsev S.1
1Department of Biological Chemistry, David Geffen School of Medicine at University of California, Los Angeles, CA, USA
sryazant@ucla.edu

Cryo-electron microscopy (cryo-EM) and single-particle 3D reconstruction can produce spectacular results with an appropriate sample, equipment, and hundreds of thousands of particles. Available cryo-EM techniques have greater success with large molecular complexes such as ribosomes, viruses, etc. But many biologically important proteins have a molecular weight in the range of 10 to 100 kDa, which is not optimal for cryo-EM. Negative staining can be used for structural analysis of 60 to 160 kDa proteins, when other options are limited. For instance, many native, full-length immunoglobulins (class G in particular, IgG) are not crystallized and therefore X-ray structure may not be obtained. There are no complete structures for human IgG subclasses available. We applied a single-particle 3D reconstruction approach to negatively stained samples of human IgG2 (hIgG2), and produced a 3D model with resolution of 1.78 nm [1]. The model shows details known from X-ray data from isolated Fab and Fc subunits [2]: Fab and Fc subunits; variable and constant parts; some individual domains. The presence of these details validates our model. Since V- and C-parts of Fab have separate densities, we estimated that structural “units” as small as 25 kDa may be distinguished by this technique. The orientation of the subunits in space is crucial for IgG effector functions. Based on orientation of Fab subunits within hIgG2 molecule we determined that our hIgG2 sample was in fact an A/B isoform. For the first time, the existence of hIgG2 isoforms was confirmed by direct observation. Understanding of the 3D structure of hIgG is essential for meaningful design of modern IgG-based therapeutics. Other examples of 3D reconstruction for the 60-160 kDa proteins will be presented and discussed – in all cases 3D structures obtained from negatively stained samples were validated by independent methods. The resolution was from 1.2 to 2 nm by FSC=0.5 criterion. Negative staining increases the signal-to-noise ratio and therefore makes alignment procedures in 3D reconstruction robust, which facilitates better resolution. Based on our experience, a reasonable 3D model from negatively stained protein samples may be obtained within a few weeks using ~10K particles, non-cryo basic EM equipment, free software (EMAN), and cheap multi-core PC computer(s). We will discuss limitations and advantages of this technique in detail.

References
1. Ryazantsev S, Tischenko V, Nguyen C, Abramov V, Zav'yalov V. (2013) Three-dimensional structure of the human myeloma IgG2. PLoS One., Jun 7;8(6):e64076.

2. Kratzin HD, Palm W, Stangel M, Schmidt WE, Friedrich J, et al. (1989) The primary structure of crystallizable monoclonal immunoglobulin IgG1 Kol II. Biol Chem 370: 263–272.

Author thanks Anne Hawthorne for editing manuscript and support; Vladimir Tischenko, Christopher Nguyen,Vyacheslav Abramov, Vladimir Zav'yalov for participation in this project.

Fig. 1: 3D reconstruction of negatively stained human IgG2 isoform A/B. (a) Model projections and corresponding class-averages. (b) FSC plot indicating 1.78 nm resolution at FSC = 0.5. (c) hIgG2 3D model segmented and visualized in Chimera. (d) Comparison of EM [1] and X-ray [2] (pictured in gray) Fab structures at 1.78 and 1 nm resolution respectively.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1934 An epigenetic metabolic small molecule, O-acetyl-ADP-ribose, modulates the SIR-nucleosomes pre-heterochromatin

Tung S. Y.1, Tsai H. C.2, Tsai S. P.1, Lee S. P.1, Shen H. H.2, Liou G. G.2, 3, 4
1Insitute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, ROC, 2Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli 35053, Taiwan, ROC, 3Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan, ROC, 4Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 10617, Taiwan, ROC.
bogun@nhri.org.tw

It is not possible to clearly visualize how chromatin condenses to heterochromatin in vivo. However, in an in vitro system for the budding yeast Saccharomyces cerevisiae, the requirements for pre-heterochromatin filament formation mirror those found in vivo (1). The Sir2, Sir3 and Sir4 proteins mediate silencing at the telomeres and silent mating type loci in yeast. The assembly of silent heterochromatin requires the deacetylation of histone amino terminal tails by Sir2, followed by the association of Sir3 and Sir4 with the hypoacetylated histone tails, and then the recruitment of more SIR complexes along the chromosome fiber to form extended silent heterochromatin domains (2-4). Here we report that the nucleosomes and the Sir2, Sir3 and Sir4 proteins, which are required for in vitro filament assembly, are also components of these filaments, confirming that the filaments are SIR-nucleosome filaments. We show the individual localization patterns of the Sir proteins on this SIR-nucleosome filament. Furthermore, we show that the epigenetic metabolite, O-acetyl-ADP-ribose (AAR) binds to not only Sir2 but Sir3 also. We also demonstrate that AAR plays a specific and essential role in promoting the formation of this SIR-nucleosomes pre-heterochromatin (Figure1).

References

1. Onishi, M., Liou, G.-G., Buchberger J. R., Walz, T. and Moazed, D. 2007. Role of the conserved Sir3-BAH domain in nucleosome binding and silent chromatin assembly. Mol. Cell 28:1015-1028.

2. Hoppe, G. J., Tanny, J. C., Rudner, A. D., Gerber, S. A., Danaie, S., Gygi, S. P. and Moazed, D. 2002. Steps in assembly of silent chromatin in yeast: Sir3-independent binding of a Sir2/Sir4 complex to silencers and role for Sir2-dependent deacetylation. Mol. Cell. Biol. 22:4167-4180.

3. Luo, K., Vega-Palas, M. A. and Grunstein, M. 2002. Rap1–Sir4 binding independent of other Sir, yKu, or histone interactions initiates the assembly of telomeric heterochromatin in yeast. Genes & Dev. 16:1528-1539.

4. Rusche, L. N., Kirchmaier, A. L. and Rine. J. 2002. Ordered nucleation and spreading of silenced chromatin in Saccharomyces cerevisiae. Mol. Biol. Cell 13: 2207-2222.

We thank Yu-Ching Chen, Yi-Yun Chen, Wen-Li Peng, Ai-Ru Wan, lab members of Liou lab for technical help, Dr. Chung-Shi Yang at NHRI, Dr. Yeu-Kuang Hwu at Acad. Sinica(AS) & NSRRC, Image center of Institute of Molecular Biology, AS for providing a Hitachi-H7650, a Jeol 2100 and an FEI Tecnai G2 EM, respectively, to be used. This work was supported by grants NSC-102-2311-B-400-002 & MG-103-PP-08.

Fig. 1: Figure1. Modulation of SIR-Nucleosome filament formation by AAR. Electron micrograph showing the in vitro assembly reaction containing Sir2/Sir4, Sir3, nucleosome and AAR (A);  an enzymatic defaced Sir2, Sir2H364Y/Sir4, Sir3, nucleosomes and NAD (B) orSir2H364Y/Sir4, Sir3, nucleosomes and AAR (C), respectively. Bar represents 100 nm.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1936 Structure of native oligomeric Sprouty2 and its property of electroconductivity

Chen F. J.1, 2, Lai C. C.1, Lee K. W.1, Liou G. G.1, 3
1Institute of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli, Taiwan, ROC, 2Department of Photonics & Institute of Electro-Optical Engineering, National Chiao Tung University, Hsinchu, Taiwan, ROC, 3Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC
951008@nhri.org.tw

Receptor tyrosine kinases (RTKs) signaling pathways are involved in many fundamental cellular processes, such as apoptosis, chemotaxis, differentiation, migration, proliferation and survival (1-5). Sprouty2 (Spry2) is known as an important regulator of RTK signaling pathways (3-4). Therefore, the property of Spry2 is worth to be investigated more. In this study, we find that Spry2 is able to self-assemble into oligomers with a high affinity KD value of approximate 16 nM, through the BIAcore surface plasmon resonance analysis. The three dimensional (3D) structure of Spry2 looks like a donuts shaped with two lip-covers, solved by electron microscopy (EM) single particle reconstruction approach (Figure 1). Furthermore, using energy dispersive spectrum of EM and total reflection X-ray fluorescence (TXRF) methods to analyze the elements carried by Spry2, it is demonstrated that Spry2 is a silicon and iron containing protein. The silicon may contribute for the electroconductivity of Spry2. And the electroconductivity of Spry2 exhibits concentration-dependent feature. This first report of silicon and iron containing protein and its 3D structure may provide a hint to allow us to (1) study the potential mechanism of switching electronic transfer on or off for controlling the signal transduction and (2) develop a new type of conductor or even semiconductor using biological or half-biological hybrid materials in the future.

References

1. Bache KG, Slagsvold T, Stenmark H (2004) Defective downregulation of receptor tyrosine kinases in cancer. EMBO J 23: 2707-2712.

2. Haglund K, Di Fiore PP, Dilic I (2003) Distinct monoubiquitin signals in receptor endocytosis. Trends Biochem Sci 28: 598-603.

3. Ishida M, Ichihara M, Mii S, Jijiwa M, Asai N, Enomoto A, Kato T, Majima A, Ping J, Murakumo Y, Takahashi M (2007) Sprouty2 regulates growth and differentiation of human neuroblastoma cells through RET tyrosine kinase. Cancer Sci 98: 815-821.

4. Mason JM, Morrison DJ, Basson MA, Licht JD (2006) Sprouty protein: multifaceted negative-feedback regulators of receptor tyrosine kinase signaling. Trends Cell Biol 16: 45-54.

5. Schreiber SL, Bernstein BE (2002) Signaling network model of chromatin. Cell 111: 771-778.

We thank SP Lee, SP Tsai, YC Chen, YY Chen & labmates of Liou lab for technical help; Institute of Molecular Biology, Acad. Sinica(AS), Dr. Chung-Shi Yang at NHRI, Dr. Wei-Hau Chang at AS, Dr. Yeu-Kuang Hwu at AS & NSRRC for providing FEI Tecnai G2, Hitachi H7650, Jeol 1400 & Jeol 2100F EM, respectively, to be used. This work was supported by grant MG-101-PP-08 & NSC-99-2311-B-400-002-MY3.

Fig. 1: Different views of representative 3D reconstitutional model of Spry2. The overall dimensions of donut-like part have a thickness of approximately 4.2 nm, an internal diameter around 5.0 nm & external diameters around 13.4 and 10.0 nm, measured from two vertices or close points of lobs, respectively. The estimated resolution of density map is 25 Å.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-1981 Conformational changes leading to DNA delivery in T7 bacteriophage upon receptor interaction

Cuervo A.1, González-García V.1, Pulido M.1, Chagoyen M.2, Arranz R.1, Castón J. R.1, Fernández J. J.1, García-Doval C.1, Valpuesta J. M.1, Camacho A.3, van Raaij M. J.1, Martín-Benito J.1, Carrascosa J. L.1, 4
1Department of Macromolecular Structure. Centro Nacional de Biotecnología, CSIC. Darwin 3, Cantoblanco, 28049 Madrid, Spain., 2Systems Biology Department, Centro Nacional de Biotecnología/CSIC, Darwin 3, Cantoblanco, 28049 Madrid, Spain., 3Centro de Biología Molecular “Severo Ochoa”/CSIC, Cantoblanco 28049 Madrid, 4Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia), Cantoblanco, 28049 Madrid, Spain
jlcarras@cnb.csic.es

Most of bacterial viruses need an specialised machinery named the tail to deliver its genome inside the bacterial cytoplasm without disrupting cellular integrity. T7 bacteriophage is a well- characterized member of the Podoviridae bacteriophage family infecting E. coli, and it presents a short non-contractile tail that assembles sequentially in the viral head after DNA packaging. T7 tail is a complex of around 2.7 MDa composed by a tubular structure with a central channel that serves as a conduit for DNA ejection surrounded by fibers.

We used cryo-electron microscopy (cryo-EM) and image reconstruction methods together with biochemical interaction essays to determine the precise topology of the 4 proteins that form the tail complex. Further experiments allowed to identify the protein and lipid bacterial compounds used as receptor for the virus, being able to set up an in vitro ejection system for T7. Characterization of the ejection reactions by cryo-EM allowed us to build a three-dimensional model of the tail after DNA ejection. The structural analysis of the three-dimensional models before and after ejection permitted to unravel the conformational changes that take place in the tail complex during DNA delivery and to propose a model for T7 infection.

The similarities found in several components of the tail machinery, comprising the nozzle domain, as well as the gatekeeper and the connector, for different viruses from the podoviridae family suggest that the DNA ejection mechanism is conserved based on common structural arrangements.

This work was partly funded by Grant BFU2011-29038

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-2004 Three-dimensional arrangements of membrane associated E. coli polysomes

Hoffmann T.1, 2, Ortiz J. O.1, Antonoaea R.2, Hartl F. U.2, Baumeister W.1
1Department of Structural Biology, , Max-Planck Institute of Biochemistry, 82152 Martinsried, Germany, 2Department of Cellular Biochemistry, Max-Planck Institute of Biochemistry, 82152 Martinsried, Germany
thoffman@biochem.mpg.de

Translating bacterial ribosomes can form structurally ordered polysomes. The three-dimensional (3D) organization of such polysomes has been described in solution using cryoelectron tomography (CET) and template matching [1]. However, little is known about how polysomes are organized in space when translating membrane proteins, and many questions related to the ribosome association to the membrane remain open. We propose to examine the 3D arrangement of membrane associated ribosomes in situ, for an optimal preservation of polysomes architecture; and in vitro, for a better control of translation conditions. We used the mannitol permease (MtlA) as a substrate for translation-coupled translocation through the membrane. In order to form densely packed polysomes, we inserted a SecM sequence at the end of the MtlA messenger. Thus, we stalled ribosomes in situ and in vitro, while the ribosome nascent chain complexes are still attached to the membrane. In situ this transmembrane protein construct is expressed in slow- and fast-growing BL21 E. coli cells. Fast-growing cells are thinned using a Focused Ion Beam technology [2]. In vitro, a transcription-translation reaction is supplemented with inverted vesicles isolated from E. coli inner membrane. First results show that the described approaches are adequate for revealing how membrane-associated ribosomes are spatially related in the context of polysomes. Furthermore the analysis of cytosolic ribosomes reveals a fraction of particles in similar 3D organization of dense polysomes previously described in vitro [1].


Reference:
[1] Brandt et al. 2009 Cell, 136, 261-271
[2] Alexander Rigort et al. 2010 J Structural Biology, 172, 169-179

Prof. Dr. Wolfgang Baumeister
Prof. Dr. F. Ulrich Hartl
Dr. Friedrich Förster
Dr. Julio Ortiz
Elitenetzwerk Bayern: Protein Dynamics in Health and Disease
Fondation Fourmentin-Guilbert

Fig. 1: Tomogram from induced E. coli cells expressing MtlA385-SecM-Stop construct. (A,C) Slices from whole cell grown in minimal media (slow growing); (B,D) Slices of FIB-milled cell grown in rich media (fast growing). In the Image ribosomes are visible as highly contrasted particles. - (A,B) XY-slices; (C,D) XZ-slices. Bar 100 nm.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-2331 Alignment of Direct Detector Device micrographs using a local least-squares approach

Abrishami V.1, Vargas J.1, Marabini R.2, Sorzano C.1, Carazo J.1
1Biocomputing Unit, Centro Nacional de Biotecnología-CSIC, Madrid, Spain, 2Escuela Politécnica Superior, Universidad Autónoma de Madrid, Madrid, Spain
carazo@cnb.csic.es

Abstract:The recent introduction of Direct Detector Devices (DDDs) in cryo-EM represents a crucial step forward for this structural technique.. As expected, the quality of these DDD images is much better than the one obtained from Charged Coupled Devices (CCDs) but, additionally, their fast image acquisition rate makes possible the collection of “movies” composed of individual “frames”, opening the possibility to the study of how frozen hydrated specimens temporally behave as a function of electron dose rate. Indeed, biological specimens in a solid matrix of amorphous ice behave as if they were moving when being imaged, resulting in Beam Induced Movement (BIM). It turns out that BIM is a very serious experimental “resolution barrier” in cryo-electron microscopy. However, BIM “correction” is not an easy task, and several approaches have been already proposed. In this work, we present a method to correct for BIM at the image level, resulting in an integrated image where much of the BIM blurring is compensated. The methodology is based on a robust optical flow approach that can deal both with local and global movements in a very fast manner thanks to its implementation in a Graphic Process Unit (GPU). Additionally, the spatial analysis of the optical flow in between frames allow for a detailed, objective and quantitative analysis of the BIM pattern itself, providing with a new tool to evaluate this crucial effect in cryo-EM. The new approach is publically available as part of XMIPP 3.1.

Keywords: Direct Detector Devices; Single Particle Analysis; Electron Microscopy

The authors would like to acknowledge economical support from the Spanish Ministry of Economy and Competitiveness through grants AIC–A–2011–0638 and BIO2010-16566, the Comunidad de Madrid through grant CAM(S2010/BMD-2305) and the NSF through grant 1114901, as well as postdoctoral “Juan de la Cierva” grant with reference JCI-2011-10185. C.O.S. Sorzano is recipient of a Ramón y Cajal fellow.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-2332 Contrast Transfer Function (CTF) estimation benchmark

Marabini R.1, Cuenca J.2, Quintana A.2, Sorzano C.2, Carazo J.2
1Escuela Politécnica Superior, Universidad Autónoma de Madrid, Madrid, Spain, 2Biocomputing Unit, Centro Nacional de Biotecnología-CSIC, Madrid, Spain
carazo@cnb.csic.es

Electron microscope images are affected by the contrast transfer function (CTF) of the transmission electron microscope, which arises from the aberrations of the lenses and from the defocus used in imaging. The CTF introduces spatial frequency-dependent oscillations into the Fourier space representation of the image. These oscillations result in contrast changes and modulation of the spectrum amplitudes, as well as an additional envelope that attenuates high-resolution information. Estimation of the CTF and correction for its effects is thus essential for any image to faithfully represent a projection of the specimen.
We present in this work the ''CTF Benchmark'' which provides an opportunity to the researchers in the field to carry out a comprehensive evaluation of their CTF estimation methods based on a common database of image (see more details at URL: http://i2pc.cnb.csic.es/3dembenchmark)
A total of 21 different uploads were submitted, covering most of the software packages in the field including: ace, appion, bsoft, ctffind, dudelft, eman, fei, imagic, particle, sparx, spider, xmipp, etc. The main conclusions of this Benchmark are:
* In general, for quality datasets such as the ones in this Benchmark, about 40\% of the datasets are practically not limited by CTF estimation errors while, for the rest, CTF errors set the limit in between 5 to 7 A resolution at most.
* As a rule, and certainly not unexpected, estimations of the mean defocus is much better than astigmatism estimation. Although the error is lower when estimating astigmatism than when only a 1D CTF model is used, indicating that CTF defocus estimation is beneficial to achieve
high resolution.
* It is very clear the trend that when a dataset is specially suited for high resolution most software packages provide similar estimations for the CTF parameters. In other words, when a dataset is "good", is "good" for everything. Consequently, there must be image characteristics that dictate their ability to provide high resolution structural information.
* Our initial hypothesis was that micrographs with carbon support and with a high concentration of particles were going to be "the best", simply because of their increased scattering power did not hold. The "better" datasets do not have carbon, and one of the best does not have a particularly large density of particles.
*Finally, synthetic data behave similarly as experimental data with respect to CTF estimation

The authors would like to acknowledge economical support from the Spanish Ministry of Economy and Competitiveness through grants AIC–A–2011–0638 and BIO2010-16566, the Comunidad de Madrid through grant CAM(S2010/BMD-2305) and the NSF through grant 1114901, as well as postdoctoral “Juan de la Cierva” grant with reference JCI-2011-10185. C.O.S. Sorzano is recipient of a Ramón y Cajal fellow.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-2508 Ultrastructural characterization and quantification of fully de novo designed protein-only nanoparticles

Unzueta U.1,2,3, Sánchez-Chardi A.4, Rossinyol E.4, Céspedes M. V.1,5, Tatkiewicz W.1,6, Ratera I.1,6, Veciana J.1,6, Mangues R.1,5, Ferrer-Miralles N.1,2,3, Vazquez E.1,2,3, Villaverde A.1,2,3
1CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBERBBN), Bellaterra, 08193 Barcelona, Spain , 2Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain , 3Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain , 4Servei de Microscòpia. Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain , 5Oncogenesis and Antitumor Drug Group, Biomedical Research Institute Sant Pau (IIB-SantPau), Hospital de la Santa Creu i Sant Pau, C/ Sant Antoni Maria Claret, 167, 08025 Barcelona, Spain, 6Department of Molecular Nanoscience and Organic Materials, Institut de Ciencia de Materials de Barcelona (CSIC), Bellaterra, 08193 Barcelona, Spain
rossinyol@gmail.com

Fully de novo designed functional protein nanoparticles are a challenging task in innovative medicine as versatile and biologically safe vehicles for targeted drug and nucleic acid delivery. The characterization and quantification of this kind of biomaterial at nanoscale by electron microscopy imaging is limited by severe constraints, including methods to preserve their native structure and the observation without alterations or artifacts. Here, we characterize size and shape of 3 different self-assembling protein nanoparticles with biomedical applications (T22GFPH6, R9GFPH6 and T22iRFPH6) by means of AFM, TEM (negative staining and Cryo-TEM) and SEM. Moreover, nanoparticles size distribution determined by dynamic light scattering (DLS) was compared with that determined by image analysis with ImageJ from SEM images, obtaining similar results of diameters (Mean values in nm, T22GFPH6: DLS: 13.19 , SEM: 14.9; R9GFPH6: DLS: 23.08, SEM :21.28; and T22iRFPH6: DLS:13.82, SEM: 14.5). These long-term architectonic stable protein-NPs have been proved to be an excellent biomaterial for receptor mediated targeted delivery of drug and nucleic acids in tumoral cells [1-6]. We conclude that imaging from microscopy techniques is greatly useful to obtain qualitative and quantitative data of size and shape, and surface morphology of non conductor biomaterials at nanoscale as well as to complement quantitative data of other techniques.

References

[1] Unzueta et al, Non-amyloidogenic peptide tags for the regulatable self-assembling of protein-only nanoparticles. Biomaterials. 2012, 33:8714

[2] Vazquez et al, Internalization and kinetics of nuclear migration of protein-only, arginine-rich nanoparticles. Biomaterials. 2010, 31:9333

[3] Vazquez et al, Protein nanodisk assembling and intracellular trafficking powered by an arginine-rich (R9) peptide. Nanomedicine. 2010,2:259

[4] Unzueta et al, Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles. Int J Nanomedicine. 2012, 7:4533

[5] Unzueta et al, Improved perfomance of protein-based recombinant gene therapy vehicles by tuning downstream procedures.Biotechnol Prog. 2013.

[6] Unzueta et al, Sheltering DNA in self-organizing, protein-only nano-shells as artificial viruses for gene delivery. Nanomedicine. 2013.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-2729 Three dimensional electron tomography characterization of islet amyloid polypeptide aggregates in drosophila melanogaster

Xie L.1, Gu X.2, Westermark G.2, Leifer K.1
1Department of Engineering Sciences, Applied Materials Sciences, Box 534, Uppsala University, 751 21, Sweden., 2Department of Medical Cell Biology, Box 571, Uppsala University, 751 23, Sweden
ling.xie@angstrom.uu.se

In human more than 30 different proteins can misfold and form amyloid. Alzheimer’s disease (AD) and type 2 diabetes (T2D) are common disease where amyloid deposits play an important role in the pathogenesis. In AD, amyloid beta precursor protein (AβPP) deposits in brain and in T2D form islet amyloid polypeptide (IAPP) amyloid in islets of langerhans that leads to destruction of the insulin producing beta cells. [1]

There are mouse and rat models that facilitate studies on AβPP and IAPP aggregation and subsequent development of respective disease, however, it is a long process that extend over many months. Therefore, we and others have established Drosophila melanogaster models that enable studies of amyloid protein misfolding and cellular effects [2].

Structural analysis on the misfolded protein aggregates provide data important for understanding the driving force of protein aggregation and how one protein can adopt different structures dependent on the biological environment. We have applied transmission electron microscopy (TEM) to study the structure of IAPP aggregates formed in Drosophila melanogaster. As shown in figure 1, we detected highly ordered IAPP aggregates in Drosophila melanogaster expressing human IAPP. However, from single 2D TEM image (as shown in figure 1), we are not able to determine the structural information in Z direction. Therefore, we have applied electron tomography technique to study the structural information in Z direction. The tilt series were acquired from 60˚ to -60˚, and double tilt series were carried out in order to minimize the elongation effect in Z direction. [3] The IMOD software was used for image alignment and reconstruction. [4]

In summary, IAPP aggregates detected in the drosophila melanogaster exhibit a spherical shape in the reconstructed tomogram, and spheres are arranged in a body center cubic structure. The individual spheres have a diameter of 17 nm and BCC structure is shown in figure 2 with a distance of 25 nm between.

References

[1] Sipe JD, et al., Amyloid, 2012,19:167

[2] Schultz SW, et al., PLoS One. 2011; 6(6): e20221

[3] Midgley PA, et al., Ultramicroscopy, 2003 96:413

[4] Mastronarde DN J Struct Biol, 1997,120:343

Fig. 1: Figure 1. 2D TEM image of Amyloid peptides aggregates.

Fig. 2: Figure 2. (Left) 2D TEM image of Amyloid peptides aggregates, (Right) Segmented protein aggregates in 3D volume by isosurface method, (Right)showing as BCC structural distribution in 3D. Scale bar is 20 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-2882 Three-dimensional reconstruction of the S885A mutant of the human mitochondrial Lon protease

Kereïche S.1, Kováčik L.1, Pevala V.2, Ambro L.2, Bellová J.2, Kutejová E.2 3, Raška I.1
1Institute of Cellular Biology and Pathology, 1st Faculty of Medicine, Charles University in Prague, Czech Republic, 2Department of Biochemistry and Structural Biology, Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia, 3Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
skere@lf1.cuni.cz

The Lon protein is a protease belonging to the superfamily of ATPases Associated with diverse cellular Activities (AAA+). Its main function is the control of protein quality and the maintenance of proteostasis by degrading misfolded and damaged proteins, which occur in response to numerous stress conditions [1]. Lon protease has been also shown to participate in regulation of levels of transcription factors that control pathogenesis, development and stress response, e.g. [2]. Furthermore, it seems to play an important role in aging [3], and it is supposed to be involved in mtDNA replication, translation, or repair [1, 4, 5]. We focus our interest on the structure of human mitochondrial Lon (hLon) protease whose altered expression levels are linked to some severe diseases, such as epilepsy, myopathy, or lateral sclerosis [5].

During the last decade, sub-structures of bacterial and human Lon have been resolved by X-ray scattering (e.g. [6-8]), and more recently a 3D structure of an E. Coli Lon dodecamer active at physiological protein concentrations was resolved with electron microscopy [9]. At the moment, it is assumed that Lon subunits assemble into oligomeric structures whose conformations are supposed to differ at ATP, ADP, and protein substrate binding [1]. However, neither the full 3D structure of the Lon holoenzyme nor the mechanism of Lon action is known [4, 5].

Here, we present the first 3D structure of an ADP-bound Lon S885A mutant obtained as a result of preliminary negative staining studies (Fig. 1). The S885A mutant has a point mutation on the proteolytic domain, which completely disables its proteolytic function. 2D classification of the collected dataset revealed classes with regular hexameric arrangement (Fig. 1B), 3D refinement with C6 symmetry applied revealed that the Lon mutant was formed as a hexameric ring of a 120 Å diameter having 90 Å in height. Its resolution was estimated at 19 Å by the FSC=0.5 criterion. We will also present an update of this mutant structure obtained by cryo-electron microscopy.

References

1 Lee I. and Suzuki CK. (2008) Biochim Biophys Acta 1784, 727-735. 2 Mizusawa, S. and Gottesman S. (1983) PNAS 80, 358-362. 3 Bota D. A.et al. (2002) FEBS Let 532,103-106. 4 Ambro L. et al. (2012) J Struct Biol 179, 181-192. 5 Venkatesh S. et al. (2012) BBA-Mo. Cell Res, 1823, 56-66. 6 Duman R. E., Löwe J. (2010) . Mo. Biol 401, 653-670. 7 Botos I. et al. (2004) J Biol Chem, 279, 8140–8148. 8 García-Nafría J. (2010) Protein Sc. 19, 987-999. 9 Vieux E. F. et al. (2013) PNAS 110, E2002-E2008.

The work was supported by the grants: P302/12/G157 and 13-32339P from the Czech Science Foundation, Prvouk/1LF/1 and UNCE204022 from the Charles University in Prague, Slovak Research and Development Agency (Grant APVV-0123-10) and by the Slovak Grant Agency (Grant VEGA 2/0113/14).

Fig. 1: Preliminary results from negative stain. (A) Micrograph of negatively stained Lon particles. Scale bar: 50 nm. (B) Typical class averages. (1) top-view showing a hexameric arrangement of the Lon protease, (2)-(3) side-views. Scale bar: 5 nm. (C)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-2920 Efficient automatic detection of filaments in cellular electron tomograms based on reduced representation templates

Page C.1, Volkmann N.1
1Sanford-Burnham Medical Research Institute
niels@burnham.org

Electron tomography is the most widely applicable method for obtaining 3D information by electron microscopy. It has become a powerful tool for elucidating 3D architectures of biological samples at resolution of about 4-6 nm and is the only method suitable for investigating polymorphic structures such as organelles, cells and tissues. However, in addition to the relatively low resolution, electron tomograms inevitably suffer from a low signal-to-noise ratio and some data-collection artifacts. These factors significantly hamper development of algorithms for reliable detection of structural features, which poses a severe barrier to progress in the field. As of today, the tasks of extracting information from these highly complex cellular tomograms are, for the most part, painstakingly carried out manually. Apart from the subjectivity of the process, the time consuming (and tiring) nature of this manual task all but precludes the prospects of the high throughput necessary to take full advantage of the method’s potential.

Here, we present a novel tool for the detection of filaments in cellular tomograms that is based on reduced representation templates. Reduced representations consist of small sets of 3D points that capture the characteristics of the underlying structure. The use of these representations results in a reduction of computational complexity that allows scanning large volumes in real space in a relatively short time. This approach is specifically useful for detecting structures with higher order such as filaments and bundles. The use of reduced representations allows efficient adjustment of the scoring function for variations in signal-to-noise level, background, and surrounding environment (crowding), all factors that significantly hamper reliable detection using traditional correlation-based template matching. As a result, the approach is capable of matching or even exceeding the detection performance of a human operator.

Funding was provided by NIH grant P01 GM098412

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-3117 Structural and functional characterization of Odontogenic Ameloblast-associated (ODAM) and Amelotin (AMTN) proteins

Fouillen A.1,3, Kholmogorova S.1, Wazen R. M.1, Moffatt P.2, Baron C.3, Sygusch J.3, Nanci A.1,3
1Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, Montréal, Quebec, Canada, 2Shriners Hospital for Children, Montreal, Quebec, Canada and the Department of Human Genetics, McGill University, Montreal, Quebec, Canada, 3Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec, Canada
aurelien.fouillen@gmail.com

Introduction: The junctional epithelium (JE) is the portion of the gingiva that attaches to the tooth enamel. This specialized structure seals off the periodontal tissue from the aggressive environment of the oral cavity. A loss of its integrity leads to periodontal disease followed by bone and ultimately tooth loss. The adhesion mechanism of the JE to mineralized tooth surfaces is provided by an epithelial structural complex consisting of an atypical basal lamina (BL) and hemidesmosomes. The interaction with a mineralized matrix instead of connective tissue therefore requires special components. Two proteins, odontogenic ameloblast-associated (ODAM) and amelotin (AMTN), encoded by members of the secretory calcium binding phosphoprotein gene cluster, have been identified by our laboratory as novel components of this BL. Proteins from this cluster stabilize Ca and PO4 ions in tissue fluids and regulate their deposition onto extracellular matrices. ODAM and AMTN were shown to co-localize at cell-tooth interfaces suggesting that they might interact (Fig1). Problematic: In the literature, little information is available on the physicochemical properties and structure of ODAM and AMTN. They contain some post-translational modifications but they do not possess structural homologues. Our aim is to structurally characterize these proteins and their potential interactions. Methods: Proteins were produced using bacterial or eukaryotic systems. They were purified using His-tag affinity and gel-filtration chromatography and then characterized by atomic force microscopy (AFM) and transmission electron microscopy (TEM). For AFM studies, proteins at 0,1mg/ml on 50mM Na2HPO4 were incubated 5minutes on HOPG, rinsed three times with distilled water, and air dried. Results: With gel-filtration, we have obtained AMTN complexes of 5 monomers and ODAM complexes of ≈20 monomers. AFM images of ODAM and AMTN alone presented globular complexes with average sizes of approximately 32 nm and 36 nm and average heights of ≈2.5 nm and 2.8 nm, respectively. Mixing the proteins resulted in globular complexes with an average size of 84 nm and an average height of 8 nm, suggesting the existence of interactions between them (Fig2). Conclusion: Our data show for the first time structural images of ODAM and AMTN. They suggest that AMTN and ODAM are capable of self-assembly and that protein-protein interactions occurs among them. Exploring the molecular arrangement and interactions of proteins in this BL is essential to understand how the JE maintains its unique adhesive relationship with the tooth.

Supported by CIHR, NSERC, RSBO, Shriners of North America

Fig. 1: Immunofluorescence (A, B) and Immunoperoxydase (C, D) preparations for ODAM (B, D) and AMTN (A, C) in the junctional epithelium (JE). ODAM localizes at the ameloblast– enamel interface where there is an atypical basal lamina (BL) and is also present among the cells of the JE. Labeling for AMTN is restricted to the inner BL. GE: Gingival epithelium

Fig. 2: Highly magnified AFM observation of (A) ODAM and (B) AMTN proteins alone at 0,1mg/ml on HOPG surface. (C) Changes in structure can be observed when ODAM and AMTN (0,1mg/ml) are incubated together.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-3188 Initial bridges between the two ribosomal subunits are formed within 9.4 milliseconds: A time-resolved cryo-EM study

Shaikh T. R.1,2, Yassin A.2,5, Lu Z.3, Barnard D.2, Meng X.2, Lu T. M.3, Wagenknecht T.2,4, Agrawal R. K.2,4
1Central European Institute of Technology, Masaryk University, Brno, Czech Republic, 2Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, NY 12201, 3Center for Integrated Electronics, Rensselaer Polytechnic Institute, Troy, NY 12180, 4Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY, 5Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt
shaikh@ceitec.muni.cz

The kinetics of ribosomal subunit association has been a subject of great interest for past several decades (Wishnia, 1975), however, there have been only few published reports on the kinetics of specific structural elements during this process (Fabbretti2007, Hennelly2005). The goal of the present study is to capture structures of the association intermediates of the two ribosomal subunits on a millisecond (ms) timescale.
The challenge of time-resolved cryo-EM is to rapidly mix reactants and then deposit them in a thin film of solvent within a short time, before flash freezing the EM grid into liquid ethane. We have found that spraying the mixture with an air atomizer can produce an aqueous film thin enough to allow transmission of the electron beam (Lu, 2009). The mixing, reacting, and spraying steps were accomplished by means of a monolithic, microfabricated silicon device that incorporated a mixer, incubation channel, and pneumatic sprayer in a single chip. This mixer sprayer was incorporated into a computer-controlled plunging apparatus (White2003). Three-dimensional reconstructions, obtained from the cryo-EM data collected from the ribosomal subunit association experiments, show missing densities for specific inter-subunit bridges, suggesting that the inter-subunit bridges are formed in a sequential order.

REFERENCES.

Fabbretti, A., Pon, C.L, Hennelly, S.P, Hill, W.E, Lodmell, J.S Gualerzi, C.O (2007) The real-time path of translation factor IF3 onto and off the ribosome. Mol Cell 25:285-296.
Hennelly, S.P., Antoun, A, Ehrenberg, M, Gualerzi, C.O, Knight, W, Lodmell, J.S Hill, W.E (2005) A time-resolved investigation of ribosomal subunit association. J Mol Biol 346:1243-1258.
Lu, Z., Shaikh, T.R, Barnard, D, Meng, X, Mohamed, H, Yassin, A, Mannella, C.A, Agrawal, R.K, Lu, T Wagenknecht, T (2009) Monolithic microfluidic mixing-spraying devices for time-resolved cryo-electron microscopy. J Struct Biol 168:388-395.
White, H.D., Thirumurugan, K, Walker, M.L Trinick, J (2003) A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray. J Struct Biol 144:246-252.
Wishnia, A., Boussert, A, Graffe, M, Dessen, P.H Grunberg-Manago, M (1975) Kinetics of the reversible association of ribosomal subunits: stopped-flow studies of the rate law and of the effect of Mg2+. J Mol Biol 93:499-415.

The work was supported by NIH NCRR Grant P41 RR01219, and in part by the NIH grant R01 GM61576 (to RKA) and grant CZ.1.07/2.3.00/20.0042 (to TRS) from the European Social Fund and the state budget of the Czech Republic.

Fig. 1: Cryo-EM maps of the 70S ribosomes formed within 9.4 ms. (a) Control, pre-associated 70S ribosomes which had been passed through the microfluidic mixer. (b) Reconstruction of the 70S-like particles which were associated using the microfluidic mixer. (c) Reconstruction from one ML3D class. (d) Reconstruction from another class.

Fig. 2: Cryo-EM maps of the 70S ribosomes formed within 43 ms. (a) Control, pre-associated 70S ribosomes which had been passed through the microfluidic mixer. (b) Reconstruction of the 70S-like particles which were associated using the microfluidic mixer. (c) Reconstruction from one ML3D class. (d) Reconstruction from another class.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-3332 Exploring the ultrastructure of bacterial inclusion bodies (IB) with therapeutical interest

Sánchez-Chardi A.1, Seras-Franzoso J.2,3,4, Villaverde A.2,3,4
1Servei de Microscòpia. Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain , 2CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBERBBN), Bellaterra, 08193 Barcelona, Spain , 3Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain , 4Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
Alejandro.Sanchez.Chardi@uab.cat

IBs are highly pure, mechanically stable and biocompatible protein deposits commonly observed during recombinant protein production processes in microbial hosts [1, 2]. Since these sub-micron particles retain biological activity, several potential applications in biomedicine for bacterial IBs have recently emerged [3]. In this regard, it has been  demonstrated that these protein deposits, when formed by proteins with therapeutic interest can actually rescue challenged cells in culture [4]. The present study explores the electron microscopy methods to obtain information of fine architecture (both size and shape, and surface morphology) of bacterial Inclusion Bodies (IBs). In addition, IB biological activity is studied through immunolocalization techniques of the particle forming protein (GFP in our IB model).
 The analyses have been performed using scanning electron microscopy (ultrastructure, freeze-fracture and immunolocalization in entire IBs) and transmission electron microscopy (ultrastructure of entire IBs with negative staining and ultrathin sections of IBs in Epon resin, immunolocalization of GFP in entire IBs and in ultrathin sections of Lowicryl HM20 resin).
 The fine architecture and the high content of protein of IBs were evaluated by SEM and TEM using ultrastructural morphology and immunolocalization (Figure 1). The ultrastructure also revealed differences in porosity between IBs, showing from high compact structures to highly porous IBs. In summary, this study reports several EM methods to obtain ultrastructural data of IBs characterized with conventional and non-conventional microscopy techniques and help to understand the mechanism of IB-mediated protein drug delivery for therapeutic use.

References

[1] de Marco,A., Schroedel,A.,  BMC Biochemistry, 6 (2005), 10.
[2] Garcia-Fruitos,E. et al., Microb. Cell Fact., 4(2005) 27.
[3] Garcia-Fruitos,E. et al.,Trends Biotechnol. 30 (2012) 65-70.
[4] Vazquez,E. et al, Advanced Materials, 24 (2012) 1742-1747.

Fig. 1: Representative micrographs of IBs: A) TEM negative staining; B) TEM immunolabelling of GFP in entire IB; C) TEM immunolabelling of GFP in Lowicryl section; D-E) SEM morphology using 2 different secondary detectors; F-H) SEM freeze-fracture using different substrates (Cu, Si, Au); and IBs-HeLa cell surface contacts observed with SEM (I) and TEM (J).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-3499 The Application of Atomic Force Microscopy to investigate drug effects on trypanosomatid mitochondrial DNA

Gonçalves C. S.1, Zuma A.2, De Souza W.1,2, Motta M. C.2, Cavalcanti D. P.1
1National Institute of Metrology, Quality and Technology, Inmetro., 2Institute of Biophysics Carlos Chagas Filho, UFRJ.
dpcavalcanti@inmetro.gov.br

The Trypanosomatidae family comprises a large number of protozoa, some of which are agents of serious tropical human diseases. These protozoa form an ancestral branch of eukaryotes and present unusual biological structures such as the kinetoplast, a specialized region of the unique mitochondrion, which contains DNA (kDNA). The kDNA is composed of circular molecules, which are topologically interlocked to form a single network. It has been reported that the kinetoplast constitutes a potent target for chemotherapy since it is highly affected by DNA binding drugs, intercalating agents and topoisomerase inhibitors that interfere with its structure and replication. Although we can observe the kDNA using transmission electron microscopy (TEM), it is difficult to reveal its underlying organization by TEM. Atomic force microscopy (AFM) has been extensively used to study biological samples, ranging from individual molecules to cells and tissues. AFM has been successfully applied to investigate the structure of nucleic acids as well as changes in DNA topology caused by protein binding or interactions with drugs. In this work, we used AFM to analyze the action of different classes of drugs on kDNA ultrastructural organization. The inhibitors used were nalidixic acid (a topoisomerase II inhibitor), acriflavine (an intercalating drug) and berenil (a minor-groove binding agent). The kDNA of Trypanosoma cruzi, the etiological agent of Chagas disease, was affected by all drugs tested. Our group previously analyzed the effect of these compounds on the protozoa ultrastructure by TEM. However, in order to better understand how these inhibitors promoted alterations on the kDNA arrangement, we isolated intact networks of treated and non-treated protozoa and analysed the samples using AFM. In non-treated parasites, the isolated kDNA appeared as an intact and massive network presenting its typical arrangement with fibers homogeneously distributed throughout the network (Fig. 1a). The treatment with 500 µg/ml of nalidixic acid for 48h induced the formation of thicker DNA strands (Fig 1b), while berenil (50 µM, 48h) promoted shrinkage of the network and compactation of kDNA fibers (1c). In addition, the treatment with 50 µg/ml of acriflavine for 48 h promoted the release of minicircles from the edge of the network, culminating with kDNA fragmentation and dispersion throughout the protozoan mitochondrial matrix (Figure 1d). Taken together, our results highlight the mechanism of action of different classes of inhibitors that target the kDNA structure, confirming that AFM is a powerful tool to study the structural organization of biological samples, including complex arrays of DNA.

Supported by CNPq and FAPERJ.

Fig. 1: AFM analysis of isolated kDNA network of non-treated T. cruzi (A) and protozoa treated with nalidixic acid (B), berenil (C) and acriflavine (D).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-5839 Structural Investigations of COP9 signalosome binding to Cullin-RING ligases:promiscuous interactions sharing common regulatory principles

Cavadini S.1,2, Fischer E. S.1,2, Goldie K. N.3, Böhm K.1,2, Lingaraju G. M.1,2, Bunker R. D.1,2, Pantelic R. S.3, Mohamed W. I.1,2, Stahlberg H.3, Thomä N. H.1,2
1Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland., 2University of Basel, Petersplatz 10, 4003 Basel, Switzerland., 3Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, 4058 Basel, Switzerland.
k.goldie@unibas.ch

The CUL4A-RBX1-DDB1-DDB2 ligase (CRL4ADDB2) is the primary DNA damage-sensing complex in the repair of UV light induced pyrimidine dimers1. The class of cullin-RING ubiquitin ligases (CRLs) comprises six canonical cullin families (Cul1, Cul2, Cul3, Cul4A, Cul4B, and Cul5) which together with the RING-domain proteins Rbx1 or Rbx2 mediate the ubiqutination ~20% of the proteins degraded by the proteasome2. CRLs assembly is modular, with sets of receptors and adaptors giving rise to hundreds of distinct cullin-RING E3 ubiquitin ligase complexes3. Regulation of this structurally diverse family of ligases depends on the COP9 signalosome (CSN), an 8-subunit isopeptidase that removes the covalently conjugated Nedd8 activator from the cullin4-6. In addition to its catalytic function, CSN form tight complexes with the unmodified CRL, a process implicated in preventing the CRL substrate adaptor from undergoing cycles of futile auto-ubiquitination and degradation in vivo7-9. CSN inhibition is overcome once the CRL binds a substrate. The mechanism underlying CSN binding to structurally diverse CRL complexes across families remains unclear. Here we present the structure of CRL4 family and a dimeric CRL3 in complex with CSN using single-particle electron microscopy (EM). We find that the conserved cullin C-terminus together with Rbx1 is held by CSN2 and CSN4 subunits. In the CRL1, CRL3 and CRL4 family the divergent receptors and adaptors form a variety of contacts that are unexpectedly plastic in nature involving CSN1, CSN3 and CSN helical bundle. Moreover, we show that: (i) the ligase substrates can access the CSN-bound cullin receptors in a similar fashion. (ii) Depending on the size of the substrate, CSN is released by steric means. Altogether our findings imply a model where steric repulsion by the cognate substrate allow CSN-mediated regulation of ~300 different CRL enzymes in response to different cues, without the need for dedicated interactions or common motifs.

References;

1. Scrima, A. et al. FEBS letters 585 (18), 2818-25. (2011).

2. Soucy, T. A. T. et al. Audio, Transactions of the IRE Professional Group 458, 732-736, (2009).

3. Deshaies, R. J. & Joazeiro, C. A. P. Annual review of biochemistry 78, 399-434, (2009).

4. Schwechheimer, C. et al. Science (New York, NY) 292, 1379-1382, (2001).

5. Lyapina, S. et al. Science (New York, NY) 292, 1382-1385, (2001).

6. Wei, N., Chamovitz, D. A. & Deng, X. W. Cell 78, 117-124, (1994).

7. Fischer, E. S. et al. Cell 147, 1024-1039, (2011).

8. Enchev, R. I. et al. Cell Reports, 1-23, (2012).

9. Emberley, E. D., Mosadeghi, R. & Deshaies, R. J. Journal of biological chemistry 287, 29679-29689, (2012).

Fig. 1: Cryo-EM structure and pseudo-atomic model of the CSN-N8CRL4DDB2 complex.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-5998 Single particle analysis of molecular architecture of DNA replication fork complex and switching mechanism of replication factors

Mayanagi K.1, 6, Kiyonari S.1, Nishida H.2, Ishino S.1, Fujikane R.3, Saito M.4, Kohda D.1, Ishino Y.1, Shirai T.4, Morikawa K.5
1Kyushu University, Fukuoka, Japan, 2Hitachi Ltd., Japan, 3Fukuoka Dental College, Fukuoka, Japan, 4Nagahama Inst. of Bio-Sci. and Tech. Shiga, Japan, 5International Inst. for Advanced Studies, Kyoto, Japan, 6PRESTO, JST, Japan
maya@bioreg.kyushu-u.ac.jp

DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). PCNA has a trimeric ring structure that encircles the DNA, and increases the processivity of the bound DNA polymerase by tethering it to the DNA. It is known now, that PCNA also interacts with multiple partners to control DNA replication, DNA repair, and cell cycle progression, and works not only as the platform, but also as the conductor for the recruitment and release of these factors. However, the molecular architectures as well as the mechanism of the regulation of these replication factors are not known in detail.
We have investigated the three-dimensional structure of the core components of the replisome, such as DNA polymerase B(PolB)-PCNA-DNA, and DNA ligase-PCNA-DNA ternary complexes (1- 2), by single particle analysis, and successfully could obtain atomic models of both complexes, by docking crystal structures of each components in the two maps . Both maps exhibited a novel contact between both polymerase-PCNA and ligase-PCNA, besides the authentic interaction through a PCNA-interacting protein box (PIP-box).
Mutant analysis showed that these contacts are involved in the regulation of the replication factors, such as the switching between the polymerizing and editing modes of the PolB. As we found similar multiple interactions in both complexes, we are now studying the structure of other complexes such as Hjm helicase-PCNA-DNA and FEN-PCNA-DNA, to check if such regulation mechanism can be also found in replication complex more generally.

References
(1) Mayanagi, et al., PNAS, 108, 1845-1849 (2011)
(2) Mayanagi, et al., PNAS, 106, 4647-4652 (2009)

This research was supported by JST, PRESTO; JST, BIRD; A grant-in-Aid for Scientific Research from MEXT.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-4-P-6052 Structural studies on phosphorylase kinase using cryo-electron microscopy

Li Z.1, Nadeau O. W.2, Schenk A.3, Walz T.3, Carlson G. M.2, Venien-Bryan C.1
1Institut de Minéralogie, de Physique des Matériaux, et de Cosmochimie (IMPMC) Sorbonne Universités – UPMC Univ Paris 06, UMR CNRS 7590, F-75005 Paris, France , 2Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS, 66160, USA, 3Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
zhuolun.li@impmc.upmc.fr

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑

LS-5. Cellular transport and dynamics

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-5-IN-1734 Mutual Conformational Changes of Kinesin (KIF5) and GTP-Microtubule upon their Binding and the Mechanism of the Directional Transport revealed by the Cryoelectron Microscopy and Super-resolution Microscopy (PALM).

Hirokawa N.1
1Graduate School of Medicine, University of Tokyo, Tokyo, Japan
hirokawa@m.u-tokyo.ac.jp

Intracellular transport is fundamental for cellular morphogenesis and functions not only in polarized neurons but also cells in general. We identified kinesin superfamily molecular motors (KIFs) and have been studying the mechanism of intracellular transport including the directional transport towards the axon vs dendrites. As a major mechanism for the directional transport we uncovered that the motor domain of KIF5 (a kinesin-1) recognizes axonal microtubules, which are enriched in EB1 binding sites, and selectively move towards the axon. Further, we found that axonal microtubules were preferentially stained by the anti GTP-tubulin antibody(hMB11). Super-resolution microscopy (PALM) combined with EM immunocytochemistry revealed that hMB11 was localized at KIF5 attachment sites. In addition, EB1, which binds preferentially to GTP- microtubules in vitro, recognized hMB11 binding sites on axonal microtubules. In vitro studies revealed approximately threefold stronger binding of KIF5 motor head to GTP- microtubules than to GDP microtubules. Collectively, these data suggest that the abundance of GTP-tubulin in axonal microtubules may underlie selective KIF5 localization and polarized axonal vesicular transport.
Microtubule dynamics are regulated by GTP hydrolysis by β-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the GDP state. As a next step we solved the cryo-EM structure of GTP- microtubule at8.8-A resolution by developing a novel cryo-EM image reconstruction algorithm. Significant changes were detected between GTP- and GDP- microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule binding proteins, such as EB1 or kinesin.
Furthermore , recently we　successfully revealed the 8Å cryo-electron microscopy structure of nucleotide-free KIF5 complexed with GTP-microtubule and the crystal structure of nucleotide-free KIF5 without bound microtubule. These structures illustrated mutual conformational changes induced by the binding of GTP-microtubule and KIF5. Conformational change of tubulin also strengthens the longitudinal contacts of GTP-microtubule mainly from the plus-end side. This could provide the structural keys to solve the molecular mechanisms of preferential binding of KIF5 to GTP-microtubule and cooperative binding of KIF5 to the microtubule.

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-5-IN-5787 Intracellular trafficking and secretion of immune mediators: insights from electron microscopy

Melo R. C.1
1Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil
rossana.melo@ufjf.edu.br

Secretion is an essential biological activity of all eukaryotic cells by which they release specific products in the extracellular space during physiological and pathological events. In cells from the immune system, such as eosinophils, basophils, neutrophils and macrophages, secretory mechanisms underlie the functions of these cells during allergic, inflammatory and immunoregulatory responses. Therefore, a central question to understand immune responses is: how does an immune mediator, for example a specific cytokine, travel from intracellular sites to the cell surface in order to be released upon cell activation? Our Group has been using different electron microscopy (EM) techniques including pre-embedding immunogold EM and electron tomography (ET) to understand intracellular localization and release of immune mediators from human activated leukocytes [1-5]. By using eosinophils as a model, we have identified an active secretory pathway within these cells characterized by vesicular transport of small packets of materials from the cytoplasmic secretory granules to the cell surface (piecemeal degranulation-PMD). PMD enables differential mobilization and selective secretion of granule-derived cytokines and other proteins in response to varied stimuli. Some proteins, such as major basic protein-1, are transported in the fluid phase of vesicles (Fig. 1) [3] whereas others, as recognized for the cytokine interleukin-4, are transported bound to their cognate membrane-inserted receptor [1,4]. EM has also been used to immunolocalize tetraspanins such as CD63 at granules (Fig. 2) and vesicles. We identified two morphologically distinct vesicles as transporters of immune mediators: small, classical spherical vesicles and vesiculotubular carriers (Fig. 2), which present substantial membrane surfaces and are larger and more pleiomorphic than small vesicles, as revealed by ET [1,2]. These tubular carriers seem particularly relevant for rapid delivery of preformed cytokines or other proteins from secretory granules. The identification of these previously unrecognized pools of immune mediators at vesicular compartments revealed a unique mechanism contributing to the specificity of secretion in leukocytes and adds support to a broader role for large carriers in the intracellular trafficking and release of cytokines and other mediators from the immune system. This is important to understand the pathological basis of allergic and other leukocyte-associated inflammatory diseases.

References

[1] R.C.N. Melo et al, Traffic 20 (2005) 28-33

[2] R.C.N. Melo et al, Microsc Microanal 16 (2010) 653-60

[3] R.C.N. Melo et al, Lab Invest 89 (2009) 769-81

[4] L.A. Spencer et al, Proc Natl Acad Sci USA 103 (2006) 3333-3338

[5] R.C.N. Melo et al, Allergy 68 (2013) 274-84

Supported by FAPEMIG, CNPq (Brazil) and NIH (USA).

Fig. 1: Large vesiculotubular compartments (arrowheads) are positive for major basic protein-1 (MBP-1) within a human eosinophil. Cells were isolated from the peripheral blood and prepared for immunogold electron microscopy as before [3]. Scale bar, 500 nm.

Fig. 2:  Pre-embedding immunanogold labeling of CD63 within a human eosinophil stimulated with eotaxin. Note the dense labeling at secretory granules (*) undergoing emptying of their contents (piecemeal degranulation). N, nucleus. Scale bar, 1.5 μm. .
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-5-O-2175 Role of phopholipase D2 in the organization of the Golgi complex and tubular formation

Martinez-Martinez N.1, Martinez-Alonso E.1, Martinez-Menarguez J. A.1
1University of Murcia, Murcia, Spain
narcisamm@um.es

An increasing body of evidence has pointed to the crucial importance of lipids and lipid-modifying enzymes in the biogenesis, maintenance and fission of transport carriers in the secretory and endocytic pathways. In the present study we demonstrate that phosphatidic acid generated by phospholipase D2 (PLD2) is involved in the formation of Golgi tubules. The main evidence to support this is: 1) the inhibitors of several enzymes that generate phosphatidic acid inhibit the formation of low temperature-induced Golgi tubules, obtained the highest inhibition of tubular formation for PA generated via PLD2, 2) the PLD2-depletion inhibit the formation of tubules containing resident enzymes and regulators of intra-Golgi transport in a low temperature (15ºC) model of Golgi tubulation, but do not affect BFA-induced tubules, 3) both the inhibition of PLD2 activity by the compound VU0364739 and PLD2-depletion, in cells cultured under physiological conditions (37ºC), induced the formation of tubules containing the Golgi matrix proteins GM130 and GRASP65 but not the Golgi resident enzyme, and, 4) electron microscopical analysis of PLD2-overexpressed cells showed a disorganised Golgi complex where most stacks were replaced by tubular networks. In addition, we assessed the localization of endogenous PLD2 within the Golgi membranes by cryoimmunoelectron microscopy. It was found that the enzyme is located in cisternae (61%) and peri-Golgi tubule-vesicular elements (39%). PLD2 was observed in lateral rims of the Golgi apparatus, from which the tubules grow, including COPI coated buds as well as flat portion of cisternae. Most of the immunolabelled vesicles were uncoated (80% of the labelling in vesicles) although some showed clathrin (12%) and COPI (7%) coats. Taken together, our results support the idea that PLD2 is a key regulator of Golgi morphology and tubule formation.

This work was supported by grants Consolider COAT (CSD2009-00016) and Fundación Séneca (04542/GERM/06)

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-5-O-3400 Electron microscopic examination of mechanisms of intra-Golgi transport mechanism of different cargo proteins

Beznoussenko G. V.1, Mironov A. A.1
1Istituto FIRC di Oncologia Molecolare. Milan. Italy
alexandre.mironov@ifom.eu

Mechanisms of intra-Golgi transport represent a disputable issue. The vesicular, compartment maturation, diffusion and kiss-and-run models of intra-Golgi transport compete with each other for the ‘right’ to be regarded as the paradigm. <span>Here, we compare the transport the soluble secretory proteins albumin and <span><span>a<span>1-antitrypsin (AAT) with that of supra-molecular cargoes (e.g., procollagen) that are proposed traverse the Golgi by <span>compartment progression–maturation. The soluble proteins move much faster than procollagen through the same Golgi stack, indicating the coexistence of two transport mechanisms. Albumin transport might occur by diffusion via intercisternal continuities or via rapidly shuttling vesicles. Kinetic and morphological evidence favour the former model. These data indicate multiplicity of the intra-Golgi trafficking modes, and help to explain many of the reported cargo trafficking patterns. Additionally, we have examined predictions derived from these models using several parameters and assays; namely, the kinetics of exit of cargo and post-Golgi carriers from the Golgi area, and from the Golgi per se, the volume of the Golgi, and the surface area of Golgi membranes, and the diffusion and concentration of cargo during intra-Golgi transport. Intra-Golgi transport of GFP-tagged albumin and AAT included their concentration at the trans side of the Golgi. Both blockage of formation of inter-cisternal connections and their stabilization inhibited penetration of albumin across the Golgi stacks and its concentration at the trans side of the Golgi. In contrast, not all of these influences affected transport of GFP-tagged version VSVG and PC-I. Manipulation with the ionic gradients existing through the Golgi affected concentration of albumin and AAT at the trans side, but not their penetration into the TGN. These results suggest in favour of the kiss-and-run mechanism of intra-Golgi transport. The kiss-and-run model appeared the most encompassing in the explanation of these set of experimental data.<span>

We thank FIRC

Fig. 1: Presence of albumin in intercisternal connections
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-5-O-3494 Nanoscale Protein Diffusion by STED-based Spatiotemporal Fluorescence Correlation Spectroscopy

Bianchini P.1, Cardarelli F.2, Di Luca M.3, Diaspro A.1, Bizzarri R.1,3,4
1Nanophysics, IIT—Italian Institute of Technology, Genoa, Italy, 2Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy, 3NEST, Scuola Normale Superiore and Istituto Nanoscienze - CNR, Pisa, Italy, 4Istituto di Biofisica – CNR, Pisa, Italy
r.bizzarri@sns.it

Fluorescence Correlation Spectroscopy (FCS) represents an established technique to recover single-molecule diffusion and binding properties in cells. Recently, scanning microscopy imaging was applied to add a spatial dimension to the classic, purely temporal, FCS modality: spatiotemporal FCS (stFCS) provides details about the routes that are followed by the diffusing particles or molecules in the specimen [1]. We report on the combination of spatiotemporal fluorescence correlation spectroscopy (stFCS) and stimulated emission depletion (STED) to monitor intracellular protein diffusion at spatial resolution below the optical diffraction limit (superresolution). Our method was validated both in vitro and at intracellular level by following the diffusion of fluorescent nanocapsids and of GFP bound to SV40 Nuclear Localization Signal (NLS), respectively. NLS-GFP represents a well-known model of actively nuclear-imported protein that has been the subject of intense research by some of us [2]. The relevance of our approach was demonstrated by the discovery of the persistence of complexes between nucleocytoplasmic transporters and NLS-GFP at distances >500 nm from the nuclear envelope, a phenomenon otherwise invisible at the best resolution of conventional confocal imaging mode. We should stress that, in principle, the resolution of stFCS diffusional maps is limited only by the photophysics of the fluorescent reporter in STED conditions [3]

1. Digman, M. A.; Gratton, E. Ann Rev Phys Chem 2011, 62, 645-68.

2. Cardarelli, F.; Bizzarri, R.; Serresi, M.; Albertazzi, L.; Beltram, F. J Biol Chem 2009, 284, (52), 36638-46.

3. Hell, S. W. Science 2007, 316, (5828), 1153-8.

We wish to thank Mrs. Barbara Storti for technical help with cell cultures. This work was supported by the Regione Toscana under the framework of the project "New diagnostic strategies from nano-engineered viral capsid proteins", Bando Salute 2009.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-5-O-5726 Advantage of TEM studies in the investigation of physiological role of extracellular vesicles

Kittel Á.1, György B.2, Szabó T. G.2, Pálóczy K.2, Boncz D.1, Buzás E. I.2
1Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary , 2Department of Genetics, Cell- and Immunobiology, Semmelweis Medical School, Budapest, Hungary
kittel.agnes@koki.mta.hu

Extracellular vesicles (EVs) are cell derived submicron structures surrounded by phospholipid bilayer. Their critical roles such as potential biomarkers, possible pathogenic factors in numerous diseases or therapeutic targets and therapeutic vehicles have been discussed in numerous publications in recent years [1].
However, in spite of extensive studies, even classification of these vesicles and their appropriate isolation protocols are still under debate [2]. Transmission electron microscopy (TEM) is an excellent tool for checking the purity of isolated vesicle preparations, for statistical analysis based on their size distribution, moreover, since properly fixed pellet of EVs is suitable not only for immunocytochemical but also enzyme cytochemical staining we may get acquainted with molecular details of the labelled vesicles and learn more about their physiological functions, too.
Our aim was to investigate the possible role of EVs in purinergic signaling. Although purinergic hypothesis was slowly accepted, by now it has been proved that purinergic signaling system, including the most abundantly expressed receptors in living organisms, is implicated in a huge range of physiological and pathological processes [3].
Based on our isolation protocol for EVs and by means of TEM immuno – and enzyme cytochemistry, we could present the expression of purinergic receptor P2Y12 in subpopulation of EVs released by the cells of microglia cell line BV2, and demonstrated the presence of active NTPDase1, one of the main enzymes hydrolyzing extracellular ATP.
We may hypothesize that EVs are involved in purinergic signaling and are convinced that exploration of molecular and functional details of extracellular vesicle release and action may provide important lessons for the design of future drug delivery systems.

Refenves:

1. Kittel A, Falus A,  Buzas E: Eur J Microbiol Immunol (Bp), 2, 91-6 (2013)
2. Crescitelli R, Lasser C, Szabo TG, Kittel A, Eldh M et al.: J Extracell Vesicles, (2013)
3. Burnstock G, Krugel U, Abbracchio MPIlles P: Prog Neurobiol, 2, 229-74 (2011)

This work was supported by grant NK 84043.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-5-P-1548 Studies on Biomolecular Transport through Nuclear Membranes using a Home Built Confocal Laser Scanning Microscope

Samudram A.1, Bijeesh M. M.1, Varier G. K.1, Kowshik M.1, Patincharath N.1
1Birla Institute of Technology and Science, Pilani - K K Birla Goa Campus, Goa, India
arunkarthick.biotech@gmail.com

Studies on the kinetics of biomolecular transport through nuclear membrane are of interest from a fundamental physics perspective as well as from the point of view of biological applications. The mechanism of this transport and the parameters that control the transport phenomena are not well understood. A proper understanding of the transport phenomena will help in developing methodologies which will be of interest to biomedical research, especially related to gene therapy. Confocal fluorescence microscopy is one of the most widely used imaging techniques employed to study biomolecular systems. However, because of the high cost, commercial confocal microscopes are not affordable to many laboratories. Also most commercial designs are not adaptable to different individual laboratory requirements. Here we report on the design and development of a cost effective and versatile confocal laser scanning microscope that can play a vital role in studies on biomolecular transport through membranes. Results on the nuclear transport of different sizes of FITC labelled dextran which is a model drug molecule are also reported. Studies were carried out in digitonin permeabilized cells using an in-house constructed confocal laser scanning microscopy in the time lapse imaging scheme. We specifically examined the kinetics of transport of dextrans of 4, 10, 20, 40 and 70 kDa through the nuclear membrane of live A549 cells and determined the diffusion rate constants. The intake and intracellular distribution of dextran were found to be dependent on the molecular weight of the dextrans. This analysis can provide us an estimate of the effective pore size available for nuclear transport of these biomolecules. The model can be used to study the transport of proteins and DNA molecules through nuclear membranes. The method described in this work is not restricted to nuclear transport processes alone. It may be used, for instance, to analyse translocation of different biomolecules like proteins and nucleic acids across different bio-membranes.

Authors gratefully acknowledge the financial support from DBT, DRDO and DST, Govt. of India. S. Arunkarthick would like to acknowledge the CSIR, Govt. of India for SR Fellowship.

Fig. 1: Experimental Setup of our Home Built Confocal Laser Scanning Microscope. SL- Scan Lens, X and Y- Scanning mirrors, DM- Dichroic Mirror, S- Shutter, FC-Fiber Coupler, F-Optical Fiber, BPF- Band Pass Filter, PMT- Photomultiplier Tube, M1-M4 – Mirrors and DAQ- Data Acquisition Card.

Fig. 2: Pollen Grain Serial Optical Sections by Confocal Microscope. (A) Figures A-J shows Ten confocal cross-sections of dye labeled pollen grain. 3D reconstruction of the pollen grain serial optical sections using ImageJ.

Fig. 3: Preliminary results on nuclear transport study. Time course study of nuclear accumulation of 4 kDa FITC-Dextran.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-5-P-1858 Morpho-functional analysis of the endocytic Golgi apparatus in response to treatment with 2-deoxy-D-glucose

Santler B.1, Ranftler C.1, Vetterlein M.1, Ellinger A.1, Neumüller J.1, Pavelka M.1
1Department of Cell Biology and Ultrastructure Research, Center for Anatomy and Cell Biology, Medical University of Vienna, Schwarzspanierstraße 17, 1090 Vienna, Austria
n0842383@students.meduniwien.ac.at

The Golgi apparatus is a central station in the cellular biosynthetic secretory pathway and is also involved in endocytic traffic via retrograde trafficking routes, which are necessary for the retrieval, repair and recycling of molecules. Although it is capable of several different feats, the transport and post-translational modification of secretory cargo proteins from the endoplasmic reticulum to their final destinations is one of its most important functions. Furthermore, it has the ability to take up endocytosed substances, among them wheat germ agglutinin (WGA), the internalisation of which is a complex multi-step process [1].
In mammalian cells, the Golgi apparatus is arranged into stacks, which are made up of cisternae and organized in cis-, medial-, trans- subcompartments. The Golgi apparatus is not a static organelle. It undergoes vast structural reorganizations in reaction to a variety of stimuli, such as the reduction of cellular ATP levels [2, 3]. To explore whether the Golgi apparatus retains some of its functionalities during this state of tubular-glomerular reorganization, human HepG2 hepatoma cells were treated with peroxidase-labelled wheat germ agglutinin before ATP was depleted from the cells using 2-deoxy-D-glucose. After fixation in 1% glutaraldehyde in 0.1M cacodylate buffer, internalized WGA was visualized by means of oxidation of diaminobenzidine. After post-fixation in 1% veronal-acetate-buffered OsO4, the specimens were dehydrated and embedded in epoxy resin. Thick sections of 200-250 nm were prepared for electron tomography to create 3D-models in order to study the complex architectures of the reorganized Golgi apparatus. Electron tomography was performed in a 200kV transmission electron microscope (Tecnai20, FEI); the tomograms were reconstructed using the Inspect 3D software (FEI) and 3D-models were drawn with the aid of the Amira 5.4.5 software. The dispersal of internalized WGA (Figs.1 and 2) showed that the Golgi apparatus maintains the ability to subcompartmentalize some of its structures even in a state of disorganization induced by 2-deoxy-D-glucose, which may provide further insight into the cellular reactions to metabolic stress.

 

References

1. Vetterlein, M., et al., Golgi apparatus and TGN during endocytosis. Histochem Cell Biol, 2002. 117(2): p. 143-50.
2. del Valle, M., et al.,Membrane flow through the Golgi apparatus: specific disassembly of the cis-Golgi network by ATP depletion. J Cell Sci, 1999. 112 ( Pt 22): p. 4017-29.
3. Meisslitzer-Ruppitsch, C., et al., The ceramide-enriched trans-Golgi compartments reorganize together with other parts of the Golgi apparatus in response to ATP-depletion. Histochem Cell Biol, 2011. 135(2): p. 159-71.

The authors would like to thank Mag.Beatrix Mallinger, Mrs Regina Wegscheider, Mr Ulrich Kaindl and Mr Thomas Nardelli for their technical assistance and help.

Fig. 1: 3D-model of a HepG2 cell Golgi apparatus reorganized in a glomerular structure after treatment with 2-deoxy-D-glucose. The endocytic parts containing internalized WGA are coloured in red. Yellow: WGA-negative compartments.

Fig. 2: Transitions between WGA-positive (red) and WGA-negative compartments (yellow) indicated by arrows.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-5-P-2268 Telocytes: insights on uterine ultrastructure and intercellular communication

Cretoiu S. M.1, 2, Cretoiu D.1, 2
1Carol Davila University of Medicine and Pharmacy, Bucharest, Romania, 2Victor Babeş National Institute of Pathology, Bucharest, Romania
sanda@cretoiu.ro

Background. Telocytes are cells with telopodes, described in a wide variety of human organs. Telopodes are structured as a succession of thin segments called podomers and dilated regions named podoms. They compose a labyrinthine system (by 3D convolution and overlapping) due to homo-and heterocelular interactions.
Methods. We examined telocytes by electron microscopy followed by two-dimensional reconstruction of successive microscopic fields and by time-lapse videomicroscopy
Results. Each uterine telocyte presented between 1-3 telopodes, the number of telopodes being roughly estimated at approximately 20 per 1000 µm2. Gauge measurements revealed podomers dimensions between 75-80 nm and podoms between 270-315 nm. Telocytes are coupled through: (a) close contacts and nanocontacts; (b)‘classical’ cell–cell junctions such as gap junctions; and (c) atypical homocellular junctions such as the tiny puncta adhaerentia minima and processus adhaerentes. Telopodes release extracellular vesicles: exosomes (60-100 nm vesicles) and ectosomes (diameters: 250-350 nm up to 1 μm). The diameter of secreted vesicles varied between 65 and 362 nm with most measurements around the median value of 170 nm. Time-lapse videomicroscopy of cell culture showed dynamic interactions between telopodes and smooth myocytes. Also, telopodes were snapped leaving behind small fragments containing signaling (macro)molecules (‘snow footprints’), probably helping the neighboring cells to migrate.
Conclusion. Telocytes could play a role in intercellular signaling and the control of local tissue homeostasis, confirmed by the presence of issued ectosomes in the extracellular environment.

This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number 82/2012 PN-II-PT-PCCA-2011-3.1-0553.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-5-P-2762 Cryoimmunoelectron microscopy analysis of the tubular elements of the Golgi complex of epididymal cells

Martínez-Alonso E.1, Martínez-Martínez N.1, Atienza-Guillén E.1, Martínez-Menárguez J. A.1
1Department of Cell Biology, School of Medicine, University of Murcia, 30100 Murcia, Spain
emma@um.es

The Golgi complex is usually composed of 3-9 stacked flat cisternae forming the Golgi stack or dictyosome, surrounded by vesicular and tubular elements. In most mammalian cells, stacks are laterally connected by tubules forming a continuous ribbon. Associated to the cis Golgi side of the Golgi stack, there is a tubule-vesicular system known as the cis-Golgi network (CGN). This is formed of tubules connected to the first Golgi cisterna. The trans-Golgi network (TGN) is located at the trans side and is the place where proteins are sorted, packed and delivered to their final destination. These tubules are continuous with the trans Golgi cisternae. Tubules were almost forgotten in the field for decades probably due to the difficulty of identifying them in electron micrographs. Epididymal epithelial cells have a highly developed Golgi complex (1,2,3), so that they are the perfect model to study tubular elements. In the present study, electron microscopy and cryoimmunocitochemistry were used to characterize the tubular networks associated to the stacks in this cell type. The tubular nature of most membranes associated to the stack was demonstrated in 80-nm thick sections. A wide battery of antibodies against Golgi proteins (resident enzymes, Rab, SNARE, and matrix proteins) was used to identified and characterize these elements. Rab 6 and Giantin have been proved to be good markers of the lateral tubules connecting stacks, while GM130 and KDEL receptor were specify for the cis-Golgi network. Further analysis of their structure and composition may help to integrate these elements in our knowledge of the Golgi complex.

References:

1. Hermo L and Smith CE (1998) The structure of the Golgi apparatus: a sperm’s eye view in principal epithelial cells of the rat epididymis. Histochem Cell Biol 109:431–447.

2. Robaire B and Hermo L (1988) Efferent Ducts, Epididymis, and Vas Deferens: Structure, Functions, and Their Regulation. Knobil and Neill’s Physiology of Reproduction The Physiology of Reproduction, Raven Press.

3. Robaire B, Hinton BT, and Orgebin-Crist MC (2006) The Epididymis. Knobil and Neill’s Physiology of Reproduction, Third Edition edited by Jimmy D. Neill, Elsevier.

This work was supported by grants Consolider COAT (CSD2009-00016) and Fundación Séneca (04542/GERM/06)

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-5-P-3103 A new probe for super-resolution imaging of membranes elucidates trafficking pathways

Revelo N. H.1, 2, 3, Kamin D.1, Truckenbrodt S.1, 4, Wong A. B.2, 3, 5, Reuter K.3, 6, Reisinger E.3, 6, Moser T.3, 5, 7, Rizzoli S. O.1, 3, 7
1Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, European Neuroscience Institute, Göttingen, Germany, 2International Max Planck Research School Neurosciences, Göttingen, Germany, 3Collaborative Research Center 889, University of Göttingen, Germany, 4International Max Planck Research School Molecular Biology, Göttingen, Germany., 5InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, Germany, 6Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology, University Medical Center Göttingen, Göttingen, Germany, 7Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, Germany
nataliarevelo@gmail.com

Up to now it has been difficult to establish the molecular composition of organelles involved in membrane uptake and recycling, due to the lack of suitable tools. We have developed a novel probe, named mCLING (membrane-binding fluorophore-Cysteine-Lysine-Palmitoyl Group), which binds to the plasma membrane, is efficiently taken up by endocytosis, and remains attached to membranes after fixation and permeabilization. mCLING is therefore a suitable tool to not only trace endocytosed organelles, but also to establish their molecular identity by immunolabeling methods (Fig. 1A). By conjugation to the fluorophore Atto647N, mCLING-labeled organelles were studied under high-resolution stimulated emission depletion (STED) microscopy. In cultured mammalian cells mCLING was taken up into the same organelles recycling the EGF and Transferrin receptors (Fig. 1B), and proved to be useful for co-labeling with endosomal markers (e.g. Syntaxin 6, Figure 1C). We applied mCLING to the auditory inner hair cells (IHCs) to study the mechanisms of synaptic vesicle recycling at their highly efficient active zones. It also allowed us to disclose the endosomal nature of tubular organelles described previously by electron microscopy in these cells (Fig. 1D). In primary cultured neurons, mCLING revealed molecular differences between actively and spontaneously released synaptic vesicles. Additionally, we confirmed the applicability of mCLING as membrane marker in the Drosophila larva neuromuscular junctions, and in microorganisms like yeast and bacteria. We conclude that mCLING has a great potential for the molecular study of membrane trafficking processes in a wide range of biological preparations.

Reference

Revelo N.H., Kamin D., Truckenbrodt S., Wong A.B., Reuter K., Reisinger E., Moser T., Rizzoli S.O. A new probe for super-resolution imaging of membranes elucidates trafficking pathways. J Cell Biol. Accepted, March 18 2014.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) through the Collaborative Research Center 889 ‘Cellular Mechanisms of Sensory Processing’ (to SOR and TM), by the Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain (grant to SOR and TM), and by a Starting Grant from the European Research Council, Program FP7 (NANOMAP, to SOR).

Fig. 1: A. mCLING is a novel endocytosis marker, fixable and combinable with immunostaining. B. Living cells. mCLING is endocytosed along with transferrin (Tf) and the epidermal growth factor (EGF). C. Cells fixed and immunostained for Syntaxin 6. D. IHCs. Two-color STED images. mCLING colocalizes with Syntaxin 6 and Syntaxin 16. Scale bars 2 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-5-P-3341 The effects of endoplasmic reticulum stress on the morphology of the ovarian follicles of mice.

Kubi J. A.1, Guzel E.1, Arda O.1, 2
1Cerrahpasa Medical Faculty of Istanbul University Histology and Embryology Department. ISTANBUL, TURKEY, 2Medical Faculty of Kemerburgaz University Histology and Embryology Department. ISTANBUL, TURKEY
oktayarda@yahoo.com

In eukaryotic cells, several pathways have evolved independently to ensure the integrity of the protein-folding environments in the endoplasmic reticulum (ER). The ER is the cellular organelle for synthesis, folding, and maturation of most secreted and trans membrane proteins. Processes that disturb protein folding may also cause accumulation of unfolded proteins in the ER leading to ER stress. Zona pellucida is a glycoprotein, which synthesized in ER of the ovum. It is a very important layer in the cellular relationship between ovum and follicular cells. In addition, it plays a significant role in follicular development. We used Tunicamycin (TM), which is an antibiotic that induces the ER stress. It blocks the synthesis of all N-linked glycoproteins and we studied its effects on the structure of zona pellucida and the morphology of the mice ovarian follicles. There were 24 C57BL/6 young female mice that weighed between 10-25g. We split them into three groups each having 8 mice. The control group had intraperitonial (IP) injection of basic water (pH 9) twice in each week. The other two groups were the experimental ones of 1 and 2. The first experimental group had IP injections of 5 μg of TM in 0.1ml of basic water for two weeks. The second experimental group had the same dose of TM twice per week for four weeks. We stained the histological sections with H+E, PAS+Masson and PAS+H. We used immunohistochemistry and Western blot methods to evaluate the effects of cellular stress on the morphology of the ovarian follicles. According to the anti-GRP/BiP immune reaction, the TM induced ER stress was comparatively expressed most in the first experimental group. The ER stress expression in the second experimental was relatively less than that of the 2 week group, but close to that of the control group. Immunohistochemistry and Western blot results also show low levels of ZPGs in first experimental group but high levels of ZPGs in the second experimental and control groups. Appropriate histomorphometric methods were also used to support the data. P -values for Zona pellucida and number of granulosa cells surrounding the oocytes in secondary follicles were found to be statistically significant among the groups. Consequently, it was concluded that tunicamycin induced cellular stress, and has a negative effect on the morphology of the mice ovarian follicles. REFERENCES 1. Sato E, at all.: Microscopy Research And Technique. 2006;69:427-435. 2. Liang LF, at all.: Mol. Cell. Biol. 1990, 10(4): 1507. 3. Hoodbhoy T, at all.: MCB. 2006; 26(21): 7991-7998. 4. El-Mestrah M, at all.: Biol. Reprod. 2002; 66: 866-876. 5. Schr'odera M, at all.: Mutation Research. 2005; 569: 29-63. This present work was supported by the Research Fund of Istanbul University. Project No. 23471

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-5-P-3356 Cytotoxic effects of TiO2 nanospheres after UV-light irradiation on urothelial cancer cells

Imani R.2, Erdani Kreft M.1, Iglič A.3, Veranič P.1, Hudoklin S.1
1Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, Ljubljana, Slovenia , 2Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Zdravstvena 5, Ljubljana, Slovenia, 3Laboratory of Biophysics, Faculty of Electrical Engineering, University of Ljubljana, Tržaška 25, Ljubljana, Slovenia
samo.hudoklin@mf.uni-lj.si

Urothelial cells line the urinary bladder and form the tightest permeability barrier in mammalian species, including humans. The process of normal urothelial differentiation can undergo alternative pathways, which often leads to the formation of urinary bladder cancers. Bladder cancers are clinically very relevant issue as they present the ninth most common malignancy in man, and show high recurrence rate with the traditional operational approaches. Therefore, development of novel approaches to remove the cancer cells more effective is needed. One of the viable options is photocatalysis of metal nanoparticles, which are endocytosed by cancer cells and produce cytotoxic reactive oxygen species (ROS) when irradiated with UV-light. Here, our aim was to determine cytotoxic potential of TiO2 nanospheres after UV-light irradiation on invasive urothelial cancer cells T24.

Materials&Methods. The human urinary bladder cancer cells T24 (high-grade and invasive transitional carcinoma cells) were incubated in culturing medium supplemented with TiO2 nanoparticles for 2 hours, washed and irradiated with UV-light (15 W/cm2) for 10-, 20- or 30 minutes. The cytotoxic effects of TiO2 nanospheres were evaluated 8 and 24 hours after illumination; quantitatively with Live/Dead Viability Kit (Molecular probes, Invitrogen) under light microscope (T300, Nikon), and ultra-structurally with scanning and transmission electron microscopes (840A, Jeol and CM100, Philips, respectively).

Results. TiO2 nanospheres were internalized by the T24 cells and were detected in various endosomal compartments, which were distributed beneath the plasma membrane and around the nucleus. Viability of T24 cells that contained TiO2 nanospheres, but were not irradiated, was comparable to the cells without nanospheres and irradiation. Eight hours after UV-light irradiation, the viability test showed green (live) or red (dead) staining of the cells that had contained TiO2 nanospheres. The percentage of red-coloured cells was irradiation-time dependent and was the lowest in the cultures that were irradiated for 10 minutes, higher in 20-, and the highest (> 65%) in cultures irradiated for 30 minutes (Fig. 1, upper panels). Twenty-four hours after UV-light irradiation the trend was the same, however, percentage of red-coloured cells was even higher (>85% in cultures that were irradiated for 20 minutes or longer; Fig. 1, lower panels).

Conclusions. TiO2 nanospheres that were used in the experiments could be endocytosed by the epithelial cells and are not toxic for them per se. However, in the combination with UV-light irradiation TiO2 nanospheres exhibit strong cytotoxic effects on cells, and are therefore recommended for further use as an excellent photocatalytic nano-material.

Fig. 1: Viability of T24 cells, which were pre-incubated with TiO2 nanospheres, 8- and 24 hours after irradiation with UV-light for 10-, 20- or 30 minutes. Green staining corresponds to live cells and red staining corresponds to dead cells. Scale bar: 10 µm
↑ Go to the Table of content ↑

LS-6. Microbiology and virology

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-6-IN-3167 Structural Analysis of Respiratory Syncytial Virus

Wright E. R.1, Kiss G.1, Holl J. M.1, Williams G. M.1, Alonas E.2, Vanover D.2, Lifland A. W.3, Gudheti M.3, Guerrero-Ferreira R. C.1, Nair V.4, Graham B. S.5, Santangelo P. S.2
1Emory University, Department of Pediatrics, Atlanta, GA, USA, 2Georgia Institute of Technology and Emory University, Wallace H. Coulter Department of Biomedical Engineering, Atlanta, GA, USA, 3Vutara, Inc., Salt Lake City, UT, USA, 4Research Technologies Branch, Microscopy Unit, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA, 5Vaccine Research Center, NIAID, NIH, Bethesda, MD, USA
erwrigh@emory.edu

Paramyxoviruses are pleiomorphic enveloped RNA viruses; and include the human pathogens respiratory syncytial virus (RSV), human metapneumovirus (hMPV), measles virus (MeV), and human parainfluenza viruses (HPIVs). RSV primarily infects infants, young children, and the elderly, causing severe bronchiolitis, viral pneumonia, and death. There is no vaccine against RSV.

To define the structure of RSV, we combined several state-of-the-art imaging and analysis technologies. We used cryo-electron tomography (cryo-ET) and a newly developed approach, Zernike phase contrast (ZPC) cryo-ET, to visualize the architecture of RSV in its native state. To evaluate the presence and localization of M2-1 in filamentous viruses and to correlate with the cryo-ET data, we used confocal microscopy and direct stochastic optical reconstruction microscopy (dSTORM). In addition, we utilized a proximity ligation assay (PLA) to measure the specific protein-protein and protein-RNA interactions in RSV particles.

For all imaging experiments, we either produced and purified RSV from HEp-2 cells or procured suspensions of RSV (Advanced Biotechnologies Inc.). Cryo-EM data was collected with a JEOL JEM-2200FS 200 kV, field emission TEM with an in-column energy filter, on a Gatan 4kx4k CCD camera. For the confocal microscopy experiments, images of fixed cells were taken on a LSM 710 laser scanning confocal microscope (Zeiss). Super-resolution images were recorded with a Vutara SR 200 (Vutara, Inc.) microscope. PLA was performed according to the Duolink II kit manufacturer’s instructions (Olink Bioscience).

Using cryo-ET, we classified virus particles as spherical, filamentous, or asymmetric. All three morphologies sustained a similar organization of the surface glycoproteins, matrix protein (M), M2-1, and the ribonucleoprotein (RNP). Employing computational methods, the RNP filaments were traced in 3D and their total length was calculated. The measurements emphasized that the inclusion of multiple full-length genome copies per particle was a common attribute. The RNP was associated with the viral membrane whenever the M layer was present. We used fluorescence light microscopy (fLM), dSTORM, and PLA, to provide additional evidence illustrating that M2-1 is located between the RNP and M in isolated viral particles. In addition, regular spacing of the M2-1 densities was resolved when RSV viruses were imaged using ZPC cryo-ET. Our studies highlight the complexity of RSV structure and substantiate that M and M2-1 regulate virus organization.

This work was supported in part by Emory U., CHOA, and the GRA to E.R.W.; the CFAR at Emory U. (P30 AI050409); NSF grant 0923395 to E.R.W.; and public health service grants R21AI101775 to E.R.W.; R01GM094198 to P.J.S.

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-6-IN-5834 Microbes with identity issues: the energy-conserving prokaryotic organelle and (atypical) cell wall of anammox bacteria

van Niftrik L.1
1Microbiology, Institute for Water & Wetland Research, Faculty of Science, Radboud University Nijmegen, The Netherlands
l.vanniftrik@science.ru.nl

Anaerobic ammonium-oxidizing (anammox) bacteria are recognized as major players in the global nitrogen cycle and estimated to be responsible for up to 50% of the nitrogen in the air that we breathe. In addition, anammox bacteria are extremely valuable for wastewater treatment where they are applied for the removal of nitrogen compounds. Besides their ecological and industrial importance, anammox bacteria defy some basic biological concepts. Whereas other bacteria have only one cell compartment, the cytoplasm, anammox bacteria have three independent cell compartments. The innermost, major, cell compartment is called the anammoxosome and is the location of the anammox reaction. We propose that the anammox reaction is coupled to the anammoxosome membrane, leading to the establishment of a proton motive force and subsequent ATP synthesis. To test this hypothesis, we have isolated the anammoxosome from the cell and investigate its role as the energy factory of the cell. In addition, anammox bacteria are proposed to have an atypical cell wall devoid of both peptidoglycan and a typical outer membrane. The anammox cell wall thus does not seem to resemble that of other known bacteria but instead resembles the cell wall of Archaea; with an energized outermost (cytoplasmic) membrane and a surface protein layer on top. Both anammoxosome and cell wall projects combine molecular and protein toolboxes with (cryo-)electron microscopy techniques. The results will add new insights to the fundaments of bacterial cell biology. In addition, understanding how anammox bacteria work is important for global climate change and development of sustainable wastewater treatment systems.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-6-O-2250 FIB/SEM and (serial) Electron Tomography of the enigmatic Ignicoccus hospitalis/Nanoarchaeum equitans Co-Culture

Heimerl T.1, Flechsler J.1, Wanner G.2, Rachel R.1
1Center for Electron Microscopy, University of Regensburg, Germany, 2Institute of Botany, LM University of Munich, Germany
thomas.heimerl@ur.de

On the basis of cryopreparation in combination with serial sectioning, FIB-SEM and (serial) electron tomography, we deliver a detailed look into the unusual cell architecture of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis and its relationship to Nanoarchaeum equitans. The 3D-models obtained reveal a highly complex and dynamic endogenous membrane system that is unrivaled among prokaryotes.

The hyperthermophilic Crenarchaeon Ignicoccus is unusual in many ways; above all in its ultrastructure [1]: Unlike most archaea which have a proteinaceous S-Layer as the outmost sheath in their cell envelope, Ignicoccus exhibits an outer cellular membrane (OCM). Between this outer cellular membrane and the cytoplasmic membrane, an intermembrane compartment (IMC) can be found whose volume makes up ~40% of the whole cell volume in average. In some cells it can reach an extent much larger than the volume of the cytoplasm. In the IMC, membrane-surrounded tubular and vesicular structures can be found. Apparently, these structures constrict from or fuse with the cytoplasmic membrane and interact with the outer membrane via pore complexes. All interacting structures are connected via thin filaments (~3-6 nm in diameter), that span through the whole IMC. The same structures also seem to be involved in building up the contact to Nanoarchaeum. The S-Layer of Nanoarchaeum appears to be disintegrated at the contact site, but might play a role in the initial adherence process. Intriguingly, this contact was revealed as a fusion of cytoplasms of both cells. Overall, the inner membrane system of Ignicoccus is reminiscent of the eukaryotic endogenous membrane system not only in structure and dynamics. Putatively involved homologues to designated eukaryotic proteins can be found: small GTPases (Sar1/Arf like), Coatomer proteins, Sec61β, proteins of the ESCRT-III system, a tethering complex component (Bet3) and the ATPase p97 [2, 3]. Altogether, the results are tempting to speculate about a prokaryotic origin of the eukaryotic endogenous system.

[1] Rachel et al., Archaea 1 (2002), 9
[2] Podar et al., Biology Direct 3 (2008), 2
[3] Giannone et al., PLOS One 6(8) (2011), e22942

supported by a grant of the Deutsche Forschungsgemeinschaft (DFG)

Fig. 1: (A) slice of a tomogram; CP=Cytoplasm, IMC=Intermembrane Compartment; CM= Cytoplasmic Membrane; SL= S-Layer; bar 0.5 µm; (B) detail of the contact site between Ignicoccus and Nanoarchaeum; bar 0.2 µm; (C) 3D-Model of the contact site; (D) cross section; yellow=CP of both organisms, green=OCM/IMC, purple=SL, red=filamentous structures
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-6-O-2343 Localization of a novel CO2 fixation pathway in a compartmentalized Prokaryote

Flechsler J.1, Heimerl T.1, Rachel R.1
1University of Regensburg, Centre for Elelctron Microscopy, Regensburg, Germany
Jennifer.Flechsler@ur.de

In autotrophic eukaryotes, CO2 is fixed via the Calvin Cycle, a pathway which takes place in the stroma of the chloroplasts; autotrophic prokaryotes, however, have developed at least five alternative ways of CO2 fixation which by now were all assumed to be located in the cytoplasm [1]. In the hyperthermophilic Crenarchaeum Ignicoccus, CO2 fixation is proposed to proceed via the unique dicarboxylate/4-hydroxybutyrate cycle and as we could show provided a surprise in its location [2].
Ignicoccus cells exhibit an extraordinary ultrastructure. In addition to the cytoplasmic membrane, there is an outer cellular membrane, which encases an intermembrane compartment (IMC). The IMC contains membrane-surrounded vesicular structures and tubes, derived from the cytoplasm. Altogether, the ultrastructure is reminding of the eukaryotic endogenous membrane system [3].
Another curiosity about Ignicoccus is the location of its ATP synthase. This enzyme is exclusively found in the outer cellular membrane of Ignicoccus cells, thus leading to the assumption that large quantities of ATP are available in the IMC [4].
The compartmentalized cell structure of Ignicoccus and the unique location of its ATP synthase in the outer cellular membrane raise questions about the subcellular distribution of different steps of the CO2 fixation pathway. To answer these questions we used different methods of 3D electron microscopy (serial sectioning, electron tomography) and immunolabelling of cryo-immobilized, freeze substituted cells. Immunolabelling studies revealed that the Acetyl-CoA synthetase, an enzyme that catalyzes the initial step of the CO2 fixation, is located in the IMC, tightly associated with the outer cellular membrane.
Additionally, we were able to detect the Crotonyl-CoA hydratase (step 12) and the PEP carboxylase (step 3) in the IMC of Ignicoccus. Currently, we are targeting more enzymes involved in the CO2 fixation to track down its route and to get a deeper understanding in the physiology of these highly unusual cells.

References:
[1] Fuchs, Annu Rev Microbiol. 65 (2011), p 631-58.
[2] Jahn et al., J Bacteriol 189 (2007), p. 4108.
[3] Heimerl, EMC 2012 proceedings (2012).
[4] Küper et al., PNAS 107 (2010), p. 3152.

This research was supported by grants from the DFG (Germany)

Fig. 1: Ultrathin section of Ignicoccus; cells were cryo-fixed, freeze-substituted, and embedded in Epon. Section was labeled with antibodies directed against the Acetyl-CoA synthetase; detection with goat anti-rabbit immunoglobulin 6 nm. Bar, 0.5 µm.

Fig. 2: 3D-reconstruction and visualization of different data sets of serial sections from Ignicoccus cells, prepared as described, labeled with antibodies directed against the Acetyl-CoA synthetase. Alignment, segmentation, and visualization were done using AMIRA. Red: cytoplasm; blue: vesicles in the IMC; yellowish/white: gold; light blue: Nanoarchaeum.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-6-O-2867 Anammox bacteria: Linking ultrastructure and function

Mesman R.1, van Teeseling M.1, Neumann S.1, van Niftrik L.1
1Department of Microbiology, Institute for Water & Wetland Research, Faculty of Science, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands 1
r.mesman@science.ru.nl

Anammox bacteria convert ammonium and nitrite to nitrogen gas to obtain energy for growth. Until its discovery in the early 1990s This so called anammox reaction was deemed impossible. Now, anammox is recognized to contribute significantly to oceanic nitrogen loss and is estimated to be a major source of gaseous nitrogen on Earth. In addition, anammox bacteria are extremely valuable for wastewater treatment where they are applied for the removal of ammonium. Besides their importance in industry and the environment, anammox bacteria defy some basic biological concepts. Whereas most other bacteria have only one cell compartment, the cytoplasm, anammox bacteria have three independent cell compartments, from out- to inside; the paryphoplasm, riboplasm and anammoxosome. The anammoxosome is the largest cell compartment and is proposed to be dedicated to energy transduction. As such it would be analogous to the mitochondria of eukaryotes. The riboplasm contains the nucleoid and ribosomes and the paryphoplasm has a yet unknown function. Having three cellular compartments poses challenges to protein sorting, substrate transport and cell division and it is largely unknown how anammox bacteria achieve these functions. In addition, anammox bacteria have been proposed to have an atypical cell wall devoid of both peptidoglycan and an outer membrane. By combining advanced electron microscopy techniques (such as cryo-electron microscopy and cryo-electron tomography) with immuno-localization studies, proteomics and cell fractionation we aim to gain a better understanding of the ultrastructure and function of these unique bacteria.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-6-O-3435 DNA structure inside the T5 bacteriophage capsid

LEFORESTIER A.1, SUNG B.1, DE FRUTOS M.2, LIVOLANT F.1
1Laboratoire de Physique des Solides, CNRS UMR 8502, Orsay, France 1, 2Institut de Biochimie et Biophysique Moleculaire et Cellulaire, UMR CNRS 8619, Universite Paris Sud, Orsay, France 2
francoise.livolant@u-psud.fr

Using multiple methods including cryoTEM observations of phages in suspended vitrified films or cryosections of infected bacteria, we address the question of bacteriophage T5 DNA organization in the full capsid and during encapsidation and release of the genome.

- The organization of DNA in the full capsid has been debated over the years. We propose that the hexagonally packed DNA is organized into multiple domains separated by defect walls [2]. Ongoing tomography observations will be presented.

- DNA release form the bacteriophage capsid involves physical and biological processes. It is possible to follow the ejection of DNA from the bacteriophage in vitro after interaction of the phage with its purified receptor. CryoTEM experiments let us visualize the portion of DNA kept inside the capsid at different steps of the ejection and provide information on its structure [1-3]. From these experiments, we discuss how the structure of confined DNA changes with the ionic conditions (isotropic, versus fully or partially toroidal). CryoTEM and fluorescence approaches reveal the presence of pauses during T5 ejection (moments where the ejection speed slows down to zero) allowing partially filled capsids to be imaged. We discuss the importance of ionic conditions and presence/absence of a support film in the location of these pauses [4]. We also discuss the reasons why pauses have not been detected in other phages like Lambda. Our work highlights the role of DNA organization inside the bacteriophage capsid on the stochastic and out of equilibrium nature of the ejection process.

- DNA organization during encapsidation inside the bacteria is analysed on cryosections of infected bacteria (work in progress) and comparisons are driven with capsids containing various amounts of DNA under multiple ionic conditions.

[1] A Leforestier, F Livolant, Proc. Natl. Acad. Sci. 2009, 106, 9157–9162.

[2] A Leforestier, F Livolant, J. Mol. Biol, 2010, 396, 384–395.

[3] A. Leforestier, A. Siber, F. Livolant , R. Podgornik, Biophys. J. 2011, 201, 100, 2209–2216

[4] M. De Frutos, A. Leforestier, and F. Livolant 2014, Biophys. Rev. Lett. 09, DOI: 10.1142/S1793048013500069

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-1434 The mechanism of bactericidal action of sodium hypochlorite against Staphylococcus aureus

Ujimine S.1, Tone S.2, Saito M.3, Yamada S.1,3
1Health science, Kawasaki University of Medical Welfare, Kurashiki, Japan, 2Biochemically, 3Microbiology, Kawasaki Medical School, Kurashiki, Japan
s_adagio_cantabile@yahoo.co.jp

Sodium hypochlorite (NaOCl) is used as a disinfectant. However, its bactericidal mechanism has not yet been clarified. In the present study, the bactericidal mechanism of NaOCl was examined using microscopy and gel electrophoresis techniques.

Staphylococcus aureus strain 209P was treated with NaOCl. Scanning electron microscopy images of the staphylococcal cells treated with 0.05% (w/v) NaOCl for 5 minutes showed an irregular surface with cells partially invaginated. Transmission electron microscopy images of the bacterial cells treated with 0.05% for 5 and 15 minutes showed cytoplasmic alteration, accompanied by partially irregular surfaces. When treated with a high concentration of NaOCl (0.1%), significant alteration of the cytoplasm was seen and condensation was observed in the nuclear region in some cells. By a fluorescence microscope, we clearly observed fluorescence quenching in these 0.1% NaOCl-treated cells. Based on these observations, which indicated that NaOCl damaged chromosomal DNA, we next treated chromosomal DNA from bacterial cells with NaOCl and performed agarose gel electrophoresis. Chromosomal DNA was absent from the gel comprising the DNA sample from the bacterial cells treated with 0.05% NaOCl. From these biochemical results, it became clear that NaOCl degrades the chromosomal DNA of S. aureus. Taken together with these findings, we assume that the condensation of nuclear region induced with NaOCl is mediated by the NaOCl-induced degradation of S. aureus chromosomal DNA.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-1540 The taxonomic and phylogenetic studies on Pucciniastrum and related genera

Yang T.1, Tian C. M.1, Liang Y. M.2, You C. J.1
1The Forestry Institute, Beijing Forestry University, Beijing, China, 2Museum of Beijing Forestry University, Beijing, China
yt871212@163.com

The genus Pucciniastrum was first established by Otth in 1861 with a type species Pucciniastrum epilobii. But the taxonomic treatment of this genus with the genus Calyptospora and Thekopsora was different among researchers. The genus Thekopsora was described by Magnus in 1875 based on the species Thekopsora areolata. It was distinguished from Pucciniastrum only by the position of the telia, namely in Pucciniastrum teliospores develop underneath the epidermis of plants, but within the epidermal cells of plants in Thekopsora. Calyptospora was established by Kühn in 1869, consisting of a single species Calyptospora goeppertiana. This species was distinguished from Thekopsora by the absence of the uredinia and formation of teliospores on stems (Pady 1933, Faull 1938). Although these three genera could be distinguished by the position of telia, other morphological characteristics were similar. In earlier studies, some authors did not consider the position of telia as an important taxonomic characteristic and treated Thekopsora and Calyptospora as synonyms of Pucciniastrum, thus, Pucciniastrum as broad sense (Dietel 1900, Fischer 1904, Arthur 1907-1925).
In order to get a clearer understanding of the relationship among Pucciniastrum, Thekopsora and Calyptospora, phylogenetic analyses of 28s rRNA is applied in this research along with the morphological studies. The characteristics of both uredinia and telia were observed under the light microscope and scanning electron microscope. Sequences of genera of Pucciniastreaece were used in this study. And it is also the first time to use the molecular studies on the systematic research of Pucciniastraceae.
The phylogenetic results showed that species of these three genera did not assemble in separate clades on the basis of genus. Species of these three genera were crossed and separated in three main clades which had a better correspondence with the characteristics of the ostiolar cells of the uredinia ranther than the location of the telia. That is to say, species which had well-developed and smooth ostiolar cells (Group Ⅰ), had no well-developed ostiolar cells (Group Ⅱ)and had well-developed and coarsely ostiolar cells (Group Ⅲ) formed three distinct lineage (Fig.1; 2). It was the first time to use comparative analyses of molecular phylogeny and morphology to study of the taxonomic status of Pucciniastrum, Thekopsora and Calyptospora. It suggested that it is inappropriate to distinguish these three genera according to the position of telia, and we propose a combination of these three genera and treat them as broad Pucciniastrum. Also, the result implied that the characteristics of the uredinial ostiolar cells had a closer relationship with the evolution of these genera rather than the telia.

This research is supported by the NSFC in China (NO.31070572). Thank to the herbaria HH, TSH, HMAS, HMNWFC. We are grateful to Dr. M. Kakishima and Dr. Y. Ono for their help.

Fig. 1: A tree formed based on 28S sequences with maximum parsimony method. Bootstrap values were calculated from 1000 replications. Parsimony bootstraps greater than 40% are shown. Sequence of Melampsoridium betulinum was used as out group. The bar of the picture stands for 20μm.

Fig. 2: A tree formed based on 28S sequences of species of Pucciniastraceae with maximum parsimony method. Bootstrap values were calculated from 1000 replications. Parsimony bootstraps greater than 40% are shown. Sequence of Puccinia hordei was used as out group.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-1546 A Deep-Sea Microorganism and the Origin of the Eukaryotic Cell

Yamaguchi M.1, Worman C. O.2, Mori Y.3, Furukawa H.3, Yamamoto Y.4, Higuchi K.4, Arai S.4, Murata K.5, Kawamoto S.1
1Medical Mycology Research Center, Chiba University, Chiba, Japan, 2Department of Biology, Francis Marion University, SC, USA, 3System in Frontier Inc., Tokyo, Japan, 4High Voltage Electron Microscopy Laboratory, Nagoya University Ecotopia Institute, Nagoya, Japan, 5Section of Electron Microscopy, National Institute for Physiological Sciences, Okazaki, Japan
mandn80@hotmail.com

There are only two kinds of organisms on Earth: prokaryotes and eukaryotes. Although eukaryotes are considered to have evolved from prokaryotes, there were no previously known intermediate forms between them until recently. The differences in their cellular structures are so vast that the problem of how eukaryotes could have evolved from prokaryotes is one of the greatest enigmas in biology. In 2012, we discovered a unique organism with cellular structures appearing to have intermediate features between prokaryotes and eukaryotes in the deep-sea off the coast of Japan by using electron microscopy and structome analysis [1]. The organism was 10 µm long and 3 µm in diameter, having more than 100 times volume of Escherichia coli. It had a large ‘nucleoid’, consisting of naked DNA fibers, with a single layered ‘nucleoid membrane’, and ‘endosymbionts’ that resemble bacteria, but no mitochondria. We named this unique microorganism the ‘Myojin parakaryote’ with the scientific name of Parakaryon myojinensis (“next to (eu)karyote from Myojin”) after the discovery location and its intermediate morphology. The existence of this organism is an indication of a potential evolutionary path between prokaryotes and eukaryotes, and strongly supports the endosymbiotic theory for the origin of mitochondria and the karyogenetic hypothesis for the origin of the nucleus.

[1] Yamaguchi M, Mori Y, Kozuka Y, Okada H, Uematsu K, Tame A, Furukawa H, Maruyama T, Worman CO, Yokoyama K: Prokaryote or eukaryote? A unique microorganism from the deep sea. J. Electron Microsc. 61: 423-431, 2012.

This work is the result of joint research with Yoshimichi Kozuka, Hitoshi Okada, Katsuyuki Uematsu, Akihiro Tame, Tadashi Maruyama, and Koji Yokoyama. We sincerely thank them.

Fig. 1: All Figures.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-1547 Super Support Film for High-Resolution Electron Microscopy

Yamaguchi M.1, Maruta S.2
1Medical Mycology Research Center, Chiba University, Chiba, Japan, 2Nisshin EM Ltd., Tokyo, Japan
mandn80@hotmail.com

We developed a new support film for electron microscopy using a plasma polymerization method [1]. In this method, a thin film is formed on the surface of sodium chloride crystals by applying a high voltage (2 kV, D.C.) across electrodes thorough naphthalene gas in a plasma polymerization replica apparatus (Fig. 1, step 1–4). The film is floated off in water, and picked up on an electron microscopy grid placed on a filter paper (Fig. 1, step 5–6). After drying in air, the grids are ready for use (Fig. 1, step 7).

This support film was named “Super Support Film”, and is now commercially available from Nisshin EM Ltd. (telephone and fax: 81-3-3355-3001). Super Support Film has the following features.

1) It is a three-dimensionally polymerized carbon film.

2) It shows an amorphous texture and high transparency to electrons.

3) It is mechanically strong and resists heat, chemicals, and electron bombardment.

4) It has a very smooth surface, suitable for negative staining.

Figure 2 shows a cross section of Super Support Film. Figure 3 shows a negative staining of influenza A virus using a Super Support Film-coated microgrid. Figure 4 to 6 show, respectively, an ultrathin section of a freeze-substituted yeast (Cryptococcus), a negative staining of hepatitis B core particles, and a negative staining of the bacterium Helicobacter pylori. These micrographs demonstrate the clarity and resolution possible with Super Support Film.

[1] Yamaguchi et al.: A support film of plasma-polymerized naphthalene for electron microscopy: method of preparation and application. J Electron Microsc. 41: 7-13, 1992.

Fig. 1: All Figures.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-1557 Initial Structural Analysis of Frog Virus 3 by Electron Microscopy and Tomography Shows Composition and Morphology of its Large Virion with Inner Membrane

Nemecek D.1,2, Vesely T.3, Plevka P.1, Plitzko J. M.2
1Central European Institute of Technology-Masaryk University, Brno, Czech Republic, 2Max Planck Institute of Biochemistry, Martinsried, Germany, 3Veterinary Research Institute, Brno, Czech Republic
nemecek@ceitec.muni.cz

Nucleocytoplasmic large dsDNA viruses (NCLDV) are among the largest and most complex viruses known – their capsids are 200–800 nm large and the dsDNA genome is replicated in the cell nucleus and cytoplasm with only little help of the host organism [1]. Here, we focused on ranaviruses (~200 nm in diameter) that have caused significant economic losses to fish industry and threaten biodiversity of amphibian species worldwide [2]. Despite increasing importance of ranaviruses as global pathogens relatively little is known about the structural and mechanistic features of their replication cycle. The ranaviruses exist in two infectious forms (naked capsids and enveloped virions), each with a different way of cell entry and cell egress [3]. In order to determine the structural basis for cell entry by the two respective forms of virions, we purified and imaged virions of the type ranavirus, frog virus 3 (FV3), by cryo-electron microscopy and tomography. Electron micrographs showed large capsids with three distinguishable layers enclosing the electron-dense core of packaged dsDNA (Figures 1, 2). The inner shell presumably corresponds to an internal membrane, the intermediate layer to a proteinous capsid shell and the outer layer to an external lipid envelope. Initial 3D reconstruction of FV3 revealed an icosahedral shell with large flat triangular facets between the 5-fold vertices and small turrets at the 5-fold vertices of the shell (Figure 3). The capsid diameter varies from 157 nm at the 3-fold axis and 159 nm at the 2-fold axis to 175 nm at the 5-fold icosahedral axis. Overall, the FV3 capsid exhibits similar morphology and structural features as related viruses from the family Iridoviridae, PBCV-1 and CIV [4]. Electron tomograms of FV3 virions additionally revealed small spikes at the outer surface of enveloped virions that likely correspond to viral glycoproteins. Subtomogram averaging of individual extracted virions was done in Bsoft [5] using icosahedral symmetry. The average (Figure 2) is consistent with the single particle reconstruction and further image analysis will be undertaken to identify the putative special vertex for genome ejection.

[1] La Scola, B. et al. (2003) Science, 299, 2033.
[2] Gray, M.J. et al. (2009) Dis. Aquat. Organ., 87, 243–266.
[3] Chinchar, V.G. et al. (2009) Curr. Top. Microbiol. Immunol., 328, 123–170.
[4] Xiao, C. and Rossmann, M.G. (2011) Curr. Opin. Virol., 1, 101–109.
[5] Heymann, J.B. et al. (2008) J. Struct. Biol., 161, 232–242.

This research has been supported by the Career Integration Grant (No. 618111) to DN and by the grant from Ministry of Agriculture of the Czech Republic (MZE 0002716202) to TV.

Fig. 1: Cryo-electron micrograph of FV3 virions collected at 300 kV and 4-um defocus. The virions have angular appearance and consist of a dense DNA core and multiple outer layers.

Fig. 2: The central slice of a subtomogram average of FV3 virions shows a dense DNA core and multiple outer layers.The layers are more separated under the 5-fold vertices (arrowhead) than the rest of the icosahedral shell.

Fig. 3: Isosurface rendering (1.5σ) of the reconstructed icosahedral FV3 capsid reveals small turrets above the 5-fold vertices (arrow). The icosahedral asymmetric unit and axes are depicted by the white triangle.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-1888 Evaluation of urinary catheter surfaces on Proteus mirabilis biofilm formation by SEM and EDS

Villar S.1, Scavone P.2, Iribarnegaray V.2, Zunino P.2
1Laboratorio de Microscopía Electrónica, Facultad de Ciencias, UdelaR, Iguá 4225, Montevideo, Uruguay , 2Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Av Italia 3318, Montevideo, Uruguay.
svillar@fcien.edu.uy

Catheter-associated urinary tract infections (CAUTIs) are one of the most common nosocomial infections. Half of the patients experience bacteriuria in the first 10-14 days of catheterization and the risk is significantly higher in long-term catheterization. In patients with more than 28 day of catheterization the risk of infection approaches to 100%. Proteus mirabilis is a gram negative bacterium commonly associated with CAUTI. It can cause complicated urinary tract infection (UTI). They can express several virulence factors that are related with infection as fimbriae, flagella, immune-avoidance, damage in host cells and biofilm formation. We used a clinical P. mirabilis 2921 strain and an isogenic mutant strain for an efflux protein (P. mirabilis 40) which make them inefficient to form biofilm. We assessed the capability to swarm over latex and silicone catheters (Sylkolatex 2 way Foley catheter, Teleflex Medical, USA and Silkomed ®Teleflex Medical, Germany) with both strains. The experiment was performed 10 times. Scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) was performed in the different catheters.
The strains were divided in 3 groups according to the results obtained: (0) non-crossing, (1) swarming but not able to bridge the catheter and (2) able to bridge the catheter. The results show that wild type P. mirabilis 2921 was able to bridge in all cases (10/10) in both types of catheter. In silicone, P. mirabilis 40, 50% of bacteria were non-crossing, 30 % were swarming but not able to bridge and 20 % were able to bridge the catheter. Moreover in latex, 60% of them were able to swarm but not to bridge while 40 % were able to bridge the catheter. This results show that the mutant is affected in their capability of crossing the catheter but there are also differences related to the catheter material.
EDS revealed that latex has an 8.27% of zinc among other components while silicone has mainly silicone (36.5%) and no other metals were found (Fig.1 and 2). Zinc is an essential transition metal in all organisms; bacteria are predicted to incorporate zinc into 5-6% of all proteins. Zinc proteins are involved in DNA replication, glycolysis, pH regulation and the biosynthesis of amino acids, extracellular peptidoglycan and low molecular thiols. Probably, the accessibility of zinc allows the mutant cross better the latex than the silicone catheters. SEM images revealed that wild type P. mirabilis 2921 form biofilm over the surface (Fig. 3 and 4) while the mutant only appear in small groups.
The results obtained in the present work will contribute to the understanding of CAUTI and should be taking into account in clinical practice.

Fig. 1: Latex catheter surface without bacteria. The structure in the image resembling a ball isin the internal surface of the catheter

Fig. 2: Silicon catheter surface without bacteria

Fig. 3: P. mirabilis 2921 over latex catheter. It is possible to recognize the “balls” in the latex surface (*). Bacterial biofilm is indicated by two **

Fig. 4: P. mirabilis 2921 over silicon
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2131 Viability and surface properties of the solvent-resistant Rhodococcus ruber IEGM 231

Korshunova I. O.1, Kuyukina M. S.1, 2, Ivshina I. B.1,2
1Institute of Ecology and Genetics of Microorganisms, Ural Branch, Russian Academy of Science, Perm, Russia, 2Perm State University, Perm, Russia
subunit2012@yandex.ru

Bacteria tolerant to organic solvents could be used for the bioconversion of hydrophobic compounds relevant to pharmaceutical, chemical and food industries (Schmid et al., 2001). Rhodococcus actinobacteria are promising biotechnology agents due to their great catalytic capabilities and high resistance to toxicants and harsh environmental conditions (Kuyukina, Ivshina, 2010). Organic solvents affect negatively the structural and functional integrity of bacterial cells (de Carvahlo, 2010). Using a coupled confocal laser scanning (CLSM) and atomic-force microscopy (AFM), it is possible to study surface properties of living bacterial cells. The aim of this work was to investigate the effect of cyclohexane on R. ruber cell viability, morphology and elastomechanical properties.

We studied the R. ruber IEGM 231 strain from the Regional Specialized Collection of Alkanotrophic Microorganisms (acronym IEGM, WDCM # 768; www.iegm.ru/iegmcol/strains). A coupled AFM/CLSM system (Asylum MFP-3D, Asylum Research and Olympus FV1000, Olympus Corporation) was used. Silicon cantilevers AC240TS (Olympus) were used for the AFM imaging in tapping mode in air (scan size – 20 μm). Silicon nitride cantilevers BL-TR400PB (Olympus) were used for the force measurements in water. Images were analyzed using FV10-ASW (Olympus) and Igor Pro 6.22A (WaveMetrics) softwares. Cell viability was tested using LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen). Only 40% of cells remained viable after 24 h exposure to cyclohexane and significant changes in their surface morphology in response to toxic effects of the organic solvent were revealed (Fig. 1, 2). Rhodococcal cells became smaller by 29% in length and 21% in width upon 24h–incubation with cyclohexane and restored their sizes by 8% and 33% respectively after 5 days. While the surface roughness (RMS) of viable cells increased by 31 nm at the first day and continued to increase by 46 nm after 5 days. Interestingly, dead cells were characterized by increased roughness after 24 h but their surfaces became equal to the control cells after 5 days. Average Young modules of 1.6 MPa were registered for control cells, while 1.3 MPa values were obtained for solvent-resistant cells, thus suggesting a slight decrease in cell elasticity. We suppose that an increasing cell roughness could result in higher contact area between cells and organic solvent, thus leading to enhanced solvent uptake and biotransformation. Solvent-resistant Rhodococcus strains could be used for the bioremediation of waste waters and biotechnological processes carried out in organic phases.

1.    de Carvalho CCCR, 2010. Microbiol. Monographs. 16,109-131
2.    Kuyukina MS, Ivshina IB, 2010. Microbiol. Monographs. 16,231-262
3.    Schmid A et al., 2001. Nature. 409,258-258

The work was supported by the Russian Federation President Program “Leading Scientific School” NS-4607.2014.4 grant and the grant of general committee of Russian Academy of Sciences “Molecular and Cell Biology”.

Fig. 1: Dynamic of cell viability and roughness during exposure to cyclohexane.

Fig. 2: AFM/CLSM image of live cells stained with SYTO 9 (green) and dead cells stained with propidium iodide (red) after exposure to cyclohexane.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2141 HSV-1 amplicon vectors mediate production of Rotavirus-like particles in transduced cells

Laimbacher A. S.1, Meier A. F.1, Schraner E. M.1,2, Tobler K.1, Fraefel C.1, Wild P.2, Ackermann M.1
1Institute of Virology, University of Zurich, Switzerland, 2Institute of Veterinary Anatomy, University of Zurich, Switzerland
andrea.laimbacher@uzh.ch

Rotaviruses (RVs) are important enteric pathogens of humans and animals causing severe enteritis that often leads to death, especially in the very young. The RV genome, segmented dsRNA, is encapsidated within a triple-layered virus particle, comprising four proteins: VP2 at its inner layer, VP6 forming the intermediate layer, and VP7 forming the outer layer. Finally, the VP7 layer is also interspersed by protruding VP4 spikes.

The details of RV particle formation are not completely understood and controversially discussed in the literature. In the present study, we used a minimalistic approach to generate Rotavirus-like particles (RVLP) by transduction of cell cultures with Herpes simplex virus type-1 (HSV-1) amplicon vectors. The helpervirus-free amplicon particles were engaged to deliver a DNA-cassette, encoding for a single, polycistronic mRNA, which contains the coding sequences of the three capsid proteins VP2, VP6, and VP7, separated by internal ribosome entry sites.

Transmission electron microscopy of ultrathin sections from cells fixed with glutaraldehyde and osmium tetrotxide revealed that viroplasm-like structures comprising numerous RVLPs were indeed formed within the cytoplasm of amplicon vector-transduced cells (Fig. 1). Moreover, RVLPs were isolated, purified and imaged after negative staining (Fig. 2) and immunogold labeling (Fig. 3), which confirmed the VLP's identity as RV-like.

However, Western immunoblot analysis and auxiliary immunogold labeling of purified RVLPs indicated that the observed VLPs consisted predominantly of the two inner layers VP2 and VP6, whereas VP7 was abundantly present in transduced cells, yet, hardly incorporated into the VLPs. We hypothesize that VP7 needs the assistance of further RV proteins in order to complete the third capsid layer. Most likely, VP4 and NSP4 are involved in this process. Therefore, their functions will be evaluated in future work that is aimed to define the minimal requirements for triple-layered RVLP formation.

The authors thank Didier Poncet, CNRS/INRA, Gif-sur-Yvette, France, for providing the anti-rotavirus polyclonal serum

Fig. 1: Transmission electron microscopy of ultrathin sections of cells harvested from monolayers by pelleting and fixation with glutaraldehyde and osmium tetrotxide. RVLPs assembled in viroplasm-like structures within the cytoplasm of transduced cells. Scale bar = 100 nm.

Fig. 2: Negative staining (phosphotungstic acid) of RVLPs produced with the helpervirus-free HSV-1 amplicon vector system. Two days after transduction, RVLPs were purified over a sucrose cushion. Scale bar = 100 nm.

Fig. 3: Immunogold staining of the same sample of RVLPs as in figure 2 using a polyclonal anti-rotavirus serum and a secondary antibody coupled to 12 nm colloidal gold. Scale bar = 100 nm.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2329 Characterising the effect of top domain modifications on the self-assembly and particle formation of major core protein VP7 of African horse sickness virus

Wall G. V.1, van der Merwe C. F.2, Hall A. N.2, Huismans H.1, van Staden V.1
1Department of Genetics, University of Pretoria, Pretoria, 0002, South Africa, 2Laboratory for Microscopy and Microanalysis, University of Pretoria, Pretoria, 0002, South Africa
gaylevwall@gmail.com

African horse sickness virus (AHSV) causes an often fatal disease in horses, of major economic impact in South Africa. The AHSV virion has a double-layered protein capsid, composed of a diffuse outer layer housing an icosahedral core. VP7 is the major surface protein of the core particle and forms trimers that in addition to being incorporated into the core, can also self-assemble into flat, hexagonal crystalline-like particles in infected cells. This is unique to AHSV VP7, which in contrast to the VP7 of related viruses, is highly hydrophobic and insoluble. To construct tools for the study of AHSV VP7 assembly and transport, six amino acids were inserted downstream of residues 144, 177 and 200 of the AHSV VP7 top domain, thereby yielding VP7 vector proteins, into which the marker protein eGFP was subsequently inserted and expressed from recombinant baculoviruses. These modifications are known to affect the solubility and trimerisation of the protein. A significant amount of misfolded and non-fluorescing versions of the VP7-177-eGFP and VP7-200-eGFP fusion proteins has also been documented. We aimed to investigate the effect of these top domain modifications on the self-assembly and particle formation of AHSV VP7, and to determine the fate of misfolded and non-fluorescing VP7-eGFP fusion proteins in the cell. Confocal and transmission electron microscopy (TEM) showed that wild-type (WT) VP7 self-assembled into large, flat hexagonal or rod-shaped crystalline-like particles (Fig. 1) resembling those in AHSV infected cells. VP7-144 formed small and loosely associated spindle-like structures (Fig. 2A, B). Both VP7-177 and VP7-200 formed particles resembling WT VP7, but the stable layering was affected to some extent resulting in rosette-like structures (Fig. 2C, D). VP7-144-eGFP formed small protein aggregates that associated to form large, irregularly-shaped structures or foci of fluorescence (Fig. 3A, B). VP7-177-eGFP assembled to smaller spherical foci, whereas VP7-200-eGFP assembled to small disc-shaped foci that appeared rod-like when viewed at a 90˚ angle (Fig. 3C, D). Thus, self-assembly to single or multiple site(s) was observed in the case of all constructs studied. Particle formation however was affected by these modifications, with all proteins differing with regard to the nature of the protein aggregates or particles formed at the sites of self-assembly. No differential distribution of misfolded VP7-eGFP proteins was detected, as in all cases red primary antibody signal colocalised exclusively with green auto-fluorescence (Fig. 4). This study indicates that AHSV VP7 self-assembly and particle formation are separate events, and that charge and solubility may have an important effect on the nature of the AHSV VP7 particle.

We thank Mr Flip Wege for technical assistance, and the National Research Foundation, Poliomyelitis Research Foundation, Technology Innovation Agency and MSSA Trust for funding.

Fig. 1: Confocal immunofluorescence (A, B, C) and TEM (D) images of wild-type (WT) AHSV VP7 flat, hexagonal or rod-like crystalline-like particles (depending on the angle at which the particles are viewed) within the cytoplasm of Sf9 cells. Immunolabelling was done using a VP7-specific primary antibody.

Fig. 2: Confocal immunofluorescence (A, C) and TEM (B, D) images of spindle-like VP7-144 protein aggregates (A, B) and rosette-like VP7-177 structures, composed of rigid rod-like particles (C, D) within the cytoplasm of Sf9 insect cells. Immunolabelling was done using a VP7-specific primary antibody.

Fig. 3: Confocal immunofluorescence (A, C) and TEM (B, D) images of the single large foci (A, B) formed by VP7-144-eGFP, and the disc-shaped foci formed by VP7-200-eGFP (C) that appear rod-like at a 90˚ angle (D). VP7-eGFP auto-fluoresces green and DAPI-stained nuclei are blue. Immunogold labelling was done using anti-VP7.

Fig. 4: Confocal images of Sf9 cells expressing VP7-144-eGFP and labelled with anti-VP7. VP7-eGFP auto-fluorescence green (A), anti-VP7 primary antibody was detected with a red secondary antibody (B), DAPI-stained nuclei (C), merged images (D). Green auto-fluorescence colocalised with red primary antibody signal with no differential red staining observed.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2204 Interactions of Mouse polyomavirus structural proteins with host cell structures

Hornikova L.1, Fraiberk M.1, Forstova J.1
1Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic
lenka.hornikova@atlas.cz

Mouse polyomavirus (MPyV) is a small non-enveloped double-stranded DNA virus. Virions of MPyV are composed of three structural proteins – the major protein, VP1, and two minor structural proteins, VP2 and VP3. Seventy-two VP1 pentamers create viral capsid and each pentamer is associated with one molecule of either VP2 or VP3. A great deal is known about functions of the Mouse polyomavirus capsid proteins in the virion structure as well as in early stages of virus infection. However, interactions of these proteins with cellular proteins and structures in late stages of infection are not well characterised.
We studied properties of VP1 after its individual expression and also after co-expression with VP2 and VP3 in mammalian cells using confocal and electron microscopy. Although VP1 possess nuclear localization signal, it was localised preferentially in the cytoplasm. In situ fractionation of transfected cells revealed that VP1 protein formed fibres in the cytoplasm. These fibres were sensitive to nocodazol, drug destabilizing microtubules. Moreover, in a comparison with control cells, tubulin was retained in insoluble fraction in cells transiently expressing VP1. Immunoelectron microscopy of thin sections of cells after in situ fractionation showed co-localization of VP1 and tubulin cytoskeleton. Co-expression of VP1 with VP2 or VP3 led to nuclear localisation of structural proteins. Two phenotypes of VP1 staining were observed – difuse localization and spot-like phenotype. Nuclear lamina staining (with an antibody against lamin A/C or lamin B) was disturbed in infected cells and in cells co-expressing VP1/2 and VP1/3. A possible meaning of these interactions in viral life cycle context will be discussed.

This work was supported by the European Social Fund and the state budget of the Czech Republic, project no. CZ.1.07/2.3.00/30.0061, by "BIOCEV - Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University"(CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund and by TACR - project no. 2013TA03010700.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2494 AFM identification of single biomolecules using gold and carbonic nanosized immunolabels

Nikiyan H. N.1, Davydova O. K.1, Tatlybaeva E. B.1
1Orenburg State University, Orenburg, Russian Federation
nikiyan@yahoo.com

The problem of biological molecules identification is of current importance in microbiology. It has been traditionally solved by means of antibodies, containing various labels, such as enzymes, radioactive isotopes, fluorogenic or electrochemiluminescent tags. However in most cases appropriate methods are focused on identification of a significant amount of interacting molecules and don't provide insight into their spatial distribution. One of possible solutions of the problem is to use atomic force microscopy (AFM) method. AFM is a relatively new technique that has provided novel opportunities for the surface analysis of biological specimens with nanoscale resolution and minimal effect on the sample structure. The specified possibilities of AFM allow to develop highly sensitive methods for single molecule detection that opens wide prospects for the analysis of immune and substrate specific activity.
The aim of the work was the development of an AFM method for single antigen molecules identification using gold and carbonic labels. Direct visualization and quantitative evaluation of morphometric characteristics of the antigen-antibody complexes was used as a criterion for the detection.
Protein A conjugated with colloidal gold (PrA+Au) and protein G conjugated with amorphous carbon (PrG+C) were used for labeling antibodies, being bond to Rubella virus antigens onto polystyrene microtiter ELISA plates. Images were collected by using an SMM-2000 atomic force microscope (JSC "Proton-MIET Plant", Russia) operated in contact mode.
AFM-images of the antigen, antigen-antibody complexes (Ag-At); and also specific complexes Ag-At-PrA+Au and Ag-At-PrG+C were obtained step by step during the study (see Figure 1 and 2). It was shown that except specific complexes detection, the atomic force microscope allows to describe quantitatively their distribution on microtiter plate surface. Besides, the sensitivity of the method for each tag was estimated and compared to ELISA method. The obtained results indicated high sensitivity of an offered approach and certain advantages of carbonic tags use in comparison with gold tags, because of their easier detection and unambiguous identification on the received images.
Thus, offered approach in the long term perspectives will allow to solve a problem of specific marking of single molecules and microorganisms in complex multicomponent associations.

This work was supported by The Ministry of Education and Science of the Russian Federation inthe framework of base part of government contracts (Project №148) and the Russian Foundation for Basic Research (Grant 13-04-97054).

Fig. 1: Antigen-antibody complexes labeled by colloidal gold. Scale bar is 500 nm.

Fig. 2: Antigen-antibody complexes labeled by amorphous carbon. Scale bar is 500 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2223 An investigation into the association of African horse sickness virus protein VP7 with host trafficking pathways and the role of trimer-trimer interactions in VP7 crystalline particle formation

Bekker S. M.1, Huismans H.1, van Staden V.1
1Department of Genetics, University of Pretoria, Pretoria 0002, South Africa
shani.bekker@gmail.com

African horse sickness (AHS) is a highly infectious and deadly vector-borne disease of Equidae with a mortality rate of up to 90% in susceptible horses. The etiological agent for AHS is an orbivirus from the Reoviridae family known as African horse sickness virus (AHSV). The AHSV virion is composed of two protein layers that are organized into an outer capsid and an icosahedral core particle made up of major core protein VP7 and subcore protein VP3. A unique characteristic of AHSV VP7 is that it is highly insoluble and, unlike any of the cognate orbivirus proteins, forms large flat crystals in AHSV-infected cells. The impact of the formation of these crystals or their role in AHSV replication remains to be discovered. The aim of this study was to investigate the process of AHSV VP7 crystal formation by immunofluorescence microscopy. We first characterized the localization of AHSV VP7 in different systems and studied the association of VP7 crystals with virus factories during infection. Co-localization data revealed that only a small amount of VP7 was associated with virus factories, while the majority of VP7 was sequestered into crystals (Fig. 1A). This is likely to have a negative impact on virus assembly. Next, we set out to investigate whether VP7 crystal formation resulted from interaction with host trafficking pathways or, alternatively, whether the crystals were a product of VP7 self-assembly. We investigated the co-localization of a VP7-eGFP fusion protein (Fig. 1B and C) with components of three host cellular trafficking pathways, i.e. the cytoskeleton, the aggresomal pathway, and protein degradation pathways. We then selectively blocked each of these pathways by studying the effect of chemical pathway inhibitors on VP7 distribution. We found that VP7 forms crystals in a host-independent manner and manages to evade host defences against protein aggregation (Fig. 2, 3, and 4). These results implied that the unique ability of VP7 to form crystals was driven by VP7 self-assembly. During core assembly, VP7 assembles into trimers that form a lattice that surrounds the inner VP3 subcore. We therefore suggest that VP7 crystal formation may be driven by trimer-trimer interactions. We set out to abolish VP7 self-assembly by targeting key residues that drive VP7 trimer-trimer interactions. Upon examination of the intracellular distribution of modified VP7 proteins, we found that the disruption of trimer-trimer interactions successfully abolished the formation of VP7 crystals thus proving that VP7 self-assembly drives crystal formation. Investigating the role of trimer-trimer interactions in core assembly will provide further insight into the formation of crystals as well as their role in AHSV replication.

This work was financially supported by BioPad, PRF, NRF, MSSA Trust and University of Pretoria. We thank Flip Wege for technical support with cell culture and both Alan Hall (University of Pretoria) and Sone Ungerer for technical support with confocal microscopy.

Fig. 1: Distribution of VP7 within AHSV- and recombinant baculovirus infected cells by confocal microscopy. AHSV-infected BSR cells (A), Sf9 cells expressing wild-type VP7 (B) and VP7-144-eGFP (C) with 3D stacking image of VP7 crystal on the right. Wild-type VP7 was detected with anti-VP7 (green) and VP7-144-eGFP was detected by eGFP auto-fluorescence.

Fig. 2: VP7 is not associated with microtubules in mammalian cells. Mock cells and cells expressing VP7-144-eGFP (green) were labelled with antibody against tubulin (red). Cells were untreated (top panel) or treated with colchicine, a microtubule depolymerizing drug (bottom panel). Nuclei were stained with DAPI. The same results were seen in insect cells.

Fig. 3: VP7 is not associated with the 26S proteasome in insect cells. Mock cells and cells expressing VP7-144-eGFP (green) were labelled with antibody against the 26S proteasome (red). Cells were untreated (top panel) or treated with proteasome inhibitor MG132 (bottom panel). Nuclei were stained with DAPI. The same results were seen in mammalian cells.

Fig. 4: VP7 is not ubiquinated or associated with the lysosome in insect cells. Mock cells and cells expressing VP7-144-eGFP (green) were labelled with antibody against ubiquitin (A) or incubated in the presence of LysoTracker Red Dye (Invitrogen) (B) (red). Nuclei were stained with DAPI. The same results were seen in mammalian cells.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2271 Programmed cell death as a mechanism for controlling bacterial communities in aquatic ecosystems

Silva T. P.1, Gamalier J. P.1, Zarantonello V.1, Roland F.2, Melo R. C.1
1Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil, 2Laboratory of Aquatic Ecology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil
silva.tp@outlook.com

Bacterial death is an important event associated with carbon and nutrient cycles in aquatic ecosystems [1]. Although programmed cell death (PCD) is a regulated process largely known in eukaryotic organisms [2], it is still poorly understood in aquatic bacteria. In this study, we investigated the occurrence of PCD in aquatic bacteria collected from an impacted ecosystem (Batata Lake) located in Northern Brazilian Amazon. This ecosystem was drastically impacted by bauxite tailings and it is presently divided in an impacted area and a natural area. Water samples collected from the subsurface of this lake (n= 6) were studied by fluorescence microscopy using different probes for analysis of cell density (DAPI) and bacterial viability (LIVE/DEAD BacLight). DNA fragmentation was assessed by flow cytometry using Tunel assay, a marker for identifying PCD [2,3]. In parallel, samples were processed for transmission electron microscopy (TEM) [4] to evaluate bacteria alterations. A higher density of bacteria was found in the natural area of Batata Lake (p<0,05) than in the impacted area. Our cell viability results enabled direct visualization of live and dead bacteria and revealed a higher proportion of bacterial death in the impacted area compared to the natural area (p<0,01). DNA fragmentation analysis (TUNEL assay) showed that PCD is a phenomenon occurring in bacteria in this lake, with higher frequency in the impacted area (15,52%, p<0,05). TEM revealed typical ultrastructural changes indicative of apoptosis in bacteria from both areas, such as cell retraction, cytoplasmic condensation and non-disrupted degenerating cells. Quantitative EM analysis showed that 47,14% of aquatic bacteria in the impacted area exhibited signs of apoptosis. Altogether, our data demonstrate, for the first time, that PCD occurs in aquatic bacteria from tropical ecosystems and that this event may be an important mechanism for controlling bacterial communities in aquatic ecosystems.

References

[1] L.R. Pomeroy et al, Oceanography, 20 (2007) 28-33
[2] L. Galluzzi et al, Cell Death and Differentiation, 19 (2012) 107-202
[3] D.J. Dwyer et al, Molecular Cell, 46 (2012) 561-572
[4] T.P. Silva et al, Antonie van Leeuwenhoek, 105 (2014) 1-14

This work was supported by FAPEMIG, CNPq and Furnas Centrais Elétricas S.A. (Brazil).

Fig. 1: Quantification of aquatic bacteria in a tropical ecosystem (Batata Lake, Brazil).(A) Bacteria density evaluated after staining with DAPI. (B) Live/dead bacteria were clearly observed as green (arrowhead) or red (arrow) structures. In (C), the percentage of live/dead cells are shown. (*) p< 0.001.Scale bar 10µm.

Fig. 2: Bacterial DNA fragmentation analysis by flow citometry. (A, B) Representative histograms of DNA fragmentation in bacteria collected from impacted and natural areas and prepared for TUNEL assay. In (C), the percentages of cells exhibiting DNA fragmentation are shown. (*) p< 0.001.

Fig. 3: Transmission electron microscopy of aquatic bacteria reveals apoptosis-like alterations. (A) Bacterium with typical ultrastructure. In (B, C), bacteria show cytoplasmic condensation (arrows), grouped granules (G) and changes in electron-density. Two empty bacteria are indicated (*). Cellular debris are observed (C, arrowhead). Scale bar 150nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2292 The von Willebrand Factor type A-like domains of collagen type VI exhibit antimicrobial activity

Abdillahi S. M.1, Maaß T.2, Kasetty G.3, Walse B.4, Schmidtchen A.5, Wagener R.2, Mörgelin M.1
1Department of Clinical Sciences, Division of Infection Medicine, Lund University, Lund, Sweden, 2Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany, 3Department of Clinical Sciences, Sector for Respiratory Medicine & Allergology, Lund University, Lund, Sweden, 4SARomics AB, Lund, Sweden, 5Department of Clinical Sciences, Division of Dermatology and Venereology, Lund University, Lund, Sweden
suado.m_abdillahi@med.lu.se

Collagen type VI is a ubiquitous extracellular matrix component that forms complex and extensive microfibrillar networks in all connective tissues, often associated with basement membranes. Structurally, it consists of three α-chains (α1, α2 and α3), where each α-chain contains a short triple helix and N- and C-terminal globular regions. More recently, three additional chains (α4, α5 and α6) were discovered, which may substitute for α3 in some tissues. The globular regions are homologous to the type A domains of von Willebrand factor (vWF-A). We have recently described the antimicrobial properties of this collagen against a number of Gram-positive and Gram-negative human pathogens. However, the molecular mechanisms of bacterial killing are still elusive. Therefore, in this study, we applied an in silico approach to unravel the antimicrobial activity of collagen type VI in further detail. Sequence and structural analysis showed that the vWF-A domains of all three α-chains contain numerous amphipathic amino acid motifs of putative antimicrobial nature. In addition, we also could show that recombinantly expressed vWF-A domains bind to negatively charged surfaces such as heparin and bacterial membranes. Five such motifs were finally chosen from N8, N9 and C1 domains of the α3- chain for further characterization in bacterial killing assays. The data suggest that amphipathic, heparin-binding amino acid motifs in the globular vWF A-like domains harbour the antimicrobial properties of collagen VI.

The authors gratefully acknowledge the skilful work of Maria Baumgarten. We wish to thank the Core Facility for Integrated Microscopy (CFIM), Panum Institute, University of Copenhagen, for providing an excellent electron microscopy platform/environment.

Fig. 1: S. pyogenes (AP1) were treated with α1, α2 or α3 chain of collagen VI and permeabilization was visualized by using scanning electron microscopy. Extensive membrane disruption and leakage of intracellular contents are observed in the presence of these proteins and are indicated with arrowheads. Bar represents 5 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2336 Assembly of Mouse polyomavirus virions is accompanied by disruption of PML nuclear bodies

Zila V.1, Ryabchenko B.1, Abrahamyan L.1, Forstova J.1
1Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague
vojzilla@gmail.com

Promyelocytic leukemia nuclear bodies (PML-NBs) play an important role in the intrinsic cellular response to viral infection due to their ability to interact with foreign DNA that enters the nucleus. It was suggested that polyomaviruses are able to exploit PML-NBs as their replications sites; however, the mechanism by which these viruses overcome antiviral activities of PML-NBs remains obscure. Here, we focused on the late stages of Mouse polyomavirus (MPyV) infection in mouse embryonic fibroblasts (MEFs) and analyzed the association of viral structures with PML-NBs and their morphological changes during late stages of virus progression. Confocal microscopy and in situ hybridization in cells at the late times (24 – 44 hours) post-infection revealed an accumulation of large T-antigen, viral DNA and the major capsid protein, VP1, in foci adjacent to the PML-NBs, suggesting that replication and assembly of MPyV are associated with the PML-NBs. As large amounts of viral DNA and newly synthesized VP1 protein appeared in the nuclei of infected cells at late post-infection, we started to observe an enlargement of PML-NBs and their shape alterations. To examine these events at the ultrastructural level, we performed immunoelectron microscopy of cells fixed at 36 hours post-infection. As the MPyV infection proceeds rapidly and asynchronously, we were able to observe progressive stages of virus infection at late stages in individual samples. In the nuclei of cells, where MPyV assembly started, we observed small clusters of newly formed virions adjacent to still compact PML-NBs (Fig. 1A), confirming that initiation of viral assembly centers is associated with PML-NBs. However, further examination of cells, where MPyV morphogenesis was more advanced, revealed large viral assembly factories formed around PML-NBs, whose size and integrity became apparently altered (Fig. 1B), or completely disrupted PML-NBs. Interestingly, virus progeny was readily observed in the remnants of disrupted PML-NBs (Fig. 1C, D). It is important to note here that the overall morphology of cells at 36 hours post-infection was still well preserved. These preliminary results indicate that MPyV progeny formation has a strong effect on PML-NBs integrity, and might reflect the result of modulation of PML-NBs antiviral activities by MPyV infection.

This research was supported by the Ministry of Education, Youth and Sports of the Czech Republic (Project SVV-2014-260081) and by the project BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund.

Fig. 1: PML-NBs are disrupted during assembly of MPyV. The ultrathin sections of MEF cells fixed at 36 h post-infection (A-D) or non-infected cells (E and F) and embedded in LR-White resin were immunolabeled with anti-PML antibody, followed by incubation with secondary antibody conjugated to 10 nm gold particles. Arrowheads point to selected virions.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2462 Can transmission electron microscopy help in study of bacteria-phage interaction?

Tusek Znidaric M.1, Naglic T.1,2, Ravnikar M.1,2, Peterka M.2, Dreo T.1,2
1National Institute of Biology, Večna pot 111, SI 1000 Ljubljana, Slovenia, 2Centre of Excellence for Biosensors, Instrumentation and Process Control, Velika pot 22, SI-5250 Solkan, Slovena
magda.tusek.znidaric@nib.si

Orchids are very popular ornamental plants and some bacterial diseases can cause large economic losses at orchid nurseries. Soft rot of orchids caused by Dickeya spp. (ex. Erwinia chrysantemi, Enterobacteraceae) is such a bacterial disease with symptoms of watery spots and rapidly rotting leaves. Since no effective chemical protection exists and elimination of diseased plants has limited efficiency, plant protection with specific bacteriophages represents an alternative disease management strategy. The aim of the study was to isolate of target bacteria and specific bacteriophages against them and to determine bacteria and phage morphology.

Pathogenic bacteria were isolated from diseased Phallenopsis orchids and were identified as Dickeya spp. by classical microbial methods, real-time PCR assay and sequencing of fliC gene (Dreo et al., 2012). During phage isolation (Naglič et al., 2013) the morphology of present particles was checked by transmission electron microscopy using negative staining method. Host-pathogen interaction and confirmation of bacteriophage efficiency were made by plaque assay method and with mixing suspension of bacteria and suspension of phage isolate; ultrathin sections were prepared with fixed and embedded pieces of plaque border and pellet of mixed suspension obtained with centrifugation. Sections were examined with transmission electron microscopy (Philips CM100) and visualized with CCD cameras (BioScan 792 and ORIUS SC 200, Gatan).

Based on morphology (polihedral shape of phage head and short tail), the plaque forming bacteriophage (Fig. 1A) supposed to belong to the family Podoviridae which was later confirmed with molecular methods. Figure 2 shows ultrathin sections of plaque forming phages; pieces from plaque border demonstrated the synthesis of new phages and lysis of bacterial cells.

Further research is focused on confirming bacteriophages efficiency in vivo and possible application of bacterophages in plant protection. In such complex research, combination of different methods are desired and transmission electron microscopy was shown to enable to prove the presence of phage particles and help in determination of phage taxonomy. It is also of great importance to follow events in host-pathogen interaction using electron microscopy because it is the only method to visualize whole phage particles.

References:

Dreo et al., 2013, 12th Symposium on Bacterial Genetics and Ecology, 9-13 June 2013, Ljubljana, Slovenia. Conventus Congressmanagement & Marketing, pp. 139.

Naglič et al., 2013, Slovenian Conference on Plant Protection with International Participation, Bled, 5.-6.3-2013, pp. 273-277

The study was financially supported by P4-0165 – Biotechnology and system biology of plants, and COBIK, the Centre of Excellence for Biosensors, Istrumentation and Process Control.

Fig. 1: A. Negative staining of isolated bacterophage (Podoviridae); B. Suspension of bacteria Dickeya spp. and bacteriophages (Podoviridae).

Fig. 2: A. Control bacteria. B. Bacteria from border part of plaque with bacterial residues (arrows); C. Bacteria with phages attached on cell wall (arrows); D. Bacteria with new phages inside the cell (arrows).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2579 Plasmodium falciparum invasion of human erythrocyte, a multi-tools story.

Hanssen E.1, Dekiwadia C.1, Baum J.2, Ralph S.1
1The University of Melbourne, 2Walter and Eliza Hall Institute
ehanssen@unimelb.edu.au

Erythrocyte invasion by merozoites forms of the malaria parasite is a key step in the establishmentof human malaria disease. To date, efforts to understand cellular events underpinning entry have been limited to insights from non-human parasites, with no studies at sub-micrometer resolution undertaken using the most virulent human malaria parasite, Plasmodium falciparum. This leaves our understanding of the dynamics of merozoite sub-cellular compartments during infection incomplete, in particular that of the secretory organelles. Using advances in Plasmodium falciparum merozoite isolation and new imaging techniques we present a three-dimensional study of invasion using electron microscopy, cryo-electron tomography and cryo-X-ray tomography. This study has enabled us to shade some light on some of the controversies concerning the architecture of the malaria parasite. We describe the core architectural features of invasion and identify fusion between rhoptries at the commencement of invasion as a hitherto overlooked event that likely provides a critical step that initiates entry. Given the centrality of merozoite organelle proteins to vaccine development, these insights provide a mechanistic framework to understand therapeutic strategies targeted towards the cellular events of invasion.

Moreover the range of techniques presented gives insights into the application of electron microscopy and fixation methods and provides a framework to on the best technical choice for different applications

The authors thank Carolyn Larabell, and Mark A. Le Gros (University of California, San Francisco) and Christian Knoechel (Lawrence Berkeley National Laboratory) for assistance and advice with X-ray tomography.

Fig. 1: Three-dimensional representation of a merozoite highlighting the apical organelles. The nucleus is depicted in red, the mitochondrion and apicoplast in Blue and yellow, the three apical rings are shown in purple, rhoptries in light blue and micronemes and dense granules (not differentiated here) are shown in multiple colours. Scale bar 200 nm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2708 Correlative Microscopy of a Metal Accumulating Biofilm

Steinacher R.1, Adlassnig W.1, Sassmann S.1, Reipert S.1, Puschenreiter M.2, Lang I.1, Lichtscheidl I. K.1
1University of Vienna, Core Facility Cell Imaging and Ultrastructure Research, 2University of Natural Resources and Life Sciences, Institute of Soil Research
wolfram.adlassnig@univie.ac.at

Biofilms are of crucial importance for the remediation of contaminated waters. Cyanobacteria of the genus Phormidium form dense mats in alpine creeks. The bacterial filaments are covered by a gelatinous sheath that adsorbs a variety of elements. In a copper contaminated creek in the Austrian Alps, an extensive, Phormidium dominated biofilm accumulates 3.9 ± 1.8% copper, thereby completely remediating the water of the creek by immobilising the copper. This study investigates the structure and ultrastructure of the biofilm with special regard to the localisation and speciation of the copper. The biofilm was chemically fixed or plunge frozen; further preparation included cryo-substition with ultrathin sectioning as well as freeze drying, conventional sectioning and analysis of whole mounts. Microscopic techniques comprised confocal microscopy, polarised and phase contrast light microscopy, X-ray microanalysis as well as scanning and transmission electron microscopy. The biofilm exhibits an extraordinary thickness of up to 22 cm with only the top layer containing living cells. It consists almost exclusively of filamentous Phormidium growing in clearly distinct layers (Fig. 1). Other bacteria like Bacillus are restricted to the surface of the biofilm. The copper is not evenly distributed in the biofilm but occurs as distinct crystals, probably consisting of the secondary copper mineral Sampleite [NaCaCu5(PO4)4 ∙ H2O] with a diameter of about 10 µm between the bacterial filaments (Fig. 2). Furthermore, copper is also found in the sheaths of the bacteria. Here, transmission electron microscopy suggests that this copper is not simply adsorbed but occurs in abundant submicroscopic electron-dense particles at the surface of the sheaths. These results show that the process of copper immobilisation by Phormidum biofilms is far more complex than simple passive adsorption and includes processes of biomineralisation. The speciation of the copper as mineral particles within the biofilm indicates that the immobilisation of metals is permanent which is confirmed by analysis of old, subfossile layers of the biofilm. These findings do not only enlighten the mechanism of metal immobilisation by Phormidium but also encourage its use for bioremediation of mine waste waters.

Thanks are due to K. Hallbeck and to the ŒAD/Appear project BioRem (Appear 43).

Fig. 1: Cross section of the biofilm after Dapi staining. The filamentous Cyanobacteria (yellow-white) form several clearly distinct layers. Non-photosynthetic microorganisms (blue) are restricted to the surface (CLSM).

Fig. 2: Between the Cyanobacteria, globular, copper rich mineral particles are found (SEM).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2753 Application Perspectives of Localization Microscopy in Virology

Cremer C.1,2,3, Kaufmann R.4,5, Gunkel M.3,6, Polanski F.2, Mueller P.3, Dierkes R.7, Degenhard S.8, Wege C.8, Hausmann M.3, Birk U.1,3
1Institute of Molecular Biology (IMB), D-55128 Mainz, Germany, 2Institute of Pharmacy and Molecular Biotechnology (IPMB), University Heidelberg, D-69120 Heidelberg, Germany, 3Kirchhoff Institute for Physics (KIP), University Heidelberg, D-69120 Heidelberg, Germany, 4Division of Structural Biology, Wellcome Trust Centre for Human Genetics, Oxford, United Kingdom, 5Department of Biochemistry, University of Oxford, Oxford, United Kingdom, 6BioQuant Center, University Heidelberg, D-69120 Heidelberg, 7Institute of Molecular Virology, Westfälische Wilhelms-Universität Münster, D- 48149 Münster, Germany, 8Institute of Biology, University of Stuttgart, D-70550 Stuttgart, Germany
c.cremer@imb-mainz.de

Localization Microscopy approaches allowing an optical resolution down to the single molecule level in fluorescence labeled biostructures have already found a variety of applications in cell biology, as well as in virology. Here we focus on some perspectives of a special localization microscopy embodiment, Spectral Precision Distance/Position Determination Microscopy (SPDM)1,2, most advantageous for the analysis of both viral pathogens as well as virus-derived nanotools. SPDM permits the use of conventional fluorophores or fluorescent proteins together with standard sample preparation conditions employing an aqueous buffered milieu, and monochromatic excitation. Thereby, SPDM allowed super-resolution imaging and studies on the aggregation state of modified tobacco mosaic virus (TMV) particles on the nanoscale with an accuracy of better than 8 nm, using standard fluorescent dyes in the visible spectrum. To gain an improved understanding of cell entry mechanisms during influenza A virus (IAV) infection, SPDM was used in conjunction with algorithms for distance and cluster analyses to study changes of the distribution of virus particles themselves or of the distribution of infection-related proteins, the hepatocyte growth factor receptors (HGFR), in the cell membrane on the single molecule level. Not requiring TIRF (total internal reflection) illumination, SPDM was also applied to study the molecular arrangement of gp36.5/m164 glycoprotein (essentially associated with murine cytomegalovirus infection) in the endoplasmic reticulum inside cells with single molecule resolution. On the basis of the experimental evidence so far obtained, we finally discuss additional application perspectives of localization microscopy approaches for the fast detection and identification of viruses by multi-color SPDM and combinatorial oligo fluorescence in situ hybridization (COMBO-FISH), as well as SPDM techniques for optimization of virus-based nanotools and biodetection devices.

1Cremer C et al. (1999) Principles of Spectral Precision Distance confocal microscopy for the analysis of molecular nuclear structure. In: Handbook of Computer Vision and Applications (ed. B. Jähne et al.), Ch. 41, Vol. 3, Academic Press San Diego, New York: 839-857

2Cremer C et al., Application Perspectives of Localization Microscopy in Virology, Histochem. Cell Biol., DOI 10.1007/s00418-014-1203-4

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2771 Interaction of Tick-borne encephalitis virus with human neural cells

Bílý T.1,2, Vancová M.1,2, Palus M.1,2,3, Růžek D.1,2,3,4, Nebesářová J.1,5, Grubhoffer L.1,2
1Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, CZ-37005 České Budějovice, Czech Republic, 2Faculty of Science, University of South Bohemia, Branišovská 31, CZ-37005, České Budějovice, Czech Republic, 3Department of Virology, Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic, 4Faculty of Science, Masaryk University, Kotlářská 2, CZ-60200 Brno, Czech Republic, 5Faculty of Science, Charles University in Prague, Viničná 7, CZ-12843 Praha, Czech Republic
thomass@paru.cas.cz

Tick-borne encephalitis virus (TBEV) causes serious infections of the central nervous system of humans. There are more than 10,000 cases of tick-borne encephalitis reported in Europe and Asia every year. TBEV is a representative of Flavivirus genus within the Flaviviridae family. The genome consists from single-stranded positive sense RNA [1]. Nucleocapsis is approximately 50 nm in outer diameter and is surrounded with lipid envelope.


The production of virions is associated with dramatic alterations of the endoplasmic reticulum and formation of special compartments, called microenvironments or replication factories. Typical flavivirus-induced structures are convoluted membranes and induced vesicles [2, 3, 4]. Neural cells represent the main target for TBEV. In our study, primary neurons and astrocytes were infected with TBEV (strain Neudoerfl) and the morphological changes in the infected cells were investigated by electron tomography. The samples were prepared by high pressure freezing and freeze substitution method. Single axis electron tomography was done over a tilt from range -65 to 65 with 0.65 degree step (JEOL 2100F equipped with high tilt stage and Gatan camera Orius SC 1000) by means of Serial EM software [5]. Tomograms were aligned, reconstructed and 3D models were generated by manually masking the area of interest using IMOD software package [6].


Interestingly, we observed unique tubule-like structures in the endoplasmic reticulum of the infected cells. The 3D reconstructions revealed their detailed organization. The diameter of tubule-like structures observed in TBEV-infected astrocytes and neurons was different (22.0 nm ± 1.3 nm,n = 51).


Tubule-like structures have origin in viral activity it was confirmed by examination of non-infected cells.

1. B.D. Lindenbach, C.M. Rice, Virology, 4th ed. (2001), Philadelphia, New York, pp. 991-1042

2. L Mirion et. Al, Virol (2013), 87:11 6469-6481.

3. S. Welch et. Al, Cell Host & Microbe 5 (2009), 365-375

4. D.K. Offerdahl, D.W. Dorward, B.T. Hansen, M.E. Bloom, PLoS ONE 7 (2012), Issue 10, 1-14

5. D.N. Mastronarde, J. Struct. Biol. (2005), 152:36-51

6. J.R. Kremer, D.N. Mastronarde, J.R. McIntosh, J. Struct. Biol. (1996), 116:71-6

This work was supported by the ASCR (Z60220518, P302/12/2490), CSF project P502/11/2116 and P302/12/2490, AdmireVet project CZ.1.05./2.1.00/01.006 (ED006/01/01), and TAČR (TE 01020118).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2841 Cryo-electron tomography of the novel Pseudomonas “STACK” structure on vitreous sections and whole plunge-frozen bacteria

Delgado L.1, Martinez G.1, Mercade E.2, Lopez-Iglesias C.1
1Cryo-Electron Microscopy, Scientific and Technological Centers of the University of Barcelona, Spain., 2Laboratory of Microbiology, Faculty of Pharmacy of the University of Barcelona, Spain.
clopeziglesias@ub.edu

Cryo-electron tomography of vitrified specimens has enabled the study of cells and their constituents in their natural hydrated state, facilitating the validation and furthering our knowledge of prokaryotic structures. On the one hand, Cryo-electron tomography of vitreous sections (CETOVIS) provides the means to study the structure of cells at high resolution disclosing molecular details. On the other hand, Cryo-electron tomography of plunge-frozen bacteria can produce images of cellular assemblies in the context of the whole cell, enabling the analysis of the spatial distribution of entire structures throughout the bacteria. Both techniques can be used complementarily in order to combine the advantages of each one in the 3D-study of cells.
In our previous work, we described a new bacterial structure, which we named “STACKS”, in the Antarctic bacteria Pseudomonas deceptionensis M1T through electron tomography after freeze-substitution and cryo-electron microscopy of vitreous sections (CEMOVIS) (Delgado et al., 2013). Now, we have used cryo-electron tomography of vitreous sections and whole plunge-frozen bacteria to reveal the 3D-structure of the “STACKS” recently described. These techniques confirmed these structures as grouped discs surrounded by a lipid bilayer membrane, usually arranged perpendicularly to the cell membrane and found in variable number and in different locations within the cell. No connection among these new structures and the plasma membrane has been observed in any of the experiments performed, and significant differences have been found between the thickness of the plasma membrane and the layer surrounding the discs, suggesting that the “STACKS” are not invaginations of the plasma membrane.
Delgado, L., Carrión, O., Martínez, G., López-Iglesias, C., Mercadé, E., 2013.  PloS One 8, e73297.


↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-2897 Comparison of freeze fracture images of mixed bacterial/yeast biofilm in cryo-SEM with high pressure freezing fixation

Hrubanova K.1, 2, Nebesarova J.3, Ruzicka F.4, Krzyzanek V.1
1Institute of Scientific Instrument ASCR, Brno, Czech Republic, 2Brno University of Technology, Brno, Czech Republic, 3Biology center ASCR, Ceske Budejovice, Czech Republic, 4Masaryk University, Brno, Czech Republic
hrubanova@isibrno.cz

Microscopic organisms include bacteria and yeasts have been studied in this project. Besides the planktonic way of living, microbes are able to adhere to surfaces or interfaces and to form organized communities, a so-called biofilm, which are embedded in a matrix of extracellular polymeric substances that they produce; visualization and quantification of this microscopic formation is the main goal of this study. In medicine the biofilm formation allows microorganisms to colonize the surface of implants and it also protects the microbial cells from attacks by the immunity system as well as from the effect of antibiotics. Therefore, the biofilm is considered to be important virulence factor in these microorganisms. The characteristic features of the biofilm infections, especially high resistance to antifungal agents, complicate therapy [1]. Understanding of the biofilm structure can contribute to understanding the biofilm formation and basic biochemical mechanisms underlying this process. It may help to develop more efficient treatment strategy for biofilm infection.

Yeast like Candida albicans and bacteria like Staphylococcus epidermidis have been recently recognized as an important cause of serious biofilm infections associated with implanted medical devices. The multi-layered biofilms formed by these microorganisms were observed by cryo-scanning electron microscope (cryo-SEM) using freeze-fracturing technique [2] and high pressure freezing (HPF) as a fixation method. The freeze-fracture technique consists of physical breaking apart (fracturing) a rapidly frozen biological sample; structural details exposed by the fracture plane may be then visualized by cryo-SEM. Our samples (mixed cultures of C. albicans and S. epidermidis) were cultivated in BHI medium at 37°C for two days on sapphire discs; fractured after high pressure freezing (EM PACT2, Leica Microsystems), then followed short sublimation of ice contamination (Alto 2500, Gatan) in two steps; at first, the sublimation takes 1 minute at -96°C (Figure 1 A, B); in the second phase, the sublimation was 7 minutes at the same temperature settings (Figure 1 C, D). In both cases, the same place of the sample was imaged at low temperature in the field emission SEM JSM 7401F (JEOL). The sublimation time reveals substances in our sample with different chemical composition that is related with the ability of sublimation; we are able to recognize water, cultivation medium, extracellular matrix and cells of our microbes.

References:
[1] R. M. Donlan and J. W. Costerton, Clin. Microbiol. Rev. 15 (2002), p. 167.
[2] K. Dobranska in “Characterisation of bacterial/yeast biofilms by scanning electron microscopy”, et al., (EMC 2012 Proceedings, Manchester) (2012), p. 671.

The authors acknowledge the support by the grants CZ.1.05/2.1.00/01.0017 and LO1212 (EC and MEYS CR), TE01020118 (TACR) and 14-20012S (GACR).

Fig. 1: Figure 1 A, B: Cryo-SEM image of biofilm of C. albicans and S. epidermidis after short 1 minute sublimation at -96˚; C, D: images after longer 7 minutes sublimation at -96˚C; there are compared fields of interest (the presence of extracellular matrix) after short and longer sublimation time in blue boxes.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3008 DIAGNOSIS OF VIRAL DISEASES IN FARMED ANIMALS AND WILDLIFE USING TRANSMISSION ELECTRON MICROSCOPY

COOLEY W. A.1, EVEREST D. J.1
1Animal Health and Veterinary Laboratories Agency, Weybridge, UK
bill.cooley@ahvla.gsi.gov.uk

Transmission electron microscopy (TEM) has provided contributions to virology and the discovery, detection and diagnosis of various viral infections. Virus diagnosis by TEM is based on the visualization and morphological identification of virus particles. Therefore, for new or unknown pathogens that may occur in the context of bio-terrorism attacks, or as a result of the manifestation of new pathogens, TEM remains the only method that can provide a quick assessment of all pathogens present in a sample, providing an “open view”. The AHVLA provides a rapid viral diagnostic service for various diseases affecting farmed livestock, wildlife species, captive and zoological animals through veterinary surveillance. Amongst the most common viruses we diagnose are the poxviruses which can infect both vertebrate and invertebrate animals and are often detected from cattle, sheep and goats causing external scabby lesions. The virus group is well known as it includes smallpox (variola). Poxviruses are also regarded as the major contributor responsible for the decline in the UK of the indigenous Red Squirrel (Sciurus vulgaris) due to its susceptibility to Squirrel pox virus first reported in 1981 (1). TEM is used to detect ‘enteric viruses’ which are an important, but diverse group of viruses found in the intestinal tract of animals (and humans). We commonly detect rotavirus and adenovirus. The open view occasionally detects mixed viral infections with an example being both parapox and calicivirus detected in a scab from a Grey Seal. Calicivirus is also the cause of viral haemorrhagic disease (VHD) which is a highly infectious and often fatal disease that affects wild and domestic rabbits and was a notifiable disease in the UK for several years during the 1990s. TEM therefore remains essential for certain diagnostic aspects of Virology (and bacteriology). It was and still is necessary for new virus characterization (e.g. Schmallenberg Virus) and for the initial identification of unknown viral agents in particular outbreaks. The nature of the samples to be analyzed can be tremendously diverse, from body fluids, biopsies, scabs, warts, gut and faecal samples. Additionally the results by TEM are often regarded by many as the 'Gold Standard', as viral particles are actually observed. The “open view” approach permits rapid and “catch-all” detection of viruses and makes it especially useful as demonstrated here for the identification and diagnosis of various animal viruses, as well as being used in the initial identification of unknown viral agents in particular disease emergencies and outbreaks and/or in suspected bioterrorism.

1. Scott, A.C., Keymer, I.F. and Labram, J. (1981). Parapoxvirus infection of the red squirrel (Sciurus vulgaris). Vet. Rec. 109, 202.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3050 Genome packaging in EL and Lin68, two giant phiKZ-like bacteriophages of P. aeruginosa

Sokolova O. S.1, Shaburova O. V.2, Sedov A.1, Pechnikova E. V.3, Krylov V. N.3
1M.V. Lomonosov Moscow State University, Moscow Russia , 2I.I. Mechnikov Research Institute of Vaccines and Sera, RAMS, Moscow, Russia, 3A.V. Shoubnikov Institute of Crystallography RAS, Moscow, Russia
sokolova184@gmail.com

A unique feature of the giant phage phiKZ of Pseudomonas aeruginosa is the way of packaging its genome onto a spool-like protein structure, called the inner body. Until recently, no similar structures have been detected in other members of the genus of phiKZ-like phages. Here we performed a comparative structural study of giant phages: EL, Lin68 and phiKZ, using cryo-electron microscopy, image processing, and bioinformatics methods. We obtained the first 3D reconstruction of the EL phage, consisting of a capsid (Fig. 1), helical tail and a hexagonal baseplate. A careful examination revealed that the EL capsid is 145 nm wide along its 5-fold axis, similar to the phiKZ (1). The hexagonally packed DNA strands are clearly visible in the cryo-images of the capsid (Fig. 2A). The distance between centers of separate DNA strands is 3.09±0.19 nm (Fig. 2B), which is slightly more than 2.8 nm measured in phiKZ (2). To find and to visualize the location of the inner bodies, phage particles, frozen in vitreous ice, were irradiated with increasing doses of electrons. High-energy electrons, bombarding the sample, cause obvious radiation damage to the specimen, resulting in selective boiling and protein degradation in those area that are in contact with the DNA (3, 4). According to characteristic boiling patterns, the shape and position of the inner body in EL were identified and they appear different from those for phiKZ and Lin68 (Fig. 3). Thus, the internal organization of capsids explains how the shorter DNA of the EL phage fits into a capsid, which has the same external dimensions as phiKZ and Lin68. The genome size of the giant phages correlates with the overall dimensions of the inner body, proving it to be a crucial feature for genome packing. The similarity in the structural organization of genome packaging in EL and other phiKZ-like phages indicates that EL is phylogenetically related to phiKZ-like phages, and that, despite the absence of DNA homology, EL, phiKZ, and Lin68 descend from a common ancestor.
References:
1. Fokine et al, 2005, J Mol Biol 352, 117-124.
2. Fokine et al, 2007, Structure 15, 1099-1104.
3. Conway et al, 1993, J Struct Biol 111, 222-233.
4. Fujiyoshi, 1989, J Electron Microsc (Tokyo) 38 Suppl, S97-101.

This study has been partially financed by the Russian Foundation for Basic Research grant (#13-04-01326 to OS)

Fig. 1: Overall structure of EL bacteriophage head and neck, calculated by single particle analysis. Bar - 100 nm.

Fig. 2: (A) The hexagonal DNA packaging within the EL capsid, marked with white arrows. (B) The distribution of the distances between DNA rods in nm (N=28).

Fig. 3: . The positions of the inner body differ in giant phages from different species: (A) phiKZ; (B) Lin68; (C) EL. Vertical rows: 1 - low dose; 2 - high dose, phage capsid positioned in the same orientation as in 1; 3 - high dose, different orientation of the capsid.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3054 Killing bad bugs by photodynamic treatment within seconds and observing photo-induced morphological changes by TEM

Gollmer A.1, Maisch T.1, Eichner A.1, Baeumler W.1, Schroeder J.2
1Department of Dermatology, University Hospital Regensburg, Germany, 2Department of Pathology, University Hospital Regensburg, Germany
anitagollmer@googlemail.com

The threatening evolution of multi-resistant microorganisms makes new antibacterial strategies indispensable. Methicillin Resistant Staphylococcus aureus (MRSA) is one of the most prominent species of Gram-positive multi-resistant bacteria. More than 11000 people died in USA in 2011 as a consequence of MRSA infection according to the Center of Disease Control and Prevention. Pseudomonas aeruginosa which are Gram-negative multidrug-resistant bacteria cause 8% of all healthcare-associated infections. The spread of multi-resistant microorganisms has not only become a major problem in medicine but also in food industry. Endospores of bacteria can survive harsh conditions due to the production of a protective capsule and are resistant to many disinfectants and antiseptics, which usually destroy vegetative bacteria. Bacillus anthracis has been recognized as agent for bioterrorism. B. atrophaeus has been reported to be less susceptible to germicides than B. anthracis.
An innovative and novel approach to combat multi-resistant microorganisms is the photodynamic inactivation of microorganisms (PIM). The microorganisms are incubated with photosensitizers (PS) which can absorb visible light and transfer charges or energy to e.g. molecular oxygen which is then converted into reactive oxygen species (ROS). These ROS can subsequently damage cellular structures such as proteins and fatty acids by oxidative stress and lead to death of these microorganisms. However, the cellular processes that occur in these microorganisms upon PIM have not yet been fully understood. Owing to their small diameter (~ 1µm) transmission electron microscopy (TEM) offers the possibility to investigate morphological changes upon PIM due to its high resolution power.
This study focuses on the investigation of ultra-structural changes upon PIM of multi-resistant species such as MRSA, P. aeruginosa and B. atrophaeus using a new generation of PS based on vitamins and using TEM. MRSA shows a destruction of the outer peptidoglycan layer as well as lamellar structures that are formed out of the cell membrane. For P. aeruginosa it is demonstrated that outer membrane vesicles are formed. The photodynamic treatment of endospores results in a disruption of the outer coat and in diffusion of macromolecules out of the inner core. For all microorganisms we achieve a 6 log10 steps reduction of viability under our experimental conditions.
We demonstrate morphological changes that bacteria and endospores undergo when inactivated within seconds by new PS. The aim is to get a better understanding of photosensitized reactions of ROS with bacterial components to further improve methodologies, in particular the efficacy of PIM by studying mechanisms of microbial inactivation using TEM.

We thank Heiko Siegmund and Claudia Grafe for excellent assistance with TEM and Ewa Kowalewski for her excellent scientific advice in microbial inactivation. A. Gollmer is supported by a DFG grant (GO 2340/1-1).

Fig. 1: TEM image of MRSA before photodynamic inactivation with its thick peptidoglycan layer and its cytoplasma.

Fig. 2: Electron microscopic image of MRSA upon photodynamic treatment with a new photosensitizer based on vitamins. MRSA shows a destruction of the peptidoglycan layer as well as lamellar structures that are formed out of the cytoplasmic plasma membrane.

Fig. 3: Imaging of B. atrophaeus endospores before photodynamic treatment with its protective coat, outer membrane, cortex and core with its nucleoid using TEM.

Fig. 4: TEM imaging of B. atrophaeus endospores upon photodynamic treatment. The photodynamic treatment of endospores results in a disruption of the outer coat and in diffusion of macromolecules out of the inner core.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3079 Ultrastructure of dormant and germinating Bacillus spores studied by using conventional and various cryo preparation methods

Dittmann C.1, Han H.2, Schertel A.3, Laue M.1
1Advanced Light and Electron Microscopy, Robert Koch Institute, Nordufer 20, D-13353 Berlin, Germany, 2Department of Systemic Cell Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Straße 11, D-44227 Dortmund, Germany, 3Carl Zeiss Microscopy GmbH, Training, Application and Support Center (TASC), Carl-Zeiss-Straße 22, D-73447 Oberkochen, Germany
lauem@rki.de

Many bacteria are amazingly persistent under unfavourable environmental conditions. How they accomplish this remarkable tolerance is not known in many cases. Bacteria of the genera Bacillus and Clostridium are producing particular survival stages termed spores. They can tolerate even harshest environmental conditions, e.g. complete drought for many years, without losing their capability to germinate and to form vegetative bacteria. The molecular and structural basis of this remarkable resistance and of the „metamorphosis“ into a dividing bacterium is only partially understood. Therefore we investigated both, dormant and germinating spores using Bacillus subtilis as a model and compared results selectively with results obtained from studies using other species of Bacillus (including Bacillus anthracis) and Clostridium. Since dormant spores are difficult to prepare for ultrastructural research, basically because they possess complex molecular barriers, we have used a combination of different preparation methods, including high-pressure and self-pressurized freezing, freeze-substitution, cryo-electron microscopy of vitreous sections (CEMOVIS) and cryo-focussed ion beam-scanning electron microscopy.

Our studies revealed a couple of structures in dormant spores which were unknown so far. We have analysed them in more detail and followed their fate during germination and outgrowth into a vegetative bacterium in relation to morphological stages and physiological events, such as calcium release or water uptake. One remarkable structure that we have discovered was a crystalline-like region in the core of the dormant spore (Fig. 1). The regions were specifically labelled by anti-DNA antibodies which indicate presence of DNA in those regions. During germination the crystalline-like regions disintegrate into anti-DNA positive filaments, which are dispersed within the cytoplasm of the developing bacterium during outgrowth. Other remarkable structures visible in dormant spores were membrane-like structures that are localised directly below the biomembrane which delimitate the spore core (Fig. 2). By electron tomography we could show that these structures are not continuous with the biomembrane. During germination the membrane-like structures disappear during the brief period when water is taken up by the spore core which is characterized by core swelling and a change in the light optical refraction of the spore. It seems that the membrane-like structures are integrated within the biomembrane of the spore to allow core swelling by re-hydration of the plasma which is a prerequisite for functioning of biosynthesis. In summary our studies provide new aspects for the understanding of bacterial adaptation to resist unfavourable environmental conditions.

Fig. 1: Ultrathin section through a spore of Bacillus subtilis with crystalline-like regions (arrows) within the core. Inset shows a similar region by CEMOVIS. Bar = 100 nm.

Fig. 2: Ultrathin section through a spore of Bacillus subtilis with membrane-like structures (arrows) directly below the core biomembrane and crystalline-like region (asterisk). Inset shows membrane-like structures by CEMOVIS. Bar = 100 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3244 Ultrastructural changes and death in cultured aquatic bacteria induced by ultraviolet radiation

Gamalier J. P.1, Silva T. P.1, Zarantonello V.1, Dias F. F.1, Vidal L. O.2, Roland F.2, Melo R. C.1
1Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil, 2Laboratory of Aquatic Ecology, Department of Biology, Federal University of Juiz de Fora
jugamalier@hotmail.com

Aquatic bacteria play crucial roles in the cycling of organic matter and energy flow in aquatic ecosystem [1]. Ultraviolet radiation (UV) is a non-ionizing radiation, which corresponds to 9% of solar radiation that falls on the aquatic ecosystems [2]. It shifts bacterial communities’ composition and may induce cellular changes. Because of the increasing incidence of UV on aquatic ecosystems, studies focused on the effects of this radiation on bacteria at both cellular and community levels are very important [3]. Here,we investigated the occurrence of death processes and ultrastructure of cultured aquatic bacteria exposed to UV. For bacterial cultures, samples were collected from Funil Reservoir (Brazil, RJ), serially diluted and seed in two non-selective solid media (R2A and TSA). Bacterial colonies were transferred to non-selective liquid media (TSB) and in stationary phase were exposed to ultraviolet radiation (UVR) during 3 h. For this experiment was used UVR in wavelength range corresponding to UV-A+ UV-B (emission peaks: 365/312nm). Hourly samples were collected and the following parameters were analyzed: (i) growth curves by optical density through spectrophotometry and cell density through fluorescence microscopy using DAPI; (ii) cellular viability using fluorescent markers for membrane integrity (Live/dead Baclight kit); and (iii) ultrastructural changes by transmission electron microscopy (TEM) [4]. Our density analyses show that bacteria exposed to UVR showed a decreased bacterial density (p<0.05). Cell viability evaluation showed that UVR induced a significant increase of the aquatic bacteria mortality in culture (p<0.05). On the third hour of exposure to UVR, we detected a higher number of non-viable cells in comparison with those found after the first hour of radiation exposure and controls (p=0.037). Moreover, our data also demonstrated that the ultraviolet exposure time negatively interferes with the aquatic bacterial growth and positively with aquatic bacterial mortality. TEM revealed that bacteria exposed to UVR had higher proportion of cellular damages (51.38%) compared to control cells (9.54%). Damaged bacteria showed loss of cellular envelope integrity, shape changes and cellular elongation. Moreover, 11.9% of bacteria in death process presented emptying of cell components. Altogether, the results of the present work demonstrate that the UV affects fresh water bacterial communities in culture by inducing ultrastructural changes and cell death.

References

[1] L.R. Pomeroy et al, Oceanography, 20 (2007) 28-33
[2] J.M. Anderson et al, Edward Arnold, 108 (1981) 13-35
[3] G.J. Herndl et al, Nature, 361(1993) 717-719
[4] T.P. Silva et al, Antonie van Leeuwenhoek, 105 (2014) 1-14

This work was supported by CAPES, CNPq and Fapemig.

Fig. 1: Bacterial death curve obtained by cell and optical density analyses during ultraviolet exposure. In (A and B), compare cell death curve in UVR-treated group and control. Bacterial cultures were submitted to ultraviolet radiation. A sharp decline of cell density (A) and optical density (B) is detected from the first hour in UVR-exposed bacteria.

Fig. 2: Visualization of live/dead aquatic bacteria in culture by fluorescence microscopy. In (A), Live bacteria are stained in green while dead cells are seen in red. (B) Bacterial viability in UVR-treated group and control. After 3 h, of UVR exposition, the frequency of dead bacteria is greater compared with 2 and 1 h (*p<0.05).

Fig. 3: Ultrastructure of aquatic bacteria. (A) A intact bacteria show nucleoid areas (N) and typical cellular, as highlighted in (Ai). (B-D) Damaged bacteria are observed after exposition to UVR. Observe bacterial changes, such as cell elongation and condensation (B), disintegration of the cell envelope (C, arrow) and emptying of cell components (D).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3125 Electron microscopy computation and stereometrical characterization of bioorganic compounds

Hovnanyan K. O.1, Sargsyan K. A.1, Hovnanyan N. L.1
1Institute Molecular biology of NAS RA, 7 Hasratyan str.,0014,Yerevan, RA,
hovkarl@mail.ru

As a result of progress in microscopic instrumentations, мicroscopists feel that they are only in the beginning of a new era of subatomic microscopic imaging [1].].The biologist could study the components of cells. Moreover, the great value has definition of elementary structure of the sample, and also computation and characterization the received images[2-3].

The aim morphometric and stereometric computations analysis TEM, SEM have been carried software programs structures of bioorganic(virus,bacteria, yeast, protozoa).

The investigation objects were the rotaviruses, virus-symbionts, bacteria, candida, entamoeba. The identifications of organic and inorganic particles conducted by means of transmission (TEM) and scanning electronic microscope (SEM) and SEM microanalysis. For preparing electronic-microscopic preparations of biological samples were standart methods. Computer morphometric and stereometric analysis of electronic microscopic pictures was performed according to the softwar computer programs.

TEM the virus-symbionts of entamoeba are shown to have stick-type form: surface square - 11036 nm2. The application of direct TEM method allows visualization of the surface of E. coli: there is a large number of fimbria. Computer stereometric TEM analysis of these fimbria points out that their diameter is 8-15 nm. Morphometric SEM analysis of the C. guilliermondii(Cg) after x-ray radiation allow to consider the relation of squareof destructive structures to the total square cell of Cg is equal to 26,2773 :73,723. The length of ribonucleoprotein helical-like nanoparticles and crystal body in the vegetative and cyst forms of protozoa was found to 300 nm (diameter - 40 nm)and intracitoplasmatic cylindrical structure of hematophage Ent.histolytica (Fig.1,2). The computer three-dimensional visualization has allowed to transform the image of the plane of cellular structures in the measurement using for building of cordinate of brightness, and also with the help softwar service of programs to show certain colour spectra of the one-colour image.

. References1.Peter W. Hawkes Advances in Imaging and Electron Physics: Aberration-corrected microscopy. Publisher: Elsevier Science.(2008),590p.

2. Hovnanyan K.O., Davtyan H.H.,SargsyanK.A., Trchunyan A.A. Reports of NAS RA.110(3),(2010), p.276-286.

3. Popov V.I., Deev A.A., Klimenko O.A., Kraev I.V., Kuz'minykh S.B., Medvedev N.I., Patrushev I.V., Popov R.V., Rogachevsky V.V., Khutsian S.S., Stewart M.G., Fesenko E.E. Neurosci. Behav. Physiol. 35(4), (2005), p. 333-41.

This work was supported by a research grant awarded from the Ministry of Education and Science of Republic of Armenia

Fig. 1: TEM.Computation of ribonucleoproteid spiral aggregates of chromatoid body of Entamoeba according to the softwar computer programs

Fig. 2: TEM Intracitoplasmatic cylindrical structure of hematophage Ent.histolytica computer analysis according to the softwar computer programs.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3135 Morphological study of Legionella pneumophyla sg 1.

Kolenc M.1, Keše D.1, Kogoj R.1, Steyer A.1, Šest I.1, Poljšak-Prijatelj M.1
1Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
marko.kolenc@mf.uni-lj.si

Legionella species are obligate or facultative intracellular bacteria infecting human macrophages and are naturally associated with protozoa, mainly with free-living amoebae, which may act as transmission vectors to humans (1). Legionella pneumophila (L.pn.) sg.1 is the most pathogenic, causing more than 80 % of cases of Legionnaires’ diseases in Europe (2). It possesses a specific epitope located on the LPS molecules which are detectable by monoclonal antibodies. Legionella are Gram-negative coccobacilli that measure 0.3-0.9 µm in width and 2-20 µm in length. Elongated filamentous forms may be seen after growth on special α-BCYE culture media (3). Inside host cells L.pn. differentiates into a replicative form and when nutrients become limited into a transmisive form. Bacteria in replicative phase are avirulent and not flagellated in contrast, transmissive phase bacteria are virulent, flagellated and highly mobile (4). The purpose of our study was to analyse the ability of intracellular replication of different subgroups of L.pn.sg.1 in the human monocytic cell line (MM6). L.pn.sg.1 subgroups Philadelphia, Knoxwille, Benidorm, Oxford and Bellingham were grown on α-BCYE agar at 35°C in a moist environment. After up to 72 hours of incubation, plates were inspected for typical growth and appropriate concentration of 3x108 legionellae/ml were prepared. MM6 cells were cultured in 75-cm3 vented culture flasks in RPMI 1640 medium to the concentration of 2 x 107 and infection of MM6 cells with each L.pn. subgroup were performed after 8h (5). The bacteria from infected MM6 were prepared for negative staining. In addition Lpn isolate was also processed for immuno-labeling technique for negative staining using the protein A/colloidal gold technique with specific patient antiserum which showed strong reaction with Lpn strain observed in fluorescence microscope. In addition infected MM6 cells were prepared for embedding in epoxy and acrylic resin. Ultrathin sections were examined in JEOL JEM 1200 EXII electron microscope, equipped with Gatan CCD camera. Ultrathin sections cut from LR White embedded Lpn were immuno-labeled using specific antiserum and protein A/Gold (10 nm). The highest efficiency in intracellular multiplication were seen at more virulent L.pn. subgroup Pneumophila, Knoxwille and Benidorm.
References:
1. Moliner C, et al. Journal of Medical Microbiology. 2010; 59: 273–284.
2. Levin AS. Expert Rev Anti Infect Ther. 2009; 7:57-68.
3. Diederen BM. J Infect. 2008; 56:1-12.
4. Gomez-Valero L et al. Infect genet Evol. 2009; 9: 727-39.
5. Neumeister B. Methods Mol Biol. 2004; 268: 141-51.

Fig. 1: Direct negative staining of L. pneumophila.

Fig. 2: Immuno-negative staining (a,b) and gold immuno-labeling on thin sections of bacteria embedded in LRW (c,d).

Fig. 3: Human monocytic cell line (MM6), 3h post infection with L. pneumophila.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3183 Differential targeting of measles virus antibodies to measles protein using immuno-gold labelling methods

Choi K.1, Kim Y.1, Kim A.1, Kim K.1, Hong K.2
1Division of Respiratory Viruses, Center for Infectious Diseases, National Institute of Health, Korea CDC, Osong, Korea, 2Division of High-risk Pathogen Research, Center for Infectious Diseases, National Institute of Health, Korea CDC, Osong, Korea
cemovis@gmail.com

In the family of paramyxoviridae, measles virus, is highly pleomorphic and infectious virus. Measles virus is enveloped virus that enclose nucleocapsid composed of the ssRNA genome and the nucleoprotein, matrix protein and has two types of glycoproteins(fusion and attachment). It is positively necessary to early diagnosis and proper treatment, because it has high infectivity. So, here we study to more effectively detect of measles virus components as like protein F, M and protein N using immuno-gold labelling(IGL) for lower leveled virus particles(Fig. 1). We have used conventional negative staining method to identify the virus particle in lysate of measles virus-infected cells and used IGL method to detection of the biomarker(protein F, M and N). For IGL analysis of viral components, carbon-film supported nickel grids were glow-discharged and incubated on the cell lysate drops from Triton X-100 and ultrasound. After immuno-gold labelling and the stained grids were observed with an Libra-120 transmission electron microscope at 120kV. We have observed the structure of the each viral component(Fig. 2) and confirmed the presence of biomarkers by indicated gold particles(Fig. 3). In this results, we observed that protein F is distributed in the surface of the virus particles and protein M is located in the bundle of complexify protein. However, protein N is coupled to the linearly arrayed proteins. More improved IGL technique may be useful for detection and identification of viral biomarkers in diagnosis.

This study was supported by KNIH intramural research fund no. 2014-NG45001-00.

Fig. 1: Schematic diagram of the IGL method for detection of viral components.

Fig. 2: Negative stained TEM images of component of measles virus.

Fig. 3: IGL images for anti protein F(A), protein M(B) and protein N(C). Scale bar=200nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3390 Biocorrosion and Microscopy

Zapata Peñasco I.1, Garibay Febles V.1, Mendez Mendez J.2, Mendoza Perez J.2, Piña Angeles J.2
1Laboratorio de Microscopía de Ultra-Alta Resolución del Instituto Mexicano del Petróleo (IMP). Programa de Ingeniería Molecular -IMP, 2 Centro de Nanociencias y Micro-Nanotecnología del Instituto Politécnico Nacional (IPN). Escuela Nacional de Ciencias Biológicas-IPN
izapata@imp.mx

The microorganisms play a major role in the processes of hydrocarbon production; they are distinguished for having different impacts due to the production of metabolites, such as enzymes, organic acids, polymers, gases, and biomass. The forty percent of all internal pipeline corrosion cases in the gas industry is microbiologically influenced corrosion (MIC) (1). Metal surfaces are rapidly colonized by planktonic-bacterium which generates in short time a mature biofilm (2). The MIC is induced by biofilms of iron and manganese reducing (5,7), sulphur-oxidizing, fermenting, slime-formers, and sulphate-reducing bacteria (2,3,4). The environmental scanning electron microscopy (ESEM), the confocal laser scanning microscopy (CLSM) and the atomic force microscopy (AFM) allow biofilm observation in real time without introducing distortion of the samples(6). The AFM has been used to obtain information about surface topography of bacteria and biofilm formation patterns (8,9). The aim of this work is to present some applications of microscopy in the study of MIC in materials of oil production installations. The physical properties of anaerobic biofilm involved in corrosion were determined by different microscopy techniques. The growth of the biofilm was measured through time by AFM Multimode-3100 Veeco Microscope with NanoscopeR IV Scanning Probe Controller (tapping methodology), ESEM (Scanning Electron Microscopy) and EDS (Energy-Dispersive X-ray Spectroscopy) with a FEI/Philips XL30 Microscope. The EDS analysis was applied to determine chemical composition of materials. A NanoScope Analysis software (2010) was used to measure the forces in order to estimate Young’s modulus (nN/nm2). The expression of the lux genes involved in the biofilm development was quantified. The results showed that surface roughness (nm) increased during biofilm development; meanwhile the tensile elasticity became greater in nN per nm2. The lux genes expression augmented substantially during the microbial growth and the development of the biofilm. The evaluation of mechanical properties of bacteria over different materials can give important information for controlling and monitoring biocorrosion in oil production industry. References: 1. Zhu X.Y., et al. 2003. Appl Environ Microbiol. 6:5354-5363; 2. Beech I.B. Gaylarde C.C. 1999. Microbiologia. 30:177-190; 3. Watkins-Borenstein S. 1994. Industrial Press USA.; 4. Videla H.A., 1996. CRC Press; 5. Mehanna M.,et al. 2009. Corros Sci 5:2596-2604; 6. Videla H.A., Herrera L.K. 2005. Int Microbiol. 8:169-180; 7. Bagge D.et al. 2001. Appl Environ Microbiol. 67(5):2319-2325; 8. Touhami A. et al. 2006. J Bacteriol. 188(2):370-377; 9. Mangold S.et al. 2008. Appl Environ Microbiol. 74(2):410-415.

The authors are grateful to Mexican Petroleum Institute and National Polytechnic Institute.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3416 Juruaçá virus induces an exacerbated fatal inflammatory response in CNS neonate infected mice

Ferreira N. C.1, Simão T. P.1, Rodrigues A. D.1, Picanço-Diniz C. W.2, Diniz J. P.1
1Laboratório de Microscopia Eletrônica, Instituto Evandro Chagas - Belém, Pará, Brasil, 2Laboratório de Investigações em Neurodegeneração e Infecção, Universidade Federal do Pará - Belém, Pará, Brasil
joseantonio@iec.pa.gov.br

Many studies have been conducted to understand the neuropathogenesis of viral encephalitis however, no experimental studies so far investigated in detail neuropathological features associated with virus infections of Picornaviridae family of virus isolated from bats in the Amazon region. The Juruaçá virus, one of these agents, has been partially characterized by other as a member of the Picornaviridae family. Although this virus did not cause cytopathic effect (CPE) in primary cultures of CNS cells, it has been associated with brain lesions with reactive gliosis in neonatal mice suggesting that this viral agent may kill neonatal mice by an exacerbated inflammatory response. The aim of this study is therefor to investigate the immune response in the CNS induced by Juruaçá virus in albino BALB/c newborn mice. To that end, we performed immunosorbent, immunohistochemical and immunofluorescence assays, to study expression of cytokines and microglial morphological changes. Our results demonstrated the presence of viral antigens in different cell types of the CNS, and the presence of reactive microglia distributed throughout the brain and anterior spinal cord. A gradient of microglial morphological changes including frequent amoeboid shapes suggesting an intense inflammatory response was observed mainly in the cerebral cortex, but also in olfactory bulb, anterior olfactory nucleus, midbrain and forebrain near the lateral ventricle. The production of anti-inflammatory cytokine (IL-10) decreased over time, whereas pro-inflammatory cytokine (IL- 6, TNF-α and IFN-γ) increased significantly from 8th day post-infection (dpi) onwards. The activation of glial cells, especially microglia, followed by subsequent production of proinflammatory cytokines coincided with the intensification of clinical signs. Taken together the results add a new piece of evidence that Juruaçá virus may kill neonate mice by inducing a fatal exacerbated inflammatory response.

CAPES; Ministério da Saúde-MS; UFPA

Fig. 1: Anti-Juruaçá immunolabeled sections from uninfected (A) and infected (B) mice brain on 8th dpi. Anti-IBA1 immunolabeled sections from uninfected (C) (orange arrow, long branches homeostatic microglia), and infected (D) mice at 12th dpi (white arrows, short branches activated microglia; black arrows, amoeboid phagocytic microglia).

Fig. 2: Cytokines production (A) IL-6, (B) TNF-α, (C) IFN-γ and (D) IL-10 in the CNS of albino BALB/c mice, infected with Juruaçá virus after 4th, 8th and 12th dpi. Control: uninfected group; Infected:infected group; 4: 4 dpi; 8: 8 dpi; 12: 12 dpi. Two-way ANOVA, post-test Bonferroni for Multiple Comparison, (***) = p<0.001, (**) = p<0.01 e (*) = p<0.05.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-3478 Automated multi-scale image acquisition for efficient particle detection and analysis using the mini-TEM

Sintorn I.1,2, Kylberg G.2, Nordström R.2, Stepan P.3, Kolarik V.3, Drsticka M.3, Coufalova E.3
1Centre for Image Analysis, Uppsala University, Sweden, 2Vironova AB, Stockholm, Sweden, 3Delong Instruments, Brno, Czech Republic
ida.sintorn@it.uu.se

Introduction
The miniTEM microscope is designed for fast, simple and cost-effective imaging and analysis of biological samples as well as other nano-sized particles. It is a novel desktop-top TEM that requires no special equipment, and can sit on any table in any lab or office. It runs at 25keV which enables imaging of biological samples with a thickness of up to at least 100nm. In order to make the miniTEM as easy to use an ordinary light microscope it has incorporated automatic functions and procedures for microscope alignment, automatic camera and microscope settings for optimal imaging, and image analysis methods for automatically searching for objects of interest at one or several scales. Here we will illustrate how built in automatic multi-scale imaging and analysis can be used to search for and identify virus particles in nsTEM and to automatically extract population statistics of nanoparticles.

 

Multi-scale search for virus particles
To identify viruses in a sample is an often tedious and time consuming task, requiring an expert to manually/visually perform the analysis at the microscope. In the miniTEM, we will incorporate multi-scale analysis methods that mimic how a specialist searches for viruses in clinical samples. This is illustrated in Figure 1 on some of the first images acquired in the miniTEM microscope. In low magnification images, grid-squares suitable for further search at higher magnification are identified. That is, squares that are broken or too cluttered are discarded. At medium magnification, regions corresponding to the size of single viruses or small clusters of viruses are detected. These regions are then imaged at high magnification, where individual virus particles can be detected and identified.

 

Automatic image acquisition for nanoparticle analysis
Two applications where nsTEM analysis is performed to gather population statistics is inorganic nanoparticle size and shape analysis as well as packing ratio of virus like particles used for drug delivery. Some of the first example images of inorganic nanoparticles and virus-like particles acquired in the miniTEM microscope prototype are shown in Figure 2. To get such statistical information typically means that a specialist acquires a number of images of the sample. Particles in the images are then often manually measured or annotated to gather population statistics. The miniTEM instrument will contain methods for automatically acquiring a number of images at random positions in the grid, avoiding mesh-bars. In addition, methods for automatically detecting and extracting morphological measures will be incorporated in the software.

This is part of the miniTEM project funded by EU and EUREKA through the Eurostars programme.

Fig. 1: Illustration of how multi-scale analysis is used to automatically search for virus particles. At low magnification good quality grid squares are detected. At medium magnification small objects possibly corresponding to single viruses or clusters of virus particles are detected, and high magnification images are only acquired of those objects.

Fig. 2: Low and high magnificationexample images of inorganic nanoparticles (left) and virus-like particles(right) acquired with the miniTEM microscope
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-5786 Beta- lactam antibiotics stimulate biofilm formation in non-typeable Haemophilus influenzae by up-regulating carbohydrate metabolism

Webster P.2, Wu S.1, Gunawardana M.3, Baum M. M.3
1Life Sciences Division, University of California, Berkeley, CA, USA., 2Center for Electron Microscopy and Microanalysis, University of Southern California, Los Angeles, CA, USA, 3Oak Crest Institute of Science, Pasadena, CA, USA
pwebster@usc.edu

Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. Using combined approaches we show that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We show that sub-inhibitory concentrations of beta-lactam antibiotic stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were upregulated in response to antibiotic exposure. A modified Schiff stain was used to demonstrate the presence of glycogen around bacterial cells in biofilms exposed to antibiotic. The results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended.

This work was made possible by support from the National Science Foundation (NSF #0722354), the NIH/NIDCD (5 P-30 DC006276-03), and by the Ahmanson Foundation. NTHi strains were provided by Prof G. Ehrlich (Drexel University, PA).

Fig. 1: Sub-inhibitory concentration of ampicillin result in an increase in dead NTHi bacteria in newly formed biofilms. A: no antibiotic, B: with 170 ng/mL ampicillin. Image is a Z-stack reconstruction of NTHi biofilms with Live/Dead stain. Intact bacteria stain green, damaged bacteria stain red.

Fig. 2: SEM images of NTHi biofilms formed on Thermanox slides. The biofilms are composed of bacterial cells aggregated into poorly defined partitions and covered with a layer of amorphous material.

Fig. 3: SEM of NTHi biofilms formed on Thermanox and with 170 ng/mL ampicillin. The biofilms are thick mats of amorphous material with few detectable bacteria.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-5788 Immunolabeling bacterial biofilms

Webster P.1, Kerwin J.2, Wu S.3
1University of Southern California, Los Angeles, CA, USA, 2UCLA, Los Angeles, CA, USA, 3Lawrence Berkeley National Laboratory, Berkeley, CA, USA
pwebster@usc.edu

Bacterial biofilms are composed of bacterial cells embedded in an extracellular matrix (ECM) of proteins, DNA and polysaccharide. Immobilization of extracellular antigens is an essential prerequisite for immunocytochemical studies. Immunolabeling is performed at ambient temperature so the optimal approach for preparing biofilms is by high pressure freezing, freeze substitution and embedding in a low temperature resin such as Lowicryl HM20 [1].

We used this approach to probe in vitro formed biofilms of non-typeable Haemophilus influenzae (NTHi), a human respiratory tract and middle ear pathogen. To identify suitable target proteins for study we performed a proteomic analysis of the ECM to identify biofilm-specific proteins, when compared with a similar list from planktonic (or non-biofilm) bacteria. Antibodies to biofilm proteins were applied to sections. However, most polyclonal antibodies reacted non-specifically to bacterial proteins, most likely because they were produced using bacteria-containing adjuvants. In order to successfully label biofilm sections we could only use polyclonal antibodies that had been targeted to purified bacterial proteins, or monoclonal antibodies to similar proteins expressed by eukaryotic cells.

Monoclonal antibodies were visualized with a secondary, polyclonal antibody followed by protein A-gold. To overcome the non-specific binding of secondary antibodies to bacterial proteins we added suspensions of lysed bacteria to antibody blocking solutions, an effective way to remove binding to bacterial proteins.

Eighteen biofilm-specific proteins were identified in ECM and all proteins were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, present in bacteria and biofilm ECM. The amount of labeling present was affected by the age of the biofilm and the location within the biofilm. The different labeling patters could be illustrated by quantitative analysis, performed using a free stereology program [2].

Identification of biofilm-specific proteins present in the ECM of immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation.

1. Webster et al (2004) Ultrastructural preservation of biofilms formed by non-typeable Hemophilus influenzae. Biofilms 1: 165-182.

2. Tschanz etal (2011) A simple tool for stereological assessment of digital images: the STEPanizer. J Microsc 243: 47-59.

The work was supported by the NSF (grant #0722354), the NIDCD (5 P-30 DC006276-03), the Fritz Burns Foundation, the Hope for Hearing Foundation, the Deafness Research Foundation, the Hearst Foundation, and the Capita Foundation. Prof T. Murphy provided anti-OMP P2 (YKA) antibodies.

Fig. 1: A cross section through an cryo-preserved NTHi biofilm. Bacteria and extracellular material are all preserved, including the top of the biofilm. The section was labeled with anti-OMP P2 polyclonal antibodies and 10nm protein A gold. The antibody labeling is low in this upper region of the biofilm

Fig. 2: At the base of of the biofilm the anti-OMP P2 label is more abundant, and is associated with bacterial cells and small vesicles in the ECM. These structures can be easily removed by conventional preparation protocols but are retained by cryo-preservation approachesl. Amorphous material at the base of the biofilm does not label.

Fig. 3: Monoclonal antibodies (mabs) to a DNA-directed RNA polymerase show abundant labeling at the base of the biofilm. Label is mostly associated with the amorphous material between cells and at the base of the biofilm. the mabs were visualized using a RxM secondary in PBS containing 1% fish skin gelatin and lysed bacteria, and 10nm protein A-gold.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-5792 Microscopic analysis of Salmonella-Containing Vacuole’s (SCV): isolated using a novel method by pre-labelling bacteria with nanoparticles prior to infection.

Singh V.1, Tedin K.1
1Centre for Infection Medicine, Institute of Microbiology and Epizootics Freie University, Berlin Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany
vikash.singh@fu-berlin.de

Salmonellosis is one of the most common food borne diseases in humans and present a major public health and economic burden worldwide. Salmonella serovars are facultative intracellular pathogen which employs a Type III secretion system (TTSS) to inject virulence factors directly into its host cell to enable the pathogen to invade and establish an intracellular replicative-niche called the Salmonella- Containing Vacuole (SCV).

A major problem in determination of interacting host proteins and compartments with the pathogen containing phagosome has been the isolation of these intact phagosomes. A number of methods column based methods, including buoyant density gradient centrifugation and even magnetic cell separation has been previously applied but these methods often resulted in cross-contamination or rupturing of the labile SCV membrane. Here, we report the use of carbon coated paramagnetic cobalt nanoparticles for pre-labelling of bacteria prior to infection to label the phagosomes from within (Figure 1). After lysis of host cells at different times post-infection, application of a mild magnetic field allows efficient collection of intact pathogen containing phagosomes, without additional concentration steps.

On examining the isolated SCV’s by confocal microscopy using antibodies against well characterized SCV markers LAMP-1 and LAMP-2, nearly 80-90% of the purified SCV’s showed positive staining for these markers (Figure 2). These observations suggest that this method of SCV isolation is rapid and yielded SCV’s which are intact and nearly free from other cellular contaminants. The microscopic results were further confirmed by Western blotting analysis of the purified SCV’s. This method can also be applied for isolating pathogen containing compartments for other pathogens thereby yielding specific in depth knowledge between host –pathogen interactions.

We would like to thank Dr. Maik Lehmann for helping me with the Electron Microscopy and Prof. Dr. Lothar H Wieler for helpful discussions and comments. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through Priority Program grant SPP1136 and Graduate Research Training grant 1121.

Fig. 1: Labelling of bacteria with nanoparticles. (a) & (b) represents TEM images of magnetic nanoparticles, attached to bacteria surface marked. (c) & (d), represents intracellular Salmonella c) unlabeled (control) and d) labelled with nanoparticles (marked with arrow heads) infected in THP-1, human macrophage cell line.

Fig. 2: Microscopic analysis. a) & b) Intracellular Salmonella associated with SCV markers LAMP-1 & LAMP-2 respectively. c) & d) Purified SCV’s also harbors these markers, suggesting that SCV’s are intact and free of any contamination. e), f) & g) close-up view of the isolated SCV.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-5794 The von Willebrand Factor type A-like domains of collagen type VI exhibit antimicrobial activity

Abdillahi S. M.1, Maaß T.2, Kasetty K.3, Baumgarten M.1, Walse B.4, Schmidtchen A.5, Wagener R.2, Mörgelin M.1
1Department of Clinical Sciences, Division of Infection Medicine, Lund University, Lund, Sweden, 2Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany, 3Department of Clinical Sciences, Sector for Respiratory Medicine & Allergology, Lund University, Lund, Sweden, 4Department of Clinical Sciences, Division of Dermatology and Venereology, Lund University, Lund, Sweden , 5SARomics AB, Lund, Sweden
suado.m_abdillahi@med.lu.se

Collagen type VI is a ubiquitous extracellular matrix component that forms complex and extensive microfibrillar networks in all connective tissues, often associated with basement membranes. Structurally, it consists of three α-chains (α1, α2 and α3), where each α-chain contains a short triple helix and N- and C-terminal globular regions. More recently, three additional chains (α4, α5 and α6) were discovered, which may substitute for α3 in some tissues. The globular regions are homologous to the type A domains of von Willebrand factor (vWF-A). We have recently described the antimicrobial properties of this collagen against a number of Gram-positive and Gram-negative human pathogens. However, the molecular mechanisms of bacterial killing are still elusive. Therefore, in this study, we applied an in silico approach to unravel the antimicrobial activity of collagen type VI in further detail. Sequence and structural analysis showed that the vWF-A domains of all three α-chains contain numerous amphipathic amino acid motifs of putative antimicrobial nature. In addition, we also could show that recombinantly expressed vWF-A domains bind to negatively charged surfaces such as heparin and bacterial membranes. Five such motifs were finally chosen from N8, N9 and C1 domains of the α3- chain for further characterization in bacterial killing assays. The data suggest that amphipathic, heparin-binding amino acid motifs in the globular vWF A-like domains harbour the antimicrobial properties of collagen VI.

 We wish to thank the Core Facility for Integrated Microscopy (CFIM), Panum Institute, University of Copenhagen, for providing an excellent electron microscopy platform/environment.

Fig. 1: Antibacterial effect of vWF-A domains of collagen VI α-chains. S. pyogenes were treated with collagen VI α-chains for 2h at 37°C and visualized by scanning electron microscopy. Extensive membrane disruption and leakage of intracellular contents are observed in the presence of these proteins and are indicated with arrowheads. Scale = 5 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-5806 Fine structural changes of Erwinia carotova in presence of Ag nanoparticles

Zavala G.1, Guevara P.2, Morales-Luckie R. A.2
1Unidad de Microscopía Electrónica, Instituto de Biotecnología. Universidad Nacional Autónoma de México , 2Universidad Autónoma del Estado de México-Universidad Nacional Autónoma de México, Centro Conjunto de Investigación en Química Sustentable (CCIQS), San Cayetano de Morelos, Toluca, Estado de México, México
gzp55@yahoo.com.mx

Fine structural changes of Erwinia carotova in presence of Ag nanoparticles. Paulina Guevara(1), Raúl A. Morales-Luckie(1) and Guadalupe Zavala(2). Universidad Autónoma del Estado de México-Universidad Nacional Autónoma de México, Centro Conjunto de Investigación en Química Sustentable (CCIQS), San Cayetano de Morelos, Toluca, Estado de México, México (1). Unidad de Microscopía Electrónica, Instituto de Biotecnología. Universidad Nacional Autónoma de México (2). gzavala@ibt.unam.mx The purpose of this study was to evaluate the morphology of Erwinia carotova grown in presence of Ag nanoparticles using transmission electron microscopy (TEM) protocols. Nanoparticles and biotechnology. Spherical nanoparticles obtained by green method synthesis (AgNO3 reduced by Citrus paradisi extract) were applied to bacterial culture in Muller Hilton medium. Dilutions of stable, single spread particles and in average size in 10 nanometers range were made according a minimal inhibitory concentration. Silver minimal dose (0.0157 ppm) inhibits bacterial growth of fresh cultures in control conditions incubation for 24 h at 35°C. Morphological changes in bacteria related Ag nanoparticles. Metal nanoparticles are related to morphological changes in cytosol of E. carotova similar to the protein aggregation under control thermal or inclusion bodies (IBs) formation in E coli related to culture conditions including chemical or thermal. In the present study Ag nanoparticles affect cytosol organization of Erwinia carotova, produces aggregation in highly hydrated dense structures with a porous structure, rough or smooth, spherical, cylindrical or ellipsoidal teardrop shapes and 50 to 700 nm in size. References. Castellanos-Mendoza A. et al. Influence of pH in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli SUBMMITED TO APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. Bio-synthesis of gold nanoparticles by human epithelial cells, in vivo. Larios-Rodriguez E, et al. Nanotechnology. 2011 Sep 2;22(35):355601 Epigenetic inheritance based evolution of antibiotic resistance in bacteria. Mike Adam et al. BMC Evolutionary Biology, Vol. 8, pp. 52, 2008.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-6070 Morphological study of antimicrobial actinomycete producing isolates from marine sediments

Srivibool Rattanaporn. -.1
1Institute of marine Science, Burapha University, Chonburi. 20131. Thailand
rattanap@buu.ac.th

Actinomycetes are gram positive bacteria in which many bioactive compounds are generally produced. Over past decade information on the diversity of actinobacteria in marine habitats has grown considerably. In this study, morphological and chemical characteristics of wall chemotype were investigated for a rapid method of basically classification. The location of sediment sampling areas were in Chonburi and Bang-pakong mangrove forests in the east and Nakhon Si-thamarat mangrove in the west of the Gulf of Thailand, including marine shallow water coastal area in the east coast. The sediment samples were pre-treated with dry heat at 100o C for 1 h before dilution and spreading on selective medium plates, incubated at 30o C for 4 weeks. Morphological study was observed both under light microscope and scanning electron microscopy. The results revealed that most active isolates from Chonburi mangrove area were Streptomyces with rectiflexibile, spiral and hook spore chain types, while the isolates from Bang-pakong and the west side of the Gulf manifested various different morphological types. The active isolates from marine sediments mostly produced single spore chain type on short or long sporophores or produced in a bundle of Micromonospora and Salinispora, respectively; including a few white spore mass Streptomyces. The electronmicrographs of many isolates could reveal more different morphological detail to consider they were same or different species. Apart of morphological and chemical characteristic studies, some of representative actinomycetes were selected to identified by 16S rRNA gene sequencing. The active isolates from mangrove and marine sediments are moderately diverse in genera, but clearly shown they are morphologically diverse and are rich sources to screen for valuable bioactive compounds.

 

Acknowledgements: The financial support from the NRCT through the Burapha University budget was gratefully acknowledged.

Fig. 1: Single spores are formed on short sporophores of substrate hyphae of Micromonospora  from marine sediment on ISP2 medium, 7 days old, 3000X.

Fig. 2: Single spores are formed on short sporophores of substrate hyphae of Micromonospora on ISP2 medium, 7 days old, 3000X.

Fig. 3: Spiral spore chain type, rugose, of Streptomyces on ISP2 medium, 7 days old, 7000X.

Fig. 4: Rectiflexibile and hook spore chains are formed on arial mycelia of Streptomyces isolated from mangrove sediment,7 days old, 2000X.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-5913 Fluorescence amplified detection of residual bacteria in the root canal space

Herzog D.1, Niazi S.2, Hirvonen L.3, Cook R.1, Koller G.1, Mannocci F.4, Foschi F.4, Festy F.1
1Biomaterials, Biomimetics and Biophotonics Research Group, King's College London Dental Institute at Guy's Hospital, London SE1 9RT, UK., 2Department of Microbiology, Dental Institute, King's College London, London SE1 9RT, UK, 3Department of Physics, Strand Building, King’s College London, London WC2R 2LS, UK., 4Department of Restorative Dentistry, King's College London, London SE1 9RT, UK.
dylan.herzog@kcl.ac.uk

Bacteria remaining in the root canal (RC) space after treatment can lead to a persistent or secondary infection, leading to treatment failure and the need for re-intervention in around 24% of cases [1-2]. Currently, no standard method exists for detection of bacterial presence within the RC space. Using in situ (scouting the RC with a fibre-probe) and ex situ (RC sampling with paper points) approaches, we aim to develop bench side diagnostics to optically detect and quantify the amount of remnant bacteria (Fig. 1a-b).

Primarily and as a proof of concept for detection, fluorescent beads were used to simulate live (green) and dead (red) bacteria (Fig. 2). For the ex situ principle, paper points were immersed in fluorescent beads, which were resolved and distinguished from each other and the paper point using two-photon microscopy (Fig. 2a-b). For the in situ approach, fluorescent beads where added directly to RCs of extracted teeth and detected with an endoscope (Fig. 2c).

Secondly, fluorescent dyes for bacterial staining were evaluated and optimised for incubation time, sensitivity and specificity. Using confocal microscopy, we identified that calcein AM positively stains a mixed species oral biofilm at a clinically relevant incubation time, with minimal background staining. It was further confirmed that both ex situ and in situ approaches were able to perform detection of the in vitro grown bacterial biofilms.

Preliminary studies have focused on developing and optimising the ex situ approach. Real-time detection of stained bacteria was achieved using a spectrometer coupled to a wide field fluorescence microscope. Spectral unmixing was used to distinguish between the distinct calcein AM emission and paper point autofluorescence (Fig. 3a-b). The methodology was validated in vivo by detection of bacteria from samples acquired during RC treatments (Fig. 3c).

This work has identified the potential for our technique to be applied as a powerful tool in dental clinics for the efficient and effective detection of remnant bacterial bio-burden, minimising failure and future need for root canal re-intervention.

[1] Lumley, P. J., Lucarotti, P. S. K. & Burke, F. J. T. Ten-year outcome of root fillings in the General Dental Services in England and Wales. International endodontic journal 41, (2008), p. 577–85.
[2] Chávez de Paz, L. Redefining the persistent infection in root canals: possible role of biofilm communities. Journal of endodontics 33, (2007), p. 652–62.

Dr. Fred Festy, Dr. Federico Foschi, Prof. Francesco Mannocci, Dr. Liisa Hirvonen, Dr. Chris Chong, Dr. Garrit Koller, Dr. Sadia Niazi, Dr. Richard Cook, Hina Gosrani, Peter Pilecki and Richard Mallet

Fig. 1: Diagram showing the a) in situ incubation and detection and b) ex situ incubation and detection.

Fig. 2: Two-photon microscopy image of a paper point tip immersed in green and red fluorescent 1 µm beads. Paper fibers were visualised using SHG (blue). a) Front view and b) side view of a rendered 3D stack. c) Endoscopic recording of the root canal: green fluorescent beads (yellow arrow) are visible on reflected blue background (red arrow).

Fig. 3: Spectral readings of a) comparison of the normalised spectra of calcein emission and paper point autofluorescence. b) Detection of calcein peaks on an in vitro paper point sample, spectra taken every 500 µm starting from the tip. c) Detection of calcein peaks on an in vivo sample taking during a RC treatment.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-5921 Biofilms in biomedical devices: strategies of bacteria to avoid biocides and antibiotics

Reyes Ábalos A. L.12, Villar Arias S.12, Scavone Guillermo P.3
1Department of Scanning Electron Microscopy and Microanalysis (SMEByM) Faculty of Science (UdelaR) , 2Department of Genetics, Institute for Biological Research Clemente Estable (IIBCE) , 3Department of Microbiology, Institute of Biological Research Clemente Estable (IIBCE)
reyesabalos@gmail.com

The biomedical devices are composed of different biomaterials however over 60% of infections acquired in medical centers are due to the presence of biofilms (1). Although bacterial biofilms are clearly observed by scanning electron microscopy (SEM) and appear as "dead parts of microorganisms", laser confocal microscopy show that a biofilm is a three dimensional structure covered by exo-polysaccharides with feed channels for bacterial nourishment and proliferation (2). This cover prevents the arrival and effective action of biocides and antibiotics and allows acquire resistance to them over the time. Thus, the biofilm becomes a strategy for survival in a harsh environment. Common strains in biofilms in dialysis devices are Staphylococcus aureus, S. epidermidis, Pseudomonas, Escherichia coli. In this work we analyze the response of biofilms to different biocide treatments: ozone, peracetic acid and UV. Peracetic acid is a mixture of acetic acid and hydrogen peroxide. The compound eliminates microorganisms by oxidation and subsequent rupture of the cell membrane, by the hydroxyl radical (HO). Peracetic acid was used cold, in a 0.09% solution of water during 15 minutes approximately. Ozone is an allotrope of oxygen formed by three atoms of it. Its role as a disinfectant is recognized by its high oxidant potential. Due to its instability, it has to be produced in the site of application through especial generators that produce O3 gas in water circulating by the system of dialysis. Concentrations used were from 0.08 to 0.4 ppm with a time of application that varied between 4 to 6 hours. The last kind of tubes analyzed was those that are part of the osmosis system disinfected by UV-C. Tubes of PVC, PPT and PE of a great variety of marks were used as negative controls (Fig. 1). All tubes analyzed in this work were treated with O3, peracetic acid, both treatments and also we assessed tubes that were under the action of UV in the osmosis equipment. The concentrations and times were those indicated above. In all cases, we found biofilms of different morphologies, showing that the concentrations, times of applications and procedures did not work efficiently to eliminate biofilms of the systems (Fig.2). New approaches to avoid the colonization of biomedical devices by bacteria are necessary to ensure an appropriate protocol of dialysis.

(1) Treter J, Macedo AJ (2011). Catheters. A suitable surface for biofilm formation. In Méndez-Vilas A. ed. Science against microbial pathogens: communicating current research and technological advances. Spain: Formatex Research Center. pp. 835-842
(2) Costerton JW, Stewart PS, Greenberg EP (1999). Bacterial biofilms: a common cause of persistent infections. Science. 284 (5418):1318-22.

Sectorial Commission of Scientific Research
Faculty of Sciences, University of Uruguay

Fig. 1: (SEM) Tubes of polyvinylchloride (PVC) and, polyethylene PE of a great variety of marks were used as negative controls, with a thin layer of pure gold (120 seconds).

Fig. 2: (SEM) Tubes of polyvinylchloride (PVC) and polyethylene (PE) treated with O3, peracetic acid, and UVC. All tubes show contamination with biofilms. These biomaterials were metallized with a thin layer of pure gold (120 seconds).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-5972 Dielectrophoresis for single-virus force spectroscopy

Korneev D V Generalov V M Zaitsev B N
State Research Center of Virology and Biotechnology “Vector”, Koltsovo, Novosibirsk region, Russia
korneev_dv@vector.nsc.ru

It is possible to use the atomic force spectroscopy technique for manipulation of single virus particles [1]. To do this a virus particle needs to be attached to the tip of atomic force microscope’s probe. A dielectrophoresis (DEP) is a phenomenon in which a force is exerted on a dielectric particle when it is subjected to a non-uniform electric field [3]. The direction and magnitude of DEP force are the functions of frequency of the electric field. These functions are defined by electrical properties of the particle. It is possible to use positive DEP (a movement of particles to electrodes) for attaching a single virus particle to the conductive AFM probe’s tip for the force spectroscopy measurements.

A curvature radius of conventional gold-coated AFM probe’s tip is not more than 35 nm (Fig. 1). During the experiments the small (60 nm) virus-like particles and virions with size from 100 nm (influenza virus) to 350 nm (vaccinia virus) were used. The AFM tips (CSG01/Au, NT-MDT, Russia) were flattened by the scanning of silicon or sapphire surfaces in contact mode with high load, high scan speed, and a large scanning area. This procedure creates a flat area at the tip [2]. The AFM probe’s tips were examined by transmission electron microscopy (JEM 1400, Jeol, Japan) with custom-made TEM holder.

It has been proved that this method is useful for attaching the virus particles (influenza, vaccinia, etc.) to the tip of AFM probe. The results were verified by a TEM examination of the AFM tips.

REFERENCES

1. D. Alsteens, E. Pesavento, G. Cheuvart, V. Dupres, H. Trabelsi, P. Soumillion, Y.F. Dufrene, Controlled manipulation of bacteriophages using single-virus force spectroscopy, ACS Nano, 2009, 3(10), pp. 3063-3068.
2. C.L. Cheung, J.H. Hafner, C.M. Lieber, Carbon nanotube atomic force microscopy tips: Direct growth by chemical vapor deposition and application to high-resolution imaging, PNAS, 2000, 97(8), pp. 3809–3813.
3. H.A. Pohl, Dielectrophoresis, Cambridge University Press, Cambridge, 1978.

Fig. 1: A virus-like particle on the tip of AFM probe

Fig. 2: A flat area at the tip of AFM probe
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-6010 Structural investigation of polyvalent staphylococcal bacteriophage phi812K1-420 mutant by single-particle electron microscopy

Novacek J.1, Benesik M.1, Pantucek R.1, Plevka P.1
1CEITEC, Masaryk University, Brno, Czech Republic
jiri.novacek@ceitec.muni.cz

Staphylococcus aureus is a major causative agent of human and animal diseases. The increasing number of pathogenic strains resistant to antimicrobial drugs is a serious public health problem that can be solved by applications of phage therapy as a suitable alternative to antibiotics treatment. Polyvalent staphylococcal bacteriophage phi812 is a member of SPO1-like viruses from family Myoviridae. Phage phi812 and its mutants kill 75% MRSA, 95% MSSA and act against another staphylococcus species. Mutant phage phi812K1-420 is most perspective for phage therapy because has broadest host range. The phi812K1-420 virion consist of 1060 A diameter icosahedral head containing 138 kb phage genome and 2100 A contractile tail with the baseplate. Herein, we have employed electron cryo-microscopy to provide first description of the phi812K1-420 three-dimensional structure. The icosahedral symmetry was employed to obtain the initial reconstruction of the phage head. The symmetry relaxation was performed in order to determine an asymmetric phage head structure. The helical reconstructions of the tail sheath and the c6 symmetry reconstructions of the baseplate have been performed for phi812K1-420 in its normal extended state and for the urea treated bacteriophages in order to determine the structural model of the virion during the infection. From the comparison of the phage models prior and during infection, the structural changes related to the bacterial infection are monitored in order to implicate the structural mechanism by which the staphylococcal cell wall is recognized.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-6012 Cryo-electron Microscopy of Rhinovirus Uncoating Intermediates: Geometry of Membrane Attachment and Conformation of the RNA Genome

KUMAR M.1
1Max F. Perutz Laboratories, Medical University of Vienna, Austria
mohit.kumar@univie.ac.at

Cryo-electron Microscopy of Rhinovirus Uncoating Intermediates: Geometry of Membrane Attachment and Conformation of the RNA Genome
Mohit Kumar, Shushan Harutyunyan, Heinrich Kowalski and Dieter Blaas

Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical University of Vienna, Austria.

Human rhinoviruses (HRVs) are the main cause of the common cold. During uncoating they undergo conformational changes, first converting into the subviral A-particle and finally, on releasing the RNA genome, into the (empty) B-particle. Mimicking in vivo conditions of uncoating in the late endosome we attached the A-particle to the liposomes and demonstrated that the RNA was transferred into the liposomal lumen upon acidification; arrival of the RNA was shown with RT/PCR (1) and fluorescence correlation spectroscopy. Single particle 3D cryo-electron microscopy reconstruction (cryo-EM 3DR) of membrane bound A-particles showed that they are attached via one of the 30 two-fold icosahedral axes (2). In Enteroviruses, such as poliovirus, it has been shown that the RNA was released close to a 2-fold axis when native virus was heated to 56°C. When the RNA was crosslinked with psoralen within native HRV-A2 followed by exposure to 56°C, particles representing an intermediate stage different from the classical A- and B- subviral particles accumulated. Cryo-EM 3DR revealed a rod-like internal density, which presumably represents the condensed form of the viral RNA. One end of this ‘rod’ was near a viral icosahedral 2-fold axis (4). This suggests that the rhinoviral RNA was in the process of being released but got stuck because double stranded regions could not be unfolded as a consequence of the crosslinking. It remains to be seen whether such condensed RNA plays any role during uncoating under more physiologic conditions. A mechanism imparting directionality to the genome release process might be common to many icosahedral non-enveloped single stranded RNA viruses.

References:
1. Gerhard Bilek, Nena M. Matscheko, Angela Pickl-Herk, Victor U. Weiss, Xavier Subirats, Ernst Kenndler, and Dieter Blaas (2011). Liposomal Nanocontainers as Models for Viral Infection: Monitoring Viral Genomic RNA Transfer through Lipid Membranes. J. Virol; 8368–8375.

2. Mohit Kumar, Dieter Blaas (2013). Human Rhinovirus Subviral A Particle Binds to Lipid Membranes over a Twofold Axis of Icosahedral Symmetry. J. Virol .87; 1309–11312.

3. Shushan Harutyunyan, Mohit Kumar, Arthur Sedivy, Xavier Subirats, Heinrich Kowalski, Gottfried Köhler, Dieter Blaas (2013). Viral Uncoating Is Directional: Exit of the Genomic RNA in a Common Cold Virus Starts with the Poly-(A) Tail at the 3’-End. PLOS Pathogens; 9.

I would like to thank my professor Dr. Dieter Blaas for his continuous support during my PhD. My PhD committee, Dr. Thomas Marlovits and Prof. Kristina Djinovic-cargo for their important inputs and discussion. I would also like to thank Prof. Heinrich Kowalski and Prof. Holland Cheng for their valuable suggestions and support, and finally my colleagues from the lab.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-6031 Novel lateral contact is essential for structural integrity of helical Barely stripe mosaic virions.

Pechnikova E. V.1, Clare D. K.2, Skurat E. V.3, Makarov V. V.4, Solovyev A. G.4, Sokolova O. S.1,3, Orlova E. V.2
1A.V. Shubnikov Institute of Crystallography RAS, Moscow, Russian Federation, 2Institute of Structural and Molecular Biology, UCL and Birkbeck, London, UK, 3Department of Biology, Moscow State University, Moscow, Russian Federation, 4A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russian Federation
eugenia.pechnikova@gmail.com

Barley stripe mosaic virus is a part of Hordeivirus genus, which includes plant viruses with tripartite +RNA genome. Three genomic RNAs are incapsidated separately. Virus spreads via contacts between plants, via seed and pollen and course diseases from light mosaic to lethal necrosis. BSMV virion has a shape of a rigid rod with helical distribution of RNA and coat protein.
Structure of members of Hordeivirus genus is poorly studied, whereas structure of close Tobamovirus genus is well-discribed. The aim of this work is to determine the structure of BSMV using methods of electron microscopy and image processing (Clare and Orlova,2010).
It was estimated that the helix has 5 turns per period and 111 subunits per period. The angle between subunits was ~16.2°, and helical rise between subunits was ~1.18Å.
Multivariate Statistical Analysis (MSA) of obtained electron microscopical data showed that there was a structural heterogeneity in the dataset: diameter of viral particles varied. The images of wide and narrow particles (with diameter of 224Å and 216 Å correspondingly) were extracted and used for 3d reconstruction. It turned out that narrow particles have slightly different helical parameters: 106 subunits per period, the angle between subunits was ~17°, and helical rise between subunits was ~1.24Å. The resolution of the final electron density map was 5.7 Å for a wide particle and 5.1Å – for a narrow. On the vertical slice of both structures there is visible a groove between protein subunits, where RNA is located (Fig. 1). The reached resolution makes it possible to trace the α-helices in the coat protein of BSMV and the sites of RNA-protein interactions are determined (Fig. 1). The flexible fitting of homology model of BSMV coat protein (CP) showed that there is a loop at a high radius. The loop protrudes from hydrophobic core and makes a contact with the next subunit (Fig. 2). To analyze the functional importance of interacting arm formed by the BSMV CP internal loop, we constructed two CP mutants, BSMV-del10 with a ten-residue deletion in the loop and BSMV-IY/GG, in which two residues Ile86 and Tyr91 involved in the contact with neighboring CP subunit were replaced with Gly residues (Fig. 3a). Recombinant viruses carrying the mutant CP gene were able to systemically infect N. benthamiana plants and accumulated to levels similar to recBSMV (Fig.3a,b). However, virions were not found in tissues infected with recBSMV-del10 and recBSMV-IY/GG, while readily detected in recBSMV-infected plants (Fig. 3c,d), showing that both mutants were unable to form stable virions. These data demonstrate the crucial importance of the inter-subunit interacting arm in formation and/or maintaining the structure of BSMV virions.

This work was supported by grants: EMBO ASTF 118 – 2012 and RFBR 13-04-01326

Fig. 1: Structure of wide BSMV particle. a) Surface of wide BSMV particle. b) Vertical slice of wide BSMV particle. c) Horizontal slice of wide BSMV particle d) Fourier shell correlation

Fig. 2:  Loop protrudes from hydrophobic core at a high radius and makes contact with next subunit.

Fig. 3: Deletion analysis of role of the loop in virion formation.a) The sequences of loop region in recBSMV, with loop deletion and with double point mutation.b) recBSMV without modifications of coat proteinc) recBSMV-10deld) reBSMV-IY/GG,scale bar – 200nm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-6-P-6034 The role of bacterial consortium in in-situ bioremediation of tannery waste water and detoxification study of chromium in HepG2 cell line

Srivastava S.1,2, Jaiswal P. K.1,3, Thakur I. S.1
11. School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India , 22. Amity School of Earth and Environmental Sciences, Amity University, Gurgaon, India , 33. The research institute of the McGill University health centre 1650 cedar Avenue Montreal Canada
shailisrivastava05@gmail.com

The role of bacterial consortium in in-situ bioremediation of tannery waste water and detoxification study of chromium in human hepatoma cell line
Shaili Srivastava1, 2, Prashant Kumar Jaiswal 1,3 and Indu shekhar Thakur1
1. School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India
2. Amity School of Earth and Environmental Sciences, Amity University, Gurgaon, India
3. The research institute of the Mcgill University health centre 1650 cedar Avenue Montreal Canada
Abstract
Tanneries are responsible for environmental pollution as it uses huge amount of chromium sulphate (CrIII) and pentachlorophenol (PCP) in the leather tanning processes to inhibit the growth of microorganism. Effluent contaminated by metals and chlorinated organic compounds are difficult to remediate. Chromium sulphate [Cr (III)] and pentachlorophenol (PCP) are widely used as tanning agent and biocide for the leather preparation in tanneries. Pentachlorophenols are highly toxic and recalcitrant compound. Chromium (III) may be converted in to chromium (VI) form in aquatic environment which is highly toxic and carcinogenic in nature. Bacterium consortiums obtained from tannery soil sediment were used for bioremediation and bioconversion of PCP and chromium from the tannery waste water. A bacterium isolated from tannery consortium was identified as Serratia sp. by 16S rDNA analysis. Bacterium reduced Cr(VI) to Cr(III) by a homodimer enzyme, chromate reductase, with a monomer molecular mass of 40 kDa. The potency of Serratia sp. for degradation of pentachlorophenol was determined by HPLC after formation of tetrachlrohydroquinone and chlrorohydroquinone. Bioremediation of chromium and PCP were tested in bioreactors in sequential way where bacterium treated effluent subsequently treated by fungi showed reduction of chromium (82%) and PCP (85%) after 120 hrs. Biosorption of chromium was determined by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The toxicity of Cr(VI) was also determined in vitro HepG2 cell line by IC50, Ethoxyresorufin-O-deethylation activity, Fluorescence-activated cytometry sorter analysis and Confocal laser scanning microscopy indicated detoxification of chromium after adsorption by bacterium.

We thanks the Council of Scientific and Industrial Research, Government
of India,NewDelhi, for providing a Research Associate fellowship
and a contingency grant.We also thanks to advanced instrumentation facility of Jawaharlal Nehru
University, and All India Institute of Medical Sciences New Delhi, India, for providing the TEM, SEM-EDX, fluorescent microscope facility.

Fig. 1: Figure 1 (a) Scanning Electron Microscopy (SEM) of exposed cell of bacterium (Serratia sp.) after tannery effluent treatment (b) SEM of tannery effluent treated fungal mycelium of Aspergillus niger .
↑ Go to the Table of content ↑

LS-7. Invertebrates and parasitology

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-7-IN-5799 Tracing malaria pathogenesis with cryo-electron tomography

Cyrklaff M.1, Srismith S.1, Burda K.2, Frischknecht F.1, Lanzer M.1
1University of Heidelberg, School of Medicine, Germany 1 , 2AGH, Kraków, Poland 2
cyrklaff@me.com

Severe malaria risks health and lives of mainly children in subtropical areas (WHO 2011) and is related to Plasmodium falciparum parasites invasion and asexual proliferation cycles in host erythrocytes. However, individuals with mutated hemoglobin such as sickle cell, thalassemia, and other hemoglobinopathies are protected from severe outcomes of malaria.

Central to Plasmodium pathogenesis is the cytoadhesion of infected erythrocytes to the endothelium and intracapillary sequestration (Voss 2006). The blood stages parasites produce cyto-adhesins (PfEMP1) that are transported through the cytosol of host erythrocyte and presented on the surface. This poses a challenge for the parasite as the entire transport machinery must be assembled de novo.

Using cryo-electron tomography, we have viewed into whole, intact erythrocytes infected with P. falciparum (Cyrklaff 2011, Cyrklaff 2012) (Fig 1). The tomograms revealed that the parasitic assemblies in host erythrocyte, such as Maurer’s clefts, tubulo-vesicular networks and knobs are interconnected via elaborated networks of actin filaments (Fig 1A). The parasite majorly remodels the actin networks from what was seen in uninfected erythrocytes (Fig 1B) and used it to its own benefits. Numerous vesicles were attached to the networks and we proposed that the parasite uses the vesicular transport along actin filaments to facilitate presentation of the adhesins at the erythrocytic surface.
Markedly, the actin networks appeared incomplete in Plasmodium infected sickle cells (Fig 2). This presents a likely explanation for the protective mechanism of sickle erythrocytes, whereby the parasite was not capable of remodelling the host actin cytoskeleton. We attributed this to differences in actin polymerization dynamics in the presence of denatured hemoglobin, the process that we also verified in vitro (Cyrklaff 2011).
The mechanism of interference in the transport of malicious adhesins to cell surface is similar for homo- and heterozygotic hemoglobinopathies. The latter are of particular medical interest, as the protection against malaria is not compromised by disorders associated with strong phenotypes such as sickle disease and thalassemia major.

What we have learned from natural protection we want to apply in practice. We try to replicate the protective role of sickle cells in normal erythrocytes by interfering with the parasitic export through host cytoplasm. This concept is worth pursuing as, when successful, it would eliminate the problem of resistance, similarly to the fact that there is no parasites resistant against the structural hemoglobinopathies.

WHO 2011; Lives at Risk: Malaria in Pregnancy
Voss et al 2006 Nature 439:1004
Cyrklaff et al 2011 Science 334:1283
Cyrklaff et al 2012 Trends in Parasitology 28:479

This work is supported by the grant from the Bill and Melinda Gates Foundation (Grand Challenges Explorations; grant number OPP1069409)

Fig. 1: Figure 1: Sections through cryo-electron tomograms and surface rendered views of (A) P. falciparum-infected erythrocyte containing normal HbAA hemoglobin; (B) uninfected HBAA erythrocyte; PM, erythrocyte plasma membrane (dark blue); K, knobs (red); V, vesicles (cyan); MC, Maurer’s clefts (cyan); filaments (yellow)-arrowheads. Scale bars: 100 nm

Fig. 2: Figure 2: Section through cryo-electron tomogram and surface rendered view of P. falciparum-infected erythrocyte containing mutated HbCC hemoglobin; colours and marking as in Fig 1. Scale bar: 100 nm

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-7-IN-6080 A structural understanding of how to build, divide and change a trypanosome parasite cell

Gull K.1
1Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
keith.gull@path.ox.ac.uk

The precise shape and form of single celled parasites enabled their early classification as etiological disease agents based solely on microscopy. The changes in shape and form of the kinetoplastid parasites (amastigote, promastigote, epimastigote, slender/stumpy trypomastigotes) have evolved for particular pathogenicity niches and coordinate with cell functions at different lifecycle stages.

The cytoskeleton defines their shape and form, providing a clear rationale for the importance of understanding how it functions to define each form. Leishmania, Trypanosoma brucei, T. cruzi still represent major threats to world health and agricultural development. Trypanosomes in sub-Saharan Africa cause Human African Trypanosomiasis and the livestock disease nagana.

We have studied the structural organization of these parasites using light and electron microscopy techniques ranging form immunofluorescence, ImmunoEM, EM tomography, SEM, serial block face SEM etc. The T. brucei trypomastigote cell form possesses a single flagellum that is attached to the side of the cell body. The Basal Body duplicates in the cell cycle, performs a 3D traverse around the old flagellum axis, facilitating kinetoplast (mitochondrial genome) segregation and new Flagellar Pocket formation. Concomitantly, the new flagellum extends outside of the cell with its tip attached to the side of the old flagellum via the mobile, transmembrane Flagella Connector junction. Internally, a Flagellum Attachment Zone (FAZ) filament and associated microtubule quartet extend forming a seam in the sub-pellicular microtubule array. Double transmembrane complexes then assemble to cross-link the flagellum to the cell body. I will describe this process and then show how it is modified bloodstream form parasites and in Leishmania parasites that can reduce the motile flagellum to a short, immotile sensory 9+0 and 9v sensory flagellum in the amastigote form. In addition I will describe, using freeze fracture approaches, how these cytoskeletal structures define membrane domains that characterize the cell body, flagellum and flagellar pocket.

These structural features will be discussed in relation to the pathogenicity features of these parasites.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-7-O-1606 Cryo-tomography reveals the morphological difference between Plasmodium rhoptries

Lemgruber L.1,3, Kudryashev M.2, Stahlberg H.2, Frischknecht F.3
1Laboratório de Microscopia, Diretoria de Metrologia Aplicada às Ciências da Vida - DIMAV, Instituto Nacional de Metrologia, Qualidade e Tecnologia, INMETRO, 2Center for Cellular Imaging and Nano Analytics (C-CINA), Biozentrum, University of Basel, 3Parasitology, Department of Infectious Diseases, University of Heidelberg Medical School
llemgruber@gmail.com

Rhoptries are organelles that have a key role in Apicomplexa biology. During infection, these organelles take part in several essential and complex processes that include host cell entry and parasites development. Several proteins have been characterized and localized in distinct areas of these organelles, mainly in Toxoplasma gondii and Plasmodium falciparum. Particular in Plasmodium a genus that contains several species, it is believed that the morphology of the rhoptries is the same. And so the localizations of the proteins would not present any difference. In order to clearly settle this question, we used cryo-electron tomography to study the morphology of the merozoites rhoptries of the two main Plasmodium species used in experiments: P. berghei and P. falciparum. The reconstructed tomograms revealed that P. berghei rhoptry presented a cylinder bulb with a narrow, long neck, similar to the rhoptry of T. gondii. The rhoptry apertures were located within the apical rings. In some tomograms, a close proximity was observed with the apicoplast. The density of the rhoptry was homogeneously with both the neck and the bulb being electron dense, with the bulb probably containing the majority of the lipidic content (it started to bubbled before any other part of the merozoite). In P. falciparum, the rhoptry presented a more tear-shape morphology, with the neck being more short and wider than in P. berghei, with the neck also opening within the apical rings. The rhoptries were closer to the subpellicular microtubules. The rhoptries appeared to be less electron dense than the ones from P. berghei.

LL is supported by a Pronametro fellowship from INMETRO. The German Research Foundation, the Cluster of Excellence CellNetworks and European Research Council founded this work.

Fig. 1: 1 – A section from a cryo-tomogram from a Plasmodium berghei merozoite, showing the cylinder bulb from one rhoptry. Note the rhoptry’s long, narrow neck. Arrowheads points to the neck of the other rhoptry. 2 – A cryo-electron micrograph from a Plasmodium falciparum merozoite. Arrows point to the tear-shape rhoptries. Bar = 30 µm.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-7-O-2302 Structural changes of cell lines after the real and simulated inoculations with Cryptosporidium muris oocysts

Melicherová J.1, Valigurová A.1
1Department of Botany and Zoology, Brno, Czech Republic
janka.melicherova@gmail.com

    In the present study, two types of cell lines, HCT8 and HT29, were used for an in vitro cultivation of the gastric parasite Cryptosporidium muris (Cryptosporidiidae, Apicomplexa). Evaluations of structural changes of both cell lines were carried out after 24, 48 and 72 DPI using combined approaches of light, electron and confocal laser scanning microscopy. So far, we succeed to detect sporozoites released from oocysts, free sporozoites gliding on the cell line surface that were equipped with a typically prolonged apical end and seemed to search for appropriate infection site, and few structures closely resembling full or already emptied cryptosporidian parasitophorous sacs. Evidently, newly formed round cells or gaps in a discontinuous layer characteristic for young cell cultures were frequently preferred and attacked by invading sporozoites. Interestingly, after 24 DPI, both cell lines started to embrace unexcysted oocysts of C. muris. These oocysts were found to be completely or partially enveloped by projections of individual host cells. The experimental inoculation with polystyrene microspheres was designed in order to verify whether this behaviour of cell lines is provoked by oocysts of C. muris or it represents their innate reaction to foreign objects in general. The microspheres were found only occasionally to be covered by a tiny filamentous projections arising from host cells or remnants in old cell cultures. Direct comparison and evaluation of both cell lines inoculated either with C. muris oocysts or with polystyrene microspheres confirmed that the enclosing of oocysts by HT29 and HCT8 cells was induced by the parasite. Based on present data, we consider this to be a natural adherence of biological garbage to the surface of polystyrene microspheres.

Financial support was provided by a postdoctoral grant GPP506/10/P372.

Fig. 1: HT29 cell lines started to embrace unexcysted oocysts of C. muris.

Fig. 2: The polystyrene microsphere weren’t enveloped by HCT8 cell lines.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-7-O-3221 The role of macromolecules in the mineralization process of the calcareous sponge Paraleucilla magna class calcarea

Rossi A. L.1, Bulou H.2, Campos A. P.3, Barroso M. S.4, Klautau M.4, Archanjo B. S.3, Borojevic R.4, Farina M.4, Werckmann J.3
1Centro Brasileiro de Pesquisas Fisicas, Rio de Janeiro - RJ - Brasil , 2Institut de Physique et de Chimie des Matériaux de strasbourg,Strasbourg, France, 3Instituto Nacional de Metrologia, Qualidade e Tecnologia, Duque de Caxias , Brasil., 4Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil
werck@ipcms.unistra.fr

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-7-O-3479 Structural and chemical aspects of the osmoregulatory system of Trypanosoma cruzi

Girard-Dias W.1,2, De Souza W.1,3, Miranda K.1,3
1Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho and Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens – Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2Plataforma de Microscopia Eletrônica Rudolf Barth – IOC / Fiocruz, 3Laboratório de Biologia estrutural, Diretoria de Programas, Instituto Nacional de Metrologia, Qualidade e Tecnologia-Inmetro
wendell@biof.ufrj.br

Trypanosoma cruzi is a protozoan parasite that has a complex life cycle, involving different hosts. During the course of the infection the parasite faces environments where extreme variations in the concentration of ions and osmolytes in the extracellular milieu are found. To cope with these fluctuations, the parasite has developed adaptation mechanisms that involve signaling pathways and remodeling of parasite organelles, as the contractile vacuole complex (CVC) and acidocalcisomes, which are electron-dense acidic organelles rich in calcium, polyphosphate and other cations, and shown to be involved in several functions as calcium homeostasis and osmoregulation. The structural and chemical aspects of these organelles have been intensely investigated by different techniques. At the electron microscopy level, two-dimensional analysis of thin sections of chemically-fixed cells has been one of the most commonly used techniques, despite the known potential of generating artifacts during chemical fixation and the subsequent steps of sample preparation, which is one of the main limitations to study these organelles by electron microscopy. In contrast, more sophisticated techniques, such as cryofixation followed by freeze substitution that are known to preserve the samples in a more close-to-native state, have not been widely applied to T. cruzi. Therefore, the use of these techniques is an alternative for structural and chemical preservation that, when combined with electron tomography and X-ray microanalysis, allow the obtainment of the three-dimensional information on the distribution of elements. In this work, we employed high-pressure freezing followed by freeze substitution techniques to study the 3D structure of the CVC and acidocalcisomes and analytical electron tomography to visualize the 3D distribution of the acidocalcisome elements. The structural preservation of the CVC was highly improved, showing a characteristic tubular structure in a polarized position of the parasite (Fig. 1). Three-dimensional analytical electron tomography (Fig. 2) showed a heterogenic distribution of cations (calcium, magnesium e.g) in contrast to anions (phosphorus and oxygen) that were homogeneously distributed within the acidocalcisome matrix (Fig. 3). Altogether, results show that cryofixation and electron tomography can significantly contribute to the comprehension of the spatial organization of the osmoregulatory system of this protozoan and the employment of analytical electron tomography can define the 3D distribution of ions inside organelles.

References:
P. Rohloff and R. Docampo, 2008. Exp. Parasitol., 118: 17-24.
R. Docampo et al, 2005. Nature Rev Microbiol, 3: 251-261.
R. A. Steinbrecht and K. Zierold, 1987. Springer, Berlin.

This work was supported by CNPq, FAPERJ, FINEP and CAPES (Brazil).

Fig. 1: A, Scheme of T. cruzi epimastigote. B and C, CVC at systole fase. D and E, CVC at diastole fase. F (flagellum) and K (kinetoplast). Scale bars: 200 nm.

Fig. 2: Analytical electron tomography of T. cruzi whole cells showing the 3D distribution of phosphorus, magnesium, calcium and sulphur inside the acidocalcisomes.

Fig. 3: Analytical electron tomography of T. cruzi whole cells showing the 3D distribution of phosphorus, magnesium and calcium in the acidocalcisome (arrow) segmented by threshold. Note the segregation of calcium and magnesium. F (flagellum) and K (kinetoplast). Scale bar: 500 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1445 The antihelminthic effects of a plant-derived compound, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), against Fasciola gigantica and Schistosoma mansoni

Lorsuwannarat N.1, Wanichanon C.1, Sobhon P.1
1Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand
natcha.lor@gmail.com

                     Fasciolosis and schistosomiasis by the trematodes in Fasciola and Schistosoma spp. are recognized as major global parasitic diseases that cause health problems in animals and humans as well as economic losses worldwide. At present, effective vaccines are not yet available; therefore, anthelmintic drugs including triclabendazole (TCZ) and praziquantel (PZQ) are the main method of control of the infections. However, resistances to these drugs have emerged and may pose a serious problem as no other effective drugs are yet available. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PB) is a compound derived from the roots of many plants, especially those in the Plumbaginaceae family, which is used as a traditional medicine for the treatments of several ailments including parasitic infections. However, there are only a few scientific reports of its potential and no data of its mechanism as an anthelmintic agent. The objective of this study was to investigate the in vitro anthelmintic activities of PB against Fasciola gigantica and Schistosoma mansoni, and its effect on the structure of the tegument and associated structures by light (LM), scanning (SEM), and transmission electron microscopy (TEM).
                     Based on the measurements of relative motility and survival index, PB showed more antihelmithic effect than TCZ and PZQ. When examined by LM and SEM, PB caused more damage in the tegument on male than female flukes. PB caused similar tegumental alterations as those observed in TCZ or PZQ treatments, but with greater severity, comprising of swelling, blebbing and rupturing of the tegument, loss of spines, and eventually erosion, lesion and desquamation of the tegument. When observed by TEM, PB-treated flukes exhibited markedly swollen mitochondria, followed by disruption of the apical plasma membrane, dilatation of basal infolds, depolymerization of microtrabecular and cytoskeletal networks, and formation of vacuoles throughout the tegument syncytium, followed by the breaking-down and detachment of the whole tegument. Over a long periods of incubation, the tegumental cell bodies, subtegumental musculature, and surrounding parenchymal tissue showed degeneration and necrotic changes, while TCZ and PZQ showed less effect at comparable doses and times. A test by MTT assay indicated that PB reduced mitochondrial activity, thus this may be the initial tripping point that triggered the cascade of structural changes in the tegument and underlying structures that eventually lead to parasites’ death.

This research was supported by a grant from Mahidol University to Prasert Sobhon, and the Thailand Research Fund-Mahidol RGJ Ph.D. Scholarship to Natcha Lorsuwannarat.

Fig. 1: Light micrographs (LM) of (a) untreated and (b) 100 μg/ml PB-treated 4-week-old juveniles of F. gigantica at 24 h, (c) untreated and (d) 10 μg/ml PB-treated adult S. mansoni at 3h

Fig. 2: Scanning electron micrograph (SEM) of the tegumental surfaces of adult male S. mansoni from untreated control group

Fig. 3: Transmission electron micrographs of the 4-week-old juveniles of F. gigantica in untreated group after 24 h incubation in 0.1% DMSO. 

Fig. 4: Newly excysted juvenile liver fluke (NEJ) of Fasciola hepatica after in vitro incubated with (A) 0.1% DMSO as control, and (B) the purple formazan granules appeared throughout the tissues of control NEJ incubated 4 hour with MTT (Magnification approximately 300x).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1512 The effect of plumbagin from Plumbago indica root against motility of adult Haemonchus placei.

Saowakon N.1,2, Sobhon P.2
1Institute of Science, Suranaree University of Technology, Nakon Ratchasima 30000, Thailand , 2Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
naruwan@sut.ac.th

Although, the commercial anthelmintic drugs can inhibit nematode infection, but they have documented cases of resistant populations including Haemonchus spp. in Thailand. The drug discovery is urgent. Reportedly, the anthelmintic effect of purified plumbagin of Plumbago indica inhibited the motility of nematode and some trematodes. Therefore, this work aimed to investigate the anthelmintic effect of plumbagin (PB) of P.indica root on adult Haemonchus placei on relative motility (RM) assay and histopathological changes. Two hundred and forty flukes were divided in to 6 treatment groups (n=10 per group). Groups 1, 2, 3, and 4 flukes were incubated with M-199 medium containing plumbagin in the serial concentrations; 0.01, 0.1, 1.0, and 10 mg/ml, respectively. Group 5, as incubated with medium mixed with albendazole (ABZ) at 10 mg/ml as the positive control and group 6, and they were incubated with medium containing 0.1% DMSO as the negative control. Flukes were evaluated the RM values on 3, 6, 12 and 24 h incubation using scoring under the stero-microscopy. Then, they were collected from each observation time to run tissue processing for histopathological changes using H&E staining. The results showed that RM values of PB-treated groups at the concentration 10 mg/ml in H.placei were progressively decreased more than ABZ-treated group since 3 to 12 h exposure, and few activity of P. cervi was observed at 24 h exposure. Observation under the stereo-microscope, adult H.placei were partial move on 3 h and 6 h incubation in PB at concentration 10 mg/ml. At 12 h and 24 h exposure, they were dead which were confirmed by vital dye staining. The swelling tegumental surface of H.placei was occurred, but their tegument did not peel off. The slowly motility of H.placei was observed after 12 h and 24 h incubation with plumbagin at 0.1 and 1 mg/ml. Light microscopic observation showed similar tegumental layer as normal group of H.placei. But, the parenchymal cells and vitelline cells in deeper of parasites-treated plumbagin were found apoptotic appearance. These results suggest that plumbagin of P.indica could be against the motility of adult stage of H.placei.

Acknowledgements: This work was supported by the National Research Council of Thailand, Mahidol University and Suranaree University of Technology (SUT1-102-57-24-24).

Fig. 1: Figure 1. Light microscopes of PB-treated adult H.placeiat 10 mg/ml. The tegumental (T) layer is covered by theexternal sheath. The vitelline (Vit) gland show apoptotic damages (arrow), whereasthe gastrointestinal (G) tract has still normal appearance.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1532 3D structural analysis of malaria parasite-infected red blood cells by SBF-SEM

Sakaguchi M.1, Miyazaki N.2, Fujioka H.3, Kaneko O.1, Murata K.2
1Nagasaki University, Nagasaki, Japan, 2National Institute for Physiological Sciences, Okazaki, Japan, 3Case Western Reserve University, Cleveland, USA
miako@nagasaki-u.ac.jp

Malaria caused by Plasmodium parasites remains a major infectious disease in tropical and subtropical parts of the world, and 300-500 million malaria cases and about 1 million deaths are recorded annually. P. falciparum, that causes most severe malaria, has a complex life cycle involving asexual multiplication in the red blood cells (RBCs) in human host and sexual reproduction in the mosquito host. In the blood stage, P. falciparum grows through ring, trophozoite, and shizont stages to produce daughter cells within 48 h, and remodels the host RBC. To transport the parasite proteins to the infected RBC (iRBC) membrane surface, membranous structures called Maurer’s clefts are constructed in the iRBC cytoplasm acting as a sorting compartment in trafficking. The cytoadherent ligand PfEMP1 is exposed at protrusions called knobs on the surface of the iRBC to adhere to several receptors on the vascular endothelium and to evade the host immune system and hence elimination from human body.

The molecular biology of Plasmodium infection is well studied, but the ultrastructural morphological characterization is still not sufficient. Although ultrastructural 3D reconstruction of the whole Plasmodium-iRBC by electron tomography and FIB-SEM has been reported recently [1-4], the quantitative 3D analysis of the whole structure has little been performed. In this study, we used SBF-SEM to image the 3D structure of multiple whole Plasmodium-iRBCs. The 3D organization showed that Maurer’s clefts are not sub-compartments or membrane extensions of the parasitophorous vacuole membrane but independent membrane structures as reported by using other microscopy techniques [1, 2, 5]. Moreover, we will discuss the quantitative analysis of the numbers and/or sizes of their cell components at the ultrastructural level.

References

[1] Hanssen et al., Int J Parasitol 40: 123-134 (2010)
[2] Hanssen et al., J Struct Biol 173: 161-168 (2011)
[3] Weiner et al., Cell Microbiol 13: 967-977 (2011)
[4] Medeiros et al., PLoS One 7: e33445 (2012)
[5] Spycher et al., Mol Microbiol 68: 1300-1314 (2008)

We thank M. Nagayoshi for helping with the 3D reconstruction. This work is supported by MEXT Grants-in-Aids for Scientific Research 24590508 (M.S.).

Fig. 1: SBF-SEM slice including P. falciparum-infected red blood cells.

Fig. 2: 3D reconstruction of the whole P. falciparum-infected red blood cell.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1628 Production and Characterization of Monoclonal Antibody Against Recombinant Leucine Aminopeptidase (rFgLAP) of Fasciola gigantica

Chantree P.1, Changklungmoa N.2, Songkoomkrong S.1, Wanichanon C.1, Sobhon P.1
1Department of Anatomy, Faculty of Science, Mahidol University, Ratchathewi, Bangkok 10400, Thailand, 2Department of Pathobiology, Faculty of Science, Mahidol University, Ratchathewi, Bangkok 10400, Thailand
pathaninchan@gmail.com

Fasciola gigantica is a prevalent trematode parasite of fasciolosis in tropical regions, where it causes serious losses of domestic animals, especially cattle, sheep and goat. The parasite can also infect humans. During development, there are various proteases that the parasites use for migration, invasion, processing of nutrients, and evasion from the hosts’ immune responses. During feeding, the parasites utilize endopeptidases, including cathepsins B, L and D, to digest the host hemoglobin into short peptides. These are then digested by exopeptidases into free amino acids that are absorbed and utilized by the parasites. Leucine aminopeptidase (LAP) is in a family of metalloexopeptidases, which cleave short peptide fragments at the N-terminals. Fasciola gigantica possesses a member of these enzyme and because of its important role in digestion, invasion and migration through the host’s tissues, LAP is considered as a target for vaccine and immunodiagnostic candidate for fasciolosis. In this study, The recombinant protein (rFgLAP) was expressed in prokaryotic expression system. It was then used for immunization of BALB/c mice to produce MoAbs. Reactivity and specificity of this monoclonal antibody was assessed by indirect ELISA and immunoblotting. This MoAb reacted specifically with 56.7 kDa of rFgLAP. Localization of this antigen by immunohistochemistry methods showed that this antigen was presented in the apical cytoplasm of caecal epithelial cells. These findings suggested that FgLAP may be a new candidate for immunodiagnosis for fasciolosis which will be developed by sandwich ELISA method.

This research was supported by a research grant from Mahidol University to Prasert Sobhon and RGJ- Ph.D. Scholarship from Thailand Research Fund to Pathanin Chantree

Fig. 1: Localization of FgLAP protein in metacercaria (MET)stage of F. gigantica by immunoperoxidase and immunofluorescence technique showing positive immunoreactivity in caecal epithelial cells.

Fig. 2: Localization of FgLAP protein in newly excysted juvenile (NEJ) stage of F. gigantica by immunoperoxidase and immunofluorescence technique showing positive immunoreactivity in caecal epithelial cells.

Fig. 3: Localization of FgLAP protein in adult F. gigantica by immunoperoxidase technique The negative control (A). (B) Sections stained with MoAb showing positive immunoreactivity in caecal epithelial cells. .C,D) High magnification of the tall caecal epithelial cells showing intense positive staining in the apical part of the gut epithelial cells.

Fig. 4: Localization of FgLAP protein in adult F. gigantica by immunofluorescence technique The negative control (A). (B) Sections stained with MoAb showing positive immunoreactivity in caecal epithelial cells. .C,D) High magnification of the tall caecal epithelial cells showing intense positive staining in the apical part of the gut epithelial cells.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1704 Co-localization of crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) in the eyestalk of the black tiger shrimp, Penaeus monodon

Pratoomthai B.1, Chayaburakul K.1, Anantasomboon G.1, Withyachumnarnkul B.2
1Anatomy Unit, Faculty of Science, Rangsit University, Paholyotin Road, Pathumthani, 12000, Thailand, 2Centex Shrimp, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
maew_199@hotmail.com

In crustacean, crustacean hyperglycemic hormone (CHH) is involved in the control of important physiological process such as the sugar metabolism, molting and reproduction. Molt-inhibiting hormone (MIH) controls molting by inhibit the synthesis and secretion of ecdysteroid or molting hormone. Both hormones are produced and secreted from X-organ sinus gland complex in the eyestalk optic lobe. It has been reported that CHH/MIH peptides are expressed within the same cell in the optic lobe of some species. Therefore, the purpose of this study is to localize CHH/MIH in the optic lobe of the black tiger shrimp, Penaeus monodon. Eyestalk of P. monodon (20g BW) were fixed in Davidson’s fixative, dehydrated and embedded in paraffin. Polyclonal antibodies against CHH and MIH were used as the primary antibodies, and FITC-conjugated goat anti-rabbit/mouse IgG was used as the secondary antibody for immunofluorescent staining and sections of the optic lobe were examined under laser-scanning confocal microscope. The results revealed that numerous neurosecretory cells in the medulla terminalis of the optic lobe contained CHH and few cells contained MIH. Some contained both CHH and MIH, but some contained neither one of the two hormones, which are undistinguishable by morphological criteria, but clearly distinguishable by specific antibodies. The sinus gland and the axonal tract of the eyestalk also contained both CHH and MIH. Therefore CHH/MIH co-localization may exist, though not always being the case, in this economic penaeid shrimp.

This study was funded by the Centex Shrimp, Mahidol University, Rangsit University and by the NSTDA, Ministry of Science and Technology of Thailand.

Fig. 1: Photomicrograph by laser-scanning confocal microscope showing immunofluorescence of MIH (green) and CHH (red) in the X-organ (XO) (A) and co-localization of MIH and CHH (orange) in sinus gland (B) of the Penaeus monodon optic lobe.

Fig. 2: Photomicrograph by laser-scanning confocal microscope showing immunofluorescence of MIH (green) (A), CHH (red) (B), and co-localization of MIH and CHH (orange) (D) in the X-organ of the Penaeus monodon optic lobe. In negative control (without antibody) sections, no fluorescence signal were observed (C).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1722 Morphological diferences between African camallanid nematodes revealed by SEM

Masova S.1, Barus V.1, 2
1Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic, 2Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Květná 8, 603 65 Brno, Czech Republic
masova@sci.muni.cz

Fish nematodes are important group of parasites, because it can cause several serious diseases. They can infect any part of the fish body, including the body cavity, internal organs, deeper layers of the skin or fins, and external muscle layers as adults or as larvae. During ichtyoparasitological research in several African countries carried out from 2005 to 2013 were revealed, among other parasitic species, nematodes referable to species Procamallanus laeviconchus (Wedl, 1862) from family Camallanidae (Camallanoidea). SEM of their outer morphology revealed, that under this name is several morphospecies hidden. Procamallanus laeviconchus sensu lato is quite common nematode of various African fish species and has a Pan-African distribution. It has indirect life cycle, crustaceans (copepods) serve as intermediate hosts. Catfishes seem to be the most frequent definitive hosts. Specimens were recovered mainly from stomachs of catfishes from families Mochokidae, Clariidae and partially also from Bagridae (Siluriformes), and citharinids from Citharinidae (Characiformes).
For study of important, however poorly recognizable morphological structures, light microscopy (LM), scanning electron microscopy (SEM) and partially also environmental scanning electron microscopy (ESEM) were used. Samples were prepared by standard methods for SEM and examined using a Quanta TM 250 FEG SEM at an accelerating voltage of 10 kV or JEOL JSM-7401F FE SEM at an accelerating voltage of 4 kV.
All studied procamallanid specimens from different host fish species were medium-sized nematodes with thick, roughly transversely striated cuticle. Some of them showed features as oval mouth with peribuccal flange forming 6 bifid lobes (Fig. 1). Mouth of all samples were usually surrounded by six flat, crescent-shaped elevations, however variously elevated. All samples had 8 submedian cephalic papillae arranged in 2 circles, each formed by 2 papillae, however they differ in number of small small finger-shaped processes on the conical tail.
Procamallanus spp. are mutually recognizable by mouth opening with or without oral flange (Figs. 1, 2) and by number and shape of projections on tail tips of females. Examination of these nematodes by SEM showed that the material comprise several species new to science.

This study was supported by the Department of Botany and Zoology, Faculty of Science, Masaryk University and by the Czech Science Foundation (project No. P505/12/G112).

Fig. 1: A. Head region of nematode from Distichodus niloticus with peribuccal flange and crescent-shaped elevations. B. Head region of nematode from Clarias gariepinus without peribuccal flange and low crescent-shaped elevations.
Fig. 2:
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1769 Behavior of the Lysosome related organelle during differentiation of Giardia intestinalis

Midlej V.1, 2, De Souza W.1, 2, 3, Benchimol M.1, 2, 3
1Universidade Santa Úrsula, Rio de Janeiro, Brazil, 2Instituto de Biofísica Carlos Chagas Filho, UFRJ , Rio de Janeiro, Brazil, 3Instituto de Metrologia – Inmetro, Rio de Janeiro, Brazil
marlenebenchimol@gmail.com

Giardia intestinalis is an unicellular parasite that commonly causes diarrheal disease all over the world. Giardia is an eukaryotic cell that presents two nuclei with nuclear membranes, an endomembrane system consisting of the endoplasmic reticulum (ER) and peripheral vesicles (PVs) and cytoskeleton structures such as the adhesive disk , median body and the funis. During its life cycle the protozoan presents two developmental stages: the flagellated trophozoite which attaches to the microvillus border of the small intestine and is responsible for the disease symptoms, and the cyst which is the resistant infective form. The transformation of trophozoites into cysts is known as the encystation process and characterized by the appearance of large vesicles named encystation secretory vesicles (ESVs). Although typical lysosomes are also not found, this parasite presents a large number of peripheral vesicles (PVs) that show acid phosphatase activity, and accumulate macromolecules ingested by the protozoan. Enzyme cytochemistry, showed that acid phosphatase, SH-containing proteins, and glucose-6-phosphatase, are localized in the PVs1. Furthermore, PVs also contains cysteine endoproteases that are orthologous to the cathepsin L and cathepsin B found in lysosomes of higher organisms and are therefore useful markers of cell compartments where protein degradation takes place2. These data suggest that the PVs fulfill all criteria to be identified as early and late endosomes, as well as lysosomes, representing an ancient structure that later on, during evolution, was separated into distinct compartments. In the present study we decided to analyze further the endomembrane system of G. duodenalis during the process of encystation. In order to analyze the behavior of PVs during the differentiation process of the parasite, G. intestinalis were induced to encyst in vitro. Lucifer yellow and Acridine orange markers were used to track the PVs during encystment. Moreover, acid phosphatase cytochemistry technique was performed. The results were observed using fluorescence microscopy and transmission electron microscopy (TEM), respectively. Biochemistry analysis of phosphatase activities was performed, measuring the rate of p-nitrophenol (p-NP) production. Our data show a fluorescence decrease during encystment process when Lucifer yellow and Acridine orange dyers were used. The same results were observed during cytochemical localization of acid phosphatase activity; a reduction in the electron dense stain was noted in parasites after 21h pos-encystment induction.

References

1.Lanfredi-Rangel et al., J.Struct. Biol., 1998, 123, 225. 

2.Ward et al., Cell, 1997, 89, 437.

This work was supported by CNPq, FAPERJ, PRONEX, INMETRO and AUSU.

Fig. 1: Cytochemistry for acid phosphatase in giardia trophozoite (a) and 21h-encysted (b) analyzed by TEM. The encysted cell is identified by ESV; the peripheral vesicles (PV) are indicated with arrows. The decrease of acid phosphatase staining is noted (b). Moreover, plasma membrane phosphatase detection (arrowhead) is only observed in encysted cell.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1859 Using environmental scanning electron microscopy (ESEM) as a non-invasive method to studying fixed parasites

Masova S.1, Nedela V.2, Tihlarikova E.2
1Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic, 2Institute of Scientific Instruments of the ASCR, v. v. i., Královopolská 147, 612 64 Brno, Czech Republic
masova@sci.muni.cz

Scanning electron microscopy (SEM) is popular and for taxonomy of parasites very important and not substituted method in many times. However sometimes taxonomists have only one specimen and cannot use classical SEM, because their sample (poor conductor) have to be fixed, dehydrated and coated before it can be observed. This method condemns samples for destroying and do not allow other using of it, e.g. for molecular study or depositing as type material in museum. Moreover, the specimen preparation is often long and slow. Environmental scanning electron microscopy (ESEM) brings two main advantages: elimination of speed of sample preparation and non-invasivity.
We have made ESEM observation on several groups of already fixed parasites in 4% formaldehyde solution or 70% ethanol: crustacean (Ergasilus sp.), nematode (Contracaecum osculatum) and others. Observations were made with the experimental environmental ESEM AQUASEM-II redesigned in the Institute of Scientific Instruments of the Academy of Sciences of the Czech Republic by Ionization and YAG-BSE detectors. The samples were cooled down to 2°C and observed in an high pressure water vapour environment of 650–700 Pa. Samples were placed on a Peltier cooled specimen holder to a drop of water. Consequently the water was slowly evaporated from the sample, see Fig. 1.
Combination of SEM and ESEM techniques brought two slightly different views. Conventional SEM has better contrast, more details in microstructure and resolution, however in specific case, for example at nematodes; it is ESEM almost comparable with SEM. In this study, we showed that ESEM allows the examination of specimens at high pressure conditions without any previous treatment in fully hydrated state and can be used effectively in taxonomical studies of the parasites, where valuable unique specimens sometimes exist.

This study was supported by the Department of Botany and Zoology, Faculty of Science, Masaryk University, and by projects No. P505/12/G112 and No. GA 14-22777S.

Fig. 1: Sequential drying of Contracaecum osculatum specimen documented by ESEM AQUSEMII. Scale bars: 100 µm. Observation parameters were: cooling temperature 2°C, pressure of water vapour 680 Pa, distance between the sample surface and the second pressure limiting aperture 2.7 mm, accelerating voltage 20 kV and probe current 95 pA.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1910 Improved preservation of Toxoplasma gondii by High Pressure Freezing and Freeze-Substitution

Travassos de Lima R.1, De Souza W.1, Attias M.1
1Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
mattias@biof.ufrj.br

Toxoplasma gondii, the agent of Toxoplasmosis is an obligatory intracellular parasite of warm-blooded animals, including humans [1]. The ultrastructure of T. gondii was described mainly based in scanning (SEM) and transmission (TEM) electron microscopy of chemically fixed specimens. Figure 1 displays a ultrathin section of a typical tachyzoite form fixed with 2.5% glutaraldehyde in 0.1M cacodylate buffer followed by post-fixation in 1%OsO4 in the same buffer. Chemical fixatives typically have a slow rate of penetration and usually cells suffer hypo or hyperosmotic shock during fixation. High pressure freezing (HPF) followed by freeze substitution can avoid these artifacts, bringing a more realistic view of the ultrastructure. Tachyzoites from culture cells were inserted by
capillarity in cellulose capillaries and quickly frozen in a Balzers HPM010. Freeze substitution was carried out in a Leica AM EFS2. Four protocols were tested. Best results were obtained with 0.1% tannic acid in absolute acetone at -70oC for 24h, rinsed in acetone at -90oC, followed by 0.1%uranyl acetate, 1% OsO4,and 0.5% H2O. Temperature was raised at 1oC per hour up to 4oC; samples rinsed in absolute acetone and embedded in Epoxy resin. General organization of the cell was confirmed, except for ribosomes that were not randomly scattered in the cytoplasm, but rather formed clusters of
polysomes. Rhoptries did not have the spongy and biphasic appearance, but were homogeneously stained. These two features probably are more faithful to the real ultrastructure of T. gondii because of the speed of immobilization resulting from HPF. However, impregnation of the fixatives in internal membranes under low temperatures did not result in contrast, so the inner membranes were clear and the cytoplasm was very electron dense (Figure2). The inner membrane complex internal space was also electron lucent. Addition of tannic acid enhanced preservation of the microtubules.
Although, new information was brought by this fixation protocol, it also had some artifacts, as the tendency of the outer membrane to detach from the surface. In conclusion, routine chemical fixation, reliably preserves the ultrastructure of Toxoplasma gondii, but high pressure freezing and freeze substitution techniques have revealed some novel aspects of the ultrastructural organization of this parasite.

Acknowledgements: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas de Amparo à Pesquisa (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Fig. 1: Toxoplasma chemically fixed. Rhoptries (R) are biphasic and ribosomes (*) are scattered homogeneously in the cytoplasm. M-mitochondrion. 

Fig. 2: HPF fixed, freeze substituted Toxoplasma. Rhoptries  (R) are homogeneously stained and ribosomes form clusters (*). M-mictochondrion, C-conoid.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1923 Trypanosomes associated with the woylie, a critically endangered Australian marsupial

Clode P. L.1, Botero A.2, Thompson A.2
1Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Crawley WA 6009, Australia, 2School of Veterinary Sciences, Murdoch University, Murdoch WA 6150, Australia
peta.clode@uwa.edu.au

Introduction: Trypanosomes are parasitic protozoa that will typically exploit a bloodsucking insect and the blood and/or tissues of a vertebrate as a host. Natural mixed infections of different species and genotypes of trypanosomes occur frequently in a variety of hosts. Their interaction may affect or modify parasite infection dynamics through co-operation or competition, either by reducing or enhancing parasitemia, virulence and pathogenicity in the host.

Results: Our studies have revealed the presence of three different species of trypanosomes naturally occurring within nine species of West Australian marsupials [1]. However, the woylie Bettongia penicillata, which has undergone a massive population decline (~90% over 10 years) and has been listed as a critically endangered species since 2006 [2, 3], is the only marsupial that has been found to be highly infected with trypanosomes and also to harbour mixed infections.

In woylies only single-species infections were found in the blood, however, co-infections with two or three trypanosome species were found in individual tissues such as heart, skeletal muscle, lung and oesophagus. Infected tissues displayed significant levels of inflammation and damage. Interestingly, significant differences in Trypanosoma species and prevalence rates of mixed infections were found when comparing animals from a stable population at Karakamia Sanctuary, to an unstable, declining population in the Upper Warren region.

While T. copemani is known to naturally infect other native Australian marsupials, the species found to be dominant in declining woylie populations appears to be pathogenic. T. copemani is shown to have similar biological behaviour in the host to the pathogenic T. cruzi, which is responsible for Chagas disease in humans. Both species are capable of invading cells and colonising different tissues in the host, and are able to produce an inflammatory process that can result in significant damage to vital organs including the heart and liver. Parallel in vitro studies have further confirmed the capacity of T. copemani to invade cells.

Conclusion: Our results show that the marsupial B. penicillata is highly susceptible to co-infections within it’s tissues by trypanosomes, which is atypical for Australian marsupials. We suggest that interactions between these different trypanosome species is playing an important role in the rapid population decline of the woylie.

1. Botero, A. et al. Int J Parasitology: Parasites & Wildlife 2, 77–89 (2013).

2. Wayne, A. F. et al. Wildl. Res. 40, 169 (2013).

3. Wayne, A. Oryx -The International Journal of Conservation. In press, (2014).

The authors acknowledge use of the facilities at the Centre for Microscopy, Characterisation & Analysis, UWA, which is funded by State and Commonwealth governments; and funding from the West Australian Government's State NRM Program.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-1950 Existence and localization of COX 1 in the ovary of the giant freshwater prawn, Macrobrachium Rosenbergii

Soonklang N.1, Sumpownon C.2, Stewart P.3, Wanichanon C.2, Sobhon P.2
1Department of anatomy, Preclinical Science, Faculty of medicine Thammasat University, Thailand , 2Department of anatomy Faculty of Science Mahidol University, Thailand, 3Faculty of science education and engineering, University of the sunshine coast, Australia
nansoon13@hotmail.com

Several physiological processes are regulated by prostaglandins (PGs). In mammals, PGE2 is the most abundant type of prostanoid. PGE2 is involved in many reproductive processes including ovarian follicular function, ovulation, luteolysis and parturition in numerous species.

In crustaceans, several studies reported that PGs are involved in the regulation of reproduction. However, the mode of PG biosynthesis in arthropods and enzymes involved in these pathways has remained unresolved.

The key enzymes in the synthesis of PGs from arachidonic acid is prostaglandin endoperoxide G/H synthesis or cyclooxygenases (COX). COX is present all the vertebrate and has been used as a marker enzyme for the synthesis of PGs. The aim of this study is to verify the existence of COX and therefore PGs, in the reproductive system of decapods, using female freshwater prawns Macrobrachium rosenbergii as a model. The results revealed that COX1 immunoreactivity was detected in the cytoplasm around the nuclear membrane of oocyte stage 1 and 2 by using immunohistochemistry technique. However, there was no immunoreactivity detected in oocyte stages 3 and 4. The presence of COX1 immunoreactivity in the ovary suggests that COX is involved in the synthesis of PGs, Which may exercise control over the ovarian maturation in this species.

References

Botting, R.M. (2006). Journal of Thermal Biology. 31, 208-219.Varvas, K., Kurg, R. Hansen, K., Jarving, K., Jarving, I., Valmsen, K., Lohelaid, H., Samel., N. (2009). Insect Biochemistry and Molecular Biology. 39, 851-860.

 This research was supported by a Distinguished Research Professor Grant (co-funded by Thailand Research Fund, Commission on Higher Education, and Mahidol University) Ph.D. Scholarship to Assoc. Prof. Chaitip Wanichanon and Miss Chanudporn Sumpownon.

Fig. 1: Immunoperoxidase micrographs of developing oocytes of M. rosenbergii stained with COX1Ab. A and B: micrograph showing positive immunostaining at Oc1 and Oc2 in low, middle, and high magnification, respectively. C: Control section showing no immunostaining in low, middle, and high magnification, respectively.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2065 Localization of the catepsin L isoforms in the salivary glands of the hard tick Ixodes ricinus by transmission electron microscopy

Schrenková J.1,2, Vancová M.1,2, Nebesářová J.1,3, Kopáček P.1
1Institute of Parasitology, Biological Centre of ASCR, v.v.i, Ceske Budejovice, Czech Republic, 2Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic, 3Faculty of Science, Charles University in Prague, Vinicna 7, 128 43 Praha 2, Czech Republic
jana.schrenkova@gmail.com

An enzymatic cascade is responsible for the blood digestion in ticks. It contributes to the cleavage of hemoglobin molecules into shorter fragments and individual amino acids. Almost all of these enzymes occur strictly in the digestive cells inside the gut epithelium. Cathepsin L, however, is the only one peptidase which is expressed in other tissues too (Sojka et al., 2008). Apart from the gut, it can be localized also in the salivary glands, ovaries and Malpighian tubules. Although the function of the cathepsin L and its both isoforms (IrCL1, IrCL3) in the gut seems to be obvious, its role in the salivary glands of tick remains unclear. Detailed immuno-localization of this enzyme in the salivary glands during feeding should help us to find out its function. The specific antibodies against these peptidases were prepared by affinity chromatography and used for their detailed immunogold labeling of thawed cryosections according to Tokuyasu (Tokuyasu, 1973). Using transmission electron microscopes, we localized both isoforms of cathepsin L. It has been shown that both isoforms of cathepsin L are presented in type III acini, inside secretion granules of e cells. It has also been measured up that the amount of both isoforms increases during feeding. To further clear up the position of cathepsin L in the tick physiology, concretely in the digestive cascade, it might be appealing to use these and other methods, such as electron tomography or FIB/SEM, for co-localization of other members of the cascade.

References:
Tokuyasu K. T., 1973. A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol. 57: 551-565.

Sojka D., Franta Z., Horn M., Hajdušek O., Caffrey C. R., Mareš M., Kopáček P., 2008. Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases. Parasit. Vectors.,1: 7.

The authors acknowledge funding from the Technology Agency of the Czech Republic, project TE01020118.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2079 Microscopy analyses of trilobite exoskeletons, new insight into an old topic

Rak S.1, Laibl L.2, Burdikova Z.3
1Department of Zoology, Faculty of Science, Charles University, Viničná, Prague, Czech Republic, 2Charles University, Faculty Institute of Geology and Palaeontology, Albertov, Prague, Czech Republic , 3Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Prague, Czech Republic
deiphon@geologist.com

Trilobites are well known as an extinct arthropod taxa from the Palaeozoic Era. Worldwide palaeontologists studied them for more than hundred years and described many forms and types. My study is concentrated on their structures and ontogeny and main target is to analyse their life as well as feeding habits based on morphology. During my study I am trying to applicate microscopy analyses on two extraordinary preserved trilobites: on ontogenetic stage and cephalic spine of another taxon. The exceptionally preserved ontogenetic stages of Cyrtosymbole? sp. have been discovered in the locality near Brno. Material, preserved in muddy limestone, contains several protaspid specimens as well as disarticulated meraspid cranidia and pygidia. Thanks to SEM we are able to study microstructures and morphology. The protaspides are approximately 1 mm long and 0.9 mm wide, sub-ovoid in dorsal view. Protocranidium is sub-trapezoidal, with glabella reaching ca 80 % of protocranidial length. Trunk is semicircular in outline, composed of at least four segments. The morphology of the protaspides is typically proetoid and corresponds to a benthic post-metamorphic stage. This material represents the first Famennian ontogenetic stages of trilobites which has been discovered at the area of the Bohemian Massif and it represents the youngest known ontogenetic trilobite stages from the Czech Republic. Other object of my study is the cephalic spine of very rare trilobite Ancyropyge sola have been found for the first time in Czech Republic and its structure is poorly known based on Canadian finds. Thanks new microscopy photographs (ESEM) we can interpret its right screw-structure.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2105 The search for magnetoreceptive cells in the honeybee Apis mellifera

Boyd A.1, Saunders M.1, House M.2, Baer B.3, Cowin G.4, Mathes F.5, Shaw J.1
1Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Perth, Australia, 2Biomagnetics Group, The University of Western Australia, Perth, Australia, 3Centre for Integrative Bee Research (CIBER), The University of Western Australia, Perth, Australia, 4Centre for Advanced Imaging, The University of Queensland, Brisbane, Australia, 5School of Earth and Environment, The University of Western Australia, Perth, Australia
jeremy.shaw@uwa.edu.au

Evidence that a diverse range of organisms can utilize the Earth’s magnetic field to orient and navigate has accumulated over a period of 50 years. Much of the evidence currently available is based on behavioral data, with the exact mechanistic basis of magnetoreception remaining one of the great unsolved mysteries in biology. Just as magnetotactic bacteria use magnetic iron oxide nanoparticles to take advantage of the Earth’s magnetic field for orientation, it has been hypothesized that a similar mechanism could be present inside specialized cells in higher animals. Future research on magnetoreception requires to complement behavioral studies by proximate approaches to identify the mechanistic basis of magnetic field detection at the cellular level. Novel correlative microscopic approaches that bridge a range of length scales now provide the necessary approaches to determine the location, structure and function of these elusive cells.

The honeybee Apis mellifera is known to exhibit magnetoreceptive behavior and represents an ideal model system for elucidating the cellular basis of this sense in animals. In theory, a magnetoreceptive system could function using only a small number of cells located virtually anywhere in the body, which has presented researchers with the problem of searching for a potentially rare cell type of unknown location and structure; the classic ‘needle in a haystack’ problem.

Here we used a broad range of imaging and analytical techniques to initially characterize the particulate composition of honeybee body parts. Using a specially developed filtration technique, the bulk particle fraction of the honeybee was isolated (Fig.1). The particles’ structure and composition have then been characterized using a variety of techniques. In parallel, 2D and 3D imaging techniques such as scanning electron microscopy (SEM), magnetic resonance imaging (MRI) and X-ray micro-computed tomography (micro-CT) were attempted in order to provide overall anatomical detail and potentially pinpoint the location of magnetoreceptor cells (Fig. 1). Our ultimate aim is to trace the location of the particles back to their original anatomical position and provide a detailed description of cell ultrastructure and function. This knowledge is crucial to guide future research to clarify the importance and function of magnetoreception in honeybees.

The ARC (DE130101660). The University of Western Australia RDA and UWA-UQ schemes. The National Imaging Facility and the Australian Microscopy & Microanalysis Research Facility.

Fig. 1: Anatomical information achievable by (A) Optical, (B) SEM and (C, D and E) MRI imaging in the honeybee. (E) An MRI section through the abdomen reveals the internal anatomy, including highly contrasting material beneath the cuticle (arrowheads). (F) SEM has also been used to image magnetically responsive particulates extracted from whole bees.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2195 Biological effect and subcellular localization of aromatic diamidines in Trypanosoma cruzi

Batista D. G.1, Kumar A.2, Branowska D.2, Ismail M. A.2, Hu L.2, Boykin D. W.2, Soeiro M. C.1
1Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz,Rio de Janeiro, 962 RJ, Brazil, 2Department of Chemistry, Georgia State University, Atlanta, 30302 Geogia, USA
denisegama@ioc.fiocruz.br

Trypanosoma cruzi, an intracellular protozoan of the Trypanosomatidae family, is the etiological agent of the Chagas disease that is a tropical neglected illness that is the leading cause of heart disease in Latin America where it affects approximately 12 million people living in very poor social conditions. Today, it still represents a serious public health problem in these affected areas claiming for care and resolution of its current challenges, including the imperative need to sustain public policies related to the transmission control and the requirement for new chemotherapic agents. Pentamidine and related di-cations are DNA minor groove-binders with broad-spectrum anti-protozoal activity. In this context, our aim was investigate the tripanocidal activity in vitro of six di-cationic compounds – DB1582, DB1627, DB1645 DB1646, DB1651 e DB1670 – against bloodstream trypomastigotes (BT) of Trypanosoma cruzi and their cellular targets by fluorescence microscopy. Our results demonstrated that DB1645, DB1582 and DB1651 were the most active against BT showing IC50 values ranging between 0.15 and 6.9 µM. The compounds displayed toxicity after 24 h of incubation with 96 µM less than 20%. Following treatment for 72 h, DB1627, DB1645, DB1651 and DB1670 resulted in 36, 21, 45 and 22% loss of cellular viability at 96 µM drug concentration, while both DB1582 and DB1646 gave a <20% reduction. DB1645, DB1582 and DB1651 were also the most effective against intracellular parasites, with IC50 values ranging between 7.3 and 13.3 µM. Due to the characteristics of the tested compounds, blue fluorescence is emitted when excited by UV light, the amidines could be localized in both cellular structures of both BT and amastigotes through fluorescence microscopy. When the parasites were treated for 1 h with 10 µg/ml of each compound, we observed that in the treated parasites the diamidines presented a striking localization within the kDNA (asterisk) and at much lower levels in the parasite and host cardiac cells nuclei (thick white arrow). Interestingly, DB1582 and DB1651 were also localized in several punctated non-DNA containing organelles (thin white arrows) distributed within the BT and intracellular forms, which is related to acidic compartments (acidocalcisomes) localization and morphology (thin arrow). Our data suggest that the localization of these amidines in these organelles may be a consequence of their intracellular accumulation in these cellular sites (storage sites) and/or due to their primary or secondary drug targets. These studies may aid future design and synthesis of novel agents that could be used for Chagas disease therapy.

The present study was supported by grants from Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro, Conselho Nacional Desenvolvimento Científico e Tecnológico, FIOCRUZ, PDTIS, CAPES and CPDD.

Fig. 1: Intracellular localization of the compounds in bloodstream (A–E, G) and amastigote (F) forms of Trypanosoma cruzi after 1 h of incubation of each diamidine. DB1582, DB1627, DB1645, DB1646, DB1651 and DB1670.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2240 Detection and immunolocalization of the enzyme constitutive nitric oxide synthase (cNOS) in promastigotes forms of the Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis

Furtado R. R.1,2,4, Rodrigues A. D.3, 4, Farias L. S.1,4, Silva E. O.1,4
1Laboratório de Parasitologia e Laboratório de Biologia Estrutural, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Pará, Brazil, 2Laboratório de Leishmanioses, Instituto Evandro Chagas, Secretaria de Vigilância em Saúde do Ministério da Saúde, Ananindeua, Pará, Brazil, 3Laboratório de Microscopia Eletrônica, Instituto Evandro Chagas, Secretaria de Vigilância em Saúde do Ministério da Saúde, Belém, Pará, Brazil, 4Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Brazil
luishsf@gmail.com

American cutaneous Leishmaniasis is a parasitic disease, widely spread in most countries of Latin America and caused by different species of the genus Leishmania. This protozoan is an obligate intracellular parasite that developed mechanisms to subvert the microbicidal activity of macrophages, such as regulation of superoxide and nitric oxide (NO) production. During Leishmania infection, the nitric oxide plays a crucial role killing parasites in vitro and in vivo. In this work, were analyzed the constitutive Oxide Nitric Synthase (cNOS) activity and NO production by Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis. Leishmania- cNOS was identified in promastigotes by immunolocalization and analyzed by Transmission Electron Microscopy (TEM)(Fig.1) and confocal microscopy (Fig.2). NO production was measured in the supernatants of promastigotes cultures as nitrite form by Griess reaction. Immunolocalization by TEM showed cNOS in the glycossomes-like vesicles and immunofluorescence assay showed presence of cNOS enzyme in both parasites species. To confirm the enzyme activity, nitrite measure showed that L. braziliensis promastigotes was able to produce NO and has a higher production when compared to L. amazonensis. In conclusion, a correlation between the expression of cNOS and NO production by L. braziliensis and L. amazonensis suggest a possible virulence factor, which could be related to a regulatory mechanism of host cell NO production that confer parasites resistance to NO damages.

CAPES, CNPq/UFPA e Instituto Nacional de Biologia Estrutural e Bioimagem (INBEB)

Fig. 1: (A-D) Immunolocalization by transmission electron microscopy (TEM) of cNOS in Leishmania braziliensis promastigotes (A and C), and Leishmania amazonensis promastigotes (B and D). Note presence of anti-cNOS colloidal gold nanoparticles conjugated (arrows).

Fig. 2: Indirect immunofluorescence assay by confocal microscopy of L. amazonensis and L. braziliensis promastigotes on STAT growth phases. Note the presence of cNOS in the parasites. Bars 10 μm.

Fig. 3: Nitric oxide production measured in the supernatants of promastigotes cultures of L. amazonensis and L. braziliensis at different growth phases, logarithmic (LOG) and stationary (STAT). Both parasites are able to produce NO. However, the significant difference was between the two LOG phases. *p<0.05
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2247 Ultrastructure of Proventriculus and Midgut of Graphosoma lineatum (Linneaus, 1758) (Heteroptera: Pentatomidae)

Amutkan D.1, Suludere Z.1, Candan S.1
1Gazi University, Ankara, Turkey
damlamutkan@gazi.edu.tr

Graphosoma lineatum is a species of the Pentatomidae family known as the striped shield bugs. This species is distributed throughout Europe including Turkey and preferentially feed on the generative organs of Apiaceae family and found on umbels. Due to agricultural and economical losses resulting from the insect damage to the crops, it is important to examine digestive system of G. lineatum. In this study, proventriculus and midgut of G. lineatum are examined in detail using light microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM). The connection of proventriculus and midgut is indistinguishable. In general, proventriculus and all parts of midgut have similar properties. Midgut is differentiated into three distinct portions: Anterior, median, posterior. Anterior region is similar to a wide elongated sac. Median region is narrower and tubular in shape. Posterior region is a short dilated portion. Proventriculus and midgut which have role in digestion of the nutrients are surrounded by trachea and muscles. Both SEM images and cross section images illustrate that outer and inner surfaces of proventriculus and anterior midgut regions are indented. Cells of inner surface are simple cylindrical epithelium. Apical membranes of these cells are abundant in microvilli and there are many basement membrane invaginations. A continuous basal lamina lies down under the epithelium. Cells are rich in mitochondria especially under the microvilli and also abound with rough endoplasmic reticulum and lysosomes. There are many spherocrystals that are rod or spherical in shape and varisized lipid droplets. Outer surface of median region is less indented than proventriculus and anterior midgut regions. Cells of median region of the midgut vary in height and have microvillus towards lumen. In addition cells which have great numbers of vacuoles and different electron densities are observed. The posterior midgut assists to absorption of water in nutrients before passing to hindgut. Its outer surface is quite smooth. Epithelium cells in membrane structure are shorter than those of previous parts. Cells contain plenty of lysosomes and lipid granules. Lateral membrane foldings among cells are distinguished. In addition basal membrane infoldings are observed.

Fig. 1: General view of proventriculus and all region of midgut of Graphosoma lineatum-Stereomicroscope

Fig. 2: Longitudinal section of the proventriculus and anterior region of midgut of Graphosoma lineatum-Light Microscope

Fig. 3: The outer surface of the median region of midgut of Graphosoma lineatum-SEM

Fig. 4: Thin section of the posterior region of midgut of Graphosoma lineatum-TEM
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2248 Ultrastructure of Rectum of Graphosoma lineatum (Linneaus, 1758) (Heteroptera: Pentatomidae)

Amutkan D.1, Suludere Z.1, Candan S.1
1Gazi University, Ankara, Turkey
damlamutkan@gazi.edu.tr

Graphosoma lineatum (Linneaus, 1758) is a species belongs to Pentatomidae family and feeds on cultivated plants belong to Apiaceae family and anise, cotton, tobacco, rice, fruit trees which grow in Turkey. Its nymphs and adults prefer the generative organs of the host plants and damage to immature or mature seeds. This damage causes a decrease in quality and quantity of seeds. Due to the economic importance it is necessary to know the ultrastructure of this species. To this respect, ultrastructure of rectum of G. lineatum was examined using light microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM). An important function of the rectum in insects is the reabsorption of water from the faeces. Reabsorbed water is recycled and added to the contents of the midgut. In this species, rectum is a wide elongated sac located at the end of hindgut. Its outer surface is surrounded by well developed trachea and muscle. Its wall is covered with monolayer cubic-cylindrical epithelium in which intense infoldings are observed. The thickness of the epithelial layer increases in the region of the nucleus. Rectum inner surface is lined by a cuticular intima that is sharp or angled protrusions. Apical membranes of epithelial cells under the cuticula, involve a small number of microvillus in different height and thickness. There are many basement membrane invaginations into cells and there is a basal lamina layer under them. Lateral membranes make folds between cells. Mitochondria with dense matrices are the most abundant organelles in the cell cytoplasm. In SEM micrographs, crystals of various sizes are seen in the lumen of the rectum.

Fig. 1: The general view of the rectum of Graphosoma lineatum-SEM

Fig. 2: The inner surface of the rectum of Graphosoma lineatum-SEM

Fig. 3: Semi-thin section of the rectum of Graphosoma lineatum

Fig. 4: Thin section of the rectum of Graphosoma lineatum-TEM
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2251 Morphology and ultrastructure of the midgut in Poecilimon cervus Karabag, 1950 (Orthoptera, Tettigoniidae) based on light, scanning and transmission electron microscopes

Polat I.1, Suludere Z.1, Candan S.1
1Gazi University, Ankara, Turkey
irmakyilmaz@gazi.edu.tr

The alimentary system of insects is divided into three main regions: foregut, midgut and hindgut. Midgut is a tubular channel which is between foregut and hindgut. Its main role is absorbing nutritions and excreting enzymes. In this study, morphology and ultrastructure of the midgut of Poecilimon cervus were examined in detail by using of light microscope, scanning (SEM) and transmission electron microscopes (TEM). P. cervus (Orthoptera, Tettigoniidae, Phaneropterinae) is an endemic species that lives in Kızılcahamam-Ankara, İskilip-Çorum and Tosya-Kastamonu in Turkey. Approximately 10 adult male of Poecilimon cervus were collected from oak forests in Kargasekmez, Kızılcahamam, Ankara in June of 2013 and were dissected under the stereomicroscope. Dissected midgut tissues were prepared separately for the light microscope, SEM and TEM. Midgut of P.cervus is a long, thin channel located between foregut and hindgut. In cross sections of midgut, simple columnar epithelium is observed. Midgut cells have numerous, long, untidy microvilli at the apical side of the cell. In TEM examinations, it is clear that the apical plasma membrane is more electron lucent considering other side of the cell. Generally, the nucleus of the cell is round in shape but in some cells it seems ovoid in shape. The nucleus is situated near the basal plasma membrane and it has distinct nucleolus. Around the nucleus there are numerous mitochondria and rough endoplasmic reticulum. Intercellular gap at the lateral side is enlarged in some regions. We hope that this study about the midgut structure of P.cervus will be basis for following works on the insect digestive tract.

This study is a part of Irmak Polat’s PhD thesis. We express our thanks to Mustafa ÜNAL (Abant Izzet Baysal University, Turkey) for the identification of Poecilimon cervus.

Fig. 1: General view of midgut of Poecilimon cervus, SEM.

Fig. 2: Midgut cells of Poecilimon cervus, light microscope.

Fig. 3: Midgut cells with long microvilli, TEM.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2254 Structure of the Malpighian tubules in Poecilimon cervus Karabag, 1950 (Orthoptera, Tettigoniidae)

Polat I.1, Suludere Z.1, Candan S.1
1Gazi University, Ankara, Turkey
irmakyilmaz@gazi.edu.tr

In insects, Malpighian tubules are excretory organs which are responsible for osmoregulation by regulating ion and water balance. They absorb water and solutes, like mineral salts, from the surrounding haemolymph and then transfer them to the gut lumen. In this study, the Malpighian tubule structure of Poecilimon cervus Karabag, 1950 (Orthoptera) was observed with the use of light microscope, scanning (SEM) and transmission electron microscope (TEM). P. cervus is an endemic species that belongs to Tettigoniidae (Orthoptera) and lives in Kızılcahamam-Ankara, İskilip-Çorum and Tosya-Kastamonu in Turkey. Approximately 10 adult of P. cervus were collected from oak forests in Kargasekmez, Kızılcahamam, Ankara in 2013 and that Malpighian tubules were dissected under the stereomicroscope. Dissected tissues were prepared separately for SEM, TEM and light microscope. The Malpighian tubules of P. cervus are long, blind tubules extending from midgut-hindgut junction and they are scattered all over the haemocoel. Malpighian tubule lumen opens into the gut lumen. In cross sections of Malpighian tubules, relatively large 4-6 epithelian cells were observed in the transmission electron microscope. There is a basal lamina at the base of cuboidal cells and apposed to the basal lamina muscle cell layer is observed. Numerous infoldings of the basal plasma membrane and several mitochondia in between are present. Apical membrane of the Malpighian cell is differentiated into long, narrow apical cell projections called microvillus. In apical cytoplasm at the base of microvillus there is a great number of mitochondia. In the majority of the observed Malpighian cells, there are so many lipid droplets of all sizes. The Malpighian cells are rich in mitochondria, rough endoplasmic reticulum and spherites in their cytoplasm. Spherites are described as a side of biological accumulation of minerals and they play an important role in regulating the composition of the internal environment. Spherites composed of concentric strata are seen in vacuole. In this study, we aimed to reveal the ultrastructure and histology of Malpighian tubules of P. cervus.

This study is a part of Irmak Polat’s PhD thesis. We express our thanks to Mustafa ÜNAL (Abant Izzet Baysal University, Turkey) for the identification of Poecilimon cervus.

Fig. 1: Malpighian tubules surrounding the digestive tract, stereomicroscope.

Fig. 2: Cross section of Malpighian tubule of Poecilimon cervus, light microscope.

Fig. 3: Cross section of Malpighian tubule of Poecilimon cervus, TEM.

Fig. 4: General view of Malpighian tubule cell, TEM.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2258 The Morphology, Histology and Ultrastructure of the Female Reproductive System of Eurydema ventrale (Heteroptera: Pentatomidae)

Özyurt N.1, Candan S.1, Suludere Z.1
1Gazi University, Ankara, Turkey
nurcanozyurt@gazi.edu.tr

Eurydema ventrale is an important pest of Cruciferae and Capparaceae species. This species is distributed throughout Europe including Turkey. Due to agricultural and economical losses resulting from the insect damage to the crops, it is important to examine female reproductive system of E. ventrale. In this study, structure of the female reproductive system of E. ventrale is studied using light, scanning and transmission electron microscopes to contribute for a better understanding of its biology. The female reproductive system of E. ventrale consists of the paired ovaries with ovarioles, a pair of lateral oviducts, a common oviduct, a spermatheca and the paired accessory glands. Each ovariole consists of terminal filament, tropharium, vitellarium, and pedicel. The ovarioles in the ovary are connected to each other by terminal filaments forming a compact bunch shaped structure. Apical part of ovariole is formed by a tropharium region with large trophocytes and small prefollicular cells. The latter are located at the basal part of the tropharium. Trophocytes contain a large nuclei, many ribosomes and mitochondria in the cytoplasm. The centre of the tropharium, termed the trophic core makes a connection with trophocytes and oocyte in the vitellarium. Trophocytes and oocytes communicate with the core by means of long cytoplasmic processes and by nutritive cords, respectively. The other region of ovarioles in E. ventrale is constituted by vitellarium. The vitellarium is characterized by oocytes in three different development stages: Previtellogenesis, vitellogenesis and choriogenesis. Undeveloped oocyte in the previtellogenesis stage is surrounded by a single columnar epithelium in which has a great number of rough endoplasmic reticulum and mitochondria. On the oocyte surface in the vitellogenesis stage, variable shaped polygons are clearly seen and abundant organelles are seen in the follicular cells. During choriogenesis numerous rough endoplasmic reticulum and ribosome appear in the cytoplasm of the follicular cells. At this stage of egg maturation, there is a chorion layer which is composed of two well-differentiated layers: a narrow electro-dense endochorion, and a thick exochorion. At all stages, lipid droplets and protein granules are found in oocyte. Each ovariole connects through a pedicel to the lateral oviduct. Lateral oviducts unit together in the middle line of the abdominal cavity, forming the common oviduct, which is very short relative to the total size of the reproductive system. The common oviduct opens ventrally the genital chamber. The oocytes leave the terminal portion of the ovariole towards the lateral oviduct and are fertilized when going through the genital chamber.

Fig. 1: General view of female reproductive system in Eurydema ventrale

Fig. 2: Tropharium and vitellarium in Eurydema ventrale

Fig. 3: The oocyte surface in the choriogenesis stage of Eurydema ventrale

Fig. 4: The oocyte in the previtellogenesis stage of Eurydema ventrale
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2260 Ultrastructure of the Male Reproductive System of Codophila varia (Heteroptera: Pentatomidae)

Özyurt N.1, Candan S.1, Suludere Z.1
1Gazi University, Ankara, Turkey
nurcanozyurt@gazi.edu.tr

Codophila varia (Fabricius, 1787) (Heteroptera: Pentatomidae) is economically important species in most of Europe countries and in Turkey where it feeds on umbelliferous and graminaeous culture plants and also on weeds. The aim of present study is to investigate the reproductive morphology and histology of the male reproductive system of C. varia. Adult males of C. varia were collected from Ankara, in July 2011, Turkey. Structure of the male reproductive system of C. varia is studied morphologically and histologically using both light and scanning electron microscope. The male reproductive system of C. varia consists of two testes, two vas deferentia, two seminal vesicles, two ectodermal sacs, one ejaculatory bulb, one ejaculatory duct and a pair of accessory glands. Paired testes lie on either side of the digestive tract. The testes are lined with the tunica propria and peritoneal sheath with embedded tracheoles. The red pigmented testis is roughly oval and consist of seven testes tubules which enter the vas deferens. Three development zones were noted within the testes tubules; the growth zone, the maturation zone, the differentiation zone. The spermatozoa migrate to the vas deferens, they are transferred to the seminal vesicle. Vas deferens, which extend posteriorly from the testes to the seminal vesicle, are also red pigmented. The vas deferens and the seminal vesicle are same. The walls of vas deferens and seminal vesicle consist of an inner layer of simple epithelium which is surrounded by a network of muscle fibers extending in various directions. The seminal vesicle receives and stores the spermatozoa. Seminal vesicle are connected with the anterior medial portion of the ejaculatory bulb, which is covered by the investing epithelium and thrown into folds. Ejaculatory duct extends from the base of the ejaculatory bulb to the aedeagus. There are multiple accessory glands in C. varia which are situated in the posteroventral region of the male, milk white in color and sack-shaped. The accessory glands consist of three different parts: the muscle layers or muscularis, the secretory epithelium and the lumen. It is continuous with the aedeagus and covers the cuticle. In this study, we tried to explain the morphology and histology of male reproductive system of C. varia for obtaining the key information for future research about the reproductive biology of the Heteroptera.

Fig. 1: Spermatogonia in Codophila varia testis

Fig. 2: Spermatocytes and spermatids in Codophila varia testis

Fig. 3: Spermatids and spermatozoa in Codophila varia testis

Fig. 4: Bulbus ejaculatorius, ductus ejaculatorius and accessory glands in male reproductive system of Codophila varia
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2270 Correlative Light and Electron Microscopy in Borrelia Research

Strnad M.1 2, Vancová M.1, Rego R.1, Grubhoffer L.1 2, Nebesářová J.1 3
1Institute of Parasitology, Biology Centre of the ASCR, v.v.i., Branišovská 31, 37005 České Budějovice, Czech Republic, 2Faculty of Science, University of South Bohemia, Branišovská 31, 37005 České Budějovice, Czech Republic, 3Faculty of Science, Charles University in Prague, Viničná 7, 128 43 Praha, Czech Republic
martin.strnad.cze@gmail.com

Correlative light and electron microscopy (CLEM) allows us to combine wide field images collected from light microscopy with the high resolution of electron microscopy. CLEM is especially convenient for visualization of rare and dynamic events, which cannot be followed by electron microscopy on its own, such as membrane trafficking, signalling and cell division. Although the use of CLEM in host-parasite research is not very common today (Loussert et al., 2012), it fulfils all criteria to become a method of choice for visualization/investigation of infectious diseases where only a small number of pathogenic organisms is required to cause illness. We apply correlative fluorescence scanning electron microscopy to image the spirochete B. burgdorferi, the causative agent of Lyme disease, which is the most common tick-borne infection in the Western world. We examine and more fully characterize the events involved in the progression of B. burgdorferi through the tick salivary glands, using a genetically modified strain carrying the GFP reporter gene. Unlike the mode of penetration through the tick gut (Dunham-Ems et al., 2009), the way how B. burgdorferi transverse the salivary gland barrier remains unclear. Our results help to possibly shed new light on the adaptation of the Lyme disease spirochete within the tick vector and its transmission to a new mammalian host.

1. S.M. Dunham-Ems, M.J. Caimano, U. Pal, et al., J. Clin. Invest. 119 (2009), p. 3652.
2. C. Loussert, C. L. Forestier and B. M. Humbel, Methods Cell Biol. 111 (2012), p. 59.

The study is supported by the Technology Agency of the Czech Republic (TE01020118).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2421 Ultraestructural alterations induced by sesquiterpene lactone on Trypanosoma cruzi

Da Silva C. F.1, Batista D. J.1, Siciliano J. A.1, Batista M. M.1, Lionel J.1, De Souza E. M.1, Hammer E. R.1, Silva P. B.1, De Meri M.2, Adams M.2, Zimmermann S.2, Hamburger M.2, Brun R.3, Schühly W.4, Soeiro M. C.1
1Laboratório de Biologia Celular, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil, 2Department of Pharmaceutical Sciences, Pharmaceutical Biology, University of Basel, Switzerland , 3Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland, 4Institute of Zoology, University of Graz, Graz, Austria
franca@ioc.fiocruz.br

American trypanosomiasis also known as Chagas disease (CD) is a neglected disease caused by the intracellular protozoan Trypanosoma cruzi. Sesquiterpenes lactones (STL) like cynaropicrin, are terpenoid compounds characteristic of the Asteraceae family, exhibiting a wide variety of chemical structures with pharmacological effects in a large number of biological test systems including anti-inflammatory, anti- tumour and antimicrobial effects. As part of multidisciplinary study to identify novel anti-T.cruzi candidates, bloodstream trypomastigotes (BTs – Y strain) were incubated at 37°C for 24 h in the presence of increasing doses (0-6.65 µg/mL) of cynaropicrin. Our data showed that this SL exhibited an EC50 value of 1 ± 0.2 µg/mL, being lower than the reference drug, benznidazole (EC50 3 µg/mL). Aiming to analyze the cellular targets of this compound, transmission electron microscopy analysis of BTs treated or not for 2 h at 37°C with the corresponding EC50/24h was performed. The samples were fixed for 60 min at 4 °C with 2.5% glutaraldehyde 2.5 mM CaCl2 in 0.1 M cacodylate buffer, pH 7.2 and post-fixed for 1 h at 4 °C with 1% OsO4, 0.8% potassium ferricyanide 2.5 mM CaCl2 using the same buffer. Next, treated and untreated parasites were routinely processed for TEM and examined using a Zeiss EM10C electron microscope (Oberkochen, Germany). Ultrastructural analysis demonstrated that while untreated parasites exhibited normal morphology like mitochondrion, kinetoplast and flagelum (Fig. 1B), cynaropicrin treated BTs showed intense intracellular vacuolization, occurrence of large multivesicular profiles and membrane projections (Fig. 1C), which can be suggestive of autophagy, a type II programmed cell death (PCD) mechanism. Biochemical studies are underway in order to better characterize the molecular events associated to the triggering of T.cruzi cell death by SLs agents.

PDTIS/Fiocruz, CNPq, FAPERJ, CAPES and Fiocruz.

Fig. 1: Chemical structure of cynaropicrin (A).Transmission electron micrographs of cynaropicirn effect on bloodstream trypomastigotes: Untreated parasites display typical morphology (B), while treated parasites (C) show vacuolization (*) and plasma membrane projection (arrow).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2489 Mitosome behavior during the life cycle of the pathogenic protozoan Giardia intestinalis

Midlej V.1, Penha L.1, Silva R.1, De Souza W.1, 2, 3, Benchimol M.1, 2, 3
1Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro – Brazil, 2Instituto Nacional de Metrologia e Qualidade Industrial – Inmetro, Rio de Janeiro – Brazil, 3Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens-INBEB
vmidlej@hotmail.com

The mitosome is a double-membrane bounded organelle found in few unicellular eukaryotes, one of which is the human intestinal parasitic protozoan Giardia intestinalis. The discovery of mitosomes in Giardia was strongly supported by the identification of the protein machinery components responsible for iron-sulfur (Fe-S) cluster assembly (IscS and IscU proteins) in this organelle. G. intestinalis also lacks mitochondria and peroxisomes and has been considered to be among the earliest-branching eukaryotes. This flagellated protozoan grows in vitro as trophozoites and under some conditions differentiates into cysts, characterized by the absence of externalized flagella, a rounded shape and the presence of a cyst wall. Using antibodies that recognize two proteins present in the mitosome, heat-shock protein 70 (mit-HSP70) and giardial chaperonin 60 (GiaCpn60), we used confocal laser scanning and electron tomography microscopy, western blots and qRT-PCR to analyze the presence and distribution of the mitosomes during both the cell cycle and the process of the trophozoite-to-cyst transformation. At early stages of the differentiation process (~12 h), there was a significant decrease in the extent of labeling in the cells and the numbers of mitosomes, which almost disappeared after 21 h, but were recovered during the cyst stage. This was confirmed by an mRNA expression analysis, thus indicating a process that modulates the formation of mitosomes during the G. intestinalis life cycle. Electron microscopy tomography, which allows for three-dimensional reconstruction, revealed the presence of both rounded and elongated mitosomes.

This work was supported by CNPq, FAPERJ, CAPES, PRONEX, INBEB and AUSU

Fig. 1: Confocal microscopy of Cpn60 during Giardia differentiation. Immunofluorescence using the antibodies anti-Cpn60 (green) and anti-CWP1 (red). Vegetative cells are observed (a-c). In 21h-encysted cells (d-f), no Cpn60 labeling is seen; the cells present a positive CWP1 signal. In the cysts (g–i), the Cpn60 and CWP1 signals are observed.

Fig. 2: Confocal microscopy of HSP70 during Giardia differentiation. Immunofluorescence using the antibodies anti-HSP70 (green) and anti-CWP1 (red). Vegetative cells are observed (a-c). In 21h-encysted cells (d-f), no HSP70 labeling is seen; the cells present a positive CWP1 signal. In the cysts (g–i), the HSP70 and CWP1 signals are observed.

Fig. 3: Electron tomography and 3D reconstruction of mitosomes in Giardia. Mitosomes (arrows) are seen in vegetative (a–c) and 21h-encysted (d–f) cells. No differences are seen between the mitosomes of the vegetative and 21 h-encysted cells. Elongated and ovoid organelles were observed. VD, ventral disc; Ax, axonemes; M, mitosome; N, nucleus.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2555 Nucleolar assembly during mitosis of the epimastigotes of Trypanosoma cruzi

Nepomuceno T.1, Lara R.1, Hernandez R.1, Segura L.1, Jimenez L.1
1National Autonomous University of Mexico
sjng7@yahoo.com

Nucleolar assembly, often termed nucleologenesis, is a cellular event that requires the synthesis and processing of ribosomal RNA, in addition to the participation of pre-nucleolar bodies (PNBs) formed during early telophase. In mammal and plant cells, the biogenesis of the nucleolus has been described in detail, but in unicellular eukaryotes is a poorly understood process. In this study, we used light and electron cytochemical techniques to investigate the pathway of nucleolus re-building during closed mitosis in epimastigotes of Trypanosoma cruzi, the protozoan parasite that causes American trypanosomiasis. Uranyl-lead conventional contrast, silver impregnation specific for nucleolar organizer regions and EDTA regressive procedure to preferentially stain ribonucleoprotein revealed a single electrodense nucleolus which is intensively impregnated with silver mainly into fibrillar component. Additionally, we observed fibers of nucleolar chromatin surrounded by argyrophilic elements in interphase. Early in mitosis, the AgNOR proteins are maintained but are dispersed throughout the nucleoplasm. During the normal course of the division, the nucleolar material is divided asymmetrically and migrates to opposite poles of the elongated nucleus. Late in mitosis, when the two nuclei have been formed, the AgNOR proteins are concentrated in the NOR and the nucleolus is assembled. In contrast with other eukaryotic cells, the classical perichrosomal sheath and PNBs were not visualized. We suggest that the formation of the nucleolus during mitosis of epimastigotes of T. cruzi occurs as a continuous process that does not require the concentration of nucleolar material within intermediates nuclear bodies such as the characteristic PNBs.

Tomás Nepomuceno-Mejía was a recipient of a postdoctoral scholarship from DGAPA - UNAM, México. This work was also partially supported by Grants DGAPA UNAM PAPIIT IN-227810; CONACyT 180835.

Fig. 1: AgNOR light microscopy

Fig. 2: Uranyl-lead standar electron microscopy

Fig. 3: AgNOR electron microscopy

Fig. 4: EDTA electron microscopy
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2575 Morphometric Analysis of Granulomas in Nectomys squamipes Brants, 1827 (Rodentia: Sigmodontinae) naturally infected with Schistosoma mansoni Sambon, 1907 (Digenea: Schistosomatidae).

Amaral K. B.1, Dias F. F.1, Silva T. P.1, Malta K. K.1, Gentili R.2, Neto S. F.2, Melo R. C.1
1Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, MG, Brazil. , 2Laboratory of Biology and Parasitology of Wild Mammals Reservoirs, Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil.
katia.amaral@oi.com.br

The semi-aquatic rodent Nectomys squamipes has been found naturally infected with Schistosoma mansoni and has a wide geographical distribution that coincides with endemic areas of human schistosomiasis in Brazil. Due to its susceptibility, high abundance and water-contact patterns, it contributes significantly with the epidemiology of the disease [1]. The pathological effects of schistosomiasis occur mainly because of the immunological reactions of the host due to the eggs of the parasite that are not excreted and are deposited on the tissues and later on are encapsulated to form the granulomas, which undergo several evolutional stages according to the phase of the infection [2]. In this process, the eosinophils, leukocytes of the innate immune system, are actively recruited and secrete cytotoxic products that cause damage to parasites [3]. In this study, we investigated the role of eosinophils in the formation of different stages of granulomas in this naturally infected rodent. Animals (n=3) were captured and immediately euthanized by deep anesthesia (animal ethical approval # L-0049/08). Liver fragments were processed for light microscopy. For the morphometric analysis, Histoquant software was used to measure parameters as area, perimeter, major and minor diameter of granulomas with visible eggs in the center. Liver sections showed inflammatory infiltrates and periovular lesions in pre-granulomatous stages, characterized by exudative reaction (7,93%) and exudative-necrotic reaction (1,22%) and a clear predominance of exudative-productive (48,78%) and productive (42,07%) granulomas. However, no significant differences were found in the areas of all types. Some granulomas around eggs containing well-preserved miracidium showed an acute inflammatory reaction, with many polymorphonuclear cells, mainly eosinophils. They presented the largest numbers of eosinophils (absolute numbers) and eosinophils per area (density of cells). More frequently, an inflammatory reaction of chronic type was seen, with cells as eosinophils, macrophages and fibroblasts and collagen deposition in varying amounts around empty eggshells or eggs containing degenerated miracidia. Finally, we observed that the degree of impairment of liver tissue (calculated as the sum of the area occupied by granulomas and that occupied by inflammatory infiltrates) was very small (mean = 5.05%), which explains the lack of symptoms observed in these rodents during schistosomiasis infection.  

References:
[1] Gentili, R. et al. Oecologia Australis, 14(3) (2010) 711-725.
[2] Lambertucci, J.R. 2010. Memórias do Instituto Oswaldo Cruz, 105(4) (2010) 422-435.
[3] Melo, R.C.N. et al. Journal of Leukocyte Biology, 83 (2008) 229-235.

Support by FAPEMIG, CNPq and CAPES.

Fig. 1: Granulomas in liver of Nectomys squamipes infected with S. mansoni. In (A-D) different stages of granulomas around parasite eggs are seen. Liver sections were stained with Hematoxilin-eosin and analyzed through light microscopy. In (E and F) the frequency and area of granuloma types are shown. (a, b, c) indicate p<0,001. Scale bar: 50 µm.

Fig. 2: Eosinophils in liver granulomas of Nectomys squamipes. In (A) observe many eosinophils with characteristic nucleus (arrow) and granules (arrowhead) surrounding the Schistosome egg, as noted in detail (Ai). In (B) the graph shows the eosinophil abundance, while in (C) is presented the eosinophil density. Scale bar: 25µm (A), 5 µm (Ai).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2663 Immunofluorescence imaging helps to elucidate the role of membrane-bound enzymes of Spodoptera frugiperda (Lepidoptera) in adaptive mechanism against plant protease inhibitors

Oliveira C. F.1, Marques P. P.1, Macedo M. L.2
1Universidade Estadual de Campinas, Campinas, Brazil, 2Universidade Federal do Mato Grosso do Sul, Campo Grande, Brazil
petruspm@gmail.com

Some insect pests are able to adapt to presence of plant defense compounds. Is important understands these adaptive mechanisms in order to design effectives strategies of pest control. The fall armyworm (Spodoptera frugiperda) (Lepidoptera: Noctuidae) is an important pest in Brazil, attacking several crops of economic importance, as such corn and cotton. The adaptive mechanism of the fall armyworm against plant protease inhibitors is triggered by transcription of resistant trypsins, but the physiological role and location these enzymes are unknown. In this work, we used immunofluorescence techniques to investigate the presence and spatial location of Entada acaciifolia trypsin inhibitor (EATI) in S. frugiperda larval tissues to elucidate details about this phenomenon. Different tissues from larvae chronically fed with EATI were separated, rinsed and analyzed by western blot: midgut, gut epithelial membrane, fat body, Malpighian tubules, hemolymph and frass. For immunofluorescence studies, these larvae were euthanized in cold heptane, frozen in liquid nitrogen, embedded in Tissue Tek and sectioned at 8 µm in a cryostat. Sections were washed in BSA 1% and primary antibody anti-EATI was applied in 1:200 concentration overnight, washed again in PBS and incubated with secondary fluorescent antibody (1:300) in the dark for 1 hour. Fluorescent stain DAPI (1:1000) was then applied for 5 minutes. Images were acquired with microscope Eclipse (Nikon) and camera Coolsnap (Media Cybernetics). Western blot detected EATI in frass, midgut and gut epithelium. The immunofluorescence studies showed the presence of the inhibitor in the gut lumen, along the peritrophic membrane, on the surface of the gut epithelium and minority in fat body. With exception of fat body, the presence of EATI in others compartments follows the spatial location of digestive enzymes, especially trypsins. The digestive process in S. frugiperda involves the secretion of enzyme vesicles by a microaprocrine process. These vesicles are solubilized in the lumen, releasing active enzymes. In regards to trypsin secretion, a fraction these enzymes remains incorporated into the peritrophic membrane, with an unclear participation on digestive process. We suggest that the S. frugiperda mechanism to bypass the effects of protease inhibitors involves the immobilization of sensitive trypsins into peritrophic membrane associated with the release of soluble resistant enzymes into lumen.

The authors are thankful to the supported receive from FAPESP, CNPq and FINEP.

Fig. 1: Location of EATI in the gut of Spodoptera frugiperda larvae. A- Staining with haematoxilin and eosin, B- immunofluorescent visualization of EATI associated at peritrophic membrane (PM) and C- basal membrane (BM).D- Minoritary presence of EATI in fat body. The nuclei are marked in blue. Bar=50µm in A and 20µm in B,C,D
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2751 Optimization of microscopic procedures for study of monogenean anatomy

Hodová I.1, Vaškovicová N.1, Gelnar M.1, Valigurová A.1
1Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
hodova@sci.muni.cz

The outer body surface of parasitic Platyhelminthes, including monogenean parasites, is covered by a tegument consisting of a syncytial layer, cell bodies that are situated below the syncytium and muscle layers connected to the syncytium by several cytoplasmic processes. The subtegumental position of nuclei is generally thought to protect them from host responses. The family Diplozoidae (Monogenea) includes blood-feeding gill ectoparasites of freshwater fish. They exhibit extraordinary body architecture and life cycle involving a permanent fusion of two larval worms and their subsequent transformation into one individual (permanent copula).
Developmental stages of Paradiplozoon bliccae comprising diporpa, juvenile and adult were investigated for their anatomy using a combined approach of light, confocal and electron microscopy. The freeze-etching method proved to be a strong tool to visualize the membranous structures of tegumentary cells and muscles of the body wall. Direct labelling of filamentous actin with fluorescent phalloidin demonstrated the organization of the major muscular structures. The body wall musculature is well-developed and highly organized, with circular, intermediate longitudinal and inner diagonal muscle fibres. Wavy muscle fibres were observed. The buccal suckers and the pharynx represent the most dominant muscular structures of the worm’s forebody while the hindbody bears the prominent haptor with four pairs of clamps serving for parasite’s strong attachment to the host gills. The bundle of the muscles attached to every clamp controls the mobility of the clamp´s skeletal jaws.

This research was financially supported by the Czech Science Foundation Grant No. GAP506/12/1258.

Fig. 1: The forebody musculature of Paradiplozoon bliccae (adult stage). CLSM.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2902 Three-dimensional analysis of the biogenesis of hemozoin crystals during intra-erythrocytic cycle of Plasmodium chabaudi

Wendt C.1, Rachid R.1, de Souza W.1, 2, Miranda K.1, 2
1Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho and Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens – Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. , 2Diretoria de Metrologia Aplicada a Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia – Inmetro, Rio de Janeiro, Brazil.
camilawendt@biof.ufrj.br

Malaria is a disease caused by protozoan parasites from the genus Plasmodium with the highest impact on public health in endemic areas. Morbidity and mortality of malaria results from the asexual replication of Plasmodium in the erythrocyte of the mammalian host. In the course of infection, different developmental stages of the parasite are formed as it progresses from the ring stage to the trophozoite and then to the replicating schizont stage. During its intra-erythrocytic development, malaria parasites internalize massive amounts of hemoglobin from the red blood cell in order to obtain free amino acids and to regulate osmotic pressure. Hemoglobin is digested in a compartment with acidic pH termed the food vacuole, producing aminoacids and others byproducts, namely heme. It is known that free heme can generate free-radicals, causing molecular and cellular damage. In order to avoid these effects, free heme is immobilized and stored in a crystal form known as hemozoin or malaria pigment. Although hemoglobin uptake and heme crystallization are physiological steps used as target for many antimalarial drugs, the fine mechanisms underlying hemoglobin crystallization are still under discussion. In this work we studied the mechanism of hemozoin nucleation in the different stages of the intra-erythrocytic cycle of the rodent parasite Plasmodium chabaudi by transmission electron tomography of cryofixed and freeze substituted cells. Cryofixation of samples generally provided a better preservation of the cells and their hemozoin crystals (figure 1). Electron tomography showed the three-dimensional dispersion of hemozoin crystals within the food vacuole and the cytoplasm of the parasite (figure 2). Results showed that large amounts of hemoglobin are internalized during the early stages after invasion (ring stage and early throphozoite), whereas in late stages (throphozoite), assembly of several vesicles containing hemoglobin spread through the parasite cytoplasm were observed. Small food vacuoles concentrated near the membrane of the parasite were also frequently seen. In the late (schizont) stage, the hemoglobin containing vesicles were drastically reduced and larger food vacuoles were seen, containing large amounts of hemozoin (figure 2). Taking together, these results provide new insights on the mechanisms of hemoglobin uptake and degradation in rodent malaria parasites.

This work was supported by CNPq, FAPERJ, FINEP and CAPES (Brazil)

Fig. 1: Serial electron tomogram of an early throphozoite (A-D) and a schizont (E-H) form of P. chabaudi submitted to high pressure freezing and freeze substitution. The morphology of several cell components could be observed through the different Z-sections, including hemozoin crystals (arrows). N: nucleus. Scale bar: 300nm

Fig. 2: Dispersion of hemozoin crystals in P. chabaudi schizont. Two different food vacuoles are seen throughout z sections(A-C). Vacuoles occupy small portion of the cell volume and are completely filled with hemozoin(D-H). Yellow: parasite membrane, blue:nucleus, gray:food vacuole, purple:hemozoin crystals, red:vesicles with hemoglobin. Scale bar: 300nm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-2906 Microscopy Techniques Unveil the Mechanism of Action of Berenil, a DNA Binding Drug, on Trypanosoma cruzi

Zuma A. A.1, Cavalcanti D. P.2, Thiry M.3, de Souza W.1,2, Motta M. C.1
1Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil , 2Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO), Rio de Janeiro, Brazil, 3Department of life Sciences, GIGA-Neurosciences, Unit of Cell and Tissue Biology, University of Liege, Belgique
alinezuma@gmail.com


The protozoa Trypanosoma cruzi, the aetiological agent of Chagas disease, belongs to the Trypanosomatidae family that presents as a main characteristic a single mitochondrion with an enlarged portion termed kinetoplast. This structure contains the mitochondrial DNA (kDNA) that is composed of interlocked maxi and minicircles that are released from this network for replication. Since the kDNA arrangement is unique in nature and represents a hallmark of kinetoplastids, it constitutes a valuable target in chemotherapeutic and cell biology studies. In this work, we analyzed the effects of berenil, a minor-groove binding agent that targets preferentially the kDNA, on the proliferation and ultrastructure of T. cruzi, using different microscopy approaches. For this purpose, cells were cultivated in medium containing different drug concentrations (2, 10, 20 and 50 μM) and samples were collected after each 24 hours for counting on Neubauer’s chamber and for analysis by optical and electron microscopy. The presence of dyskinetoplastic cells, which lost partially or totally the kDNA, was revealed after DAPI staining and cell viability was verified using MTS/PMS method based on mitochondrial viability. Our results showed that berenil promoted a slight effect on parasite growth and its viability was not affected. However, this compound caused significant changes at ultrastructural level as revealed by transmission electron microscopy, when comparing control and treated cells, such as mitochondrial swelling, including loss of matrix, and strong changes on kDNA arrangement. Furthermore, membrane profiles were observed in the middle of the kinetoplast network, as well as an electron-lucid area close to the kDNA. In order to investigate if such areas corresponded to uncatenated minicircles, we used the TdT technique that specifically recognizes DNA, however no labeling was detected in this kinetoplast region. Using atomic force microscopy, we observed that the isolated kDNA presented a more compact arrangement after berenil treatment, when compared to control cells. Taking our results together we can assume that berenil impeaches the minicircle decatenation of the network, thus impairing DNA replication and culminating in the appearance of dyskinetoplastic cells. Since berenil affected directly the kDNA topology, our data reinforce the idea that the kinetoplast represents a potential target for chemotherapy against trypanosomatids.

Supported by CNPq and FAPERJ.

Fig. 1: Figure 1: Non-treated Trypanosoma cruzi, showing the bar shape kinetoplast, the nucleus and the mitochondrion. n = nucleus, ht = heterochromatin, m = mitochondrion, k = kinetoplast, f = flagellum, gc = Golgi complex, cy = cytostome.

Fig. 2: Figure 2: Transmission electron microscopy of parasite treated with 20 µM berenil for 48 hours. Note the electron-lucid area close to the kDNA network (arrowhead).

Fig. 3: Figure 3: TdT technique applied on parasite treated with 20 µM berenil for 48 hours. Gold particles were observed in the compact kDNA, but not at the kinetoplast electron-lucid area (arrowhead).

Fig. 4: Figure 4: Atomic force microscopy of the kDNA after treatment with 50 µM berenil for 72 hours. Note that the network periphery is more compact (white arrow).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-3141 Orangutan faeces under scanning electron microscopy

Foitová I.1, 2, Hodová I.1, Nurcahyo W.3
1Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic, 2Orangutan Health Project, UMI - Saving of Pongidae Foundation, Brno, Czech Republic, 33Department of Parasitology, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta, Indonesia
hodova@sci.muni.cz

Orangutans are in an extremely precarious state, with the Sumatran orangutan (Pongo abelii) critically endangered and the Bornean orangutan (Pongo pygmeaus) endangered with rapidly declining populations. There is only limited information available on orangutan parasites in general. Studies focusing on the parasites of orangutans in their natural habitat are even rarer, making any new information especially valuable. The most extensive record of orangutan parasites comes from a study which summarizes the data based only on standard coprology analyses often with not clear identification to the parasite species level. We would like to present scanning electron microscopy (SEM) as important tool in parasites identification to the species level which has been used in recently described new pinworm species and identification of Ascaris sp. parasitizing Sumatran orangutan. The use of the SEM methodology in nematodes was found to be necessary for studies of cephalic structures and the caudal end in males, where the study of form and distribution of cloacal and caudal papillae by light microscopy produces inaccurate results. The locality of investigation, the village Bukit Lawang (former home to a rehabilitation centre for Sumatran orangutans) is situated on the southwestern border of the Gunung Leuser National Park, northern Sumatra, Indonesia. From the fresh faeces of semi-wild orangutans, nematodes were collected and immediately fixed. These nematodes were examined under a light and scanning electron microscope for morphometric analysis.

Financially supported by the UMI- Saving of the Pongidae Foundation and GA P505/11/1163.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-3264 Ultrastructural characterization of Ovarian Follicle atresia in Rhodnius prolixus infected by Trypanosoma rangeli

Machado G. S.1, Freitas S. C.2, Santos-Mallet J. R.2, Feder D.1, Gomes S. A.1
1Universidade Federal Fluminense-UFF, Niterói, Rio de Janeiro, RJ, Brazil, 2Instituto Oswaldo Cruz - FIOCRUZ - Rio de Janeiro, RJ, Brazil
suzetearaujo@id.uff.br

The parasites can alter host behavior and physiology as way of increase its own probabilities to complete its life cycle. These alterations can interfere in insect host reproductive success. Although the pathogenic effects of Trypanosma rangeli on R. prolixus are already described in the literature, the impact of this parasitic infection on the reproductive biology has not yet been fully evaluated. In order to identify reproductive patterns caused by an infection in the insect vector, in this work we characterized the influence of T. rangeli infection on R. prolixus oogenesis. Our previous results demonstrated that the time mating was lower in pairs with infected females when compared with the control pairs. The oviposition in infected females was also lower when compared to the control group. Furthermore, a smaller number of eggs hatched were observed in infected females, compared to the control group. Ultrastructural characterization by transmission electron microscopy of thin sections of infected ovarian epithelium indicated nuclear fragmentation with spread chromatin (karyorrhexis) (Figure 1 A-B) and mitochondrial morphology alteration or mitochondrial swelling (Figure 2 A-B) suggesting follicular atresia or ovarian follicles degeneration. We are now investigating the programmed cell death (PCD) mechanisms by TUNEL labeling, autophagic vesicles by monodansylcadaverin, a lisossomotropic fluorescent compound useful for identifying cadaverin protein and also assessing proteolytic enzymes involved in R. prolixus reproductive disorder caused by T. rangeli infection.

Key-words: Trypanosoma rangeli, Rhodnius prolixus, follicle atresia, parasitc infection, reproduction.

Supported by: FAPERJ, PROPPI-UFF and IOC-FIOCRUZ

Fig. 1: Figure 1: A- Nuclear chromatin organization of ovarian follicle of Rhodnius prolixus control; B - Nuclear fragmentation with spread chromatin (karyorrhexis) of R. prolixus infected by T. rangeli.

Fig. 2: Figure 2: A- Appearance of mitochondria in ovarian follicle of Rhodnius prolixus: control insects; B- mitochondrial swelling in ovarian follicle of R. prolixus infected insects.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-3336 Ultrastructural aspects of Toxoplasma gondii egress

Caldas L. A.1,3, Seabra S. H.2, Attias M.3, De Souza W.1,3
1Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Rio de Janeiro, Brazil, 2Centro Universitário da Zona Oeste, Rio de Janeiro, RJ, Brazil, 3Instituto de Biofísica Carlos Chagas Filho; Universidade Federal do Rio de Janeiro, Brazil
mattias@biof.ufrj.br

The obligate intracellular protozoan parasite Toxoplasma gondii is capable of infecting almost every nucleated warm blooded cells and has raised medical and veterinary importance. Nevertheless, its egress from host cell is still poorly understood. Herein, we investigated some general aspects of the dynamics of egress by confocal and elecron microscopy. For Field Emission Scanning electron microscopy (FESEM), between 30 and 48 hours post-infection, LLCMK2 monolayers were washed and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, post-fixed for 1 h with 1% OsO4 in 0.1 M cacodylate buffer pH 7.2 and 0.8% potassium ferrocyanide, dehydrated in ethanol, critical point dried in CO2, sputtered with carbon and observed in a Jeol 6340 field emission scanning electron microscope. Some samples had the surface detached by adhesive tape, which allowed the observation of the host cell interior. For confocal microscopy, the monolayers were pre-incubated with 50 mM ammonium chloride and 3% BSA in PBS pH 8.0 for 45 min. The samples were then incubated with primary antibodies at a 1:100 dilution for 1 h, rinsed, and incubated with 1:400 secondary antibodies at room temperature for 1 h. Observation was carried out in a Zeiss 510 LSM 510 NLO. By FE-SEM, in natural egress, we observed the detachment of one of the parasites from the residual body with the parasitophorous vacuole membrane and the tubular network still present (Figure 1). Comparing natural egress with calcium ionophore induced egress in the second one tubular disassembly precedes parasite detachment. The participation of the host cell cytoskeleton was also checked, indicating a dual role in T. gondii egress: it can be a substrate for intracellular gliding of the parasite, contributing to its escape, and also a barrier for T. gondii during egress since escape from host cells occurred preferentially at actin free zones (Figure 2). 

Acknowledgements: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas de Amparo à Pesquisa (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Fig. 1: A rosette of T. gondii inside a parasitophorous vacuole, from which parasites, after 30hpi, begin to detach (arrow). Bar. 1μm

Fig. 2: T. gondii induced egress after cytochalasin D treatment. Parasites (blue) escape through actin (red) free zones (arrows).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-3496 Different Uptake Mechanisms Dependent on Actin and Tubulin Dynamics Occur During Cryptococcus neoformans Internalization by Peritoneal Macrophages.

Guerra C. R.1, Seabra S. H.2, de Souza W.3, 4, Rozental S.1
1Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, 2Biology and Health Sciences Collegiate, State University Center of Zona Oeste, Campo Grande, Brazil, 3National Institute of Metrology, Quality and Technology, Duque de Caxias, Brazil, 4National Institute of Structural Biology and Bioimages, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
carolineguerra@gmail.com

Cryptococcosis caused by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for its pathogenesis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood.

We investigated the mechanism of C. neoformans phagocytosis by peritoneal macrophages using laser scanning confocal microscopy (LSCM) and electron microscopy techniques, as well as flow cytometry (FACS) quantification. Five C. neoformans strains differing in serotypes and capsule size - H99, B3501, ATCC28957 and acapsular mutants CAP59 and CAP67 - were allowed to interact with peritoneal macrophages, previously adhered on glass cover slips, in a ratio of 25:1 yeast per macrophage, for 2 hours. Interactions occurred in the presence or absence (control) of cytoskeletal dynamics inhibitors: cytochalasin D, latrunculin B, nocodazole or placlitaxel and then cells were processed for FACS, LSCM and transmission and scanning electron microscopy (TEM and SEM, respectively). For LSCM, actin filaments were labeled with AlexaFluor® 488 phalloidin and α-tubulin was labeled with anti-α-tubulin AlexaFluor® 546 conjugate.

Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both 'zipper' (receptor-mediated) and 'trigger' (membrane ruffle-dependent) phagocytosis mechanisms (Fig.1). Actin filaments surrounded phagosomes of capsular and acapsular yeasts (Fig. 2), and the actin depolymerizing drugs inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis.

Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells.

This research was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Fig. 1: Uptake of Cryptococcus neoformans acapsular strain CAP59 after interaction with peritoneal macrophages. Scanning electron microscopy showed both trigger-like (A) and zipper-like (B) uptake structures. Scale bars represen 1 μm (A) and 2 μm (B).

Fig. 2: Actin is recruited to the phagosome area during yeast uptake. Confocal laser scanning microscopy of internalized Cryptococcus neoformans strain H99 identified by bright field (arrow in A) can be visualized in the context of host cell actin (red) and microtubule (green) cytoskeletons. Host cell DNA is labeled with DAPI (blue). Bar represents 5μm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-3497 Elucidating the development of Wolbachia in the larval stages of Dirofilaria immitis

Kozek W. J.1, Cangani C. C.1, Santiago N.1, Subkorndej P.2
1University of Puerto Rico, San Juan, Puerto Rico, 2University of Georgia, Athens, GA, USA
wieslaw.kozek@upr.edu

All filarial nematodes of medical importance harbor intracellular, mutualistic endosymbionts bacteria belonging to clades C, D and F of the Wolbachia group. Lack of safe and effective chemotherapeutic agents against the adult filariae suggests that an alternate, indirect treatment directed against Wolbachia may be effective in controlling and eventually eradicating filaria infections. Since Wolbachia are vertically transmitted during the life cycle of filariae, we traced the location of Wolbachia in the larvae of Dirofilaria immitis maturing in the mosquito vector, to elucidate the role that Wolbachia may play during development of the larvae. Aedes aegypti (Liverpool strain) were infected by feeding on a dog blood containing microfilariae. Groups of six to ten mosquitoes were killed on day 3, 5, 7, 9 and 14, their malpighian tubules, and larvae developing therein, were isolated and processed for examination by transmission electron microscopy. Results obtained indicate that the Wolbachia were limited to the lateral chords, as in the adult worms, and appeared to be actively reproducing before each molt in the early larval stages. In the third stage, Wolbachia were widely, but sparsely, distributed in the lateral chords. The results of this first report tracing the development of Wolbachia in filarial larval stages suggest that Wolbachia may actively produce components required for the synthesis of the cuticle of successive larval stages. In the third larval stage Wolbachia appear to enter a period of latency and are reactivated again when the third stage is introduced into the final host.

Supported, in part, by the RCMI NIH Award RR-003051 to the University of Puerto Rico and NIAID/NIH Filariasis Research Reagent Resource Center (FR 3) at the University of Georgia.

Fig. 1: Wolbachia in the hypodermal cell of a 12-day D. immitis larva. X10,000.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-5712 The sperm of Triatoma brasiliensis and Triatoma shelocki (Insecta, Reduviidae, Triatominae)

Baffa A. F.1,2, Silva E. R.2, Santos-Mallet J. R.1, Freitas S. C.1
1Setor de Entomologia Médica e Forense, Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz – FIOCRUZ, Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ. 21040-360., 2Laboratório de Insetos aquáticos, Departamento de Zoologia, Instituto de Biociências, Univeridade Federal do Estado do Rio de Janeiro, Av. Pasteur, 458, Urca, Rio de Janeiro. 22290-240.
sfreitas2@gmail.com

Spermiogenesis in insects has demonstrated a number of changes in different groups of Hemiptera, providing diversity adequate to justify descriptive studies that may support analysis taxonomy, reproductive biology and phylogeny. The objective of this paper is to obtain more data on the morphology of spermatozoa of T. brasiliensis and T. sherlocki, which can be used in phylogenetic analyzes of Hemiptera. For SEM, drops of sperm suspension were spread on histological glass slides and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 1 hour at room temperature. After drying, the glass slides were mounted on metal stand, covered with a thin layer of gold and observed under scanning electron microscope, model JEOL JSM 6390 LV of Electron Microscopy Platform of Oswaldo Cruz Institute, FIOCRZ, RJ. For TEM, males were dissected in saline solution and seminal vesicles transferred to 2.5% glutaraldehyde in sodium cacodilate buffer at room temperature, washed in same buffer and post-fixed in 1% OsO4 in buffer for 1h. The seminal vesicles were dehydrated in a graded series acetone and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under Transmission Electron Microscopy Jeol JEM 1011 of Electron Microscopy Platform of Oswaldo Cruz Institute, FIOCRZ, RJ. The sperm are long and thin, formed by the nucleus, inserted at the anterior end and flagellum composed of double axial wire and two mitochondrial derivatives (Fig. 1 and 2). The flagellum consist of the axoneme, which follows the pattern of microtubule arrangement 9 + 9 + 2, 9 accessories, 9 doubles and 2 central microtubules, and the two mitochondrial derivatives that flank the axoneme forming a heart-like structure (Fig. 2). In mitochondrial derivates could be seen bridges adhering the tank between mitochondrial derivatives and microtubules (Fig. 2). In the transition between nucleus and flagellum could be seen the centriole adjunct, parallel to the nucleus (Fig. 3). The mitochondrial derivates terminate before the end of the axoneme, therefore, the end of the flagellum is composed only by the axoneme (Fig. 4). The morphology of the sperm of T. brasiliensis and T. sherlocki is similar to those of other insects already studied, differing in the shape of the structures. Some characteristics observed in the sperm of two species can be considered as markers shared between families of Heteroptera.

CAPES and Electron Microscopy Platform of the Oswaldo Cruz Institute, RJ.

Fig. 1: Nucleus and flagellum of the sperm. Arrowhead showing junction of nucleus and flagellum. 

Fig. 2: Cross-section of the flagellum showing the axoneme (Ax) consisting of nine microtubule accessories, nine doublets and one central pair, and mitochondrial derivatives (MD) with bridges between microtubules and mitochondrial derivates (arrowhead). N: nucleus.

Fig. 3: Cross-section of the nucleus-flagellum transition region, showing the nucleus (N) and the portion of the centriole adjunct (CA).

Fig. 4: Posterior extremity of sperm. Axoneme (Ax); mitochondrial derivates (MD).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-5713 Localization of acid phosphatase activity in sperm of Triatoma brasiliensis (Insecta, Reduviidae, Triatominae)

Freitas S. C.1, Santos-Mallet J. R.1
1Setor de Entomologia Médica e Forense, Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz – FIOCRUZ, Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ, 21040-360.
sfreitas2@gmail.com

The cytochemical study is useful to determine the functional role of different elements of the spermatozoa, in its movement and in the fertilization process, and particularly to detect the role of enzymes in sperm development. The present study analyses the localization of acid phosphatase im spermatozoa of T. brasiliensis, which is the most important Chagas disease vector in the semiarid areas of Northeast Brazil. Adult males were dissected and its seminal vesicles removed and fixed in 1.5% glutaraldehyde solution in 0.1 M sodium cacodylate buffer at pH 7.2 during 30 min at 4 ° C, and washed in the same buffer for an equal period, followed by incubation at 37°C for 1 h in the medium: Tris-maleate buffer 1mM, pH 5,0, sodium beta-glycerophosphate 1mM, saccharose 5% (p/v) and CeCl3 2mM. Controls were incubated in the medium without sodium beta-glycerophosphate, but otherwise prepared in the same manner. The vesicles were post-fixed in 1% osmium tetroxide in cacodylate buffer for I h. Dehydration was carried out in an ethanol series and propylene oxide and embedding in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and observed under Transmission Electron Microscopy Jeol JEM 1011 of Electron Microscopy Platform of Oswaldo Cruz Institute, FIOCRZ, RJ. In spermatozoa stored in the seminal vesicles, the phosphate of lead deposits are located specifically in axonema, on the radial spokes extending from the central pair of microtubules to the outer pairs 9 and the connection of accessory microtubules (Fig. 1-4). The intensity of the response varies from one to another axonema and also between the fibers of a single axonema. These results indicate that acid phosphatase appears to be involved in phosphate metabolism important for flagellar motility, since this enzyme activity begins only after acquiring all its axonema microtubules and connecting fibers and to be fully equipped for movement.

CAPES and Electron Microscopy Platform of the Oswaldo Cruz Institute, RJ.

Fig. 1: Transverse section of spermatozoa. In the axoneme, the lead deposits are very dense on the radial spokes (arrow) between the central pair of microtubules and the nine peripheral doublets. N: nucleus.

Fig. 2: Transverse section of spermatozoa. In the axoneme, the lead deposits are very dense on the radial spokes (arrow) between the central pair of microtubules and the nine peripheral doublets. MD: mitochondrial derivates.

Fig. 3: Longitudinal section of axoneme. Lead phosphate deposits are evident on the radial spokes (arrow). N: nucleus.

Fig. 4: Prepared control, with spermatozoa showing no enzymatic reaction product (arrow). Ax: axoneme.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-5732 Toxicicity parasite cell response by CdTe nanoparticles

Gomes S. A.1, Vieira C. S.1, Marques W. A.1, Almeida D. B.4, Pacheco J. P.1, Menna-Barreto R. S.3, Santos-Mallet J. R.2, Cesar C. L.4, Feder D.1
1Laboratório de Biologia de Insetos, GBG, Universidade Federal Fluminense-UFF, Niterói, Rio de Janeiro, RJ, Brazil, 2Laboratório de Transmissores de Leishmanioses, Setor de Entomologia Médica e Forense, IOC-FIOCRUZRio de Janeiro, RJ, Brazil, CEP: 21040-360., 3Laboratório de Biologia Celular, IOC-FIOCRUZ-Rio de Janeiro, RJ, Brazil- CEP: 21040-360, 4Laboratório de Aplicações Biomédicas de Lasers, Departamento de Eletrônica Quântica, Instituto de Física GlebWataghin, Universidade Estadual de Campinas, Campinas, SP, Brazil, CEP: 13083-970
mdfeder@id.uff.br

Nanotoxicity by quantum dots (QD) has been extensively studied in prokariote and eucaryote cells. The toxicity of QDs is associatedwith their physicochemical properties. Several attempts have been made to reduce particle size by (a) selecting the capping of the nanoparticles; (b) using minimal doses; and (c) modulating nanoparticle size. All these factors are importantfor cell toxicity response and consequently to the use of QDs as fluorescent marker. The potential effects of CdTe in T. cruziepimastigotes were evaluated by our group showing that high doses of QDs (200 µM) led to a decrease in T. cruzi growth patternsthroughthedecreasing ofthe percentage of duplicated-DNA parasites (14.5%) and increased of the percentage of fragmented-DNA parasites (36.7). Ultrastructural data showed extensive mitochondrial swelling and abnormal chromatin condensation, moreover endoplasmic reticulum profiles surrounding the subcellular structure and myelin-like structures, were also observed leading us to suggest autophagy. To confirm the hypothesis parasites were incubated with monodansylcadaverin (MDC) a lisossomotropic fluorescent compound useful for identifying cadaverin protein, present in the autophagic vesicles and observed by fluorescence microscopy. The images revealed many autophagic vacuoles within parasite cells, which could to confirm the autophagy developing.At the same time, experiments were performed with 3 methyladenine (3MA) a phosphatidylinositol 3 kinase (PI3K) inhibitor that showed a dose-dependent reversion of cell death. Taken together our results show that high QDs concentrations are toxic to T. cruzi, inducing cell death by autophagy.

Supported by: FAPERJ, PROPPI-UFF and IOC-FIOCRUZ.

Fig. 1: A- T. cruzi control; b- T. cruzi treated with 200μM of QDs + 100 μM of monodansylcadaverine. These head arrows represent vacuoles MDC (+) expressing the cadaverine protein labeled, visualized by Zeiss Axioplan Microscope (fluorescence).

Fig. 2: Transmission electron microscopy analysis of Trypanosoma cruzi CdTe labeled A- T. cruzi control presenting typical morphology, B and C T. cruzi labeled with 200 uM QDs. Bars = 1 µm

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-5904 Induction of calcium-dependent events relating to cell invasion in Toxoplasma gondii tachyzoites.

González-del Carmen M.1, Cariño-Calvo L.2, Trujillo J. U.1, Huerta M. J.1, Valdés A.1, Gonzalez S.3, Mondragon R.4
1Facultad de Medicina, Universidad Veracruzana Av. Hidalgo, Esq. Carrillo Puerto s/n, Col. Centro, C.P. 94740 Cd. Mendoza, Veracruz, México, 2Facultad de Ciencias Químicas, Universidad Veracruzana, Zona Cordoba-Orizaba, 3Unidad de Microscopia Electrónica del CINVESTAV, 4Departamento de Bioquímica, CINVESTAV-IPN Av. Instituto Politécnico Nacional No 2508. Col. San Pedro Zacatenco, Del. Gustavo A. Madero., México D.F. C.P.07360
manugonzalez@uv.mx

INTRODUCTION

Toxoplasma gondii is an intracellular protozoan parasite which affects humans causing encephalitis, chorioretinitis and death. The tachyzoite has structures that allow it to perform cell invasion. During the invasion, tachyzoite adheres to the target cell membrane through proteins from micronemes and then projects a dynamic structure called conoid [1,2]. Subsequently the parasite enters the cell and is housed in a parasitophorous vacuole proliferating by endodyogeny. After several cycles of replication, the parasites leave the infected cell by mechanisms such as conoid extrusion and secretion, which enables the parasite to invade neighboring cells. This replication cycle results in cell destruction and is responsible for the major clinical manifestations of toxoplasmosis. Ethanol is a well-characterized inductor calcium-dependent events in different cell models. In this work we used ethanol and we analyze the morphological changes that occur in isolated T. gondii tachyzoites.

METHODS

Cell culture. The cell model used for our invasion, proliferation and egress assays was HEP-2 cells (human epithelial laryngeal carcinoma cells) (Hep-2,ATCCCCL-23).

Parasites and conoid extrusion. The RH strain of T. gondii was maintained in BALB/c mice, purified and incubated with 0.5M ethanol, fixed, and processed for electron microscopy and immunofluorescence.

Eletronic microscopy. The samples were fixed, dehydrated and infiltrated in resin or processed for scanning electron microscopy. Thin sections obtained were contrasted with uranyl acetate and examined in a transmission electron microscope (Jeol 2000 EX).
Immunodetection. MIC2 protein detection was performed without permeating in extracellular tachyzoites. The samples were fixed and incubated with specific primary antibodies and analyzed using confocal microscopy.

RESULTS

The exposure of tachyzoites to ethanol induces reversible conoid extrusion, clearly observed the projection of the apical part of tachyzoites (Fig. 1). We also observed the presence of vesicle components accumulating in the back of tachyzoites, associated with some changes in the plasma membrane. We could detected that these vesicular components presenting on its surface was always associated with the extruded conoid(Fig. 1). For immunofluorescence, we showed that the secreted products during extrusion conoid micronemes come from. These results suggest a similar mechanism of secretion and induction for extrusion.

REFERENCES

[1] Mondragón y Frixione . J. Euk. Microbiol. 43 (1996) 120-127.

[2] González-Del Carmen, et al. Cell Microbiol 11(2009):967-82

This study was supported by grant CONACYT # 165282 and UV-PTC-672 from PROMEP-SEP to MGC and by grant CONACyT #155459 to RMF.

Fig. 1: Conoid extrusion induced by incubation with ethanol. Tachyzoites were incubated ethanol and analyzed by scanning electron microscopy. Conoid projected (arrows) and the presence of secreted elements accumulate in the posterior end (arrow head) is observed. Scale =2 μm

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-5916 Morphological analyses with transmission electron microscope of Cryptosporidium obtained from raw water supplies

Afzan M Y1, Hizrri M A1
1Department of Biomedical Science, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia
afzan_y@yahoo.com

Cryptosporidium, an enteric protozoan pathogen is hidden enemy in surface water listed as reference pathogen perils for drinking water supplies under WHO guidelines of Safe Drinking-water. In Malaysia, data involving intestinal protozoa infections in raw water supplies are few in numbers. This study provides morphological analysis of Cryptosporidium oocysts obtained from raw water supplies by transmission electron microscopy. The samples were processed by using membrane filtration technique using flat-bed membrane filtration technique. Then the processed samples were observed under direct microcopy. We successfully detected the occurrence of Cryptosporidium by its size which is approximately 4 µm and its rounded shape. The positive samples with Cryptosporidium oocysts were pooled together and preserved with 4% glutaraldehyde for transmission electron microscopy processing. The findings showed that the oocyst is ovoid in shape and covered by thick-doubled cell wall which contains sporozoites. Through this study, an improved understanding on morphological aspect of Cryptosporidium oocyst obtained from raw water will help in controlling and combating the occurrence of this parasite. Therefore, the use of transmission electron microscope serves as confirmation tool to confirm the occurrence of Cryptosporidium in raw water supplies. Besides, on the technical aspects, transmission electron microscopy is the appropriate tool to study the biological and characteristic of other parasites.

The study would not be possible without the support of RAGS grant no 13-010-0073. The authors of this paper also would like to thank Kulliyyah Allied Health Sciences for the financial support, Integrated Centre of Research Animal, IIUM and PAIP Department for technical support, and final year project-students Ceria group for their kindness help. 

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-5917 Characteristic of Ascaris lumbricoides in stool of humans by scanning electron microscopy

Mardhiah M1, Nurul Munirah A1
1Department of Biomedical Science, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia, Kuantan, Pahang, Malaysia
mardhiahmm@gmail.com

Helminth infections in human are usually caused by intestinal helminths include Ascaris lumbricoides that can be diagnosed through detection of eggs and larvae in the stool of human sample. It has been reported that high prevalence of Ascaris lumbricoides was recorded in regions such as Central sub-Saharan Africa, Southeast Asia, Latin America and Oceania. The external environment is crucial for the egg of Ascaris lumbricoides maturation period before they are passed into the soil and need up to 4 weeks to develop into the infective stage. The disease diagnosis through the examination of worm eggs or larvae in stool samples is usually intended to identify the occurance of Ascaris lumbricoides. Direct microscopy examination and staining method are not sufficient to study the morphology and characteristic of Ascaris lumbricoides. Therefore, the aim of this study is to characterized the egg of Ascaris lumbricoides by using scanning electron microscope method for better images and identification of the egg of Ascaris lumbricoides can be made. Human stool had been collected and were examined by scanning electron microscopy. The results showed that the egg of Ascaris lumbricoides exhibited rough and wrinkle surface. Besides, many granules were detected on the surface of the egg. Hence, the use of scanning electron microscope is a promising method which serve an alternative method to study the morphology and characteristic of the surface structure of this parasite other than staining method.

The author of this paper would like to thank Kulliyyah Allied Health Sciences for the financial support and guidances. 

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-5933 The apicomplexan parasite Babesia divergens internalizes Band 3, Glycophorin A and Spectrin during invasion of human red blood cells

Repnik U.1, Gangopadhyay P.2, Bietz S.2, Przyborski J. M.2, Griffiths G.1, Lingelbach K.2
1Department of Biosciences, University of Oslo, Oslo, Norway, 2Department of Parasitology, Philipps University Marburg, Marburg, Germany
urska.repnik@ibv.uio.no

Shared first authors: Repnik U. and Gangopadhyay P.

Shared last authors: Griffiths G. and Lingelbach K.

 

Plasmodium falciparum and Babesia divergens are obligate intracellular parasites that belong to the phylum Apicomplexa. P. falciparum invades human RBC, is transmitted by mosquitoes and is a causative agent of malaria, while B. divergens infects bovine and, occasionally, human RBC, is transmitted by ticks, and causes a hemolytic disease termed babesiosis.

The mammalian RBC is normally unable to endocytose or phagocytose and the process of the RBC invasion by either parasite is incompletely understood. Initially both parasites are surrounded by the RBC plasma membrane-derived parasitophorous vacuolar membrane (PVM) that is formed during invasion. We have investigated the formation and fate of the PVM in a B. divergens strain adapted to human RBC. In addition to the ultrastructural analysis, the presence of host RBC membrane proteins and lipids in the PVM was analyzed by whole mount or on-section immunofluorescence and immunogold labeling. For labeling, we used chemically fixed samples, as well as cells that were high pressure frozen, freeze substituted and embedded in LR White resin.

Our results demonstrate that protein and lipid components of the RBC plasma membrane are present in the initial PVM. Integral membrane proteins band 3 and glycophorin A, and the cytoskeletal protein spectrin were found to be associated with the PVM of B. divergens, whereas they were absent from the PVM of P. falciparum at the ring or the trophozoite stage. We provide evidence that the biophysical properties of the RBC cytoskeleton per se do not preclude the internalization of cytoskeletal proteins by invading parasites.

This work has been funded by the German Research Council (DFG) SFB593 and SPP1580. We thank the Electron Microscopy Unit for Biological Sciences, Department of Biosciences, University of Oslo. We gratefully thank Heinz Schwarz for setting up HPF and FS in the EM lab and Andreas Brech, University of Oslo, for the use of Leica HPM100.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-5934 Ultrastructural aspects of the tunic wound repair in Styela plicata and Ciona intestinalis (Tunicata)

Di Bella M. A.1, De Leo G.1
1Dept. Biopathology and medical and forensic Biotechnology, University of Palermo, Italy
m.antonietta.dibella@unipa.it

After an injury or surgery of integumentary tissues that are in permanent contact with the environment, an orchestrated process begins to protect the organisms against pathogens and heal the wound to restore the tissue continuity and functioning. The tunic is the intermediary tissue between the exterior and the interior of the body of ascidians, marine organisms belonging to the subphylum of tunicates representing the sister group of vertebrates [1]. It is a specialized tissue covering the mantle epithelium or epidermis, and consists of a leathery or gelatinous matrix containing microfibrils of polysaccharides linked to proteins, and free living cells randomly distributed within it. Apart from its role as a support and mechanical protection, the tunic owning to its cellular component, is also involved in many biological functions [2 and references therein]. Tunicates lack an adaptive immune system but they rely on a robust innate immunity which consists of both humoral and cellular responses [3,4]. Tunic damage caused by the environment and by encrusting organisms, triggers repair mechanisms to prevent microbes from gaining access and spreading throughout the body. A simple defence reaction leading to wound healing, maintenance of tunic tightness, and neutralization of foreign microorganisms occurs. The present study reports on ultrastructural aspects of both Styela plicata and Ciona intestinalis tunic physical wounding occurring in the natural habitat. The most remarkable differences here reported between intact and wounded tunics, are: a) thinning of the tunic b) high presence of bacteria and protozoans within the tunic c) inflammatory aspects Moreover, the presence of natural AMP molecules was immunolocalized in the extracellular space of the wounded tunic. The study aims to compare the cellular reactions in naïve S. plicata and C. intestinalis, significative animal models for the study of the phylogenetic relationship with vertebrates.

[1] Delsuc, F., Brinkmann, H., Chorrot, D., Hervé, P., 2006. Nature 439 (7079), 965-968.

[2] Burighel P., Cloney R.A., 1997. Urochordata: Ascidiacea. In: Harrison, F.W., Ruppert, E.E. (Eds.), Microscopical Anatomy of Invertebrates. Wiley-Liss, New York, Vol. 15, pp. 221-347.

[3] Azumi, K., De Santis, R., De Tomaso, A., Rigoutsos, I., Yoshizaki, F., Pinto, M.R., Marino, R., Shida, K., Ikeda, M., Ikeda, M., Arai, M., Inoue, Y., Shimizu, T., Satoh, N., Rokhsar, D.S., Du Pasquier, L., Kasahara, M., Satake, M., Nonaka, M., 2003. Immunogenetics 55 (8), 570-581.

[4] Shida, K., Terajima, D., Uchino, R., Ikawa, S., Ikeda, M., Asano, K., Watanabe, T., Azumi, K., Nonaka, M., Satou, Y., Satoh, N., Satake, M., Kawazoe, Y., Kasuya, A., 2003. Biochem. Biophys. Res. Commun. 302 (2), 207-218.

The support from the Italian Ministero della Università e della Ricerca (MIUR) and the University of Palermo research grant, is gratefully acknowledged.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-5997 Outer surface identification of Trichostrongylus eggs obtained from goat faecal samples in Malaysia

Afzan MY1, Syahmeiyah WI1, Hazirah NH1
1Department of Biomedical Science, Kulliyyah of Allied Health Sciences, International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia
afzan_y@yahoo.com

Despite the betterment done by the government especially the Ministry of Health regarding the infectious diseases, the issue of gastrointestinal parasites especially among livestock is still a major concern in Malaysia. Indeed there were studies done pertaining the prevalence of gastrointestinal nematodes in livestock, nevertheless they were not as much as studies done in human. Trichostrongylus which known as hairworm commonly affects cattle, goats and other ruminants which can lead to gastritis to animals. Trichostrongylus eggs laid by adult females in the large intestine of the host will be shed together with the faeces to the environment. Hence, this study provides a better understanding on the outer surface identification and morphological characteristics of Trichostrongylus eggs obtained from goat feacal samples by using scanning electron microscopy. Faecal samples were collected per rectum from 42 goats in two farms, Haji Kassim’s farm and Pasfa’s farm which both are located in Kuantan, Pahang, Malaysia. The results showed that the eggs are ovoid or elongated shape. The outer surface of Trichostrongylus eggs were appeared rough, crease and ruffled structure. The rough surface of Trichostrongylus eggs may imply that these forms could be resistant form for these eggs to survive in the outer environment and remain infective for several months. In conclusion scanning electron microscopy examination would facilitate evaluation and identification of outer surface structure of Trichostrongylus and other parasites as well.

The study would not be possible without the support of RAGS grant no 13-010-0073. The authors of this paper also would like to thank Kulliyyah Allied Health Sciences for the financial support, Integrated Centre of Research Animal, IIUM and final year project-students Ceria group for their kindness help.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-7-P-6057 New Insights on the Cytoskeleton of Giardia lamblia using Helium Ion Microscopy and Ultra High Resolution Scanning Electron Microscopy

Gadelha A. P.1, de Souza W.1 2
1Instituto Nacional de Metrologia, Qualidade e Tecnologia, Rio de Janeiro, RJ, Brazil, 2Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
anagadelha@gmail.com

Giardia lamblia is a pathogenic protozoan that causes intestinal disorders in humans. This unicellular organism, although considered one of the earliest eukaryotic cells, presented a complex microtubular cytoskeleton formed by an adhesive disk, four pairs of flagella, funis and a median body. The organization of Giardia cytoskeleton at the ultrastructural level has been analyzed by different microscopy techniques, including high resolution scanning electron microscopy. Nevertheless, recent advances in scanning microscopy technology, which resulted in the development of ultra-high resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM), have opened a new venue to the detailed characterization of new cellular structures. Here we studied the organization of the cytoskeleton of trophozoites of Giardia lamblia using UHRSEM and HIM in membrane extracted cells. Giardia disk was arranged in a spiral organization with cross-bridges measuring around 25 nm in length and connecting their elements. HIM showed a compacted arrangement the microtubules of the ventral disk periphery that were maintained even after break up of the cross-bridge (Fig. 1A-1B). The banded collar, found in the barea area, presented three segments and was associated to the axonemes and the disk microtubules (Fig; 1C). The microtubular sheets of the funis presented a lattice-like array (Fig. 1D). Images of marginal plates showed that these structures were associated with a network of filaments not identified before (Fig. 1E), being the last one spread out in all dorsal surface of the trophozoite. Cells showed a set of filaments oriented parallel to the main axis of the cell body that extended through all cell periphery (Fig. 1F). Taken together, these data revealed the presence of new structures of the cytoskeleton of Giardia lamblia and contribute to the understanding organization of Giardia trophozoites.

FAPERJ, CNPq, INMETRO, UFRJ.

Fig. 1: A-B. HIM images showed a compacted arrangement the microtubules of the ventral disk periphery (arrows in A and * in B). C. The lattice-like array of the funis was visualized (F). D. The banded collar had three segments (arrowhead). E. Marginal plates (MP) were associated with a network of filaments (large arrows). AX: axoneme.
↑ Go to the Table of content ↑

LS-8. Plant science and mycology

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-8-IN-2535 MEMS (microelectromechanical systems) technology in combination with high resolution live cell imaging

Agudelo C. G.2, Sanati Nezhad A.2, Ghanbari M.2, Naghavi M.1, Packirisamy M.2, Geitmann A.1
1University of Montreal, Montreal, Canada, 2Concordia University, Montreal, Canada
anja.geitmann@umontreal.ca

Microdevices based on microfluidics and MEMS (microelectromechanical systems) technology have been employed for numerous biological applications, thereby exploiting the possibility to create 3D structures with dimensions comparable to those of cells (few micrometers). Microdevices can be used to miniaturize laboratory functions (Lab-on-a-chip), and they offer the possibility of isolating or quantitatively measuring different aspects of a complex process, which is not necessarily possible in bulk plate assays. We developed an experimental platform with the specific aim to study tip growing cells, the TipChip. The device allows fluid-flow driven positioning of pollen grains or fungal spores at the entrances of serially arranged microchannels harboring microscopic experimental setups. The tip growing cells, pollen tubes, filamentous yeast or fungal hyphae, can be exposed to chemical gradients, microstructural features, integrated biosensors or directional triggers within the modular microchannels. We demonstrated that the device is compatible with high resolution Nomarski optics and fluorescence microscopy. Using this platform we were able to answer several outstanding questions on pollen tube growth. We established that unlike root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, pollen tubes were found to be able to elongate in air raising the question how and where water is taken up by the cell. Finally, we quantified the invasive properties of pollen tubes. To reach its target in the in vivo situation, the pollen tube needs to exert significant penetrative forces. Using the TipChip we tested the pollen tube's ability to navigate mechanical obstacles and to exert penetrative forces by guiding them through microscopic gaps made of elastic polydimethylsiloxane (PDMS) material. Based on the deformation of the gaps the invasive force exerted by the elongating tubes was determined using finite element methods.

This work was financed by a team grant from the Fonds Québecois de la Recherche sur la Nature et les Technologies (FQRNT) to AG and MP.

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-8-IN-5950 Fungal cell networking: dynamics and mechanisms

Read N. D.1
1Manchester Fungal Infection Group, University of Manchester, Manchester, UK
nick.read@manchester.ac.uk

The majority of fungi are microscopic moulds (filamentous fungi) that grow by means of tip growing tubular ‘cells’ called hyphae. Most filamentous fungi form complex, interconnected, networks of fungal hyphae and these networks are generated by the fusion of genetically identical hyphae or cell protrusions. The interconnected state allows the fungal colony to function as a coordinated individual by the sharing and exchange of nutrients, water, signalling molecules, nuclei and other organelles (Read et al., 2010). During colony initiation in the fungal model Neurospora crassa, germinating asexual spores (conidia) form specialized hyphae called germ tubes that are involved in colony establishment. They also develop specialized cell protrusions termed conidial anastomosis tubes (CATs) that generate fused networks of spore germlings. My group is using the CAT system in N. crassa as a model to study self-signalling and self-fusion between cells in filamentous fungi (Read et al., 2012). I will show how we are combining the live-cell imaging of proteins with mutant analyses to study the cell biology and mechanistic basis of CAT induction, chemotropism and fusion (e.g. Lichius et al., 2014). Prolific cell fusion continues as the fungal colony develops and matures. I will finish my presentation by showing with time-lapse imaging how the complex coenocytic (supracellular), hyphal network formed in the mature colony facilitates the long distance and highly dynamic movement of nuclei and other organelles.

Lichius A, Goryachev AB, Fricker MD, Obara B, Castro-Longoria E, Read ND (2014) CDC-42 and RAC-1 regulate opposite tropisms in Neurospora crassa. J Cell Sci 127: 1953-1965

Read ND, Fleißner A, Roca GM, Glass NL (2010) Hyphal Fusion. In Cellular and Molecular Biology of Filamentous Fungi (ed KA Borkovich & D Ebbole), pp. 260-273. American Society of Microbiology

Read ND, Goryachev AB, Lichius A. (2012). The mechanistic basis of self-fusion between conidial anastomosis tubes during fungal colony initiation. Fungal Biol Rev 26: 1-11.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-8-O-2619 Towards understanding the biogenesis of photosynthetic membrane in the model cyanobacterium Synechocystis PCC 6803 using on-section labeling

Bučinská L.1,2, Nebesářová J.2,3, Maldener I.4, Flötenmeyer M.5, Sobotka R.1,2
1Department of Phototrophic Microorganisms, Institute of Microbiology, Academy of Sciences, Trebon, Czech Republic, 2Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic, 3Biology Centre, Institute of Parasitology, Academy of Sciences, Ceske Budejovice, Czech Republic, 4IMIT, Microbiology/Organismic Interactions, Department of Biology, University of Tübingen, Tübingen, Germany, 5Max Planck Institute for Developmental Biology, Tübingen, Germany
lenka.bucinska@gmail.com

Cyanobacteria form a very important bacterial phylum that emerged at the dawn of life on Earth. These organisms evolved an efficient photoautotrophic metabolism based on oxidation of water molecules and the reduction of CO2 to organic matter using energy of photons. This unique process involves an endogenous thylakoid membrane system and requires two large chlorophyll-protein assemblies called photosystem I and II (PSI and PSII). In the last decades an amazing amount of progress has been achieved in understanding the structure of photosystems, as well as the mechanisms by which they are assembled from individual components. In contrast, we have very limited knowledge regarding the membrane compartment(s) in which the core photosystem subunits are synthesized and assembled into mature photosynthetic complexes. It appears that synthesis and early stages of photosystem biogenesis are inherently related to biogenesis of the whole thylakoid membrane system, and there is an urgent need to localize these processes in the cell.
The unicellular cyanobacterium Synechocystis PCC 6308 provides a unique combination of molecular genetics and physiological characteristics, and is frequently used as a tool for photosynthetic studies. Recently, a distinct region called PratA-defined membrane (PDM) has been identified in Synechocystis in the vicinity of the plasma membrane, and appears to be a likely site for both commencement of PSII biogenesis, and the terminal steps of chlorophyll biosynthesis.
At this time, available data regarding PDM are inconclusive, however in an attempt to clarify this, we have commenced localizing the proteins critical for synthesis of chlorophyll-binding PSII subunits such as YidC insertase, factors like Sll0933 and Ycf48 known to assist in PSII biogenesis, and enzymes related to chlorophyll biosynthesis. Additionally, we are trying to trace the steps of thylakoid membrane biogenesis, by analyzing the ‘greening’ of the nitrogen depleted/repleted cells.
Employing various Synechocystis mutants as controls, we prepared Tokuyasu cryo-sections and localized selected proteins by specific antibodies. To date, our data has not highlighted any specific membrane region where biogenesis could occur, as the analyzed proteins are quite regularly scattered thru the thylakoid membranes. We have however observed an intriguing membrane structure, including putative vesicles protruding from the plasma membrane. Our data contradict the current model based on PDM, and indicate both that the biogenesis of thylakoid membranes could involve vesicular transport, and that the synthesis of photosystems might be located in hypothetical biosynthetic rafts in thylakoids.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-8-O-2928 Automatic analysis of calcium spiking profiles in root-microbe symbioses: introducing the CaSA software

Russo G.1, Spinella S.2, Sciacca E.2, Bonfante P.1, Genre A.1
1Dipartimento di Scienze della Vita e Biologia dei Sistemi, Università di Torino, Italy;, 2Dipartimento di Informatica, Università di Torino, Italy.
andrea.genre@gmail.com

Repeated oscillations in cellular calcium (Ca2+) concentration, known as Ca2+ spiking signals, have been described in plants within a limited number of signaling pathways. Among them, the common symbiotic signaling pathway (CSSP) mediates the recognition of two symbiotic microbes, arbuscular mycorrhizal (AM) fungi and nitrogen fixing rhizobia, by their legume hosts.

Due to the complexity and variability of the Ca2+ spiking patterns we have developed automated Ca2+ spiking analysis (CaSA) software that performs i) automated peak detection, ii) statistical analyses based on the detected peaks, iii) autocorrelation analysis of peak-to-peak intervals to highlight major traits in the spiking pattern.

We used Medicago truncatula root organ colture (ROCs) expressing the 35S:NupYC2.1 cameleon and confocal microscopy for all the FRET experiments, in order to test CaSA on two experimental cases. In the first, CaSA highlighted unpredicted differences in the spiking patterns induced in Medicago truncatula root epidermal cells by exudates of the AM fungus Gigaspora margarita as a function of the phosphate concentration in the growth medium of both host and fungus. In the second study we compared the spiking patterns triggered by either AM fungal or rhizobial symbiotic signals. CaSA revealed the existence of different patterns in signal periodicity, which are thought to contribute to the so-called Ca2+ signature.

On this basis we propose CaSA as a useful tool to complement microscopy observation for studying oscillatory biological phenomena including - but not limited to - Ca2+ signaling.

We are grateful to Mireille Chabaud, Björn Sieberer and David Barker (LIPM, Toulouse, France) for sharing their records of Nod factor-induced Ca2+ spiking, and to David Barker for critical revision of the text.

Financial support for this research was granted to PB by Regione Piemonte BioBITs Project (Converging Technologies 2007, area: Biotechnology-ICT) and by the PRIN Project 2011 Pro-Root.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-8-O-3484 Imaging plant cell walls

Talbot M. J.1, Liu Q.1, Walford S.1, Barrero Sanchez J. M.1, White R. G.1
1Commonwealth Scientific and Industrial Research Organisation, Division of Plant Industry, Canberra, ACT 2601, Australia
rosemary.white@csiro.au

Plant cell walls are integral to plant development and growth, and are important bioproducts in timber, fibres, food and biofuel. Two cell wall types we are interested in are those of cotton fibres and cereal seeds. Cotton fibres are extensions from approx. 30% of the cotton seed epidermal cells, and their number per seed, length, thickness and strength are important qualities for industry. Fibres begin as epidermal protrusions just before the flowers open, which is when they are first identifiable, and we have developed a simple protocol for scanning electron microscope (SEM) imaging and quantification of cell size at this stage. We image methanol-fixed, critical-point-dried tissue uncoated in variable pressure mode using both backscattered and secondary electrons, which provide both cell outline and topological information, respectively (1,2). Later, during fibre elongation, we analyse cell wall changes in different mutant lines by imaging acridine orange-stained resin sections using the confocal laser scanning microscope. This method is less prone to the laser polarisation artefacts commonly seen after staining with cellulose-specific fluorescent dyes. Cotton fibres are dead at maturity, and cell wall thickness, presence of a cell lumen and fibre twisting properties are important quantities assessed using a combination of methods, including cryo-SEM to measure fibre wall thickness. We have modified a protocol for measuring animal hair cross sections that is fast and avoids artefacts caused by wetting dry fibres before cryo-SEM imaging, and enables measurement of all three properties simultaneously.

Cell wall properties are also integral to cereal quality, especially in the starchy endosperm that makes up the bulk of the crop. Our focus is on the role of cell walls and wall enzymes in seed dormancy and germination. We showed previously that the barley coleorhiza, which is the protective tissue surrounding the embryo roots, is the first tissue in which hydration-induced gene expression changes are detected (3), and we also see changes in several cell wall-related genes. Localisation of cell wall components with antibodies to specific carbohydrate residues revealed correlated changes in callose (b1-3 glucan), mixed-link glucan (b1-3,1-4 glucan) and xyloglucan in the coleorhiza of germination-ready seeds. Unmasking cell wall components by treating resin-embedded sections with enzymes to remove specific cell wall carbohydrates allowed increased staining of coleorhiza and other embryo tissues.

1. Talbot MJ, White RG (2013) Plant Methods 9: 36 doi:10.1186/1746-4811-9-36

2. Talbot MJ, White RG (2013) Plant Methods 9: 40 doi:10.1186/1746-4811-9-40

3. Barrero JM, Talbot MJ, White RG, Jacobsen JV, Gubler F (2009) Plant Physiology 150: 1006-1021

This work was supported by Cotton Breeding Australia, a joint venture between CSIRO and Cotton Seed Distributors.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-8-O-3516 Dynamics of auxin concentration, distribution and transport, and NIT2 expression reveal their involvement in microspore embryogenesis

Rodríguez-Sanz H.1, Solís M. T.1, López M. F.2, Gómez-Cadenas A.2, Risueño M. C.1, Testillano P. S.1
1Pollen Biotechnology of Crop Plants group. Centro de Investigaciones Biológicas (CIB) CSIC. Ramiro de Maeztu 9, 28040 Madrid, Spain., 2Dep. Ciencias Agrarias y del Medio Natural, Univ.Jaume I, Campus Riu Sec, 12071, Castellón, Spain.
TESTILLANO@CIB.CSIC.ES

Microspore embryogenesis constitutes an intriguing system in which a cell, the microspore, is reprogrammed from its gametophytic program towards an embryogenic pathway by stress treatments, giving rise to an embryo and a plant, a widely used method to obtain double-haploid plants with many applications for plant breeding. The auxin indole-3-acetic acid (IAA) is a major coordinating signal in the regulation of plant development. Despite the abundant data on the auxin involvement in plant growth and development, no information on the role of endogenous IAA on microspore embryogenesis is available.

In this work IAA levels and distribution, expression of BnNIT2, responsible of conversion of indol-3-acetonitrile to IAA, and effects of inhibition of IAA transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-Chlorophenoxy) isobutyric acid (PCIB) treatments were analyzed during Brassica napus microspore embryogenesis, a system in which the process is induced and embryo developed in vitro from individual isolated cells without addition of plant growth regulators. Experimental approach included immunofluorescence using anti-IAA antibodies and confocal analysis, BnNIT2 expression analysis by qPCR, quantification of IAA levels by LC/ESI-MSMS, and treatments with NPA and BCIP.

Results indicated de novo synthesis of IAA at early stages of microspore embryogenesis, progressive IAA increase with microspore-embryo development, and differential distribution pattern in embryo regions at late developmental stages. BnNIT2 gene was up-regulated during microspore embryogenesis. Both, the inhibition of the IAA transport by NPA and the inhibition of IAA action by PCIB negatively affected the microspore-embryo development. NPA also modified the IAA distribution pattern in embryos and at cellular level IAA accumulated in small cytoplasmic compartments suggesting that auxin transport involving the secretory pathway and/or the endoplasmic reticulum could operate during microspore embryogenesis. Taken together, results indicated that endogenous auxin biosynthesis, action and transport are involved in microspore embryogenesis initiation and development.

Rodríguez-Sanz H, Solís MT, López MF, Gómez-Cadenas A, Risueño MC, Testillano PS (2014) Auxin biosynthesis, action and transport are involved in stress-induced microspore embryogenesis initiation and development. Submitted.

Supported by projects funded by Spanish MINECO (BFU2011-23752) and CSIC (PIE 201020E038). HRS is recipient of a predoctoral FPI grant (BES-2009-014245) of MINECO.

Fig. 1: IAA immunofluorescence in microspore embryogenesis and BCIP-NPA effects. A: Vacuolated microspores. B: Early embryo. C: Globular embryo. D: Torpedo embryo. E-G: Control (E), BCIP (F) and NPA-treated (G) cultures. H: IAA immunofluorescence in NPA-treated embryo.  Bars A-C: 20µm, D: 40µm, E-G: 10mm, H: 10µm.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-1740 Immunofluorescence localization and tertiary structure modeling of Tobacco rattle virus PSG strain replicase in tobacco and pepper generative and vegetative organs.

Otulak K.1, Kozieł E.1, Garbaczewska G.1
1Warsaw University of Life Science-WULS SGGW, Faculty of Agriculture and Biology, Department of Botany, Warsaw, Poland
katarzyna_otulak@sggw.pl

Tobacco rattle virus (TRV) is a member of the genus Tobraviruses and has the widest host range of any plant virus. Over 100 plant species are infected in nature, and under laboratory conditions more than 400 species have been infected. TRV is transmitted by parasitic plants, nematodes and partially through seed. TRV not only infects vegetative organs and causes decrease of crop production plant such as tobacco, potato and pepper, but also it has enormous impact on seeds and pollen development. Abnormalities in generative processes have immense influence on infected plants seed production. We demonstrated the structural model of TRV PSG replicase prepared in bioinformatic programs such as: Jmol and Scrach Protein Predictor to present three-dimensional structure of the RNA dependent RNA polymerase of Tobacco rattle virus, and to indicate the localization one of the most antigenic epitope in TRV replicase. The significant epitope was used to production specific antibodies against chosen PTKSGDADTYNANSDR-Cys amino-acids sequence of large subunit RdRp of TRV PSG. The aim of this work was an indicating the potential regions of TRV replicase deposition in the context of crucial meaning of viral replication process, using immunofluorescence detection and immunogold labelling localization. The epitope of large subunit TRV PSG RdRp detection was demonstrated during viral infection in diagnostic host plants, but not only in vegetative organs, but also in ovaries and anthers of tobacco and pepper flowers. The large subunit TRV polymerase was detected in primary cortex parenchyma and phloem in roots of both host as well as in and around vascular bundles of infected leaf blades. The strong fluorescence signal indicated the presence of RdRp of TRV-PSG was shown in ovaries tissues (especially in ovary wall, vascular bundle and placenta) and inside ovules cells. The replicase of TRV PSG was detected in young and mature anther tissues. The strong TRV polymerase fluorescence signal was observed also in tobacco pollen tubes. This results shows new and interesting view on the role of TRV replicase in plant-virus interactions in different parts of host plant connected with pathogenic changes in vegetative and generative organs.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-1761 The level of branching activity: a critical parameter to promote the accumulation of starch-like glucans in a branching enzyme mutant of Arabidopsis complemented with the E. coli GlgB ortholog

Boyer L.1, Ndjindji O.2, Lancelon-Pin C.2, Wattebled F.1, Roussel X.1, D'Hulst C.1, Pontoire B.3, Putaux J. L.2
1UGSF, UMR CNRS 8576, Université Lille 1, Bât. C9, F-59655 Villeneuve d'Ascq, France, 2CERMAV, UPR CNRS 5301, ICMG FR 2607, BP 53, F-38041 Grenoble Cedex 9, France, 3UR1268 BIA, INRA, F-44300 Nantes, France
christine.lancelon.pin@cermav.cnrs.fr

Starch and glycogen are the two main storage polysaccharides that accumulate in living cells. Although they are both made of α(1→4)-linked glucose residues branched in α(1→6) position, they differ in structure and properties. The main difference lies in the distribution of the α(1→6) linkages in the macromolecules. In glycogen, these branching points are homogenously distributed whereas in amylopectin (the major and semicrystalline fraction of starch), they are concentrated in amorphous lamellae. The crystalline regions of amylopectin are mainly composed of linear glucans that intertwine into parallel double helices. In starch metabolism, the isoamylase-type debranching enzymes (that specifically cleave the α(1→6) linkages) control the distribution of the branching points by removing those in excess or misplaced, allowing the interlacing of the chains, an essential event to confer crystallinity to amylopectin [Wattebled et al., Plant Physiol. 138 (2005), 184; Wattebled et al., Plant Physiol. 148 (2008), 1309]. This work aimed at establishing the implication of branching enzymes (that introduce the α(1→6) linkages in the glucans) in the branching point distribution in amylopectin. The Arabidopsis be2- be3- branching enzymes double mutant [Dumez et al., Plant Cell 18 (2006), 2694] has been transformed allowing the expression of the E. coli branching enzyme (GlgB) natively involved in glycogen synthesis in this bacterium. Several transformed plants harboring different levels of GlgB activity were cultivated. Strips of freshly cut leaves harvested at the end of the day were fixed with glutaraldehyde, post-fixed with OsO4 and embedded in Epon resin. Ultrathin sections were cut with a diamond knife, post-stained with periodic acid thiocarbohydrazide silver proteinate (PATAg) and observed by transmission electron microscopy (TEM). Typical images of plastids from the wild-type and three transformants with different levels of GlgB activity are shown in Figure 1. The glucans were specifically stained with PATAg and morphological differences can clearly be seen by comparison with the well-formed starch granules in the wild-type specimen (Fig. 1a). These observations complement chain length distribution profiles (established by HPAED-PAD after complete enzymatic debranching of the molecules) and determination of crystallinity levels (determined by X-ray diffraction) of insoluble polysaccharides. Altogether, our results indicate that replacing the endogenous plant branching enzymes by a protein of bacterial origin can result in the production of a polymer with characteristics close to those of the wild-type amylopectin. Our results allowed to establish a relation between the level of branching enzyme activity and the structure of the synthesized polysaccharides.

The authors gratefully acknowledge funding from Agence Nationale de la Recherche.

Fig. 1: TEM images of ultrathin sections of leave plastids from wild-type (a) and transformed (b-d) Arabidopsis, positively stained with PATAg. The level of ClgB activity in the three transformants varies from high (b) to intermediate (c) and low (d).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-1836 Structure and functions of the glandular trichomes of the vegetative and reproductive organs in some Asteraceae species

Muravnik L. E.1, Kostina O. V.1
1Laboratory of Plant Anatomy and Morphology, Komarov Botanical Institute of Russian Academy of Sciences, St. Petersburg, Russia
lemur50@mail.ru

The present work is devoted to morphological, histochemical and ultrastructural research of the trichomes covering a leaf, petiole, phyllary, and peduncle in six Senecioneae species (Doronicum macrophyllum, D. orientale, Ligularia dentata, Senecio integrifolius, S. viscosus и Tussilago farfara). The secretory structures of each species have specific morphological features. In D. macrophyllum and D. orientale a head contains one layer of the secretory cells (from two to five cells) and the long stalk (Fig. 1). Trichomes of Ligularia dentata are mainly 1-seriate, 2-seriate ones are found rarely. In S. viscosus 2–4 secretory cells form two layers of the head (Fig. 2). Leaf trichomes differ by the stalk height (from 3 to 7 cells). In S. integrifolius the trichomes of a phyllary are smaller; they have one-celled head and a short stalk. In T. farfara the trichome of the leaf and peduncle consist of a head on a long 2-seriate stalk. 3–5 layers of the secretory cells are in the head and 10–12 layers are in the stalk.

All studied trichomes are capable to auto fluoresce in UV radiation without application of the markers. Yellow colour is typical for the walls of the head cells, whereas the red auto fluorescence is found in the chloroplasts of the stalk cells. The yellow-green fluorescence of phenylpropanoids is revealed in the secretory cells in the presence of Natural or Wilson reagents. Phenylpropanoids are located in the subcutical space and in the cell wall. Using the histochemical dyes, in the trichome secretory cells it is possible to find the total, acid and neutral lipids, tannins, polyphenols, terpenoids and sesquiterpene lactones. The most intensity of the colouring is characteristic for 2–3 upper layers of the secretory cells; the stalk cells are colourless.

General ultrastructural features are revealed in the secretory cells of the trichomes in all studied species (Fig. 3). The upper cell wall is heterogeneous: numerous light lamellas are under a cuticle. Secretion is accumulated in the periplasmic space as the small drops. In cytoplasm of the secretory cells abundant smooth endoplasmic reticulum is formed. During maturation the dark contents are accumulated in it. Cisternae of the rough endoplasmic reticulum are rare. In the terminal head cells the leucoplasts have a various form and extended surface. They are rounded, oval, amoeboid, and with cup-shaped invaginations. Tubular membrane elements, plastoglobuli and amorphous inclusions are found in the plastid stroma. Elements of endoplasmic reticulum are met in proximity to the outer membrane of the leucoplast envelope. In the secretory cells situated under the terminal ones and also in all stalk cells there are the chloroplasts. Lipid drops are typical for cytoplasm of the head cells.

We appreciate the Core Centre “Cell and Molecular Technology in the Plant Science” at the Komarov Botanical Institute (St. Petersburg) for provision of equipment for light and electron microscopy. This work was supported by Russian Foundation of Basic Research (grant 13-04-00797).

Fig. 1: Trichomes of the peduncle in Doronicum orientale (A-F) and D. macrophyllum (G-I). Scale bars: A, G – 250 µm; B-F, H-I – 50 µm.

Fig. 2: Trichomes of the leaf in Senecio viscosus. Scale bars: A – 100 µm; B-D – 50 µm.

Fig. 3: Ultrastructure of the secretory cells in the trichomes of some Asteraceae species. A, D, G – Tussilago farfara; B, E, H – Doronicum macrophyllum; C, F, I – Senecio viscosus. A-C. The upper cell wall of the secretory cells. D-F. Smooth endoplasmic reticulum in the secretory cells. G-I. Leucoplasts in the secretory cells. Scale bars: A-I – 1 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-1837 Two different methods of fruit cell size measurements

Pecinar I. M.1, Pekić Quarrie S. V.1, Bertin N. I.2, Rancic D. V.1, Cheniclet C. I.3, Stikic R. I.1
1University of Belgrade, Faculty of Agriculture, Belgrade-Zemun, Serbia, 2INRA,UR1115, Plantes et systèmes de culture horticoles, Site Agroparc Domaine St Paul, Avignon Cedex 9, France, 3University of Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, F-33140 Villenave d’Ornon,
ilinka@agrif.bg.ac.rs

Cell size is a structural component of fleshy fruit such as tomato berry, contributing to important trait such as fruit size. There are currently a number of methods for measuring cell sizes most rely either on tissue sectioning or digestion of the tissue with cell wall degrading enzymes to release single cells. In this study, we used two tomato plants, cv. Ailsa Craig (wild type) and its ABA deficient mutant flacca. We performed histological analysis and calculate pericarp cell size distribution from slides for light microscopy made according to standard paraffin procedure (Ruzin, 1999). In addition, we measured tomato pericarp mean cell area at ripe fruit stage using the method of cell separation by pectinase solution described in Bertin et al. (2002). At least five measurements per pericarp section were done on five fruits replicates for each genotype. Pericarp sections were observed with a Leica DMLS microscope; images were acquired with a Leica DC300 digital camera and measured by Leica IM1000 software. Cell size of macerated tissue were measured by the public domain Image J software (Rasband, 1997-2009, http://rsbweb.nih.gov/ij), using the “analyze particles” tool, after manually adjusting the segmentation threshold. For both genotypes a minimum of 500 cells were measured and it was found that each of them displayed a distribution of cell size. In ripe fruits, differences between genotypes were clearly visible; cell sizes were larger and more heterogeneous in wild type than in flacca. By both methods the mean size of cells in flacca was about 50% smaller than in wild type. Comparing these two methods for cells measuring we could say that pectinase is less time consuming and could give quantitative trait such as cell number, but histological cross sections provides real view of cell size distribution.

This study was supported by Bilateral Cooperation between Serbia and France 2012-2013, Serbian Ministry of Education and Science (No TR 31005) and FP7-AREA (No 316004, 2013-2016).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-1909 Determination of micro-mechanical properties of apple cells (Malus domestica) by means of bio-atomic force microscope (bio-AFM) and analysis of the multidimensional images

CARDENAS S.1, CHANONA J.1, MENDEZ J.2, CALDERON G.1, PEREA M.2, NERI E.1
1Escuela Nacional de Ciencias Biologicas-IPN, 2Centro de Nanociencias y Micro y Nanotecnologías-IPN
stefany_fany03@hotmail.com

Morphometric parameters and micro-mechanical properties of vegetable cells can be considered as an index of quality and ripeness of climacteric fruits. After harvest, the cells undergo biochemical changes that modify their cell size, texture and flavor which are key quality features that influence consumer acceptability of fruits and functional properties. The aim of this work was to study the mechanical properties of apple cells by indentation with a bio-atomic force microscope (bioAFM) to contribute to the understanding of the maturity process on climacteric fruits. In this study was carried out the isolation of parenchymatic cells from the central cortex of the unripe apple (Malus domestica) by dissolution in mannitol (0.3M) at 45 °C and 30 min according to Schaffer et al., (2009). Isolated cells were contrasted to be observed in microscope (Nikon Eclipse 50i, Japan). Images obtained were analyzed in order to characterize the size and cellular morphology (about 300 cells were used). The values of cellular area had a normal distribution with a mean of 352.2±86.16 μm2, while diameter and circularity values ​​were not normally distributed with a mean of 245±36.7 μm and 0.68±0.12 respectively (Figure 1). Also, mechanical properties were studied by means of a bio-AFM (Bioscope Catalyst, Bruker, USA) to indent apple tissue by means of microcantilevers with pyramidal tips (ScanAsyst, radius: 20 nm). Force curves were obtained in several areas of cells and their Young's modulus were obtained by two fit mathematical models (Hertz and Sneddon). Figure 2 shows the force curves obtained during the indentation. Furthermore, multidimensional images of height, error, DMT modulus (e.g. Figure 3 and 4), deformation, dissipation, and adhesion mapping images were obtained. Otherwise, the Young´s modulus was calculated from the force curves and a mean of 0.281±0.125 MPa was obtained to cells analyzed, which it is within the range for biologic materials as reported by Zdunek et al., (2013). An optimizing of the fit models showed that the best fit was the Sneddon model with an R2average of 0.99. The study of cellular mechanical of vegetable cells by atomic force microscopy imaging and the mechanical properties obtained from Bio-AFM could be important for understanding the maturity process that occur during the storage and postharvest handling of climacteric fruits.

Schaffer R.J., McAtee, A.P., Hallet I.C., Jhonston J.W. (2009) A rapid method of fruit cell isolation for cell size and shape measurements. BioMedCentral Plant Methods 9, 5:5

Zdunek A., Kurenda A., (2013) Determination of the Elastic Properties of Tomato Fruit Cells with an Atomic Force Microscope. Sensors, 13, 12175-12191

This research was funded through projects SIP-IPN: 20140387 and 20141662, at the Instituto Politécnico Nacional-Mexico.

Fig. 1: Isolated apple cells used for morphometric characterization by means of image analysis, stained with methylene blue.

Fig. 2: Typical curve force (red line) and fitted with Sneddon model (green line) for isolated apple cells

Fig. 3: Mapping image of peak force error signal of cell wall unripe apple

Fig. 4: Mapping image of DMT modulus signal of cell wall unripe apple

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-1913 Ultrastructural changes in Barbacenia purpurea Hook. (VELLOZIACEAE) seedlings influenced by temperature

Louro R. P.1, Andrade I. F.1,2, Santiago L. M.2
1Laboratório de Ultraestrutura Vegetal, CCS, Instituto de Biologia, Dep. Botânica, Universidade Federal do Rio de Janeiro, RJ, Brasil , 2Laboratório de Biodiversidade e Biotecnologia, Departamento de Botânica, Universidade Federal do Estado do Rio de Janeiro, RJ, Brasil
louro@biologia.ufrj.br

Barbacenia purpurea Hook. (Velloziaceae) is a nurse plant of Sugar Loaf and Urca Natural Monument, a rocky mountains complex, localized in Rio de Janeiro, southeastern Brazil, within Atlantic Rainforest, considered a biodiversity hotspot. Because their high tolerance to adverse conditions, adult nurse plants play an important role on seedling establishment in stressful environments and dynamism of forest expansion. However, B. purpurea seeds germination and seedlings development are inhibited under hottest conditions, making such events possible only during humid months. Since global climate change may compromise the successional dynamic of Sugar Loaf and Urca Natural Monument, this work aim to investigate the influence of temperature on ultrastructure of B. purpurea seed germination. Thirty seeds were imbibited in sterilized water, distributed in plate dishes and incubated under 30ºC, 35º C and 40ºC in growth chamber for 3 h, 3 days and 7 days. For ultrastructural analysis, seeds were fixed with 2.5% glutaraldehyde and 4% paraformaldehyde in 1. 25% PIPES buffer (pH 7.3), postfixed with 1% osmium tetroxide, dehydrated in a graded acetone and embedded in Spurr's resin. Ultrathin sections were post-stained with 1% uranyl acetate in absolute ethanol and lead citrate. It was demonstrated that 100% of seeds germinated 3 days after exposition under 30ºC and 35ºC. Seven days after incubation under 30ºC, 100% of normal seedlings were produced. However the temperature increase to 35ºC induced 100% of seedlings with anomalous morphology and the increased to 40ºC inhibited 100% of seeds germination. Ultrastructural analyses demonstrated that under 30ºC, for 3 hours, 3 days and 7 days, respectively, both endosperm and embryo cells reduced their storage of starch grains, proteins and lipid bodies, in contrast to seeds germinated under 35ºC, where such reserves structures were slightly consumed, and under 40ºC, where they did not alter both in number or size in relation to the first steps of seed germination (3 hours). These results suggested that the influence of temperature on Barbacenia purpurea seeds germination might be related to the inhibition of storage resources consumption and mobilization, which would affect seed germination and seedling morphology.

The authors would like to thank the Universidade Federal do Estado do Rio de Janeiro for financial support of the research through a graduate fellowship provided to the second author.

Fig. 1: Transmission electron microscope of seed endosperm cells of Barbacenia purpurea Hook. 3 hours after incubation under 30o C, evidencing starch grains, protein bodies and lipid droplets.

Fig. 2: Embryo epidermal cell 3 hours after incubation under 30o C showing cell wall, plastids, mitochondria, lipid droplets, endoplasmic reticulum and part of nucleus.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-1951 The diversity of the labellum and gynostemium trichomes in Polystachya Hook. (Orchidaceae)

Narajczyk M.1, Łuszczek D.1, Kubiak J.2, Grochocka E.2
1Laboratory of Electron Microscopy, University of Gdansk, Gdansk, Poland, 2Department of Plant Taxonomy and Nature Conservation, University of Gdansk,Gdansk,Poland
magdalena.narajczyk@biol.ug.edu.pl

Polystachya Hook. (Orchidaceae) is a large epiphytic mainly African genus with 197 species. The species representing Polystachya section Caulescentes Kraenzl. are plants with non-pseudobulbous, reed-like, clustered stems leafy in upper part, simple or paniculate, many-flowered inflorescences. The flowers are characterized by a prominent mentum and 3-lobed lip with a callus. The section includes 18 species divided into 3 subsections. Histochemical studies conducted by Davies et al. (2002, 2009) showed some morphological diversity of labellar trichomes in Polystachya. The authors analyzed two representatives of the section Caulescentes, Polystachya bennettiana Rchb.f. and P. caloglossa Rchb.f.

The aim of the study is to observe the diversity of labellar trichomes in two other species representing Polystachya section Caulescentes (Polystachya albescens subsp. imbricata (Rolfe) Summerh. and Polystachya laxiflora Lindl.), to compare the trichome types occurring in different parts of the labellum and the gynostemium as well as to determine the taxonomic value of the studied characters.

The epidermis of the labellum and gynostemium of two species of Polystachya section Caulescentes were studied. The trichomes from the different parts of the labellum (basal, central with callus and apical) were analyzed using scanning electron microscope.

The analysis reveals the structure of labellar and gynostemial surface of two representatives of Polystachya section Caulescentes. No farinaceous, pollen-like material called pseudopollen is observed. The labella (Fig. 1, 3) and gynostemia (Fig. 2, 4) of both studied species are furnished with similar type of trichomes. They are two-celled (occasionally three-celled) food-hairs with clavate apical cell and occur on the labellum and the gynostemium.

The trichomes of both species analyzed in this study as well as in two other representatives of the section Caulescentes (Polystachya caloglossa and P. bennettiana) studied by Davies et al. (2004) are of similar type, however, there is some diversity in the length and shape of the food-hair regarding on the particular species and parts of the labellum. The trichomes of P. albescens ssp. imbricata are generally longer and slender than the trichomes of P. laxiflora. The labellum and gynostemium of the latter species are more densely covered by the food-hairs.

References

Davies K.L., Roberts D. L. Turner M.P. 2002. Pseudopollen and Food-hair Diversity in Polystachya Hook. (Orchidaceae). Annals of Botany 90: 477-484.

Davies K.L. 2009. Food-hair form and diversification in orchids. In: Kull, Arditti & Wong (eds.). Orchid biology: Reviews and Perspectives, X: 159-184. Springer

Fig. 1: Trichomes of the apical part of the labellum of Polystachya albescens ssp imbricata (Rolfe) Summerh

Fig. 2: Gynostemium trichomes of Polystachya albescens ssp imbricata (Rolfe) Summerh.

Fig. 3: Callus (labellum structure) trichomes of Polystachya laxiflora Lindl.

Fig. 4: Gynostemium trichomes of Polystachya laxiflora Lindl.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-1964 Ultrastructure studies of Polystachya vulcanica (orchidaceae).

Richert M.1, Kubiak J.2, Narajczyk M.1, Mytnik-Ejsmont J.2
1Laboratory of Electron Microscopy, University of Gdansk, Poland, 2Department of Plant Taxonomy and Nature Conservation, University of Gdansk, Poland
malwina.richert@biol.ug.edu.pl

Orchidaceae is one of the largest families of Angiosperms and one of the most specialized lines of flowering plant evolution. Orchids are plants distributed all over the world with exceptional species diversity, unique and often spectacular flowers and various relationships with both pollinators and fungi.

Polystachya vulcanica Kraenzl. is one of 197 species of the pantropical genus Polystachya Hook. classified within exclusively African section Cultriformes Kraenzl. including one-leaved plants. The species is endemic to the Albertine Rift (part of Democratic Republic of Congo, Rwanda, Burundi and Uganda), only known from Kahuzi-Biega, Nyungwe-Kibira, the western Virunga Volcanoes, Kigezi and Ruwenzori, at 1300–2400 m.a.s.l.

The species is an epiphyte growing in montane forest, on mossy branches or sometimes a lithophyte occurring on mossy rocks. It flowers from January to April and from August to December.

Polystachya vulcanica is a plant up to 20 cm tall with filifrom pseudobulbs tightly clustered with a terminal single, linear, semiterete leaf. Its inflorescence is shorter than the leaf, 1- to 5-flowered, flowers are medium-sized, creamy-white, flushed with rose, lip and petals wine-red or purple (fig.1).

Fragments of the lip and gynostemium of Polystachya vulcanica were prepared to transmission electron microscopy. The ultrastructural observations based on ultrathin sections shown that epidermal cells predominantly have centrally located vacuoles and cytoplasm concentrated near the cell wall (fig.2). What is significant, cell wall and cuticle of epidermis form characteristic more or less regular folds. Between these folds, above the cuticle, there are visible secreted materials (fig.3). Specific construction of cell wall and cuticle, such as presence of numerous bubbles within, may to facilitate the secretion and indicate possible pathway of this activity. Substance outside epidermal membranes may play role to attract insects.

Fig. 2: Ultrastructure of the epidermal cells with cuticle of Polystachya vulcanica lip.

Fig. 3: Epidermal outer cell wall and cuticle with secreted materials.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-1992 VISUALIZING SYMBIOTIC INTERFACE BIOGENESIS IN LIVING ARBUSCULAR MYCORRHIZAS

Genre A.1, Chabaud M.2, Ivanov S.3, Russo G.1, Zarsky S.4, Bisseling T.3, Barker D.2, Bonfante P.1
1University of Torino, Italy, 2LIPM CNRS/INRA Toulouse, France, 3Wageningen University, The Netherlands, 4Charles University Prague, Czech Republic
andrea.genre@unito.it

Arbuscular mycorrhizas (AM) are symbiotic associations involving up to 90% of terrestrial plants and symbiotic fungi belonging to Glomeromycota. AM fungi enhance the absorption of plant nutrients and give them resistance against pathogens by colonizing the root with inter- and intracellular hyphae and arbuscules, the highly branched structures where the nutrient exchange takes place. A symbiotic interface compartment is developed by the host cells around intracellular fungal structures and is considered as a hallmark of the biotrophic condition of AM fungi, and enables fungal development inside the plant cell space, while preserving its integrity. This presentation focuses on the plant perception of AM fungi and their accommodation within the host cell. Our results, largely based on in vivo confocal microscopy, demonstrate that the process of interface construction takes place upon recognition of the AM fungus and adhesion of a hyphopodium to the root epidermis. Epidermal cells contacted by the fungus show repetitive oscillations (spiking) of nuclear calcium concentration. These oscillations are a central element in the signaling pathway that controls the symbiosis. Activation of this pathway leads to the assembly of the prepenetration apparatus (PPA), a columnar cytoplasmic aggregation, containing all the elements of the secretory pathway. By taking advantage of a range of fluorescent protein markers we show that the proliferation of the host plasma membrane takes place within the PPA, leading to the assembly of the perifungal membrane and symbiotic interface, in advance of hyphal tip growth.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-2000 Imaging of vessel wall degradation by Ophiostoma novo-ulmi in micropropagated elms using AFM PeakForce quantitative nanomechanical mapping and SEM observations

Ďurkovič J.1, Mamoňová M.2, Lagaňa R.2, Kučerová V.3
1Department of Phytology, Technical University, 960 53 Zvolen, Slovakia, 2Department of Wood Science, Technical University, 960 53 Zvolen, Slovakia, 3Department of Forest Protection and Game Management, Technical University, 960 53 Zvolen, Slovakia
jaroslav.durkovic@tuzvo.sk

The ascomycetous fungus Ophiostoma novo-ulmi Brasier is the causative agent of the current Dutch elm disease (DED) pandemic, which has ravaged both European and North American Ulmus populations. The pathogenic fungus spreads within the secondary xylem vessels of infected trees, causing the formation of vessel plugs due to tyloses and gels, which ultimately results in foliar wilting and subsequent tree death. Recently we found that the primary targets of O. novo-ulmi ssp. americana × novo-ulmi attack were medium-molecular weight macromolecules of cellulose, resulting in the occurrence of secondary cell wall ruptures and cracks in the vessels (Fig. 1) but rarely in the fibers of the examined Dutch elm hybrids 'Groeneveld' and 'Dodoens'. In this study, the newly developed atomic force microscopy (AFM) technique, PeakForce quantitative nanomechanical mapping, was used to reveal vessel wall topography in the infected trees. Quantitative images for nanomechanical properties of cell walls such as adhesion, deformation, the reduced Young’s modulus of elasticity, and dissipation, were also obtained. This new technique of imaging provided direct control of the maximum loading force and the deformation depth in vessel wall samples keeping indentations small, while eliminating damaging lateral forces in order to preserve both the tip and sample. High-resolution and non-destructive imaging (Fig. 2) resulted from the short range repulsive forces which dominated the interaction between tip and sample. Microscopic observations of vessel wall degradation were supplemented with size exclusion chromatography and 13C nuclear magnetic resonance (NMR) measurements. The NMR spectra revealed that a loss of crystalline and non-crystalline cellulose regions occurred in parallel.

This work was supported by the Slovak scientific grant agency VEGA (1/0132/12).

Fig. 1: SEM images of O. novo-ulmi ssp. americana × novo-ulmi and elm secondary cell wall cracks. (A) Tyloses occlude earlywood vessel lumens, and fungal hyphae growing inside the vessel, cross-section. Scale bar = 100 μm. (B) Fungal hyphae inside the earlywood vessel and radial cracks in the secondary cell wall (arrows), radial section. Scale bar = 50 μm.

Fig. 2: Flatten height (A) and adhesion (B) AFM images of the elm vessel wall surface upon infection by O. novo-ulmi ssp. americana × novo-ulmi. Scale bars = 10 μm. L, lumen; CW, cell wall.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-2038 Fluorescence in situ hybridization (FISH) for the investigation of repetitive DNA sequences in lymegrass genome

Anamthawat-Jonsson K.1
1Institute of Life and Environmental Sciences, University of Iceland, Reykjavik, Iceland
kesara@hi.is

Fluorescence microscopy is essential for the study of genes, chromosomes and genomes. The method called fluorescence in situ hybridization (FISH) is used here to map repetitive DNA sequences on chromosomes of plants species in the tribe Hordeeae, family Poaceae, that have the Ns-genome in its genomic constitution. Two types of Ns-specific repetitive DNA sequences from Leymus Hochst. and Psathyrostachys Nevski have been isolated in my group: (A) dispersed retroelement-like repeats isolated from tetraploid northern North American and amphi-Pacific species L. mollis (Trin.) Pilger and octoploid European species L. arenarius Hochst. (Figure 1A, Bödvarsdóttir & Anamthawat-Jónsson 2003, Genome 46: 673-682); and (B) sub-telomeric satellite repeats isolated from tetraploid western North American species L. triticoides (Buckley) Pilger (Figure 1B, Anamthawat-Jónsson et al. 2009, Genome 54: 381-390) and two diploid Psathyrostachys species (unpublished results). These sequences have been analysed molecularly and mapped on chromosomes. The sequences, particularly the dispersed repeats, have been used to confirm the presence of Ns-genome in a wide range of species in Triticeae genera including Leymus, Psathyrostachys, Hordelymus (Jessen) Harz and Hystrix Moench. The results consistently show that all genomes in the polyploid Leymus species examined are Ns. The Ns-specific dispersed repeats may have spread predominantly and rapidly across genomes following allopolyploidisation, thus homogenising the nuclear genomes. The Ns-genome may have been evolutionarily diverged into a variable array of Ns-karyotypes, the variation which can be identified using satellite repeats. Furthermore, polymorphisms in Ns-genome specific sequences can be used to reveal relationships among taxonomically related species and to differentiate closely related species having different geographical distributions.

I would like to thank Ægir Thór Thórsson, Sæmundur Sveinsson, Sigrídur Klara Bödvarsdóttir and Tidarat Puangpairote for the good work on molecular cytogenetics of lymegrass.

Fig. 1: Fluorescence in situ hybridization (FISH) mapping of red-fluorescing pLm44 dispersed retroelement-like repeat (A) and green-fluorescing sub-telomeric satellite repeat Lt1-1 (B) on chromosomes of Leymus triticoides. The double FISH in both images represents localization of the 18S-25S ribosomal loci. The scale bar represents 5 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-2043 Study of storage compounds in beech embryos during dormancy breaking

Eliášová K.1, Vondráková Z.1
1Institute of Experimental Botany AS CR, Prague, Czech Republic
eliasova@ueb.cas.cz

The common beech (Fagus sylvatica L.) is one of the most important broadleaved species in European forestry. At harvest, beechnuts are in deep physiological dormancy as seeds of many temperate trees. Dormancy as a mechanism preventing germination during unsuitable ecological conditions is controlled by the genetic factors and regulated by phytohormones. Seeds can be stimulated to dormancy breaking and to germination by cold stratification. The control of dormancy breaking is recently studied in the different levels – environmental, biochemical, molecular etc.
Our study is aimed to the changes in the amount and distribution of storage compounds in the beech embryos during stratification. Beechnuts belong to the group of non-endospermic seeds with reserves stored predominantly in the cotyledons. The major mobilization of storage compounds as starch, proteins and oils within storage tissues commences after protrusion of the radicle. Nevertheless, some partial mobilization of storage proteins starts with the uptake of water by imbibition of the dry seed.
Paraffin sections were observed under the transmission light microscope Jenaval, Zeiss. We detected the storage proteins using protein specific stains – Ponceau-xylidin, amido black 10B and Commassie brilliant blue R250. Storage proteins were deposited in the vacuoles predominantly in the cells of cotyledons. In dormant embryos the most intensive protein labelling was observed in storage vacuoles which filled up the cotyledon cells and partially also in the cytoplasm. After imbibition of the seeds, storage vacuoles in the external parts of the cotyledons diminished and their content exhausted. Later on the small vacuoles fused to form large central vacuoles. During stratification we observed strong labelling in the cytoplasm. The central vacuoles remained free of storage proteins.
Starch was stained using Lugol solution. Abundance of starch grains were observed in cotyledons, as well as in the embryo axes. We did not recognize any differences in the distribution or amount of starch grains in dormant and non-dormant embryos. Besides these storage compounds the immense amount of calcium oxalate (CaOx) crystals was observed under the polarized light. CaOx appeared as druses or small prismatic crystals in cotyledons excepting the region of vascular tissues. Small druses were rarely found also in the embryo axes.
According to our results, storage proteins localization and utilization only is linked with stratification. Other storage compounds will be probably used during germination.

The research was supported by the Ministry of Agriculture, project QI102A256.

Fig. 1: Storage tissue in cotyledon of dormant beech seed. Cells are filled with protein storage vacuoles (red colour). Paraffin section stained with Ponceau-xylidine / Azur II; scale bar = 50 µm.

Fig. 2: Storage tissue in cotyledon of beech seed released from dormancy. Proteins were present in small bodies or in the cytoplasm (red colour). Paraffin section stained with Ponceau-xylidine; scale bar = 50 µm.

Fig. 3: Starch grains (dark dots) in the cells of beech embryo cotyledon surrounding the vascular bundle. Paraffin section stained with Lugol (I/KI) and Azur II; scale bar = 50 µm.

Fig. 4: Druse crystals of calcium oxalate (arrow) in vacuoles of cotyledon storage cells of the beech embryo. Paraffin section in the polarized light; scale bar = 20 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-2066 Identification of the genetic traits responsible for 'Stabilstroh' phenotype.

Muszynska A.1, Börner A.1, Melz G.2, Röder M. S.1, Rutten T.1, Hoffie K.1, Benecke M.1, Melzer M.1
1Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), D-06466 Gatersleben, 2Monsanto Saaten GmbH Zweigniederlassung Nienstädt, D-31688 Nienstädt
muszynska@ipk-gatersleben.de

Lodging, the state of permanent displacement of tillers from their upright position, decreases grain quality and increases the costs of harvesting, qualifying this phenomenon as one of the most serious problems in cereal crop production. Therefore lodging resistance is an important agronomic trait, especially in rye (Secale cereale L.), where yield losses due to lodging can be as high as 75%. ‘Stabilstroh’, a recently identified genotype of rye, not only has the best lodging resistance, but simultaneously it is characterized by the longest tillers among the German cultivars of rye hybrids.

In order to identify the genetic traits responsible for the 'Stabilstroh' phenotype histological and ultrastructural investigations were focused on the most prone to lodging basal internodes of segregating F2 population (‘304/1’) and its parental lines: ‘ms135’ (‘Stabilstroh’) and ‘R1124’ (wild type). Analyses of tissue distribution, cell size, and cell wall thickness using Light Microscopy, Scanning Electron Microscopy, and Transmission Electron Microscopy revealed sclerenchymal and inner periclinal cell walls of epidermis to be thicker, more lignified, and more structured in the 'Stabilstroh' genotype as compared to the wild type. 'Stabilstroh' is also morphologically characterized by many pronounced stem invaginations and a significantly higher ratio of sclerenchyma to parenchyma tissues (sc/pa ratio), important factors enhancing mechanical stability of the crop stem. Not only do these features improve mechanical properties of lodging resistant genotype, but they are also responsible for increased biomass production.

The quest for QTLs (Quantitative Trait Loci) for improved lodging resistance is based on the inheritance of microsatellite (SSR) markers linked to the traits affecting mechanical stability of tillers, including stalk invaginations, thickness of cell walls, lignin content, and sc/pa ratio.

Fig. 1: A standard phloroglucinol staining of lignin in wild type (A) and 'Stabilstroh' (B). Hand cross-sections of wild type (C) and 'Stabilstroh' (D) under SEM. Basic fuchsin staining of cell walls in: wildtype (E) and 'Stabilstroh' (F). SI- stem invagination; Vb –vascular bundle; Pa- parenchyma; Sc- sclerenchyma
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-2463 Visualization of silica phytoliths in Sorghum bicolor (L.) Moench. by fluorescence microscopy

Soukup M.1, Cigáň M.2, Lux A.1
1Department of Plant Physiology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska dolina B-2, 842 15 Bratislava, Slovakia , 2Institute of Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska dolina CH-2, 842 15 Bratislava, Slovakia
soukup.em@gmail.com

Silica phytoliths are microscopic biominerals of amorphous silica (SiO2 ∙ nH2O) formed by specialised plant cells. Their morphology is very variable among the species and they are often used as a taxonomic marker. They serve as a protection against herbivores and pathogens and support mechanical properties of plant tissues. Their structural features offer many possibilities of application also in industry and technology [1, 2]. However, small dimensions and optical properties of phytoliths obstruct their microscopic observations. Recent imaging techniques require lengthy preparation of samples, phytolith isolation, or a work with toxic chemicals. Tracking the changes in biomineralization process caused by various exogenous or endogenous factors thus might be complicated. Our study was focused on elaboration of a novel phytolith visualization technique by fluorescence microscopy that could facilitate further silica biomineralization studies.
Experiments were performed with Sorghum bicolor L. plants characteristic by regularly distributed silica phytoliths in root endodermal cells. Plants were grown for three days in hydroponics containing distilled water and sodium silicate solution in final concentration of 2.5 mol.dm-3. Samples were prepared from seminal root basal regions 20 – 30 mm from the root-shoot junction. Rhizodermis and cortical tissues were mechanically removed and samples were placed on slides into a mounting solution of NaOH with pH adjusted to 12. Observations were performed by fluorescent microscope Axioskop 2 plus (Carl Zeiss) with excitation filter TBP 400 + 495 + 570 nm, dichroic beamsplitter TFT 410 + 505 + 585 nm and emission filter TBP 460 + 530 + 610 nm. Spectrofluorimetric properties were analysed by spectrofluorimeter FSP 920 (Edinburgh Instruments).
Phytoliths in Sorghum roots are formed as dome-shaped structures of amorphous silica nested in inner tangential walls of endodermal cells. Their diameter and axial dimensions ranged 8.51 ± 2.49 µm and 7.08 ± 1.17 µm, respectively. Number of phytoliths counted from 7 to 14 per cell. Silica phytoliths emitted blue fluorescent signal, which was clearly distinguishable from cell wall background and was time-stable for more than 10 minutes. Despite this fact, the fluorescence emission spectrum induced by 400 nm excitation wavelength did not show any remarkable peaks and reaches maximum at 475 nm.
Fluorescent visualization of silica phytoliths induced by basic pH provided suitable features for their localisation and morphological studies. Preparation of samples was quick and simple, and might be applicable for high throughput screenings.

This study was supported by grants 1/0817/12 from Slovak grant agency VEGA, by Slovak research and development agency APVV according to contract n. APVV-0140-10, and by Comenius University grant UK/394/2013.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-2246 Effects of kojic acid on the cell wall of filamentous fungal Curvularia pallescens

Pereira J. L.1,3, Rodrigues A. D.2,3, Farias L. S.1,3, Santos A. S.4, Silva E. O.1,3
1Laboratório de Parasitologia e Biologia Estrutural, Instituto de Ciências Biológicas, Universidade Federal do Pará, Brazil, 2Laboratório de Microscopia Eletrônica, Instituto Evandro Chagas, Brazil, 3Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Brazil, 4Laboratório de Desenvolvimento e Planejamento de Fármacos, Universidade Federal do Pará, Brazil
luishsf@gmail.com

Curvularia pallescens is an endophytic fungi and melanin producer that occasionally causes a variety of human infections (Figure 1). Currently, there are some studies about antifungi effects of bioproducts which have low toxicity. The hydroxy-2-hydroxymethyl-γ-pyrone (Kojic acid) is a secondary metabolite synthesized by some species of fungi from Aspergillus, Penicillium and Acetobacter genera. The kojic acid has several applications mainly as tyrosinase inhibitor, enzyme that works in biosynthetic pathway for melanin formation in mammalian and fungi cells. Thus, this study evaluated the kojic acid effect in Curvularia pallescens morphology cultivated and treated with 50, 100 e 200 μg/ml of kojic acid. The morphological analysis by scanning electron microscopy (Figure 2), performed at Zeiss Leo 1400, transmission electron microscopy (Figure 3), analyzed at Zeiss Leo EM 900 and confocal microscopy (Figure 4), observed at Zeiss LSM 5 Pascal. After incubation, treated cells showed accumulation of many cytoplasmic vesicles like lipid droplets as well external vesicles. In addition, it was showed an accumulation of lipid vesicles in the cytoplasm and cell wall. Furthermore, severe disruption of this wall and subsequent release of these retained vesicles were observed (Figure 3). These results suggested that kojic acid disturbs the structure of the cell wall that could be causing the death of fungus. Further studies are needed to identify the mechanisms that induce these alterations and cell death.

CAPES, CNPq/UFPA, Ministério da Saúde-MS e Instituto Nacional de Biologia Estrutural e Bioimagem (INBEB)

Fig. 1: Curvularia pallescens growth on GPY medium. (A) forward side of culture plate (B) backward side of culture plate (C) conidiation process.

Fig. 2: Scanning electron microscopy of the Curvularia pallescens. (A) untreated culture; (B, C and D) treated with 50, 100 and 200 µg/mL of KA respectively; Observe intense externalization of vesicles in hyphae (brown) and wilted conidia (green). Scale bars (B-D) 10 µm and (A-C) 15 µm.

Fig. 3: Transmission electron microscopy of Curvularia pallescens using osmium-imidazole method. (A) untreated control; (B, C and D) treated with 50, 100 and 200 µg/mL of KA respectively. Note the presence of lipid bodies (*) and lipid vesicles with strong eletrodense stain (arrows). Scale bars: 2 µm.

Fig. 4: Confocal microscopy of Curvularia pallescens incubated with BODIPY®. (A) untreated control; (B, C and D a) treated with 50, 100 and 200 µg/mL of KA respectively. Observe the presence of lipid bodies extra and intracytoplasmic, marked in green. Scale bars: 10µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-2514 RELIEF, SCHLIEREN AND PHASE-CONTRAST MICROSCOPY OF PLANTS: Label-free imaging and refractive index matching

Pelc R.1,2,3, Hostounský Z.4, Kim C. S.2
1Inst. Physiol., Acad. Sci., Prague, Czech Rep., 2Warm-Temperate Forest Res. Ctr. KFRI, Jeju Island, Korea, 3Jeju Technopark JBRI/HiDI, Korea, 4Stentor Inst., Hostivice-Palouky, Czech Rep.
radek.pelc@seh.oxon.org

INTRODUCTION

Detailed investigation of microscopic anatomy and its physiological implications often requires the use of specialized staining procedures and complex imaging equipment. In some cases, however, staining is impractical or even impossible, and contrasting by purely optical means represents a convenient alternative. The present paper highlights the advantages and pitfalls of several microscopic optical contrasting modalities suitable for imaging unstained plant cells/tissues and specimens derived from them.

APPARATUS

Relief- and schlieren-contrast imaging highlighted in the present paper utilize a shifting asymmetric (edge) diaphragm in the condenser and as such represent the simplest and historically also the oldest microscopic optical-contrasting modalities [1,2]. They are not directly available in most commercially available microscopes (see [3] for a rare exception), and one possible adaptation is depicted in Fig. 1. The rim of a partly pulled out objective Wollaston prism holder (itself a DIC-­Nomarski imaging accessory) served as the schlieren diaphragm (simple modulator) at the objective back focal (Fourier) plane (Fig. 2).

RESULTS & DISCUSSION

Optically thick objects such as leaf replicas in transparent acrylate resin are typically best rendered in relief or schlieren contrast (Fig. 2), often superior in image quality to Hoffman modulation contrast, itself a more complex variant (requiring special objectives) of schlieren imaging. Other examples of relief-contrast (off­-axis illumination) microscopy may be found elsewhere (e.g., [4]). Objects of medium optical thickness such as spores of the field horsetail (Equisetum arvense) or osmotically swollen (burst-open) pollen grains of yew or juniper are most conveniently imaged by Hoffman modulation contrast or apodized phase contrast. However, schlieren imaging yields superior contrast (especially when low-power objectives are used), comparable even to DIC-Nomarski microscopy (Fig. 3). Optically thin objects such as refractive-index matched stellar trichomes of olive (Olea europaea) or Elaeagnus sp. leaves ideally require the use of low-transmittance phase-contrast microscopy. Refractive-index matching (achieved by embedding, e.g., in acrylate resin or glycerol) is often essential to reliably visualize fine microscopic structures normally filled with air, such as the spokes of the seed-bearing ‘parachutes’ in the common dandelion (Taraxacum officinale), and makes it possible to determine their refractive index.

[1] Töpler A. (1866) Annalen der Physik 203 (4): 556-580
[2] Axelrod D. (1981) Cell Biophys. 3 (2): 167-173
[3] Hostounský Z. & Pelc R. (2007) Adv. Physiol. Educ. 31 (2): 232-235
[4] Pelc R., Hostounský Z. & Otaki T. (2008) J. Biomed. Opt. 13 (5): 054067

RP was supported by LC06063 and RVO:67985823 grants, and international NRF/KOSEF fellowship 2010. Some images were acquired on a microscope lent by Nikon CZ.

Fig. 1: Adaptation of Nikon D-CUD condenser to relief/schlieren imaging. Relief diaphragm (RD) made of black paper is used for imaging. SD, schlieren diaphragm. Fig. 2. Abaxial leaf replica of rhododendron (Daphniphyllum macropodum) in transparent resin (image ID: 2010-02-24_*). Fig. 3. Pollen grains of yew (Taxus baccata) in water (image ID: 2009-08-29_*)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-2561 Effects on the morphology and ultrastructural organization of the red alga Pterocladiella capillacea following exposure to ultraviolet radiation (UVA+UVB) and copper treatment

Schmidt E. C.1, Felix M.1, Kreusch M.1, Pereira D.1, Costa G.1, Simioni C.1, Chow F.2, Ramlov F.1, Maraschin M.1, Bouzon Z. L.1
1Plant Cell Biology Laboratory, Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina 88049-900, CP 476, Florianópolis, SC, Brazil, 2Institute of Bioscience, Department of Botany, University of São Paulo, 05508-090, São Paulo, SP, Brazil
edcash@ccb.ufsc.br

The red seaweed Pterocladiella capillacea, an alga which occurs along the southeastern and southern Brazilian coastline, is an excellent source of agar. Ultraviolet radiation has become an increasing concern since discovering the ozone hole in Antarctica, which has resulted in the release of such atmospheric pollutants as chlorofluorocarbons, halocarbons, chlorocarbons, dioxins and carbon dioxide. At the same time, ecosystems around the world have also been polluted by the presence of heavy metals, and algae can, by their participation in many food chains, contribute to the contamination of other organisms. In particular, copper is found in low concentrations in algae species, and it is essential for metabolic processes such as photosynthesis. Based on its contribution to the important supply of agar, Pterocladiella capillacea was evaluated in this study to determine the effects of ultraviolet radiation (PAR+UVA+UVB), PAR+copper in different concentrations, and the combined effects of PAR+UVA+UVB + copper on morphology and cell organization. Control samples of P. capillacea stained with Periodic Acid-Schiff exhibited a positive reaction in the cell wall and floridean starch grains. On the order hand, P. capillacea-treated plants revealed a decrease in the density of floridean starch grains in the cells. When observed by transmission electron microscopy, P. capillacea control cells showed a somewhat vacuolated cortical region filled with numerous chloroplasts and some starch grains, all surrounded by a thick cell wall. The chloroplasts assumed the typical internal unstacked organization of red algae, i.e., evenly spaced thylakoids. Electron-dense lipid droplets described as plastoglobuli were observed between the thylakoids. However, after exposure to PAR+UVA+UVB for 3 h per day during a 7-day period, P. capillacea was observed to undergo ultrastructural changes, including irregularly shaped cortical cells with increased vacuolation and increased cell wall thickness with concentric layers of microfibrils. In addition, chloroplasts showed visible changes in ultrastructural organization, including irregular morphology (Figs. 1a-b). Plants of P. capillacea exposed to PAR + copper in different concentrations showed fewer ultrastructural changes; the thylakoid membranes showed no demonstrable ultrastructural damage (Fig.1c). However, the combination of PAR+UVA+UVB+ copper caused more dramatic changes than did PAR+UVA+UVB and PAR+copper treatments, with the cortical cells showing the greatest reduction in cytoplasmic cell volume. In addition, the chloroplasts were degenerated and disrupted (Fig.1d).

The authors acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) for the financial support of Éder C. Schmidt (process 473088/2013-4).

Fig. 1: Figure 1: Transmission electron microscopy micrographic images of P. capillacea. a. Detail of cortical cell with many starch grains (S) and thick cell wall (CW). b. Observe the irregular shape chloroplast (C) and a presence of vacuoles (V). c. Observe the absence of changes in chloroplast. d. Note the degenerated and disrupted chloroplast.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-2572 Leaf anatomy of Suaeda edulis and Suaeda nigra ; two halophytes with contrasting C3 and C4 metabolism from México.

Zavaleta-Mancera H. A.1, Noguez-Hernández R.2, Carballo-Carballo A.3
1Botánica, Colegio de Postgraduados, Campus Montecillo, Montecillo, Estado de México, México., 2Departamento de Preparatoria Agrícola, Universidad Autónoma Chapingo, Estado de México, México. , 3Producción de Semillas, Colegio de Postgraduados, Campus Montecillo, México.
arazavaleta@colpos.mx

A new species of Suaeda sect. Brezia, Suaeda edulis Flores Olv. & Noguez was recently described [1]. And it is found in saline lakes of Estado de México, Guanajuato, Jalisco, Michoacán, Distrito Federal, Tlaxcala and Puebla which are d with S. nigra and S. mexicana [1] which are edible herbs known as “Romerito” and harvested from wild population or from cultivated plants.

The objective of the present research was to contribute to the anatomical knowledge of S. edulis (C3) and S. nigra (C4), using Scanning Electron Microscopy. This research is part of a “Romerito network” for the conservation, knowledge and sustainable use of this halophyte herb.

Materials and methods. Fragments of the central part of mature leaves of S. edulis (from Montecillo Estado de Mexico, Guanajuato, Puebla and Distrito Federal) and S. nigra (from San Luis Potosi), were fixed in 2.5 % glutaraldehyde in 0.1 M pH7 phosphate buffer 0.1 M for 48 h, in vacuum. Cross sections 3 mm thick, were washed and dehydrated in a series of ethanol and then critical point dried (Samdri 780A), and coated with gold with a Ion Sputter (JFC-1100 Jeol, Japan) and observed with a SEM (JSM-6390, Jeol, Japan) operating at 30 Kv.

Results. The leaf cross section of S. edulis appears elliptic to oblong with a Brezia type C3 anatomy (Fig. 1). It has three vascular bundles aligned and equally distributed in the centre of the leaf; the mesophyll contains several layers of elongated parenchyma cells with numerous chloroplasts (Fig. 2). In contrast, S. nigra shows a round shape with the presence of a typical Salsina-type C4 anatomy, a bundle sheath or Kranz cells with abundant chloroplasts, surrounding five vascular bundles aligned in an arc shape (Fig. 3). Chloroplasts of the Kranz cells have similar size to those of the mesophyll (Fig. 4). The epidermal cells of S. edulis are thinner (20-30 µm) than those of S nigra (50-70 µm). The mesophyll of S. nigra is formed by a single layer of thin palisade cells. In contrast to S. taxifolia and others species of Suaeda with Salsinia type C4 [2], the internal parenchyma to the bundle of S. nigra contains round and abundant chloroplasts. 

References.

[1] Noguéz Hernández R., Carballo Carballo A. and Flores Olvera H. Botanical Sciences (2013)19-25.

[2] Smith M.E. Photosynthetic performance of single-cell C4 species (Chenopodiaceae). Thesis Master of Science in Botany. Washington State University, USA (2007) 64p.

The present research was supported by the National System of Phytogenetic Resources for Food and Agriculture of Mexico (Sistema Nacional de Recursos Fitogenéticos para la Alimentación y la Agricultura, SINAREFI) grant IMP-ROM-13-3.

Fig. 1: Leaf cross section of S. edulis from Montecillo, Estado de México. Typical Brezia type C3 anatomy. e: epidermis; me: mesophyll;; vb: vascular bundle.

Fig. 2: Mesophyll of S.edulis.. Typical Brezia C3 anatomy. e: epidermis; me: mesophyll; vb:vascular bundle.

Fig. 3: Leaf cross section of S. nigra from San Luis Potosí. Salsinia type C4 anatomy. e:epidermis;me: mesophyll, palisade cells; bc: bundle or Kranz cells; vb: vascular bundle.

Fig. 4: Bundle cells of S.nigra, Salsinia type C4 anatomy. e: epidermis; me: mesophyll, palisadecells; bc: bundle or Kranz cells, ch: parenchyma.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-2646 Element Analysis by EDX in Aquatic Carnivorous Plants

Koller-Peroutka M.1, Hefel B.1, Adlassnig W.1, Adamec L.2, Sassmann S.1, Lichtscheidl I. K.1
1University of Vienna, Core Facility Cell Imaging and Ultrastructure Research, Althanstraße 14, A-1090 Vienna, Austria, 2Institute of Botany AS CR, Section of Plant Ecology, Dukelská 135, CZ-379 82 Třeboň, Czech Republic
wolfram.adlassnig@univie.ac.at

Aquatic carnivorous plants use different types of glands within their trap leaves in order to secrete digestive enzymes and absorb prey-derived nutrients. Since the glands are the physiologically most active part of the trap, enhanced contents of physiologically important elements can be expected here. Furthermore, nutrient uptake from prey via the trap lumen should further increase the content of nutrients within these glands. It remains unclear if these elements are accumulated or immediately transported to other parts of the plant.
We used X-ray microanalysis (EDX) to study element contents and distributions in two genera of aquatic carnivorous plants, Utricularia (Lentibulariaceae) and Aldrovanda (Droseraceae). Plants were cultivated in the greenhouse, frozen in liquid nitrogen, dehydrated via freeze-drying in order to avoid element translocations, carbon coated and analysed in a Philips XL20 scanning electron microscope with attached EDX. Contents of N, P, S, K, Mg, Zn and other biologically relevant elements were compared between epidermal cells and various gland types in young but fully mature traps.
In Aldrovanda vesiculosa, significantly higher contents of N and Mg, but lower contents of P and S, were found in the quadrifid (A) glands compared to the epidermis. The elemental composition of the quadrifid glands was similar to that in the digestive glands (B), but clearly different from the external sessile glands outside the trap (C). In Utricularia purpurea, the quadrifid glands (D) contained higher contents of N, P, Ca and Zn. Generally, A. vesiculosa contained more N but less Mg, S, P and K than U. purpurea in the epidermis and especially in the quadrifid glands.
These results indicate different physiological functions of the quadrifid glands of Aldrovanda and Utricularia in spite of their similar morphology. Furthermore, the different functions of the various gland types in Aldrovanda are reflected by their different elemental content. The relevance of these findings for the physiology of nutrient uptake in aquatic carnivorous plants is discussed.

Thanks are due to Dr. M. Weidinger for kind support.

Fig. 1: Glands of Aldrovanda vesiculosa (A-C) and Utricularia purpurea (D )
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3048 White light confocal and Lambda square mapping: a doorway to understanding autofluorescence in phototrophic microorganisms

Roldán M.1, Monteagudo J.2, Hernández-Mariné M.3
1Servei de Microscòpia, Universitat Autònoma de Barcelona, Edifici C, Facultat de Ciències, 08193, Bellaterra, Spain, 2Leica Microsistemas, C/ Nicaragua, 46. 08029 Barcelona, Spain., 3Dep. Productes Naturals, Biologia Vegetal i Edafologia, Unitat de Botànica. Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028, Barcelona, Spain.
monica.roldan@uab.es

In this study we apply spectrofluorometric-imaging microscopy in a confocal microscope using a supercontinuum laser as an excitation source and a custom-built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. This set up enables two-dimensional maps of fluorescence intensities as a function of the excitation and emission wavelengths. These detailed maps, called lambda-squares, are especially useful in samples with many unknown fluorescences. An example is the environmentally-important micro-communities formed by photosynthetic organisms that cause damage to cultural heritage. Cyanobacteria are the primary and most abundant of such photosynthetic colonizers. They have chlorophyll a and accessory pigments, such as carotenoids and phycobilins, which allows them to absorb a wide range of light radiation. For some years now these communities and their spectral emission properties have been studied using confocal laser scanning microscopy (Roldán et al. 2006). Using this technique together with the Lambda-scan feature makes it possible to analyse the autofluorescence emission spectra of single cells. In addition, confocal microscopy with white light laser applied on photosynthetic organisms, permits to obtain the shape of the emission spectra and the position of their maximum, allowing us to identify the photosynthetic pigments. For the Cyanobacteria the presence of phycoerythrin, helps them to survive in dim environments; their maximum excitation and emission were very similar in the different species studied, while the shape of the spectra varied. In the Bacillariophyta phylum, the species essentially had the same maximum excitation and emission, corresponding to the presence of chlorophyll a and c. However, the shape of the excitation spectra was slightly different. Finally, for the Chlorophyta phylum, or green algae, the shape of the excitation and emission spectra and the maximum of chlorophylls a and b were fairly constant in all species. These communities are complex in terms of their pigment composition, which is indicative of the microbial composition, and also due to the different physiological states of the cells (Roldán et al. 2014). The fact that some species may have broader absorption spectra could imply an advantage for ecological success in these hostile environments. In conclusion, this technique can be used not only to discriminate between different phyla but also to determine their photosynthetic plasticity and physiology in natural environments.

References
Roldán, M. et al. (2006). Appl Environ Microb 72, 3026-3031.
Roldán M. et al. (2014). Appl Environ Microb (In press).

Fig. 1: Lambda-square map of Nostoc sp. from Nerja cave (Málaga, Spain). Orthogonal spectral image with the excitation and emission spectra in a point of Lambda-square map.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3175 Microscopic morphology of ascospore of thermotolerant yeast, UBU-strains

Pukahuta C 1, Kaewsai K 1, Ruamsook S N.1
1Department of Biological Sciences, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand
hed2546@yahoo.com

Yeast is an eukaryotic microorganism. Yeast’s cell is spherical, oval or lemon shape. The diameter of yeast’s cell is around 5 µm. Asexual reproduction of yeast is budding. Some species of yeast reproduce sexual stage by mating of two compatible haploid cells. After fusion of cytoplasm, the mated cell becomes diploid cell. Then diploid cell divides by meiosis into four haplYeast is an eukaryotic microorganism. Yeast’s cell is spherical, oval or lemon shape. The diameter of yeast’s cell is around 5 µm. Asexual reproduction of yeast is budding. Some species of yeast reproduce sexual stage by mating of two compatible haploid cells. After fusion of cytoplasm, the mated cell becomes diploid cell. Then diploid cell divides by meiosis into four haploid cell. Haploid cell develops to be ascospore or basidiospore depend upon its genetic material. Ascospore will be formed in the ascus or sac. We had isolated the new strains of thermotolerant yeast that can produce ethanol. We also identified them by their morphology and biochemical characters. The characteristics of ascospore such as shape, size, color, decoration and number of ascospore per ascus, are essential for identification the sample into species. The experiment inducing ascospore-formation was conducted for the required stage. The optimal condition was found to be cultivation on V-8 juice medium at pH 7 under 25 degree celcius. 23 out of 25 isolates could produce ascospore in above condition. Then the microscopic morphology of thermotolerant yeast, UBU-strains at their ascospore-formation stage was revealed by light microscope and scanning electron microscope. Two out of 23 isolates, UBU-1-11 and UBU-2-16 produced ascospores as shown in fig 1 and fig 2. The microscopic morphology of the thermotolerant yeast at vegetative stage and sexual stage will be described in details.

Funding provided by National Research Council of Thailand for CP and logistic support by Faculty of Science, Ubon Ratchathani University were thanked.

Fig. 1: Ascospore formation of thermotolerant yeast strain, UBU-1-11, on V-8 juice medium at 25 oC at day 12 under light microscope.

Fig. 2: Ascospore formation of thermotolerant yeast strain, UBU-2-16, on V-8 juice medium at 25 oC at day 12 under light microscope.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3200 Cryofixation of Candida albicans Biofilms Ensures the Preservation of Its Extracellular Polymeric Matrix

Fonseca B. B.1, Vila T. V.1, Cunha M. M.2, De Souza W.1,3, 4, Rozental S.1
1Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Brazil, 2Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Brazil, 3National Institute of Metrology, Quality and Technology, Brazil, 4National Institute of Structural Biology and Bioimages, Federal University of Rio de Janeiro, Brazil
bastos3@gmail.com

The structural complexity of the Candida albicans biofilm and the presence of an extracellular matrix (ECM) are thought to be associated with the biofilm resistant behavior. The ECM makes it resistant biofilm overlying cells, since for the antifungal action he needs first then penetrate the biofilm to reach the cells. Electron microscopy techniques have been used to study in vitro fungal biofilm structure and the effectiveness of antifungal drugs, but chemical fixation protocols usually fail to preserve the ECM. Since cryofixation techniques are today standard for successfully fixation of biological samples, we applied high pressure freezing and plunge freezing on biofilm samples for ECM preservation.
Thus, in this work, we evaluated the efficacy of high pressure freezing (HPF) and plunge freezing (PF) techniques, both combined with freeze substitution (FS) in preserving C. albicans biofilms for scanning electron microscopy. C. albicans biofilms were formed on 5 mm sections of central venous catheters (CVC), afterwards samples were subjected to HPF or PF followed by FS, then warmed to 4°C, critical point dried in CO2, attached to metal stubs and coated with gold for observation in a FEI Quanta 250 Microscope (Netherlands) or FEI Magellan Microscope (Netherlands).
These techniques provided a better preservation of biofilm structure, including the ECM not seen after chemical sample handling. PF samples also presented a perfectly preserved biofilm, with huge amount of ECM around and nearby cells (figure 1). Interestingly, sample carriers for HPF produced fractures in the yeasts surface (figure 2), enabling the visualization of intracellular features such as cell wall thickness and the plasma membrane.
Our results describe cryofixation protocols that enable good preservation of biofilms for SEM and offer novel insights about ECM ultrastructure and organization on C. albicans biofilms. This is of outstanding importance as chemical fixative methods usually used for SEM led to the total extraction of ECM leading to a reductionist observation of biofilm complexity.

This research was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Fig. 1: Candida albicans 44A biofilms formed on the surface of catheters cryofixed by PF followed by FS. Arrows indicate the extracellular matrix (A to D).

Fig. 2: Candida albicans 44A biofilms (A to D) formed on the surface of catheters cryofixed by HPF followed by FS.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3250 Unbiased estimation of selected mesophyll structural parameters of Norway spruce needles under ambient and elevated CO2 concentration

Kubínová Z.1, Janáček J.2, Lhotáková Z.1, Kubínová L.2, Albrechtová J.1
1Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Czech Republic, 2Department of Biomathematics, Institute of Physiology, AS CR, v.v.i., Prague, Czech Republic
kubinova@natur.cuni.cz

The Norway spruce (Picea abies L. Karst.) is an abundant conifer in European temperate forests playing an important role in the global carbon cycle. To reveal the effect of elevated CO2 concentration on Norway spruce needle anatomy, selected structural parameters were measured using unbiased methods including a 3D method for chloroplast counting in cells.

The trees were planted in glass domes with ambient or elevated (700 ppm) CO2 concentration at the CzechGlobe experimental site Bílý Kříž (Beskid Mts., Czech Republic). Sun needles were collected from upper shoots of the crowns and stored frozen until processing. Cross-sections were cut off in a systematic uniform randomly chosen positions along the needle. Images were captured by a Leica SP2 AOBS confocal laser scanning microscope and analysed using Ellipse software.

Needle volume density of mesophyll (mesophyll volume per needle volume) was estimated by the point counting method (Weibel, 1979) and needle volume by the Cavalieri principle (Gundersen and Jensen, 1987). The estimated number of chloroplasts per mesophyll cell was calculated by the ratio of the chloroplast number and the mesophyll cell number per needle volume, while both values of these quantities were determined by application of the disector probe (Sterio, 1984; Gundersen, 1986) on series of optical sections.

Our previous study showed that the commonly used profile counting of chloroplasts on 2D sections yielded 10 times underestimated value compared to unbiased estimation of chloroplast number per cell obtained by 3D stereological approach (Kubínová et al., 2014). The pilot results of the volume density of mesophyll in sun needles did not prove to be significantly affected by CO2 concentration. Needles grown in ambient and elevated CO2 were 0.72±0.04 SD (standard deviation) and 0.73±0.05, respectively. The pilot results of the number of chloroplasts per mesophyll cell showed a trend of a higher number of chloroplasts per cell under elevated CO2, however, this parameter is still being measured.

References

Gundersen HJG. 1986. Stereology of arbitrary particles—a review of unbiased number and size estimators and the presentation of some new ones, in memory of Thompson, William, R. Journal of Microscopy 143, 3–45.

Kubínová Z, Janáček J, Lhotáková Z, Kubínová L, Albrechtová J. 2014. Unbiased estimation of chloroplast number in mesophyll cells: advantage of a genuine three-dimensional approach. Journal of Experimental Botany 65 (2), 609-620.

Sterio DC. 1984. The unbiased estimation of number and sizes of arbitrary particles using the disector. Journal of Microscopy 134, 127–136.

Weibel ER. 1979. Practical methods for biological morphometry. In: Stereological methods, vol. 1. New York: Academic Press, 1–415.

Supported by the Czech Science Foundation (P501/10/0340), the Academy of Sciences of the CR (AV0Z50110509 and RVO:67985823) and by the Charles University in Prague (SVV 260076/2014).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3276 Ultrastrutural studies of melanosomes and cell wall on the Sporothrix schenckii complex

Cunha M. M.1, Santos L. P.2, Rozental S.2, Marco S.3, 4, Lopes-Bezerra L. M.1
1Laboratório de Micologia Celular e Proteômica, Cell Biology Department, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil, 2Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil, 3Institut Curie, Orsay, France, 4INSERM U759, France
marcelcunha@uerj.br

Expression of melanin pigments is associated with virulence in pathogenic fungi of the Sporothrix schenckii complex by conferring resistance to antifungal drugs and to host immune system. Also, Highly melanized S. schenckii and other fungi has been reported as being more virulent against mouse models. This is a strong motivation for the study of the structural characteristics around melanin production and its role in the cell biology of pathogenic fungi.
We studied by scanning electron microscopy and STEM tomography the contribution of melanin to cell wall structure and the presence of this pigment in organelles.
Analyses of the surface morphology of mycelia by scanning electron microscopy showed that heavily melanized areas of fungal colonies of S. brasiliensis have a matrix-like material covering and connecting conidia and parts of hyphae (figure 1A). On S. schenckii samples such structure was not observed (figure 1B).
STEM tomography of 300 nm thick slices of resin embed S. schenckii yeasts showed an electron-dense layer localized in the cell wall of the fungus and electron-dense content inside cytoplasmic organelles. In transmission electron microscopy studies, melanin is usually easily observed as an electron-dense material in the cell or tissue. Segmentation based on threshold levels indicated that the electron-dense layer and the electron-dense content of cytoplasmic organelles have gray values on a same specific range. We hypothesized that such electron-dense material observed in both organelles and on the cell wall is melanin, and that melanin in S. schenckii could be produced in melanosomes and exported to cell wall, as observed in other pathogenic fungi.
This study and further observation of melanized and non-melanized fungi may shed light on how melanin participates to cell wall assembly in melanized S. schenckii.

The authors thank prof. Marcia Amorim and the Laboratory of Electron Microscopy of Instituto de Química da UERJ. Financial support: Ciência Sem Fronteiras proc. 014/2012; CAPES, CNPq and FAPERJ.

Fig. 1: Extracelular matrix associated to conidia (arrowhead) and linking conidia and hyphae (arrow) in S. brasiliensis (A). In B, S. schenckii showed no extracelular matrix covering cell features. Bars = 1 µm.

Fig. 2: A, B and C shows slices after STEM tomography of S. schenckii and WBP reconstruction, spaced 50 nm in depth. Electrondense material is observed in organelles (arrow) and in an external layer of the cell wall (arrowhead). Segmentation shows cell membranes (D), eletrondense material (Melanin, E) and their association (F), bar = 400nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3283 Sun and shade chloroplast ultrastructure of Norway spruce needles 3D-visualized using dual-axis TEM tomography and serial sectioning

Radochová B.1, Michálek J.1, Janáček J.1, Čapek M.1, Lhotáková Z.2, Bílý T.3, Nebesářová J.3, Kubínová L.1, Albrechtová J.2
1Institute of Physiology ASCR, Prague, Czech Republic, 2Charles University in Prague, Czech Republic, 3Biology centre ASCR – Institute of Parasitology, České Budějovice, Czech Republic
albrecht@natur.cuni.cz

The most common method used for visualization of chloroplast ultrastructure is transmission electron microscopy (TEM). However, due to the size of chloroplasts (diameter of about 3 to 8 µm), there is only a very limited extent of three-dimensional (3D) structural information, when standard ultrathin sections (70-80 nm) are used. Both serial sectioning and electron tomography offer a powerfull tool for 3D reconstruction and visualization of chloroplast ultrastructure described in detail in the abstract of Radochová et al. (IMC2014).

In the present study, dual-axis TEM tomography and serial sectioning were used for visualization of the spatial arrangement of chloroplast ultrastructure. Norway spruce (Picea abies L. Karst.) needles were sampled at the experimental site Bílý Kříž (Beskids Mts., Czech Republic). Samples were chemically fixed, processed using microwave tissue processor and embedded into Spurr´s epoxy resin. Electron tomography projections of two orthogonal axes were acquired from 200 nm thick sections using JEOL JEM-2100F microscope equipped with Gatan camera Orius SC 1000 and Serial EM automated acquisition software. Tomograms were processed by specialized computer program (IMOD package, Boulder Laboratory, Colorado). Serial sections (at least five consecutive sections) were viewed in JEOL JEM-1010 microscope and acquired image data were aligned using special software (Link MRC). Chloroplast components (e.g. thylakoids, starch grains and plastoglobuli) were traced in reconstructed series of images (from both electron tomography and serial sectioning) in a process called interactive segmentation and then visualized using 3D plug-in in Ellipse software.

Based on a previous quantitative study on chloroplast ultrastructure using ultrathin sections and TEM, we observed differentiation into two types of chloroplasts within one sun-exposed needle. The chloroplasts from the light exposed side of the sun needle appeared to have less and lower grana (GT) and more plastoglobuli, which can indicate high light stress (Fig. 1). The chloroplasts from the light unexposed side of the sun needle showed more grana, less plastoglobuli and higher starch accumulation. The 3D-visualization using dual-axis electron tomography and serial sectioning (Fig. 2) enabled comparison of spatial arrangement of thylakoid membranes of the two types of chloroplasts from light exposed and unexposed sides of the sun needle.

This work was supported by Czech Science Foundation (P501/10/0340), by the Academy of Sciences of the Czech Republic (RVO: 67985823) and by Technology Agency of the Czech Republic (TE01020118).

Fig. 1: Norway spruce chloroplast differentiation into two types of chloroplasts within one sun-exposed needle. The chloroplasts from the light exposed side of the sun needle appeared to have less and lower grana (GT) and more plastoglobuli (arrows) comparing to the chloroplasts from the light unexposed side of the sun needle.

Fig. 2: 3D visualization of Norway spruce chloroplast from the light unexposed side of a sun needle, acquired via dual-axis electron tomography. Thylakoids in green, starch grain in yellow and plastoglobuli in brown.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3313 Ultrastructural characterization of Sporothrix schenckii complex species by scanning electron microscopy.

Borba-Santos L. P.1, Fonseca B. B.1, Rozental S.1
1Laboratório de Biologia Celular de Fungos, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
rozental@biof.ufrj.br

Sporothrix schenckii complex species are the etiological agents of sporotrichosis, cosmopolitan subcutaneous mycoses that are endemic in Latin America countries, such as Brazil. The complex was recently created and groups the dimorphic species: Sporothrix schenckii sensu stricto, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix mexicana, Sporothrix pallida and Sporothrix. luriei. Although the disease and its etiological agent have been described over a century ago (Schenck, 1898), still little is known about the ultrastructural aspects of the fungus. Therefore, this study aims to characterize the ultrastructural aspects of S. schenckii complex species in the filamentous form, as well as yeasts, by scanning electron microscopy.
The vegetative and reproductive mycelia of fungus adhered to the coverslips obtained in microculture technique and yeasts growth in Brain Heart Infusion were processed and visualized in a scanning electron microscope FEI Quanta 250. Diameters of sessile conidia and yeasts were measured using the Image J software (NHI, USA) and the area was calculated with an elliptical. Differences in the cell size were analyzed by one-way ANOVA (Tukey’s test) and statistical significance was accepted when p < 0.05.
S. schenckii specie showed subglobose sessile conidia and elongated yeast; S. brasiliensis showed globose sessile conidia and globose yeast; S. globosa elliptical sessile conidia and globose yeast. Additionally, S. luriei showed sessile conidia with largest area, followed by S. globosa, S. brasiliensis, S. pallida, S. mexicana and S. schenckii. Therefore S. schenckii presented the lowest conidia. Yeasts showed larger areas than sessile conidia, and S. globosa showed the lowest yeasts.
The species morphology in the filamentous form (hyphae and conidia) showed tenuous differences (Figure 1), except for the S. luriei specie which showed a characteristic morphology with very elongated conidia. However, considering the morphology of the filamentous form and yeast together, it was found that different species exhibit characteristic morphological profiles. Furthermore, differences between sizes of infectious particles may reflect differences in the interaction of the fungus with the host cells. Thus, the different species will present naturally a varied interaction dynamic.
Basics features of cellular biology of S. schenckii complex species remains unknown. Thereby, ours results extend the knowledge about this pathogenic fungus, whose medical importance has increased in recent decades.

This work was supported by CNPq, FAPERJ and CAPES.

Fig. 1: Scanning electron microscopy images of Sporothrix schenckii complex species in filamentous form (Bars= 5 µm).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3323 Comparative analysis of chloroplast organization and metabolism in the red macroalga Gelidium floridanum exposed to cadmium, copper and lead

Bouzon Z. L.1, Santos R. W.1, Pereira D. T.1, Costa G. B.1, Simioni C.1, Chow F.2, Ramlov F.1, Maraschin M.1, Schmidt E. C.1
1Federal University of Santa Catarina, Brazil, 2University of São Paulo, Brazil
zenilda.bouzon@ufsc.br

Heavy metals, such as lead, copper, cadmium, zinc, and nickel, are among the most common pollutants found in both industrial and urban effluents. High concentrations of these metals cause severe toxic effects, especially on organisms living in the aquatic ecosystem. Cadmium (Cd), lead (Pb) and copper (Cu) are the heavy metals most frequently implicated as environmental contaminants, and they have been shown to affect development, growth, photosynthesis and respiration, and morphological cell organization in seaweeds. This study aimed to evaluate the effects of 50 and 100 μM of Cd, Pb and Cu on growth rates, photosynthetic pigments, biochemical parameters and ultrastructure in Gelidium floridanum. To accomplish this, apical segments of G. floridanum were individually exposed to the respective heavy metals over a period of 7 days. After 7 days of experimentation, control, Cd-, Cu-, and Pb-treated G. floridanum showed altered morphological features with reduction in the amount of branches, noticeable discoloration, algal necrosis, and reduction in the content of photosynthetic pigments. When observed by transmission electron microscopy, control samples of G. floridanum showed cortical cells to be somewhat vacuolated, mostly filled with chloroplasts (Fig. 1a) and a large quantity of floridean starch grains close to the chloroplasts. However, after culturing G. floridanum in 50 and 100 μM of Cd for 7 days, cortical cells appeared more vacuolated with increasing cell wall thickness, exhibiting concentric layers of microfibrils. Chloroplasts showed a few changes in ultrastructural organization (Fig. 1b), and the number of plastoglobuli increased in the chloroplasts. In contrast to Cd exposure, copper treatment caused more dramatic ultrastructure changes in G. floridanum, with cortical cells showing a large reduction in cytoplasmic cell volume. The cell wall showed increasing thickness with deposition of concentric layers of microfibrils, and spots of black deposits appeared, most likely Cu. The chloroplasts were degenerated and disrupted, and the presence of plastoglobuli was observed (Fig. 1c). After Pb treatment, the cortical cells of G. floridanum showed a few changes in shape and increased cell wall thickness. In the cortical cells, an increase of vacuole volume could be observed. On the other hand, chloroplasts showed no changes in ultrastructural organization (Fig. 1d). These results indicate that Cd, Pb and Cu negatively affect metabolic performance and cell ultrastructure in G. floridanum and that Cu was more toxic than either Pb or Cd.

The authors would like to acknowledge the staff of the Central Laboratory of Electron Microscopy (LCME), Federal University of Santa Catarina, Florianopolis, Santa Catarina, Brazil, for the use of  transmission electron microscope.

Fig. 1: Figure 1: Transmission electron microscopy micrographic images of G. floridanum. a. Detail of chloroplast (C), showing the thylakoids near the mitochondria. b. Observe the irregular shape in thylakoid membranes (*). c. Note the disrupted chloroplast (C) with the presence of plastoglobuli (P). d. Observe the absence of changes in chloroplast.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3486 Characterising mutants in root-to-shoot mobile silencing: peroxide regulates long-distance cell-to-cell spread

Liang D.1, Waterhouse P. M.1, 2, 3, White R. G.1
1CSIRO Plant Industry, Canberra, ACT, Australia, 2University of Sydney, School of Biological Sciences, Sydney, NSW, Australia, 3Queensland University of Technology, Centre for Tropical Crops and Biocommodities, Brisbane, QLD, Australia
rosemary.white@csiro.au

RNAi-derived mobile silencing signals have been known for more than a decade in plants, but proteins involved with the short- and long-distance transport of these signals remain a mystery. Mobile silencing is commonly investigated by grafting tissue expressing the RNA hairpin to tissue expressing the target gene, then monitoring the development of silencing once the grafted tissues have reconnected. To overcome some of the limitations imposed by the need for grafting, we developed a dexamethasone-inducible Root-to-Shoot Silencing system (RtSS), in which green fluorescent protein (GFP) expressed throughout the plant (Arabidopsis thaliana) is silenced by an RNA hairpin expressed solely in the root following dexamethasone treatment. With this system we showed that GFP silencing spreads up from the roots slowly, cell-to-cell via plasmodesmata, rather than through the plant vascular system (1).

To explore the factors underlying signal transmission, we carried out an EMS screen for Arabidopsis mutants with altered root-to-shoot silencing. Mutants displayed four types of altered silencing: no silencing at all; an altered silencing pattern; an increased rate of mobile silencing; a decreased rate of mobile silencing. Plants that showed no silencing were assumed to be deficient in the gene silencing machinery or the dexamethasone-signaling pathway and were not analysed further. We screened 110,000 Arabidopsis plants and recovered one mutant (vasc1), with a vascular silencing pattern rather than the usual front of cell-to-cell silencing advancing upward through shoot tissues. A second mutant line showed increased movement of silencing (imos1), in which analysis of symplastic dye movement in leaves revealed plasmodesmata that were more open than in RtSS wildtype plants. In a further two mutants, silencing was initiated in roots but progressed very slowly up the hypocotyl, never reaching the shoot meristem before flowering. One of the latter mutants lacked a type III peroxidase, RCI3-2, causing reduced cell wall peroxide levels throughout the plant. Addition of H2O2 to the mutant restored silencing mobility, and addition of catalase (which breaks down H2O2) to wildtype RtSS plants slowed silencing spread. Since type III peroxidases can restructure cell walls, we speculate that RCI3-2 may regulate silencing mobility by loosening or tightening cell walls around plasmodesmata.

1. Liang D, White RG, Waterhouse PM (2012) Gene silencing in Arabidopsis spreads from the root to the shoot, through a gating barrier, by template-dependent, nonvascular, cell-to-cell movement. Plant Physiology 159: 984–1000

This work was supported by an Australian Research Council Federation Fellowship to PMW, and by matching funds from CSIRO Plant Industry.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3489 Short- and long-distance signal transmission – following tagged proteins and their mRNA across graft junctions in Arabidopsis

White R. G.1
1CSIRO Plant Industry, Canberra, ACT, Australia
rosemary.white@csiro.au

Grafting is an ancient technique in plant propagation, often used to join productive shoot systems (scions) onto disease-tolerant or otherwise vigorous root systems (rootstocks). In successful grafts, the long-distance transport systems rejoin early, supplying shoots with water and soil nutrients via the xylem, and supplying roots with the products of photosynthesis via the phloem. Grafts are also used to investigate whether endogenous compounds or the products of transgenes can be transported along certain pathways between rootstock and scion. The assumption is that for species with both internal and external phloem, bidirectional phloem transport is possible, but that for other species, this is either not possible or rare.

We investigated the capacity of the limited phloem system in Arabidopsis thaliana hypocotyls to carry macromolecules both shootward from roots and rootward from shoots. Rootstocks or scions expressing tagged proteins were grafted in the hypocotyl to non-expressing scions or rootstocks, respectively. We traced protein and mRNA from tissues expressing either green fluorescent protein (GFP), glucuronidase (GUS), or larger GFP-tagged proteins moving from transgenic scions to wild-type (WT) rootstocks, or vice versa. Free GFP (27 kDa mwt), expressed either throughout the tissue or only in phloem companion cells, moved rapidly via reconnected phloem from GFP-expressing scions to WT root tips, but moved only slowly from GFP-expressing rootstocks into the phloem of WT scions. Larger proteins such as GFP-sporamin (67 kDa) or GUS (68 kDa) also moved readily in the phloem from scion tissue into WT roots, and in WT scions on transgenic rootstocks, GUS protein was detected in phloem of many leaves. GFP associated with the endoplasmic reticulum rarely crossed the graft junction in either direction, and was detected within 2-4 cells of the junction. Similarly, transgene mRNA from scions was readily detected in WT rootstocks, but transgene mRNA from rootstocks was only detected in WT scion tissue very close to the graft junction. Clearly, non-native proteins and mRNA can move bidirectionally in phloem, although there is substantially greater movement towards the root tips. Tracer proteins were found only in phloem in the destination WT tissue and accumulated in companion cells. We speculate that similar to the introduced tracer proteins, many endogenous macromolecules may be able to move bidirectionally once they enter the phloem. Escape from the phloem may then require specific unloading signals, and the tight control of unloading may be key to regulation of development by abundant phloem-mobile proteins and RNA species.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3502 An extended analysis of the extracellular matrix of Candida albicans biofilms extracted during sample preparation process

Vila T.1, Fonseca B. B.1, dos Santos G. R.2, Bergter E. B.3, de Souza W.1, Rozental S.1
1Biophysics Institute Carlos Chagas Filho, Brazil, 2Clementino Fraga Filho University Hospital, Institute of Medical Biochemistry, Brazil, 3Institute of Microbiology Paulo de Goes, Brazil
taissavila@gmail.com

Candida albicans biofilms are formed by yeasts and hyphas adhered to a surface and enclosed in an extracellular polymeric matrix (ECM) secreted by the biofilm cells. ECM is mainly composed by polisaccharides and its water-soluble nature hampers its observation by electron microscopy. Thus, the aim of this work was to evaluate if ECM is extracted from C. albicans biofilms during routine SEM preparation techniques. Biofilms were formed in vitro on the surface of central venous catheters (CVCs) and prepared for Scanning Electron Microscopy (SEM). Samples were chemically fixed, dehydrated in an ethanol series, critical point dried in CO2 and coated with gold for observation in a scanning electron microscope. After each step of sample preparation, an aliquot of the supernatant liquid was collected for further analysis. ECM physically extracted from biofilms was used as a control sample. Negative staining (ammonium molybdate, 1%) and gas chromatography followed by mass spectrometry (GC-MS) were used to evaluate the presence of ECM in the supernatant collected. Environmental Scanning Electron Microscopy (ESEM) and Confocal Laser Scanning Microscopy (CLSM) were used to confirm that ECM was being produced during in vitro biofilm growth. SEM images confirmed that no ECM could be observed after routine preparation methodologies (Figure 1). Negative staining confirmed that ECM fibers are being released from the biofilms during sample preparation. Supernatants from all SEM preparation steps presented large amounts of fibers similar to control sample (Figure 2) and all fractions exhibited similar thickness (22- 28 nm). GC-MS analysis corroborates with this hypothesis and shows that all supernatants have the same carbohydrate content as the control sample, being mannose and glucose the main monosaccharide, present in every step of sample preparation. Additionally, analysis using ESEM mode confirmed that yeast cells were completely covered by ECM before sample preparation and the mannose/glucose nature of the ECM around the cells were demonstrated by the intense labeling of biofilms with Concanavalin-A, as shown in CLSM images (Figure 3). Here we demonstrate that chemical fixation followed by dehydration of biofilms samples lead to the total extraction of biofilm ECM. This result shows that utilization of only routine preparation methods for SEM leads to a reduced or mistaken analysis of biofilm structure. Comparing results from ESEM, CLSM and SEM we also showed that each microscopy technique has some information to offer and that the combination of several methodologies would lead to a better understanding of the whole biofilm structure.

Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Fig. 1: Candida albicans biofilms formed in vitro on central venous catheters and prepared for SEM using chemical fixation and dehydratation methodology. No extracellular matrix can be observed in the biofilm.

Fig. 2: Negative staining of the supernatants collected after each sample preparation step. (A) An aliquot of physically extracted extracellular matrix (ECM) was used as control; (B) sample from fixation step; (C) sample from Ethanol 50% step; (D) sample from ethanol 70% step.

Fig. 3: Candida albicans biofilms labelled with concanavalin-A and visualized using a confocal scanning microscopy. (A) ECM can be observed involving biofilm cells (white arrow); (B) Biofilm distribution.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-3534 Linking xylem anatomy and stem hydraulic conductivity with yield productivity in wheat

Rancic D.1, Pecinar I.1, Pekic Quarrie S.1, Bouche P.2, Morris H.2, Jansen S.2
1Faculty of Agriculture University of Belgrade, Nemanjina 6, 11080 Belgrade-Zemun, Serbia, 2Institute for Systematic Botany and Ecology, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
ilinka@agrif.bg.ac.rs

Xylem tissue plays a crucial role in long-distance water transport from roots to leaves. Since hydraulic efficiency is associated with stomatal conductance, and thus indirectly with the supporting growth and photosynthetic capacity of a plant, it could also influence grain yield in crop plants such as wheat (Triticum aestivum). In addition, hydraulic properties of the stem xylem tissue could provide information about its plasticity to environmental conditions such as water deficit. Increased drought stress and reduced food production is indeed one of the most important challenges to scientists and our society. In wheat, as in most monocots, the vascular cambium is absent and the entire plant body is the product of its primary growth, meaning that the water transport capacity of the stem is determined during early growth conditions.
The aim of this study was to determine how climate conditions during spring 2011 and 2012 would affect stem hydraulic conductance, plant growth, and grain yield in 40 different wheat genotypes. Wheat stems were sampled from field experiments during the early generative phase. The xylem pathway in wheat stems was investigated using transverse sections and quantitative vessel characteristics were measured using light microscopy. We applied an analysis of variance for all data of 2011 and 2012, and calculated pearson correlations between anatomical traits.
There was a significant correlation between 2011 and 2012 for the biomass, grain number per spikelet, grain weight per average spike, and the number of large vascular bundles. Anatomical characters correlated with yield parameters (e.g., biomass, grain number, and grain weight) included the number of vascular bundles, vessel area, and the number and size of parenchyma cells. Moreover, the theoretical hydraulic conductance was significantly correlated with biomass. Differences between genotypes will be analysed to identify the most productive genotypes, and how this selection could be used to increase wheat production under climate change.

We acknowledge the Serbian Ministry of Education, Science and Technical Development Project No TR31005, a bilateral Serbia-DAAD project (451-03-03159/2012-09/6), and the FP7 Project AREA (No 316004) for financial support.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-5970 Callose deposition plays a crucial role in barley rhizodermis cell differentiation.

Marzec M.1, Muszynska A.2, Melzer M.2, Kurczynska E.3
1Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Poland, 2Department of Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany, 3Department of Cell Biology, Faculty of Biology and Environmental Protection, University of Silesia, Poland
muszynska@ipk-gatersleben.de

<span>Symplasmic communication via plasmodesmata is a part of the information exchange system for plant cells. A multitude of molecules as ions, hormones, minerals, amino acids, sugars, proteins, transcription factors, and different classes of RNA can pass through plasmodesmata, suggesting the participation of plasmodesmata in the regulation and coordination of plant growth and development.

<span>The main aim of the presented studies was a description of symplasmic communication between different cells of the barley root epidermis during cell differentiation. For the comparable analysis the parental variety and two root hairless mutants, rhl1.a and rhl1.b, from the Mutant Collection of Department of Genetics were selected. Differences in the transport via plasmodesmata between the genotypes would suggest the importance of symplasmic isolation during the differentiation of barley root hairs.

<span>The results obtained clearly showed that symplasmic communication was limited during root hair differentiation in the parental variety, whereas in both root hairless mutants epidermal cells were still symplasmically connected in the zone of mature root hairs (Fig.1). Transmission electron microscopy analysis revealed that there are no differences in the ultastructure of plasmodemata between mutants and parental variety. However immonogold labeling of callose showed a significant higher number of gold particles at plasmodesmata in the differentiation zone of ‘Karat’ variety compared to root hairless mutant.

<span>This is the first report about the role of symplasmic isolation in barley epidermal root cell differentiation. Additionally, the presented data show that a disturbance in the restriction of symplastic communication is present in root hairless mutants.

<span>This research was supported by grants 2011/01/M/NZ2/02979 and 2013/08/T/NZ3/00811 from the Polish National Science Center and the German Acedemic Exchange Service (DAAD) ID 59755272.

Fig. 1: Transport of fluorescent probes between root epidermal cells of barley ‘Karat’ and mutants rhl1a and rhl1b at different stages of development. aez, apical elongation zone; bez, basal elongation zone; dz, differentiation zone; hc, hair cell; mz, meristematic zone; n, total number of analysed cells; nhc, non-hair cell; nfr, no fluorescence recovery.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-8-P-5982 INSECT IDENTIFICATION (Trioza Rusellae Tuthill) IN LEAVES Brosimum alicastrum Swartz IN YUCATAN, MEXICO

Andrade S.1, Ascencio A.1, Martín R.1, Tucuch J.1, Huchim E.1, Larqué A.1
1Centro de Investigación Científica de Yucatán, A.C. 130 No.43, Chuburná de Hidalgo, 97200, Mérida, Yucatán, México. sbac@cicy.mx
silvana74@gmail.com

Brosimum alicastrum Swartz, (tree commonly known as “Ramon”) is a dominant tree in the forests of Mexico and Central America, it is considered as a species of high economic importance (Larqué-Saavedra, 2013). Its high nutritional value surpasses corn, wheat, oats, and rice, among others (Sánchez and Pardo, 1977); in addition, its performance is five times greater than corn (CATIE, 1999). In this work, the insect that produces galls on the Brosimum alicastrum Swartz (Ramon) tree was identified. There is evidence that the insect is Trioza Rusellae Tuthill. In order to isolate and identify it, there were collections performed some areas of the peninsula specify in the town of Muna and Sacalum, Yucatán. Five to ten years old trees were sampled; branches with leaves that had damage gills were selected, placed in plastic bags and taken to the laboratory. They were subsequently placed in entomological cages; adults were obtained with a vacuum cleaner and nymphs with a razor and a brush (0.000) taking them out of the gills. Both samples were preserved in 70% alcohol, and Formalin fixer-Water-Alcohol (FAA). From samples in 70% alcohol nymphs and adults were mounted, they were observed in stereoscopic microscope and the insects that did not show body harm and have whole organs were selected. The samples in FAA were processed and observed with an Scanining Electron Microscope JEOL 6360 LV. Both types of samples were photographed and the different development stages were identified with entomological keys

Fig. 1: Morphology of the nymphs stages. Nymphs are similar in form to the adult except for the presence of wings, which are not developed until adulthood

Fig. 2: Morphlogy of the adult insect head. The head contains most of the sensing organs, including the antennae, ocellus or eyes, and the mouthparts.
↑ Go to the Table of content ↑

LS-9. Genetically-modified organisms and animal science

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-9-IN-5758 Visualizing zebrafish development with high-speed light sheet microscopy

Huisken J.1
1Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
huisken@mpi-cbg.de

Light sheet microscopy, such as Selective Plane Illumination Microscopy (SPIM, [1]), reduces photo-toxic effects to a minimum. Due to the illumination of the sample in a thin volume around the focal plane no tissue outside the plane of interest is exposed and bleached. In addition, the fluorescence is collected with highly sensitive cameras. Combined with novel sample mounting techniques [2], SPIM has become a powerful technique for the long-term observation of fragile biological organisms [3, 4]. SPIM benefits from the latest camera technology and is therefore constantly improving in speed and sensitivity. Over the years it has become evident that light sheet microscopy is revolutionizing bio-imaging in several ways. Lately, we have shown that three-dimensional (3D) volumes can be imaged almost instantaneously using electrically tunable lenses (ETL) in SPIM [5]. This makes SPIM the fastest fluorescence microscopy technology for non-invasive 3D imaging. Even the dynamics of the beating zebrafish heart can be captured and the myo- and endocardial tissues as well as the blood can be visualized by 3D reconstruction [6].


Experiments have become possible that run at full speed using the best possible hardware, without being limited by the fragility of the sample. The speed advantage of the SPIM over other fluorescence technique can be utilized not only to image rapid events in developing tissues but also to record a large number of views for multi-view reconstruction. One key application of light-sheet technology includes the multi-dimensional imaging of the developing zebrafish larvae over extended periods of time [2]. I will give some examples of the unique capabilities of SPIM, especially for monitoring the development of the zebrafish heart [6] and the early endoderm [7].

References

[1] J. Huisken, et al., Science 305 (2004) 1007.
[2] A. Kaufmann, et al., Development 139 (2012) 3242.
[3] J. Huisken, D.Y.R. Stainier, Development 136 (2009) 1963.
[4] M. Weber, J. Huisken, Curr Opin Genet Dev 21 (2011) 566.
[5] F.O. Fahrbach, et al., Opt Express. 21(2013):21010.
[6] M. Mickoleit, et al., under review.
[7] B. Schmid, et al., Nat Commun 4 (2013) 2207.

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-9-IN-5764 Cellular, subcellular and molecular in vivo imaging of nervous system pathology in mice

Kerschensteiner M.1
1Institute of Clinical Neuroimmunology, LMU Munich, Germany
martin.kerschensteiner@med.uni-muenchen.de

Here, I want to discuss how advances in in vivo microscopy together with mouse genetics can improve our understanding of the cellular, subcellular and molecular mechanisms that mediate neuroinflammatory tissue damage.

To illustrate this approach I will use our recent insights into the in vivo pathogenesis of immune-mediated axon damage as an example. Immune-mediated axon damage plays a crucial role in inflammatory diseases of the central nervous system (CNS) like multiple sclerosis (MS), as we know by now that the number of axons damaged by immune cells critically determines the clinical disability of MS patients. However we still understand very little about the process that leads to axon damage. Recently, we have used an in vivo imaging approach to investigate the pathogenesis of immune-mediated axon damage in an animal model of multiple sclerosis. By time-lapse imaging of fluorescently labeled axons we could follow the slow and spatially restricted degeneration of axons in inflammatory CNS lesions. This “focal axonal degeneration” appears to be a novel type of axonal degeneration that is characterized by intermediated stages that can persist for several days and progress either to the degeneration or full recovery of the affected axons.

In vivo imaging approaches now allow us to address the following key aspects of the axon degeneration process: First, to identify the molecular mechanisms that drive axonal degeneration, we can now reveal the actions of key damage mediators, in particular the influx of calcium and the release of reactive species, in vivo. Second, to better understand the relation between structural and functional axon damage in neuroinflammatory lesions, we can directly measure axonal transport in neuroinflammatory lesions. Using these examples, I hope to illustrate how recent advances in light microscopy can help us to reveal and mechanistically dissect neuroinflammatory tissue damage as it happens in the living CNS.

This work is supported through grants from the Deutsche Forschungsgemeinschaft (Transregio 128), the German Federal Ministry of Research and Education (Competence Network Multiple Sclerosis), the European Research Council under the European Union’s Seventh Framework Program (FP/2007-2013; ERC Grant Agreement n. 310932), the Hertie-Foundation and the “Verein Therapieforschung für MS-Kranke e.V.”.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-9-P-1843 Ultrastructural Analysis of Platelets in Experimental Cerebral Ischaemia

van der Spuy W. J.1, Pretorius E.2
1University of the Witwatersrand, Johannesburg, South Africa, 2University of Pretoria, Pretoria, South Africa
wendy.vanderspuy@wits.ac.za

It is widely known that oestrogen is neuroprotective [1] through various mechanisms which suggest that sex hormone levels, thrombotic mechanisms and inflammatory processes are strongly interconnected in predicting the outcome and consequences of cerebral ischaemia [2]. Cerebral ischaemia is associated with parameters of altered blood coagulation which may lead to the occlusion of blood vessels; including increased thrombin activity, elevated fibrinogen levels, altered fibrin network ultrastructure, increased platelet counts and even resistance to fibrinolysis [3,4]. Because platelet ultrastructure is altered in conditions like thrombosis and associated with stroke [5,6], the question arises what insight ultrastructural analyses of platelet morphology may provide into the role of oestrogen during ischaemic insult.

A hyperglycaemic modification to the two-vessel occlusion model for inducing experimental cerebral ischaemia was established in Sprague Dawley rats, divided into three experimental groups (males, cyclic and acyclic females). Subsequent to termination at four intervals, neural tissue integrity levels were correlated to corresponding platelet ultrastructure so as to determine whether there is an association between cerebral ischaemia and altered platelet ultrastructure.

It is apparent in the results that under the influence of oestrogen in cyclic females, there was indeed lesser neural tissue damage and a reduced overall degree of inflammation evident in chemical analysis and platelet ultrastructure when compared to males and acyclic (ovariectomised) females. Mirroring the biochemical ischaemic cascade [2], inflammation is shown to peak early in the morphological evidence. Platelet morphology suggests that the largest shock due to cerebral apoptosis indeed takes place within the first 24 hours after insult. At this time males and acyclic females displaying necrotic ultrastructure, whereas cyclic females are rescued by oestrogen’s neuroprotective and anti-inflammatory effects. At 48 hours, the largest disruption of the blood brain barrier takes place, and this is again evidenced by the extension of platelet pseudopodia.

In conclusion, physical neural injury is closely shadowed, if not preceded, by inflammatory changes in the coagulation system, particularly manifested in platelet ultrastructure. Ultrastructural platelet study may be used successfully to follow the progression of events of cerebral ischaemia and possibly assist in the assessment of treatment strategies and their effects on haemostasis [7].

[1] Front Neuroendocrinol 2009; 30:201-11
[2] Rev Neurosci 2012; 23:269-278
[3] Arterioscler Thromb Vasc Biol 2007; 27:2507-13
[4] Stroke 2009; 40:1499-1501
[5] J Thromb Thrombolysis 2011; 31:507-13
[6] Pathophysiol Haemos Thromb 2008; 36:251-8
[7] Funding: Medical Research Council (MRC) of South Africa grant to EP

Fig. 1: Platelet morphology representation of cyclic females. A: pre-ischaemia control minimally activated, Post-ischaemic reperfusion B: 2h post-reperfusion indicative of inflammation, C: 24h post-reperfusion demonstrating anti-inflammatory effects of oestrogen, D: 48h post-reperfusion inflammation around second BBB disruption. (Scale bar = 200nm)

Fig. 2: Platelet morphology representation of males. A: pre-ischaemia control minimally activated, Post-ischaemic reperfusion B: 2h post-reperfusion indicative of inflammation, C: 24h post-reperfusion 70% of platelets necrotic due to low oestrogen levels, D: 48h post-reperfusion inflammation around second BBB disruption. (Scale bar = 200nm)

Fig. 3: Platelet morphology representation of acyclic females. A: pre-ischaemia control displaying thrombotic preparedness, Post-ischaemic reperfusion B: 2h post-reperfusion soothed inflammation, C: 24h post-reperfusion 60% of platelets necrotic, D: 48h post-reperfusion inflammation around second BBB disruption. (Scale bar = 200nm)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-9-P-6036 Micromirror structured illumination for drosophila brain imaging and physiology

Pedrazzani M.1, Nutarelli D.1, Benrezzak S.1, Tchenio P.1,2, Preat T.2
1Laboratoire Aimé Cotton, Orsay, France, 2Genes and Dynamics of Memory Systems, Paris, France
melanie.pedrazzani@u-psud.fr

Cellular and neural network dynamics during memory formation remain poorly known. Drosophila melanogaster is a unique model to better understand them. Its main advantages are the small size of its brain which gives an optical access to the whole brain after microsurgery, the tractability of global analysis of the whole network and the availability of powerful genetic methods while Drosophila melanogaster demonstrate remarkable learning abilities. Genetically encoded fluorescent reporters have given a special place to optical microscopy in drosophila neurobiology research because they allow in vivo analyses of biochemical processes, with good temporal resolution in targeted well-defined cells owing to the binary UAS-Gal4 system. In this field, current optical implementations rely on commercial optical set-up, usually confocal microscope. Integrated analysis of most brain functions, like memory for instance, would benefit from a global 3D monitoring of activity of the neurons involved in the function which is out of reach of current confocal approaches. To take advantage of the potential temporal resolution of the reporter, I have developed a wide-field microscope based on structured illumination by a micromirror matrix technology. This microscope exhibits both high speed imaging and optical sectioning ability. I will present results on mushroom body physiology, a brain area known to be the memory center for olfactory associations, obtained with recently developed genetically encoded reporters, especially the calcium reporter Gcamp6.

↑ Go to the Table of content ↑

LS-10. Human health and disease

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-10-IN-1566 Cancer-associated fibroblasts: the new potential target for cancer therapy

Smetana K.1
1Charles University, 1st Faculty of Medicine , Institute of Anatomy, Prague, Czech Republic
karel.smetana@lf1.cuni.cz

Incidence of malignant tumor is increasing with ageing of population worldwide. Tumors are highly complex tissues, where cancer-associated fibroblasts (CAF), inflammatory cells and blood vessels support the activity of malignant cells. From this point of view, the intercellular interactions seem to be in the center of interest of cancer cell biologists. CAF origin is not fully understood. However, among other sources, they can be formed by induction of transition of normal fibroblasts by TGF-β1 and/or endogenous lectin, galectin-1. These cells usually express smooth muscle actin, but it is not obligatory. CAF differ from normal fibroblasts in expression of almost 600 genes. They are functionally very active because CAF cultured with the normal epithelial cells significantly changed their phenotype to be similar to cancer cells. Interestingly, the fibroblasts under the influence of CAF acquire properties of mesenchymal stem cells. To complete the study of intercellular crosstalk in the tumor we also studied the opposite situation, where normal fibroblasts were cultured with normal/malignant epithelial cells.  Normal fibroblasts cultured under the influence of epithelium acquire properties of CAF but for the limited time only.  On the other hand, the activation of CAF by cancer cells seems to be more stable.  The results demonstrated that IGF-2, BMP-4, CXCL-1, IL-6, IL-8 can play an important role in cancer cell-mesenchymal interaction. Blocking of cancer cell-CAF interaction by targeting of these molecules can have some therapeutic potential in future. Some similarity between the cancer and wound healing has been also observed. The presented data show how the combination of microscopy with genomic approach increases the volume of informations to improve the complexity of cancer microenvironment research.

References:
Dvorankova et al.: Histochem Cell Biol 137: 679, 2012
Kolar et al.: Biol Cell 104: 738, 2012
Smetana Jr. et al.: Folia Biol 59: 207, 2013
Strnad et al.: Histochem Cell Biol 133: 201, 2010
Valach et al.: Int  J Cancer 131: 2499, 2012

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-10-IN-2164 MUSCLE CELL FATE: A COMPLEX BALANCE AMONG APOPTOSIS, NECROSIS AND AUTOPHAGY

Falcieri E.1
1Department of Earth, Life and Environmental Sciences, University of Urbino Carlo Bo, 61029 Urbino and IGM-CNR, Rizzoli Orthopedical Institute Bologna; Italy
elisabetta.falcieri@uniurb.it

Apoptosis plays a crucial role in muscle pathology, in denervation and disuse. On the other hand, autophagy, a form of organelle/molecules deletion, is a mechanism of cell survival diffusely described in literature. In this work we studied in vitro muscle cell response to a variety of experimental conditions, all leading to final cell death. C2C12 murine myoblasts were conventionally cultivated in flasks and, after achieving monolayer confluence, myogenic differentiation was induced (D’Emilio et al., 2010). Both on myoblasts and myotubes, cell death was induced with H2O2, UVB, (oxidant agents), staurosporine (PKC blocking agent), cysplatin (DNA cross-linker), starvation or etoposide (topoisomerase II inhibitor). Cell response was investigated by means of a variety of morpho-functional approaches. TEM or SEM were carried out to identify possible chromatin changes, organellar component involvement or surface blebbing, all cell death-related patterns. TUNEL reaction allowed to highlight in situ DNA fragmentation, generally well demonstrated by DNA agarose gel electrophoresis too, but not so easily identifiable in muscle cells, even if in the presence of other apoptotic patterns (Salucci et al., 2013).
Myoblasts appeared more sensitive than myotubes to all treatments. In particular, myoblasts treated with H2O2, UVB, cysplatin, starvation or etoposide showed characteristic apoptotic features, with chromatin condensation and DNA cleavage, even if in the absence of the classic cup-shaped dense patches. Interestingly, in multinucleated myotubes apoptotic and normal nuclei appeared inside the same syncytium: this behavior could support the common observation of a better resistance of myotubes. Their nuclear-dependent “territorial organization”, could indeed determine a progressive “myonuclear death”, later than that of mononucleated myoblasts.
Cells treated with staurosporine, evidenced late apoptotic features and secondary necrosis. After the majority of stimuli, autophagic granules -frequently containing mitochondrial remnants- could be diffusely revealed in myotube cytoplasm, both by TEM and LC-3 immunolabeling. We hypothesize that they could preserve muscle cell integrity, counteracting chemical treatments, most of which activate death pathways involving mitochondria. Therefore, autophagy, an important mechanism for mitochondria degradation (Sandri et al., 2013), plays a central role in muscle biology and, in particular, in cell death-resistance of differentiated cells.

dr. S.Salucci, dr. S.Burattini, dr. M.Battistelli, dr. V.Baldassarri, dr. D.Curzi and dr. F.Giordano are thanked for, precious, continuous collaboration.

Fig. 1: C2C12 myoblasts (A,B,C,D,E) and myotubes (F,G,H,I,L) after H2O2 (A,B), staurosporine (C,H), cysplatin (D), starvation (E), UVB (F,G) and etoposide (I,L). n=nuclei; ap=apoptotic nuclei. A,C,I Bar=2µm; B,D, Bar=5µm; E,G, Bar=10µm; F,L, Bar=1µm; H, Bar=20µm.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-10-O-2045 Confocal Laser Scanning Microscopy for the detection of intracellular bacterial communities in children

Scavone P.1, Robino L.2, Araujo L.2, Algorta G.2, 3, 4, Zunino P.1, Pírez M. C.4, Vignoli R.2
1Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Av Italia 3318, 2Departamento de Bacteriología y Virología, Facultad de Medicina, UDeLaR, Instituto de Higiene, Alfredo Navarro 3051, Montevideo, Uruguay, 3Laboratorio Central, Centro Hospitalario Pereira Rossell (CHPR). Br Artigas 1550, 4Departamento de Pediatria, Centro Hospitalario Pereira Rossell, Facultad de Medicina, UDeLaR.Br Artigas 1550
pscavone@gmail.com

Urinary tract infection (UTI) is the third cause of medical consultant in childhood. 3-5% of children until the age of 11 are in risk of having UTI. Uropathogenic Escherichia coli (UPEC) is the most common etiological agent, being responsible for 80-90% of cases. Different virulence factors are related with the capability of UPEC of causing UTI and recently it was demonstrated that it can produce intracellular bacterial communities (IBC) in the vesical epithelium in children. Until now, the only technique available for the detection of IBC in urine cells is confocal laser scanning microscopy (CLSM).

The objective of the present work was to determine the presence of intracellular bacteria in children with UTI caused by E. coli, to characterize its virulence attributes and to establish the relationship with the clinical presentation and recurrent UTIs.

The study included 106 children with E. coli UTI assisted in a Children’s Hospital in Uruguay (HP-CHPR) between June-November 2012. Urine samples were analyzed by confocal microscopy for exfoliated urothelial cells with intracellular bacteria. Phylogenetic group and 24 virulence factors of E. coli were determined using multiplex-PCR.

CLSM images allow us to classify at least three different intracellular reservoirs. Intracellular bacteria isolated (IBI) was characterized by the presence of bacteria inside the cell but separated each other (Figure 1). Small intracellular bacteria communities (sIBC) were defined as a group of at least five bacteria (Figure 2) and the presence of a bigger group was classified as intracellular bacterial communities (IBC) (Figure 3). The presence of intracellular bacteria was detected in 37/106 (34,9%) samples, 26 cases as IBC, 11 as intracellular bacteria isolated (IBI). We analyzed 49 medical records: 22 with IBC/IBI and 27 with none IBC/IBI. The presence of IBC/IBI was associated with the absence of certain virulence factors (P pili and siderophores, p0.006 and 0.035 respectively). IBC formation was associated with the presence of lower urinary syndrome and absence of fever (p0.000), not associated with recurrent UTIs. IBI's presence was associated with recurrent UTI (p0.01).

A new type of UPEC strains with low virulence factors but IBC/IBI producers could be described. The fail of antibiotic therapy could be related with the presence of intracellular bacteria. CLSM should be employed as a gold technique for the detection of IBC in particularly in patients with recurrent UTI. It will also necessary to establish if IBI/sIBC/IBC are different steps from the same phenomena or if they are independent process.

Fig. 1: Intracellular bacteria isolated (IBI). CSLM image; in red uroplakin III from urothelial cell, in green E. coli. The bar represents 7 um

Fig. 2: Small Intracellular bacteria communities (sIBC). CSLM image; in red uroplakin III from urothelial cell, in green E. coli. The bar represents 7 um.

Fig. 3: Intracellular bacteria communities (IBC). CSLM image; in red uroplakin III from urothelial cell, in green E. coli. The bar represents 7 um.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-10-O-2054 Investigating deposit-crowded cells by SIM microscopy to quantify the progress of age related granule-accumulationin human RPE cells.

Schock F.1,2, Best G.1,2, Celik N.2, Bakulina A.6, Hagmann M.1,2, Heintzmann R.4,5, Sel S.2, Birk U.3,7, Hesser J.6, Dithmar S.2, Cremer C.1,3,7
1Kirchhoff Institute for Physics, University of Heidelberg, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany, 2Department of Ophthalmology, Universityhospital of Heidelberg, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany, 3Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany, 4Institute for Physical Chemistry, University of Jena, Lessingstr. 10, 07743 Jena, Germany, 5Institute of Photonic Technology, Albert-Einstein-Straße 9, 07745 Jena, Germany, 6Experimental Radiation Oncology, University Medical Center Mannheim (UMM), University of Heidelberg, Theodor Kutzer Ufer 1-3, 68167 Mannheim, Germany, 7Department of Physics, University of Mainz, Staudingerweg 7, 55128 Mainz, Germany
florian.schock@kip.uni-heidelberg.de

Age related macular degeneration (AMD) is closely connected to the non-reversible accumulation of degradation products in the inside of human retinal pigment epithelium (RPE) cells[1]. There they form granules with a typical volume of around (0,6 +/- 0,5)μm³. To better understand the mechanisms of AMD formation, granules are analyzed quantitatively, especially their size, number and composition. Conventional light-microscopy is usually unable to resolve single granules reliably. Also it is practically impossible to manually identify and characterize up to over 100 granules in a single cell for a statistically relevant number of cells. As solution we used Structured Illumination Microscopy (SIM)[2,3] to resolve the granules inside the cells and to distinguish between different deposit materials. Furthermore, we introduce an algorithm that separates individual granules even in cells with high granule density. Besides, the algorithm determines characteristic quantitative parameters of the granules.
We present these characteristic parameters gained by analyzing over 200 RPE cells in histological samples of human donors of different age. All work on human tissue was done according to the Declaration of Helsinki.

[1] V. L Bonilha; Age and disease-related structural changes in the retinal pigment epithelium,Clinical Ophthalmology 2008, 2:413-424
[2] G. Best, R. Amberger, D. Baddeley, T. Ach, S. Dithmar, R. Heintzmann, C. Cremer (2011); Structured illumination microscopy of autofluorescent aggregations in human tissue, Micron 42;
[3] T. Ach, G. Best, S. Rossberger, R. Heintzmann, C. Cremer, S. Dithmar ;Autofluorescence imaging of human RPE cell granules using structured illumination microscopy; British Journal of Opthalmology, BJO Online First, published on July 3, 2012 as 0.1136/bjophthalmol-2012-301547

Fig. 1: SIM-Image of human RPE. Age of donor was 53years. Color-code is according to the excitationwavelengths 488nm, 568nm and 671nm.

Fig. 2: Left: 2D-Image of a single RPE-cell imaged by SIM. Right: Automatically separated granules of the left Image. The single granules are colored randomly.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-10-O-2239 The Vasa vasorum of the human great saphenous vein: three-dimensional vascular arrangement and vessel optimalities expressed, analysed by scanning electron microscopy and 3D-morphometry of vascular corrosion casts

Herbst M.1, Hoelzenbein T.2, Minnich B.1
1University of Salzburg, Department of Cell Biology, Division of Animal Structure & Function, Salzburg, Austria, 2Paracelsus Medical University Salzburg, Salzburg, Austria
markus.herbst@stud.sbg.ac.at

“Vasa vasorum” (VV) derives from Latin and literally means “vessels of the vessels”. Hence, the VV are a network of small arterioles, venules and capillaries which supply the outer two layers of the wall tissue of large blood vessels with oxygen and nutrients [1, 2]. The largest blood vessels in the body (e.g. the human great saphenous vein, the aorta, etc.) depend on this supporting network to maintain their health and function. Thus, the Vasa vasorum are an important part of the blood circulatory system [2, 3].

In the present study VV were studied in explanted segments of the human great saphenous vein (Vena saphena magna, HGSV), taken during harvesting for coronary bypass grafts or extirpation of varicose vein segments at the University Clinics for Vascular and Endovascular Surgery (PMU Salzburg), using vascular corrosion casting (VCC), scanning electron microscopy (SEM, FEI/Philips XL-30 ESEM) and 3D-morphometry (M3).

The aim of this study was the examination of the three-dimensional arrangement of the Vasa vasorum in healthy and pathological (varicose) conditions. Moreover, it was intended to identify the most vital segments of the HGSV in order to improve the results of bypass surgeries.

A meticulous analysis of the whole delicate microvascular system of the VV of the HGSV and its spatial arrangement (Fig. 1) is presented. It is one of the first studies yielding detailed quantitative data on the geometry of the HGSV’s Vasa vasorum and the optimality principles (minimal lumen volume, minimal pumping power, minimal lumen surface and minimal endothelial shear force) underlying the design of this microvascular network.

Arterial feeders originating from nearby arteries were found to approach the HGSV every 15mm, subsequentially forming a rich capillary network within the adventitia and the outer two thirds of the media in normal HGSV. In HGSV with intimal hyperplasia capillary meshes of the VV were found to extend into the inner layers of the media.

Measurements of spatial branching-off angles in bifurcations and consecutive optimality calculations showed that in both, the medial and distal part of the HGSV, data are homogenously distributed close to the theoretical optimum of vessel diameters.

We gratefully thank Christine Radner, BSc. for her technical assistance, Dr. Wolf-Dietrich Krautgartner for his support at the scanning electron microscope and Univ.-Prof. Mag. Dr. Alois Lametschwandtner for useful advices at corrosion casting of the delicate specimens.

Fig. 1: The Vasa vasorum run predominantly parallel to the longitudinal axis (LA) of the HGSV. Vessels having a longitudinal arrangement are defined as orders 1 & 3. Orders 2 & 4 indicate vasa with a circular arrangement. Arterial vasa (A) are coloured in red, venous vasa (V) in blue & capillaries (c) in orange. Arrows indicate the direction of blood flow.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-10-O-2790 A multiscale approach to study the composition of coronary thrombus in acute myocardial infarction

Tessarolo F.1,2, Bonomi E.2, Piccoli F.3, Morat F.3, Caola I.3, Caciagli P.3, Bonmassari R.3, Nollo G.1,2
1Bruno Kessler Foundation, Trento, Italy , 2University of Trento, Trento, Italy , 3APSS, Provincial Health Trust Trento, Italy
tessarolo.f@gmail.com

Thrombus aspiration (TA) is a recommended technique in the treatment of myocardial infarction allowing thrombus removal from the culprit artery via a specific catheter. A reduced mortality in patients affected by myocardial infarction with ST elevation (STEMI) has been associated to the use of TA in conjunction with primary percutaneous intervention (PCI) (De Luca et al. 2008; Burzotta et al. 2009). Noteworthy, TA allows to collect the biological aspirate in a minimally invasive way for the characterization of its morphology and composition that can trace the evolution of the pathology from the lesion of the atherosclerotic plaque to the reperfusion of the ischemic cardiac tissue (Silvain et al. 2011). We aimed at developing and applying a multiscale approach to determine coronary thrombus composition by means of optical and electron microscopic techniques on a series of 57 thrombi collected during PCI with TA on STEMI patients.
Each thrombus was immediately fixed (2% glutaraldehyde in 0,1M phosphate buffer) and a digital colour image (10x) was obtained for the qualitative macroscopic classification into “red”, “mixed” and “white” thrombus (Figure 1). The sample was subsequently prepared and imaged by SEM in high vacuum mode (2000x) for percent quantification of platelets, red blood cells (RBCs), white blood cells (WBCs) and fibrin (Figure 2) according to a method we developed for thrombi in hemodialysis catheters (Lucas et al. 2013). Scanned samples were than rehydrated and processed for permanent histology. Platelet, RBCs and WBCs were quantified on Carstair’s stained sections by setting specific colour thresholds in the La*b* colour space (Figure 3).
Macroscopic classification according to two senior and a young cardiologists gave 8(14%) “white”, 20(35%) “mixed” and 29(51%) “red” thrombi. Inter-observer agreement was good for “red” and “white” thrombi, but lower for the identification of mixed thrombi. The mean (SD) histological composition was 18%(20%), 47%(24%) and 35%(20%) for platelet, RBCs and WBCs respectively. Features quantification on SEM images was feasible in 53/57 samples giving a mean (SD) SEM composition of 31%(25%), 40%(22%), 26%(19%), and 3%(3%) for platelets, RBCs, WBCs and fibrin respectively. Sub-groups analysis showed an agreement between macroscopic classification and both SEM and histological composition: white thrombi presented a mean prevalence of platelets (42% at histology and 58% at SEM) while red thrombi were mostly composed of RBCs (59% at histology and 42% at SEM). Intermediate features were found for mixed thrombi.
Method here presented deserves high potential for understanding the mechanisms of thrombus formation in STEMI and for investigating correlations between composition and thrombus age or drug treatments.

We thank the Cardiology Division of the Trento Hospital.

Fig. 1: Macroscopic classification of aspirated coronary thrombi. Representative image of a white (a), mixed (b), and red (c) sample.

Fig. 2: SEM images of the thrombus surface. Representative fields of view with a prevalence of platelets (A), RBCs (B), WBCs (C, stars), and fibrin (D). High vacuum mode, 10 KV, SE detector, 2000x original magnification.

Fig. 3: RBCs, fibrin, and platelets on a mixed thrombus section stained with Carstair’s method (left). Representative colours associated to the three components of interest are shown. Binary images after specific threshold for RBCs (a), fibrin (b), and platelets (c). Inset d) reports about components not recognized in the three previous features.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-1614 Investigation of the presence of biofilms in patients with cochlear implant by scanning electron microscopy

Dag I.2, Incesulu A.1, Kaya E.3, Acar M.4, Veziroglu L.5
13Department of Otorhinolaryngology, Eskisehir Osmangazi University School of Medicine, Eskisehir, Turkey, 2Electron Microscope Laboratory, Eskisehir Osmangazi University, Eskisehir, Turkey , 3Department of Otorhinolaryngology, Yunusemre State Hospital, Eskisehir, Turkey
idag280@gmail.com

Investigation of the presence of biofilms in patients with cochlear implant by scanning electron microscopy
Armagan Incesulu1, Ilknur Dag2, Ercan Kaya3, Mustafa Acar4, Leman Veziroglu5
1,3Department of Otorhinolaryngology, Eskisehir Osmangazi University School of Medicine, Eskisehir, Turkey
2Electron Microscope Laboratory, Eskisehir Osmangazi University, Eskisehir, Turkey
4,5Department of Otorhinolaryngology, Yunusemre State Hospital, Eskisehir, Turkey
Abstract
Biofilms are organized microbial communities that are playing an increasing role in otolaryngologic diseases such as chronic or recurrent otitis media, cholesteatoma, adenoiditis or tonsillitis. Moreover, biofilm infection may also be problem in the prosthetic device. Development of biofilm formation may due to the device itself, host or both. They are difficult to eradicate owing to their resistance to immunologic defense mechanisms and antibiotics.
Various cultural techniques are available to detect biofilm-producing microorganisms, but the using the electron microscopic methods may offer detailed insight into the ultrastructure of biofilm and their environment. Despite this, limited data are available from this area.
Cochlear implants are highly acceptable rehabilitation method in the person with severe to profound hearing loss. Although the complication rate of the cochlear implantation is very low, biofilm formation is considered as a main reason of the flap problems or local infection in this patient.
In this project, our objective is to investigate the evidence of biofilms in patients who underwent surgery for cochlear implantation. For this purpose, specimens were taken during the surgery (42 tissue samples; 21 mastoid and 21 middle ear mucosa).
Our findings support the hypothesis that biofilms may play a significant role in otolaryngologic infections. 12 of the 21 patients (% 57) with cochlear implant demonstrated findings of a biofilm. However, bacterial microcolonies were not evenly distributed over the entire surface of the specimen, but rather located in some parts of the specimen, mostly located in small depressions between cells of normal-appearing mucosa. For the biofilm studies with SEM, high magnifications are very important. Sometimes, for the samples which seem to be negative at low magnifications, as magnification was increased, biofilm presence was encountered. On the contrary, sometimes, for the samples which seem to be positive at low magnifications, as magnification was increased, rough surface structure of tissue or erythrocytes were observed. Nevertheless, further investigations should be performed in order to determine that whether biofilm formation may be an important factor in the pathogenesis of these infections.

Acknowledgement

This work was supported by a grant from Eskisehir Osmangazi University (Project no. 201141045).

Fig. 1: Figure 1. Scanning electron micrograph of middle ear tissue covered with biofilm. All specimens were removed from patients with cochlear implant.

Fig. 2: Figure 2. Higher magnifications of same picture. Arrows indicate the extracellular material connected to the bacteria.

Fig. 3: Figure 3. This image shows a mastoid tissue sample of same patient. Arrow indicate the biofilm.

Fig. 4: Figure 4. This image shows the surface of a middle ear of a patient with cochlear implant. The specimen was used as a control in our study. Note the relatively smooth surface and lack of organisms.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-1834 Molecular morphological changes in the glomerulus associated with the elevation of O-GlcNAcylation in diabetic nephropathy

Akimoto Y.1, Miura Y.2, Toda T.3, Fukutomi T.4, Sugahara D.1, Wolfert M. A.5, Wells L.5, Boons G.5, Hart G. W.6, Endo T.2, Kawakami H.1
1Dept. Anatomy, Kyorin Univ. Sch. Med., Tokyo, Japan , 2Res. Team for Mech. Aging, Tokyo Metropol. Inst. Gerontol., Tokyo, Japan, 3Adv. Med. Res. Ctr., Yokohama City Univ., Yokohama, Japan, 4Dept. Pharmacol. Toxicol., Kyorin Univ. Sch. Med., Tokyo, Japan, 5Complex Carbohyd. Res. Ctr., Univ. Georgia, Athens, USA, 6Dept. Biol. Chem., Johns Hopkins Univ. Sch. Med., Baltimore, USA
yakimoto@ks.kyorin-u.ac.jp

Modification (O-GlcNAcylation) of proteins by O-linked N-acetylglucosamine occurs in the nucleus and in the cytoplasm. O-GlcNAcylation is particularly relevant to chronic human diseases including diabetes, cancer, neurodegenerative disorders, and cardiovascular disease.

Increased flux through the hexosamine biosynthesis pathway promotes the O-GlcNAcylation, and has been implicated in the development of insulin resistance and diabetes complications. In our previous immunohistochemical study, we demonstrated that the O-GlcNAcylation level increased in various tissues including kidney from diabetic GK rats, which is an animal model of type 2 diabetes.

To identify marker proteins that change in their extent of O-GlcNAcylation in the diabetic kidney from GK rats, we separated total kidney proteins by two-dimensional gel electrophoresis. O-GlcNAcylated proteins were detected by the immunoblot using anti-O-GlcNAc antibody. Selected proteins that changed markedly in the O-GlcNAc level were identified by Mass Spectrometry analysis. The localization and the quantity of these O-GlcNAcylated-proteins were analyzed by in situ Proximity ligation assay (PLA). O-GlcNAcylated proteins that changed significantly in the degree of O-GlcNAcylation were identified as cytoskeletal proteins (α-actin, α-tubulin, α-actinin 4, myosin) and mitochondrial proteins (ATP synthase, pyruvate carboxylase). Results of immunoprecipitation and immunoblot studies, as well as in situ PLA demonstrated that the extent of O-GlcNAcylation of the above proteins increased in the diabetic kidney. Immunoelectron microscopy revealed that α-actinin 4 increased in the foot process of podocytes and the proximal tubules. To further examine the changes of the O-GlcNAcylation of glomerular proteins accompanied with diabetic nephropathy, we isolated glomerulus from kidney and performed proteomic analysis.

It was revealed that some glomerulus-specific proteins including synaptopodin were O-GlcNAcylated. To elucidate the role of O-GlcNAcylation of glomerular proteins in the diabetic nephropathy the morphological changes of the glomerular epithelial cells were examined under the various conditions in vitro.

References

1) Hart, G.W., Housley, M.P., Slawson, C.: Nature 2007, 446:1017-1022

2) Degrell P, Cseh J, Mohás M, et al.: Life Sci 2009, 84:389-393

3) Akimoto, Y., Miura, Y., Toda, T., et al.: Clin Proteomics 2011, 8:15

This study was supported in part by Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology (C-24590260 to YA), from Japan Diabetes Foundation (to YA), from Kyorin University School of Medicine, Kyorin Medical Research Award 2013 (to YA) and by NIH R01 DK61671 (to GWH).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-1841 Ultrastructural Investigation of the Experimental Relationship between Breast Cancer and Thrombosis – Preliminary Study

van der Spuy W. J.1, Augustine T. N.1
1University of the Witwatersrand, Johannesburg, South Africa
wendy.vanderspuy@wits.ac.za

Thromboembolic complications have been identified as the second most common cause of death in breast cancer patients [1,2]. The interaction and result thereof, between circulating tumour cells and platelets is complex and reciprocal: platelet activation and aggregation has been implicated in facilitating coagulation-mediated metastasis; tumour-derived cytokines and growth factors have been implicated in thrombocytosis [3] and malignancy itself is associated with increased risk of thromboembolism [1,2]. Platelet and fibrin network morphology is altered in human disease conditions such as diabetes, stroke and cancer and associated with thrombosis [4,5]. It is thus understood that the in vivo relationship between breast cancer cells and platelets may lead to changes in platelet morphology and function, increasing susceptibility to thrombosis.

The reciprocal interactions between tumour cells and coagulation ability in vitro were investigated, by assessing ultrastructural alterations in platelet and fibrin network morphology, through the establishment of a co-culture system (MCF-7 luminal phenotype breast cancer cells cultured with blood plasma of healthy female individuals). Co-cultures were implemented for 5 to 30 min after which platelet and fibrin network coagula were prepared as per published protocol [5] on glass coverslips.

Utilising scanning electron microscopy, we found that that enhanced fibrin network formation and platelet aggregation appears to take place concurrently [6,7]. Changes in morphology from normal were visible as early as 5 min in co-culture, with platelets displaying pseudopodia extension and fibrin networks increasing in density. At 10 and 15 min, distinguishable morphology deteriorated drastically, with platelets spreading their hylomeres and fibrin plaque formation. By 20, 25 and 30min there was almost no semblance of normal characteristics in both platelet and fibrin network preparations, with dense plaque formation in both. This induced deterioration of cellular and fibre characteristics resembles that of previously studied blood plasma preparations of severe inflammation- and thrombosis- prone patients [4,5].

In conclusion, our preliminary results seem to indicate that the in vitro environment closely mimics the in vivo at an ultrastructural level, evidencing that it may indeed be the reciprocal interaction between tumour cells and the coagulation system which induces thrombosis, regardless of other systemic factors. The more rigid the construction of platelet and fibrin networks, the more impaired fibrinolysis would be. These results offer picturesque confirmation that breast cancer patients would be increasingly susceptible to thrombotic-related consequences [8].

[1] Int J Haematol 2000; 73:137-44
[2] Lancet Oncol 2002; 3:425-30
[3] J Thromb Haemost 2011; 9:237-49
[4] Microsc Res Tech 2009; 72:679-83
[5] J Thromb Thrombolysis 2011; 31:507-13
[6] Am J Med 1990; 88:601-6
[7] Crit Rev Oncol Hematol 2006; 60:144-51
[8] Funding: National Research Foundation of South Africa to WJS (NRF 87935), Carnegie Large Research Grant to TNA (001.408.8421101)

Fig. 1: Platelet ultrastructure. Platelet rich plasma was co-cultured with MCF-7 breast cancer cells. Ultrastructure was studied preparing platelet coagula from plasma. A: Control platelet, unexposed to cells. Experimental platelets B: 5min co-culture, C: 15min co-culture, D: 30min co-culture (Scale bar = 1µm)

Fig. 2: Fibrin network ultrastructure. Platelet rich plasma was co-cultured with MCF-7 breast cancer cells. Ultrastructure was studied preparing fibrin coagula through addition of thrombin to plasma. A: Control fibrin networks, unexposed to cells. Experimental networks B: 5min co-culture, C: 15min co-culture, D: 30min co-culture (Scale bar = 1µm)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-1903 Experimental stem cell therapy of skeletal muscle diseases: role of transplanted bone marrow cells in regenerative myogenesis

Cizkova D.1, Vavrova J.2, Micuda S.3, Filip S.4, Dolezelova E.3, Bruckova L.1, Mokry J.1
1Department of Histology and Embryology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Czech Republic , 2Department of Radiobiology, Faculty of Military Health Sciences in Hradec Kralove, University of Defence, Czech Republic, 3Department of Pharmacology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Czech Republic, 4Department of Oncology and Radiotherapy, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Czech Republic
cizkovad@lfhk.cuni.cz

Recently discovered ability of the adult bone marrow cells (BMCs), incl. hematopoietic and mesenchymal stem cells, to contribute to injury–induced skeletal muscle regeneration has raised new possibilities in treatment of skeletal muscle diseases. However, mechanisms by which BMCs participate in regenerative myogenesis have still remained to be fully elucidated. To extend our knowledge of experimental stem cell therapy of skeletal muscle diseases and investigate the role of exogenous adult BMCs in the skeletal muscle regeneration, we intravenously transplanted mouse lacZ+ or GFP+ freshly isolated BMCs into whole-body lethally irradiated immunocompetent mice 7 hours after or 4 weeks before the cardiotoxin-induced injury of the recipients’ tibialis anterior muscles. Seven to 33 days after the toxin injection, injured muscles were excised and fixed in 4% paraformaldehyde, processed for X-gal histochemistry to detect lacZ+ cells, embedded into GMA resin (to obtain 1 µm thin sections) or paraffin or fixed in 2% paraformaldehyde, frozen in methylbutane, chilled in liquid nitrogen, cryosectioned and examined for GFP fluorescence. The presence of lacZ gene in injured muscles was determined by qPCR. The skeletal muscles of recipients injured 7 h before the transplantation did not regenerate, nevertheless, X-gal positivity was predominantly identified in desmin- and nestin- multinucleated cells resembling foreign body giant cells located in the injured areas, 14 and 33 days after grafting. On the contrary, the recipients’ muscles injured 4 weeks after the transplantation fully regenerated. X-gal or GFP positivity was observed in the regenerating muscles excised 7 days after the injury in numerous inflammatory cells, in some newly formed myoblasts and myotubes and in some cells of the endomysium and in the regenerated muscles examined 28 days after the toxin injection in some endomysial cells and rarely in newly formed myofibers. qPCR verified presence of transplanted lacZ+ BMCs in injured recipients’ muscles. Our results confirmed ability of intravenously transplanted exogenous BMCs to settle in the injured skeletal muscle and participate in the skeletal muscle regeneration. They generated blood cells that infiltrated endomysium and took part in the cleaning reaction, the first step of the regeneration process, and moreover, they contributed to new myofibers formation.

This work was supported by the grant PRVOUK P37/06.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2028 NEW ANTIOXIDANTS IN THE PREVENTION OF UVB-INDUCED KERATINOCYTE APOPTOSIS

Salucci S.1, Burattini S.1, Curzi D.1, Buontempo F.2, Martelli A.2, Falcieri F.1,3, Battistelli M.1
1DiSTeVA, University of Urbino Carlo Bo, Urbino, 61029, Italy, 2Department of Biomedical and Neuromotor Sciences; University of Bologna; Bologna, Italy, 3IGM, CNR, Rizzoli Orthopaedic Institute, Bologna, 40136, Italy
sara.salucci@uniurb.it

Skin cells can respond to UVB-induced damage either by tolerating it, or restoring it through antioxidant activation and DNA repair mechanisms or, ultimately, by undergoing programmed cell death when damage is massive. Nutritional factors, in particular, food antioxidants, have attracted much interest because of their potential use in new preventive, protective, and therapeutic strategies for chronic degenerative diseases including skin inflammation (Vitale et al., 2013).
Hydroxytyrosol (HyT), a polyphenol present in virgin olive oil, well tolerated by organism after oral administration, shows a variety of pharmacological and clinical benefits such as anti-oxidant, anti-cancer, anti-inflammatory, and neuro-protective activities (Hu et al., 2014). These properties have been also reported for its synthetic derivatives. In fact, in an our previous study we have already demonstrated the HyT and its esthers anti-apoptotic effect against pro-oxidant chemical agents in different cell models (Burattini et al., 2013).
Here, the possible protective effects of HyT and its derivatives against UV-induced apoptosis were investigated in HaCat cell line. Human keratinocytes were pre-treated with antioxidants before UVB exposure and their effect evaluated by means of ultrastructural and molecular analyses. After UVB radiation, a known cell death triggers (Salucci et al., 2012), typical morphological apoptotic features, absent in control condition (Fig. A) and in HyT alone treated cells (Fig.B), appear. In particular, nuclear chromatin condensation (Fig. C, D) cytoplasm shrinkage and vacuolization (fig. D) can be observed at Electron Transmission Microscopy. Moreover, in control cells (Fig. F) and in the case of HyT administration alone (Fig. G) no labelled nuclei can be detected after TUNEL reaction, they diffusely appeared after UVB radiation exposure (Fig.H, I). An evident ultrastructural apoptotic patterns (Fig. E) and TUNEL positive nuclei (Fig. L) decrease can be observed when antioxidants were administrated before cell death induction. These data have been confirmed by molecular analyses (Fig. M, N, O). In fact, after UVB radiation, both intrinsic and extrinsic pathways appeared activated as well as PARP, a specific protein to identify nuclear damage directly correlated to apoptotic nuclear changes. Antioxidant compound administration before UVB-induced cell death showed an evident caspase activation decrease according to morphological analyses described above. In conclusion, our preliminary results demonstrate that natural antioxidant compounds are able to prevent apoptotic cell death in human keratinocytes exposed to UVB, attributing to these molecules an important role in preventing skin damage.

Dr. F. Giordano, A. Valmori, O. Rusciadelli and L. Bedini.

Fig. 1: Fig. 1: control (A,F,1) , HyT alone (B, G,2) , UVB (C, D, H, I,3), HyT+UVB (E, L,4) conditions, observed at TEM (A-E) and after TUNEL reaction (F-L). In M,N,O western blotting analyses of caspase- 9, -8 and -3, respectively. Bars: 2µm for A, B, E; 1µm for C, D; 25µm for F-L.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2734 Does Lapatinib increase pulmonary toxicity when concurrently used with Radiation Therapy? An experimental study with Wistar-albino Rats

Yetmen O.1, Güzel E.1, Eser M.1, Karaca C.1, Coban I.2, Süzer O.1, Bese N S.1
1Istanbul University Cerrahpasa Faculty of Medicine, Istanbul, TURKEY, 2Istanbul University Faculty of Veterinary Medicine, Istanbul, Turkey.
elifguzelctf@yahoo.com

Lapatinib (L) is an oral receptor tyrosine kinase inhibitor which is used for the treatment of metastatic breast cancer. Adjuvant usage of L is being investigated in clinical phase III trials. There is no data regarding the side effects of combination of RT and L which may be a problem when L is used in the adjuvant setting. Lung is the most radiosensitive organ to observe late effects of RT. We evaluated if concurrent administration of L has any impact on the development of radiation induced pulmonary fibrosis in rats (RIPF).
Fourty female Wistar-albino rats (WAR) were divided into 4 experimental groups (G). G1 (control) did not receive any treatment, G2 (RT) received RT to whole thoracic region, G3 (L) received L without RT, G4 (L+RT) received L with RT. A total dose of 30 Gy in 10 fractions was given to both lungs with an anterior field at 2 cm depth. L equivalent to 1500 mg/day, 60 kg adult dose, were calculated according to the mean weight of rats, orally administrated with a feeding tube twice daily including the weekends until WAR were sacrificed. WAR were anesthetized and sacrificed 16 weeks after RT which was shown to be a sufficient period for the development of RIPF in rats. Paraffin sections (5 µm thick) of lungs were stained with hematoxylin-eosin and Masson’s trichrome. A semiquantitative comparative analysis was performed among 4 groups by scoring the pulmonary injury between 0 and 4 according to the infiltration of inflammatory cells into the alveolar spaces, alveolar wall thickening and architectural deformation across the entire lung section. Each criterion had a possible score of 0 to 4, where 0 no injury; 1 minimal injury, 2 mild injury, 3 moderate injury, and 4 marked injury. The total lung injury score was calculated by summing the scores of these three parameters. Statistical analysis was performed.
Control group demonstrated normal pulmonary architecture. Lungs of the rats which received RT (G2) revealed inflammatory cell infiltration and increased amount of collagen fibers in the interstitial area leading to fibrosis, which thereby result in thickening of the alveolar septa and shrinkage of the size of the alveoli. RT damaged the lung architecture and G2 had a significantly lower lung histological injury score than did G1 (p<0.05). G3 showed minimal alveolar septal thickening and infiltration of inflammatory cells into the alveolar spaces which were not significantly different than G1. The histopathological findings in the group which L treatment was given together with RT (G4) was similar to those in G2 and was statistically significantly different compared to both the control group and G3 (p<0.05) (Figure 1).

This study shows that addition of Lapatinib to RT does not increase radiation induced pulmonary fibrosis in rats.

Fig. 1: Representative photomicrographs. A,B; thin alveolar walls. C,D; areas of intensivefibrosis and thickened alveolar walls. E; lapatinip alone had no adverse effect on thetissue structure. F; lapatinip+RT did not worsen the effects of RT on lung.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2237 Histopathological Changes of Rabbit Gingiva Following Application of Tricalcium Phosphate Cement

Kasim M.1, Yucel D.2, Uslu S.3, Arbak S.2, Biren S.1
1Marmara University, Faculty of Dentistry Department of Orthodontics Istanbul, Turkey, 2Acibadem University, School of Medicine, Department of Histology and Embryology, Istanbul, Turkey, 3Acibadem University, Vocational School of Pathology, Istanbul, Turkey
denyucel@gmail.com

Introduction: Application of miniscrew with bone cement is a promising method that may extend the limits of force application for orthodontic movement. Tricalcium Phosphate (TCP) is a kind of biodegradable material and used for bone replacement and augmentation. Tricalcium Phosphate cement has a potential to be used with orthodontic miniscrews. It will be in contact with bone also with the surrounding soft tissue during application with miniscrews. In this study, the early effects of the cement on the surrounding gingiva tissue was investigated by histological examinations of epithelium and connective tissue.
Materials and Methods: 8 male of New Zealand white rabbits were divided into two groups; as cement treated (n=7) and control (n=1) group. For cement treatment, a defect of 1.2 mm in diameter was created by punch at the level of lower left incisor gingiva, and the formed cavity was filled with TCP. The animals were sacrified after 24 hour of surgery, and the gingival tissues were dissected for histological examinations. Tissue samples were fixed in 10 % formalin solution for 24 hour before tissue processing. Following paraffin embedding, gingival tissue sections of 5-μm were stained with hematoxylin-eosin (H&E) for evaluation of epidermis and dermis, Gomori methenamine silver and Masson’s Trichrome staining techniques in order to investigate the dermal extracellular matrix related molecules, especially collagen fibers.
Results: Gingival epithelium of control group described a parakeratinized stratum corneum whereas other layers of epithelium, stratum granulosum, startum spinosum, as well as stratum basale reflected a normal morhology. In addition, the cells of stratum spinosum were polygonal in shape and the ratio of nucleus to cytoplasm was normal. In the epithelium of TCP- treated gingival tissues, the stratum corneum, stratum granulosum and stratum basale layers were quite similar to those of the control group. However, there was a decrease in the ratio of the nucleus to cytoplasm of the stratum spinosum cells related to an increase in the amount of cytoplasmic mass. Histopathological examination of the dermal extracellular matrix related molecules, particularly collagen fibers, did not reveal any significant difference in terms of organization of collagenous fiber network in both groups.
In conclusion, we could suggest that TCP treatment resulted in early histopathological changes in gingival epithelium whereas no prominent change had been encountered in collageneous fiber network of the gingival connective tissue. In the future studies, further histopathological changes and the self-repair mechanism of the gingival tissue upon removal of biodegradable TCP will be investigated.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2245 The Role of The Cholinergic Anti-Inflammatory Pathway in Endotoxemia Induced Multiple Organ Damage in Rats: Histological and Biochemical Investigations

Acikel Elmas M.1, Kolgazi M.2, Ozgur F.3, Beyazoglu O.3, Kocagoz A.3, Celik M.3, Yuksel M.4, Ercan F.1, Alican I.3
1Marmara University, School of Medicine, Dept. of Histology and Embryology, Istanbul, Turkey, 2Acibadem University, School of Medicine, Dept. of Physiology, Istanbul,Turkey, 3Marmara University, School of Medicine, Dept. of Physiology, Istanbul,Turkey, 4Marmara University, Vocational Health School, Medical Laboratory, Istanbul, Turkey
acikelmerve@gmail.com

Introduction: Endotoxemia is one of the important causes of death in clinics which trigger septic shocks and multiple organ damage. The cholinergic anti-inflammatory pathway is a physiological neuroimmune mechanism that regulates innate immune function and controls inflammation. The functional activity of this pathway can be modulated through its neuronal (efferent vagal neurons and higher brain structures) and non-neuronal (alpha7 nicotinic cholinergic (α7nACh) receptors on cytokine-producing cells) cholinergic components.

Aim: The aim of the study is to investigate the anti-inflammatory role of the cholinergic pathway in endotoxemia induced multiple organ damage and the interaction between pathway and nitric oxide synthase (NOS) and cyclooxygenase (COX) systems.

Materials and Methods: Endotoxemia was induced in male and female Sprague-Dawley rats (250-300 g; n=8/group) by intraperitoneal (ip) administration of 10mg/kg Escherichia coli (LPS; serotype 0111:B4) following a 16 hour starvation period. Control group was received physiological saline solution (1ml/kg; ip). The treatment groups were injected either nicotine (1mg/kg; ip), or nicotine + aminoguanidin (AG; 8mg/kg, ip) or nicotine + nimesulide (NIM; 10mg/kg, ip) for 3 days before LPS administration. At the 24th hour after LPS induction, blood, liver and kidney samples were collected. Samples were stored at -70 oC for the measurement of malondialdehyde (MDA), glutathione (GSH) levels, and myeloperoxidase (MPO) activity. Formation of reactive oxygen species in tissue samples was monitored by using chemiluminescence method. ALT, AST and BUN levels were measured in serum. In addition, the tissue samples were fixed with 10% neutral formaldehyde and processed for routine paraffin embedding. Hematoxylin and Eosin stained sections were evaluated histologically. Data were analyzed with Student’s t test and Mann Whitney-U test.

Results: Tissue damage scores, serum ALT, AST, BUN, MDA and MPO levels and chemiluminescence levels increased, GSH levels decreased (p˂0.05-0.001) in LPS group comparing to control group. Nicotine treatment ameliorated microscopic scores and biochemical parameters of inflammation in all tissues and serum (p˂0.05-0.01). Nicotine+AG and Nicotine+NIM treatments attenuated all tissue and serum inflammation parameters which increased with LPS induction except kidney GSH levels. While Nicotine+NIM treatment reduced microscopic damage in kidney, Nicotine+AG treatement reduced only liver damage.

Conclusion: The stimulation of cholinergic anti-inflammatory pathway with nicotine had beneficial effects on endotoxemia model in rats. iNOS or COX-2 inhibition did not changed the nicotin effects. In some cases, iNOS and COX-2 inhibition contributed positive nicotine effects.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2444 Therapeutic efficacy of PMAsh microcapsules on breast cancer cells

Colone M.1, Kaliappan S.2, Cavalieri F.2, Tortora M.2, Ponticelli G. S.1, Calcabrini A.1, Stringaro A.1
1Italian National Institute of Health, Rome, Italy, 2University of Tor Vergata, Rome, Italy
marisa.colone@iss.it

Various drug delivery systems such as nanoparticles, liposomes, microparticles and implants have been demonstrated to significantly enhance the preventive/therapeutic efficacy of many drugs by increasing their bioavailability and targetability.

Herein, we report the preparation of capsules from hydrogen-bonded polymer multilayers that are cross-linked through disulfide (S-S) bonds. These capsules are both stable at physiological pH and amenable to deconstruction under reducing conditions or via thiol-disulfide exchange, such as those occurring within cells. We employed a pair of polymers that form stable multilayers when alternately deposited at moderately acidic conditions, pH 4, poly(methacrylic acid) containing thiol moieties, PMASH. The resulting single-component PMA hydrogel capsules are colloidally stable in a range of conditions, including the presence of blood serum proteins. Furthermore, and of particular importance for biomedical applications, the capsules degrade in the presence of intracellular concentrations of a natural reducing agent, glutathione (1, 2).

In this study we provide PMAsh biocompatibility by MTT test on a cell line of human breast adenocarcinoma (SKBR3). These studies have shown that PMAsh are not toxic. To evaluate the potential of PMASH capsules as a carrier to deliver anticancer drugs, we incorporated doxorubicin (DOX) into the capsules. DOX has an intrinsic fluorescence spectrum (excitation at 480 nm, emission at 550-650 nm) that can be exploited to monitor localization of the drug. The amount of DOX loaded into the PMASH capsules ( 2 1012 molecules/caps) was determined by UV

Fig. 1: SKBR3 cell viability evaluation after incubation with PMAsh (25, 50, 75, 100 particles/cell) for 24, 48 h.

Fig. 2: CLSM observations show the effective internalization of PMAsh-DOX capsules inside the cells.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2581 Evaluation of duodenal mucosa with scanning electron microscopy and light microscopy after dose dependent isotretinoin treatment in young male rats

THOMAZINI B. F.1, DOLDER H.1
1Department of Structural and Functional Biology, State University of Campinas, Campinas-SP, Brazil
bruna.fth@gmail.com

Isotretinoin is chemically known as 13-cis-retinoic acid and is part of the broad group of compounds related to vitamin A. This therapy shows great efficiency in cases of severe acne and is related to the induction and control of epithelial differentiation and mucus secreting tissues. The isotretinoin pharmacological activity for this treatment is to block the secretion of sebaceous cells. The daily dose is calculated according to the patient's weight, and ranges from 0.5 to 2mg/kg/day for 16 to 35 weeks. The duodenum mucosa is made up of villi and crypts. The villi epithelium consists mostly of absorptive cells (enterocytes) and goblet cells, and arises from the crypt epithelium, which in turn contains some absorptive cells, goblet cells, enteroendocrine cells, Paneth cells and stem cells. The aim of this study is to investigate alterations in duodenal mucosa using two different dosages of isotretinoin, one of which is that suggested for humans. For this study, 20 young male Wistar rats were separated in four groups. The drug was diluted in soybean oil, and offered by gavage every day for 60 days. G1: control with water; G2: control with soybean oil; G3: 1mg/Kg of isotretinoin; G4: 5mg/Kg of isotretinoin. A volume of 2mL of solution was given to each animal. After the treatments, the animals were euthanized by application of xylazine and cetamine. Portions of the duodenum were collected and fixed with Karnovsky´s fixative. Part of these samples were treated following the usual protocol for light microscopy with Hematoxilin-Eosin staining technique. Other samples were treated following the usual protocol for analyses with scanning electron microscopy. After fixation, the samples were treated with osmium tetroxide. The next step was critical point drying, followed by gold sputter coating. The analysis of the both samples from the control groups (G1 and G2) showed a perfect mucosa, with the expected structure. The villi are in contact with the lumen and the crypts at the base of the villi. The connective tissue supporting the villi, and containing lymphatic vessels and blood vessels can be observed with no alterations. Just below the crypts, a connective tissue layer and a muscular layer were defined. This last structure has an internal circular muscle layer and an external longitudinal muscle layer. The villi and the crypts could be clearly defined. In the treated groups (G3 and G4), modifications were not found compared with the control group (Figure 1). This result suggests that the treatment proposed does not cause modifications in the general structure of the duodenum mucosa. Further evaluation is needed for more complete conclusions.

To CAPES for the support.

Fig. 1: Duodenal mucosa. a, b, e, f: Scanning Electron Microscopy.; c, d, g, h: Light Microscopy, Hematoxilin-Eosin staining. G1: control with water; G2: control with soybean oil; G3: 1mg/Kg; G4: 5mg/Kg. V: villi; C: crypt; T: connective tissue; M: muscular layer. Bar: 100µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2582 Duodenal mucosa structure observed in light microscopy under different staining techniques after dose dependent isotretinoin treatment in young male rats

THOMAZINI B. F.1, CANHASSI G. S.1, DOLDER H.1
1Department of Structural and Functional Biology, State University of Campinas
bruna.fth@gmail.com

Isotretinoin or 13-cis-retinoic acid therapy shows great efficiency in cases of severe acne and is related to the induction and control of epithelial differentiation and mucus secreting tissues. The isotretinoin is part of the broad group of compounds related to vitamin A and its pharmacological activity for the treatment is to reducing the activity of sebaceous glands. The suggested dosage for humans is 0.5 to 2mg/kg/day for 4 to 8 months. The substance is absorbed in the small intestine and the duodenum is the first segment of this organ. The duodenum structure is made up of villi and crypts. The villi epithelium consists mostly of absorptive cells (enterocytes) and goblet cells, and arises from the crypt epithelium, which in turn contains some absorptive cells, goblet cells, enteroendocrine cells, Paneth cells and stem cells. The aim of this study is to investigate alterations in duodenal mucosa using two different dosages of isotretinoin, one of which is that suggested for humans. For this study, 20 young male Wistar rats were separated in four groups. The drug was diluted in soybean oil, and offered by gavage every day for 60 days. G1: control with water; G2: control with soybean oil; G3: 1mg/Kg of isotretinoin; G4: 10mg/Kg of isotretinoin. A volume of 2mL of solution was given to each animal. After the treatments, the animals were euthanized by application of xylazine and cetamine. Portions of the duodenum were collected and fixed with Karnovsky´s fixative. The samples were treated following the usual protocol for analyses with light microscopy. To observe structure, Hematoxilin-Eosin staining was used, as well as the following cytochemical techniques: Reticulin, Masson's trichrome and Alcian Blue-PAS combination. The analysis of the samples showed a perfect mucosa, with the expected structure. Hematoxilin-Eosin stain showed villi and crypt structure with the villi in contact with the lumen and the crypts at the villi´s base. In the Masson's trichrome sample, the connective tissue can be observed supporting the villi, and containing lymphatic vessels and blood vessels. Just below the crypts, a connective tissue layer and a muscular layer were defined. With Reticulin samples, the reticulin fibers can be observed providing support for the structures. The Alcian Blue-PAS combination technique, demonstrated goblet cells in all of the absorptive tissue, more concentrated in the crypt in relation to the villi. All of these techniques showed that in the treated groups (G3 and G4), no alterations were found compared to the control groups (G1 and G2) (Figure 1). This result suggests that the treatment proposed does not cause modifications in the general structure of the duodenal mucosa. Further evaluation is needed for more complete conclusions.

To CAPES for the support.

Fig. 1: Duodenal mucosa structure using light microscopy. G1: control with water; G2: control with soybean oil; G3: 1mg/Kg; G4: 10mg/Kg. a: Hematoxilin-Eosin staining; b: Reticulin technique; c: Masson's trichrome; d: Alcian Blue-PAS combination. V: villi; C: crypt; M: muscular layer; *: connective tissue. Bar: 50µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2608 Effect of an adapted physical exercise on satellite cells from skeletal muscles of old mice: ex vivo and in vitro analyses at light and electron microscopy

Cisterna B.1, Giagnacovo M.2, Costanzo M.1, Zancanaro C.1, Pellicciari C.2, Malatesta M.1
1University of Verona, 2University of Pavia
barbara.cisterna@univr.it

During aging, a progressive loss of skeletal muscle mass and a concomitant decrease in muscle strength and endurance take place. This condition, termed sarcopenia, has important health-care and socioeconomic implications, since it contributes to frailty, disability and premature death (1). The mechanisms leading to sarcopenia are probably manifold and still incompletely elucidated, although the decline in muscle regeneration efficiency is thought to play a crucial role. Still in the absence of specific therapies, many studies have stressed the importance of physical exercise as an effective approach to prevent/limit the sarcopenic drive (2,3).
In this study, the effects of an adapted exercise on the number and myogenic properties of satellite cells (SCs) were investigated in quadriceps femoris and gastrocnemius muscles of old male mice (28 months). Both muscles contain a high proportion (about 90%) of type II fibers, which are specifically affected by sarcopenia (4). We compared old exercised mice (OE) versus old sedentary mice (OS), using adult sedentary mice (12 months, A) as a control: SCs were identified (Figs. 1) and quantified ex vivo; in addition, the proliferation and differentiation potential of SC-derived myoblasts from the same groups of mice was studied in vitro (Figs. 2 and 3). Ultrastructural morphology and immunocytochemical techniques at light and electron microscopy were used with special attention to some molecular markers of SC activation, and to some protein factors involved in RNA transcription and splicing.
Our results demonstrated that: 1) physical exercise induces an increase in the total number and in the activated fraction of SCs compared with sedentary old sample; 2) myoblasts from exercised muscles show morphological features and nuclear activity quite similar to myoblasts from adult subjects, whereas myoblasts from non-exercised muscles exhibit structural and functional alterations suggestive of a reduced metabolic activity; 3) myotubes differentiated from myoblasts of exercised muscles resemble the myotubes from adult myoblasts, whereas myotubes from non-exercised muscles show marked morphological alterations.
The present study provides convincing evidence that physical exercise not only induces numerical increase and activation of SCs in old animals but can also improve their capability to differentiate into structurally and functionally correct myotubes. Adapted physical exercise may actually represent a non-pharmacological approach to stimulate SCs in the attempt to enhance muscle quality even at very advanced age.
1. Ryall et al. Biogerontol 9:213, 2008
2. Zancanaro et al. Eur J Histochem 51:305, 2007
3. Bautmans et al. Acta Clin Belg 64:303, 2009
4. Larsson et al. Acta Physiol Scand 103:31, 1978

Acknowledgements - MC is a PhD student of the Doctoral Program “Multimodal Imaging in Biomedicine” (University of Verona).

Fig. 1: Fluorescence microscopy: the nucleus of a SC after immunolabelling for Pax7 (a SC molecular marker) (a) and MyoD (a marker of SC activation) (b); nuclear DNA was stained with Hoechst 33258 (c). Bar, 10 µm

Fig. 2: Immunoelectron microscopy: nuclei of SC-derived myoblasts from A, OS ad OE mice after labelling for activated RNA-polymerase II. Bars, 500 nm. Quantitative evaluation demonstrates similar values (mean±SE) in A and OE mice, while OS mice show significantly lower values (Mann Whitney test).

Fig. 3: Phase contrast microscopy: the myotubes differentiated from myoblasts of OS mice show irregular shape and clustered nuclei, whereas the myotubes of OE mice resemble those of adults (A). Bar, 10 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2726 Glioblastoma cancer stem cells: organization in 3D structures

Bozzuto G.1,2, Narayanan A.3, Galli R.3, Molinari A.1
1Istituto Superiore di Sanità, Rome, Italy, 2National Council Research, Rome, Italy, 3S. Raffaele Scientific Istitute, Milan, Italy
giuseppina.bozzuto@iss.it

Particular poor prognosis for patients in advanced stages of solid tumours have opened the possibility that tumours include a population of cells responsible for the initiation of tumour development, growth and its ability to metastasize and reoccur. Because these cells share some similarities with stem cells, they are referred to as cancer stem cells (CSCs). CSCs possess inherent properties of self-renewal and differentiation, along with expressing certain genes related to a mesenchymal phenotype. These features favour the promotion of tumour recurrence and metastasis in cancer patients. Treatment of malignant gliomas represents one of the most formidable challenges in oncology. Despite treatment with surgery, radiation therapy, and chemotherapy, prognosis remains poor, particularly for GBM. Quiescent, or slowly proliferating, CSCs may contribute to chemotherapy resistance as this therapy acts mainly on rapidly cycling cell populations. Issues regarding CSC movement are important in neurosphere biology as cell–cell or cell–environment interactions may have significant impacts on CSC differentiation and contribute to the heterogeneity of the neurosphere. Despite the growing body of literature data on the biology of brain tumor stem cells, floating CSC-derived neurospheres have not been fully characterized from a morphological and ultrastructural point of view. Thus, to better understand the mechanisms underlying GBM CSC biology, the behavior of the CSCs was followed in living conditions by time-lapse videomicroscopy and by scanning electron microscopy (SEM). Several CSC cell lines isolated from glioblastoma patients were analyzed. The invasive potential was assessed by transwell chamber invasion assay and was compared with SEM observations carried out after 3 and 20 hours after deposition on a MatrigelÔ film. After 3 hours from seeding, CSC cells invaded the film organizing long cords constituted by bipolar elongated cells (Figure 1). Moreover, satellite cells with peculiar surface morphology (Figure 2) were present in all CSC lines analyzed. In particular experimental condition, CSCs formed characteristic units composed by a flat epithelioid cell housing other CSCs with globular morphology (pseudo-niche) (Figure 3). After 20 hours, CSC agglomerates displayed the surface completely covered by the MatrigelÔ film (Figure 4), only satellite cells lined outside the structure. The results obtained in this study indicated (i) the ability of CSC populations to self organize in ordered structures; (ii) the presence of satellite cells whose location suggest a specific role still to be clarified; (iii) the presence of pseudo-niche like structures, i.e. the pluripotentiality of the population of glioblastoma isolated stem cells.

Fig. 1: Bipolar elongated CSC cells invaded the film organizing cords.

Fig. 2: Satellite cell.

Fig. 3: Pseudo-niche constituted by flat epithelioid cell housing CSCs with globular morphology.

Fig. 4: CSC agglomerates covered by Matrigel™ film.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2740 Effects of Turkish propolis on endotoxin induced uveitis on rats

Erturkuner S. P.1, Yaprak Sarac E.1, Gocmez S. S.2, Ekmekci H.3, Ozturk Z. B.3, Sever O.4, Seckin I.1, Keskinbora K.5
1Istanbul University, Cerrahpasa Faculty of Medicine, Histology and Embryology Dept, 2Namık Kemal University, Faculty of Medicine, Pharmacology Dept, 3Istanbul University, Cerrahpasa Faculty of Medicine, Biochemistry Dept, 4Namık Kemal University, Faculty of Medicine, Ophtalmology Dept, 5Bahcesehir University, School of Medicine, Ophtalmology Dept
drpelin@yahoo.com

Uveitis is a chronic inflammatory eye disease which may be accompanied by some systemic diseases, often considered as idiopathic. LPS-induced inflammation is an experimental animal models of acute uveitis. Propolis is a natural substance collected by honeybees from buds and exudates of certain trees which has antioxidant, antibacterial, antiviral and anti-inflammatory effects [1,2]. The aim of this study is to investigate the effects of propolis on endotoxin induced uveitis (EIU) by immunohistochemical, ultrastructural and biochemical methods.

24 male Wistar albino rats were divided into four groups (n=6). A single dose of LPS (150 µg/kg/ip) was administered to 12 animals intraperitonally (ip). Water extract of propolis (WEP) from Adapazarı region of Turkey (50mg/kg/ip) was administered to 6 of them after LPS injection. Control group was injected with saline ip. After 24 hours, the aqueous humor of both eyes of animals were collected under anesthesia for biochemical analysis of inflammatory markers, namely TNF-α ve HIF-1α levels. The right eyeballs were fixed with formalin and paraffin-embedded for immunohistochemical staining of Nf-kb p65 and left eyeballs were fixed with 4% glutaraldehyde and araldite-embedded for ultrastructural analysis.

Immunoreactivity against anti-Nf-κB p65 at corpus ciliare was significantly decreased in EIU group treated with Turkish propolis extract (Figure1-2). Moreover, ELISA analysis of HIF-1α and TNF-α levels in aqueous humour was significantly decreased in same group (Figure 3). Ultrastructural analysis of retinal regions showed that EIU group treated with Turkish propolis extract has less vacuoles and mitochondria degeneration at retinal pigment epithelium (RPE) than EIU group (Figure 4). The intercellular spaces of the inner nuclear layer and outer limiting membrane were compatible with control group; no polymorphonuclear cells were seen in both intravascular and extravascular spaces and no stasis symptoms were detected at the capillary lumen (Figure 4).

Our report is the first study that established anti-inflammatory effect of Adapazarı/Turkish propolis on LPS-induced uveitis rat model. As a marker of inflammation, NF-κB reactivity was increased significantly in corpus ciliare and coroid of eyes of LPS-induced rats, accompanied by TNF-α induction of HIF-1α, whereas the levels were markedly decreased in Propolis treated and LPS-induced group. We think that Adapazarı/Turkish propolis might be a new class of bioavailable dietary supplement for the treatment of inflammatory opthalmic diseases such as uveitis.

References

1- Jin XH, et al. Invest ophthalmol Vis Sci 2006:47,2562-2568.

2- Kumazawa S, et al. Food Chemistry 84 2004:329–339.

Fig. 1: Immunohistochemical staining of Nf-κB p65 in rats. A. Negative control, B. Control group injected with saline, C. 50mg/kg/ip Turkish propolis injected group D. 150 µg/kg/ip LPS group without propolis-treatment, E. LPS group treated with propolis.

Fig. 2: Statistical analysis of H-SCORE results of Nf-κB immunostainings. One-way ANOVA was performed with Tukey-Kramer Multiple Comparisons Test between all groups and *P < 0.0001 is considered extremely significant.

Fig. 3: Statistical analysis of biochemical assay of HIF-1α and TNF-α in aqueous humor of rats. All groups are compared with Tukey-Kramer Multiple Comparisons Test. *P < 0.05 is considered significant compared to control group. #P < 0.01 is considered significant compared to Propolis group.

Fig. 4: Electron micrographs of retina layers. Control group: 1) x3000, 2) x6000,3) x25000. LPS group: 4) x6000, 5) x5000, 6) x5000. LPS+Propolis group: 7) x5000, 8) x12000. Propolis: 9) x7500,10) x6000.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2777 Histopathological and Mechanical Evaluation of Rabbit Bone Tissue After Application of Bioabsorbable Tricalcium Phosphate Bone Cement Treated Miniscrew

Kasim M.1, Ates M.1, Uslu S.2, Yucel D.3, Arbak S.3, Biren S.1
1Marmara University, Faculty of Dentistry Department of Orthodontics Istanbul, Turkey, 2Acibadem University, Vocational School of Pathology, Istanbul, Turkey , 3Acibadem University, School of Medicine, Department of Histology and Embryology, Istanbul, Turkey
musiuslu@gmail.com

Introduction: Tricalcium phosphate (TCP) cement is a bioabsorbable material used in clinical applications as bone substitute material. In orthodontic treatments the presence of bone cement over miniscrew is an alternative approach to fix the screw and enhance bone healing. In this study, the effect of TCP cement treatment over miniscrew was investigated both on different bone healing stages and primary stability of orthodontic miniscrew.
Method: 36 male of New Zealand white rabbits were used and 4 miniscrews were implanted on each tibia of both legs. Animals were divided into four groups (in each group n=9) based on post-operation periods, and the cement treated (a) and untreated (b) miniscrews were implanted on right and left tibia in all groups, respectively. Tibias were dissected after 24h (Group 1), 2 weeks (Group 2), 4 weeks (Group 3), and 8 weeks (Group 4) of implantation. Samples were decalcified for 14 days after fixation. Sections of samples were stained with hematoxylin-eosin to evaluate histomorphometrical analysis based on thickness, number and areas of trabecules by image analysis system. Biomechanical stability was evaluated by measuring maximum torque, maximum pull-out load and tensile strength. Specimens were loaded at a cross-head speed of 1 mm/min using a universal testing machine. The force needed to remove the miniscrew was measured using a digital torque by DID-05-E Digital Torque Screwdriver.
Results: Histopathological evaluations revealed any inflammation, foreign substance reaction and necrosis in all groups. According to histomorphometrical analysis based on thickness, number and areas of trabecules, the measurements of Group 2, 3 and 4 for untreated and cement-treated tissues were almost similar. In Group 1 vascularization was prominently higher in the untreated tissues compared to the cement-treated tissues. Specimens were loaded at a cross-head speed of 1 mm/min using a universal testing machine to evaluate the pull-out strength and shear strength. The force needed to remove the miniscrew was measured by DID-05-E Digital Torque Screwdriver. A statistically significant difference was found between the pull-out strengths of the groups (p<0.01). The pull-out strengths of the miniscrews placed with TCP were significantly greater than the untreated groups. In shear tests, there was no statistically significant difference among the groups.
In conclusion, the usage of bioabsorbable cement on the miniscrew could be recommended to be used in orthodontic treatments. Eventhough the bone healing stages were similar among all groups according to histopathological evaluation, the mechanical test results revealed that the treatment of cement with miniscrew enhances the stability of the implant.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2782 Platelet Ultrastructure of Complicated Chron’s-Like Colitis Associated With Hermansky-Pudlak Syndrome

Caliskan T.1, Eskazan A. E.2, Erturkuner S. P.3, Isıldar B.3, Erzin Y. Z.1, Baslar Z.2, Soysal T.2, Tasyurekli M.3, Celik A. F.1
1Istanbul University, Cerrahpasa Faculty of Medicine, Gastroenterology Dept, 2Istanbul University, Cerrahpasa Faculty of Medicine, Hematology Dept, 3Istanbul University, Cerrahpasa Faculty of Medicine, Histology and Embryology Dept
basakisildar@gmail.com

Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder consisting of a triad of oculocutaneous albinism, platelet dysfunction and accumulation of ceroidlike depositions in tissues[1]. We report five patients with HPS which have various clinical symptoms associated with gastrointestinal complications related to chronic granulomatous colitis.
Case1:28-year old female patient whose HPS was diagnosed in 2001.Performed colonscopy showed mucosal ulcers, pseudopolyps and biopsy results showed granulomatous colitis.
Case2:28-year-old male patient whose HPS was diagnosed in 2009.Performed colonoscopy showed edema from anal canal up to the cecum, biopsy results showed granulomatous colitis.
Case3:28-year-old male patient whose HPS was diagnosed in 2011. Colonoscopy doesn’t perform yet, but clinical symptoms were compatible with Chron’s Disease.
Case4:32-year-old female patient whose HPS diagnosed in 2013. Performed colonoscopy showed compatible results with ulcerative colitis with typical distribution of inflammatory regions, biopsy results showed inflammatory bowel disease.
Case5:33-year-old female patient whose HPS diagnosed in 2013 is the sister of case1.Colonoscopy doesn’t performed yet. There are no signs of inflammatory bowel disease.
The bloods which were obtained from the patients, separated citrated platelet-rich plasma (C-PRP), and fixed samples of platelets for examination at the electron microscope were modified from Hayat M E. et al..
Each case of platelets have increased spreaded glycogen and plenty of dense bodies. Eventhough in case 4 glycogen stores and alpha granules were seen. In case 3 and case 5, the platelets size were larger than the others. Each case of platelets were seen irregular in shape.
The platelets ultrastructural examinations will keep going by applying various aggregating agents (fibrinogen, collogen, adenosine diphosphate, epinephrine) to the C-PRP of the patients which are treating with various medicines.
References
1.Erzin Y, Cosgun S, Dobrucalı A, Tasyurekli M, Erdamar S, Tucer M. Complicated granulomatous colitis in a patient with Hermansky-Pudlak syndrome, successfully treated with infliximab.Acta Gastro-Enterologica Belgica, Vol.LXIX,April-June 2006.
2.Hayat M E.Principles and Technics of Electron Microscopy,Biological Application.Vol.1 Von Nostrand,Reinhold Company Melborn 1970.

Fig. 1: DB (dense bodies), AG (alpha granules), grey arrow (glycogen), G (glycogen store), OCS (open canalicular system), black arrows: a canaliculus associated with outside.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2961 Effects of the recreational use of PDE5 inhibitors on the corpus cavernosum of young healthy rats

Simsek A.1, Tugcu V.3, Erturkuner S. P.2, Alkan F.2, Ozbek E.3, Tasci A. I.1
1Bakırkoy Dr. Sadi Konuk Training and Research Hospital, Department of Urology Istanbul, Turkey, 2Cerrahpasa Medical Faculty, Department of Histology and Embryology, Istanbul, Turkey, 3Okmeydani Training and Research Hospital, Department of Urology, Istanbul, Turkey
drpelin@yahoo.com

Objective:Phosphodiesterase type 5 inhibitors (PDE5) are widely used for the treatment of erectile dysfunction. However, these drugs have recently become popular among men without erectile dysfunction as a means of enhancing sexual performance and improving sexual desire. The aim of this study was to investigate the histopathological and ultrastructural effects of PDE5 inhibitors on the corpus cavernosum in young, healthy male rats.

Methods:24 four-month-old male Wistar albino rats were divided into four groups (n=6). The first group was the control group. Sildenafil citrate, vardenafil hydrochloride and tadalafil was administered respectively the second, third and fourth groups. All drugs were administered for 4 weeks. Penile tissues were fixed with 4% glutaraldehite and araldite-embedded for ultrastructural analysis and fixed with formaldehite and paraffin-embedded for collagen measurements.

Results:Electron microscopic analysis indicated that the number of active fibroblasts and macrophages and the synthesis of new collagen fibers increased in treated rats. Cavernous tissue collagen levels were significantly higher in the sildenafil citrate, vardenafil hydrochloride, and tadalafil treated groups than the controls (46.16±4.9, 42.06±2.4, 41.07±2.4, and 29.20±3.3, respectively) (p<0.001). Young men who use these drugs to enhance performance in the absence of erectile dysfunction may experience irreversible damage to the corpus cavernosum.

Conclusion: However, more studies are needed to evaluate the molecular mechanisms by which PDE5 inhibitors affect the corpus cavernosum.

Fig. 1: Ultrastructure of corpus cavernosum A.Control:Endothelial basement membrane.X5000 B.Sildenafil citrate:Collagen bundles, fibroblasts, RER.X40.000 C.Vardenafil hydrocloride:Collagen.X6000 D.Tadalafil: Fibroblasts,RER.X12.000
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-2973 A SEM characterization of the perimplant oral mucosa adhering to dental abutments

Tessarolo F.1,2, Tomasi C.3, Piccoli F.4, Caola I.4, Nollo G.1,2
1Bruno Kessler Foundation, Trento, Italy, 2University of Trento, Trento, Italy, 3University of Gothenburg, Goteborg, Sweden, 4APSS, Provincial Health Trust, Trento, Italy
tessarolo.f@gmail.com

The establishment of an efficient soft tissue seal around a dental implant is a prerequisite for implant success. The formation of the tissue barrier is the result of a wound healing process that has been largely documented in animal models (Berglundh et al., 2007). However, data on human oral mucosa are scarce. Recently, we proposed a novel human model to evaluate the morphogenesis of the mucosal attachment to implants (Tomasi et al., 2013 ). In this study we aimed at characterizing and quantifying tissue adhering to the implant surface by SEM.
Patient informed consent was obtained and after implant installation, a custom-designed experimental abutment was connected to the implant. Soft tissue biopsies and titanium abutment were collected after 12 weeks of tissue healing by the use of a circular cutting device. The whole system was placed in 4% formalin and transferred to a 70% ethanol solution after 48 h. After fixation, the tissue was separated from the experimental abutment using the previously described “fracture technique” (Berglundh et al., 2007).
A total number of 6 experimental abutments were processed for high vacuum SEM to qualitatively and quantitatively reveal biological remnants. Samples were washed twice in phosphate buffer, dehydrated, dried, and gold sputtered. BSE images were acquired at a 15 KeV showing compositional contrast between titanium surface and biologic debris. One set of low magnification (80x) high resolution (2576x1936 pixels) images was acquired per each of the four abutment facets (Figure 1). Each set was digitally processed creating a single high resolution 1.5 mm wide image strip per abutment side. To quantify different morphologies within the biological remnants, a 150x150 µm squared grid was superimposed to the image strips. The observation of each single cross point allowed to quantify the percent of the following micro-morphological features: a) plaque, b) uncovered titanium, c) epithelium-like tissue, d) connective-like tissue (Figure 2).
A minimal amount of plaque was found in the coronal side below the mucosal margin, followed by a uncovered titanium area where no bacteria were present. More apically, single or few layers of nucleated cells formed the epithelium-like tissue. In the deepest area, a thicker connective-like tissue showing fibrous morphology was present. Quantitative results showed a mean (SD) amount of 2%(2%), 52%(27%), 20%(13%), 26%(18%) for plaque, titanium, epithelium-like and connective-like tissue respectively, thus showing a mature healed mucosa in strict contact with the abutment surface (Figure 3). SEM investigation and quantification of tissue adhering to the healing abutment can complement histological findings in describing the healing process of human oral mucosa.

Supported by Dentsply Implants IH, Molndal, Sweden.

Fig. 1: Image acquisition and processing for features quantification on the experimental abutment surface. a) Set of images acquired by SEM at 80x magnification covering the whole surface of the abutment facet. b) High resolution strip obtained from image montage. Identification of the mucosal margin and supra and sub mucosa areas.

Fig. 2: Representative images for the four micromorphological features found on abutment surface: a) plaque, b) titanium, c) epithelium-like, d) connective like. SEM, original magnification 250x.

Fig. 3: High magnification images of the abutment surface covered by epithelium-like (a) and connective-like (b) tissue. Close interaction of tissue cells with titanium surface is indicated by white arrows. SEM, original magnification 4000x.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3041 Impact of Cross-Platform Telemicroscopy for Ultrastructural Pathological Diagnosis and Research

Schroeder J. A.1, Siegmund H.1, Grafe C.1, Hofstaedter F.1
1Central EM-Lab, Department of Pathology, University Regensburg, Germany
josef.schroeder@ukr.de

Transmission electron microscopy (TEM) is still used as an ancillary tool, quality control method or gold standard to complement, support or confirm the results of specific histopathological diagnoses. For “second opinion” consultation of difficult cases we used over years an interactive remote TEM operation via Internet with a server-client architecture (“Ultrastructural Telepathology”). This contribution focuses on the presentation of an alternative web-based approach.

The routine sample processing is performed by computer controlled tissue processors (LYNX/Leica); microwave-assisted processing allows a “same-day diagnosis”. Resin sections are examined in the LEO-912AB/Zeiss TEM equipped with a side-entry digital camera (TRS; 2k x2k pixel) image acquisition system (iTEM/OSIS, Muenster). We applied for remote camera operation and online image sharing with external experts the IT-software program called TeamViewer (www.teamviewer.com) running on a WIN7 server.

The TeamViewer software allow cross platform (Windows, Mac, Linus, iOS, Android) global connections with other computers (even through firewalls and routers). It was used in the “Meeting Mode” for online sharing of the TEM images captured by the microscope camera [Fig. 1], in the “Remote Mode” to remote control of the CCD-camera and the TEM using the GUI of the iTEM software from external computers via Internet. We experienced a fast and high quality image and file transfer to the remote expert, the remote camera and microscope operation was performed with negligible time delay (1-3sec.) depending on the available bandwidth and daytime of the link.

Additional discussion and communications tools like annotations, object measurement, databank access combined with optional parallel live video (webcams) and voice transfer of the consultation participants increases significantly the telepresence cooperation and networking [Fig. 2].

Our centralized EM unit is integrated into the diagnostic service of the Pathology Department, external responders, and research institutions. The received samples cover a broad spectrum of materials and diseases, including rapid virus detection in skin lesions or in urine of kidney TXT-patients (DD: acute rejection/polyoma), a number of neoplastic and especially non-neoplastic conditions of the kidney, muscle, nervous system, skin, cilia defects, storage diseases, liver biopsies, respiratory diseases, toxic lesions, microsporidia, opportunistic infections, and more.

The necessary expert consultations in a number of difficult diagnoses of some above mentioned samples showed good performance saving time and money. This TeamViewer based, cross-platform mediated telepathologic networking, opens new ways for remote pathological EM diagnosis, research, and teleteaching.

Fig. 1: The Internet connected TEM at the University Regensburg. On the right monitor note the TeamViewer panel, the iTEM camera and EM control software as well as the patient’s databank; left the currently consulted sample (detail of a human liver sample, abnormal mitochondria).

Fig. 2: Monitor display at the remote expert site. Note right the TeamViewer control panel with webcam images of the consulting partners (telepresence), left the transmitted EM image as seen in Fig.1.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3067 Inhibition of Microtubule Polymerization by Estradiol Dimers: Direct Targeting or Mediated Action?

Darmostuk M.1, Jurasek M.2, Drasar P.2, Ruml T.1
1Department of Biochemistry and Microbiology, Institute of Chemical Technology, CZ-166 28 Prague, Czech Republic, 2Department of Chemistry of Natural Compounds, Institute of Chemical Technology, CZ-166 28 Prague, Czech Republic
darmosta@vscht.cz

Cancer cells are known for their uncontrolled proliferation due to the defects of the cell cycle checkpoints. Microtubules are considered as one of the best targets for anticancer drugs because of their principal role in cell division. Recently, it was shown that various estradiol derivatives target microtubules of cancer cells and inhibit their proliferation. One of such derivatives, 2-methoxyestradiol, is already registered drug by Entremed Inc. and is under clinical trials [1]. However, this endogenously present derivative is prone to rapid degradation by gastrointestinal and liver enzymes. To increase the bioavailability of potential drugs, dimers of different estradiol and testosterone derivatives were synthesized by our group. We studied their cytotoxicity, effect on cell cycle, apoptosis and microtubules assembly in vitro. Then the most promising compounds were conjugated with fluorescent dye and there localization was studied by spinning-disc confocal microscopy. Our studies revealed, that such compounds do not interact directly with microtubules, preferentially localizing in endoplasmic reticulum. Instead, obtained results suggest that tested estradiol derivatives affect microtubules polymerization by other mechanism than direct binding. The mechanism is the subject of our ongoing study.  

1.  Bruce JY, Eickhoff J, Pili R, Logan T, Carducci M, Arnott J, Treston A, Wilding G, Liu G. A phase II study of 2-methoxyestradiol nanocrystal colloidal dispersion alone and in combination with sunitinib malate in patients with metastatic renal cell carcinoma progressing on sunitinib malate. Invest New Drugs. 2012 Apr; 30(2):794-802. 

Financial support from specific university research (MSMT No 20/2014)

Fig. 1: Localization of estradiol dimer conjugated with BODIPY (green) in HeLa cells after 4 h of incubation. Microtubules were visualized by antibodies against alpha tubulin (red), nuclei were stained by DAPI (blue).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3087 Effects of pyridoxine administration on pancreatic islet cells in streptozotocin-induced diabetic rats

Kaya Dağıstanlı F.1, Yılmazer S.1, Tunçdemir M.1, Hatemi H.2
1Istanbul University Cerrahpasa Medical Faculty, Medical Biology Department, Istanbul, Turkey, 2Istanbul University Cerrahpasa Medical Faculty, Internal Medicine Department, Istanbul, Turkeyl
drselmayilmazer@gmail.com

In this study our aim was to investigate the effects of pyridoxine which has neurotrophic effects and is known to be deficient in diabetes mellitus on pancreatic islet cells of streptozotocin (STZ)- induced diabetic rats.
Adult female Wistar rats (n=24) each weighing 200-250 g. were used. The first group was the control group. The second and third groups were injected single dose of streptozotocin (i.p, 50 mg/kg). Second group was not given any treatment. The third group received, 5 mg/kg/day prydoxine for a period of one month. The pancreatic tissue sections were immunostained with insulin, glucagon, somatostatin, pdx-1, PCNA and synaptophysin antibodies. Body weight and blood glucose levels of the animals in all groups were measured. All values were analyzed with statistical methods.


Blood glucose levels of STZ+Pyridoxine groups, were significantly decreased (p<0,001) when compared with the untreated STZ group. The number of insulin, pdx1 and PCNA immunopositive cells within the islet was detected to be higher in the STZ+ Pyridoxine group than the STZ group (p<0.001). In the treated and untreated STZ diabetic groups number of glucagon and somatostatin immunopositive cells significantly increased (p<0.001) compared to the control group. The glucagon and somatostatin cells were centrally localized in the islets of STZ group, whereas their localisation in the STZ+Pyridoxine group were similar to the control group. Synaptophysin immuno reactivity was detected in all of the endocrine cells of islets in all groups.


We concluded that pyridoxine induced beta cell proliferation.This effect might depend on the neuronal like properties of pancreatic B cells.

1. Tamai H : Nippon Rinsho 57: 2362-2365 ,1999
2. Victor M , Adams RD :Am. J Clin Nutr 4: 346-355 ,1996
3. Nawale RB et al : Indian J. Biochem. Biophys. : 43 (337-44), 2006

Fig. 1: Statistical evaluation of the immunopositive cell numbers in pancreatic islets.a,,d,f,g,h,i,*p<0,001, hp<0,01 and c,gp<0,05; compared to control group, b,fp<0,001 and bp<0,01 ; compared to STZ+Pyridoxine group.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3204 The effects on apoptotic and autophagic cell deaths of Colchicum baytopirum CD Brickell extract in HeLa cells

Ozturk M.1, Dagdeviren O.2, Arıcan G.2, Kayacan S.1, Tuncdemir M.1, Sutlupınar N.3
1Istanbul University Cerrahpasa Medicine Faculty Medical Biology Department 1, Istanbul University Science Faculty Biology Department 2, Istanbul University Pharmacy Faculty Pharmacognosy Department 3
mozturksezgin@gmail.com

Natural products play an important role in investigation of novel anti-cancer agents and the development of more effective drugs. In elemination of cancer cells, the effective treatment models are suggested such as the stimulation of both apoptotic and autophagic cell death types in the same time by using different agents (1). Colchicine is a natural product which is obtained from plants of the genus Colchicum, a member of Colchicacea family (2) . We studied with Colchicum baytopiorum CD Brickell (Cb) which is an endemic plant species of Antalya-Turkey (3). It contains colchicine and its derivatives including demecolcine, 2-demethyldemecolcine, 3-demetylcolcine and cornigerin. The aim was to determine the role in both apoptotic and autophagic cell death pathways of the Cb extract on HeLa cell line.

The cytotoxic effect of Cb plant extract (0.1mg/ml) on HeLa cells was determined by using MTT assay. Cell viability was measured by trypan blue. To identify its molecular targets, the expression of genes which are involved in apoptosis and/or autophagic cell death (Bif and BNIP3 for both apoptotic and autophagic cell death; Atg12, Atg5 and DAPk for autophagic cell death; Bcl-xL, Bad, Puma, Noxa, Fas, Akt, TNFR1 and caspase-3,-8,-9 for the apoptotic cell death) were analyzed by qRT-PCR. Immunocytochemical analysis was performed by using active caspase-3 and t-Bid antibodies, then evaluated semiquantitatively.

We observed that there is a significant difference by means of cell cytotoxicity values between the control and the Cb extract treatment groups after an incubation period of 48 hours (p<0.001). According to qRT-PCR results, the expression levels of Bif(2,5), BNIP3(5,4), Atg12(5,7), Atg5(14,3), DAPk(2,3),Beclin-1(31), Bcl-xL(3,1), Bad(2,2), Puma(2,5), Noxa(4,2), Fas(2,4), Akt(2,6), TNFR1(25,1) and Caspase-3(5,7) ,-8(2,1) ,-9(2,3)genes were significantly increased(as fold) after the extract treatment. Active caspase-3 and t-Bid immunopositive cells were detected higher number in the Cb extract treated group compare to the control group.

This study shows that the treated dose of the Cb extract induces the crosstalk mechanisms between apoptotic and autophagic cell deathin HeLa cells, as well as upregulates some of genes in both of cell deaths. We suggest that this endemic plant extract seems to be a promising new therapeutic approach in cancer.

1- Zhou J, et al. Vitexin 6, a novel lignan, induces autophagy and apoptosis by activating the Jun N-terminal kinase pathway. Anticancer Drugs (2013) 24(9):928-36
2- Le Hello C. The pharmacology and therapeutic aspects of colchicine. Alkaloids (2000) 53:288-352
3- Akan H, Eker I. Check-list of the genus Colchicum in the flora of Turkey. Turk J Bot (2005) 29: 327-31

This study was supported by Istanbul University Research Fund Project: 17492

Fig. 1: Immunohistochemistry of t-bid (a,b) and active caspase-3 (c,d). (C:Control group, Cb:Colchicum baytopirium group)

Fig. 2: Cytotoxic activity (MTT) of Cb extract throughout 48h on HeLa cell cultures.*p<0.001 compared to control group. (C:Control group, Cb:Colchicum baytopirium group)

Fig. 3: Analysis of distribution of t-Bid and active caspase-3 expression in HeLa cells.*p<0.001 compared to control group. (C:Control group, Cb:Colchicum baytopirium group)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3226 Preparation and Characterization of Guar Gum Nanoparticles for Colonic Drug Delivery

Tanwar U. K.1, Randhawa G. S.1, Pruthi V.1
1Indian Institute of Technology Roorkee, India
umeshdbt@iitr.ac.in

Despite technological challenges polymeric nanoparticles have shown great promise for the development of drug administration and are now viewed as a spy to check cellular machinery so as to combat various dreadful life threatening diseases. Apart from their sub cellular size, biocompatibility with tissue and cells these polymeric nanoparticles hold good promise as an efficient drug deliverers in a controlled and sustained release manner. Guar gum, used in the present investigation, is a naturally abundant non-ionic hydrophilic polysaccharide involving low cost in its processing. Guar gum has emerged as a potential candidate for the same due to its unique drug release retarding property and susceptibility to microbial degradation in the large intestine. In the present study, our focus was on development of a new pharmaceutical formulation for colon targeted drug delivery using guar gum nanoparticles. The drug loaded guar gum nanoparticles were prepared by nano-precipitation method. 5-Fluorouracil, an anticancer agent, was used as a model drug in this study. The nanoparticles were characterized using Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The nanoparticles were found to be 50-100 nm in size and spherical in shape. The uniformity distribution was checked by the Zeta-sizer. In-vitro drug release pattern was studied using HPLC. The results obtained using drug loaded guar gum nanoparticles were found to be quite encouraging for development of new colon targeted drug delivery system. Thus, data obtained could be exploited to reduce the systemic side effects and provide effective and safe therapy in colorectal cancer treatment.

Keywords: Nanoparticles, Guar gum, Drug delivery, 5-Fluorouracil, Colorectal cancer

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3320 Effects of nicotinamide on differantiation of exocrine cell into beta cell in neonatal STZ diabetic rats

Kaya Dagistanli F.1, Ozturk M.1
1Medical Biology Department, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey
fkaya@istanbul.edu.tr

The pancreas develops from Pdx1 expressing progenitors that emerge from the foregut endoderm to form ventral and dorsal buds. Pdx-1 is necessary for pancreatic development and beta cell maturation. The formation of the pancreas and its subsequent differentiation to the exocrine and endocrine cell types results from the ordered activation of a large number of genes (1). Ngn3 is an important regulator of pancreatic endocrine cell formation. Notch signaling regulates ngn3 gene expression negatively (2). Nicotinamide (NA) is a precursor of nicotinamide adenine dinucleotide that protects beta cells from several toxic agents. It is known to increase the mitotic index of beta cells after pancreatectomy, and is a potent inducer of endocrine differentiation of human fetal pancreatic cells in vitro (3,4). We investigated the relationship in between expression of notch1, jagged1, ngn3 and pdx1 with pancreatic beta cell regeneration and/or differentiation of NA treated neonatal diabetic rats.

Three groups were performed. The first group was the control group. The second group was STZ diabetic (100 mg/kg i.p on the second day after birth; n2-STZ). The third group received, 500mg/kg/day NA for 5 days (n2-STZ+NA) by starting from third day. The pancreatic tissue sections were immunostained with insulin, pdx-1, notch1, jagged1 and ngn3 antibodies and also double immunostained with insulin and PCNA antibodies. Body weight and blood glucose levels of the animals in all groups were measured. All values were analyzed with statistical methods.

The increase of the blood glucose levels in n2-STZ+NA group were significantly decreased by NA treatment (p<0.01). The number of insulin/PCNA double-positive cells significantly increased in the n2-STZ+NA group compared with the other groups (p<0.001). n2-STZ group had lower number of insulin and pdx-1 positive cells compared to NA treated diabetic group in islet. We found that immunopositive insulin, pdx1 and ngn3 cells were located in small cell clusters or scattered in exocrine tissue and close to ducts in n2-STZ+NA. We did not observe the expression of ngn3 in any islet. There was significant difference between the numbers of notch1 and jagged1 immunopositive cells in islets when the n2-STZ+NA group was compared with the other groups.

In conclusion, we showed that NA treatment stimulates duct epithelium or acinar cell differentiation into the beta cell via up regulation of ngn3 and pdx1, and down regulation of notch1 in exocrine pancreas.

1-Bernardo et al. Mol Cell Endocrinol. 2008;294:1-9. 2- Qu et al. Dev Biol. 2013;376:1-12

3- Otonkoski et al. J Clin Invest 1993; 92,1459–66

4- Sandler S and Andersson A. Diabetologia 1986; 29,199–202

This study was supported by the Scientific Research Projects Coordination Unit of Istanbul University.

Fig. 1: Figure 1. Immunolocalisation of jagged1 in the pancreas of all groups. A, Control; B, n2-STZ; C, n2-STZ+NA groups.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3347 Short- and long-term treatments with DPP-4 inhibitor in type 2 STZ-diabetic neonatal rats: effects on beta cell regeneration and apoptosis

Argun-Kurum G.1, Kaya-Dagistanli F.1, Ozturk M.1
1Istanbul University, Cerrahpasa Medical Faculty, Medical Biology Department, Istanbul, Turkey
gamzeargun@gmail.com

Dipeptidyl peptidase-4 (DPP-4) is an enzyme responsible for the degradation and inactivation of incretins such as GLP-1 and GIP. Vildagliptin (VG), a new inhibitor of DPP-4, increases glucose depended insulin secretion by increasing GLP1 and regulates plasma glucose levels (1,2). We aimed to observe the effects of long and short term VG treatment on possible beta cell regeneration, apoptosis and regulation of islet morphology in neonatal streptozotocin (STZ) diabetic rats.

In this study three groups including control group, diabetic (n2STZ) group and treatment (n2STZ+VG) group were recruited for 36 neonatal rats. STZ (100 mg/kg,ip) were injected to n2STZ and n2STZ+VG group in the second day after birth. VG (60 mg/kg/day,orally) was administered to n2STZ+VG group during 8 and 28 days. The pancreatic tissue sections were immunostained using insulin, glucagon, somatostatin and PCNA antibodies. TUNEL method was performed for apoptosis. Body weights and blood glucose levels were measured at 10th and 30th days. All data were analyzed with statistical methods.

Blood glucose levels in n2STZ groups were significantly increased compared to control in 10 (p<0,001), n2STZ+VG group were significantly lower (p<0,01) compared to n2STZ groups in 30 days old rats. Islet size and number in n2STZ groups were detected decreasing compared to control and n2STZ+VG groups in both term groups. Immunopositive insulin cells and beta cell clusters were scattered in exocrine tissues and duct epithelia, and area of insulin (+) cells and islets size increased in n2STZ+VG groups compared to n2STZ groups in both 10 and 30 days old rats (respectively p<0,001, p<0,05). In n2STZ groups, glucagon and somatostatin immunopositive cells were significantly increased within the islets compared to n2STZ+ VG and control groups in both short and long term groups (p<0.001). In 10 and 30 days old rats, number of PCNA immunopositive cells within the islets in n2STZ+ VG group was significantly higher than n2STZ and control groups (p<0,001). In 10 days old rats, apaptotic cells number of islets in n2-STZ+ VG and control groups was lower than n2-STZ group (p<0,001). Apoptotic cells were observed within exocrine tissue cells and duct epitelium, but there was no significant difference in 30 days old rats for all groups.

The results show that vildagliptin as a DPP4 inhibitor promotes beta cell neogenesis from duct epithelium or acinar cells by inducing some of the endocrine progenitor cells, induces islet cells proliferation by increasing the expression of PCNA, and reduces apoptosis in the islets, also regulates morphological reorganization of the islets in the STZ diabetic neonatal rats.

1-Ahrén B, et al. Obes Metab.13(9):775-83,2011
2-Duttaroy A, et al. Eur J Pharmacol.15;650(2-3):703-7,2011

This study was supported by The Scientific Research Projects Coordination Unit of Istanbul University as the project no:BAP-28090

Fig. 1:  Immunolocalization of insulin in the pancreas of all groups. A,D Control Group. B,E n2STZ Group, C,F n2STZ+VG Group. Upper: 10 days old rats, bottom: 30 days old rats. Scale bar = 20 µm.

Fig. 2:
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3398 The antidiabetic effects of the Fruits of “Laurocerasus officinales Roemer” on Pancreatic Islands of Streptozotocin-induced Diabetic Rats

Eser M.1, Senturkoglu S.1, Tuncdemir M.1, Oztürk M.1, Balci H.1, Karaca C.1, Uslu E.1, Atıkeren P.1, Karabulut E.1, Islam A.2
1Istanbul University Cerrahpasa Faculty of Medicine, Istanbul, TURKEY, 2Ordu University Faculty of Horticulture, Ordu, TURKEY
medihao@yahoo.com

The incidence of diabetes mellitus is increasing Nowadays. The growing number of oxidative stress and the diabetes is related with each other. Climbing number of free radicals causes several tissue damages by interacting with lipids, proteins and nucleic acids. To encounter with the effects of the defective radicals, the organism has some enzymatic and non enzymatic defense systems. However, the results of the researches points out a decrease in the antioxidant defense systems.
The fruit of “Laurocerasus officinales Roemer” is widely used as a herbal treatment for diabetes In the Black Sea region of Turkey. Locally they call it “taflan”. It contains considerable amount of antioxidants. This is proven scientifically with many studies, but there is no animal studies about it till now. We aimed to investigate its anti diabetic effects on the islets of Langerhans of rat pancreas.
We prepared four groups of Albino rats. There were eight rats in each grout. The 1st group was The healthy control group. The 2nd group was The diabetic control. The 3rd group was The taflan + STZ + taflan and The 4th group was The taflan. The 2nd group received only STZ. The 3rd group had taflan for one month. They received intraperitoneal injections of STZ and then fed with taflan for a month. The 4th group fed with taflan. 3rd and 4th group had taflan during each day from 8 AM to 4 PM. The 1st and 2nd groups had normal pallets all day, but the 3rd and the 4th groups had a pallet diet as ad libitum during 4 PM to 8 AM.
Serum glucose levels:The values were high in the 2nd group (≥ 200 mg/dL.). The other groups were low (≤ 200 mg/dL). Body weight: There was weight loss in the 2nd group and there were no significant changes in the other groups. HDL, LDL, VLDL, CHOLESTEROL TRIGLYCERIDE:HDL was low. LDL, VLDL, cholesterol and triglyceride were high in the second group. HDL was high in the 1st, 3rd and 4th groups. LDL, VLDL, Cholesterol and triglyceride were low in the 1st, 3rd and 4th groups. Tissue; MDA and SOD values: MDA was high in 2nd group and it was low in 1st, 3rd and 4th groups. SOD was low in 2nd group and it was high in 1st, 3rd and 4th groups. Immunohistochemistry findings: Insulin antibody staining was negative in the 2nd group. It was positive in 1st, 3rd and 4th groups. Glucagon was positive in all groups. Electronmicroscopy observation: 2nd group: Secretory granules diminished. GER dilated and the crista of mitochondria vanished. 3rd group: Similar morphological features as 1st and 4th control groups.
Conclusion: The fruit of Laurocerasus officinalis Roemer have protective effects against the development of the experimental diabetes and its complications in rat.

This study was supported by “İstanbul Üniversitesi Bilimsel Araştırma Projeleri (BAP)” Istanbul University Scientific Research Projects. Project Number: 4118

Fig. 1: Immunohistochemical staining for insülin (X10). A; Islet cells of the healthy control group. B; Islet cells of the diabetic control group. C; Islet cells of the taflan + STZ + taflan group. D; Islet cells of the taflan group.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3401 Evasion of the parasite Trypanosoma cruzi in Chagas disease can be related to prostaglandin E2 release by lipid bodies own parasite

Toledo D. A.1,2, Roque N. R.2, Teixeira L.1,2, Freire-de-Lima C. G.3, Bozza P. T.2, D'Avila H.1, Melo R. C.1
1Federal University of Juiz de Fora, Juiz de Fora, Brazil, 2Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, 3Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
danielkbssa1@hotmail.com

Lipid bodies (LBs) are complex organelles, rich in lipids and delimited only by a monolayer of phospholipids [1]. LBs are present in all eukaryotic cells, being sites for synthesis of inflammatory mediators in cells from the immune system [2,3]. Accumulation of LBs in the cytoplasm of pathogens in response to interactions with host cells has been attracting great interest, but the meaning of this accumulation is not yet understood. In this study, we investigated LB formation within the intracellular parasite Trypanosoma cruzi, the causal agent of Chagas’disease, during different situations of interaction with host cells (macrophages) and the potential role of these organelles as sites for synthesis of inflammatory mediators, such as prostaglandin E2 (PGE2). First, LBs were identified in both trypomastigotes and amastigotes forms of the parasite after staining with different lipid probes (Osmium tetroxide, BODIPY and Oil red O) (Figure 1). Second, stimulation of cultured trypomastigotes with fatty acids (arachidonic acid (AA), oleic acid (OA)) induced significant formation of LBs (mean ± SEM: 4.67 ± 0.208 in control and 8.19 ± 0.19 in stimulated with 7.5 μM of AA, n = 6) (Figure 2). Third, PGE2 was detected in newly formed, stimulated LBs within the parasite (mean ± SEM: 16.02 pg/mL ± 3.76 in control and 101.5 pg/mL ± 10.52 in stimulated with 1.5 μM of AA, n ≥ 3) in a mechanism dose and time-dependent. Transmission Electron Microscopy (TEM) of infected macrophages revealed that LBs formed in the parasite were significantly larger and more electron-dense in cells infected in vivo (heart macrophages) compared to LBs formed in response to the in vitro infection (peritoneal macrophages) (0.03 μm2 ± 0.00 in vitro and 0.04 μm2 ± 0.00 in vivo, n = 90 and 66 LBs analyzed, respectively). Our study demonstrates that LBs within the parasite Trypanosoma cruzi are organelles able to respond to stimuli and to produce PGE2, with potential roles during the infection process and maintenance of the parasite in the host cell. These findings bring a new focus on LBs and help to understand the functional capabilities of these organelles during inflammatory responses induced by infectious diseases.

References

[1] D. J. Murphy et al, Protoplasma, 249(3) (2012) 541-585

[2] H. D'Avila et al, Mediators of Inflam., 1 (2012) 1-11

[3] R. C. N. Melo et al, J. Histochem. Cytochem., 59(5) (2011) 540-556

This work was supported by CAPES, CNPq and Fapemig.

Fig. 1: Lipid Bodies (LBs) in trypomastigote forms of parasite Trypanosoma cruzi, observed by light microscopy. (A) LBs appear as rounded and darks structures in the cytoplasm after staining with osmium tetroxide. (B) Marking with fluorescence probe for neutral lipids BODIPY® reveals LBs green in cytoplasm of trypomastigotes forms.

Fig. 2: Difference in the numbers of lipid bodies (LBs) between trypomastigotes stimulated and not stimulated. LBs appear in large amount in the cytoplasm of trypomastigotes after stimuli with arachidonic acid and staining with osmium tetroxide (A) compared to not stimulated (B).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3429 Electromagnetic Waves Emitted By Mobile Phones Effect On The Testis Morphology Of Rat, Cell Death and Blood-Testis Barrier: Evaluation for Infertility

Tok O. E.1, 2, Şehirli A. Ö.3, Ercan F.2
1Department of Histology and Embryology, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey, 2Department of Histology and Embryology, Faculty of Medicine, Marmara University, Istanbul, Turkey, 3Faculty of Pharmacy, Marmara University, Istanbul, Turkey
olguenistok@gmail.com

The electromagnetic waves (EMW) emitted by commonly used mobile phones are reported to have effects on many tissues. In this study, we aimed to show effects of EMW emitted by mobile phone with DSC 1800 carrier frequency which has the highest SAR value 1.79 W/kg on the cell proliferation, cell death and blood-testis barrier of rat testis. Wistar-albino rats were used in this study and were formed to five experimental groups as 1) Control, 2)Stand by Fetal, 3)Stand by, 4)EMW Fetal and 5)EMW (n=6). Testes of rats in all experimental groups were taken at postnatal 60th day under ether anesthesia. Body and testis weights of the rats were weighed, diameter and area of seminiferous tubules were measured, presense of proliferative and apoptotic cells were determined and quantitative analysis of ZO-1 were done. To establish the ultrastructural morphology we used transmission electron microscopic techniques. In the tissues ratios of MDA and GSH; in the serum levels of LH, FSH and testosterone were biochemically analyzed. While body weight of rat was decreased in only EMW group, testis weight and seminiferous tubule area were decreased in EMW Fetal and EMW groups. Seminiferous tubule diameters were decreased in all experimental groups. However apoptotic index were significantly increased in Stand by, EMW and EMW Fetal, proliferative index were significantly decreased. In EMW and EMW Fetal groups irregular dispersion of ZO-1 and significantly decreased levels of ZO-1 protein were shown. In electron microscopic examinations small vacuols between and inside the cells were determined in Stand by and EMW Fetal groups; big and great number of vacuols were shown in EMW group. While levels of GSH were significantly decreased in all experimental groups, levels of MDA were significantly increased. However no changes were minitored between serum FSH and LH levels of experimental groups, serum testosterone levels were significantly decreased in EMW and EMW Fetal groups. However it has less effects on stand by mode, cell phones may couse of infertility of rats via decreasing testosterone levels, inducing cell death and breaking down the blood-testis barrier.

Key Words: Cell phone, Electromagnetic waves, Rat, Testis

The study was supported by Marmara University Research Fund (SAG-C-YLP-031210-0270).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-3445 The effects of electromagnetic waves on urinary bladder morphology and urothelial barrier function in developing rats.

Tok O. E.1, 2, Kıran D.2, Şehirli A. Ö.3, Ercan F.2
1Department of Histology and Embryology, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey, 2Department of Histology and Embryology, Faculty of Medicine, Marmara University, Istanbul, Turkey, 3Faculty of Pharmacy, Marmara University, Istanbul, Turkey
olguenistok@gmail.com

The potential biological effects of electromagentic waves (EMWs), have become a great concern in the public. In the present study the biological effects of Digital Cellular System (DCS) 1800-MHz radiation from a common digital mobile phone which has the highest specific absorbtion rate (SAR) value 1.79 W/kg on the urinary bladders of male wistar albino rats are investigated. The study was performed in five different groups. 1) Control group; 2) Stand-by fetal group (rats exposed to EMW emmited by mobile phone on stand-by mode from embryonic day 14 until parturition); 3) Stand-by group (rats exposed to the same EMW mode as Stand-by fetal group from embryonic day 14 until postnatal day 60); 4) EMW fetal group (rats exposed to EMW emmited by mobile phone on speaking mode from embryonic day 14 until parturition); 5) EMW group (rats exposed to same EMW mode as EMW fetal group from embryonic day 14 until postnatal day 60). The exposure time was 2 hours per day for all groups. All of the animals in experimental groups were sacrified under ether anesthesia at postnatal day 60. The effects of EMWs exposure were examined in terms of urothelial morphology, barrier function, inflammatory cell infiltration and oxidative damage. The barrier function of urothelium was assessed using zonula occludens 1 (ZO-1) and E-cadherin immunohistochemistry and ruthenium red (RR) staining for transmission electron microscopy. Luminal urothelial morphology was evaluated with scanning electron microscope. EMW group showed desquamation of urothelial cells and degradation of the glycosaminoglycan layer, inflammatory cell infiltration and increased number of total mast cells. Decrease in the immunreactivity of ZO-1 and E-cadherin were detected in the EMW group. The diffusion of the RR to the intercellular spaces was detected in all EMW exposed groups. Finally increase in the malondialdehyde and decrease in the glutathione levels were observed in all experimental groups comparing to control group. Exposure intensity and time correlate with adverse effects in developing period of urinary bladder. These changes can lead to urinary bladder inflammatory disorders.
Key Words: Cell Phone, Electromagnetic Waves, Urinary bladder, ZO-1, e-cadherin

The study was supported by Marmara University Research Fund.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-5728 Adaptation of JADAS as an Automatic Particle Acquistion Tool for Quantitative Analysis of Vaccines by Transmission Electron Microscopy

MacLellan-Gibson K.1, Renault L.2, Costa A.2, Fleck R. A.3
1National Institute for biological standards and Control, Blanche Lane, South Mimms, Potters Bar, EN6 3QG, 2Cancer Research UK, South Mimms, Potters Bar, EN6 3QG, 3Kings College London, 3New Hunts House, London, SE1 1UL
roland.fleck@kcl.ac.uk

NIBSC routinely employs imaging, including; electron microscopy (EM) in the testing and evaluation of biological medicines. However, as biological medicines develop so do the challenges of ensuring their safety. Today’s emerging and postulated therapies present an ever-moving target with greater demands on basic research, control and standardisation. The largely artefact free, high-resolution information threshold provided by cryo-EM techniques has been core to the evaluation of a number of current and postulated therapies [i.e., human papilloma virus (HPV) vaccine, measles vaccine, gene therapy vectors, Bordetella pertussis and Meningitis vaccines]. However, each of these studies, despite offering higher information thresholds than were achievable with more traditional room-temperature processing techniques remain demanding in operator time and lack quantitative robustness able to inform against variation in vaccine state.

EM remains a powerful tool for evaluation of these biological medicines due to its lack of bias in identifying variations in samples. If this “catch-all” analytical capability could be extended to allow automated acquisition of larger data sets with digital classification of particles it is theoretically possible that EM could be employed as a “gold standard” quality control procedure for biological medicines. This could have a significant impact on novel vesicle type vaccines which are sensitive to osmotic variation.

NIBSC has adapted an automated particle acquisition software suite; JEOL Automated Data Acquisition System (JADAS) which was developed to acquire images of ice-embedded macromolecular complexes under low dose conditions with the intention of further processing the data for 3D macromolecular reconstruction. The software offers the facility to acquire large quantities of high quality data without operator intervention. We have adapted the software to allow for both room temperature and cryo-EM data acquisition and have carried out extensive evaluation of the HPV and Neisseria meningitides, group B, outer membrane vesicle vaccines. The data shows for the first time the potential of EM as a quantitative analytical tool for the quality control of complex biological medicines and vaccines.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-5761 The Effects of Various Drugs Practices Over Proliferation On MDAH-2774 Human Ovary Cell Culture

Ayla S.1, Bilir A.2, Tanriverdi G.3, Erturkoglu S.3, Soner B. C.4, Sofuoglu K.1, Oktem G.5
1Zeynep Kamil Gynocology and Maternity Training and Research Hospital 1, 2İstanbul University, Istanbul Medical Faculty, Histology and Embryology Department 2, 3Istanbul University, Cerrahpasa Medical Faculty, Histology and Embryology Department 3, 4Istanbul University, Cerrahpasa Medical Faculty, Histology and Embryology Department 3, 5Necmettin Erbakan University Meram Faculty of Medicine Department of Pharmacology 4,6Zeynep Kamil Gynocology and Maternity Training and Research Hospital 1, 7Ege University, Medical Faculty, Histology and Embryology Department 5
suleayla@hotmail.com

In this study, effects of resveratrol as a natural polyphenol compount, gemcitabine as an antimetabolite which has nucleoside structure, analogous of deoxycytidine and para aminophenol derivated paracetamol were investigated with single and combined applications in monolayer MDAH-2774 human ovarian cancer cell line. Drugs were evaluated in cell culture with respect to cell proliferation, cell cytotoxycity (trypan blue dye exclusion test), synthesis phase of cell cycle and cell structure in 24, 48, 72, 96 h. Resveratrol has diminished both cell proliferation and cell cycle synthesis phase indication in monolayer cell cultures (p<0,05). Structural changes were also observed in electron micrographs. Gemcitabine has demonstrated a decreasing effect in cell proliferation and marked reduce according to other drug groups were observed in cell cycle synthesis phase indication in monolayer cell cultures (p<0,05). Structural changes observed in electron micrographs showed more impairment in respect to resveratrol. Even paracetamol has shown a decreasing effect in cell proliferation compared to control group in monolayer cell cultures (p<0,05), this effect increased with respect to other drugs. Similar effects were observed in both control and paracetamol group in cell cycle synthesis phase indication. No structural changes were observed in electron micrographs in both the control and paracetamol group. All combination groups showed similar effects that were mainly more effective in respect to single usage of resveratrol and gemcitabine in monolayer cell culture. As a result, the effects of gemcitabine, resveratrol and paracetamol were investigated in monolayer MDAH-2774 human ovarian cancer cell line and a decrease in cell number in cell cycle synthesis phase, prevention of cell proliferation and destruction of cell structure were observed.

Fig. 1: Control ,Tumor cells marked with Brdu (red colored) (a), Resveratrol(R) fewer marked cells (b), Paracetamol(P): large number of marked cells (c), Gemcitabine(G) less marked cells (d); R+G: less marked cells (e), P+G: Cells that have lost their cell extensions, round and unmarked cells (f); x40 ,96 th hour.

Fig. 2: Control (a), Resveratrol(R): Degenerated tumor cells with round appearance (b), Paracetamol(P): Tumor cells that seems more degenerated (c), Gemcitabine(G) : More spoilt tumor cells (d); R+G: Cells with spoilt structure (e), P+G: Cells with spoilt structure (f), round and apoptotic; a,b,c,d,f x 750, e x1000, 96 th hour.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-5825 Effects of the Different Periodontal Treatment Methods on the Root and Cementum

Bozbay E.1, Dominici F.1, Gokbuget A. Y.2, Guida L.3, Cintan S.2, Aydin M. S.4, Pilloni A.1
1Department of Dentistry and Maxillofacial Surgery, Section of Periodontics, School of Dentistry, Sapienza University of Rome, Rome, Italy, 2Department of Periodontology, Faculty of Dentistry, Istanbul University, Istanbul, Turkey, 3Department of Odontostomatological, Orthodontic and Surgical Disciplines, Second University of Naples, Naples, Italy, 4Department of Histology and Embryology, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey
mehmetserifaydin@gmail.com

Cementum is a component of the periodontium, and its major role is to serve as the site of attachment for principal collagen fibers. Periodontal disease is the local inflammation of the supporting tissues of teeth, it causes destruction of gingival tissues, bone loss and loss of connective tissue attachment to cementum. It is generally accepted that removal of the pathogenic microorganisms that form plaque and calculus is the major goal of the periodontal treatment. The teeth treated by hand curettes (HC) and ultrasonic scaler (US) can present a surface without cementum and the open dentinal tubules. US with new shaped tips and airpolishing (AP) devices as alternative to HC designed for subgingival access have been developed for minimal root damage. The aim of the this study is to compare the effect of in vivo root instrumentation using a new piezoelectric US instrument, HC and air polishing by glycine powder, under routine clinical conditions, on the thickness and surface characteristics of cementum.
Twenty-seven patients with teeth clinically and radiographically diagnosed by chronic advanced periodontitis and scheduled for extraction treated in four different methods. The teeth were instrumented subgingivally at one approximal site either with manual instruments, air-polishing, piezoelectric ultrasonic scaler, piezoelectric ultrasonic scaler following air-polishing. After instrumentation the teeth were immediately extracted and cut horizontally into two sections. The teeth were sectioned perpendicularly to the root axis with a microtome and stained with hematoxylin & eosin. Six parts of cementum of all sections, including mesial and distal areas of each tooth, were analysed and each measure was reported as a mean value of five quantifications. The root surface characteristics of teeth were analyzed by scanning electron microscopy.
The results showed that all periodontal treatment methods used in this study remove cementum although using AP alone removed less cementum than HC and US. AP device was more effective at the apical part of the treated area. However, in using of US device following AP the cementum loss was lower on the apical sites than coronal sites. This study showed that US devices and subgingival AP preserve more tooth structure compared to HC, while AP is producing a smooth root surface.

This study was supported by Marmara Istanbul University Scientific Research Committee (#32987).The participants volunteered for the study after receiveing verbal and written information and a signed informed consent approved by the Sapienza, University of Rome Ethical Committee. (Resolution 2821 from the National Health Council, Health Ministry, Italy, 27/09/2013).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-5832 Protective effect of beta-glucan on renal ischemia and reperfusion injury in young and aged rats

Esrefoglu M.1, Tok O. E.1, Aydin M. S.1, Iraz M.2, Selek S.3, Ozer O. F.3, Kocyigit A.3
1Bezmialem Vakif University Medical Faculty, Dept. of Histology and Embryology, Istanbul, Turkey , 2Medeniyet University Medical Faculty, Dept. of Pharmacology, Istanbul, Turkey , 3Bezmialem Vakif University Medical Faculty, Dept. of Medical Biochemistry, Istanbul, Turkey
drmukaddes@hotmail.com

Ischemic acute renal failure (ARF) is a common clinical event leading to development of chronic kidney disease and a high mortality. ARF has a higher incidence in elderly people than younger ones. β glucans are glucose polymer groups possessing protective effects against oxidative damage through an effective free-radical scavenger function. In this study, effects of β glucan on renal ischemia/reperfusion injury were investigated in young and aged Sprague Dawley rats. 56 female rats were randomly assigned to two main groups as young (4 months old) and aged groups (16 months old). Groups were designed as follows: Young and aged sham, young and aged I/R, young and aged β glucan, young and aged I/R+β glucan. I/R were performed as described previously. β glucan was administered by gavages at a dose of 50 mg/bw/day for 10 days prior to the surgery. At the end of the experiment, following collection of blood samples from the heart, rats were sacrificed and kidneys were removed Creatinin, blood urea nitrogen, oxidant/antioxidant status, thiol, myeloperoxidase (MPO), paraoxanase (PON), catalase (CAT) and aryl esterase (ARES) were measured in serum. Histopathological changes including tubular degeneration, vacuolization, tubular necrosis, glomerulosclerosis, hemorrhage, capillary dilatation and congestion and intercellular edema were evaluated.

Mean HDSs of sham operated young and old groups were 0.5±0.83 and 1±0.57; respectively whereas 8±1.67 of young and 4.86±1.34 aged I/R groups. Mean HDSs of young β glucan I/R group was 1.83±0.75 and of aged β glucan I/R group was 2.75± 1.16. A significant difference was detected between young sham and young I/R group (P<0.001). Serum urea and creatinin levels of young and aged of sham group and β glucan administered groups were all lower than those of I/R and β glucan+ I/R groups. Significant differences in creatinin levels were detected between young and aged β glucan administered groups and I/R performed groups (P<0.001, P<0.01; respectively). Mean CAT activities and MPO levels of young and aged β glucan administered groups were lower than those of I/R and β glucan+ I/R groups. PON activities of sham operated young and aged rats were higher than those of I/R and β glucan+ I/R rats. Thiol levels of young and aged I/R groups were higher than those of β glucan+ I/R groups. Differences in total oxidant and antioxidant status among groups were not significant.

As a conclusion, β glucan found to be protective against renal I/R injury in young and aged rats. However, we suggest that oxidative stress should have been evaluated in tissue samples rather than sera since 2 hours, full duration of the experiment, probably is not sufficient for indicating oxidative damage in serum. Tissue analysis of the experiment are continuing.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-5960 GABA Immunoreactivity in the Testis Tissue of Wistar and Genetic Absence Epilepsy Rats

Gürsoy D.1, Çilingir Ö. T.1, Şirvancı S.1
1Department of Histology and Embryology, Faculty of Medicine, Marmara University, Istanbul, Turkey
duygugrsy@gmail.com

Introduction: Gamma-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system. Besides existence in the brain tissue, GABA is also found in nonneuronal tissues such as testis. There is evidence that GABA has a role in the release of testosterone. GABAergic system in the testis was also shown to have a negative effect on the spermatogonial stem cell proliferation.
Aim: Studies have shown that reproductive hormones are affected in patients with epilepsy. Based upon the knowledge that the changes in these hormones may result in infertility in epilepsy, the present study aimed to investigate the possible morphological and GABAergic system alterations in the testis tissue of genetic absence epilepsy rats from Strasbourg (GAERS).
Materials and methods: Adult male Wistar rats and GAERS were used in the present study. Animals were perfused with 4% paraformaldehyde and the testes tissue were routinely processed for paraffin embedding. The sections were stained with hematoxylin and eosin for morphological observation. Other sections were processed for GABA immunohistochemistry.
Results: Qualitative observations revealed that GAERS testis showed less sperm in the seminiferous tubules compared to the Wistar controls. GABA immunoreactivity was observed in the seminiferous tubules and interstitial areas of Wistar rats and GAERS.
Conclusion: Previous studies demonstrated the presence of GABA, glutamic acid decarboxylase (GAD) and GABA receptor subunits in the seminifeorus tubules. Our results also demonstrated the presence of GABA in the testis tissue of both strains. We suggest that the alterations in GABAergic system in absence epileptic rats may also affect the gonadal system, resulting in decreased sperm production.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-6000 ANALYSIS OF THE VARIATION AND ACCURACY OF QUANTIFIED SECOND HARMONIC GENERATION IMAGING OF COLLAGEN IN HUMAN LUNG TISSUES

Kable E. P.1, Tjin G.2,5, Kable S. H.3, Burgess J. K.2,4,5
1Australian Centre for Microscopy and Microanalysis, The University of Sydney, Australia, 2Woolcock Institute of Medical Research, Sydney Australia, 3School of Chemistry, The University of New South Wales, Sydney, Australia, 4Discipline of Pharmacology, The University of Sydney, Sydney, Australia5 , 5Central Clinical School, Faculty of Medicine, The University of Sydney, Sydney, Australia2
eleanor.kable@sydney.edu.au

Second harmonic imaging (SHG) is a non-invasive technique used for studying biological samples for over a decade. Fibrillar collagen I, a very potent generator of SHG signal, is a component of the ECM and the most abundant mammalian protein.  The contribution of extracellular matrix (ECM) remodelling to the pathogenic changes in chronic obstructive pulmonary disease (COPD) is complex and not well understood. Collagen I, a component of the ECM altered in COPD airways has second harmonic generation (SHG) properties. The SHG signal is coherent, propagating both forward (F) (primarily organized/mature collagen fibrils) and backward (B) (primarily disorganized/immature collagen fibrils) parallel to the incident light. Formalin fixed and paraffin embedded tissues of 30µm thickness was used in the experiments. F/B SHG ratio was used to determine the proportion of organized to disorganized collagen, with lower variation in F/B ratio between sampling regions within the same patient and between patients in the same disease group (Figure 1) compared to analysing F and B data alone. The F/B ratio was independent of laser power drift, regions analysed within a tissue and tissue orientation during analysis. Using this method we identified a significant difference in collagen organization in airway tissue between COPD and non-diseased patients.  We have developed a robust optimization and calibration methodology that will allow direct comparison of data obtained at different times and from multiple microscopes that is directly adaptable for use with other tissue types. We report a powerful new tool for advancing our understanding of pathological ECM remodelling that may help uncover new therapeutic targets in the future.

References.

1. Williams, R.M., Zipfel, W.R., and W.W. Webb, Interpreting second-harmonic generation images of collagen I fibrils. Biophys J, 2005. 88(2): p. 1377-86.
2. Tjin, G., Xu, P., Kable, S.H., Kable, E.P.W., and Burgess, J.K., Quantification of Collagen I in airway tissues using Second Harmonic Generation. J Biomed Opt, (2014). 19(3), 036005; doi: 10.1117/1.JBO.19.3.036005.
3. Schindelin, J., et al., Fiji: an open-source platform for biological-image analysis. Nat Methods, 2012. 9(7): p. 676-82

Cardiopulmonary transplant team and pathologists at St. Vincent’s Hospital.
Surgeons and pathologists at RNSH, Concord and Strathfield Hospitals and Rhodes Pathology.
J.K. Burgess is supported by a NHMRC Career Development Fellowship #1032695.

Australian Microscopy and Microanalysis Facility

Fig. 1: Variation of Area F/B ratio values between regions (solid black lines, n=3 each patient), patients (distance between solid red lines, mean individual deviations from the averaged region variations from each patient for non-diseased or COPD) and disease (distance between blue dotted lines; relative difference of the means for non-diseased vs COPD).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-10-P-6013 The Role of Klc2 in Hearing and Deafness

Ebrahim S.1, Ingham N.1, Steel K. P.1
1King's College London, London, United Kingdom
seham.ebrahim@kcl.ac.uk

Our hearing organ, the organ of Corti (OC), is exquisitely sensitive- able to detect one hundred trillion units of sound intensity, across a range of frequencies that spans several orders of magnitude. In normal hearing, sound, a mechanical stimulus is converted to an electrical signal that triggers neural activation. This is accomplished by specialized sensory hair cells in a process called mechanotransduction, which relies on a combination of extracellular protein filaments, transmembrane complexes including ion channels, actin-based stereocilia and actin-associated intracellular motor proteins. The sophisticated micromechanics underlying our ability to hear would not be possible without the unique morphologies and remarkable patterning of hair cells and non-sensory support cells of the OC (Figure 1). Each cell type within the OC has a characteristic cytoskeletal architecture that is defined by different arrangements and dynamics of actin filaments and microtubules. Accordingly, mutations in actin as well as a number of actin regulatory proteins and microtubule-associated proteins have been associated with hearing disorders. We have recently identified, from the Wellcome Trust Sanger Institute Mouse Genetics Project screen for hearing defects, that the mutation of the cytoskeleton-associated Klc2 gene, which encodes kinesin light chain 2 (KLC2), causes a progressive hearing loss in mice. Kinesins are a family of molecular motor proteins that move along polarized microtubules to transport macromolecular cargo using energy obtained from ATP hydrolysis, and kinesin light chains are involved in cargo-binding. We are currently using a combination of microscopy techniques to characterize the Klc2 mutant mouse, to elucidate the role of Klc2 in progressive hearing loss. The localization of Klc2 in the inner ear is being investigated using immunofluorescence together with confocal fluorescence microscopy; the apical morphology of the OC in the Klc2-/- mouse is being assessed using scanning electron microscopy; and the ultrastructure of hair cells and synapses interrogated using transmission electron microscopy. Together, these data will provide novel insights into the role of Klc2 in progressive hearing loss, but importantly also add to existing knowledge on the molecular bases of normal hearing and deafness.

Fig. 1: A scanning electron micrograph of the apical surface of the OC, showing the unique architecture of hair cells, and precise checkerboard patterning of hair cells (HC) and support cells (SC )
↑ Go to the Table of content ↑

LS-11. Physiology and pathology

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-11-IN-5848 Immune cell biology using super-resolution microscopy

Davis D. M.1
1Manchester Collaborative Centre for Inflammation Research, University of Manchester
daniel.davis@manchester.ac.uk

Standard optical microscopy is fundamentally limited in the details that can be observed due to diffraction (the way that light bends around objects). Super-resolution fluorescence microscopy overcomes this limitation and gives us the ability to explore cell biology on the nanometre-scale. Here, I will demonstrate how explorative research using super-resolution microscopy has already led to several new ideas about how immune cells interact with other cells, how they recognize signs of disease, and how these discoveries seed new ideas for drug design.

We have recently found, for example, that the immune synapse clears and excludes molecules above a size threshold, and as a consequence of this, drugs that target synaptic cytokines or cytotoxic proteins must fit these dimensions. Another finding is that, various cell types, including immune cells, can be connected by thin membrane tethers termed membrane nanotubes. Membrane nanotubes may facilitate a new mechanism for intercellular communication. We have found, for example, that microRNAs traffic from macrophages into cancer cells to dampen unwanted cellular proliferation. One broad theme emerging from high- and super-resolution microscopy is that interactions between immune cell receptors, kinases and adaptors are at least in part controlled by transient interactions between nano-scale assemblies. This is a significantly different concept from a linear cascade of individual protein-protein interactions depicted in textbook diagrams of immune receptor signaling pathways. Importantly, our preliminary observations indicate that the surface of alveolar macrophages might be organised differently in disease settings, which is likely to impact signal integration in these cells.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-11-O-1568 microRNA miR-142-3p inhibits breast cancer cell invasiveness: characterization of altered morphology and functional properties

Götte M.1, Schwickert A.1, Kemper B.2, Riethmüller C.3
1Münster University Hospital, Münster, Germany, 2Center for Biomedical Optics and Photonics, Münster, Germany, 3Serend-ip GmbH, Münster, Germany
mgotte@uni-muenster.de

MicroRNAs (miRNAs) are pivotal post-transcriptional regulators of gene expression. These endogenous small non-coding RNAs play significant roles in tumorigenesis and tumor progression. miR-142-3p expression is dysregulated in several breast cancer subtypes. We aimed at investigating the role of miR-142-3p in breast cancer cell invasiveness applying a range of microscopic techniques combined with functional analysis. Supported by transcriptomic Affymetrix array analysis and confirmatory investigations at the mRNA and protein level, we demonstrate that overexpression of miR-142-3p in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells leads to downregulation of Integrin-αV, RAC1, WASL (N-WASP) and CFL2, molecules implicated in cytoskeletal regulation and cell motility. ROCK2, IL6ST, KLF4, PGRMC2 and ADCY9 were identified as additional targets in a subset of cell lines. Decreased matrigel invasiveness was associated with the miR-142-3p-induced expression changes. Confocal immunofluorescence microscopy, nanoscale atomic force microscopy and digital holographic microscopy revealed a restructuring of the intracellular actin cytoskeleton as well as a reduced cell volume and size. A more cortical actin distribution and a loss of membrane protrusions were observed in cells overexpressing miR-142-3p. Luciferase activation assays confirmed direct miR-142-3p-dependent regulation of the 3’UTR of ITGAV and WASL. siRNA-mediated depletion of ITGAV and WASL resulted in a significant reduction of cellular invasiveness, highlighting the contribution of these factors to the miRNA-dependent invasion phenotype. Our data identify ITGAV and several additional cytoskeleton-associated molecules as novel invasion-promoting targets of miR-142-3p in breast cancer. With its tumor suppressive potential, miR-142-3p is a promising candidate for future approaches of miRNA-based anti-metastatic cancer therapy.

This study was supported by Innovative Medizinische Forschung, Medical Faculty, Münster University [I-Gö 111110, to M.G.], the Germany Federal Ministry for Education and Research (BMBF), research focus program “Biophotonics” [FKZ13N10937, to B.K.], and BMBF-IB [01DJ130022A, to M.G. and C.R.).

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-11-O-2631 Expression of Amelotin, Odontogenic-Ameloblast Associated and Secretory Calcium-Binding Phosphoprotein-Proline-Glutamine-rich 1 in teeth of Laminin-332 deficient mice

Wazen R. M.1, Moffatt P.2, Adair-Kirk T.3, Nanci A.1, 4
1Faculty of Dentistry, Université de Montréal, Montréal, QC, Canada, 2Shriners Hospital for Children, Montréal, Montréal, QC, Canada, 3Department of Medicine, Washington University School of Medicine, St. Louis, MO, 4Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, QC, Canada
rima.wazen@umontreal.ca

The epithelial ameloblasts are separated from maturing enamel by an atypical basal lamina (BL) enriched in laminin (Lm)-332. This heterotrimeric protein (α3, ß3 and γ2 chains) provides structural integrity to BLs and exerts effects on various epithelial cell processes including cell adhesion. A distinctive feature of this BL is that it binds to mineral rather than connective tissue. In a separate abstract, we demonstrate that three secreted proteins amelotin (AMTN), odontogenic ameloblast-associated (ODAM), and secretory calcium-binding phosphoprotein-proline-glutamine-rich 1 (SCPPPQ1), members of the SCPP gene family, are novel constituents of this BL. This atypical BL has therefore adapted by integrating these specialized molecules. Objective: To explore how the three proteins participate in structuring the BL and mediating cell-mineral adhesion, we have used a mouse model of Lm-332 deficiency, which expresses a doxycycline (Dox)-controllable human Lm-γ2 transgene under the cytokeratin 14 promoter on the Lm-γ2 knockout background (PLoS One 7(9):e45546). Method: Mandibles and maxillae from normal, Lm-332 mice on-DOX (expressing Lm-γ2) and off-DOX for 1 or 2 weeks (Lm-332 deficiency) were paraffin-embedded and sections were processed for immunohistochemistry using rabbit antibodies to rat AMTN, ODAM, or SCPPPQ1, and human Lm-γ2. Results: Incisors from both on- and off-Dox mice showed severe histological changes at the level of the enamel organ. In addition, as seen in decalcified samples, the organic matrix of forming enamel was altered and residual matrix was found throughout the maturation stage. CT-scans and scanning electron microscope analyses showed that these mice exhibited a hypomineralized enamel layer and occlusal surface wear in molars. In normal mice, all three proteins localized discretely in the region of the BL, and no Lm-332 was detected in the maturation stage using the Lm-γ2 antibody, confirming the species specificity of the antibody. In both on- and off-Dox mice, the distribution of AMTN, ODAM and SCPPPQ1 labeling was altered, accumulating as intensely stained focal patches within the cell layer rather than at the cell apex. Human Lm-γ2 was also detected in these patches indicating that off-Dox mice are only partially Lm-γ2 deficient. Conclusion: The expression of human Lm-γ2 chain in these mouse models causes alterations that are similar to those in teeth of patient with junctional epidermolysis bullosa due to Lm-332 mutations. Considering that rodent AMTN, ODAM, SCPPPQ1 proteins only show partial homology with the human proteins, this suggests that proper molecular ratios and interactions between AMTN, ODAM, SCPPPQ1 and Lm-332 are required for structuring the atypical BL and mediating adhesion to the tooth surface.

CIHR, NIH/NHLBI P01HL029594, Shriners of North America

Fig. 1: Light micrographs illustrating the histological appearance of the maturation stage of amelogenesis in (A) wild type and (B) Lm-332 deficient mice off-DOX. In Lm-332 off-DOX mice, the enamel organ is disorganized and enamel matrix residues (*) are still present in the maturation stage.

Fig. 2: Immunohistochemistry for rat AMTN (A), ODAM (B), SCPPPQ1 (C) and human Lm-γ2 in Lm-332 off-DOX mice. Expression is found at the cell-tooth interface and as focal patches (*) in the disorganized enamel organ.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-1431 Detection of Survivin in the Skin of Male Albino Rats at Different Ages: Histological and Immunohistochemichal Study.

Ghallab A. M.1, Salama N. M.2, Sayed S. S.2, Ghaith S. M.3
1Histology Department, Faculty of Medicine, Zagazig University, Egypt, 2Histology Department, Faculty of Medicine, Cairo University, Egypt, 3Histology Department, Faculty of Dentistry, MSA Universiy, Egypt
aymanghallab@yahoo.com

Background and Aim of work: The epidermis is a self-renewing stratified squamous epithelium that forms the outer most component of the skin. A balance between epidermal cell proliferation, differentiation and apoptosis preserve epidermal homeostasis. A potential inhibitor of apoptosis has been recently identified as survivin. This study was designed to detect the presence of survivin in normal skin of male rats at different stages of postnatal development.
Materials and Methods: This study included 30 male albino rats equally classified, according to their ages, into 6 groups: zero, five, ten, fifteen, twenty and sixty day- old. Skin specimens were obtained from the back of all animals, processed, sectioned and submitted to H&E and immunohistochemical staining for survivin. The area percent and the optical density of survivin in the epidermis, dermis and hair follicles were detected using image analyzer and were statistically analyzed.
Results: Survivin immunopositivity was detected mainly in the nuclei of basal as well as prickle and granular cell layers of epidermis. In the dermis, survivin immunostaining was seen in the fibroblasts, cells of sebaceous glands as well as in the germinal, inner root sheath and outer root sheath of hair follicles. The area percent of survivin in both epidermis and dermis was higher at young ages then gradually decreased towards adulthood with a second rise demonstrated at age of sixty days.
Conclusion: Survivin exists in normal skin at different stages of postnatal development. Its existence is greatly confined to cells involved in mitosis. Thus, its anti-apoptotic role seems to be connected to the highly proliferating cells. Understanding the role of survivin in the skin would help to approach new strategies in prevention and therapeutics of skin cancer and other skin inflammatory diseases.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-1508 Pre-suckling calcium supplementation prevents bone microstructural defect in lactating rats

Suntornsaratoon P.1,2, Krishnamra N.1,2, Charoenphandhu N.1,2
1Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand, 2Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand
mai_panan@hotmail.com

Breastfeeding places a stress on maternal calcium homeostasis because of the increased calcium demands for milk production, leading to massive bone resorption and osteopenia. Ordinary calcium supplement is often not effective to prevent this maternal bone loss since fractional calcium absorption in the intestine is apparently low. Previous investigations showed that high plasma prolactin, especially the suckling-induced prolactin surge, enhanced intestinal calcium absorption; therefore, it was hypothesized that pre-suckling calcium supplementation is the potential regimen for preventing of lactation-induced bone loss. Day-7 lactating Sprague-Dawley rats were daily administered 4 times per day for 14 days with water (Vehicle) or various doses of CaCl2 solution (i.e., 1, 2 and 4 mg/kg/dose) at 90 min prior to suckling. Thereafter, maternal femora, tibiae and L5–6 lumbar vertebrae were collected for measurement of bone mineral density (BMD) and bone mineral content (BMC) by using dual energy X-ray absorptiometry (DXA). BMD and bone microarchitecture in the tibiae or femora of 5- and 7-week-old offspring were also investigated by DXA and computer-assisted bone histomorphometric analysis, respectively. The results showed 4 mg/kg/dose CaCl2-treated lactating rats had higher femoral, tibial and vertebral BMD and BMC as compared to vehicle-treated rats. Male and female offspring breastfed by calcium-supplemented dams showed higher femoral BMD and trabecular bone volume than those breastfed by vehicle-treated dams. In conclusion, pre-suckling calcium supplement (4 mg/kg/dose, 4 times per day, administered 90 min prior to suckling) could effectively prevent bone microstructural defect in lactating rats and also increased bone density in their offspring.

This work was supported by Thailand Research Fund-Mahidol University through the Royal Golden Jubilee Ph.D. Program (PHD/0172/2552 to PS) and NSTDA (P-10-11281 to NC).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-1513 Aberrant growth plate histology and bone cortical impairment in diabetic rats

Charoenphandhu N.1,2, Lapmanee S.1,2, Aeimlapa R.1,2, Krishnamra N.1,2
1Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand, 2Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand
narattaphol.cha@mahidol.ac.th

In young growing humans and rodents, diabetes mellitus can lead to growth retardation and low bone density, but how diabetes mellitus alters bone structure remains unclear. In the present study, we used micro-computed tomography to visualize the 3-dimensional structure of cortical envelope and evaluate volumetric bone mineral density of femora (long bone) obtained from non-obese Goto-Kakizaki rats, which exhibited type 2 diabetes mellitus with high fasting plasma glucose level (hyperglycemia) and insulin resistance. The results showed that diabetic rats had lower volumetric bone mineral density in the femora (especially in the trabecular part) and L5–6 lumbar vertebrae as compared to the wild-type rats. They also had lower femoral cortical bone area and moment of inertia, the latter of which was an indirect indicator of bone strength. In addition, the shorter femoral and tibial lengths in diabetic rats suggested that endochondral bone growth was impaired. A histomorphometric analysis further showed that the total height and hypertrophic zone height of the tibial growth plates in diabetic rats were lower than those in the wild-type rats. On the other hand, the height of epiphyseal resting zone was markedly greater in diabetic rats as compared to the wild-type rats. It could be concluded that type 2 diabetes mellitus was associated with aberrant growth plate histology and defective cortical structure. These findings could explain, in part, how diabetes mellitus led to impairment of bone structure.

We thank Dr. Dutmanee Seriwatanachai for technical assistance, National Laboratory Animal Center, NSTDA (to NK), and Faculty of Science, Mahidol University (to NC).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-1519 Defective bone microstructure in the tibial secondary spongiosa of Goto-Kakizaki diabetic rats

Wongdee K.1, 3, Suntornsaratoon P.2, 3, Aeimlapa R.2, 3, Krishnamra N.2, 3
1Office of Academic Management, Faculty of Allied Health Sciences, Burapha University, Chonburi, Thailand, 2Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand, 3Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok, Thailand
kannikar@buu.ac.th

Type 1 diabetes mellitus has been known to induce bone microstructural defect, low bone density, and osteoporosis, but the findings from type 2 diabetes mellitus (T2DM) remain controversial. Here, bone microstructure was investigated in female young growing Goto-Kakizaki diabetic rats by using computer-assisted bone histomorphometric analysis. This rat strain was non-obese and hyperglycemic, and exhibited T2DM with insulin resistance. Two doses of 10 mg/kg calcein (a fluorescent dye) were injected subcutaneously on day 7 and 1 prior to euthanasia to stain mineralizing bone surface. The tibiae were removed, sectioned, and finally processed for Goldner’s trichrome staining. The results showed that the tibial secondary spongiosa of diabetic rats had lower trabecular bone volume, trabecular thickness, and osteoblast surface than the wild-type rats, but osteoblast morphology appeared normal. Analysis of calcein labeling under a fluorescent microscope revealed lower mineralizing surface, mineral apposition rate, and bone formation rate in diabetic rats as compared to the wild-type rats. Active osteoclasts were also observed in the tibial metaphases of diabetic rats, agreeing with a histomorphometric finding that osteoclast surface was greater in diabetic rats than wild-type rats. Therefore, the present study has provided evidence that T2DM was associated with defective bone microstructure, presumably due to the suppression of osteoblast-mediated bone formation and enhancement of osteoclast-mediated bone resorption.

We thank Dr. Dutmanee Seriwatanachai for technical assistance, National Laboratory Animal Center, National Science and Technology Development Agency, and Mahidol University.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-1590 Investigation of the membrane effects and mode of action of the synthetic peptides Os and Os-C

Taute H.1, Bester M. J.1, Gaspar A. R.2, Neitz A. W.2
1Department of Anatomy, Faculty of Health Sciences, University of Pretoria, South Africa, 2Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, South Africa
helena.taute@up.ac.za

Antimicrobial peptides (AMPs) are found in all forms of life, and form part of the innate immune system. Strategies for the development of AMPs for clinical application involves the isolation and characterisation of these AMPs, then to use these peptides as templates for the synthesis of shorter peptides with greater stability, lower cost of production and greater antimicrobial activity.

The peptide Os is a synthetic defensin C-terminal derived peptide from the tick Ornithodoros savignyi. Both Os and its analogue, in which three cysteine residues were omitted (Os-C), were previously found to be bactericidal to Gram-positive and Gram-negative bacteria. Peptides investigated for clinical application must not be toxic to mammalian cells. Cell culture based studies have shown that both peptides were not cytotoxic (Prinsloo et al. 2013).

In this study the morphological effects and mode of killing of Os and Os-C was further investigated using SEM and confocal fluorescence microscopy (triple staining with DAPI - stain all cells, CTC - viable cells and FITC - cells with permeabilised membranes). Possible cytotoxicity was further evaluated using human erythrocytes which represent a typical eukaryotic membrane.

SEM of melittin (Mel), a known lytic peptide, showed distinct blebbing of the cell surfaces of E. coli and B. subtilis at 25 µM. In contrast both Os (0.77 µM) and Os-C (1.74 µM), at their minimum bactericidal concentrations (MBCs), caused leakage of intracellular content that led to a flattened morphology (fig 1).

Triple fluorescence staining showed that E. coli exposed to 2.5 µM Mel had undergone membrane permeabilisation while no viable bacteria remained. Likewise at the MBC of Os and Os-C membranes were damaged with few viable cells. At concentrations ten times lower than the MBCs, Os and Os-C caused membrane permeabilisation, however the bacteria remain viable. This suggests a dual mechanism of action that includes both the membrane and intracellular targets.

At 25 µM Mel caused 100% haemolysis with the formation of echinocytes at concentrations as low as 2.5 µM. Both Os and Os-C did not cause haemolysis up to 100 µM and SEM showed normal membrane morphology and biconcave cells (fig 2).

In conclusion, preliminary indications are that these peptides have a different mechanism of action than Mel, possibly affecting multiple targets including membranes and intracellular components. The peptides show no toxicity towards human erythrocytes. Further investigation will be done by fluorescently labelling the peptide and tracking with confocal microscopy. Cytotoxicity towards human leukocytes will also be investigated.

Thank you to the personnel of the Micrscopy Unit at the University of Pretoria for all your assistance during this project.

Fig. 1: SEM of B. subtilis treated with 25 µM Mel showing membrane blebbing (A), and 0.77 µM Os showing flattened cell morphology (B). Scale bars = 1 µm

Fig. 2: SEM of human erythrocytes exposed to 2.5 µM Mel showing the formation of echinocytes (A) and 100 µM Os showing normal morphology (B). Scale bars = 1 µm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-1604 Melatonin promotes beneficial Mitofusin 2 in obese mice kidney

Favero G.1, Stacchiotti A.1, Lavazza A.2, Rodella L. F.1, Rezzani R.1
1Anatomy Unit, Dept.Clinical and Experimental Sciences, Brescia University, Brescia, Italy, 2Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna, Brescia, Italy
alessandra.stacchiotti@unibs.it

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2021 Investigation of the effect of Sibutramine on platelets and fibrin networks of male Sprague Dawley rats

Van der Schoor C.1, Oberholzer H. M.1, Bester M. J.1, Van Rooy M.2
1Department of Anatomy, Faculty of Health Sciences, University of Pretoria, South Africa, 2Department of Physiology, Faculty of Health Sciences, University of Pretoria, South Africa
ciska.vanderschoor@gmail.com

Sibutramine is a Serotonin-Norepinephrine Re-uptake Inhibitor (SNRI) which was widely used in the treatment of obesity as it acts on the central nervous system influencing satiety as well as energy expenditure. Although now withdrawn from most markets, sibutramine is often a hidden ingredient in “natural” weight loss agents which have been shown to contain far higher concentrations of this compound than the original prescription drug. In light of the obesity epidemic and the associated comorbidities, some are of the opinion that the benefits associated with sibutramine use outweigh the possible risks. Comprehensive risk assessment of this compound is essential in order to fully define the safety and efficacy of its use.


Numerous adverse events have been associated with sibutramine use, with cardiovascular complications being most predominant. This study was aimed at investigating the effect of sibutramine on the ultrastructure of platelets and fibrin networks by using scanning electron microscopy. Male Sprague-Dawley rats, treated with a low (LD; 1,32mg/kg) and high dose (HD; 13.2mg/kg) of sibutramine for 28 days were used in this study and were compared to control animals. Blood samples were collected on the day of termination via cardiac puncture and plasma smears were prepared for the evaluation of platelet morphology. To evaluate fibrin clot structure thrombin was added to the plasma to form the coagulum.


Compared to controls, platelets from exposed animals presented with pseudopodia formation as well as membrane spreading (Fig. 1). Upon higher magnification signs of necrosis, such as membrane tears, were also evident, characteristic of over activation (Fig. 2). The fibrin clots of the sibutramine-treated animals revealed fused thick fibres with thin fibres forming a net-like architecture, covering the thick fibres (Fig. 3). Fibrin network formation was also seen without the addition of thrombin (Fig. 1C). This may be due to elevated concentrations of coagulatory factors which is associated with the over activated phenotype. These results are typical of a hypercoagulable state, as has been previously described in cases such as thromboembolic ischaemic stroke.


It can therefore be concluded that sibutramine alters the ultrastructure of platelets and fibrin networks to that typical of a hyercoagulable state. This could contribute to the increase in blood pressure and heart rate associated with sibutramine use. This effect may occur through peripheral noradrenergic stimulation, which further activates various physiological processes in response to shear stress. In depth biochemical investigations are required to identify the molecules and regulatory process involved to fully understand the mechanisms whereby sibutramine affects platelet function.

The authors would like to acknowledge the Unit for Microscopy and Microanalysis, University of Pretoria, for the use of their facilities throughout this study.

Fig. 1: Platelets from the different experimental groups. A: Control; single pseudopod is visible (arrow); B: LD Sibutramine; Activated platelet, numerous pseudopodia (thick arrows) and membrane spreading (thin arrows); C: HD Sibutramine; numerous pseudopodia (arrows), matted thick fibres (white star) and platelet interaction is visible

Fig. 2: Higher magnification of platelets from the different experimental groups. A: Control; smooth surface and open canalicular pores visible (arrows); B: LD Sibutramine; platelet membrane appears granular; C: HD Sibutramine; membrane appears necrotic (arrows) with membrane tears (white star)

Fig. 3: Fibrin networks of animals in the different experimental groups. A: Control; major thick (thick arrows) and minor thin (thin arrows) fibres; B: LD Sibutramine; fused thick fibres (thick arrows) and minor fibres forming a net-like structure (thin arrows); C: HD Sibutramine; minor fibres (thin arrows) covering the thick fibres (thick arrows)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2186 Fumonisin B1 alters the expression of cyclin-dependent kinases (CDK) and CDK inhibitors in mouse liver

Lee S.1, Yang S.1, Han K.1
1Department of Anatomy, Ewha Womans University
khhan@ewha.ac.kr

Fumonisin B1 (FB1), the most prevalent member of a family of toxins produced by fusarium verticillioides, induces cell proliferation in the liver. Cell proliferation requires a coordinated interaction of cyclin-dependent kinases (CDK) and CDK inhibitors. The purpose of this study was to examine the expression of CDKs and CDK inhibitors in FB1 treated liver. C57BL/6 mice were divided into 3 groups and received FB1 (0, 5, and 20mg/kg/day, i.p) for 5 days. Liver tissues were processed for immunohistochemistry and immunoblot analysis. Low-dose　FB1　(5 mg/kg)　did not affect AST levels. However, high-dose FB1 (20 mg/kg)　significantly increased AST serum levels and caused extensive necrosis. Both low-dose and high-dose FB1 significantly induced cell proliferation. Immunohistochemical detection of PCNA and quantification revealed that cell proliferation increased by 4.69-fold (5 mg group) and 4.86-fold (20 mg group), respectively. Expression of CDK2, CDK4, and CDK6 significantly increased in both low-dose and high-dose FB1 groups. Also, expression of CDK associated cyclins (D1 and D3) increased in both FB1 groups. Confocal microscopy showed that expression CDK2 was co-localized with PCNA in the nucleus of many hepatocytes. In contrast, expression of P18INK4C and P27KIP1 significantly decreased in both low-dose and high-dose FB1 groups. Interestingly, localization of P27KIP1 shifted from the nucleus to the cytoplasm in both FB1 groups. These results suggest that expression and intracellular redistribution of CDKs and CDK inhibitors may play an important role in FB1-induced cell proliferation in the liver.

This work was supported by the National Research Foundation of Korea (NRF-2011-0016068, 2013R1A1A2058028).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2266 In vitro electrophysiology of human myometrial telocytes

Radu B. M.1, 2, Cretoiu S. M.3, 4, Banciu A.2, Banciu D. D.2, Cretoiu D.3, 4, Popescu L. M.3, 4
1University of Verona, Verona, Italy, 2University of Bucharest, Bucharest, Romania, 3Carol Davila University of Medicine and Pharmacy, Bucharest, Romania, 4Victor Babeş National Institute of Pathology, Bucharest, Romania
dragos@cretoiu.ro

Background. Newly described telocytes (TCs) are a novel cell type in the interstitial space of human myometrium. TCs are characterized by very long and distinctive prolongations named telopodes (Tps). It was suggested that TCs could influence the contractile activity of myometrial smooth muscle cells (SMCs). Therefore, our aim was an in vitro electrophysiological evaluation of myometrial TCs.
Methods. We used whole-cell patch-clamp recordings and immunofluorescence on TCs from human myometrium.
Results. In non-pregnant myometrium, patch-clamp recordings on TCs in voltage-clamp mode revealed a hyperpolarization-activated chloride inward current with calcium dependence and the absence of L-type calcium channels. Measurements of membrane capacitance showed the in vitro electrical coupling of TCs to SMCs. In human non-pregnant myometrium, T-type calcium currents have been evoked by brief ramp depolarization in TCs electrically coupled to SMCs, but not by standard protocols of step-depolarizing pulses. Mibefradil (1 μM) inhibits the T-type calcium currents. TCs exposed to oxytocin (40 nM) present a slight oscillating activity in current clamp mode generated by action potential like protocols. Immunofluorescence of TCs in pregnant myometrium using isoform selective T-type calcium channel antibodies indicates strong expression of CaV3.1 and CaV3.2 on the cell body and in Tps. The expression in TCs from the non-pregnant uterus is less intense, being confined to the cell body for CaV3.2, while CaV3.1 was expressed both on the cell body and in Tps.
Conclusion. The presence of T-type calcium channels in human myometrium could be a key issue in understanding the excitatory-coupling events during pregnancy and labor.

This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number 82/2012 (PN-II-PT-PCCA-2011-3.1-0553).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2301 Transplantation of olfactory ensheathing cells and aFGF promotes functional recovery and remyelination in rats with chronic complete spinal cord sections

Botero L.1, Gómez R. M.2, Chaparro O.3
1Veterinary Pathology Laboratory, Veterinary Faculty, National University of Colombia 1, 2Neurosciences group. Faculty of Medicine Sabana University, Chía. Colombia 2, 3Physiology Department, Faculty of Medicine, National University of Colombia 3
lboteroe@unal.edu.co

Spinal cord injury (SCI) can lead to paraplegia or quadriplegia. Although there are no fully restorative treatments for SCI, many cellular and molecular therapies have been tested in animal models. Olfactory ensheathing cells (OECs) are known to enhance axonal regeneration and to produce myelin after transplantation and have become a prime candidate for cell-mediated repair following a variety of CNS lesions. Some grown factors like (acid fibroblast grow factor) aFGF are used to potentiate this effect. This study evaluated the effect of OEC+aFGF transplantation on spinal cord lesions in rat. Fifteen Wistar rats underwent a T8-T 10 complete spinal cord section. Sixty days post injury, nine rats were injected directly into the injury with OECs+aFGF, and 6 rats were used as controls. Functional outcome was measured using the Basso-Beattie-Bresnehan score and inclined grid test 24 hour after the treatment and up to seventy five days after transplantation when the animals were sacrifized. Samples of spinal cord tissue were studied for ultrastructural changes. The results showed a clear and progressive functional recovery of the animals treated with OEC+aFGF transplantation, compared to controls (Fig. 1). Ultrastructural evaluation exhibited severe axonal and myelin changes, like ruptured myelin sheaths, neuropil edema, very thin myelin sheaths, axonal degeneration and peri-axonal edema, in control rats (Fig 2). In transplanted rats these changes were reduced in frequency and severety. In addition, in transplanted rats there were foci of remyelinated axons (Fig. 3) that were not observed in control rats. These results suggest that OEC+aFGF transplant induces axon regeneration and remyelination and functional recovery in chronic injured rats.

Colcieencias for financial support. Dra Gloria Patricia Cardona. SIU, Antioquia University for the use of facilities for maintenance of rats.

Fig. 1: Figure 1. BBB score. Functional recovery of rats with chronic completely transected spinal cords after OEC+aFGF transplantation

Fig. 2: Figure 2. Transmission electron micrographs from the lesion site of a control animal. A) Ruptured myelin sheaths (arrows) and neuropil edema (star). B) Very thin myelin sheaths (arrows). C) Thin myelin sheaths and axonal degeneration. D) Peri-axonal edema (arrows)

Fig. 3: Figure 3. Micrographs from the lesion site of a transplanted rat with OEC+aFGF. A) Semi thin sections with toluidin blue (TB). It shows foci of axonal regeneration and remyelination. B and C Transmission electron micrographs. B) Foci of remyelination.(circle) C) Detail of the forward. Remyilinated axons by oligodendrocytes (arrows)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2419 The influence of the local treatment with simvastatin in the collagen organization during tendon healing

Oliveira L. P.1, Guerra F. D.1, Vieira C. P.1, Almeida M. S.1, Salgado C. M.1, Simões G. F.1, Oliveira A L R1, Pimentel E. R.1
1Department of Structural and Functional Biology – Institute of Biology, State University of Campinas – UNICAMP, Campinas, SP, Brazil
lelekapr@hotmail.com

Although the Achilles tendon is the strongest tendon in the body, it is also the most affected by ruptures. Tendon repair is a slow and complicated process and for these reasons researchers have investigated various treatments to accelerate the healing process and the recovery of the tendon integrity. Statins are drugs widely prescribed for the treatment of hypercholesterolemia and in the last few years studies have showed the benefits of these drugs in the recovery of bone fractures and wound healing making evident the anti-inflammatory, immunomodulatory and angiogenic effects of the statins. Considering that statins are beneficial in several injuries and that there are no studies about the effect of statins in the tendon healing, this work has investigated the influence of local application of simvastatin in the tendon after partial tenotomy.
Wistar rats were treated for 21 days after partial tenotomy and divided in the following groups: (N) intact tendon; (L) injured tendon; (LSL) injured tendon treated with local administration of a sponge of collagen soaked with simvastatin (2.2 mg/50 µl) Simvastatin was dissolved in aqueous solution of 0.5% carboxymethylcellulose (CMC); (LVL) injured tendon with local administration of a sponge of collagen soaked with an aqueous solution of 0.5% CMC. Sections with 7 µm, without stain, were analyzed through polarizing microscope for detecting birefringence and alterations in the organization of collagen fibers. Other sections were stained with toluidine blue (TB) (pH 4.0) and analyzed under light microscope to detect cells and glycosaminoglycans (GAGs). CatWalk system was used to evaluate gait recovery.
Birefringence measurements showed significant differences between the groups (Fig 1). The groups L, LVL and LSL had lower birefringence than the N group that exhibited the greatest organization of the collagen fibers (Fig 2). Birefringence analysis also showed better collagen organization in the LVL group when compared to the other two injured groups. TB stain showed intense metachromasy in the L and LSL groups indicating higher content of GAGs (Fig 3). The analysis of gait recovery showed no significant differences between the groups (Fig 4).
Therefore, LVL showed a higher birefringence than the L and LSL groups, indicating that this group had a better deposition and aggregation of collagen I in the extracellular matrix. Due to the apparently high content of GAGs in LSL, we believed that the aggregation of collagen may be impaired. In conclusion, our results suggest that local administration of simvastatin had no beneficial effects in the collagen organization and in gait recovery after 21 days of treatment. Perhaps the treatment with simvastatin needs more time to make effect on the collagen organization.

The authors thank FAPESP for the fellowship (2013/04071-0) awarded to Oliveira, L. P. and CNPq for the financial support.

Fig. 1: Longitudinal sections of the tendons from different groups observed by polarization microscopy where (a) N, (b) L, (c) LVL, (d) LSL. The larger tendon axis was set at 45° to the polarizers. N group: strong birefringence of the collagen fibers is observed. LVL group is apparently more organized when compared to the other injured groups. Bar 50µm

Fig. 2: Birefringence measurements. The largest axis of the tendon was positioned at 45°with respect to the crossed polarizers. The number of measurements (100) chosen at random in 9 sections from 3 tendons of each group. LVL group had higher birefringence value when compared to the other injured groups. GA: Gray Average.TR transection region. (*) p<0.05

Fig. 3: Tendons sections stained with TB where (a) N, (b) L, (c) LVL, (d) LSL. Observe the bigger number of cells in the injured groups and the intense metachromasy especially in the L and LSL groups. Bar 50µm

Fig. 4: Max contact intensity during gait of the rats obtained by the catwalk system. Measurements performed on animals 21 days after injury. Measurements were made on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, 15th, 17th, 19th and 21st days after injury. There are no significant differences between the groups.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2373 The effect of oxytocin treatment on streptozotocin (STZ) - induced diabetic adult rat liver.

Köroğlu P.1, Bulan Ö.1, Bingöl Özakpınar Ö.2, Uras F.2, Arbak S.3
1Istanbul University, Faculty of Science, Department of Biology, Vezneciler, Istanbul, Turkey., 2Marmara University, School of Pharmacy, Department of Biochemistry Istanbul, Turkey., 3Acıbadem University, School of Medicine, Department of Histology and Embryology, Istanbul, Turkey.
pnnar88@hotmail.com

Diabetes mellitus, a serious metabolic disorder, causes damage in many tissue and organs (1). It has a significant damaging impact on liver. This experimental study has been designed to investigate possible therapeutic and protective effects of oxytocin; a well-known antioxidant in several organs (2) on liver of STZ- induced diabetic rats which were treated with oxytocin before and after STZ administration.
4 experimental groups each containing 6 adult Wistar Albino rats were established. 1) control group: 1 ml of saline solution was injected intraperitoneally (i.p.) for 5 days, 2) STZ group: a single dose of STZ 65 mg/kg, freshly dissolved in 1 ml of saline solution was injected i.p. , 3) pre-oxytocin group: 5 µg/kg of oxytocin was injected i.p. for 5 days prior to the administration of a single dose of STZ injection, 4) post-oxytocin group: 5 µg/kg of oxytocin was injected i.p. for 5 days beginning by 28th days following the administration of single dose of STZ injection. Rats with blood glucose levels of 200 mg/dl or higher were considered to be diabetic and included in the study. Sacrification at the end of the 4th week, liver tissue samples were taken to be processed for light microscopy. Blood samples were processed for malondialdehyde (MDA), glutathione (GSH) and advanced oxidation protein products (AOPP) measurements. Paraffin sections from liver, stained with Haematoxylin and Eosin (H&E), were evaluated under a light microscope. Body weights of experimental animals were measured and all data were analyzed by Graph-Pad Prism.
Liver injury, based on disrupted arrangement of hepatocyte plates, sinusoidal dilatation, hyperemia, vasocongestion, , pyknotic nuclei in degenerated hepatocytes was scored by using a scale ranging from 0 to 3 (0: none; 1: mild; 2: moderate; and 3:severe) for each criterion. Control group reflected a normal liver parenchymal histology. In STZ group, swollen, hypertrophied hepatocytes with pyknotic nuclei, hyperemia, vasocongestion and sinusoidal dilatations reflected prominent tissue damage. Microscopic analysis of tissue sections from pre-oxytocin group demonstrated a reduction in the severity of liver parenchymal damage. Tissue degeneration in post-oxytocin group was quite similar to that of STZ group. According to the biochemical data, oxytocin treatment led to a decrease in liver tissue damage which was more prominent in pre-oxytocin group compared to post-oxytocin group.
We can conclude that oxytocin pretreatment reduced the degree of liver injury in STZ- induced diabetic rats by providing a cellular protection against oxidative stress produced by STZ-induced diabetes mellitus.

1. Szkudelski T. (2001). Physiol Res, 50: 536–546.
2. Lee J, Macbeth AH, Pagani J, Scott W. (2009). Prog Neurobiol, 88 (2):127–151.

This work was supported by Acıbadem, Marmara and Istanbul University.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2509 Acute Effects of Different Resuscitation Fluids on Renal Tissue in a Rat Model of Endotoxemia

Kandil A.1, Ergin B.2, Silke B.3, Corrina L.3, Demirci-Tansel C.1, Ince C.2
1Department of Biology, University of Istanbul, Istanbul Turkey , 2Department of Translational Physiology, Academic Medical Center, Amsterdam, The Netherlands, 3Fresenius Kabi Deutschland GmbH, Bad Homborg, Germany
aslikandil@istanbul.edu.tr

In the early stage of sepsis, impairment of the renal microcirculation is a key complication potentially leading to renal failure through hypoxia-induced tubular epithelial cell injury and acute tubular necrosis. Fluid resuscitation during sepsis is considered crucial for the preservation of adequate intravascular volume and blood pressure and thereby promotion of microvascular perfusion and renal oxygenation. Balanced fluids are able to improve renal oxygenation, oxidative stress, and renal function under septic conditions remains to be elucidated where titration of an optimal dose is still an area of uncertainty. Balanced 6% HES (130/0.4) dissolved in Ringer’s acetate solution (HES-RA; Volulyte® 6%, Fresenius Kabi) or a new experimental solution (AQIX®RS-I), or isotonic saline 0.9% NaCl were investigated as to their efficacy in renal tissue in a rat model of LPS-induced endotoxemia.
Male Wistar-albino rats were randomized in 5 groups (n=6 per group) to receive intravenous administration of 10 mg/kg lipopolysaccharide (LPS; Escherichia coli serotype: A127:B8, Sigma) or vehicle (time control) in 30 min. An amount of 20 ml/kg/hr Volulyte® 6% (Fresenius Kabi), 20 ml/kg/hr and 40 ml/kg/hr a new balanced solution AQIX®RS-I and 20 ml/kg/hr and 60 ml/kg/hr of 0.9%NaCl was continuously given for a period of 180 min after a period of 120 min a mean arterial pressure (MAP) of about 60 mmHg was reached. After the experiments, kidneys were isolated and analyzed immunohistochemically for inducible nitric oxide synthase (iNOS), fatty acid binding protein (FABP), interleukin-6 (IL-6) and myeloperoxidase (MPO) expression.
AQIX®RS20 or AQIX®RS40 administration reduced the increased levels of iNOS and IL-6 reactions, and MPO-stained leukocytes in LPS group compared with control group. AQIX®RS20 decreased the L-FABP reaction, whereas AQIX®RS40 did not decrease its reaction in LPS group. In conclusion, these results showed that AQIX®RS was effective partially on prevention of oxidative stress and inflammation in renal tissue.

Thanks to Fresenius Kabi AG

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2530 The Effects of Resveratrol on Cyclophosphamide-Induced Ovarian Damage in Rats

Ozdemir U.1, Ozyaman S.1, Ahishali B.1
1Department of Histology and Embryology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey
bahishali@yahoo.com

The preservation of fertility during cancer therapy remains a major challenge in women in the reproductive age. The adverse effects of alkylating chemotherapeutic agent cyclophosphamide on folliculogenesis in the ovary are well-known. On the other hand, resveratrol, a polyphenolic phytoalexin, has been shown to exert antioxidant, antinflammatory, cardioprotective, DNA protective and neuroprotective effects in a variety of clinical and experimental settings. This study aims to evaluate the effects of resveratrol on cyclophosphamide -induced ovarian damage in rats.

For this purpose, 28-day-old immature Wistar Albino female rats were treated with pregnant mare serum gonadotrophin (PMSG) to develop the first generation of preovulatory ovarian follicles. Then, the animals in experimental groups were treated with cyclophosphamide (100 mg/kg, i.p), resveratrol (25 mg/kg/day, i.p), cyclophosphamide + resveratrol or vehicle ethanol. Forty eight hours after PMSG injection, rats were sacrificed, the ovaries were removed and embedded in paraffin. Cleaved caspase-3 immunohistochemistry and TUNEL staining were performed on paraffin sections to determine the apoptotic process in the ovarian follicles. To compare the intensity of cleaved caspase-3 expression and the extent of TUNEL labeling between experimental groups, a semiquantitative assessment of immunostaining in granulosa and theca cells of the follicles at different development stages was performed by h-scoring on four sections obtained at regular intervals from serial sections of the ovaries.

Cleaved caspase-3 immunoreactivity and TUNEL labeling were significantly increased in granulosa and theca cells of the multilaminar primary, secondary and Graafian follicles in cyclophosphamide-treated rats. In these animals, resveratrol treatment significantly reduced the increased cleaved caspase-3 immunoreactivity in granulosa and theca cells of the multilaminar primary follicles and in cumulus and mural granulosa cells of the Graafian follicles (Figure 1). In addition, a significant decrease of the increased TUNEL labeling was observed in granulosa and theca cells of the multilaminar primary follicles and in mural granulosa cells of the Graafian follicles (Figure 2).

In conclusion, our data suggest that resveratrol treatment may provide a choice of pharmacologic approach in the preservation of fertility owing to its beneficial effects on chemotherapy-induced apoptotic process in ovarian follicles at both early and late phases of development.

This study was supported by the Research Fund of Istanbul University (28586).

Fig. 1: Representative images showing cleaved caspase-3 immunostaining of Graafian follicles from ovaries of rats in cyclophosphamide (a) and cyclophosphamide + resveratrol (b) groups. Scale bars = 100 µm.

Fig. 2: Representative images showing TUNEL labeling of multilaminar primary follicles from ovaries of rats in cyclophosphamide (a) and cyclophosphamide + resveratrol (b) groups. Scale bars = 25 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2574 Dominici staining in the observation of inflammatory infiltrate caused by a new acidic bothropic PLA2

Marques P. P.1, Guerra F. D.2, Esteves A.2, Ponce-Soto L. A.1, Marangoni S.1
1Universidade Estadual de Campinas, Campinas, Brazil, 2Universidade Federal de Alfenas, Alfenas, Brazil.
petruspm@gmail.com

Ubiquitous in nature, the phospholipases A2 (PLA2) hydrolyze phospholipids at the sn-2 position and exhibit a large variety of biological functions. In the snakes, the PLA2 are among the most toxic compounds present in the venoms, exhibiting pharmacological effects: myotoxic, neurotoxic, anticoagulant and others. Some PLA2 possess high inflammatory capacity that can occur by several different paths. The Dominici stain allows the identification of different inflammation involved cells and it can help in the identification of the inflammation pathway induced by the toxin. In the present work, Bothrops hyoprora venom was fractionated via gel filtration chromatography and a PLA2 containing pool was submitted to reverse phase HPLC, yielding a new purified acidic PLA2. Inflammatory activity was monitored by observation of edema formation and cytokines IL-1α, IL-6 and TNF-α concentration in plasma, measured with commercially available kits. Purified toxin was applied in mice gastrocnemius muscle, which was removed and fixed using a 4 % formaldehyde solution in Millonig’s buffer (0.13 M sodium phosphate, 0.1 M NaOH– pH 7.4) for 18 hours at 4º C and washed in water, ethanol dehydrated, diaphanized with xylene and paraffin-embedded. Sections of 7μm were deparaffinized and immersed in water. After that, different sections were stained by hematoxylin-eosin and by a modified Dominici stain: they were submersed for 30 minutes in a mixture of acid fuchsin and orange G (0.5% each in distilled water); after that followed a quickly rinse in 60% ethanol and a counterstain in toluidine blue 0.75% for 20 seconds and another ethanol rinse. The sections were then put in 95% ethanol until the stains differentiate. The images were captured with Zeiss Axio Scope A1 Microscope. Previous experiments showed that this toxin had no myotoxic, anticoagulant or antimicrobial capacities. Nonetheless, it presented moderate edematogenic properties and high inflammatory capacity, elevating several times the concentration of interleukins 1α, 6 and TNF-α. The microscopic observations revealed no muscle damage caused by the toxin, as expected, but it was possible to observe inflammatory infiltrate in the samples. The Dominici stain made possible to identify the cells in the infiltrate as mast cells. These results are coherent with the data obtained by interleukins tests and lead to think that the source of the increase in the cytokines concentration is mast cell degranulation, triggered by the isolated acidic PLA2 injection.

The authors gratefully acknowledge financial support received from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil).

Fig. 1: Profile of cytokines IL-1α, IL-6 (pg/ml) and TNF- α (U/ml) plasmatic concentrations along 12 hours after PLA2 injection (filled markers) and phosphate buffer injection (empty markers).

Fig. 2: Sections of the gastrocnemius muscles from mice injected with PLA2 and phosphate buffer, where: A, B, C- section stained with HE of muscle injected with phosphate buffer (A) or PLA2 (B and C). D, E, F- sections stained with Dominici of muscle injected with phosphate buffer (D) and PLA2 (E,F). Bar in A,B,: 40µm. Bar in C,D,E,F: 20 µm.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2593 The effects of ozone therapy on inflammatory response associated with traumatic spinal cord injury in cutaneous wound healing in rats

Irban A.1, Uslu S.2, Emon Tural S.3, Aydınlar Ilgaz E.4, Can O.5, Ozpinar A.5
1Medipol University School of Medicine, Department of Anesthesiology and Reanimation, Istanbul, Turkey , 2Acibadem University, Vocational School, Istanbul, Turkey, 3Numune Hospital, Department of Neurosurgery, Istanbul, Turkey, 4Acibadem University School of Medicine, Department of Neurology, Istanbul, Turkey, 5Acibadem University School of Medicine, Department of Medical Biochemistry, Istanbul, Turkey
musiuslu@gmail.com

Intraduction: At the cellular level, experimental spinal cord injury (SCI) provokes an inflammatory response, leading to delay in wound healing and low intensity of transforming growth factor-β1 (TGF-β1) in the dorsal wound-tissue specimens. Systemic application of ozone leads to delivery of super enriched oxygen at a cellular level and optimizes cell function via activating the red blood cell, immune-competent cells, the enzymatic antioxidants and radical scavengers at a cellular level. The aim of this study was to investigate the effects of ozone therapy on inflammatory response associated with SCI in cutaneous wound healing in rats.

Method: The rats were allocated to one of the three groups: Group T (trauma only,n=7): SCI was performed as described. The dorsal wound margins were apposed with a non-absorbable interrupted suture. Then, 4 mL of medical air was insufflated rectally during 1 min. via an 18G cannula once a day for 5 consecutive days. Group O (trauma+ozone, n=7): After trauma induction, instead of medical air, 4 mL of ozone (10mcg/mL) was insufflated. Group Control (no trauma,n=2): No treatment was applied. All rats were sacrificed at Day 14. Wound samples taken from the dorsum of the rats were evaluated histologically (as thickness of epithelium, thickness and regeneration of collagen fibers), immunohistochemically (expressions of TGF-β and VEGF), histomorphometrically (the number of blood vessels) and biochemically (hydroxyproline and hydroxyproline/protein levels). For statistical analysis, non-parametric ANOVA and Dunn’s test as a post-hoc were used (p<0.05).

Results: Histological evaluation of the tissue samples were revealed that vessel count was higher in ozone than in other groups (p<0.001 for each). Hydroxyproline levels in ozone group is approximately 1.8 times greater (p<0.05) when compared to others. Similarly, hydroxyproline to total protein ratios were 1.5 and 1.6 times greater (p<0.05) in ozone group when compared to others. Although, in histochemistry analyses revealed that collagenization in ozone group was lesser than in control group (p<0.001), it was significantly higher than in trauma group (p<0.001). Also, collagen organization was worse in trauma group than both ozone (p<0.05) and control (p<0.01). Although expression of TGF-β and VEGF was worse in ozone group than in control group (p<0.001); expressions of these growth factors was worse in trauma group then the others (p<0.001 for both groups).

Conclusion: Based on the results on the parameters evaluated in the study, ozone therapy reverses the inflammatory response associated with traumatic SCI, leads to better dorsal cutaneous wound healing via intensifying expressions of TGF-β and VEGF in rats.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2647 Polarization microscopy and birefringence analysis as a tool to evaluate the best treatment protocol for Achilles tendon injuries

Guerra F. D.1, Marques P. P.2, Vieira C. P.3, Oliveira L. P.3, Almeida M. S.3, Rossi Junior W. C.1, Esteves A.1, Soares E. A.1, Pimentel E. R.3
1Departament of Anatomy, Biomedical Sciences Institute, Unifal-MG, Brazil., 2Department of Biochemistry, Biology Institute, UNICAMP, SP, Brazil., 3Cellular and Structural Department, Biology Institute, UNICAMP, SP, Brazil.
dgflavia@yahoo.com.br

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-2656 The role of Notch signaling pathway in endometrial cycle

Tanriverdi G.1, Gumusel A.1, Ilvan S.1, Guzel E. E.1
1Cerrahpasa Medical Faculty of Istanbul University, Istanbul, Turkey
gamzetanriverdi@gmail.com

The human endometrium, during every reproductive cycle, undergoes extensive tissue remodelling in response to cyclical hormonal changes. Estrogens stimulate proliferation and re-establishment of the stromal and vascular components of the tissue including many proteins and several transcription factors, especially angiogenic factors, that are important for DNA replication and cell division. Angiogenesis, the formation of new blood vessels, plays an important role in the remodelling of endometrial tissue. Notch is a transmembrane receptor which belongs to a growth factor family. It is structurally and functionally conserved during evolution and it regu­lates cell fate specification, stem cell maintenance, and initiation of differentiation in embryonic and postnatal tissues. The mammalian family of Notch protein consists of four different isoforms (Notch 1-4) and its ligands can be classified into two classes as Jagged 1-2. Activation of Notch signaling pathway promotes the development of the vascular system in embryo, normal adult tissues, and cancerous lesions. Notch genes and their ligands are known to be expressed in endothelial cells, however little is known about their expression in the human endometrium, and their role in the cyclical endometrial changes. The purpose of this study was to investigate the cellular localization of Notch-1 and Jagged-1 in normal endometrium at different endometrial phases under the control of steroid hormones by immunohistochemistry.
Endometrial tissue samples were obtained from 24 women with regular menstrual cycles operated because of benign gynecological conditions. Menstrual cycle phases were determined and immunhistochemistry was applied to the sections and the immunostaining was evaluated semiquantitatively.
Notch-1 and Jagged-1 immunoreactivities were both detected on cell membranes. The expression of both proteins were correlated and they showed weak immunoreactivities in early-mid proliferative phase of the menstrual cycle in both glandular epithelium and stromal cells. On the other side, in late proliferative and early secretory phases, the expressions of the proteins were the highest among the other phases of the cycle. During the late secretory phase the expressions were weak as in the early-mid proliferative phase in the glandular epithelium and stromal cells. According to our knowledge, this is the first study which shows cyclic regulation of Notch signaling pathway in the endometrium. Notch signaling pathway is shown to have an important role in cyclic structural changes of endometrium and disregulation of these functions may lead to several endometrial pathologies.

Fig. 1: Representative photomicrographs of Notch-1 expression in the endometrium at different cycles. Weak immunoreactions belong to early-mid proliferative (a) and late secretory phases (d). Strong immunoreactions are observed at late proliferative (b) and early-mid secretory phases (c). All magnifications are X 20.

Fig. 2: Representative photomicrographs of Jagged-1 expression in the endometrium at different cycles. Weak immunoreactions belong to early-mid proliferative (a) and late secretory phases (d). Strong immunoreactions are observed at late proliferative (b) and early-mid secretory phases (c). All magnifications are X 2
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-3215 Expression levels and localizations of Notch-3 and Notch-4 proteins in different menstrual phases of human endometrial tissues

Tanriverdi G.1, Ozkan S.1, Gumusel A.1, Ilvan S.2, Guzel E. E.1
1Department of Histology and Embryology, Istanbul University Cerrahpasa School of Medicine, 2Department of Pathology, Istanbul University Cerrahpasa School of Medicine
ozkanserbay@gmail.com

Endometrium is a unique tissue characterized by the changes in a cyclic fashion taken place under the control of estrogen and progesterone hormones. It has been shown that expression levels of various types of proteins like heat shock proteins, growth factors, cytokines and several signaling molecules those get involved in endometrial changes have clear differences at various phases of menstrual cycle. Notch signaling is an evolutionarily conserved mechanism used to regulate cell fate decision. Four Notch receptors (Notch 1-4) and two ligands Jagged 1-2 have been identified in mammals. In several studies it has been demonstrated that Notch signaling is responsible for significant processes, like differentiation, proliferation and apoptosis those are finely regulated in human endometrium in order to allow a successful menstrual cycle. Notch signaling pathway has active role during the blood vessel formation in angiogenesis process for endothelial cells. One of the main morphogenetic events occurring during the menstrual cycle is formation of spiral arteries(1). In this respect, there is little known about the expression levels and localization of notch-3 and notch-4 proteins in human endometrial tissue. So the aim of this study is to investigate the expression levels of Notch-3 and Notch-4 proteins in the normal endometrial samples surgically removed from 24 women. Tissues were divided according to the menstrual cycle phases, and immunhistochemistry was applied to the sections, and the immunostaining was evaluated semiquantitatively.
In the cytoplasm of glandular epithelium cells, both Notch-3 and Notch-4 immunoreactivities were observed. The expression levels of the proteins were correlated, and they showed mild immunoreactivities in early-mid proliferative phase of the menstrual cycle in glandular epithelium. On the other hand, in late proliferative as well as early secretory phase, the expression levels of the proteins were the highest with respect to the other phases. During the late secretory phase, the expressions were mild, which is the similar situation for early-mid proliferative phase in glandular epithelium. In this study, cyclic regulation of Notch-3 and Notch-4 proteins in the endometrium is demonstrated for the first time. According to our results, cyclic expression of Notch-3 and Notch-4 proteins seems to be involved in regulation of structural changes of endometrium and any malfunctioning related to those proteins may cause several endometrial pathologies.

1- Cobellis L, Caprio F, Trabucco E, Mastrogiacomo A, Coppola G, Manente L,Colacurci N, De Falco M, De Luca A. The pattern of expression of Notch protein members in normal and pathological endometrium. J Anat. 2008 Oct;213(4):464-72.

Fig. 1: Representative photomicrographs of Notch-3 expression in the endometrium at different phases. Mild immunoreactions belong to early-mid proliferative (a) and late secretory phases (d). Strong immunoreactivities are seen at late proliferative (b) and early-mid secretory phase (c).

Fig. 2: Representative photomicrographs of Notch-4 expression in the endometrium at different phase. Mild immunoreactions belong to early-mid proliferative (a) and late secretory phases (d). Strong immunoreactions are seen at late proliferative (b) and early-mid secretory phases (c). All magnifications are X 20.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-3333 Immunological microenvironment is shaping the collagen structures evidenced by SHG imaging

Chernyavskiy O.1, Kubínová L.1, Stakveev D.2, Vannucci L.2
1Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14200 Prague 4, Czech Republic, 2Institute of Microbiology Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14200 Prague 4, Czech Republic
cernavsky@biomed.cas.cz

Experimental animal models allow reproducing phases of the carcinogenesis and tumor microenvironment evolution closely to the human pathology, in particular for colorectal carcinogenesis and chronic colitis models. Azoxymethane (AOM) induced experimental cancers, in rat and mouse, present features resembling the human pathology [1, 2], as well as the ulcerative colitis model with dextran sulfate sodium (DSS). Under conventional (CV) conditions, i.e. commensal microflora in the bowel, the mucosal immunity is persistently activated (so-called “physiological inflammation”) by the continuous solicitations exerted by the microbiota on the gut associated lymphoid tissue. Therefore, different stimulation of the mucosal immunity under the two conditions can elicit different immune environments and responses, even influencing tissue structures. Germ-free reared (GF) animals may represent a new and strong model to evaluate regulatory mechanisms of inflammation and their effects on environment constitution in comparison with the CV animals [3].
When applied in vivo, the SHG Imaging can provide important results in describing physio-pathologic events, e.g., consequent to manipulation (hyperthermia) of the melanoma microenvironment [4]. Therefore, SHG appears a tool suitable of more intensive application for biological and pathological processes evaluation, very perspective also in diagnostics. This technique allows effective investigations in vivo (or in fresh tissue samples) even without staining, offering realistic detection of structural characteristics related to biological phenomena. Digital images of confocal stacks represent suitable data for quantitative measurements and for computer three-dimensional reconstructions, performed with no need of aligning and stitching images of successive sections. Here we present a quantitative comparison of SHG image data obtained in vivo as well as on fresh tissue samples.
References
1) Reddy BS. Studies with the azoxymethane-rat preclinical model for assessing colon tumor development and chemoprevention. Environ Mol Mutagen 44 (1): 26-35, 2004;
2) Guda K, et al. Defective processing of the transforming growth factor-beta1 in azoxymethane-induced mouse colon tumors. Mol Carcinog 37(1): 51-9, 2003;
3) Vannucci L, Stepankova R, Kozakova H, Fiserova A, Rossmann P, Tlaskalova-Hogenova H: Colorectal carcinogenesis in germ-free and conventionally reared rats: different intestinal environments affect the systemic immunity. Int J Oncol 32 (3): 609-17, 2008;
4) Chernyavskiy O., Vannucci L., et al. Imaging of mouse experimental melanoma in vivo and ex vivo by combination of confocal and nonlinear microscopy. Microsc Res Tech 72 (6): 411-23, 2009.

The work was funded by GAAV IAA500200917, RVO61388971 (CZ); ENI Czech Republic, Prague (CZ), Paul’s Bohemia s.r.o, Prague (CZ), Rusconi Foundation, Varese (IT).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-3354 The Effects of Retinoic Acid on Renal Cells in Mice

Kotil T.1, Erdogan A.1, Ozdemir I.1, Mutlu H. S.1, Sarıoglu T.2, Tapul L.1
1Istanbul University, Istanbul Faculty of Medicine, Çapa, Istanbul, 2Istanbul Science University, Esentepe, Istanbul
leylakuntsal@yahoo.com

The Effects of Retinoic Acid on Renal Cells in Mice
Tuğba Kotil1, Aslı Erdoğan1, İlkay Özdemir1, Hasan Serdar Mutlu1, Türkan Sarıoğlu2, Leyla Tapul1
1İstanbul University, İstanbul Faculty of Medicine, Çapa, İstanbul
2İstanbul Science University, Esentepe, İstanbul

Vitamin A derivative Retinoic acid (RA), has effects on cell cycle, proliferation and differentiation (1). Various studies propounded that RA has critical roles on renal development and repairment of renal damage (2).Aim of this study is to investigate the effects of exogenous RA on cell proliferation and RAR alpha expression in rat kidney.

12 adult female balb-c mice were used in control and experimental group. 80mg/kg/day 13-cis RA was applied for 5 days in experimental group, but in control group only saline was given by gavage. Kidneys were embedded in paraffin and stained with anti RAR alpha and BrDU antibody.

As BrDU immunreactivity was investigated, no anti-proliferative effect was detected in RA treated group (Fig. 1). Positive RAR alpha immunoreactivity was seen especially in epithelial cells of loop of Henle and collecting duct in medulla rather than glomerular and tubular cells (Fig. 2).
Intracelluar retinoic acid level depends on enzymes that synthesize and metabolize RA, transport proteins and nuclear receptors. The finding that endogenous retinoic acid has effects on principle cells and intercalated cells of collecting duct is compatible with our study which shows RAR immunoreactivity in collecting duct cells of mice kidney treated with exogenous retinoic acid (3). Although Xu Q. et al. (4) showed that exogenous RA treatment increases mortality and fibrosis in a dose-dependent manner in endogenous RA deficient transgenic mice, retinoic acid dose which is used in our study has no significant antiproliferative effect on healthy kidney cells.
References
1. Argiles A, Kraft N, Hutchinson P, Senes-Ferrari S, Atkins RC: Retinoic acid affects the cell cycle and increases total protein concent in epithelial cells. Kidney Int 1989; 36(6): 954-959
2. Gilbert T: Vitamin A and kidney development. Nephrol Dial Transplant 2002; 17 Suppl 9: 78–80
3. Wong YF, Kopp JB, Roberts C, Scambler PJ, Abe Y, Rankin AC, Dutt N, Hendry BM, Xu Q: Endogenous Retinoic Acid Activity in Principal Cells and Intercalated Cells of Mouse Collecting Duct System. Plos One, 2011;6(2):e16770
4. Xu Q, Hendry BM, Maden M, Lu H, Wong YF, Rankin AC, Noor M, Kopp JB: Kidneys of Alb/TGF-1 Transgenic Mice Are Deficient in Retinoic Acid and Exogenous Retinoic Acid Shows Dose-Dependent Toxicity. Nephron Exp Nephrol 2010;114:e127–e132

Fig. 1: Fig. 1: RA treated group BrdU immunoreactivity

Fig. 2: Fig.2: RAR alpha immunoreactivity of Henle loop and collectory canals of RA treated group.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-3368 AFM observation of changes in the extracellular matrix of articular cartilage in Wistar rats with experimental osteoarthritis.

Miranda - Sánchez M. M.1, Fragoso- Soriano R.2, Martínez- Calleja A.1, Kouri J. B.1
1 Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F, 2Departamento de Física, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F
mmiranda67mx@yahoo.com.mx

Osteoarthritis (OA) is a joint disease characterized by progressive degeneration and loss of articular cartilage, ultimately resulting in severe pain and disability. Articular cartilage derives its functional mechanical properties from its extensive extracellular matrix (ECM) of type II collagen and proteoglycans. In OA, ECM degeneration is characterized by extensive proteolysis of the type II collagen network and proteoglycans (1,2). Relationships between these structural changes and altered cartilage mechanical properties have been observed at all stages of OA degeneration. This study investigated the changes of mechanical properties and surface roughness in OA progression.
Animal samples were obtained following the Guidelines of the Internal Committee for the Care and Use of Laboratory Animals (NOM-069-ZOO-1999). Normal articular and osteoarthritic cartilage was obtained from femoral condyles of male adult Wistar rats (120-150 g) and rats with OA induced by partial menisectomy (5,10,20 and 45 days after surgery compared with normal). Full-thickness rat normal and osteoarthritic articular cartilages from weight bearing areas were fixed with 4% PBS paraformaldehyde at 4°C, and cryosectioned in 6 µm. The samples were scanned with an atomic force microscope (AFM) (Autoprobe CP Research, Thermomicroscopes) operating in contact mode.
In normal cartilage it was observed collagen fibers in double or triple junction, which provides strength and stability to the cartilage. In OA cartilage (45 days after induction) we saw that the integrity of these junctions were lost.
Using AFM, we can get new images of microstructures that help us to understand the biological processes occurring in the OA pathogenesis.
These results are part of a comprehensive study on osteoarthritis in the Wistar rat model; which provides an insight of OA pathogenesis.

References
1.Plaas A, Osborn B, Yoshihara Y, Bai Y, Bloom T, Nelson F, et al. Aggrecanolysis in human osteoarthritis: confocal localization and biochemical characterization of ADAMTS5-hyaluronan complexes in articular cartilages. Osteoarthritis Cartilage
2007;15:719e34.
2. Lark MW, Bayne EK, Flanagan J, Harper CF, Hoerrner LA, Hutchinson NI, et al. Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints. J Clin Invest 1997;100:93e106.

This project was supported by CONACYT-Mexico, the project no. 168328

Fig. 1: Normal cartilage.Lefth 2D image, double or triple very tight binding of the collagen fibers is observed . In right side 3D image, viewed from another perspective or triple pairing arrangement of the collagen fibers

Fig. 2: OA cartilageLefth 3D image of a sample of OA cartilage 45 days after induction, where the disorder of the collagen fibers is observed, we also observed a similar formations holes in the extracellular matrix. In right side another 3D perspective in which the loose arrangement of the collagen is clearly observed
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-3388 Menisectomized mini-pigs develop articular cartilage pathology resembling osteoarthritis

Cruz R.1, Ramírez C.1, Rojas O. I.1, Casas- Mejía O.1, Kouri J. B.1, Vega- López M. A.1
1Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F., México.
mmiranda67mx@yahoo.com.mx

Osteoarthritis (OA) is a chronic-degenerative and incapacitating disease, characterized by deterioration of the articular cartilage, synovitis, and alteration in the peri-articular and subchondral bone [1]. Rodent animal models have contributed to the understanding of the basic biology of the OA and have helped to describe new candidate biomarkers for diagnosis and treatment [2]. However, rodents and humans are different in many aspects, including physiological traits and gene expression. Thus, rodent models cannot sufficiently mimic human diseases in some cases and large animal models remain needed. Pigs have been used as models for human diseases because they are similar to humans in terms of anatomy, neurobiology, cardiac vasculature, gastrointestinal tract, and genome [3]. Therefore, we analyzed the articular cartilage pathology in pigs with induced joint instability to determinate if this model could be useful for the study of OA pathogenesis.
Based in our rat OA model [4], joint pathology was induced in Vietnamese pigs (Sus scrofa domestica) by unilateral knee menisectomy and post-surgery exercise for 20 days; sham-operated pigs were used as controls. Cartilage sections were fixed with 4% paraformaldehyde in PBS and stained with safranin O-fast green to assess proteoglycan content. Immunohistochemistry was performed to determine the expression of IL-1β and MMP-3 proteins.
Histological analysis of cartilage slices from menisectomized pigs produced pathologic characteristics similar to OA such as: chondrocytes cluster formation, fibrillation and depletion of proteoglycan content, mainly in the superficial zone of cartilage (Figure 1). Metalloproteinases are enzymes involved in the degradation the cartilage extracellular matrix; therefore, we analyzed the expression of MMP-3. Immunohistochemistry studies showed an increase in MMP-3 expression in menisectomyzed pigs when compared to sham operated controls (not shown). Since MMP-3 expression is induced by pro-inflammatory cytokines, such as IL-1β, we investigated the expression of such cytokine in cartilage slices. IL-1β immunostaining increased strongly in cartilage from menisectomized pigs when compared with sham operated controls (Figure 2). Taken together, our results suggest that minipigs developed OA pathology as consequence of menisectomy and exercise and therefore could be a useful model to study the physiopathology of OA disease. However, further studies are required to validate such OA model.
References
[1] M.B. Goldring and S.R. Goldring. J Cell Physiol, 213(2007) 626.
[2] R. Cruz et al. Rec Pat Biomark, 4 (2004).
[3] N. Fan and L. Lai. J Gen Genom, 40 (2013) 67.
[4] K.A. Abbud-Lozoya and J.B. Kouri. Pathol Res Pract, 196 (2000)729.

This research was supported by CONACyT (México), grant 168328.

Fig. 1: Proteoglycan content in porcine articular cartilage. Proteoglycan content was assessed by the safranin O-fast green staining in cartilage slices from sham operated pigs (left) and menisectomized pigs (right). In menisectomized animals is evident the chondrocytes cluster formation (arrow) and fibrillation (arrowhead). Scale bar: 50 µm.

Fig. 2: Expression of IL-1β in porcine articular cartilage. IL-1β expression was determined by immunohistochemistry in cartilage slices from sham operated pigs (left) and menisectomized pigs (right). Scale bar: 50 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-3392 AIDPATH – Digital Pathology Tools from and for Academia and Industry Collaboration

Bueno G.1, Fernández M. M.1, Womack C.2, Segers D.3, Ecker R.4, Costello S.5, Della Mea V.6, Carasevici E.7, Ilyas M.8, Qiu G.8, González L.9, Laurinavicius A.10, Schaefer G.11, García-Rojo M.12, Déniz O.1
1VISILAB, UNIVERSIDAD CASTILLA-LA MANCHA, ES, 2ASTRAZENECA, UK, 3BARCO, BE, 4TISSUEGNOSTICS, AT, 5LEICA MICROSYSTEMS, IE, 6UNIVERSITA DEGLI STUDI DI UDINE, IT, 7UNIVERSITATEA DE MEDICINA SI FARMACIE GR.T.POPA IASI, RO, 8UNIVERSITY OF NOTTINGHAM, UK, 9HOSPITAL GENERAL UNIVERSITARIO CIUDAD REAL, ES, 10VIESOJI ISTAIGA VILNIAUS UNIVERSITETO LIGONINES SANTARISKIU KLINIKOS, LT, 11LOUGHBOROUGH UNIVERSITY, UK, 12HOSPITAL DE JEREZ DE LA FRONTERA, ES
gloria.bueno@uclm.es

Advances in digital pathology are generating huge volumes of whole slide images (WSI) and tissue microarray (TMA) images which are providing new insights into the causes of some of today’s most devastating diseases. They also present tremendous opportunities for developing and evaluating new and more effective treatments that may revolutionize the care of patients with cancers and other diseases. The challenge is to exploit new and emerging digital pathology technologies effectively in order to process all the heterogeneous tissue-derived data. AIDPATH project addresses this challenge through a focused research in collaboration between academia and industry. The objectives are:

1) Optimizing and standardizing digital pathology image display: Digital pathology requires make diagnosis through images displayed on electronic devices such as LCD monitors. A WSI is thousands of times larger than an ordinary photograph. AIDPATH is developing novel technology for calibrating and standardizing medical image display devices. Different metrics for ensuring image quality is being developed and integrated into WSI devices (Figure 1).

2) Advanced image analysis for whole slide imaging: The H&E, IHC and FISH stained issue sections represent the mainstay of traditional pathology diagnosis. Automated image analysis of WSI can extract specific diagnostic features of diseases and quantify individual component of these features to support diagnosis and provide informative clinical measures of disease. AIDPATH develops advanced intelligent image analysis tools to automatically extract useful image features (Figure 2).

3) Evaluation and quantification of biomarkers: IHC is a robust and commonly used methodology for identifying the expression of specific biomarkers in tissues. For analysis of marker coexpression, immunofluorescence has significant importance. The application of genetic probes in FISH is now an standard tool in pathology for genetic aberrations analysis. Objective evaluation of biomarkers is needed for disease diagnosis and identification of therapeutic targets in TMA (Figure 3).

4) Clinical evaluation of the processing tools: This will help developing efficient and innovative products to fulfil the needs of digital pathology.

AIDPATH carry out research and develop: a) state of the art medical image display technology for digital pathology, b) novel image analysis solutions and knowledge discovery tools for future pathology diagnosis and research and c) state of the art solutions for biomarker evaluation and quantification.

The authors acknowledge financial support from the EC Marie Curie Actions, AIDPATH project 612471.

Fig. 1: Image quality in digital pathology. Quality evaluation of microscopy and scanned histological images for diagnostic purposes. Example of two captured histological images of size 1800 × 1800 pixels: (a) Scanner, (b) Microscope.

Fig. 2: Advanced image analysis for WSI. AngioPath: Automatic TMA vessel segmentation and characterization based on color and morphological features. Angiogenesis research.

Fig. 3: TMA Evaluation. Automatic Handling of Tissue Microarray Cores in High-Dimensional Microscopy Images for further anlaysis. H&E Breast TMA sample digitized with the motorized microscope.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-3422 Ultrastructure of the Thyroid gland in the West African dwarf goat (Capra Hircus)

Igbokwe C. O.1, Ezeasor N. D.1, Bello U. M.2
1Department of Veterinary Anatomy, Faculty of Veterinary Medicine, University of Nigeria, Nsukka-Nigeria. , 2Department of Anatomy and Physiology, Faculty of Veterinary Science, University of Pretoria, Onderstepoort 0110, South Africa.
mail4umar@yahoo.com


The present study examined the ultrastructure of the thyroid gland of West African Dwarf (WAD) goat, in order to further understand the ultrastructural changes in the components of the thyroid glands in relation with age. Thyroids glands obtained from fifteen (15) apparently healthy WAD goats of different ages and sexes slaughtered at the local abattoirs were used for this study. Electron microscopic techniques were used to study of fixed tissue with emphasis on the follicular and parafollicular cells. Thyroid tissues showed marked regional variation in structure and ultrastructure. The results showed that the follicular cells were cuboidal in thyroid of young goats but appear flattened to columnar, especially in the thyroids of much older goat (5-7 years). These cells were characterized by the presence of markedly dilated cisternae of rough endoplasmic reticulum and well developed Golgi apparatuses, which decreased in the older goats. Microvilli were short and sparse on the follicular cells and their numbers decreased in the older goats. Different sizes of apical vesicles of varying electron density (i.e measuring from 250nm to 1600nm) were encountered, with membrane bound colloid droplets, lysosome-like bodies, abundant secretory vesicles and the presence of these vesicles appear to differ with age. Parafollicular cells were encountered in the basal position between follicular cells in all the thyroids examined. Numerous dense cytoplasmic granules were observed and they were not apparently different from that described in several mammals. This finding indicates that the morphological features of the follicular epithelial cells of thyroid gland in WAD are generally similar to that reported in some domestic animals.

Authors wish to thank University of Nigeria, Nsukka for providing the ETF-ASTD grant (No. 06-2010) for the project and Mrs. Erna Van Wilpe of the Electron Microscopic Unit, Dept. of Veterinary Anatomy and Physiology, Faculty of Veterinary Science (University of Pretoria, South Africa) for her technical support.

Fig. 1: A. Electron micrograph of a high cuboidal follicular cell taken from 3 years-old WAD goat showing a basal nucleus (N). The Golgi stacks (G) close to the nucleus. The cytoplasm is rich in organelles that included abundant mitochondria (M), Rough Endoplasmic Reticulum(RER),and secretory vesicles (S), Colloid droplets(L) (Scale 2.4µm)

Fig. 2: B. Electron micrograph of parafollicular cells taken from 2years- old WAD goat. Note the extensive cytoplasm containing numerous profiles of dense secretory granules (S) and golgi complex(G). Note the rim of cytoplasm of the follicular cell(F) abutting the colloid. (Scale 2.0µm).

Fig. 3: C. Electron micrograph of apical cytoplasm of follicular cells showing some organelles close to the colloid lumen (C). Note the abundant mitochondria (M), Rough endoplasmic reticulum (RER), Lysosomes (L), Colloid droplets (D) and presence of tight junctions (white arrow) between the cells . (Scale 5µm).

Fig. 4: D. Electron micrograph of a follicular cell showing a typical intercellular junctional complex with tight junction (T), desmosome (D). Note the presence in apical cell membrane, a few short microvilli(thin black arrow), mitochondria(M) with visble cristae, some colloid droplets (C) and few ribosomes in the cytoplasm. (Scale 2µm).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-3498 ULTRASTRUCTURAL STUDY OF THE EFFECT OF Tityus sp SCORPION VENOM ON CARDIAC TISSUE OF WISTAR RATS Rattus norvegicus.

Bolaños Burgos I. C.1, Mosquera Sanchez L. P.2, Ceballos Mendoza A. J.3, Guerrero-Vargas J. A.4
1Grupo de Investigaciones Herpetológicas y Toxinológicas de la Universidad del Cauca, Popayán - Colombia., 2Unidad de Microscopía Electronica, Universidad del Cauca, Popayán - Colombia., 3Departamento de Patología, Universidad del Cauca, Popayán - Colombia., 4Departamento de Biología, Universidad del Cauca, Popayán - Colombia.
lmosquera@unicauca.edu.co

The scorpionism is a public health problem worldwide causing serious accidents in children under 10 years. Potentially dangerous scorpions to humans belong to the family Buthidae, in Latin America these events are mainly caused by the genus Centruroides and Tityus. Only in 2008 Colombia enters the list of countries affected by severe scorpion, especially that caused by the scorpion Tityus pachyurus, however Colombia for its megadiversity has more than one dangerous species to humans, among these is Tityus sp endemic Department of Cauca south western Colombia that has the most toxic lethal dose 50 (3.5 mg / kg), reported so far.

The objetive of this study was to determine the ultrastructural level cardiotoxic effect on male Wistar rats (n = 16), weighing (200 ± 20g). A design with four treatments was applied: a control group treated with 0.9% saline (n = 4), and three sub-doses of LD50 (20%, 40% and 80%), each with four rats. After 3 hours of applying venom, the heart was extracted for ultrastructural analysis. Fixation was done in 2.5% glutaraldehyde, the post-fixation in 1% osmium tetroxide, dehydrated with alcohol in increasing concentrations (30% to 100%) and soak in LR White resin. The semithin sections were stained with 500 nm toluidine blue for light microscopy at high resolution and ultra-thin 40 nm thick, and contrasted with uranyl acetate - lead citrate for transmission electron microscopy.

The effect of the venom was observed in all treatments, the most striking pathological changes occurred in the sub-doses of 80%. The main findings of cardiac tissue consisted of edema of muscle fibers, a change that is also evident at the level of mitochondrial cristae, which have notorious separation cause by edema. Also focally, vascular congestion, karyorrhexis, karyolysis and vacuolization of sarcoplasmic that guide toward the emergence of incipient necrosis is observed. It is concluded that the venom of Tityus sp produces damage in the heart of Wistar rats at the cellular and tissue.

Grupo de investigaciones Herpetológicas y Toxinológicas de la Universidad del Cauca (GIHT).

Centro de investigaciones biomédicas de la Universidad del Cauca (CIBUC).

Albeiro Polanco, Departamento de Patología de la Universidad del Cauca.

Fig. 1: Electronic mycrography of rat heart treated with Tityus sp scorpion venom , with mitochondrial edema (asterisk); vacuolization (triangle); edema of the  cardiac fibers (arrow).

Fig. 2: Electronic mycrography of rat heart treated with Tityus sp scorpion venom , with  abundant areas of mitochondrial edema (asterisk).

Fig. 3: Electronic mycrography of rat heart treated with Tityus sp scorpion venom , with ,  karyorrhexis (rhombus); cell congestion (circles).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-3533 Hepatocytes and liver hemopoietic cells responses to mild and severe hyperoxia in newborn rats

Zara S.1, De Colli M.1, Marconi G. D.1, Di Valerio V.2, Rapino M.3, Zaramella P.4, Dedja A.4, De Caro R.5, Porzionato A.5, Cataldi A.1
1Department of Pharmacy, University “G. d’Annunzio” Chieti-Pescara, Italy, 2Department of Medicine and Ageing Sciences; University “G. d’Annunzio” Chieti-Pescara, Chieti, Italy, 3Institute of Molecular Genetics CNR, Unit of Chieti, 4Neonatal Intensive Care Unit, Women's and Children's Health Department, University of Padova, Padova, Italy, 5Department of Molecular Medicine, University of Padova, Padova, Italy
s.zara@unich.it

Premature newborns are frequently exposed to both mild and severe hyperoxia and experimental data indicate modulation of liver metabolism by hyperoxia in the first postnatal period. Conversely, nothing is known about possible modulation of growth factors and signaling molecules involved in other hyperoxic responses and no data are available about the effects of hyperoxia in postnatal liver haematopoiesis.
The aim of this study is to analyse the effects of hyperoxia in neonatal liver tissue, focusing the attention on the responses shown by hepatocytes and haemopoietic cells in terms of Vascular Endothelial Growth Factor (VEGF), Matrix Metalloproteinase 9 (MMP-9), Hypoxia-Inducible Factor-1α (HIF-1α), endothelial Nitric Oxide Synthase (eNOS), and Nuclear Factor-kB (NF-kB), expression along with apoptotic event occurrence.
Exposure of newborn rats to room air (controls), 60% O2 (mild hyperoxia), or 95% O2 (severe hyperoxia) was performed for the first two postnatal weeks. The results were obtained by means of  immunohistochemical, TUNEL and Western blot analyses.
Severe hyperoxia increases hepatocyte apoptosis and MMP-9 expression and decreases VEGF expression. Reduced content in reticular fibers is found in moderate and severe hyperoxia. Moderate hyperoxia specifically induces in hepatocytes upregulation of HIF-1α and downregulation of eNOS and NF-kB expression. Postnatal hyperoxia exposure upregulates VEGF (both moderate and severe hyperoxia) and eNOS (severe hyperoxia) expression in haemopoietic cells.    
In conclusion, our study reveals different effects of hyperoxia on hepatocytes and haemopoietic cells, with growth factors and intracellular mechanisms being differently involved. Postnatal hyperoxia shows detrimental action on hepatic tissue. Decreased VEGF expression may play a role in severe hyperoxia whereas some other changes seem to drive response to moderate hyperoxia, such as increased HIF-1α expression, and decreased expression of eNOS and NF-kB. Conversely, postnatal hyperoxia exposure increases liver haemopoiesis and upregulates VEGF and eNOS expression. Thus, it may be hypothesized the involvement of VEGF and eNOS in the liver haemopoietic response to hyperoxia.     

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5824 VASCULAR COMPLICATIONS LINKED TO ALZHEIMER’S DISEASE

Bester J.1, Buys A. V.2, Lipinski B.3, Kell D. B.4, Pretorius E.1
1Department of Physiology, Faculty of Health Sciences, University of Pretoria, South Africa, 2MicrMicroanalysis Unit, University of Pretoria, South Africa, 3Joslin Diabetes Center, Harvard Medical School, Boston, USA, 4School of Chemistry and The Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK
janette.bester@up.ac.za

Introduction: Current literature suggests that vascular components play a fundamental role in

neurological diseases like Alzheimer’s disease (AD) (Kovacic and Fuster, 2012). One such

component is erythrocytes (RBCs) that are highly deformable, which contributes to assisting

blood flow in the microcirculation (Mohandas and Gallagher, 2008). Abnormalities in RBCs and

their flow can contribute to AD by obstructing oxygen delivery to parts of the brain that are

already in a compromised state. Closely linked to hematological pathology in AD are increased

iron levels that may play an important role in the pathogenesis of the condition(Barnham and

Bush, 2008). Increased iron levels cause oxidative stress, as they participate in

oxygen-dependent free radical formation (Castellani et al., 2012). This free radical stress may

have an impact on RBCs and may possibly cause extensive and accumulative damage to these

cells, ultimately compromising their functioning. Aims: Determine if there is iron overload

present in AD and if this has an effect on the structure of the RBCs. Methods: 25 AD patients

and 40 healthy control individuals were studied and results from light microscopy, scanning

electron microscopy, atomic force microscopy and confocal microscopy were correlated.

Results: RBC ultrastructure showed a changed morphology in the presence of iron overload.

These changes might impair the oxygen carrying capacity and compromise hemorheology of

the RBCs, and additionally cause a strain on the already challenged brain function of these

individuals. Conclusions: Iron overload are present in AD patients, this may cause the condition

to progress faster than in AD individuals who do not have iron overload, particularly due to the

additional hydroxyl radical load.

References:

Kovacic,J.C.,andFuster,V.(2012). Atherosclerotic risk factors, vascular cognitive impairment, and Alzheimer disease. Mt. SinaiJ.Med. 79, 664–673.

Mohandas,N.,andGallagher,P.G.(2008) .Red cell membrane:past,present,and future. Blood 112, 3939–3948.

Barnham,K.J.,andBush,A.I.(2008). Metals in Alzheimer’s and Parkinson’s diseases. Curr.Opin.Chem.Biol. 12, 222–228.

Castellani,R.J.,Moreira,P.I.,Perry,G.,andZhu,X.(2012). The role of iron as a mediator of oxidative stress in Alzheimer disease. Biofactors 38, 133–138.

We would like to acknowledge Dr Prashilla Soma who drew our blood

samples and Dr Wiebren Duim (Neurologist) who gave us insights regarding the Alzheimer’s

patient sample. Also, we are in debt to the family members of the patients who gave informed

consent for the study.

Fig. 1: Red blood cell (RBC) of a healthy individual with a typical discoid shape. Scale: 1μm

Fig. 2: Discoid shaped red blood cell from an Alsheimer's patient with normal serum ferritin levels. Scale: 1μm

Fig. 3: Irregular shaped red blood cells from Alzheimer's patient with increased serum ferritin levels. Scale: 1μm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5830 Immunohistochemical selection of tumor antigens as potential targets for non-small cell lung cancer immunotherapy.

Blanco R.1, Renfigo C. E.2, Cedeño M.1, Blanco D.3, Frómeta M.1, Carr A.1, Renfigo E.1
1Center of Molecular Immunology, Havana, Cuba, 2Manuel Fajardo General Hospital, Havana, Cuba, 3National Institute of Oncology and Radiobiology, Havana, Cuba
rances@cim.sld.cu

Abstract

Introduction. Lung carcinoma is the leading cause of cancer related mortality worldwide. For this reason, some research efforts are focusing in the evaluation of a variety of tumor-associated antigens (TAAs) for diagnosis, prognosis and therapy monitoring as well as for lung cancer immunotherapy. In line with this, adequate methods to identify which patients are most likely to benefit from the targeted drugs against the identified TAAs are needed.

Aims. To evaluate the immunohistochemical detection of the N-glycolyl GM3 ganglioside (NeuGcGM3), the epidermal growth factor receptor (EGFR) and its ligand (EGF) in non-small cell lung carcinoma (NSCLC) using four cuban monoclonal antibodies.

Materials and Methods. Five micrometer serial sections from 64 routinely processed, formalin-fixed and paraffin-embedded archival samples with diagnosis of NSCLC cancer were obtained. For EGFR and EGF antigens, the slides were pre-treated with 0.4% pepsin in 0.1N hydrochloric acid solution at 37°C for 30 minutes. The samples were incubated with ior egf/r3 (anti-EGFR), CB-EGF1, CB-EGF2 (anti-EGF) and 14F7 (anti-N-glycolyl GM3 ganglioside) Mabs followed by a peroxidise avidin-biotin system. All markers were evaluated for percentage of positive cells (0-100%) and the intensity of reaction (0-3+). The results in agreement with two observers were considered as final.

Results. The reactivity of 14F7 Mab was evidenced in 61/64 (95.3%) of NSCLC samples. The pattern of staining of this Mab was finely granular and was located on both cell membrane and cytoplasm of malignant cells (Figure 1). In one of positive cases (1.6%) an additional nuclear staining of 14F7 Mab was detected.

The immunodetection of EGFR by means of the ior egf/r3 Mab was evidenced in 37/59 (62.7%) of NSCLC samples. The pattern of staining of ior egf/r3 Mab was finely granular and was mainly located in the plasmatic membrane of malignant cells (Figure 2), although their cytoplasm was also decorated.

The expression of EGF was observed in about 70% of NSCLC samples, using CB-EGF1 or CB-EGF2 Mabs. The pattern of staining of these Mabs was finely granular and mainly located in cytoplasm; although a membrane staining was also observed (Figure 3). An additional extracellular staining was detected. A slight increase in the intensity of reaction was observed with CB-EGF1 Mab, although a significant correlation was detected when the reactivity of these two Mabs were compared (p<0.0001, rs=0.5429; Spearman test).

Conclusions. Our data permit to consider the development of diagnostic kits using ior egf/r3, CB-EGF1 and 14F7 Mabs in order to achieve a better selection of patients to specific therapies.

Acknowledgement. Financial support was provided by the Center of Molecular Immunology.

Financial support was provided by the Center of Molecular Immunology.

Fig. 1: Figure 1. Hematoxylin and eosin staining of lung adenocarcinoma (A). Note: the intense reaction of 14F7 Mab located on both cell membrane and cytoplasm of malignant epithelial cells (B) (Brown color). Counterstained with Mayer’s Hematoxylin (Blue color). White bar = 50 µm.

Fig. 2: Figure 2. Hematoxylin and eosin staining of lung adenocarcinoma (A). Note: the intense reaction of ior egf/r3 Mab mainly located in cell membrane and also in the cytoplasm of malignant epithelial cells (B) (Brown color). Counterstained with Mayer’s Hematoxylin (Blue color). White bar = 50 µm.

Fig. 3: Figure 3. Hematoxylin and eosin staining of lung adenocarcinoma (A). Note: the intense immunostaining with CB-EGF1 Mab located on both cell membrane and cytoplasm of malignant epithelial cells (B) (Brown color). Counterstained with Mayer’s Hematoxylin (Blue color). White Black bar = 100 µm.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5835 Melatonin, quercetin and resveratrol attenuates oxidative stress and hepatocellular injury in streptozotocin-induced diabetic rats

Elbe H.1, Esrefoglu M.2, Vardi N.1, Taslidere E.1, Ozerol E.3, Tanbek K.3
1Inonu University, Faculty of Medicine, Department of Histology and Embryology, Malatya, TURKEY , 2Bezmi Alem Vakif University, Faculty of Medicine, Department of Histology and Embryology, Istanbul, TURKEY, 3Inonu University, Faculty of Medicine, Department of Biochemistry, Malatya, TURKEY
drmukaddes@hotmail.com

Aim: In this study we aimed to investigate healing effects of melatonin, quercetin and resveratrol on hepatocellular injury in streptozotocin-induced experimental diabetes via histological and biochemical methods.

Material and Methods: Thirty-five adult male Wistar albino rats divided into 5 groups each containing 7 rats as follows: Group 1: Control, Group 2: Diabetes (Streptozotocin 45 mg/kg/single dose/ip), Group 3: Diabetes+Melatonin (10 mg/kg/30 days/ip), Group 4: Diabetes+Quercetin (25 mg/kg/30 days/ip) and Group 5: Diabetes+Resveratrol (10 mg/kg/30 days/ip). Melatonin, quercetin and resveratrol dissolved in 4% ethanol. Initial and final blood glucose levels and body weights were measured. At the end of the experimentation, rats were sacrified by ketamine anesthesia. The tissue samples were fixed in 10% formalin. Following routine tissue process, livers were embedded paraffin. Paraffin blocks were cut at 5 µm, mounted on slides stained with hematoxylin-eosin, Periodic acide schiff and Masson’s trichrome. Histopathologic damage score was calculated in regard to congestion, sinusoidal dilatation, inflammation, fibsosis and loss of glycogen. Maximum score was 15. Tissues were examined using a Leica DFC280 light microscope and a Leica Q Win Image Analysis system (Leica Micros Imaging Solutions Ltd., Cambridge, UK). Tissue biochemical (oxidant/antioxidant) parameters as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) were examined.

Results: The diabetic rats had significantly higher blood glucose levels than the control group (p<0.05). In diabetic rats, body weights were significantly decreased when compared with the control rats (p<0.05). There was no significant difference in body weights among diabetic groups (p>0.05). The control group was normal in histological appearence. However, histopathological alterations were detected such as congestion, sinusoidal dilatation, inflammation, fibrosis and loss of glycogen content in the diabetes group. On the other hand, in treatment groups, histopathological changes markedly reduced. The levels of MDA increased and the enzyme activities of CAT, SOD and GSH levels decreased in the diabetes group, while melatonin, quercetin and resveratrol treated diabetic rats showed an increase of CAT activity and GSH level and a decrease of MDA levels. There was no significant difference in SOD activity among treatment groups (p>0.05).

Conclusion: In view of the histological and biochemical findings, we conclude that STZ-induced hepatocellular injury should be prevented by melatonin, quercetin and resveratrol administration probably via their antioxidant actions.

This study was supported by a grant from Scientific Research Fund of Inonu University.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5846 Vascular aging and melatonin beneficial effects

Favero G 1 Franceschetti L 1 Rossini C 2 Stacchiotti A 1 Rodella L F 1 Rizzoni D 2 Rezzani R 1
Division of Anatomy and Physiopathology, Department of Clinical and Experimental Sciences, University of Brescia, Brescia, Italy 1 Clinica Medica, Department of Clinical and Experimental Sciences, University of Brescia, Brescia, Italy 2
alessandra.stacchiotti@unibs.it

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5875 Transmission electron microscopy (TEM) characterization of diabetic nephropathy in streptozotocin-induced diabetes in rats

Petrushevska M.1, Zafirov D.1, Labacevski B.1, Labacevski N.2, Trojacanec - Pavlovska J.1
1Institute of pharmacology and toxicology, Faculty of Medicine, Skopje, R. Macedonia, 2Institute of pathology, Faculty of Medicine, Skopje, R.Macedonia
mapet85@yahoo.com

Characteristic pathological changes in the glomeruli in diabetic nephropathy (DN) include expansion of the mesangial matrix and thickening of the glomerular basement membrane (GBM). Streptozotocin (STZ) induced DN in rats represents an excellent model for evaluation of drug treatment efficacy due to the progressive development of severe glomerular sclerosis and tubule-interstitial fibrosis. Additionally, this model reflects high similarity of the intrarenal enzyme distribution with the one in humans. Electron microscopy represents a unique method for analysing kidneys on ultrastructure level. The aim of this study was to confirm kidney changes in rats with STZ-induced DN and to estimate the effect of the perindopril (PER) treatment.
DM was induced by a single IP injection of STZ (60 mg/kg) in 75 Wistar rats. The control group received only IP injection of citrate buffer, pH 4.5. The rats with blood glucose levels  11 mmol/L, under fasting condition were included in the study and were left in diabetic condition for 4 wks to develop DN. The diabetic rats (n=50) were randomly assigned to two experimental groups (STZ and STZ+PER). In the STZ+PER group (n=25), PER was administered (6 mg/kg/daily) from week 4 to week 12. Changes in kidney structures were analysed in a double-blinded manner. Glomeruli were carefully graded in a sequential manner, to avoid grading the same glomeruli twice. The detected changes by light microscopy were analysed and confirmed by TEM. Kidney tissue samples with a size of 1-2mm2 after de-paraffinization and rehydration procedure were post-fixed in 1% (v/v) OsO4 for 1h and then embedded in Durcupan™ resin. Semi-thin sections were stained with Toluidine blue, while ultrathin sections obtained from ultra-microtome (PT-PC PowerTome, Ultramicrotomes, RMC Products), were contrasted in autostainer (QG-3100 Automated TEM Stainer, RMC Products) by uranyl acetate and lead citrate. Samples were then analyzed on TEM (Jeol, JEM 1400) attached to digital camera (Veleta TEM Camera, Olympus) and controlled by iTEM software v.5.2.
The renal tissue examination (STZ group, after 8 wks), have shown presence of a moderate degree of glomerulopathy characterized with basement membrane thickening, expansion of the mesangial matrix, arteriolar hyalinosis and insudative protein deposits that obstruct some of the capillaries (Fig. 1). The ultrastructure analysis have identified uneven fusion of the podocytes and widening of the mesangial matrix with GBM thickening due to deposition of basal membranaceous sclerotic material (Fig. 2, Fig. 3).
The detection of the observed changes on the ultrastructure kidney level outweighs the expense of EM for routine diagnostic purposes and adds its value as irreplaceable technique for diagnosis of renal diseases.

Fig. 1: Histopathological features of kidney from untreated diabetic rat at 12 wks with damaged glomeruli, thickened GBM, altered tubular epithelium with clear cytoplasm due to intracellular glycogen accumulation and areas of partial tubular dilatation. Sections were stained with PAS reagent. Magnification × 100 and × 200.

Fig. 2: Histopathological features of kidney diabetic rat treated with perindopril at 12 wks. Sections were stained with PAS reagent. Magnification × 100 and × 200.

Fig. 3: An electron micrograph from STZ-induced DN rats. Untreated diabetic rat at 12 wks., uneven fusion of the podocytes as well as widening of the mesangial matrix with sclerotic GBM thickening (magnification × 20,000)

Fig. 4: An electron micrograph from diabetic rat treated with perindopril at 12 wks.,showing mild thickening of the GBM and regenerated podocyte (magnification × 15,000)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5899 Association of protein kinase B (Akt) isoforms with subcellular compartments in the left and right ventricles of rat heart adapted to hypoxia

Zurmanova J.1, Hornikova D.1, Kolar F.2, Elsnicova B.1
1Department of physiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic , 2Institute of Physiology, Academy of Science of the Czech Republic, Prague, Czech Republic
Jitka.Zurmanova@seznam.cz

Chronic hypoxia results in pulmonary hypertension and right ventricle hypertrophy. On the other hand, it increases myocardial resistance to acute ischemia/reperfusion injury (Kolar et al., 2007). Akt signalling plays an important role in cardioprotective mechanisms (Ravingerova et al., 2007). Akt1 and Akt2 are known as major isoforms and their localization in cardiomyocytes appear to be crucial for their action in the area of mitochondria and sarcolemma. We aimed to assess the subcellular localization of Akt1 and Akt2 and their co-localization with either mitochondria or sarcolemma by quantitative fluorescence microscopy in hearts of rats adapted for 3 weeks to intermittent hypobaric hypoxia (7000 m, 8h/day). Frozen section method of indirect immuno-fluorescence staining combined with marker counterstaining was used. Cryosections were incubated with rabbit primary polyclonal antibodies against rat Akt1 (Cell Signalling) and Akt2 (GenScript). As counterstaining, either mitochondrial compartment or sarcolemma markers were used. Mitochondria were stained with MitoProfile Total OXPHOS Rodent Antibody Cocktail (Abcam), WGA-TMRM conjugate (Molecular Probes) was used as sarcolemma marker. Pearson correlation coefficients indicating the level of colocalization of both Akt isoforms with the two subcellular compartments were calculated using ICA plugin of Fiji ImageJ software.

Our results showed thatAkt2 was not associated with sarcolemma and its association with mitochondrial compartment was higher compared to Akt1 in both ventricles. Adaptation to hypoxia decreased (p<0.005, n=6) mitochondrial association of Akt1 in the hypertrophied right ventricle only while increasing its association with sarcolemma in both ventricles.

We conclude that Akt1, but not Akt2, subcellular localization is affected by chronic hypoxia with significant differences between right and left ventricles.

Ostadal B, Kolar F. Cardiac adaptation to chronic high-altitude hypoxia: beneficial and adverse effects. Respir Physiol Neurobiol. 2007 Sep 30;158(2-3):224-36.
Ravingerova T, Matejikova J, Neckar J, Andelova E, Kolar F. Differential role of PI3K/Akt pathway in the infarct size limitation and antiarrhythmic protection in the rat heart. Mol Cell Biochem. 2007 Mar;297(1-2):111-20.

The study was supported by the Grant Agency of the Czech Republic grant number 14-04301P and by Ministry of education of Czech Republic grant SVV-260087/2014.

Fig. 1: The overall pattern of Akt1 fluorescent signal in different cell compartments. Akt1 isoform is more associated with mitochondria than with sarcolemma. Under hypoxic condition Akt1 detach from mitochondria in RV (p<0.0022, n=6) and translocate to plasma membrane in both ventricles (p < 0.0001, n=6).

Fig. 2: Akt1 colocalization with mitochondria in hypertrophied right ventricle of rat. After adaptation to hypoxia Akt1 isoform (green) detached from mitochondria (red) in right ventricle (p<0.0022, n=6). A: tissue section of control samples B: sections of samples adapted to hypoxia

Fig. 3: Akt1 colocalization with plasma membrane. After adaptation to hypoxia, Akt1 (green) seems to translocate to plasma membrane (red). Pearson’s coefficient increased significantly (p < 0.0001, n=6). Nevertheless the absolute values of coefficient are very low and further evidence is necessary. A: control sample B: sample adapted to hypoxia
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5901 Participation of p53 in the remodeling of the actin cytoskeleton in metastases

Reyes-Ábalos A. L.12, Villar Arias S.12
1Service Scanning Electron Microscopy and Microanalysis (SMEByM) Faculty of Science (UdelaR), 2Departamento de Genética del Instituto de Investigaciones Biológicas Clemente Estable (IIBCE)
reyesabalos@gmail.com

The HL-60 human cell line corresponds to promyelocytic myeloid leukemia. HL-60 has the ability to differentiate throughout the myeloid series with the incorporation of exogenous retinoic acid (ATRA) or dimethylsulfoxide (DMSO). The induction of differentiation of HL-60 modifies the typical nuclear granulocytes ovoid to polylobulated. The HL-60 cell line has the translocation t(5;17) (q35;q21) involving the gene encoding the nuclear retinoic acid receptor (RARalpha and nucleophosmin gene (NPM) (Cho et al. 2011). The product of the t(5; 17) shares the ability to act as ligand-dependent transcriptional activator of genes responsive to retinoic acid (Hummel et al. 1999). ATRA is included in chemotherapeutic protocols of cancer in acute promyelocytic leukemia (LAP, M3). All the process of differentiation was assessed by SEM, TEM and light microscopy (Fig_1). We also investigated the relation of the p53 protein and the cytoskeleton remodeling during differentiation HL-60 to granulocytes, analyzing the dynamic of p53 distribution and its relation with polymeric actin through specific antibodies against p53 and actin and confocal laser microscopy (Fig_2). Preliminary results obtained show that differentiated HL-60 cells with ATRA or DMSO present modifications in the location and degree of intensity in p53 and actin signals. The redistribution of p53 and its association with the actin cytoskeleton in differentiated HL-60 cells may due to a change in cellular environment where colonization capacity might be reduced. In contrast, actin is present all over the cells in undifferentiated HL-60 as well as p53, although this protein may inhibits cell spreading through an inhibition of cell polarization (filopodia formation) according to Roger et al. (2006) (Fig_3). In addition, filopodia formation does not appear to be inhibited, despite p53 is expressing. A possible explanation could be that p53 or its regulator proteins are mutated in this cell line. The characteristics forming the identity of the metastatic process, is heterogeneity and instability of cells, therefore, to determine the existence of this p53 and actin distribution patterns in other tumoral cells lines is necessary to confirm the hypothesis of Roger et al. (2006).

Gallagher R, Collins S, Trujillo J (1979) Characterization of the continuous, differentiating
myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood 54: 713-733
Cho SR, Park SJ, Kim HJ, Park IJ, Choi JR, Jung HJ, Park JE (2011) Acute promyelocytic leukemia
with complex translocation t(5;17;15)(q35;q21;q22): case report and review of the literature.
Pediatr Hematol Oncol. 33:e326-9
Roger L, Gadea G, Roux P (2006). Control of cell migration: a tumour suppressor function for
p53? Biol. Cell. 98: 141-152

Sectorial Commission of Scientific Research (CSIC) Montevideo - Uruguay
Faculty of Science, University of the Republic (UdelaR), Montevideo - Uruguay

Fig. 1: (A-C) TEM of differentiated HL60-ATRA ultrathin sections (D-F). SEM of HL60-ATRA metallized with a thin layer of pure gold in which light are observed invaginations (G-I) Images obtained in inverted microscope illustrating a complex morphology exhibiting HL60-ATRA

Fig. 2: (CM) Patterns distribution of p53 in HL60-ATRA cells differentiated and cells undifferentiated (control HL60-PBS) (A1, B1, C1) merge of single cell z-stack slices 3D reconstruction of HL60-ATRA (A2, B2, C2) p53 (red) (A3, B3, C3) actin cytoskeleton; (A4, B4, C4) DAPI (blue) bar=4 microns

Fig. 3: (CM) Patterns distribution of p53 in HL60-PBS cells undifferentiated (A1) single cell z-stack slices DAPI (blue), (A2) p53 (red), (A3) actin cytoskeleton (A4) merge; (B, C, D, E) cell polarization (arrow filopodia formation). Images obtained in epifluorescence microscope illustrating a complex morphology, bar = 4 microns
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5902 Quantitative assessment of microvascular networks using optical imaging and image analysis technologies

Kostromina E.1
1Moscow State University, Moscow, Russian Federation
rumed07@hotmail.com

Microvascular networks are designed to deliver blood to certain tissue areas and adjust local blood flow to tissue metabolic needs. Morphological and functional heterogeneity of microvessels plays an important role in vasomotor reactions and regional blood flow regulation. To study the mechanisms of microvascular remodeling, we explored the potential of optical microscopy techniques for 2D and 3D imaging of microvasculature [1] in combination with blood flow measurements and quantitative image analysis [2].

Using data obtained by intravital microscopy, we estimated the distribution of microcirculatory parameters and analysed microvascular network architecture in the rat mesentery (Fig. 1) and skeletal muscle (Fig. 2). Wall shear stress values estimated from the luminal diameter and blood flow velocity measurements in terminal arterioles varied significantly and correlated with wall-to-lumen ratio. The data provide evidence of a role for shear stress and circumferential wall stress in the development and adaptation of microvascular networks under normal physiological conditions. Ex vivo microscopic visualization was used to generate high resolution 3D images of the perfused fraction in terminal vascular beds in rodents. Tissue specimens taken after iv injection of fluorescein isothiocyanate-dextran were dissected, fixed, subjected to optical clearing, and used for subsequent confocal microscopic examination. Sequential optical sections collected at 1-5 μm intervals along the z axis were used to generate 3D-images of the microvascular tree (Fig. 3). To perform a comparative assessment of total microvascular density in healthy animals and in animals with vascular abnormalities, immunohistochemical localization of vascular wall elements [3] was used (Fig. 4). The method of ex vivo microscopic visualization allows evaluation of the vascular microarchitecture and perfusion levels across a microvascular tree in tissue specimens with a thickness of up to 1-2 mm. Quantitative assessment of microvascular networks using 2D and 3D optical imaging techniques will be a useful tool for studying the mechanisms of microvascular adaptations both in healthy and diseased tissues.

References:
1. Ahlgren, U. and E. Kostromina, Imaging the Pancreatic Beta Cell, in Type 1 Diabetes - Pathogenesis, Genetics and Immunotherapy, D. Wagner, Editor. 2011, InTech. p. 269-92.
2. Shinkarenko, V., et al., in New Methods and Instruments for Microscopy in Biology and Medicine, V. Shinkarenko, Editor. 1987: Wetzlar. p. 170-175.
3. Kostromina, E., et al., Glucose intolerance and impaired insulin secretion in pancreas-specific STAT3 knockout mice are associated with microvascular alterations in the pancreas. Endocrinology, 2010. 151(5): p. 2050-9.

Fig. 1:  In vivo microscopic image of rat mesenteric microvessels. A-arterioles, C-capillaries. Scale bar=50 µm. Objective 25x.

Fig. 2:  Intravital microscopy of the rat cremaster muscle microcirculation. A-arterioles, V-venules. Scale bar=100 µm. Objective 6.3x.

Fig. 3:  Ex vivo confocal microscopic image of perfused blood vessels after iv injection of fluorescein isothiocyanate-dextran.

Fig. 4:  Endothelial cells are visualized by PECAM-1 immunostaining of frozen tissue sections. Scale bar=50 µm. Objective 40x.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5909 Protective Effects of Quercetin in Cisplatin Induced Urinary Bladder Damage in Rats

Çadırcı S.1, Köroğlu M. K.2, Ercan F.2, Şener G.1
1Marmara University, School of Pharmacy, Department of Pharmacology, İstanbul, Turkey , 2Marmara University, School of Medicine, Department of Histology and Embryology, İstanbul, Turkey
kutaykoroglu@hotmail.com

Introduction: Cisplatin (CIS) is commonly used as a chemotheropotic agent however it is associated with numerous side effects such as urinary cytem cytotoxicity. Quercetin (QT) is a naturally occurring flavonoid present in fruits and vegetables. A number of studies have evaluated its biological properties and found it to have potential benefits for human health, including antimicrobial, antiviral, antioxidative, anti-inflammatory, and anti-apoptotic activity.
Aim: The aim of the study is to investigate possible protective effects of QT in cisplatin induced damage in urinary bladder tissues.
Materials and Methods: Male Sprague Dawley rats were used in the study and four experimental groups (250-300 g; n= 8/each group) were formed as: 1- saline applied control, 2- QT applied control, 3- CIS and 4- CIS + QT groups. Following a single dose of CIS (7 mg/kg i.p.), either saline or QT (20 mg/kg, orally) was administered for 21 days. After decapitation of rats, urinary bladder were removed for histopathological and biochemical evaluation. The urinary bladder tissue samples were fixed with 10% neutral buffered formaldehyde and processed for routine paraffin embedding. Hematoxylin and Eosin stained sections were evaluated semiquantitatively. In order to examine oxidative tissue injury, 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDA) and glutathione (GSH) levels, and superoxide dismutase (SOD) and caspase 3 activities were analyzed biochemically. Data were analyzed statistically.
Results: Urothelial damage was increased in CIS induced rats and decreased with QT treatment. In the CIS treated group, increase in 8-OHdG and MDA levels and caspase 3 activity and decrease in GSH level and SOD activity were also reversed by QT treatment.
Conclusion: According to the results, quercetin exerts beneficial effects against cisplatin induced oxidative damage through its antioxidant and antiapoptoic effects.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5911 Protective effects of exercise and caloric restriction on cardivascular system in aged rats: Morphological and biochemical study

Macit Ç.1, Çadırcı S.1, Köroğlu M. K.2, Ercan F.2, Şener G.1
1Marmara University, School of Pharmacy, Department of Pharcmacology, İstanbul, Turkey , 2Marmara Universtiy, School of Medicine, Department of Histology and Embryology, İstanbul, Turkey
kutaykoroglu@hotmail.com

Introduction: Aging is defined as a progresivelly decreasing ability to maintain homeostasis and increasing risk to many cardivascular system diseases. At an older age, accumulation of altered macromolecules and membranes may impair cell functioning and accumulation of damaged organelles may increase the yield of reactive oxigen species and accelarete aging. It is known that exercise delays the aging processes and protects the cardivascular system. Due to aging body mass index increases and adipose tissue amount increases in the body.
Aim: The aim of the study was observed the protective effects of exercise and caloric restriction on cardivascular system in aged rats.
Materials and Methods: Sprague Dawley rats were used in the study. Five experimental groups (n= 8/each group), were formed as: 1-control (3 month old, standart diet), 2-aged (15 months old, standart diet), 3-aged + caloric restriction (15 months old, caloric restriction), 4-aged + exercise (15 months old, standart diet, swiming exercise for 3 months) and 5-aged + caloric restriction + exercise groups. At the end of the study aorta and heart tissues were taken from the animals for morphological and biochemical studies. The tissue samples were fixed with 10% neutral buffered formaldehyde and processed for routine paraffin embedding. Hematoxylin and Eosin stained sections were evaluated semiquantitatively. In order to examine oxidative tissue injury, 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDA) and glutathione (GSH) levels, and superoxide dismutase (SOD), nitric oxide synthase (NOS) and caspase 3 activities were analyzed biochemically. Data were analyzed statistically.
Results: Aging altered the histological appearance of the tissues and caused oxidative damage assessed by increased MDA and 8-OHdG levels and decreased GSH levels and SOD activity. Furthermore, due to aging, the heart and aorta tissue caspase-3 activity is increased, while NOS activity is decreased. When aged rats were exposed to exercise, caloric restriction, or both procedures, histopathological and biochemical changes were reversed in varying amounts.
Conclusion: In conclusion, exercise and caloric restriction may protect the cell and tissues in the cardiovascular system via balancing oxidant-antioxidant status, nitric oxide metabolism and inhibiting apoptosis.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5922 The Anti-inflammatory Effects of The Cholinergic Pathway on Pancreaticobiliary Duct Ligation-Induced Acute Pancreatitis in Rats

Kolgazi M.1, Kolbasi B.2, Guleken Z.3, Atsız A.4, Başıbüyük C. S.4, Boz İ. B.4, Küçükali B.4, Ercan F.2, Yeğen B. Ç.3
1Acibadem University, School of Medicine, Dept. of Physiology, Istanbul,Turkey, 2Marmara University, School of Medicine, Dept. of Histology and Embryology, Istanbul, Turkey, 3Marmara University, School of Medicine, Dept. of Physiology, Istanbul, Turkey, 4Marmara University, School of Medicine, 5th Year Student, Istanbul, Turkey,
bircankolbasi@yahoo.com

Introduction: Acute pancreatitis is an inflammatory condition that may lead to multisystemic organ failure. The "cholinergic anti-inflammatory pathway" modulates the immune system by acting via the alpha7 receptors expressed on macrophages and immune cells.
Aim: The aim of this study was to investigate the effects of cholinergic anti-inflammatory pathway on acute pancreatitis induced by pancreaticobiliary obstruction in rats.
Materials and Methods: Acute pancreatitis was induced in Wistar albino rats by pancreaticobiliary duct ligation (PBDL). Half of the animals, before they were induced with pancreatitis, were subjected to bilateral cervical vagotomy using capsaicin. All groups were treated intraperitoneally with either nicotine (1 mg/kg/day) or saline for 3 days. After decapitation, lung, liver and pancreas tissues were collected for histologic evaluation and measurement of myeloperoxidase (MPO) activity, malondialdehyde (MDA) and glutathione (GSH) levels. For lung tissue (1) vascular congestion, (2) fibrosis and interstitial edema, (3) alveolar structural disturbance and inflammatory cell infiltration, for pancreas tissue (1) edema, (2) acinar necrosis and (3) inflammatory cell infiltration, for liver tissue (1) vacuolization and picnotic nucleus in hepatocytes, (2) sinusoidal congestion and (3) Kupffer cell infiltration were taken as the scoring criteria. Based on a semiquantitative scale, the histological scores of the organs were calculated as the sum of the scores (0–3) given for each criterion. The maximum calculated score was 9. Student’s t-test was used for statistical analysis. Values of p<0.05 were regarded as significant.
Results: Acute pancreatitis with PBDL increased microscopic damage scores in lung, liver and pancreas tissues. Nicotine treatment and cervical vagotomy significantly reduced microscopic damage scores compared with PBDL group in all tissues (p<0.05-0.001). Nicotine treatment reduced MPO activity and MDA levels which were increased with PBDL in all three tissues, and prevented PBDL-induced GSH depletion in the liver and pancreas tissues (p<0.05-0.001). Similarly, capsaicin-induced denervation decreased MPO activity and MDA levels in liver and pancreas tissues (p<0.05-0.001).
Conclusion: Stimulation of the cholinergic system with nicotine reduced neutrophil infiltration and lipid peroxidation and prevented GSH depletion in the PBDL-induced acute pancreatitis model. In addition, capsaicin-sensitive vagal afferent neurons have a role in this cholinergic anti-inflammatory mechanism. Further experimental and clinical studies are required to elucidate the regulatory role of the cholinergic system in acute pancreatitis.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5932 Resveratrol Protects Against Experimental Induced-Reye's Syndrome by Prohibition of Oxidative Stress and Restoration of Complex I Activity

Abdeen A. A.1, Sarhan N. I.2
1Departemnt of Pharmacology ,Faculty of medicine ,Tanta University , 2Departement of histology and cell biology FAculty of medicine Tanta university
nsarhan2006@hotmail.com

Reye's syndrome is the main contraindication of using aspirin in
young children during fever-causing illnesses. The safety of paracetamol as a classical
alternate therapy is now questionable due to increasing evidence of its correlation to
increased incidence of autism. Therefore, this study was designed to investigate if
resveratrol could provide protection against Reye's syndrome induced by 4-pentenoic
acid in Wister albino rats. Methods and results: Compared to rats with untreated
Reye's syndrome; 1 hour pre-treatment by low dose resveratrol (10 mg/kg by oral
gavage) resulted in marked amelioration in liver functions in the form of significant
decrease in serum transaminases (AST, ALT) and plasma ammonia levels, shortening
of prothrombin time and increase in serum albumin levels. In addition, resveratrol
prohibited oxidative stress markers as significant increase in GSH and decrease in
MDA with restoration of complex I activity in liver tissues. The classical
histopathological presentation in Reye's syndrome of microvesicular steatosis by light
microscope and mitochondria distortion by electron microscope; has been improved by
resveratrol pre-treatment. The efficient protection by resveratrol was determined by
normalization in serum levels of AST and albumin as well as complex I activity, GSH
and MDA. Conclusion: It could be concluded that pre-treatment with resveratrol in low
dose could protect against Reye's syndrome partially via prohibition of oxidative stress
and restoration of complex I activity. This may provide the opportunity to reconsider
aspirin therapy for infants and young children. However, the verification of such result
in clinical practice remains a real challenge.

Fig. 1: Induced Reye's syndrome(group II) showing multiple microvesicular steatosis (►) and mononuclear cellular infiltrate (→). (×200). [

Fig. 2: Induced Reye'ssyndrome (group II) showing multiple microvesicular steatosis (→) and extracellular vacuoles with wide separation of thecells (*). Notice nuclear fragmentation (►). (×1000).

Fig. 3: Hepatocytes of induced Reye's syndrome (group II) showing crescents of chromatin margination inside the nucleus (N) with focal swelling (*) and disrupted nuclear envelope (wavy arrow), few mitochondriawith destroyed cristae (►) and multiple vacuoles (→). Notice also focal disruption of the plasma membrane (curved arrow).(×11700|). 

Fig. 4:  Hepatocytes of induced Reye's syndrome (group II) showing irregular contoured nucleus (N), Few mitochondria (M), large sized cytoplasmic lipid droplets (L), multiple vacuoles (►) and electron dense bodies (→).(×11700).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5936 Protective Effects of Resveratrol in Cisplatin Induced Testis Damage in Rats

Özyılmaz Yay N.1, Şener G.2, Ercan F.1
1Marmara University, School of Medicine, Department of Histology and Embryology, İstanbul, Turkey , 2Marmara University, School of Pharmacy, Department of Pharmacology, İstanbul, Turkey
nagehanozyilmaz@hotmail.com

Introduction: Cisplatin is commonly used as a chemotheropotic agent however it is associated with numerous side effects such as reproductive cytotoxicity. It causes spermatogenic cell death and DNA damage in spermatozoa via the formation of reactive oxygene species (ROS). Resveratrol (3,5,4'-trans-trihydroxystilbene), a natural phytoalexin, is a potent antioxidant agent, presents in a wide variety of dietary sources including grapes, plums and peanuts.
Aim: The aim of the study is to investigate possible protective effects of resveratrol in cisplatin induced testis damage.
Materials and Methods: Male Sprague Dawley rats were used in the study and four experimental groups (250-300 g; n= 7/each group) were formed as: 1- saline applied control, 2- resveratrol applied control, 3- cisplatin and 4- cisplatin + resveratrol groups. Following a single dose of cisplatin (7 mg/kg i.p.), either saline or resveratrol (10 mg/kg, orally) was administered for 5 days. After decapitation of rats, testes were removed for histopathological and biochemical evaluation. The testis tissue samples were fixed with 10% neutral buffered formaldehyde and processed for routine paraffin embedding. Hematoxylin and Eosin stained sections were evaluated semiquantitatively as atrophic, degenerative, regressive or normal seminiferous tubules. Oxidative injury was examined by measuring malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) activity. Data were analyzed statistically.
Results: Degenerated and atrophic tubule numbers were increased in cisplatin induced rats and resveratrol treatment decreased the degenerated and atrophic tubules significantly. In cisplatin treated group, increase in MDA level and MPO activity and decrease in GSH level were also reversed by resveratrol treatment.
Conclusion: Resveratrol is protective against cisplatin induced testis injury in rat and may be a promising agent in alleviating the systemic side effects of cisplatin.

This study was supported by Marmara University Research Fund (SAG-C-YLP-090414-0076).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-5939 Apocynin Attenuates Testicular Ischemia-Reperfusion Injury in rats: Morphological and Biochemical Study

Şener T. E.1, Yakıncı Ö. F.2, Yüksel M.3, Özyılmaz Yay N.4, Ercan F.4, Akbal C.1, Şimşek F.1, Şener G.2
1Marmara University, School of Medicine, Department of Urology, İstanbul, 2Marmara University, School of Pharmacy, Department of Pharmacology, İstanbul, 3Marmara University, Vocational School of Health Related Professions, İstanbul, 4Marmara University, School of Medicine, Department of Histology & Embryology, İstanbul
nagehanozyilmaz@hotmail.com

Introduction: Apocynin (4-hydroxy-3methoxy-acetophenone), naturally occurring methoxy-substituted catechol, extracted from the roots of Apocynum cannabinum (Canadian hemp) and Picrorhiza kurroa (Scrophulariaceae) is well known an inhibitor of NADPH oxidase.
Aim: This study was designed to examine the possible protective effect of apocynin, a NADPH oxidase inhibitor, against torsion-detorsion (TD) induced ischemia/reperfusion (I/R) injury in testis.
Material and Methods: Male Wistar albino rats were divided into sham-operated control, and either vehicle, apocynin 20 mg/kg- or apocynin 50 mg/kg-treated TD groups. In order to induce I/R injury, left testis was rotated 720 degrees clockwise for 4 hours (torsion) and then allowed reperfusion (detorsion) for 4 hours. Testicular morphology was examined by light microscopy. Left orchiectomy was done for the measurement of tissue malondialdehyde (MDA), glutathione (GSH) levels, myeloperoxidase (MPO) activity, and luminol, lucigenin, nitric oxide (NO) and peroxynitrite chemiluminescences (CL).
Results: I/R caused significant increases histological damage and luminol, lucigenin, nitric oxide and peroxynitrite chemiluminescence demonstrating increased reactive oxygen and nitrogen metabolites in tissue. As a result of increased oxidative stress tissue MPO activity, MDA levels were increased and antioxidant GSH was decreased. On the other hand, apocynin treatment reversed histopathological alterations, as well as all these biochemical indices that were induced by I/R.
Conclusison: Findings of the present study suggest that NADPH oxidase inhibitor apocynin by inhibiting free radical generation and increasing antioxidant defense exerts protective effects on testicular tissues against I/R.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-6030 Acute Effects of Resuscitation Crystalloid Fluids on Renal Tissue in a Rat Model of Hemorrhagic Shock and Liver Resection

Kapucu A.1,2, Kandil A.1, Ergin B.2, Demirci-Tansel C.1, Ince C.2
1Department of Biology, University of Istanbul, Istanbul Turkey , 2Department of Translational Physiology, Academic Medical Center, Amsterdam, The Netherlands
aysegulkapucu@hotmail.com

A rapid metabolization of acetate or lactate present in resuscitation fluids for correction of acidosis following shock without excessive edema formation can be considered an important expectation when resuscitating with balanced crystalloid solutions. However whether this actually occurs and what the respective contribution to the control of acid base of either acetate or lactate in crystalloid solutions is, especially during conditions of critical illness and cardiovascular compromise is uncertain. In this study we aimed to compare the efficacy of an acetate-gluconate based fluid Plasmalyte (PL) versus other crystalloid fluids (Ringer Lactate solution; RL, Ringers Acetate; RA and Saline (0.9% NaCl)) to demonstrate its superior ability to resuscitate in a hemorrhagic shock and resuscitation model with liver dysfunction.
Male Spraque dawley rats were randomized in 6 groups (n=6 per group). Liver resection (LR) was achieved by ligaturing the hepatic arterial portal venous and removing the ligatured 70% part of liver. For hemorrhagic shock (HS) after stabilization, the animals were bled from the left femoral artery catheter at a rate of 1 ml/min using a syringe pump till reaching a mean arterial pressure (MAP) of 30 mm Hg. This pressure was maintained for 1 hour by re-infusing or withdrawing blood. At the end of this phase, the animals were resuscitated for 60 minutes with intravenous administration of RL, RA, PL, %0,09 NaCl until a target of MAP=65 mmHg is reached. Also, this study was consisted two groups as time control and an LR+HS without resuscitation. After the experiments, kidneys were isolated and analyzed immunohistochemically for inducible nitric oxide synthase (iNOS), liver fatty acid binding protein (L-FABP), interleukin 6 (IL-6) and Tumor necrosis factor-alpha (TNF-α) expression. Saline, RA, and RL administration after LR and HS reduced the increased levels of iNOS, TNF-α and IL-6 reactions. PL did not decrease iNOS and IL-6 reactions, whereas it decreased TNF-α reactions in LR+HS group. All fluids were decreased L-FABP reactions in LR+HS group. In conclusion, these results demonstrated that these crystalloid fluids have a role on oxidative stress and cytokine expression in kidney tissue in a hemorrhagic shock and resuscitation model with liver dysfunction.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-11-P-6063 Binucleation of rat right ventricle cardiomyocyte induced by chronic modulation of nitric oxide system and cold acclimation

Hmaid A.1, Markelic M.1, Otasevic V.2, Korac B.2, Jankovic A.2, Korac A.1
1Faculty of Biology, University of Belgrade, Belgrade, Serbia, 2Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Belgrade, Serbia
amalhmaid@yahoo.com


The higher ratio of binucleated cardiomyocytes in adult mammal’s heart has been postulated to be an adaptive response to increased metabolic activity, including cell hypertrophy. Although the nitric oxide (NO) is considered to be involved in heart remodeling, the mechanisms of right ventricle hypertrophy induction after chronic treatment with NOSs inhibitors or chronic cold acclimation are still obscure. Hence, we aimed this study to examine whether these treatments affect ratio of binucleated cardiomyocytes in rats right ventricle and structure of cardiomyocytes nuclei. Rats acclimated to room temperature (22±1 °C) and cold (4±1 °C), respectively, were treated with 2.25% L-arginine, a substrate for NOSs, or with 0.01% Nω-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOSs, for 45 days. Untreated groups acclimated to room temperature or cold served as controls. The cardiomyocyte’s nuclear cross-sectional area was measured morphometrically, and the ratio of binucleated cardiomyocytes was estimated. In order to explore the potential link between cardiomyocyte proliferation and binucleation we performed immunohistochemistry for proliferating cell nuclear antigen (PCNA). All treatments independent of ambiental temperature increased ratio of binucleated cardiomyocytes, which was statistically significant in L-NAME-treated animals only. Binucleated cardiomyocytes showed immunopositivity for PCNA. At the same time, cardiomyocyte’s nuclear cross-sectional area was decreased in all treated and cold-acclimated animals, with significantly higher percent of nuclei with cross-sectional area bellow 20 µm2. These results suggest that cardiomyocytes nuclear state alterations involving decreased nuclear size and increased presence of binucleation play important role in the right ventricle remodeling, especially during chronic L-NAME-treatment.

This work was supported by Serbian Ministry of Education, Science and Technological Development, Grant # 173055.

↑ Go to the Table of content ↑

LS-12. Advances in immunohistochemistry and cytochemistry

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-12-IN-1756 Distribution of membrane lipids observed by quick-freezing/freeze-fracture replica labeling electron microscopy

Fujimoto T.1
1Nagoya University Graduate School of medicine
tfujimot@med.nagoya-u.ac.jp

The biological membrane is a highly dynamic two-dimensional structure. Lipids not only form the structural basis of the membrane, but also play many functional roles. In comparison to proteins, however, relatively little is known about how lipids distribute in the membrane. This is mainly because most microscopic methods used for proteins are not appropriate for determining lipid distribution at a small scale. Proteins can be immobilized with chemical fixatives like aldehydes so that various immunohistochemical techniques can be applied. In contrast, most membrane lipids do not react with aldehydes and remain mobile even in fixed cells. This ‘unfixability’ makes it difficult to determine lipid distribution even though specific probes are available for many membrane lipids.

We have been working on an electron microscopic method using freeze-fracture replica labeling combined with quick-freezing (QF-FRL). In this method, cells are fixed physically to avoid the problem of chemical ‘unfixability’ of lipids. Briefly, cells are subjected to quick-freezing to stop molecular motion instantaneously; then a membrane is split into two leaflets by freeze-fracturing, after which membrane molecules are immobilized by vacuum evaporation of platinum and carbon; to this physically-stabilized membrane preparation (i.e., freeze-fracture replica), specific probes are applied to label target molecules (Figure 1).

By using QF-FRL, we can observe not only the two dimensional distribution of membrane lipids (and proteins as well) but also the asymmetry that lipids show between the outer and inner leaflets. Because membranes are retained in a stable form in the freeze-fracture replica, they can be subjected to various chemical treatments that may perturb membrane structures when applied to native membranes. I would like to present several results obtained using QF-FRL and discuss the strength and weakness of this technique in comparison with other methods available today.

The study was supported by Grants-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government.

Fig. 1: The outline of quick-freezing/freeze-fracture replica labeling
↑ Go to the Table of content ↑
Type of presentation: Invited

LS-12-IN-3538 PNCs associate with structure and function of the nucleolus

Huang s.1, Wang c.1, Marugan j.2, Zheng w.2, Pantnaik s.2, Southall N.2, Titus s.2, Wen J.2, Frankowski K.3, Schoenen F.3
1Northwestern University Feinberg School of Medicine, 23NIH Chemical Genomics Center, NIH, National Center for Advancing Translational Sciences, 3Specialized Chemistry. Center, University of Kansas, Lawrence, KS
s-huang2@northwestern.edu

The perinucleolar structure is a nuclear body forming in highly malignant cancer cells. To address its structure and function, we employed chemical-biology approaches, in which small molecules that significantly reduce PNC prevalence were identified and used as chemical probes. Compound ML246 disassembles PNCs effectively in the nM range, and inhibits soft agar growth and invasion capability of cancer cells in vitro. In a PC3M human metastatic prostate cancer mouse model, ML246 significantly inhibits the growth of the tumors and blocks the metastasis of PC3M cancer cells into lungs. In a pancreatic cancer model, ML246 blocks metastasis to lung and liver. Thus, PNC disassembly corresponds well with inhibition of malignant behavior of cancer cells both in vitro and in vivo. Cellular and molecular analyses of the mechanism of action show that ML246 induces significant and reversible changes in the nucleolar structure as evaluated both by light and electron microscopy. Correspondingly, ML246 treatment induces reduction of pol I and pol III transcription. However, there is little change in pol II transcription. Gene array analyses show biased impact on gene expression upon treatment. These studies suggest that PNC function directly associates with nucleolar and pol III activities. Experiments are currently underway to identify the cellular pathways by which ML246 reduces PNC, disrupts nucleolar structure, regulates transcriptions, and blocks malignant behavior, so as to tease out the functional relation among all of them.

We would like to thank the support from NIH grant (GM078555) and grants from Robert Lurie comprehensive Cancer Center.

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-12-IN-6066 Correlative video-light-electron microscopy: development, impact and perspectives.

Rizzo R.1, Parashuraman R.1, Luini A.1
1Institute of Protein Biochemistry, National Research Council (CNR), Via Castellino 111, Naples, Italy
a.luini@ibp.cnr.it

GFP-based video microscopy provides profound insight into biological processes by generating information on the ‘history’, or dynamics, of the cellular structures involved in such processes in live cells. A crucial limitation of this approach, however, is that many important structures may not be resolved by light microscopy. Like more recent super-resolution techniques, Correlative video-Light-Electron Microscopy (CLEM) was developed to overcome this limitation. CLEM integrates GFP-based video and electron microscopy through a series of ancillary techniques such as proper fixation, hybrid labeling and retracing, and so provides the needed resolution. Another key characteristic of CLEM is that, by virtue of its electron microscopy component, it defines the context of the object of interest through the staining of bulk membranes and proteins and hence delineates all of the organelles and structure that surround or contact the structures of interest. This feature has no equivalent in light-based super-resolution techniques, which can only detect the fluorescent molecule used as marker. CLEM ‘multiplies’ the power of video microscopy, and is having an impact in several areas cell and developmental biology. Future developments of CLEM will, in our view, derive from combining again information deriving from GFP-based video microscopy (with its unique power to provide insight into the spatial-temporal organization of a process), with other types of information based on different imaging technology. In particular, correlating GFP-based video microscopy with the detection of large multi-molecular complexes, or with the localization of individual lipid species, will have significant impact in cell biology. Techniques that are suitable to achieve these aims are being developed. The potential, limitations and perspectives of correlative approaches aimed at integrating the unique insight generated by video microscopy with information from different forms of imaging will be discussed.

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-12-IN-6088 Visualizing the genome in living cells

Misteli T.1
1National Cancer Institute, NIH
mistelit@mail.nih.gov

In higher organisms, genomes are housed and function in the cell nucleus. While we have learnt a great deal in recent years about the sequence of genomes and the machinery that reads genome information, insights into how genomes function in the context of the architectural framework of the cell nucleus in a living cell are only now emerging. Several key concepts such as the existence of nuclear architectural proteins, the presence of distinct nuclear compartments, the non-random organization of genomes, and the dynamic nature of nuclear architecture are now recognized as driving genome function. Importantly, aberrations in nuclear architecture are now known to lead to diseases ranging from cancer to pre-mature aging. We are using various imaging modalities to elucidate the cell biological properties of the genome including mapping of the genome in 3D space, development of diagnostic applications based on genome organization and the visualization of genome function in living cells. In particular we have developed a novel diagnostic approach for breast cancer based on changes in higher order genome organization. We have also used Deep Imaging methods to follow the formation of chromosome translocations in living cells. Our observations establish a spatial and temporal framework for the formation of chromosome translocations and they identify several molecular pathways as contributors to translocation etiology. They also represent a illustrative example of the power of Deep Imaging as a new tool in cell biology.

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-12-IN-6093 Molecular scales: Imaging signals of disease with mass spectrometry

Heeren R. M.1,2
1FOM-AMOLF, Science Park 104, 1098 XG Amsterdam, The Netherlands, 2M4I, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands
heeren@amolf.nl

Molecular microscopy is a field of many scales; length, size, color, thickness, weight and many more. The study of molecular signaling processes related to disease on each of those scales requires the detection and analysis of the molecules involved as well as the evaluation of their spatial organization. This allows the detailed molecular typing of cells under duress and can provide critical insight in the activated pathways related to the progression of disease. Molecular imaging in the post-genome era provides insight in metabolomic and proteomic changes during the progression of disease on different scales; from single cells to whole biological systems.

Multimodal molecular imaging with mass spectrometry provides direct insight into the spatial organization of a wide variety of molecules on complex surfaces. It is truly a multiplex and label free imaging technique that takes advantage of all analytical capabilities embedded in modern mass spectrometers. This molecular histology technique allows for direct structural analysis of molecules liberated from the surfaces under study. As such, it enables direct targeted and untargeted analysis of endogenous metabolites, oligosaccharides, lipids, peptide and proteins, as well as exogenous compounds. The subsequent identification and quantification can contribute to disease specific molecular disease profiles.

The distribution of several hundreds of molecules on the surface of histological tissue sections can be determined directly in a single imaging MS experiment. This enables molecular pathway analysis as well as the role of the different molecular signals and their behavior under a drastically changing chemical or biological environment in these pathways. Imaging mass spectrometry has evolved to bridge the gap between different disciplines such as MRI, PET, fluorescence imaging and histology. In this contribution we will discuss applications of new MS based chemical microscopes in biomedical tissue analysis in various diseases. This lecture will highlight innovative applications of multimodal imaging MS that elucidate the way in which local environments can influence molecular signaling pathways on various scales.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-12-O-1881 Rapid immunohistochemical and ultrastructural diagnosis of plant virus diseases by microwave assisted sample preparation for transmission electron microscopy

Zellnig G.1, Zechmann B.1
1University of Graz, Institute of Plant Sciences, Graz, Austria
guenther.zellnig@uni-graz.at

The rapid diagnosis of plant virus diseases can be of great significance in order to identify the viral agent and to limit the spread of the diseases. Transmission electron microscopy (TEM) is a powerful method to diagnose viral diseases and to study the distribution of viruses within plant cells and tissues. Current protocols for both ultrastructural and immunological detection of viral diseases with TEM in plants take several days and are therefore not suited for the rapid diagnosis of such diseases. This study describes a method that allows the rapid ultrastructural and cytohistochemical detection of Zucchini Yellow Mosaic Virus (ZYMV) in Cucurbita pepo and Tobacco Mosaic Virus (TMV) in Nicotiana tabacum within about half a day and is based on microwave assisted plant sample preparation for TEM, negative staining methods, and immunogold labelling of viral coat protein.
With the help of microwave irradiation sample preparation for ultrastructural and cytohistochemical investigations was reduced from about 75h each to 136min and 89min, respectively. After cutting and contrasting of the sections typical ultrastructural alterations such as cylindrical inclusions in the cytosol could be observed in ZYMV infected plants whereas large areas of virions accumulating in the cytosol were visible in TMV-infected plants (Fig.1). In addition, negative staining of viral particles in the sap of the remaining ZYMV- and TMV-infected leaves revealed typical rod shaped virions with an average length and width of 707nm and 12nm for ZYMV and 280 and 17nm for TMV. These data were in accordance to the ultrastructural symptoms of ZYMV and TMV and the size range reported for the virions in the literature. Cytohistochemical labelling of TMV-coat protein with primary and secondary gold conjugated antibodies on sections prepared for cytohistochemical investigations was performed in 100min and identified virions within the cytosol as TMV in the TEM (Fig.1d). Comparison of gold particle density by image analysis revealed that samples prepared with the help of microwave irradiation yielded significantly higher gold particle density as samples prepared conventionally at room temperature.
This study clearly demonstrates that microwave assisted plant sample preparation for ultrastructural investigations, negative staining methods, and cytohistochemical localization of viral coat protein are well suited for the rapid diagnosis of plant virus diseases in altogether about half a day by TEM. As these protocols could also be applied in the fields of medical pathology they are an important application for the rapid diagnosis of virus diseases in human, animals and plants.

The authors gratefully acknowledge funding from the Austrian Science Fund (P20619, P22988).

Fig. 1: Cells of C. pepo (a-b) and N. tabacum (c-d) after microwave assisted sample preparation. Cylindrical inclusions (arrows in b) and parallel aligned virions (arrows in c,d) appeared within ZYMV- and TMV-infected plants, respectively. Gold particles were present in the areas of virions (d). C=chloroplasts, M=mitochondria, V=vacuoles. Bars=1µm.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-12-O-1902 Affinity labeling of thin sections – still a versatile tool for correlative light and electron microscopy

Schwarz H.1, Humbel B. M.2
1Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tuebingen, Germany, 2Electron Microscopy Facility, University of Lausanne, Biophore, CH-1015 Lausanne, Switzerland
heinz.schwarz@tuebingen.mpg.de

Abstract

In correlative microscopy, light microscopy provides the overview and orientation in the complex cells and tissue, while electron microscopy offers the detailed localization and correlation to subcellular structures. High quality electron microscopical preparation methods provide optimal preservation of the cellular ultrastructure. From such preparations serial thin sections are collected and used for comparative histochemical, immunofluorescence and immunogold staining.

In light microscopy histological stains identify the orientation of the sample. Immunofluorescence labeling facilitates to identifying the region of interest, namely, the labeled cells expressing the macromolecule under investigation whereas colloidal gold visualize the label within the cellular architecture at high resolution provided by electron microscopy.

 

References

Schwarz, H., Hohenberg, H. and Humbel, B.M. (1993) Freeze-Substitution in virus research: A preview. Chapt. 13, pp. 349-376 in: Immuno-Gold Electron Microscopy in Virus Diagnosis an Research (Hyatt, A.D., Eaton, B.T. eds.) CRC Press, Boca Raton, USA.

Schwarz, H., and Humbel, B.M. (2009) Correlative light and electron microscopy. Chapt. 21, pp. 537-565 in: Handbook of Cryo-Preparation Methods for Electron Microscopy (Cavalier; A., Spehner, D. and Humbel, B.M. eds.). CRC Press, Boca Racon,FL, USA.

Schwarz, H., and Humbel, B.M. (2014) Correlative light and electron microscopy using immunolabeled sections. In: Electron Microscopy: Methods and Protocols (Kuo, J., ed.) Methods Molecular Biology 1117, 559-592. DOI 10.1007/978-162703-776-1_12, Humana Press/Springer Science+Business media, New York 2014, USA.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-12-O-2512 Application of quantum dots to target, visualize and study biomedical processes on ultrastructural level

Zeuschner D.1, Mildner K.1, Rehberg M.2
1Max-Planck-Institute for Molecular Biomedicine, Muenster, Germany, 2Walter Brendel Centre of Experimental Medicine, Maximilians-Universität, Munich, Germany
d.zeuschner@mpi-muenster.mpg.de

Quantum dots (Q-dots) harbour unique chemical properties and are a valuable instrument for Correlative Electron Microscopy as they emit specific fluorescence signals, that can directly be documented and linked to an electron dense signal in the same sample processed for Electron Microscopy.
Q-dots are of small size and very robust, and are used as a tool for a variety of biomedical applications.
We have found recently while studying inflammatory processes in vivo that systemically injected Q-dots reach the microvasculature and even penetrate biological barriers depending on their surface chemistry (Ref1). Carboxyl Q-dots for example enter predominantly endothelial caveolae and get enriched in perivascular macrophages (Fig1 and 2). Moreover under pathophysiological conditions their behaviour is more differentiated, directly modulating inflammatory responses also in the extracellular environment (Ref2).
These findings suggest considering Q-dots as drug vehicles for modulating inflammatory processes.
Furthermore the in vitro application of Q-dots in pre-embedding protocols has a great potential to label the cellular volume in 3D and to bridge light microscopy imaging to ultrastructural resolution. Mainly by using a high pressure freezing, rehydration protocol for initial fixation dynamic membrane compartments are immobilized in their most ‘intact’ condition. (Ref3, 4). We are currently investigating the appearance and labelling efficiency of different membrane vehicles in a correlative approach by applying Q-dots (Fig3 and 4).

References:
1) Rehberg M, Praetner M, Leite CF, Reichel CA, Bihari P, Mildner K, Duhr S, Zeuschner D, Krombach F. Quantum dots modulate leukocyte adhesion and transmigration depending on their surface modification. Nano Lett. 2010 Sep 8;10(9):3656-64.
2) Rehberg M, Leite CF, Mildner K, Horstkotte J, Zeuschner D, Krombach F. Surface chemistry of quantum dots determines their behavior in postischemic tissue. ACS Nano. 2012 Feb 28;6(2):1370-9.
3) Donselaar E, Zeuschner D, Posthuma G, Humbel B M & J W Slot.
Immunogold labeling of cryosections from high-pressure frozen cells. Traffic. 2007 May;8(5):471-85.
4) Jimenez N, Post JA. A novel approach for intracellular 3D immuno-labeling for electron tomography. Traffic.  2012 Jul;13(7):926-33.

Fig. 1: Cross-sectioned venule in mouse cremaster muscle: injected carboxyl Q-dots are taken up by caveolae of the endothelium.Scale bar: 100nm

Fig. 2: Perivascular macrophages get enriched in carboxyl Q-dots applied systemically - note that mitochondria and ER are not reached by Q-dots.Scale bar: 100nm

Fig. 3: HUVECs were high pressure frozen, rehydrated and processed for pre-embedding labelling -overview illustrates integrity of different membrane compartments after HPF-rehydration and mild permeabilization.Scale bar: 500nm

Fig. 4: HUVECs were high pressure frozen, rehydrated and processed for pre-embedding labelling– Weibel Pallade bodies are well preserved in long branching networks. Scale bar: 200nm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-12-P-1877 Monitoring the intracellular transfer of chitosan nanoparticles by transmission electron microscopy

Costanzo M.1, Galimberti V.2, Cisterna B.1, Biggiogera M.2, Malatesta M.1, Zancanaro C.1
1Dept of Neurological and Movement Sciences, University of Verona, Italy, 2Dept of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Italy
manuela.costanzo@univr.it

Chitosan-based nanoparticles (chiNPs) are biocompatible polymeric drug carriers; they are able to prolong drug activity by stabilizing and modulating the release of the encapsulated agent, which allows its delayed delivery after NP administration (1). Due to their polycationic nature, chiNPs easily interact with the cell membrane thus rapidly crossing endothelial linings and entering cells; they are also able to cross the blood brain barrier (2).
Based on these features, we choose chiNPs as promising drug delivery systems for targeting the hypometabolizing D-Ala(2)-D-Leu(5)-enkephalin (DADLE) to the central nervous system (3). The induction of a hypometabolic state is of potential interest for surgical procedures, for preserving organs for transplantation, or for neuro- and cardio-protection. However, in the attempt to design a suitable drug delivery strategy, preliminary studies on target cells are required to elucidate the uptake mechanisms of NPs and their intracellular fate, with special reference to their degradation pathway.
Transmission electron microscopy is a valuable tool to investigate the intracellular trafficking pathway of NPs and clarify their interaction with organelles; however, due to their moderate homogeneous electron density, chiNPs are almost indistinguishable from the cytosolic milieu. To overcome this difficulty, cytochemical and immunocytochemical approaches were used to make chiNPs unequivocally detectable at the ultrastructural level. Neuronal cultured cells were administered FITC-labelled chiNPs (Fig. 1), and then submitted to DAB photo-oxidation. The resulting reaction product was easily visualized after osmication in epoxy resin-embedded samples (Fig. 2), revealing that chiNPs are internalized by endocytosis and can escape endosomes thus avoiding lysosomal degradation. In acrylic resin-embedded samples DAB precipitates were also visible without osmication; these specimens proved to be optimal for combining DAB photo-oxidation with immunoelectron microscopy, thus allowing the precise identification of the chiNP degradation sites (4).
DADLE-loaded chiNPs were also recognized in neuronal cultured cells by immunogold labeling with an anti-enkephalin antibody and this approach also allowed to label DADLE molecules released from chiNPs into the cytoplasm (Fig. 3) (5).
Preliminary tests ex vivo envisaged promising application of these ultrastructural techniques to also detect chiNPs in explanted tissues and organs, after systemic administration.
1. Jallouli et al. Int J Pharm 344:103, 2007
2. Jaruszewski et al. Nanomedicine 8:250, 2012
3. Malatesta et al. Rev Environ Sci Biotechnol 6:47, 2007
4. Malatesta et al., Micron 59:44, 2014
5. Malatesta et al. Histochem Cell Biol 2013 (DOI 10.1007/s00418-013-1175-9013-1175-9)

This work and BC fellowship were funded by Fondazione Cariverona, project Verona Nanomedicine Initiative. MC and VG are PhD students (at the University of Verona and Pavia, respectively).

Fig. 1: Fluorescence microscopy: FITC-labelled chiNPs are distributed in the cytoplasm of a neuronal cell. DNA was stained with Hoechst 33258. Bar, 10 µm.

Fig. 2: Transmission electron microscopy (epoxy resin embedding): a finely granular electron dense product is evident in an endosome-enclosed FITC-labelled chiNP after DAB photo-oxidation. Bar, 150 nm.

Fig. 3: Immunoelectron microscopy (acrylic resin embedding): a chiNP is labelled with the anti-DADLE antibody; some labelling also occurs in the cytosol. Bar, 150 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-12-P-2180 Chromatin alterations in pollen: an approach to understand initial mechanism of pollen embryogenesis in Hordeum vulgare

Pandey P.1, Houben A.1, Kumlehn J.1, Melzer M.1, Rutten T.1
1Leibniz Institute of Plant Genetics and Crop Plant Research
pandey@ipk-gatersleben.de
Pollen embryogenesis (POEM) is an efficient process for the production of doubled haploid plants and represents a convenient model for studying the process of plant cell proliferation in general and embryogenesis in particular. Uninucleate pollen is known to be the most amenable stage for the induction of POEM. Though exact mechanisms are unclear, successful induction of embryogenesis may depend on the epigenetic predisposition of microspores. To elucidate the cellular mechanisms causing gametophytic and embryogenic processes taking place in pollen grains, we have investigated chromatin alterations in both of these pathways. Since gametogenesis is accompanied by distinct chromatin alterations within the nuclei of the vegetative and generative cells (Pandey et al. 2013), it deemed likely that the switch from a gametophytic into an embryogenic pathway is also associated with epigenetic modifications. Immunolabeling confirmed this assumption. Not only did induction of POEM lead to chromatin modifications, typical patterns proved to be very different from those found during gametogenesis. While modifications were restricted to the nucleus during gametogenesis, upon induction of POEM, also the cytoplasm was concerned. Most prominent were the redistributions of the chromatin alterations H3K9ac, H3K4me2 and H3K27me3, all of which play a profound role in transcriptional activity. Inhibition of histone deacetylation by Trichostatin A, not only prevented the redistribution of H3K9ac into the cytoplasm but also that of H3K27me3 and H3K4me2, suggesting an interdependency of these modifications. Since all observed epigenetic alterations take place prior to the first pollen mitosis, they are the earliest known indicators of effective embryogenic induction. Further studies will be required to reveal causal relationships between particular epigenetic signatures and the commencement of POEM. The increased understanding of those mechanisms may eventually contribute to the development of improved haploid technology. References: Pandey et al. (2013) Cytogenet Genome Res DOI: 10.1159/000351211.

I gratefully acknowledge funding from the DAAD/Siemens Post Graduate Program. Katerin Kumke and Ingrid Otto for their technical support.

Fig. 1: Label of H3K27me3 in VN of gametogenic pollen(a), in cytoplasm after 2 days of induction(c), in nuclei after treatment of pollen culture with Trichostatin-A inhibitor(e)Label of H3K9ac in cytoplasm of GN of gametogenic pollen(b), in cytoplasm after 2 days of induction(d), in nuclei after treatment of pollen culture with Trichostatin-A inhibitor (f)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-12-P-3324 Effects of Retinoic Acid on Mice Skin

Kotil T.1, Erdoğan A.1, Özdemir İ.1, Mutlu H. S.1, Tapul L.1
1İstanbul University, İstanbul Faculty of Medicine, Histology and Embryology Department, İstanbul, Türkiye
erdoganas88@gmail.com

Retinoic acid (RA) is a regulator of epidermal cell growth and differentiation (1). RA shows its effects by binding RA receptors (RAR) and retinoid X receptors (RXR) (2). Aim of this study was to investigate the effects of excessive retinoic acid on cell proliferation and RAR alpha expression in skin.
We used a total of 12 adult female balb-c mice in control and experimental group. In experimental group 80mg/kg/day 13-cis RA was applied for 5 days. 5 days later 50mg/kg BrdU injection was done intraperiteonaly. Skin biopsies were fixed with %10 formalin, embedded in paraffin. Collagen fibers were stained with Van Gieson. For immunohistochemistry, sections were stained with anti RAR alpha and BrdU antibody and evaluated with light microscopy.
Type III collagen (thin) filaments were observed in RA applied group in sections stained with Van Gieson. There was no difference between experimental group and control group in BrdU labeling. In control group, RAR alpha immunoreactivity was observed intensively in dermis. In experimental group, weak labeling was seen in dermis.
Schwartz et al. (3) stated that type III collagen synthesis which is found with type I collagen fibers is increased with topical treatment of RA. Type III collagen fibers participate in type I collagen synthesis (4). We observed that type III collagen fibers stained with yellow with Van Gieson stain in RA treatment group. All-trans RA represses RAR alpha in cells transfected with RAR alpha and increases collagen synthesis by repressing increased MMP-1 synthesis through RAR alpha pathway (5). We observed that RAR alpha labeling is decreased in RA treatment group. Our findings are compatible with the studies of Watson et al. (5) and stimulative effect of RA on collagen synthesis may be through RAR alpha receptor and MMP synthesis. Further studies are needed to investigate the effects of different retinoic acid receptors and MMP types on this process.
References
1. Darwiche H, Celli G, Tennebaum T et al. Mouse skin tumor progression results in differential expression of retinoic acid and retinoid X receptors. Cancer Res, 1995,55: 2774-2782.
2. Roos TC, Jugert FK, Merk HF, Bickers DR. Retinoid metabolism in the skin. Pharmacological Reviews 1998,50(2): 315-333.
3. Schwartz E, Cruickshank A, Mezick JA, Kligman LH. Topical all-trans retinoic acid stimulates collagen synthesis in vivo. Dermatol, 1991, 96: 975-978.
4. Liu X, Wu H, Byrne M, Krane S, Jaenisch R. Type III collagen is crucial for collagen I fibrillogenesis and for normal cardiovascular development Developmental Biology, 1997, 94: 1852–1856.
5. Watson RE, Arjuna Ratnavaka J, Brooke RC, Yee-Sit-Yu S, Ancian P, Griffiths CE. Retinoic acid receptor alpha expression and cutaneous aging. Mech Ageing Dev. 2004, 125(7): 465-473.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-12-P-3520 Comparing immunolabelling properties in HeLa cells embedded in LR White, Lowicryl, or LR Gold resins after chemical or cryo fixation

Philimonenko A. A.1, Philimonenko V. V.1, Janda P.2, Hozák P.1
1Institute of Molecular Genetics, Prague, Czech Republic, 2J. Heyrovsky Institute of Physical Chemistry, Prague, Czech Republic
tolja@img.cas.cz

Ultrastructural immunolabelling brings important information about localization of biological molecules inside the cell compartments and is an indispensable tool of cell biology (Roth, 1989; Roth et al., 1981; Roth and Taatjes, 1998). For optimal result, it is necessary to preserve well both the fine structure and the antigenic properties of the sample. The choice of optimal technique for the sample preparation is therefore crucial. During the preparation of samples for post-embedding immunolabelling, the antigenic properties of the biological material are influenced by a number of factors, the most important being the fixation method, dehydration procedure, and the resin properties (Skepper, 2000; Stirling, 1990). Classical epoxy resins are in most cases not suitable for subsequent immunolabelling, therefore, a number of acrylic resins have been formulated, which, along with milder fixation, help to overcome the drawbacks of epoxy resins in the antigen preservation and accessibility (Acetarin et al., 1986; Carlemalm et al., 1985).
We compared four acrylic resins - LR White, LR Gold, Lowicryl HM-20, and Lowicryl K4M with regard of immunolabelling efficiency on ultrathin sections. Antigens were detected using standard immunogold technique in either chemically fixed or high-pressure frozen and freeze-substituted HeLa cells after embedment into each of the listed resins. We observed significant differences in immunolabelling densities between studied resins; however, the influence of the resin type was fixation-dependent and antigen-dependent. We recommend using LR White as a standard starting option, keeping in mind that individual optimizing of sample preparation conditions and resin choice may be needed for some antigens. Additionally, the influence of resin surface nanostructure on the immunolabelling efficiency will be discussed.

Acetarin, J.D., E. Carlemalm, and W. Villiger. 1986. J.Microsc. 143:81-88.
Carlemalm, E., W. Villiger, J.A. Hobot, J.D. Acetarin, and E. Kellenberger. 1985. J.Microsc. 140:55-63.
Roth, J. 1989. Methods Cell Biol. 31:513-551.
Roth, J., M. Bendayan, E. Carlemalm, W. Villiger, and M. Garavito. 1981. J Histochem Cytochem. 29:663-671.
Roth, J., and D.J. Taatjes. 1998. Histochem Cell Biol. 109:545-553
Skepper, J.N. 2000. J.Microsc. 199:1-36.
Stirling, J.W. 1990. J Histochem Cytochem. 38:145-157.

This work was supported by the TACR (TE01020118); the project „BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University“ (CZ.1.05/1.1.00/02.0109) from the European Regional Development Fund; the IMG institutional grant (RVO68378050).

Fig. 1: Relative labelling densities (LD) of four antigens in the nucleoplasm of HeLa cells after chemical fixation and embedment into Lowicryl HM20, LR Gold or LR White. LD for Lowicryl HM20 was set as 100%. * - P<0.05, ** - P<0.01.
↑ Go to the Table of content ↑

LS-13. Embryology and development biology

↑ Go to the Table of content ↑
Type of presentation: Invited

LS-13-IN-2100 Major causes of age-related chromosome segregation errors at meiosis I in oocytes

Sakakibara Y.1, Kitajima T.1
1RIKEN Center for Developmental Biology (CDB)
tkitajima@cdb.riken.jp

Chromosomes must be properly segregated during meiosis to transmit the correct set of the parental genome into gametes. Incorrect chromosome segregation produces aneuploid gametes, fertilization of which results in pregnancy loss and congenital diseases such as Down’s syndrome. However, it is known that the frequency incorrect chromosome segregation is extremely high at meiosis I in oocytes (20-40% in humans), compared to other cell divisions. Moreover, the frequency of the errors increases with maternal age. Why chromosome segregation is so error-prone and age-related in oocytes is not fully understood.
In this study, we established a high-throughput and high-resolution imaging of chromosome dynamics during meiosis I in live oocytes from naturally aged mice. Our high-throughput 4D recording approach using automated confocal microscopy (developed by Dr. Jan Ellenberg group at EMBL Heidelberg) allowed us to image >30 oocytes in a single experiment, at a high spatiotemporal resolution sufficient to detect nearly 100% of kinetochores and chromosomes at every timepoint from germinal vesicle breakdown to chromosome segregation and thus robustly track all the kinetochores throughout meiosis I. This approach yielded the datasets of >200 oocytes from aged mice, including >10 oocytes that underwent chromosome segregation errors at meiosis I. Thus, these datasets provide the first quantitative analysis of ‘at-risk’ single chromosome dynamics and a comprehensive resource to identify the major causes of age-related chromosome segregation errors at meiosis I in oocytes.

We thank Dr. Jan Ellenberg at EMBL Heidelberg for the macro for automated confocal microscopy.

Fig. 1: High-throuput live imaging of mouse oocytes expressing the kinetochore marker 2mEGFP-CENP-C (green) and H2B-mCherry (red).
↑ Go to the Table of content ↑
Type of presentation: Invited

LS-13-IN-2727 Imaging chromosome segregation in live mouse oocytes and early embryos

Ellenberg J.1
1EMBL, Cell Biology & Biophysics Heidelberg, Germany
schattsc@embl.de

Imaging chromosome segregation in live mouse oocytes and early embryos

The first meiotic division of mammalian oocytes is highly specialized. It has to segregate homologous chromosomes rather than replicated sister chromatids and achieve an extremely asymmetric division – polar body extrusion – to preserve the maternal nutrients for embryonic development. It is a very important division to understand, since errors in female meiosis I are the leading cause of aneuploidies that can lead to infertility, early embryonic lethality or severe developmental problems. Over the last years we have developed and applied live cell imaging approaches to quantitatively analyze the key steps of meiosis I in mouse oocytes, focusing on spindle assembly, chromosome biorientation and spindle relocation to the cortex. More recently, we have achieved efficient RNAi-mediated gene knock-down in oocytes despite the maternal load and have developed new, lower light and faster light-sheet based imaging systems tailored to the mouse oocyte and early embryo, that now also allow us to study the first embryonic divisions with high spatio-temporal resolution and understand the transition from meiotic to mitotic divisions and the origin and fate of aneuploidy in the early embryo.                                                               

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-13-O-3059 Posterior neural plate in axolotl undergoes a gastrulation-like involution and forms posterior trunk/tail muscles and tail spinal cord but no tail notochord

Kurth T.2, Taniguchi Y.1,2, Tazaki A.2, Tanaka E.2, Epperlein H. H.1,2
1TU Dresden, Department of Anatomy, Dresden, Germany, 2TU Dresden, Center for Regenerative Therapies, CRTD, Dresden, Germany
thomas.kurth@crt-dresden.de

Previous studies revealed profound rearrangements of the posterior neural plate in embryos of amphibians [1-5] and higher vertebrates [6,7]. As first shown by Bijtel, the posterior neural plate, although forming the posterior end (named “reg.3”) of the neuroepithelium of the neurula, has a mesodermal bias and gives rise to tail and posterior trunk muscles. Because morphogenesis and fate of the posterior plate had never been characterized in detail, we studied both issues in embryos of the axolotl (Ambystoma mexicanum). We grafted reg.3 from a GFP+ donor (stage 15) orthotopically into a white (d/d) host of the same stage and analysed the labelled descendants during tail development. We found that after neural tube closure reg.3 descendants undergo a gastrulation-like involution movement. As a result, the posterior half of reg. 3 gives rise to posterior trunk and anterior tail somites whereas the anterior half forms posterior tail somites and tail spinal cord. The border between tail and trunk somites does not seem to be prespecified within plate reg.3 but to rely on epigenetic events because tail presomitic mesoderm grafted into the trunk can form belly muscles which are not formed in the tail. Despite its gastrulation-like movement, reg.3 does not give rise to the notochord of the tail. This develops from involuted mesoderm underlying reg.3 in the neurula (stage 15). These results indicate that different mechanisms are involved in forming central and lateral mesoderm of the tail and that the posterior neural plate gives rise to both mesoderm and neuroectoderm.

[1] JH Bijtel, Roux Arch. EntwMech. Organ. 125 (1931) 448-486

[2] HH Chuang, Roux Arch. Entw. Mech. Organ. 143 (1947) 19-125

[3] P Ford, Proc. Zool. Soc. Lond. 119 (1947) 609-32

[4] AS Tucker and JMW Slack, Curr. Biol. 5 (1995) 807-813

[5] AS Tucker and JMW Slack, Development 121 (1995) 249-262

[6] M Catala, MA Teillet, and NM Le Douarin, Mech. Dev. 51 (1995) 51-65

[7] M Cambray and V Wilson, Development 134 (2007) 2829-2840

Financial support by DFG (EP 8/11-1) is gratefully acknowledged.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-13-O-3177 High-resolution episcopic microscopy (HREM) for phenotyping mouse embryos with gene deletions

Geyer S. H.1,2, Szumska D.3, Rose J.1, Wilson R.2, Mohun T.2, Weninger W. J.1
1Centre for Anatomy and Cell Biology & MIC, Medical University of Vienna, Austria , 2 MRC National Institute for Medical Research, London, UK, 3Wellcome Trust Centre for Human Genetics, Oxford, UK
stefan.geyer@meduniwien.ac.at

The genetic, physiologic and anatomic similarity to humans in combination with short reproduction times makes the mouse the most important model organism for studying the genetic regulation of developmental processes and the genesis of congenital diseases. The International Mouse Phenotyping Consortium (IMPC) plans to generate mouse strains carrying a deletion of each of the approximately 20 000 genes of the mouse genome. Individuals of the strains are phenotyped for gaining information of the function of the out-knocked gene. However, first results suggest that about one third of all knock out strains will produce homozygous offspring, which die during embryogenesis or in the perinatal period. Thus the “Deciphering Mouse Developmental Disorders” (DMDD) project was launched that aims at providing phenotyope information of embryos of such strains. In this presentation we will present pilot data from the DMDD project obtained by employing the “High resolution episcopic microscopy” (HREM) method for phenotyping E14.5 embryos.

HREM is a post-mortem three-dimensional (3D) imaging technique based on digital images, which show physical sections through histologically embedded and sectioned specimen. We fixed a total of 92 knock out embyos in Bouins fixative, dehydrated them in ethanols, and embedded them in plastic resin dyed with eosin. Using a microtome the blocks were cut in 3 µm thick sections. During sectioning, images of the block surface were captured after each cut. For this a microscope equipped with a GFP-filter set and a digital camera was aligned with the photo-position of the microtome. Since the block comes to rest at this position after each cut, a series of aligned digital images, which consisted of 2,000 to 3,500 single images was created. This series was converted into a volume data set with a voxel size of 3 x 3 x 3 µm3.

The quality of single HREM images nearly matches the quality of images captured from histologial sections. Thus phenotype screens of E14.5 mouse embryos can largely rely on virtual resections (Figure). In our pilot study, we developed a protocol, that is based almost solely on such sections. It enabled us to diagnose macroscopic malformations, as well as subtle, but potentially lethal tissue defects, which would escape their diagnosis with alternative 3D imaging techniques. Our results demonstrate that HREM is an optimal technique for phenotyping E14.5 mouse embryos with gene deletions.

Fig. 1: Figure: Sagittal virtual resection through HREM data of an E14.5 mouse embryo.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-13-O-3309 How can the in vitro culture affect the spindle assembly and chromosome segregation in mammalian oocytes?

Kovacovicova K.1, Anger M.1
1Central European Institute of Technology, Veterinary research institute, Brno, Czech Republic
kovacovicova@vri.cz

the chromosomal segregation errors in comparison with mitotically divided cells. This could lead to spontaneous abortion, embryonic lethality and serious congenital malformations. It has been previously shown, in mammalian oocytes, that many of quantitative and qualitative parameters, such a spindle microtubule morphogenesis, structure, assembly, cytoplasmic position and distribution, are affected by in vitro culture. The aim of our study is comparison of the spindle assembly and metaphase plate formation between in vitro and in vivo matured mammalian oocytes. To obtain physiologically relevant results we used multichannel live cell imaging confocal microscopy combined with high-resolution microscopy of fixed samples. Combination of those techniques allowed us to detect and monitor several parallel processes in every single cell. The spindle assembly and spindle bipolarization (the formation of bipolar spindle) are the results of counteraction of the kinesins (plus end-directed motoric proteins) and dyneins (minus end-directed motoric proteins). We discovered that the balance between those two groups of molecules, which is crucial for correct assembly of the spindle in meiosis I and meiosis II, is influenced by maturation in vitro. This was reflected by dramatic changes of spindle morphology and function observed in meiosis II. Since spindle is playing important role in faithful chromosome segregation, our results are suggesting that techniques frequently used in assisted reproduction techniques (ART) might contribute to chromosome segregation errors.

This work was supported by CSF Grant P502/12/2201 and MEYS Grants ED1.1.00/ 02.0068 and CZ.1.07/2.3.00/20.0213.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-13-O-3444 Development of reef-building corals

Laissue P. P.1, Boguslaw O.2, Smith D. J.1
1School of Biological Sciences, University of Essex, Colchester, United Kingdom, 2School of Engineering and Computing Sciences, Durham University, Durham, United Kingdom
plaissue@essex.ac.uk

Reef-building corals are the most biodiverse marine ecosystem, estimated to harbour a million species altogether. They also support the economic and food requirements of over half a billion people, and are a reserve of pharmaceutical and biotechnological products. Yet numerous factors, many man-made, threaten this important biome. Over 60% of the world’s reefs are in severe decline; all but the most remote reefs will be impacted in the next 50 years. Robust conservation strategies based on scientific knowledge of coral biology are required. However, corals are elusive, and despite decades of ecological, physiological and ‘-omics’-based studies, there is a remarkably wide gap in our understanding of fundamental mechanisms of coral growth.

To complement imaging techniques traditionally used in this area, such as histology and electron microscopy of fixed samples, we demonstrate the power of live imaging for long-term developmental studies. Live imaging of stony corals comes with a distinct set of problems. Firstly, a solid calcium carbonate skeleton scatters and absorbs light. Secondly, corals are highly sensitive especially to bright light, so minimisation of phototoxicity is central. Thirdly, fluorescent protein tagging is not possible, while strong autofluorescence and susceptibility often preclude the usage of live dyes. We counter these problems by means of sample preparation, low intensity for illumination and high sensitivity for detection, and the use of spectral signatures for label-free imaging.
We show long-term development of reef-building Acropora sp. using brightfield, widefield, confocal and light-sheet fluorescence microscopy. We report new growth structures, and show the dynamic interplay of the three main components – coral tissue, symbiotic algae and calcified skeleton. Development of polyps and the growing edge is described. Distinct zones, representing successive developmental stages, can be defined based on architectural components and dynamic behaviour. The approach yields a wide range of skeletal, tissue- and cyto-architectural parameters which can be used to measure the effect of different environmental conditions on coral morphology. It also enables the identification of mechanisms leading to coral disease, and both are important factors informing coral preservation programs.

Fig. 1: A) Brightfield image of coral surface with retracted polyps and brown symbiontic algae. Scalebar: 500µm. B) Light-sheet fluorescence image of extended polyp with symbiontic algae. Scalebar: 500µm. C) Confocal view of developing polyp. Coral tissue green, symbiontic algae red. Scalebar: 200µm. D) Mature polyp. View, colours and scalebar as in C).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-1395 Prenatal exposure to oral bitter leaf results in protein and DNA loss in the prefrontal cortex in Wistar rats

balogun w. g.1, cobham a. e.2, olajide o. j.3, ishola a. o.4, imam a.5, adeyemo k. a.6, enaibe b. u.7
1Department of Anatomy, College of Health Sciences, University of Ilorin, P.M.B 1515 Ilorin, Nigeria
ballonogodie@yahoo.com

Background: Bitter leaf is widely consumed by pregnant women in Africa for the treatment of many diseases during the various phases of pregnancy. But whether this treatment is deleterious to developing prefrontal cortex requires clarification.
AIM: This study investigated some histological effects of prenatal exposure of aqueous bitter leaf extract on the developing prefrontal cortex.
METHODS: Twenty-five pregnant Wistar rats with an average weight of 200g were randomly divided into five groups (n=5). The experimental groups were administered bitter leaf (400mg/kg) on the gestational days 1-7 (group B), 8-14 (group C), 15-21 (group D) and 1-21 (group E) while the control (group A) was given normal saline from gestational days 1-21. After parturition, the litters in each group were weighed and sacrificed by euthanized on postnatal day 35. The brain was weighed and the prefrontal cortices were excised, fixed in formol calcium and processed. Tissue sections were stained with: Feulgen reaction for DNA substances and Cresyl Fast Violet for Nissl substance.
RESULTS: Using CFV, there was partial loss of Nissl substances in the litters exposed to bitter leaf on E8-E14 and E1-21 while there was more DNA loss in the litters exposed to bitter leaf on E8-E14 and E1-21
CONCLUSION: The above findings suggest that prenatal exposure of young Wistar rats to oral bitter leaf at 400 mg/kg is associated with loss of protein and DNA in the prefrontal cortex.
Keyword: bitter leaf, histological studies

to the technical staff of the department and doctor akinola o.b

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-2014 Presence of Trefoil factor 1, 2 and 3 in the embryonal epidermis of a mouse

Bijelic N.1, Belovari T.1, Baus Lončar M.2
1Department of Histology and Embryology, Faculty of Medicine, Osijek, Croatia, 2Department of molecular medicine, Institute Ruđer Bošković, Zagreb, Croatia
nikola_bijelic@yahoo.com

Trefoil factor family (TFF) protein 1, 2 and 3 are low molecular weight peptides predominantly secreted in the gastrointestinal system, but also found in many different tissues, including nervous, respiratory and urinary system. They are also present during the embryonic development [1]. These proteins have an important role in epithelial protection by promoting epithelial restitution [2,3]. The aim of this research was to determine if TFF proteins are present in the developing embryonic epidermis.
Mouse embryos were isolated at E15 to E17 developmental stages (stages 23 to 25 according to Theiler), fixed in 4% paraformaldehyde and embedded in paraffin blocks. Blocks were cut into 6µm sagittal sections and transferred onto adhesive slides. Slides were incubated overnight with primary polyclonal rabbit anti-TFF1, anti-TFF2, and anti-TFF3 antibodies at 4°C, and negative controls with PBS. Biotinylated anti-rabbit antibody was applied the next day, followed by streptavidin HRP layer, and finally DAB for the visualization of the immunocomplexes.
Embryonic epidermis showed presence of all three TFF proteins at all monitored stages. Staining was mild to moderate for TFF1 and TFF2, and moderate to strong for TFF3. At stage E15, the signal was widespread, although less pronounced in stratum basale, and mostly sparing stratum corneum. At stages E16 and 17, the signal was more restricted to stratum granulosum and stratum spinosum, while stratum corneum and stratum basale showed little or no staining.
Positive staining for TFF proteins in embryonic mouse epidermis is in line with known properties and roles of these proteins in cell migration and apoptosis. Taken into consideration that TFF1 and TFF3 were found in primary mucinous skin carcinomas, but another study found no TFF immunostaining in normal adult human skin, our findings point to possible connections between mechanisms of carcinogenesis and embryonic development [4,5]. Further research may elucidate the impact of TFFs in the development of the epidermis.

1. N. Bijelić, T. Belovari, M. Baus Lončar, Acta Histochem. 15 (2013) p.204-208
2. G. Regalo, N.A. Wright, J.C. Machado, Cell. Mol. Life Sci. 62 (2005) p.2910-2915.
3. M. Baus-Loncar, A.S. Giraud, Cell. Mol. Life Sci. 62 (2005) p.2921-2931.
4. A.M. Hanby, P. McKee, M. Jeffery, W. Grayson, E. Dublin, R. Poulsom, B Maguire, Am. J. Surg. Pathol. 22 (1998) p.1125-1131.
5. J. Madsen, O. Nielsen, I. Tornøe, L Thim, U. Holmskov. J. Histochem. Cytochem. 55 (2007), 505-513.

The authors wish to thank Ms. Danica Matic for her valuable help in the histology laboratory.

Fig. 1: TFF1 signal in the epidermis of mouse embryo (17 days), predominantly in stratum granulosum and stratum spinosum.

Fig. 2: TFF2 signal in the epidermis of mouse embryo (17 days), mostly pronounced in stratum granulosum with mild staining in stratum spinosum.

Fig. 3: TFF3 signal in the epidermis of mouse embryo (17 days), strong signal in stratum granulosum and moderate in stratum spinosum.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-2365 FEG-SEM and TEM imaging combined with EDXS analyses of cuticle differentiation during early ontogenetic development in terrestrial crustacea

Mrak P.1, Žnidaršič N.1, Žagar K.2, Čeh M.2, Štrus J.1
1Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia, 2Jožef Stefan Institute, Department for Nanostructured Materials, Ljubljana, Slovenia
polonamrak20@gmail.com

Crustacean exoskeletal cuticle is an epidermal apical extracellular matrix, based on chitin-protein fibers and hardened by calcification. Mature cuticle is organized in three principal layers: the outermost epicuticle, exocuticle and the inner endocuticle. In adult isopods exo- and endocuticle are mineralized by calcite and by amorphous calcium carbonate and calcium phosphate. In isopods the cuticle is formed during ontogenetic development in the female brood pouch - marsupium and periodically renewed during molting of adults. The integrative part of cuticle formation is calcification, which is still poorly investigated in the forming cuticle of terrestrial isopods.

In our study a combination of complementary microscopic methods was applied to investigate the exoskeletal cuticle differentiation in sequential developmental stages of marsupial larvae mancae of isopod Porcellio scaber in comparison to adults. Ultrastructure and elemental composition of methanol fixed intact and transversely fractured cuticles were analysed by FEG-SEM imaging in LEI mode, supplemented by EDXS analyses. Next, methanol fixed resin embedded cuticles were prepared for correlative LEI imaging and EDXS analyses of the sample block face in combination with TEM of the corresponding ultrathin section. Methanol fixation was performed to preserve the mineral phases in the cuticle as suggested in the literature. Data were compared to conventionally aldehyde-fixed and resin embedded samples.

Our results suggest that exoskeletal cuticle calcification occurs already in marsupial larvae mancae. In advanced marsupial mancae the cuticle displays all three principal layers, lamellar sublayers in exo- and endocuticle and a network of pore canals (Fig.1). The cuticle elemental composition resembles that of adults, indicating prominent cuticle calcification in this stage (Fig.2). In earlier developmental stage, newly hatched marsupial manca, the exoskeleton displays elaborated structure of epicuticle and no distinctive structural division in exo- and endocuticle (Fig.3). EDXS analyses suggest initial calcium sequestration in the cuticle in this stage. Cuticle is a non-homogenous and dynamic matrix, thus different approaches are required for examining the cuticle during morphogenesis. Combination of applied microscopic techniques is suitable to obtain data on cuticle structure and calcification in the same sample and thus follow cuticle differentiation in progressive developmental stages. The results suggest the important role of calcification during cuticle formation in developing larvae, contributing to its support and mobility which was observed within marsupium. Elaborated cuticle also protects the larvae against physiological stress after having shed the egg envelopes.

Fig. 1: Exoskeletal cuticle in advanced marsupial manca of P. scaber. (a) TEM image of aldehyde-fixed specimen shows cuticle differentiation into epicuticle (ep), exocuticle (ex) and endocuticle (en), with lamellar sublayers and pore canals. ec-epidermal cell. (b) TEM image of methanol-fixed specimen shows lamellae of chitin-protein fibers in endocuticle.

Fig. 2: EDXS spectrum obtained from the cuticle surface in methanol-fixed advanced marsupial manca of P. scaber shows conspicuous calcium peaks, in addition to phosphorus, magnesium, sulphur, potassium, carbon and oxygen peaks. Ca peaks are evidently higher than P peaks.

Fig. 3: Ultrastructure of exoskeletal cuticle in the newly hatched marsupial manca. TEM image of aldehyde-fixed cuticle (a) and methanol-fixed cuticle (b) show elaborated epicuticle (ep) and procuticle (pro). ec-epidermal cell. In the image (b) chitin-protein fibers arranged in characteristic helicoidal pattern are clearly evident in the cuticular matrix.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-2721 Investigation Of Mitochondrial Activity And Cytoskeleton Organization In 3 Pronuclear Oocytes After Intracytoplasmic Sperm Injection

KOTİL T.1, EKTER KANTEN G.2, TUNALI G.3, KERVANCIOĞLU E.4, SOLAKOĞLU S.1
1Istanbul University, Istanbul Faculty of Medicine, Histology and Embryology Department, Çapa, İstanbul, 229 May Hospital IVF Department, Fatih, İstanbul, 3Zeynep Kamil Maternity and Children Hospital Infertility IVF Department, Üsküdar, İstanbul, 4Istanbul University Cerrahpaşa Faculty of Medicine, Department of Reproductive Endocrinology, Cerrahpaşa, İstanbul
tubakotil@msn.com

Digyny, is the presence of a third pronucleus due to failure of the extrusion of the second polar body (1). The oocyte mitochondria have critical roles such as production of ATP or regulation of Ca+2 homeostasis during oocyte maturation, fertilization and subsequent developmental processes (2,3). The cytoskeleton of the oocyte has regulatory function in meiotic spindle formation, chromosome segregation, pronuclear apposition and cytokinesis (4, 5). Mitochondrial membrane potential, distribution of F-actin and gamma-tubulin, and ultrastructure of the 3 pronuclear oocytes were investigated.
Oocytes from patients who were engaged to assisted reproduction program were collected and after ICSI process oocytes with 3 pronuclei were selected for the study. Informed consents were taken from all patients. Some oocytes were fixed by %4 paraformaldehyde and treated by anti-gamma tubulin and FITC – phalloidin antibodies for immunoflourescent study. For ultrastructural evaluation, oocytes were fixed with %1 glutaraldehyde and embedded in Epon 812. Sections were investigated by Jeol 1011 transmission electron microscope. Mitochondrial membrane potential was evaluated in fresh oocytes stained with JC-1 by fluorescent microscope.
Mitochondrial membrane potential of oocytes with three pronuclei was found to be comparable to normal zygotes (Figure 1). Gamma – tubulin were stained predominantly at the subplasmalemmal domain and microfilaments were localized at the cortical, but not perinuclear areas (Figure 2). Either partial or no cytoplasmic halo were detected. Large vacuoles were seen in cortical regions of oocyte cytoplasm. Ruptured and dilated ER cisternae were detected in some oocytes. Lipofuscin granules, degenerated mitochondria and multilamellar bodies were also seen in the ooplasm (Figure 3 and 4).
These findings suggest that mitochondrial membrane potential has no direct influence on extrusion of the second polar body. In our study quality of the oocytes with 3 pronuclei were in poor condition and most of them were showing evidences of aging. Aging features, triggered by various conditions including culture media and in vitro environment, might have caused failure in microtubule organization and microfilament distribution and such a disruption associated with the dynamics of the cytoskeleton may play a role in the formation of 3 pronuclei.
References:
1. Feenan, K. ve Herbert, M. (2006). Human Fertility, 9:3, 157-169
2. Wang, L., Wang, D., Zou, X., Xu, C. (2009). Journal of Zhejiang University Science B, 10(7), 483-492
3. Van Blerkom, J. ve Davis, P. (2007). Molecular Human Reproduction, 13(11), 759-770
4. Kim, H., Chung H.M., Cha, K., Chung, K.S. (1998). Human Reproduction, 13(8), 2217-2222
5. Veselska, R., Janısch, R. (2001). Scripta Medica (BRNO), 74(4), 265-274

Fig. 1: Fig.1: Mitochondrial membrane potential. JC-1 aggregate accumulation shows yellow-orange high membrane potential (asterisk).

Fig. 2: Fig. 2: FITC-phalloidin labeled actin filaments (green). Actin filaments located surface of the 3PN oocyte (arrows). No staining around the pronuclei (asterisk). DAPI: blue, PN: pronucleus

Fig. 3: Fig. 3: Dilated SER cisternae (asterisk) detected at the cortical region of the oocytes. PVS: perivitellin space

Fig. 4: Fig. 4: Vacuolated mitochondria (asterisk) M: mitochondria, nm: nuclear membrane, npb: nuclear precursor body, PN: pronucleus
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-2732 Phosphorylation of 4E-BP1 promotes translation at the oocyte spindle

Cerna R.1, Pesanova D.1, Prochazkova B.1, Kubelka M.1, Susor A.1
1Institute of Animal Physiology and Genetics, ASCR, Rumburska 89, Libechov, Czech Republic
cernar@iapg.cas.cz

Fully grown mammalian oocyte utilizes transcripts synthetized and stored during earlier development. In the mouse oocyte are three forms of cap-dependent translational repressors: 4E-BP1, 4E-BP2 and 4E-BP3 (Susor et al., in review process). The dominant form, 4E-BP1, inhibits cap-dependent translation by binding to the eIF4E translation initiation factor. Hyperphosphorylation of 4E-BP1 disrupts this inhibitory interaction and results in activation of cap-dependent translation (Pause et al., 1994). 4E-BP1 is highly phosphorylated after NEBD, while it is dephosphorylated after fertilization. Increased phosphorylation of 4E-BP1 (which is not detected in the cumulus cells) promotes cap-dependent translation of specific mRNAs after meiotic resumption. Our immunofluorescence analyses of the differently phosphorylated forms of 4E-BP1 in the oocytes during meiosis show even localization of 4E-BP1 and phospho-4E-BP1(T37/46) as well as spindle poles localization of phospho-4E-BP1(S65). 4E-BP1 phosphorylated on T70 co-localizes with its activator mTOR exclusively at the spindle. In addition, mTOR and CDK1 are the main positive regulators of 4E-BP1 phosphorylation after NEBD; on the other hand, inhibition of PLK1 does not affect 4E-BP1 phosphorylation. Treatment by Rapamycin, inhibitor of mTOR, results in decreased phosphorylation of 4E-BP1 on T37/46 in the whole oocyte, while T70 phosphorylation is decreased at the spindle. Our results show that 4E-BP1 phosphorylation forms promote in situ translation necessary to support spindle assembly and genomic stability.

References:
Susor, A., Pesanova, D., Cerna R., Kubelka, M., Danylevska, A., Anger, M., Toralova, T., Malik,R., Supolikova, J., Cook, M.J., Oh, J-S. (in review process).Temporal and Spatial Regulation of Translation in the Mammalian Oocyte via the mTOR/4F Pathway.
Pause, A., Belsham, GJ., Gingras, AC., Donzé, O., Lin, TA., Lawrence, JC Jr., Sonenberg, N. (1994). Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature 371,762-767.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-2764 Phosphorylation of 4E-BP1 promotes translation at the oocyte spindle

Cerna R.1, Pesanova D.1, Prochazkova B.1, Kubelka M.1, Susor A.1
1Institute of Animal Physiology and Genetics, ASCR, Rumburska 89, Libechov, Czech Republic
cerna@iapg.cas.cz

Fully grown mammalian oocyte utilizes transcripts synthetized and stored during earlier development. In the mouse oocyte are three forms of cap-dependent translational repressors: 4E-BP1, 4E-BP2 and 4E-BP3 (Susor et al., in review process). The dominant form, 4E-BP1, inhibits cap-dependent translation by binding to the eIF4E translation initiation factor. Hyperphosphorylation of 4E-BP1 disrupts this inhibitory interaction and results in activation of cap-dependent translation (Pause et al., 1994). 4E-BP1 is highly phosphorylated after NEBD, while it is dephosphorylated after fertilization. Increased phosphorylation of 4E-BP1 (which is not detected in the cumulus cells) promotes cap-dependent translation of specific mRNAs after meiotic resumption. Our immunofluorescence analyses of the differently phosphorylated forms of 4E-BP1 in the oocytes during meiosis show even localization of 4E-BP1 and phospho-4E-BP1(T37/46) as well as spindle poles localization of phospho-4E-BP1(S65). 4E-BP1 phosphorylated on T70 co-localizes with its activator mTOR exclusively at the spindle. In addition, mTOR and CDK1 are the main positive regulators of 4E-BP1 phosphorylation after NEBD; on the other hand, inhibition of PLK1 does not affect 4E-BP1 phosphorylation. Treatment by Rapamycin, inhibitor of mTOR, results in decreased phosphorylation of 4E-BP1 on T37/46 in the whole oocyte, while T70 phosphorylation is decreased at the spindle. Our results show that 4E-BP1 phosphorylation forms promote in situ translation necessary to support spindle assembly and genomic stability.


Susor, A., Pesanova, D., Cerna R., Kubelka, M., Danylevska, A., Anger, M., Toralova, T., Malik,R., Supolikova, J., Cook, M.J., Oh, J-S. (in review process).Temporal and Spatial Regulation of Translation in the Mammalian Oocyte via the mTOR/4F Pathway.
Pause, A., Belsham, GJ., Gingras, AC., Donzé, O., Lin, TA., Lawrence, JC Jr., Sonenberg, N. (1994). Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature 371,762-767.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-2807 3D analysis of the pharyngeal arch arteries of chick embryos of developmental stages HH16 – HH18.

Maurer B.1, Muratovic A.1, Geyer S. H.1, Weninger W. J.1
1Center for Anatomy and Cell Biology & MIC, Medical University of Vienna, Vienna, Austria.
barbara.maurer@meduniwien.ac.at

Knowledge about the genetic and epigenetic factors effecting the development of the cardiovascular system rests on detailed comparisons of the phenotype of normal embryos of biomedical model organisms with that of embryos of genetically engineered, and experimentally manipulated littermates. In particular the chick is a valuable model, because it offers the possibility to experimentally induce malformations by in ovo manipulation. For interpreting experimentally induced phenotypes, precise three-dimensional (3D) information about the normal situation is mandatory. This study aims at providing detailed 3D computer models and metric analysis of the pharyngeal arch arteries of normal chick embryos of developmental stages 16, 17,and 18 according to Hamburger and Hamilton (HH). From 5 embryos of each of the developmental stages HH16 to HH18 digital volume data set with Voxel sizes of 2.14 x 2.14 x 2 μm were generated with the high-resolution episcopic microscopy (HERM) data generation method. Out of the volume data, virtual, surface rendered 3D models of the lumina of the pharyngeal arch artery derivatives, and the heart were reconstructed. The models were visualized (Figure) and metrically analyzed using the visualization tools of the software package Amira following a recently developed analysis protocol. We provide detailed descriptions of the topology of the pharyngeal arch arteries and the ascending and descending aortas and descriptive statistics about their diameters. These data will serve as reference data for diagnosing malformations in manipulated embryos and provide insight into normal remodeling of the pharyngeal arch arteries between HH16 and HH18.

Fig. 1: Figure. Pharyngeal arch arteries (PAAs) of a chick embryo of HH16. View from venterolateral left. Ta = truncus arteriosus, DoA = aorta dorsalis, L1/R1 = left and right 1st PAAs, L2/R2 = left and right 2nd PAAs, L3 = 3rd left PAAs, dr4LR = dorsal roots of left and right 4th PAAs , vr4LR = ventral roots of left and right 4th PAAs.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-3003 Many functions for PLK1 in mammalian oocytes: Nuclear permeabilization, nuclear lamina breakdown, acentriolar spindle assembly and APC/C activation

Solc P.1, Kitajima T.2, 4, Yoshida S.4, Brzakova A.1, Baran V.3, Mayer A.1, Motlik J.1, Ellenberg J.2
1Institute of Animal Physiology and Genetics, Libechov, Czech Republic, 2Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany, 3Institute of Animal Physiology, Kosice, Slovakia, 4Laboratory for Chromosome Segregation, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan
solc@iapg.cas.cz

Polo-like kinase 1 (PLK1) is a protein kinase that orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, little is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live oocyte imaging, we comprehensively describe PLK1’s functions during meiosis in mouse oocytes. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and kinetochores. We demonstrate a function of PLK1 in nuclear envelope permeabilization and nuclear lamina breakdown during resumption of meiosis. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote bipolar spindle formation, as well as for stable kinetochore-microtubule attachment through BUBR1 phosphorylation. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.

Supports: P301-11-P081 and LH12057 to P.S.; JSPS KAKENHI 00376641, Nakajima F., and Uehara Mem. F. to T.S.K.; FP7/2007-2013 under agreement n°[241548] “Mitosys” and DFG EL246/4-1,2 within the SPP 1384 to J.E. T.S.K was partly supported by Human Frontier Science Program. A.B and A.M. were partly supported by P502/11/0593. V.B. was supported by APVV-0237-10. ExAM CZ.1.05/2.1.00/03.0124 to J.M.

Fig. 1: PLK1 localizes to MTOCs and kinetochores:Time-lapse imaging of meiosis I in oocytes expressing EGFP-PLK1 (green) and 3mCherry-CENP-C (kinetochores, red). Maximum intensity z-projection images at representative time points are shown. Time after induction of meiotic resumption (h:mm). Scale bar = 10 µm. Insets show magnified images on kinetochores.

Fig. 2: PLK1 with CDK1 cooperates on NE permeabilization: Time lapse of 70kDa-dextran-TRITC microinjected H2B-EGFP oocytes after induction of meiotic resumption in control, BI2536-, flavopiridol (Fl) and Fl+BI2536 medium. 70kDa-TRITC signal is pseudocolored and maximum intensity z-projection for H2B-EGFP is gray.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-3279 Oocyte Spindle Assembly Checkpoint in Space and Time

Anger M.1,2, Awadova S.1, Kovacovicova K.1
1Central European Institute of Technology, Veterinary Research Institute Brno, Czech Republic, 2Institute of Animal Physiology and Genetics, Libechov, Academy of Sciences of the Czech Republic
anger@iapg.cas.cz

Mammalian oocytes and embryos frequently suffer from aneuploidy, caused by chromosome segregation errors. Most of the time, the aneuploidy originated in meiosis or in first mitotic cycles in developing embryos would prevent further development. However, in several cases, the extra chromosomes are tolerated, which leads into severe mental and developmental disorders, such as Down syndrome. The reason, why chromosome segregation errors are so frequent in germ cells and embryos, is unknown. It seems that the problem lies in less stringent control mechanisms operating in these cells. In our study we have focused on a surveillance checkpoint mechanism called Spindle Assembly Checkpoint (SAC) during female meiosis I. Using live cell imaging multichannel microscopy we have tested, whether mouse oocytes are capable of detecting univalent chromosomes and single chromatids in meiosis I. We have also monitored the activity of SAC on every single kinetochore within individual cells throughout meiosis I. These events were detected together with chromosome movements, spindle formation, Anaphase Promoting Complex (APC) activation and polar body extrusion (PBE) simultaneously in individual oocytes at various time points during first meiotic division. Our results showed that SAC in mammalian oocytes works differently, compared to the somatic cells. In contrast to the somatic cells, single chromatids, univalents and unaligned chromosomes are unable to prolong anaphase onset. Moreover, in oocytes from aged individuals, SAC proteins are displaced from individual kinetochores with different dynamics then in young oocytes. This indicates that checkpoint mechanisms operating in oocytes, which are involved in monitoring chromosome segregation, are insufficient in prevention of propagating the aneuploidy to the embryo.

This work was supported by CSF Grant P502/12/2201 and MEYS Grants ED1.1.00/ 02.0068 and CZ.1.07/2.3.00/20.0213.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-3296 Spindle aparatus during transition from meiosis into mitosis

Sodek M.1, Novakova N.1, Nguyen N. Q.1, Anger M.1
1Central European Institute of Technology, Veterinary Research Institute, Brno, Czech Republic
sodek@vri.cz

Aneuploidy has critical consequences such as miscarriages, stillbirths and severe mental and physical defects. An improper number of chromosomes mainly results from errors in chromosomal segregation during the meiosis and the first mitotic divisions after fertilization. During early development mammalian embryo divides mitotically several times and it seems that those few first divisions are different than mitosis in somatic cells. This developmental period is also associated with the increase of chromosome segregation errors. The mechanisms behind the rise in aneuploidy level during embryonic early stages are still unclear. Employing live cell confocal microscopy we focused on the regulation of the spindle length, duration of the different cell cycle phases and the fidelity of the chromosome segregation during the first four embryonic divisions. We have combined the advantages of live cell imaging with chromosomal spreads and other methods. It gave us the unique opportunity to obtain complete picture of the cell cycle events and results of chromosome segregation in living embryos. According to our data the regulation of spindle length changes between first and second mitosis in developing embryo. Besides regulation of the spindle length we have also discovered that distribution of cohesin, the protein complex, which holds sister chromatids together, is also changing dramatically in the first embryonic mitoses. We believe that our results will help to understanding of basic developmental processes in mammalian embryos.

This work was supported by CSF Grant P502/12/2201 and MEYS Grants ED1.1.00/ 02.0068 and CZ.1.07/2.3.00/20.0213.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-3302 Vasculogenesis study using quantitative analysis from patterns obtained by video-microscopy.

Alves A. P.1, Mesquita O. N.1, Agero U. B.1
1Departamento de Física, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, BH, 31270-901, Brazil;
anafisic@gmail.com

Vasculogenesis is the process of new blood vessel formation that generates one of the first functional systems in a forming embryo. It is a dynamic process that involves changes of endothelial cells to form blood vessels, which happens concomitant with the embryo development. The own way of endothelial cells clusters selfish organize became the system ideal to study following the evolution of patterns. In order to quantify this phenomenon we use the video microcopy to obtain a movie following the vessels emergence in an embryo. We extracted embryos from chicken eggs at stage 12 HH that lay in a dish with culture medium made of agar and albumin. During the experiments, the samples are placed in a microscope incubator to control the environment at 370C degrees and 60% of humidity. A continuous movie monitoring the vessel’s growth for a period of 15 hours is made. To follow changes in the vasculogenesis patterns a rectangular region on area opaca was selected, as showed in the Fig.1. In that region were made measurements during time of fractal dimension, area and amount of clusters. The fractal dimension measured following the vessels growths increases until it reaches saturation. In the beginning of the process with just a few clusters with a random spatial distribution, the fractal dimension measured is (Df= 1, 52 ± 0, 06) a value close to results obtained for cluster-cluster interactions. This clusters self-organize to create a polygonal network. In the end of the process when the network is assembled the value measured is (Df= 1, 72 ± 0, 06) a value close to results reported for a directed percolation process in 2D. That is according with the dynamic of the process, as reported in literature after the onset of circulation the cells move collectively. With the other parameters like the number and maximum area of the clusters we can describe the aggregates dynamics. The number of cells aggregates grows as a Gaussian function, which after reaching a maximum value it decreases indicating that coalescence prevails in the system. From the maximum area of aggregates we can identify the percolation of primary plexus; there is phase transition of a non-connected region to all the regions connected. In this work, we show that sequences of interactions between cell aggregates promote the connections to build the complete vessels network, and obtain statistical parameters that can describe the vasculogenesis process.

The authors would like to acknowledge the support of the Brazilian agencies CNPq, CAPES, FAPEMIG, and INCTFcx.

Fig. 1: The rectangle selected on area opaca of embryo at stage HH-12 is followed in time to obtain the vessels emergence. The picture in the right side shows the selected area at: 0 hours, 7 hours and 14 hours.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-3330 Placental Apoptosis in Intrauterine Growth Restriction: An Ultrastructral Study

Kumar S. N.1,4, Raisuddin S.4, Vaibhav K.4, Bastia B.1, Sharma s. K.2, Borgohain D.3, Jain A. K.1
1National Institute of Pathology (ICMR), Safdarjang Hospital Campus, New Delhi -110029, INDIA, 2Regional Medical Research Centre (ICMR), Dibrugarh, Assam, India , 3Assam Medical College, Dibrugarh, Assam, India, 4Jamia Hamdard, New Delhi, India
drakjain@gmail.com

Objective: To investigate the role of placental cellular apoptosis through Bcl-2/BaX immunoreactive expression and ultrastructural alterations in placenta of tea garden workers.

Samples: Tissue specimens from 50 full term placentas of tea garden workers (TGW) and 35 normal term placentas appropriate for gestational age (AGA) of house wives were collected.

Method: Bcl-2/BaX expression was assessed by immunohistochemistry on paraffin-embedded sections; whereas, apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay and transmission electron microscopy (TEM).

Results & Conclusions: Bcl-2 protein was abundantly immunolocalized in syncytiotrophoblasts of normal term placentas of house wives, while, least abundant in term placentas of TGW. BaX was over-expressed in placental tissue of TGW than those of house wives. In accordance with the change of ratio of these two molecules, the increase of apoptotic cells was observed in placenta of TGW. These data indicate that Bcl-2 and BaX are spatio-temporally regulated during placental development and the difference in their expression is at least in part responsible for the delicate balance between cell proliferation and programmed cell death in the human placenta. Apoptosis was indicated by the morphological features, such as, condensation and margination of chromatin along an intact nuclear envelop in syncytiotrophoblast nuclei; villous surface fibrin deposits; loss of microvilli with membrane blebbing; cytoplasmic condensation; autophagocytosis of cellular debris containing nuclear fragments. From these TEM observations, it could be concluded that human placental syncytiotrophoblast undergoes apoptosis, and this process is associated with breaks in the trophoblast covering of villi. In case of low birth weight (LBW) babies placenta, typical features of apoptosis were observed including internucleosomal DNA degradation, and both nuclear (nuclear condensation and fragmentation) and extranuclear (cell blebbing) morphological alterations. The placental cellular apoptosis was confirmed by TUNEL assay. Trophoblastic apoptosis of the placenta might play an important role in the pathogenesis of LBW babies in case of TGW, and thus may be related to lowered expression of Bcl-2 and higher expression of BaX.

The financial support for the study received from Indian Council of Medical Research, New Delhi is gratefully acknowledged

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-5853 3D reconstructions of whole-mount embryonic dental epithelium from optical sections as a tool for fast and reliable morphology examination

Prochazkova M.1,2, Prochazka J.1, Klein O. D.1,3
1Program in Craniofacial and Mesenchymal Biology and Department of Orofacial Sciences, University of California San Francisco, San Francisco, CA, USA, 2Department of Anthropology and Human Genetics, Faculty of Science, Charles University in Prague, Czech Republic, 3Department of Pediatrics and Institute for Human Genetics, University of California San Francisco, San Francisco, CA, USA
michaela.prochazkova42@gmail.com

Development of tooth primordium is a well-established model for epithelial morphogenesis. Good accessibility of embryonic tooth germs for pharmacological treatment in vitro combined with molecular genetic approaches in mouse model enable comprehensive examination of the role of particular genes during epithelial morphogenesis. The analysis of two-dimensional histological sections is not sufficient to fully evaluate the variations in epithelial morphogenesis under altered genetic or biochemical conditions. Thus, 3D reconstructions of embryonic dental epithelium have been introduced as essential tool for analysis of embryonic teeth development. However, the classical method used for 3D reconstructions of dental epithelium is time-demanding and still based on histological sections (1). During this process, the tissue suffers from dehydration, which, along with mechanical sectioning and errors in digitalization of the contours, causes artifacts in the final 3D reconstruction. Here we present a new approach that allows generation of high-quality 3D reconstructions of dental epithelial structures in a short time. We used Krt14-Actin-GFP mouse line which is suitable for visualization of the epithelial cells in many tissues. The mandibles of transgenic embryos were dissected at different stages of development (E12.5 – E14.5) and fixed in 4% PFA. The tissue was subsequently cleared for two weeks following the Scale method (2). Cleared mandibles were scanned using LSM confocal microscope and the three-dimensional projection of dental epithelium was reconstructed in Imaris Bitplane software. As an example we show the wild-type molar and incisor epithelium at different developmental stages; morphological changes in molar field after disruption of Fgf and Shh signaling pathways in vitro; and the molar phenotype after specific cell line ablation using DTA transgenic system. In contrast to the classical method, optical sectioning of the tissue cleared by Scale method allows preservation of the structure in whole-mount, and therefore minimizes the artifacts. With slight variations this approach can be used in variety of epithelial structures such as hair follicles, taste papillae, and salivary glands. Taken together, the described method of 3D reconstruction is an advantageous approach that produces highly accurate 3D images of the organ of interest with reasonable time requirements.


1. R. Peterkova, M. Hovorakova, M. Peterka, H. Lesot, Three-dimensional analysis of the early development of the dentition. Aust. Dent. J. 59 Suppl 1, 55–80 (2014).
2. H. Hama et al., Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat. Neurosci. 14, 1481–1488 (2011).

Fig. 1: 3D reconstrution of mouse molar epithelium at E14.5 in Krt14-actin-GFP line (mesenchymal view). Scale bar, 150 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-5903 DEB-1 protein is required for chromosomal dynamics and homologous pairing in C.elegans meiosis.

Rohožková J.1, Fukalová J.1, Hozák P.1
1Institute of Molecular Genetics, Prague, Czech Republic
rohozkova@img.cas.cz

   Vinculin (VCL) is an actin-binding protein responsible to sense the mechanical properties of the extracellular environment. It is the main component of the focal adhesions establishing cell-cell and cell-matrix interaction. Our preliminary data locates VCL to both somatic nuclei as well to meiosis-specific structure in spermatocytes nuclei – the synaptonemal complex. This finding shows VCL as a new player in the mammal meiosis. In C. elegans, vinculin ortholog is DEB-1 protein which has ~85% amino acid sequence homology. Depletion of DEB-1 resulted into hermaphrodite sterility and “EMO phenotype” with refractive eggs that fail to hatch. However, till present there was no evidence of DEB-1 participation in meiosis.
   This model organism is well-established for the study of meiosis because adult hermaphrodite meiotic nuclei progress in the entire gonad, which allows visualizing all meiotic stages. We therefore localized the DEB-1 with a specific antibody in the whole C. elegans gonad and within rachis. As the presence of DEB-1 in meiocytes was microscopically not clearly apparent, so we produced deb-1(RNAi) knock-down worms. Our reached data suggest DEB-1 independent role in the distal part of hermaphrodite gonad independent on the ovulation activity of the contractile apparatus in the proximal somatic part. Furthermore we demonstrate clear evidence of DEB-1 engagement in meiotic prophase progression. Knock down of DEB-1 via RNAi interference impacts chromosomal pairing stabilization, attachment of chromosomes to cytoskeletal forces and formation of synaptonemal complex during prophase I, resulting in formation of synapsis between non-homologous chromosomes, meiosis delay and increased presence of univalents after diakinesis. We proceed ultra-structural visualization of meiotic chromosomes in zygotene and pachytene wild type and deb-1-/- worm gonad. Furthermore we detected DEB-1 and synaptonemal proteins (HIM-3, SYP-2) on ultrathin sections, using immuno-gold labeling method. These TEM results allowed us to describe ultrastructural localization of DEB-1 in the meiotic cells.

This experimental work is supported by the project „BIOCEV – Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University“ (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund.

Fig. 1: The deb-1(RNAi) hermaphrodites develop gonads of normal size and organization. Transition zone is present; with some transition zone-like cells (Leptotene/Zygotene onset). During diakinesis, we observed elevated numbers of DAPI-positive structures, ranging from 8 to 12 compact bodies (Diakinesis onset).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-13-P-5940 Demonstration of The Development of Tight-Junctions Between Sertoli Cells In Post-Natal Rat Testis In Days 4-12: Immunohistochemistry and Ultrastructural Observation

Kolbasi B.1, Kervancıoğlu G.2, Çetinel Ş.1
1Marmara University, School of Medicine, Dept. of Histology and Embryology, İstanbul, TURKEY, 2Kanuni Sultan Süleyman Training and Research Hospital, Infertility Center, Andrology Lab.İstanbul, TURKEY
bircankolbasi@yahoo.com

Introduction: Testicular development plays an important role in male fertility. Gonadal development begins on the 10th day fetal rat. In the 18-19 day the seminiferous tubules are filled with primordial gonocytes in the fetal testis. From postnatal day 10 primordial germ cells divide and differentiate into spermatogonia. Over the next 40days and so, testicular development demonstrates a series of successful developing germ cell layers. For a proper and continous development, the permutation and function of junctions among Sertoli cells play a crucial role. While spermatogonia transfer into spermatid through the blood-testis barrier, they also move from basal compartment to adluminal compartment.
Aim: We aimed to show the consecutive stages of tight junction morphology in the days 4-12 in the neonatal rat testis.
Materials and Methods: In this study rat testis of days 4-12 were obtained and proceeded for both light and electron microscopy. We accepted the date of birth 0. We observed the blood-testis barrier development day by day in neonatal rats by using ZO-1 immunohistochemistry and for ultrastructural examiation tissues were proceeded by ruthenium red in order to visualize the tight-junctions.
Results: In the days 4-7 the interval between Sertoli cells were wider and from the day 8 to 12 the interval became tighter and the 12th day showed the final morphology of tight junction, ZO-1 immunohistochemistry demonstrated the same morphology in accordance with electron microscopy.
Conclusion: Ruthenium red observations in electron microscopy demonstrated that tight-junction formation in neonatal rat begins by days 8-12.

↑ Go to the Table of content ↑

LS-14. Neuroscience

↑ Go to the Table of content ↑
Type of presentation: Invited

The use of confocal laser scanning microscopy to study the morphology and function of astrocytes

Grosche A.1, Grosche J.1, Pfrieger F. W.2, Pekny M.3, Reichenbach A.1
1ProRetina Stiftungsprofessur für Netzhautforschung, Institut für Humangenetik, Regensburg, 2INCI, Dept. Neurotransmission, Strasbourg, France, 3Laboratory of Astrocyte Biology and CNS Regeneration, Gothenburg, Sweden
antje.grosche@klinik.uni-regensburg.de

Information processing in our brain is a complex and delicate action. It was a well-accepted dogma in neuroscience that exclusively nerve cells are in charge of transfer and handling of information in the central nervous system communicating via transmitter release at neuronal synapsis. However, evidence is accumulating profoundly calling this into question and more and more light is shed onto the fascinating role of another abundant cell type in the central nervous system – the glia cells. Glia named for the Greek word for "glue" were thought to simply serve as a passive scaffold serving nerve cells as mechanical support and supplying them with nutrients.

Here we present methods, based on the use of confocal laser microscopy, that helped to demonstrate that astrocytes, the major glia cell type in the brain, actually speak the neuronal language. We were able to show that astrocytes use the same cellular machinery as neuronal synapses to release the prominent excitatory transmitter of the brain - glutamate. Moreover, we carefully investigated the morphology and spatial organization of astrocytes in the adult and aged brain. Combined with approaches to determine their density in the tissue, such data give an important insight into spatial organization of astrocytes – a prerequisite to understanding of the language that glia cells use and the control they exert over neuronal communication.

In summary, these very recent data resulting from the elaborate implementation of confocal laser scanning microscopy helped to redefine the role of astrocytes and glia cells in general, from passive bystanders to active partners of neurons and to develop new concepts of how our brain works.

↑ Go to the Table of content ↑
Type of presentation: Oral

LS-14-O-1429 Wiring between visual interneurons in a locust- are the monopolar cells L1 and L2 involved in motion detection pathways?

Leitinger G.1, Wernitznig S.1, Zankel A.2, Pölt P.2, Kolb D.1, Rind F. C.3
1Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Austria, 2Institute for Electron Microscopy and Nanoanalysis, University of Technology, Graz, Austria, 3Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK
gerd.leitinger@medunigraz.at

Processing of visual information in insect compound eyes starts at the synapse between photoreceptors (R-cells) and lamina monopolar cells (L-cells). In locusts, two large monopolar cells (L1 and L2) were already investigated (Nowel and Shelton 1981, Cell Tissue Res 216:377), but their connectivity pattern has not been established yet.
To disclose the wiring between the R and L-cells, we produced series of electron micrographs with serial block face scanning electron microscopy (SBEM, Denk and Horstmann 2004, PLOS Biol 2: e329; Zankel et al. 2009, J Microsc 233: 140). SBEM allowed us to automatically slice and image, i.e. cut sections from a tissue block and in each case subsequently scan the block face using a backscatter electron detector. 3D-reconstructions of L1 and L2 from one column of the first optic neuropile (Fig.1) allowed us to map their synaptic connections (Fig. 2).
The input from photoreceptor cells to both the L1 and L2 neurons was restricted to one column situated underneath one lens of the compound eye, and their axon diameters were as large as 5.5 (L1) and 3.4 (L2) µm, allowing for high conduction velocities. This suggests that the neurons may drive motion detection pathways, for which differences in illumination between neighboring columns must rapidly be calculated.
However, although L1 and L2 do not differ in their response to light (James and Osorio 1996: J Comp Physiol A 178: 183), we found fundamental differences in their connectivity pattern: Our reconstructions show that both L1 and L2 receive synapses from R- cells in the lamina (Fig. 1), but L1 receives more than twice as many synapses and contrary to L2 also exhibits feedback synapses onto the R-cells, so it could have an influence on the R-cells’ sensitivity to light.
The differences in connectivity patterns suggest that both L1 and L2 differ in the neuronal pathways they are involved in.

We thank Elisabeth Bock, Elisabeth Pritz, Peter Schönbacher, and Claudia Mayrhofer for technical help. Funded by the Styrian Government (Human Technology Interface -SMApp program)

Fig. 1: 3D-reconstruction of L1 and L2 neurons in the lamina of a locust.

Fig. 2: A photoreceptor synapse onto L1, L2, and two other, unidentified neurites.

Fig. 3: The same synapse as in Fig. 2, with L1 made transparent to display the arrangement of the neurites.
↑ Go to the Table of content ↑
Type of presentation: Oral

LS-14-O-1803 Analysis of elements in the brains of patients with Parkinson’s disease by scanning electron microscopy combined with energy-dispersive X-ray spectroscopy

Yumoto S.1, Kakimi S.2, Yamashita T.2, Nakamura R.2, Ishikawa A.2
1Yumoto Institute of Neurology, Tokyo, Japan, 2Nihon University, Tokyo, Japan
yumoto-s@viola.ocn.ne.jp

    Iron is an essential element for most life on Earth. In the human brain, iron plays a critical role in oxygen utilization, enzymatic systems, and especially neural development. In contrast, the excess accumulation of iron in nerve cells has been reported to causes neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. In this study, we analyzed the distribution of iron and other elements in nerve cells in the brains of patients with Parkinson’s disease and in those of age-matched controls to investigate the pathogenesis of Parkinson’s disease.
   Transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy (EDS) is usually applied to demonstrate elements in biological samples. However, it is impossible to analyze the localization of Fe by TEM-EDS analysis, because a high peak of the Fe spectrum emerges from components of the transmission electron microscope. Therefore, we developed a new method to demonstrate Fe using scanning electron microscopy (SEM) combined with EDS.
   Brain tissues (substantia nigra pars compacta) were taken after autopsy, fixed with 3% glutaraldehyde and 2.5% potassium dichromate, dehydrated, and embedded in Epon. Semi-thin sections (0.4 to 1-μm thick) were cut using a microtome equipped with a diamond knife, mounted on copper mesh, placed on a sample holder made of carbon, and used as samples for SEM-EDS analysis.
   <Control brains> A large number of electron-dense neuromelanin granules were present in the cytoplasm of nerve cells on SEM observation (Fig. 1A). When elements contained in neuromelanin granules (Fig 1A, arrow) were analyzed by EDS, a high peak of Fe was detected (Fig.1B, arrow). Spectra of Al, P, S, K, and Ca were also demonstrated in nerve cells. The highest peak of the Fe spectrum in nerve cells was detected in neuromelanin granules.
   <Parkinson’s disease brains> The number of neuromelanin granules reduced markedly in nerve cells. In some nerve cells, neuromelanin granules lost their electron density (Fig. 2A, arrow), and levels of Fe in these granules showed a marked decrease on EDS (Fig. 2B, arrow).
   It is likely that the levels of Fe in neuromelanin granules decrease markedly during the development of Parkinson’s disease. We also conclude that SEM-EDS analysis is a method capable of demonstrating iron in biological samples.

Fig. 1: Figure 1. Control brain. Nerve cell observed by SEM (Fig. 1A) and spectra taken by EDS (Fig. 1B). High levels of Fe are demonstrated in neuromelanin granules.

Fig. 2: Figure 2. Parkinson’s disease brain. Neuromelanin granules lost their electron density (Fig. 2A arrow), and levels of Fe in these granules showed a marked decrease (Fig. 2B).

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-1487 Ultrastructural analysis of skin biopsy in patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)

Zaletel I.1, Labudović-Borović M.1, Puškaš N.1, Kostić J.1, Trtica M.1, Lačković V.1, Bajčetić M.1
1Institute of Histology and Embryology “Aleksandar Đ. Kostić”, Faculty of Medicine, University of Belgrade, Serbia
ivan.zaletel88@gmail.com

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary stroke disease caused by the mutation in Notch 3 gene, located on chromosome 19 (19p13). This gene encodes a single-pass transmembrane receptor Notch 3, responsible for maturation of blood vessels in perinatal period and their homeostasis in adult period. Granular osmiophilic material (GOM) is a pathognomonic feature of CADASIL and represents an abnormal accumulation of Notch 3 at the cytoplasmic membrane of vascular smooth-muscle cells (VSMCs) in cerebral and extracerebral blood vessels.
The clinical picture is characterized by repeated ischemic events, cognitive disorders leading to dementia, headache, psychopathological manifestations and a wide range of various pathological events caused by vasculopathy which damages the central and peripheral nervous system, skeletal muscles, skin, heart and other organs.
Our study aimed to determine the significance of ultrastructural analysis of skin biopsy in the diagnosis of CADASIL. This study included patients in whom clinical suspicion of CADASIL was based upon the clinical picture, characteristic changes on the endocranial magnetic resonance imaging (MRI) and positive family history. After a detailed electron microscopy analysis of blood vessels in the dermis, the presence of characteristic granular osmiophilic material (GOM) was detected in 70% of patients, indicating that this method has a relatively high sensitivity level. Besides the presence of GOM deposits in indentations of altered VSMCs or between the remains of degenerated VSMCs, the morphological changes also included disruption of myoendothelial contacts, disoriented cytoskeletal elements of VSMCs and endothelial cells, thickening of VSMCs basal lamina, fibrous changes in the vascular wall and chromatin condensation and peripheral aggregation of nuclear material of VSMCs, suggesting apoptotic cell death.
Ultrastructural examination of skin biopsy is a highly specific and relatively sensitive diagnostic method for establishing a diagnosis of CADASIL. It is very important in the differential diagnosis of CADASIL, especially when genetic analysis is unavailable or limited and can be considered as a method of choice for the diagnosis of CADASIL.

This work was supported by the Ministry of Science and Technological Development of Serbia (Scientific projects No. 41002).

Fig. 1: An arteriole in the dermis. 1. Vascular smooth muscle cell (VSMC), 2. Thickened basal lamina with GOM deposit (white arrowhead) (TEM)

Fig. 2: Degenerated VSMCs (1) with disoriented cytoskeletal elements and GOM (arrowhead) in the thickened basal lamina (TEM)

Fig. 3: Small arteriole in CADASIL patient. 1. Endothelial cell 2. VSMC with chromatin condensation at nuclear periphery
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-1685 Structural characterization and functional modeling of damaged myelin sheaths in human brain caused by neurological diseases

Kurushin V.1, Shklover V.1, Filatov A.1
1Systems for Microscopy and Analisys LLC, Moscow, Russia
v.a.kurushin@gmail.com

It is well known that every human suffers lifetime axonal injuries caused by different factors: physical traumas, genetic diseases, inflammation, toxins etc., sometimes the damage takes the form of axonal swellings.
The present study aims to investigate and assess the influence of axonal swellings on nerve impulse transmission between Ranvier nodes. We studied damaged axons in prepared samples of human brain white matter obtained postmortem from schizophrenia patients.
In previous studies of electron micrographs of brain white matter slices it was shown that these characteristic changes in myelin structure (axonal swellings) are typical for schizophrenia disease. Although very important, those studies did not yield enough information on 3D properties of swellings crucial for modeling the electromagnetic signal propagation between Ranvier nodes. To collect this data we also performed volumetric study of brain white matter with nanometer resolution utilizing FEI® FIB/SEM Auto slice&view© technique as well as transmission electron microscopy methods (electron tomography) in the present study.
Based on the data obtained, we were able to incorporate true 3D shapes of axonal swelling deforming myelin sublayers into our model.
The model simulations of electromagnetic impulse propagation along axon body between Ranvier nodes has shown that such axonal swellings will cause weakening, loss, or deformation of neural pulse shape - in the same way the waveguide with distorted geometry causes signal loss or distortions. The simulation was performed on a 3D model describing the myelin layer (this area did not contain Ranvier nodes or para-nodes) as a dielectric waveguide with intracellular and extracellular liquids serving as its boundaries. The axonal swellings were supposed to have the same electromagnetic parameters as the intracellular media. In accordance with this model we performed computer simulation of electric field distribution within myelin layer and demonstrated the influence of myelin damage on signal loss during nerve impulse transmission between Ranvier nodes.

Authors want to acknowledge Natalia Uranova (Mental Health Research Center, Moscow, Russia) for helpful and critical discussions

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2122 The role of an allosteric modulator of a fibroblast growth factor receptor in neurogenesis

Kubesova A.1, 2, Palenicek T.1, 2, Tyls F.1, 2, Kaderabek L.1, 2, Novakova P.1, 3
1Prague Psychiatric Center, Prague, Czech Republic, 23rd Faculty of Medicine, Charles University, Prague, Czech Republic, 3Faculty of Science, Charles University, Prague, Czech Republic
kubesova@pcp.lf3.cuni.cz

Introduction: The synthetic peptide Fibroblast Growth Loop (FGL) is an allosteric modulator of a Fibroblast Growth Factor Receptor (FGFR) mimicking the Neural Cell Adhesion Molecule (NCAM). FGL reduces glial activation, promotes neurite outgrowth and synaptogenesis and supports the survival of different neuronal types in cell cultures [1-3]; however its effect on neurogenesis in vivo has not yet been fully determined. A recent study showed that FGL increases doublecortin immunoreactivity (a marker of neuronal precursors and immature neurons) in the dentate gyrus [4]. In this experiment we focused on the influence of short-term application of FGL on cell proliferation, survival and expression of a marker of mature neurons NeuN (neuronal nuclei).

Methods: 22 adult male Wistar rats (300±20g) were included in this experiment. Half of the animals received 4 doses of FGL (80mg/kg s.c.) during 4 consecutive days (= FGL group), the 2nd half received saline (= SAL group). On the 4th day all of the animals were injected with the S-phase marker 5-bromo-2-deoxyuridine (BrdU, 3x 50mg/kg i.p). One day after the BrdU injections, 6 animals from each treatment group (= proliferation group) were transcardially perfused. The remaining animals were transcardially perfused on the 21st day after the BrdU injections (= survival group). The rat brains were sliced (40μm) and immunohistochemically stained with mouse anti-NeuN (1:250, Millipore) and/or rat anti-BrdU (1:500, AbD Serotec). BrdU+ cells were counted in the dentate gyrus in every 12th section under a fluorescence microscope (Zeiss Axio Imager Z1). BrdU+/NeuN+ cells in the survival group were quantified using a confocal laser scanning microscope (Leica SPE). Image analysis was performed using ImageJ software and a LOCI tools plug-in. Statistical analysis (Mann-Whitney U test) was performed in the program Statistica 9.0 (Statsoft).

Results: The average number of BrdU+ cells in the SAL-proliferation group did not significantly differ from the FGL-proliferation group and in the SAL-survival group from the FGL-survival group. Three weeks from BrdU application ~80% of the BrdU+ cells survived in both the SAL group and the FGL group. The percentage of NeuN+ cells was higher in the FGL-survival group (88%) than in the SAL-survival group (78%).

Conclusion: Short-term application of FGL did not increase cell proliferation or survival in the dentate gyrus. The higher ratio of cells that differentiated into neurons might be attributed to the FGL-mediated suppressing effect on the glial cells.

References:

[1] Cambon K et al. (2004) J Neurosci. 24(17): 197-204.

[2] Neiiendam JL et al. (2004) J Neurochem. 91(4):920-35.

[3] Ojo B et al. (2011) Exp Neurol. 232(2):318-28.

[4] Ojo B et al. (2013) Neurochem Res. 38(6):1208-18

This work was supported by the grants ECGA 278006, MH CZ - DRO (PCP, 00023752), 260045/SVV/2014 and PRVOUK P34.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2134 High resolution three-dimensional imaging of synaptic vesicles by focused ion beam milling and scanning electron microscopy (FIB-SEM)

Nakanishi S
Okinawa Institute of Science and Technology, Okinawa, Japan
nakanishi_s@oist.jp
Electron microscopic studies have revealed synaptic vesicle recycling by endocytosis following neurotransmitter release by exocytosis. Imaging methods using fluorescent dyes have enabled monitoring of synaptic vesicle endocytosis and recycling in living neurons. Measurement of plasma membrane capacitance by electrophysiological recording has further elucidated mechanisms of synaptic vesicle endocytosis. However, many aspects of synaptic vesicle mobility during exocytosis and endocytosis are still a matter of debate. For example, where endocytosis occurs in synapses is not wholly understood. To analyze ultrastructural changes that occur during synaptic activity, observation of fine structure inside intact synapses is necessary. Reconstruction of synapses by serial sectioning provides good resolution, but much information is lost because sections cannot be made thinner than ~50 nm. Focused ion beam milling and scanning electron microscopy (FIB-SEM) has enabled 3D imaging of whole synapses; however, resolution has not been satisfactory. Here I present 3D images of synaptic vesicles of the calyx of Held presynaptic terminus examined by FIB-SEM, with satisfactory resolution and contrast. Reducing the probe current and acceleration voltage permitted visualization of fine ultrastructure comparable to that of TEM. It is easy to recognize the synaptic cleft, synaptic vesicles, including clathrin-coated vesicles, mitochondrial cristae, and ribosomes on rough-endoplasmic reticulum.
The author thanks Professor Tsumoru Shintake for valuable advice, Mr. Toshio Sasaki for operating the FIB-SEM, and Dr. Steven Aird for critical reading of the abstract.
Fig. 1: Top surface view of rat brainstem slice embedded in epon (left, top). Side view of specimen during ion beam milling (left, bottom). an image obtained during ion milling shows two principal neurons of the medial nucleus of the trapezoid body surrounded by the calyx of Held presynaptic terminus.
Fig. 2: FIB-SEM ion milling of a single synaptic vesicle. Siz representative serial sections out of 674 taken without drift or loss of focus. The milling thickness is estimated at ~10 nm.
Fig. 3: Resolution of FIB-SEM cut at 10 nm right) is close to that of a TEM epon section cut at 50 nm (left).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2139 Visualization of P/Q-type calcium channels: a spider toxin （ω－Agatoxin IV A）binds not only to living tissue but also to fixed tissue

Nakanishi S.1
1Okinawa Institute of Science and Technology, Okinawa, Japan
nakanishi_s@oist.jp

ω-Agatoxin IV A (AgaIVA), a peptidyl toxin from Agelenopsis sperta venom, specifically binds to P/Q-type calcium channels.  Pharmacological and electrophysiological studies showed that AgaIVA-sensitive channels are widely distributed in both the central nervous system and in neuromuscular junctions.  Using biotin-conjugated AgaIVA, it was possible to determine which cells in freshly prepared mouse cerebellar and hippocampal slices possess binding sites for this toxin (Nakanishi, S. et al., 1995).  Biotinylated AgaIVA was also applied to transcardially fixed brain slices prepared with various fixatives (4 % paraformaldehyde with 0.1 % glutaraldehyde, Zamboni's fixative, and acrolein).  AgaIVA did not bind to fixed tissues from P/Q-type calcium channel knockout mice, confirming that the binding to normal, fixed tissues was not an antifact.  With transmission electron microscopy, it was shown that the toxin also binds to fixed tissue.  Using immunoelectron microscopy, the locations of biotinylated AgaIVA binding sites were compared to those of binding sites identified with an antibody specific for the α-1A subunit of P/Q-type voltage-gated calcium channels.  Biotinylated AgaIVA binding sites visualized with FluoroNanogold-streptavidin showed a similar pattern to those visualized with antibody.  This ability of biotinylated AgaIVA to bind to fixed tissue provides a new tool to study the molecular architecture of excitatory synapses. Reference: Nakanishi, S. et al., (1995) J Neurosci Res 41: 532-539

The suthor thanks Dr. Steven Aird for critical reading of the abstract.

Fig. 1: Biotinylated AgaIVA bound to chemically fixed P/Q-type calcium channels in the medial nucleus of the trapezoid body  (left), but without toxin no binding was seen (middle).  Immunohistochemical examination using an antibody to recombinant protein (aa856-888) showed similar results (right).

Fig. 2: P/Q-type calcium channels were visualized on the presynaptic membrane (black arrow heads) with peptide-binding cytochemistry (left) and with immunoelectoron microscopy (right). Tissues shown in Fig.1 were reprocessed for TEM. Bars, 50 nm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2184 Radial glial as a progenitor cells in the neurogenic niches of Austrolebias brain fish.

Rosillo J. C.1,2, Torres M.1,2, Olivera S.3, Casanova G.4, García-Verdugo J. M.5, Fernández A.1,2
1Neurociencias- IIBCE Montevideo-Uruguay, 2Neuroanatomía Comparada- Facultad de Ciencias Montevideo-Uruguay, 3Neurobiología celular y molecular -IIBCE, 4Unidad de Microscopia Electrónica de Transmisión Facultad de Ciencias, 5Laboratorio de Neurobiología Comparada, Instituto Cavanilles, Universidad de Valencia, España
jcarlos.rosillo@gmail.com

The multiplicity of neurogenic niches in adult fish brains evidences their high postnatal capacity to repair and growth.
Such amount of sites with proliferative and neurogenic capacity is sustained by the presence of undifferentiated cells
with features of progenitor cells. The neural stem cells in vertebrate brains are not properly identified yet.
Some authors suggest that cells with radial glia features are the candidates to be the stem cells (Götz et al. 2007; Zupanc et al 2003),
the point remains not fully clarified. With the purpose to deepen into the knowledge of stem cells and/or progenitor
cells in fish brains, we have studied the distribution and structural characteristics of radial glia in the adult fish brain.
In previous works, we analyzed Austrolebias brain cell proliferation (Fernández et al. 2011). Now, we have introduced the
use of thymidine halogenated analogs chloro (CldU) and iodo (IdU)-deoxyuridines to identify different populations of
proliferating cells and those that re-enter the cell cycle, besides allowing a temporal discrimination of labelling.
We detected three types of proliferating cells: 1) cells marked 24 h after marker injection (CldU+), and located in
ventricular regions; 2) cells marked 30 days post injection (IdU+), many of them migrating; and 3) doubled marked non-
migrating cells (CldU+IdU+) that remain in the ventricular zone. The immunohistochemical analysis performed with antibodies
against Vimentin, S-100, BLBP and Glial Acidic Fibrilar Protein, showed a widespread distribution of radial glial cells
mainly located in medial zones of brain lining with the ventricular walls. In the places detected as highly proliferative,
the concentration of cells Vimentin+ was higher. As shown by transmission and scanning electron microscopy, radial glia
cells of the medial niches are communicated by connexin 43+ gap junctions along variable traits. Such cells form groups of
very elongated cells that contact apical surface of ventricular lumen and present a single cilium and different intercellular
junction complexes (Casanova et al 2014). Then, the characteristics shown by radial glia of Austrolebias neurogenic niches
suggest that these cells are highly probable candidates to be the progenitor cells that sustain neurogenesis and brain
repair.

Acknowledgments: This work received partial financial support of Program of Developed of Basic Sciences PEDECIBA.

Fig. 1: Immunohistochemistry showing proliferative markers CldU+ (red) and IdU+ (green) at the Torus longitudinalis (TL). CldU+ nuclei were detected 24 h after injection and found close to the ventricular wall. Most of the IdU+ nuclei detected 30 days after IdU administration were found at different distances from the ventricular surface .Confocal image.

Fig. 2: Transversal section of the third ventricle of brain showing cells with glial phenotypes identified by Vimentin, and Cx43, immunoreactivity. The confocal analysis showed a widespread distribution of radial glial cells mainly located in ventral medial zones of brain lining with the ventricular walls.

Fig. 3: A panoramic electron microscopy image shows the ventral ventricular region. In this region appears a kind of elongated radial cells (RG) similar to those found in others ventricular regions of the brain.

Fig. 4: Scanning microscopy image of ventral ventricular region that show different cells with phenotypes of radial glial. Note several elongated radial glial cells. (RG)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2348 The effects of ghrelin against tunicamycin-triggered endoplasmic reticulum stress in C6 glioma cells

Yaprak Sarac E.1, Tanriverdi G.1, Guzel E.1, Bolkent S.2, Koyuturk M.1
1Department of Histology and Embryology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey., 2Department of Medical Biology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey.
elifyaprak@yahoo.com

Impaired function of the endoplasmic reticulum (ER) leads to accumulation of misfolded proteins and induction of the unfolded protein response (1). The aberrant accumulation of specific misfolded proteins in intracellular inclusions or extracellular aggregates has been recognized to be crucial in the pathogenesis of some neurodegenerative diseases (2). Recent evidences suggest that astrocytes are important players in neurodegenerative disorders (3). We used an in-vitro ER stress model induced by tunicamycin (T) in C6 glioma cells which is derived from rat astrocytes. Ghrelin (G) is a natural peptide ligand of the growth hormone secretagogue receptor which is known to stimulate growth hormone secretion (4). The aim of this study was to investigate the potential protective effects of G on ER stress model.

Cells were cultured in DMEM-F12 containing 10% FBS at 37˚C with 5% CO2 in a humidified incubator. Dose and time dependent preliminary experiments were performed by 0.5, 1, 2.5, 5 and 10 µg/ml T for 2, 6, 12 and 24 hours (h). 2.5 µg/ml dosage of T was chosen and applied for 12 and 24 h. Cells were divided into four groups; control group, T mediated ER-stress group, G (100nM) treated group and G+T group (G administered one hour before T application). At the end of incubations, cells were fixed and immunocytochemical analysis was performed by using ER stress marker anti-GRP78/BiP, and BrdU antibodies. Semi-quantitative analysis of immunoreactions and proliferation percentages were statistically evaluated with ANOVA using GraphPad InStat DTCG software.

BrdU assay showed that G increased the proliferation level of cells significantly according to control group (p<0.05) while 2.5 µg/ml T decreased the level significantly (p<0.01) (Fig 1,2). When G was applied to cells before T, a significant increase in proliferation level was observed according to T group (p<0.01) (Fig 1,2). Statistical analysis of immunoreactions against GRP78/BiP showed that 2.5 µg/ml T for 12 and 24 h extremely increased the expression level of BiP significantly compared to control group (p<0.05); however, G substantially suppressed the effect of T when applied before T (p<0.01) (Fig 3,4).

According to our results, G has a protective effect on neuroglial cell proliferation suppressed by ER stress. It also affects the expression of GRP78/BiP, thus equilibrating between cell surviving and apoptotic cell death. Further research needs to be done for new approaches to applications of ghrelin as a neuroprotective agent.

References

1. Lindholm D, Wootz H, Korhonen L. Cell Death Differ. 2006;13(3):385-92.

2. Pereira CMF. ISRN Cell Biology. 2013:2013:22.

3. Maragakis NJ, Rothstein JD. Nat Clin Pract Neurol. 2006;2(12):679-89.

4. Kojima M, et al. Nature. 1999;402:656–60.

Fig. 1: BrdU immunoreactions in C6 glioma cells. Control group (A), Ghrelin group (B), Tunicamycin group (C), Ghrelin+Tunicamycin group (D). Hematoxylin counterstain, 40X.

Fig. 2: Percentage analysis of distribution of BrdU expression in C6 glioma cells.*P<0.05; compared to Control group, **P<0.01; compared to Tunicamycin and Ghrelin groups.

Fig. 3: GRP78/BiP immunoreactions in C6 glioma cells incubated for 12 (left side) and 24 (right side) hours. Control group (A,B); Tunicamycin group (C,D); Ghrelin+ Tunicamycin groups (E,F), 40X.

Fig. 4: H-SCORE analysis of intensity of GRP78/BiP expression in C6 glioma cells. *P<0.05; compared to Control group, **P<0.001; compared to Tunicamycin group.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2389 Spatial Light Modulation – Two Photon Microscopy for scanless imaging and fast multi-site acquisition of functional signals

Pozzi P.1, Gandolfi D.2, Tognolina M.2, Mapelli J.3, Chirico G.1, D'Angelo E.4
1Department of Physics, University of Milano Bicocca, Milano, Italy, 2Department of Brain and Behavioral Sciences, University of Pavia, Italy, 3Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy, 4Brain Connectivity Center, C. Mondino National Neurological Institute, Pavia, Italy
p.pozzi3@campus.unimib.it

The acquisition of millisecond-scale optical signals in biological samples is extremely useful in the study of fast biological events, such as neurons signalling or hemodynamics. However, the most used techniques for optical imaging in thick biological samples, such as confocal and two photon microscopy, are not capable of providing reliable millisecond-scale signals on a wide field of view. The main limiting factor for confocal acquisition rate is the use of galvanometric mirrors for raster scan beam displacement.
In order to allow effective parallel fast acquisition, the presence of multiple confocal excitation volumes is required. This can be achieved through the use of a Spatial Light Modulator (SLM), which can divide the coherent excitation light in multiple diffraction limited focal points across the field of view. Reported implementations of SLM based confocal microscope [1] rely on a standard galvanometric scanner for the acquisition of an image of the sample, on which locations of interest are selected to be illuminated with Spatial Light Modulation.
We present an alternative approach, allowing for the acquisition of complete three dimensional confocal images through the use of an SLM and a pixelated detector. This dramatically reduces the cost and complexity of the setup, as the microscope works without any mechanical moving part, and allows for placement of excitation volumes in locations of interest for fast parallel signal acquisition with sub-micrometer precision. Spatial light modulation – two photon microscopy (SLM-2PM) image acquisition is based on the use of the SLM to illuminate the sample with a grid of diffraction limited excitation volumes, and move such grid according to a raster scan pattern. A pixelated detector acquires the fluorescence signals of each excitation volume separately during the scan sequence, and a custom software creates the confocal image. The same process can be executed on different focal planes thorugh the use of the SLM, without the need for mechanical movement of the objective.
Images acquired with SLM-2PM have comparable quality to the ones obtained through a standard two photon microscope, and the method was successfully employed for millisecond scale calcium imaging in acute cerebellar slices.

[1] Nikolenko et Al. SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators. Frontiers Neural Circuits 2:5 (2008).

This work was supported by grants from European Union to Egidio D’Angelo(CEREBNET FP7-ITN238686, REALNETFP7-ICT270434) and by grants from the Italian Ministry of Health to Egidio D’Angelo (RF-2009-1475845).

Fig. 1: Example of high frequency acquisition: Image on the left shows granular layer in a cerebellar slice loaded with Fura-2 AM. Multiple focal points are placed on the cells indicated by red circles, obtaining the image on the center on a fast CMOS camera. Calcium signals from each cell are acquired at frequencies up to 1KHz (example on the right).

Fig. 2: SLM-2PM 3d image of a Purkinje cell, obtained without any mechanical moving part. Panel A: three confocal images acquired at planes 4 μm apart. Panel B: Projection of a total of 10 confocal images of planes.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2677 CARNOSINE NEUROPROTECTION OF THE CEREBRAL CORTEX IN MICE EXPOSED TO VANADIUM INHALATION. AN ULTRASTRUCTURAL ANALYSIS

Colín-Barenque L.1, Bizarro-Nevares P.2, Gonzalez-Villalva A.2, Zepeda A.2, Pasos F.2, Reséndiz-Avendaño S.3, Rojas-Lemus M.2, Pedraza-Chaverri J.4, Medina-Campos O. N.4, Aley P.1, Espinosa-Villanueva J.1, Fortoul T. I.2
1Dept. Neurociencias FES Iztacala.UNAM 1, 2Dept. de Biología Celular y Tisular Facultad de Medicina UNAM 2, 3Facultad de Ciencias UNAM 3, 4Dept of Biología, Facultad de Química, Ciudad Universitaria UNAM 04510, MÉXICO, D.F. 4
barenque@unam.mx

Vanadium (V) is delivered to the atmosphere by the combustion of petroleum derivates rich in this element. V can induce the formation of reactive oxygen species (ROS) in biological systems and the toxicological effects have been associated with them1. In previous studies we reported neuronal alterations in olfactory bulb and hippocampus in mice exposed to vanadium pentoxide2 (V2O5). Carnosine (beta-alanyl-l-histidine) is endogenously synthesized in brain and is endowed with antioxidant activity as a scavenger of free radicals3. The present study determined carnosine neuroprotection in mice prefrontal cortex exposed to vanadium with an ultrastructural approach. CD-1 male mice weighing 30±5g were divided into four groups. 1) V2O5 [0.02M] inhalation one hour twice for a 4-week period. 2) V2O5 Inhalation plus oral Carnosine treatment (1mg/kg/day) 3) Only Carnosine treatment (1mg/kg/day), and 4) Controls inhaled saline. Animals were sacrificed by pentobarbital overdose, perfused by intracardiac puncture with saline solution and glutaraldehyde (2%); prefrontal cortex was dissected and processed for MET. The samples were analyzed in a Zeiss EM 100. The ultrastructural examination revealed that pyramidal neuron of prefrontal cortex in carnosine group showed undamaged organelles (Fig. 1D) similar to controls (Fig. 1A). In contrast, alterations in pyramidal neuron were observed after vanadium exposure showed dark cells with shrunken soma, pyknotic nucleus and irregularly clumped chromatin, a form of necrotic neuronal death (Fig. 1B). In notably, mice vanadium exposed and treated with carnosine showed a decrease in neuronal death (Fig. 1C) compared with vanadium exposed without carnosine. These results suggest that ultrastructural alterations in pyramidal neuron death induced by vanadium are mediated by oxidative stress and that carnosine may modulate the neurotoxic vanadium action.

References 1. S.S. Soares, et al. Journal of Inorganic Biochemistry, 2007,101, 80-88.

2. T.I. Fortoul, et al. Journal Biomed. Biothech, 2011

3. J. Drozak, M. et al. Journal of Biological Chemistry, 2010, 285,9346-9356

This work was supported by DGAPA-UNAM IN-220414

Fig. 1: Prefrontal cortex. A) Pyramidal neuron (P) with nucleus (N),apical dendrite (D) of control mice, B) Dark cell (D) with shrunken soma and vacuolation (V) of exposure vanadium mice, C) Normal pyramidal neuron of exposure vanadium with treatment carnosine mice; and carnosine treatment mice D) with normal axon (A) and blood vessel (bv).Scale bar = 2 µm
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2755 Hypoxia induces modifications in the synaptic organization: Two and Three Dimensional electron microscopy study.

Capani F.1,2, Romero J.1, Holubiec M.1, Logica Tornatore T.1, Giraldez l.3, Santos G.3, Castilla R.1, Blanco E.4
11Inst. Inv. Cardiológicas Prof. Dr. Alberto C. Taquini, UBA-CONICET, Buenos Aires, Argentina, 2Departmento de Biología, Universidad Argentina J F Kenedy, 3Departamento de Ciências Biológicas (DCB), Universidade Estadual do Sudoeste da Bahia UESB , Jequié-BA-Brazil, 4Departament de Pedagogia i Psicologia, Facultat de Ciències de l'Educació, Universitat de lleida, Av. de l'Estudi General, 4, 25001 Lleida
franciscocapani@hotmail.com

Perinatal asphyxia (PA) induced short and long term synaptic and cytoskeletal alterations that has been associated with neuronal cell death following hypoxia. The lack of knowledge about the mechanisms underlying this dysfunction prompted us to investigate the changes in the synapse and neuronal cytoskeleton and related structures.

For this study we used a well established murine model of PA. Full-term pregnant rats were rapidly decapitated and the uterus horns were placed in a water bath at 37 °C for different time of asphyxia. When their physiological conditions improved, they were given to surrogate mothers. One month, 4, 6 and 18 months old after PA rats were included in this study. Modifications were analyzed using photo oxidation with phalloidin-eosin, conventional electron microscopy (EM), inmunocytochemistry and ethanolic phosphotungstic acid (E-PTA) staining combining with electron tomography and 3-D reconstruction techniques [1].

After one and two months of the PA insult, an increase in the F-actin staining in neostriatum and hippocampus synapses was observed using correlative fluorescent electron microscopy for phalloidin-eosin.[2] Mushroom-shaped spines showed the most consistent staining. Strong alterations in the dendrite and astroglial cytoskeleton organization were found at four months of PA [3]. After six months of PA, postsynaptic densities (PSDs) of the rat neostriatum are highly modified. We observed an increment of PSDs thickness related with the duration and severity of the hypoxic insult. In addition, PSDs showed and increase in the ubiquitination level. Using 3-d reconstruction and electron tomography we observed showed clear signs of damage in the asphyctic PSDs [1]. These changes are correlated with intense staining for ubiquitin. Finally, in 18 months old rat was observed a reduction in the number of synapses in the PA animals related with a decrease in BDNF staining. Overall these results demonstrate that synaptic dysfunction following PA might be produced by early changes in the actin organization and long-term misfolding and aggregation of proteins in the PSDs.

Therefore, we hypothesize that the synaptic and neuronal cytoskeleton changes induced by PA in the rat CNS could lead to the cellular dysfunction and death.

References

[1]Capani, F., et al (2009) Exp Neurol. 219, 404-13.

[2]Saraceno G.E el al (2010) Exp Neurol. 223, 615-22.

[3]Saraceno, GE et al (2012) Synapse. 66(1):9-19.

Fig. 1: Electron microscopy images of the neostriatum in control and PA animal. A- Post-synaptic densities stained with E-PTA. AP showed an increment in the PSDs thickness in comparison with the control (arrows). B- Photo-oxidation with phalloidin-eosin. After PA we observed more number of dendritic spines actin-positives. Scale bar, 1 mm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2905 Ultrastructural analysis of single labelled glial cells in Drosophila

Barti B.1, Kis V.2, Sass M.3
1Eötvös University, Budapest, Hungary, 2Eötvös University, Budapest, Hungary, 3Eötvös University, Budapest, Hungary
barti.benjamin@tdk.koki.mta.hu

The nervous system consists of two major cell types: neurons and glial cells. For a long time neurons stood in the center of interest for their important roles and special functions. In the past few years numerous researches had shown that beside the neurons, glial cells also have a significant role in the operation of the nervous system. These cells do not only fill the space between nerv cells as it was thought before but they also participate in the recyclization of the metabolits made by neurons. They regulate the synaptic process between two nerv cells and with the configuration of the blood-brain barrier they defend the whole nervous system from mechanical or molecular impacts.

However, it seems that there are still a lot of unknown functions and procedures, controlled by glial cells, even undiscovered glial subtypes may exist. For the proper investigation of these functions it is necessary to have a clear vision of the ultrastructure of those cells. Therefore we put the electronmicroscopical examination of single-labelled glial cells in the focus of our research. For model organism we used fruit-flies (Drosophila melanogaster) instead of rat or other mammals. The advantages of the fly strains are they can be kept in small place, the development of the animals are fast and last but not least their nervous system and genomic sequences are very similar to mammals in numerous ways, so the results can be referred even to human brain researches.

In this experiment with crossing of Drosophila strains we created a transgenic line where some glial cells are single-labelled in the nervous system. Those cells expresses a membrane-specific protein, the horseradish-peroxidase enzyme. The brain of those animals were dissected, fixed and the marked cells were developed with DAB-reaction. After the reaction, the whole cell membrane with its projections became visible and could be easily recognised. The samples were embedded in Durcupan epoxy resin and were cut into ultra-thin sections. After that, the complete sections were studied under electronmicroscope. To our first knowledge this is the first genetic method to label glial cells directly for electron microscopic examination.

The proper investigation of single cells in the level of the electronmicroscope is requied to have precise knowledge about neurodegenerative deseases and basic functional processes in the central nervous system, therefore our method hopefully could provide help for all the scientists working in this field.

Personally, I would like to thank for the support for the other authors (Kis, V., Sass, M.) and the Hungarian Academy of Sciences, Institute of Experimental Medicine to provided help in participating in the conferance.

Fig. 1: Single labeled glial cell visualized in Drosophila brain tissue. Light microscope image of the projections of a single glial cell. Arrowheads indicate the borders of the cell marked by horseradish-peroxidase. Scale: 10μm.

Fig. 2: Electron microscopy images of the ultrastructure of the nervous tissue. The HRP labels the membrane of the specific glial cell (white arrows), allowing observation of structures of the cytoplasm. Asterix: mitochondria, L: lipid droplet, N: nucleus. Black arrowheads mark the endoplasmic reticulum.

Fig. 3: Fluorescent images of a surface glial cell forming part of the blood-brain-barrier in Drosophila. Red Fluorescent Protein (RFP) was bound to the HRP enzyme, all cell nuclei are labeled with HisGFP. Scale: 50μm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2945 Pathological modifications of tau protein during the formation of neurofibrillary tangles in Alzheimer´s disease

Garcia-Sierra F.1, Jarero-Basulto J. J.1, Rodriguez-Cruz F.1, Torres-Cruz F. M.1, Kristofikova Z.2, Ripova D.2
1Department of Cell Biology, Center of Research and Advanced Studies of the National Polytechnic Institute, Mexico city, Mexico, 2Laboratory of Biochemistry and Brain Pathophysiology and AD Center, Prague Psychiatric Center, Prague, Czech Republic
fgs516@yahoo.com

The progression of the neurofibrillary pathology in Alzheimer´s disease (AD) was early described by Braak and Braak [1]. This progression consists in a predictable appearance and distribution of neurofibrillary tangles (NFTs) and dystrophic neurites (DN) along entorhinal, limbic and isocortical areas in the brain of AD affected individuals. It is well known that in AD, NFTs and DNs are mostly composed of tau protein which has undergone several posttranslational modifications such as abnormal phosphorylation, conformational changes and truncation [2, 3, and 4]. The timing of occurrence and possible cosegregation of these modifications during the formation and progression of the neurofibrillary pathology in AD is still under debate and we aimed to address this issue in the present study. Formalin fixed brain sections from the hippocampus and cerebral cortex of AD cases were processed for multiple immunolabeling with antibodies to distinct modifications occurring in tau protein, such as phosphorylation, truncation, ubiquitination, and conformational changes. The pattern of staining and the degree of colocalization were analyzed by using confocal microscopy. Antibodies and fluorescent markers to detect programmed cell death were also included in some experiments. Brain sections from AD cases versus age-matched control individuals were analyzed and compared. We reported the timing by which the distinct events that tau protein undergoes during its aggregation in the form of NFTs. Hence, abnormal phosphorylation was found as the earliest modification on the tau molecule that may alter its conformation. We also found that proteolytic processing of the C-terminus is a key event that links distinct conformational changes in the molecule. Our results indicate that the C-terminus of tau protein is transiently truncated from the aminoacid aspartic acid-D421 to glutamic acid-E391, leading the tau molecule to adopt distinct conformations. Apoptosis in the brain of AD cases was also associated with tau pathology, mostly when the early truncation of tau protein at the aspartic acid-D421 was the major component of NFTs. Besides this, ubiquitination was associated with this early truncation of tau, which may link two different pathways of proteolytic processing in a single substrate occurring at intermediate stages of NFTs maturation. We may conclude that this cascade of pathological molecular events occurring in the molecule of tau protein may give a better correlation with the neuropathological progression of the disease.

[1] H. Braak, E. Braak. Acta Neuropathol (Berl). 82 (1991) 239.

[2] I. Grundke-Iqbal et al., J Biol Chem. 261 (1986) 6084.

[3] G. Carmel et al., J Biol Chem. 271 (1996) 32789.

[4] M. Novak et al., EMBO J. 12 (1993) 365.

This research was supported by CONACyT grant (152535) to F. G-S

Fig. 1: Tau signatures in neurofibrillary tangles. Ubiquitination, truncation at D421 and conformational changes of tau protein are closely associated with early neurofibrillary tangles. Bar. 10 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-2997 The effect of valproic acid treatment on the dopaminergic neuron survival in a 6-hydroxydopamine rat model of Parkinson’s disease

Dagdelen M.1, Cumbul A.2, Uslu U.2, Genc E.1
1Yeditepe University Faculty of Medicine, Medical Pharmacology, Istanbul, Turkey , 2Yeditepe University Faculty of Medicine, Department of Histology and Embryology, Istanbul, Turkey
alev.cumbul@yeditepe.edu.tr

Keywords: Parkinsons’s Disease, valproic acid, dopaminergic neurons

Parkinsons’s Disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) resulting in the loss of dopaminergic innervation to the striatum. In recent years increasing number of in vitro and in vivo studies demonstrate the neuroprotective effects of valproic acid (VPA), which is a commonly used drug in the treatment of epilepsy and bipolar disorders, through many mechanisms (1). In this study, we aim to determine whether VPA protects dopaminergic neurons from 6-hydroxydopamine (6-OHDA) induced neurotoxicity.

Male Wistar albino rats (250–300 g) were assigned to 4 groups (n=4) as follows: Sham operated (S), sham operated and VPA treated (SV), 6-OHDA injected (PD) and 6-OHDA injected and VPA treated (PV). The rats were stereotaxically injected either with 6-OHDA (8μg/2μL) or saline to the SNpc. Only the rats showing rotational behaviour (≥5 contralateral turns/min) were included into the study after apomorphine (0.5 mg/kg sc) test. Two weeks following the operation, rats were intraperitoneally injected with either VPA (300 mg/kg) or saline for 10 days. Coronal sections were taken through the substantia nigra on a freezing microtome at a thickness of 16 μm. Each section was examined under Stereo Investigator version 7.5 image analysis software for tyrosine hydroxylase (TH) immunoreactive cells (2). One-way Anova followed by Tukey post-hoc test was used for statistical analysis.

The number of TH positive neurons was not different between S and SV groups. There was a pronounced loss of TH positive neurons in 6-OHDA lesioned right SNpc in PD group as compared to sham-operated groups (p<0.001). VPA treatment significantly increased the number of TH positive neurons in PV group as compared to PD group (p<0.05). However, the numbers of TH positive neurons were still significantly lower in PV group as compared to S and SV groups (p<0.01) (Figure 1 and 2).

In our study, we have demonstrated that VPA treatment may have neuroprotective effects on dopaminergic cell survival in a 6-OHDA lesioned rat model of Parkinson’s Disease.

References:

1) Kidd S.K. and Schneider J. S.,2011 Protective effects of valproic acid on the nigrostriatal dopamine system in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. Neuroscience 194, 189–194.

2) Decressac M., Mattsson B., Björklund A.,2012 Comparison of the behavioural and histological characteristics of the 6-OHDA and α-synuclein rat models of Parkinson's disease

This study was supported by Yeditepe University Research Center. Project Number: 120800002.

Fig. 1: TH immunoreactivity was decreased in PD group and increased in PV group. Photomicrographs demonstrating sections taken from right SNpc stained with TH. Groups: A; S, B; PD, C; SV, D; PV. Dopaminergic neuron is demonstrated with arrow and mononuclear cell infiltration is star. The magnification is x10. Scale bar represents 200 μm.

Fig. 2: Graphs comparing the S, PD, SV, PV groups. Data are presented as percentage of right SNpc neurons compared to total neurons in right and left SNpc. Data are expressed as mean ± SEM (*p<0.05, **p<0.01 and ***p<0.001).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-3189 The neurodegenerative phenotype Trembler-J mouse model of CMT1E shows differences associated with PMP22 mutation, T1703C, in the extracellular matrix of peripheral nerve fibers

Villar S.1, Rosso G.2, Bresque M.3, 4, Pizzarossa C.3, 4, Romeo C.3, Cal K.4, Sotelo J. R.4, Negreira C.5, Kun A.3, 4
1Facultad de Ciencias, Microscopia Electrónica de Barrido, 2Institut für Physiologie II Universitätsklinikum Münster, Alemania, 3Facultad de Ciencias, DPAN- IIBCE Unidad Asociada a Sección Bioquímica, 4Departamento de Proteínas y Ácidos Nucleicos, IIBCE, 5de Ciencias, Laboratorio de Acústica y Utrasonido
svillar@fcien.edu.uy

General information:
The extracellular matrix (ECM) of mammal peripherals nerve fibers plays an important role in Schwann cell (SC) development, proliferation, adhesion and extension. The ECM seems to be also involved in the modulation of myelination and, as a consequence, in the structural and functional nerve fibers integrity. The fiber ECM anchoring has been ensured by joints in trans from SC, connected in cis with the subcortical glial actin cytoskeleton. The structural dynamic, ensures signal transduction through the modulation of the cytoarchitecture, responding the axon to radial progress offered by Schwann cell.
Specific information:
We have recentely signaled, by Atomic Force Microscopy (AFM) and confocal microscopy (Rosso et al, 2012), molecular differences on peripheral nerve fibers ECM constitution and organization associated to Trembler-J genotype, a murine model to human Charcot-Marie-Tooth (CMT1E) neuropathy. This differences are also related to the actin cytoarchitecture, giving specific differences on mechanical properties associated to wild type (+/+) and Trembler-J (TrJ/+) genotype (Kun et al, 2012).
Our approach:
The peripheral nerve fibers from (+ / +) and (TRJ / +) genotype were studied by scanning electron microscopy (SEM). Metallized fibers showed specific ECM genotype differences, similar in appearance to that we have recently noted by AFM.
This difference appears to be supported at least partly in the absence of collagen IV in mice (TRJ / +).
The study of ECM on nonmetalized fibers also showed specific genotypic differences, apparently associated to underliging subcellular domains (node of Ranvier, Schmit-Lanterman clefts, cell nuclei), suggesting a coordinated and hierarchical structure of the basement membrane and ECM, which is present on wild type fibers and absent on (TrJ/+) that looked a more homogeneous organization of their constituents.
Along with the structural differences, the SEM microanalysis carried on nonmetalized fibers revealed significant differences in the composition and atomic ratio of both genotypes.
In our knowledge, such findings have not been reported before in the literature.

Fig. 1: Wild type fiber without gold coating showing depressions

Fig. 2: Mutant fiber (Trembler) without gold coating. Depressions are not observed
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-3241 Participation of calcium-activated potassium channels in the formation of plasmerosomes in neurons

Kaufmann W. A.1
1EM Facility, Institute of Science and Technology, Klosterneuburg, Austria
walter.kaufmann@ist.ac.at

The endoplasmic reticulum (ER) forms a continuous network of tubules and cisterns in neurons. However, recent evidence suggests that neuronal ER does not represent a uniform Ca2+ pool but rather a spatially heterogeneous system organized into subcompartments. These ER domains, or calciosomes, are usually enriched in certain isoforms of Ca2+ pumps, Ca2+ binding proteins and Ca2+ permeant channels, and are supposed to unload and refill Ca2+ independently [1]. Ca2+ release from the ER is mediated by two families of Ca2+ permeable channels, namely the ryanodine receptors (RyRs) and the inositol 1,4,5-trisphosphate receptors (IP3-Rs), each with three major isoforms. Areas of the plasma membrane overlying calciosomes also form specialized microdomains that contain unique sets of ion channels. These plasma membrane domains together with the underlying calciosomes are proposed to build functional units, termed plasmerosomes [2].

This study was undertaken (i) to unravel exact morphological parameters of subsurface cisterns (SSCs), representing particular types of calciosomes in cerebellar Purkinje cells (PCs), and (ii) to analyze the molecular composition of SSCs as well as overlying plasma membrane domains with respect to Ca2+ release channels (IP3-Rs, RyRs), voltage-gated Ca2+ channels and Ca2+ sensors such as large-conductance Ca2+ activated K+ (BKCa) channels. The morphological parameters of SSCs are established by 3D-reconstruction plane-by-plane from series of ultrathin sections by using the software CAR (Contour Alignment Reconstruction). The molecular composition is studied by means of pre- and post-embedding immunogold electron microscopy and SDS-digested freeze-fracture replica immunolabeling.

SSCs are discoid flattened cisterns, 0.4-1.5 µm wide, with a luminal depth of 4-5 nm (widening at their lateral edges), situated beneath the inner leaflet of the plasma membrane at a regular distance of 10-15 nm. IP3-Rs and RyRs are both localized to SSCs indicating that these Ca2+ release channels share a common Ca2+ pool and dispose SSCs to the generation of both Ca2+ puffs and sparks. Clustered BKCa channels are always associated with plasma membrane domains overlying SSCs and likely facilitate the generation of small transient outward currents. These findings indicate that functional units exist in cerebellar PCs resembling plasmerosomes in myocytes [3], and these units may contribute significantly to spatial signalling in central principal neurons.

[1] Verkhratsky A (2005) Physiol Rev 85:201-79. [2] Blaustein MP and Golovina VA (2001) Trends Neurosci 24:602-8. [3] Wellman GC and Nelson MT (2003) Cell Calcium 34(3):211-29.

This work was supported by a grant from Innsbruck Medical University Austria, MFI-4305, to WAK. I thank Prof. Francesco Ferraguti at Innsbruck Medical University, and Prof. Ryuichi Shigemoto, Institute of Science and Technology Austria, for continuous support.

Fig. 1: 3D-reconstruction of Purkinje cell subsurface cisterns by means of CAR (Contour Alignment Reconstruction; K. Sätzler, Univ. Ulster, UK). Green, axon terminal; purple, aspect of PC soma; dark-red, subsurface cistern; white, mitochondrion; orange and yellow, junctional and non-junctional ER, respectively.

Fig. 2: Clustering of BKCa channels at sites of subsurface cisterns in Purkinje cells. BKCa channels, immunolabeled with antibodies conjugated to 10-nm gold particles (arrowheads) in a post-embedding immunogold approach, are enriched at sites of SSCs. Scale bar = 180 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-3365 ULTRASTRUCTURAL FEATURES OF PROGENITOR CELLS IN THE Austrolebias BRAIN

Casanova G.1, 2, Rosillo J. C.2, Olivera S.2, Fernández A. S.1, 2
1Electron Microscopy Unit, Faculty of Sciences, Universidad de la República, Montevideo, Uruguay, 2Neuroanatomía Comparada, Associated Unit with Faculty of Sciences; Institute of Biological Research
casanova@fcien.edu.uy

During embryonic neurogenesis, cell-cell and cell-matrix interactions are critical for proper development of the central nervous system. In the adult brain, areas considered embryonic reservoirs called "neurogenic niches", show proliferating cells located in the vicinity of the ventricles. In previous studies, our group reported the existence of numerous proliferative zones, located on the wall that lines the ventricles of the telencephalon (VT), optic tectum (TO) and torus longitudinalis (TL) of fish of Austrolebias genus (Fernández , 2011). Different cell populations can be distinguished with both: cytosqueletal and plasma membrane specializations (Rosillo, 2010; Casanova, 2011). In spite of, some epitelial specializations at the central nervous system of higher vertebrates have been described (Garcia Verdugo, 2002), information available on this topic is scarce. Austrolebias brain showed a widespread distribution of radial glial cells mainly located in medial zones lining with the ventricular walls, as has been demonstrated using immunohistochemical analysis performed with antibodiesagainst Vimentin, S-100, BLBP and Glial Acidic Fibrilar Protein (view Rosillo et al., 2014).
In this work confocal laser, transmission electron and scanning microscopy analysis, showed that neurogenic niche cells are laterally linked through different and well developed intercellular junctional complexes. We observed extensive gap junctions between cells, as well as tight junctions sealing the ventricular wall. Often is possible to observe one or more desmosomes of different lengths, arranged in tandem, linking the plasma membrane of neighboring cells. Atypical adherens junctions were also detected. The continuity between adherens junctions and desmosomes with cytoskeletal components is evident. Cytoskeleton shows great development in "radial glia like" cells at TL, an elongated structure in the midbrain of Actinopterygii fish, linked to vision (Northmore, 1984). Using antibodies linked to fluorescent probes, we observed that cells rich in intermediate filaments, alternate with others with less developed cytoskeleton. Apically, the cells bordering the ventricular lumen of the neurogenic niches, often have microvilli and cilia. The analysis by SEM established that these are monociliated cells. The conservation of epithelial specializations in progenitor candidates cells, reinforces its functional significance. In this paper we analyze the location, ultrastructural features and molecular composition of such specializations, in the Austrolebias brain.

Fig. 1: Panoramic confocal image of cells of the telencephalic ventricular wall showing vimentin+ intermediate filaments and Cx 43 gap junctions.

Fig. 2: Panoramic TEM image of the ventricular torus longitudinalis region showing membrane specializations such as microvilli and cilia. Inset: High magnification of transversal section of a cilium.

Fig. 3: SEM micrograph showing the apical pole of cells from the ventricular telencephalic lumen which are predominantly monociliated.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-3373 Genetically Encoded Sensors of Cell Membrane Voltage for Two-Photon Polarization Microscopy

Bondar A.1 2, Kevorkova A.1 2, Jin L.3, Han Z.3 4, Cohen L. B.3, Pieribone V. A.3 4, Lazar J.1 2
1Institute of Nanobiology and Structural Biology GCRC, Academy of Sciences of the Czech Republic, v.v.i., Zámek 136, 37333 Nové Hrady, Czech Republic, 2Faculty of Science, University of South Bohemia, Branišovská 31a, 37005 České Budějovice, Czech Republic, 3Dept. of Cellular and Molecular Physiology, Yale University, New Haven, CT, USA, 4John B. Pierce Laboratory, Yale University, New Haven, CT, USA
bondar@nh.cas.cz

Changes in cell membrane voltage are a hallmark of neuronal activity. Genetically encoded fluorescent sensors of membrane voltage have recently allowed optical observations of electrical activity in isolated neurons and in living animals. Here we demonstrate a new approach to optical monitoring of cell membrane voltage that relies on two-photon polarization microscopy (2PPM). 2PPM detects changes in orientation of fluorescent moieties with respect to the cell membrane. Therefore, changes in orientation of a fluorophore that occur in response to changes in cell membrane voltage can be used for monitoring of cell membrane voltage by 2PPM. We have now applied 2PPM to known genetically encoded voltage sensors derived from the Ciona intestinalis voltage sensitive phosphatase. Our observations show that the fluorescent moiety in the investigated sensors undergoes voltage-induced reorientation that can be observed by 2PPM. Thus, the investigated sensors can be used as ratiometric 2PPM probes of cell membrane voltage. Apart from allowing us to observe changes in cell membrane voltage, our 2PPM experiments have allowed us to make insights into the mechanism of function of the existing sensors, and to outline a rational path towards development of improved sensors with faster and more robust responses. Apart from investigating existing membrane voltage sensors, we also demonstrate novel, rational approaches to development of genetically encoded voltage sensors that should take full advantage of 2PPM's strengths.

Czech Science Foundation grant P205/13-10799S to JL,
University of South Bohemia Grant Agency grant GAJU 141/2013/P
Innovative Bioimaging, L.L.C.

Fig. 1: Structure of the genetically encoded voltage sensor ArcLight, based on the Ciona intestinalis voltage sensitive phosphatase.

Fig. 2: An ArcLight expressing cell observed by 2PPM. The red-green pattern signifies a well-defined orientation of the fluorescent protein moiety.

Fig. 3: Temporal profile of changes in ArcLight fluorescence intensity and in fluorescent protein orientation induced by changes in cell membrane voltage.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-3528 Balance and coordination training and endurance training after nerve injury: morphological characteristics of sciatic nerve and soleus muscle. 

Bonetti L. V.1,2, Schneider A. K.2, Barbosa S.1, Ilha J.3, Faccioni-Heuser M. C.1,2
1Postgraduate Program in Neuroscience, Federal University of Rio Grande do Sul (UFRGS), RS, Brazil, 2Department of Morphological Sciences, Institute of Basic Health Sciences, UFRGS, RS, Brazil, 3Department of Physical Therapy, University of the State of Santa Catarina, SC, Brazil
heuser@ufrgs.br

Axonotmesis is one consequence of nerve injury and skeletal muscle inactivity occurs after these injuries. Different rehabilitation treatments have proven useful in accelerating sciatic nerve (SN) and soleus muscle (SM) regeneration. The aim of this study was analyze the effects of 4-week endurance and balance and coordination training programs on the morphology of the SN and SM fibers after crush injury of the SN. 23 male Wistar rats (3 months) were randomly divided in 4 groups: (1) sham-operated rats (Sham, n=5); (2) rats with sciatic crush (non-trained, NT, n=6); (3) rats with sciatic crush and endurance training (ET, n=6); and (4) rats with sciatic crush and balance and coordination training (BCT, n=6). For the surgical procedures, animals were anesthetized and the right SN was exposed and nerve crush injury was performed with 1 mm hemostatic forceps for 30 seconds. 48 hours after the surgery, the animals from the ET and BCT groups began specific training that lasted 4 weeks. 48 hours after the end of the training programs, the animals were anesthetized, transcardially perfused with saline solution, followed by Karnovsky solution. A short segment of the right SN 5mm distal to the crush injury site and small samples of the central part of the right SM were excised and fixed in the Karnovsky solution; postfixed OsO4, dehydrated in acetone, embedded in araldite. Sections (1µm) were stained with 1% toluidine blue. Images of the SM and the SN were captured, digitized and processed with Image Pro Plus Software 6.0. Dates were analyzed using one-way ANOVA followed by post hoc Tukey’s tests (p<0.05). In the SM, the percentages of the muscle tissue area of NT group were significantly lower and muscle connective tissue area was significantly greater than the other groups. The percentage of the muscle blood vessel area in the BCT group was significantly greater than that of the Sham and NT groups. In the SN, the percentages of the nerve tissue area of the Sham group were significantly greater and nerve connective tissue area was significantly lower than the other groups. The percentage of the nerve blood vessel area in the NT group was significantly lower than that in the Sham group and the nerve blood vessel area in the ET group was significantly larger than that in the NT group. These findings indicate that BCT and ET, when initiated in the early phase after SN injury, improve the morphological properties of the soleus muscle and sciatic nerve.

PROPESQ-UFRGS and FAPERGS

Fig. 1: Fig. 1 Digitized images. Soleus muscle (A-D). Sham(A), NT(B), ET(C), BCT(D). Bar=50 µm. Normal sciatic nerves, Sham (E), regenerating nerves, NT (F), regenerating nerves, ET (G), regenerating nerves BCT (H). Bar=20 µm. MF = muscle fiber; NF=myelinated nerve fiber; Sc=Schwann cell; *=endoneurial connective tissue; BV=blood vessel; Ld=lipid droplet
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-5707 ANALYSIS OF NEURONAL ALTERATIONS ASSOCIATED TO TM4SF2 DELETION IN KNOCKOUT MOUSE

Vezzoli E.1, Folci A.1,2, Murru L.1,2, Bassani S.1,2, Passafaro M.1,2,3, Francolini M.1,2
11) Università degli Studi di Milano, Dipartimento di Biotecnologie Mediche e Medicina Traslazionale - Via Vanvitelli 32, 20129 Milano - Italy, 22) National Research Council (CNR) – Institute of Neuroscience - Via Vanvitelli 32, 20129 Milano - Italy, 33) Dulbecco Telethon Institute - Italy
maura.francolini@unimi.it

Defective formation or function of synapses in the central nervous system (CNS) results in disorders of learning and memory, including autism and intellectual disability. X-linked intellectual disability (XLID) is a heterogeneous condition caused by single gene mutations on the X chromosome. Most of the genes mutated in XLID encodes synaptic proteins involved in actin cytoskeleton rearrangement, synaptic plasticity, synapse formation or neurotransmission, although the precise roles of most of these genes remain unknown. One of the genes responsible for XLID is TM4SF2 which encodes TSPAN7. TSPAN7 promotes filopodia and dendritic spine formation in cultured hippocampal neurons, and is required for spine stability and normal synaptic transmission. We identified PICK1 (protein interacting with C kinase 1) as a TSPAN7 partner. In vitro TSPAN7 regulates the association of PICK1 with AMPARs, and controls AMPAR trafficking. The objective of this study is to characterize the effects of TSPAN7 elimination on the structure of the cortex and hippocampus and their excitatory synapses in mice lacking TSPAN7. We analyzed the anatomy of the CNS of TM4SF2 KO mice on paraffin sections of brain; we compared the cellular architecture of the cortex and hippocampus of WT and KO mice; we did not detect any difference in the structure of these two areas. We then focused our attention on a more detailed analysis of the hippocampus by transmission electron microscopy of excitatory synapses and synaptic spines by applying quantitative morphometrical analyses. We found a significant reduction in both the length and the thickness of the PSDs of KO mice compared to WT. Golgi staining and stereological analyses also suggested that there might be a trend towards a reduction of the density of dendritic spines in mutant mice, to further investigate this aspect we used serial block-face scanning electron microscopy and 3D reconstruction to measure spine volume and density. Quantitative measurements of presynaptic boutons did not reveal significant differences in any of the samples examined. We also tested the effects of TM4SF2 gene deletion on the expression of endogenous synaptic proteins and showed a significant reduction in the levels of expression of AMPAR subunits while no reduction was observed in the levels of presynaptic proteins or inhibitory synapses markers. Moreover we explored whether the disruption of TSPAN7 resulted in alterations of synaptic transmission and plasticity in the intact network. Excitatory input was measured on acute slices from WT and KO brains. We found a difference in the frequency of mEPSCs recorded from granule cells of dentate gyrus in KO animals and a significant alteration in mEPSCs amplitude of the currents recorded from CA3 pyramidal neurons.

The authors wish to thank Gatan for 3View serial sectioning and data collection

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-5845 Functional imaging on zebrafish using digital scanned light sheet microscopy

Gualda E. J.1, Lima J.2, Feierstein C. E.2, Orger M. B.2
1Instituto Gulbenkian de Ciências, Oeiras, Portugal, 2Champalimaud Neuroscience Programme, Lisboa, Portugal
emilio.gualda@gmail.com

Even the very simplest actions can involve complex activity dynamics in networks of neurons distributed in areas throughout the brain. Understanding the nature of neuronal networks activity patterns under natural conditions is an important step in understanding how our brains generate adaptive behavior. However, our ability to make simultaneous recordings from large populations of neurons in vivo has been severely limited by the available techniques. Electrophysiology provides high temporal resolution, but typically only from a small fraction of neurons sampled blindly from a particular region. Two-photon microscopy allows single cell resolution functional imaging from large numbers of neurons, but is ultimately limited in recording speed by the need to scan each point sequentially.

Light-sheet excitation can overcome this latter limitation, since data can be acquired from many thousands of points simultaneously using an area CCD or CMOS camera. We have built a simple, compact and high speed digital scanned light-sheet microscope (DSLM) dedicated to in vivo functional imaging in larval zebrafish that will allow simultaneous experimental control of visual and other sensory stimuli, high-speed neural data acquisition and potentially monitoring behavioral output. The detection objective is mounted on a piezo actuator for fast z scanning, allowing high-speed imaging by coordinated motion of the objective and the scanned light-sheet. We also take advantage of the Hamamatsu Orca Flash 4.0 dedicated light-sheet mode in order to reduce background and improve image quality. The fish are mounted horizontally, using low-melting-temperature agarose, in an open, water-filled chamber that allows access from above for recording pipettes or tactile stimuli, and room below for visual stimulus projection. Visual stimuli are projected onto a diffusing screen below, or in front of the fish using a laser projector.

High spatial resolution knowledge of the brain’s functional architecture and an understanding of the relationship of network dynamics and behavioral output on fast timescales will be accessible using the described fast volumetric light-sheet imaging set up.

The authors acknowledge support from Fundação para a Ciência e Tecnologia, Portugal - grants SFRH/BD/80717/2011, EXPL/BBB-IMG/0363/2013, EXPL/NEU-SCC/2370/2013.

↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-5866 Phoneutria nigriventer spider venom (PNV) disrupts cytoskeleton and induces Ca2+ waves in astrocytes

Rapôso C.1, Björklund U.2, Cruz-Höfling M. A.1, Hansson E.2
1Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), 13 083-970 Campinas, SP, Brazil, 2Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, SE 413 45 Gothenburg, Sweden
cataraposa@gmail.com

Bites from genus Phoneutria (Ctenidae, Araneomorpha) are the second most frequent source of spider accidents in Southeast Brazil. In the Phoneutria nigriventer severe envenoming, systemic signs and symptoms, such as vision disturbance, spastic paralysis, tremors, profuse cold sweating and convulsions are related, suggesting that the central nervous system (CNS) is involved in the effects; however, the mechanisms of PNV in the CNS are still poorly understood. It is known that PNV induces changes in blood-brain barrier, impairs glutamate uptake, and causes astrogliosis and neuroinflammation in rats. Down-regulation of Na+/K+-ATPase in combination with changes of Ca2+ signaling in the astrocyte networks are parameters associated with neuroinflammation and abnormal glutamate uptake. Therefore, astrocytes can be a key target in central mechanisms of PNV. The present work aimed at investigating if PNV induces Ca2+ oscillations in cultured astrocytes and how this affects activated astrocytes. All experiments were done in triplicate. After confluence, cells were exposed to PNV (14000 ng/ml; 0.5, 1, 5 and 24 hours exposure), and double labeling for GFAP (immunocytochemistry) and F-actin (Alexa488- phalloidin probe) were performed. The Na+/K+-ATPase expression was accessed by immunoblotting. Ca2+ waves were measured using a fluorescent Ca2+ sensitive probe Fura-2/AM. To determine the underlying Ca2+ source of the PNV-evoked Ca2+ transients, internal stores were depleted by pre-incubation with a endoplasmic reticulum Ca2+ATPase inhibitor, thapsigargin (1 μM), and caffeine (20 mM), or incubated with a Ca2+-free buffer. Astrocytes showed impaired stress fibers 1 hour after PNV exposure, with retracted bodies and processes. After 5 and 24 hours, stress fibers and cells morphology were similar to control. Na+/K+-ATPase expression was decreased 0.5, 1 and 5 hours after PNV administration. After 24 hours, no significant alteration in Na+/K+-ATPase level was observed. Ca2+ waves were induced by PNV in astrocytes, immediately after exposure. The incubation with Ca2+-free buffer did not change the waves; however, the pre-incubation with thapsigargin/caffeine totally prevented Ca2+ waves during PNV incubation. Taking together, these results suggest that PNV directly affects astrocytes. The impairment in Na+/K+-ATPase and stress fibers might be part of the mechanisms of neuroinflammation and abnormal glutamate uptake induced by PNV. Also, PNV activates a membrane receptor, triggering waves from intracellular Ca2+ stores. Understanding the mechanisms of neurointoxication caused by PNV is of interest not only for better treating envenoming by P. nigriventer, but also for appreciating the diversity of targets triggered by PNV toxins.

Authors thank FAPESP, CNPq and FAEPEX-UNICAMP, Brazil; Edit Jacobson’s Foundation and the Sahlgrenska University Hospital (LUA/ALF GBG-11587), Gothenburg, Sweden.

Fig. 1: GFAP and stress fibers double labeling in astrocytes. Control cells showed normal morphology and actin cytoskeleton distribution (A-C). PNV impaired stress fibers and induced bodies and processes retraction after 1 hour (G-I). Five (J-L) and 24 (M-O) hours after venom exposure, cells were similar to control.

Fig. 2: Ca2+waves in astrocytes. There was an immediate astrocyte response after PNV exposure (A). When Ca2+ free medium was used, the response persisted (B). However, when cells were pre-incubated with thapsigargin/caffeine, PNV was not able to induce Ca2+ waves (C). Control was done using both Ca2+ free medium and thapsigargin/caffeine (D).
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-5931 Neurotrophic and Antioxidant Effects of Silymarin Comparable to 4-Methylcatechol in Protection against Gentamicin-Induced Ototoxicity in Guinea Pigs..

Abdeen A. A.1, Draz E. I.2, Sarhan N. I.3, Gabr T. A.4
1Department of Pharmacology, Faculty of Medicine, Tanta University, Egypt, 2Department of Forensic medicine &Clinical toxicology2, Faculty of Medicine, Tanta Universitylty ,Egypt, 3Department of Histology, Faculty of Medicine, Tanta University, Egypt , 4Department of Audiology Faculty of Medicine, Tanta University, Egypt
nsarhan2006@hotmail.com

Gentamycin is a very effective aminoglycoside. However, its usage is limited by the nephrotoxic and the irreversible ototoxic effects. This study aimed to measure the protective role of Both 4- methycatechole (a nerve growth factor) and silymarin (an antioxidant) against gentamycin induced ototoxicity. Twenty guinea pigs were divided into four groups and were treated for 19 days: group I (only saline, i.p), group II (gentamycin 120mg/kg/d, I.P), group III (4- methylcatechole 10µg/kg 2hrs before gentamycin + gentamycin 120mg/kg i.p.) and group IV (silymarin (100mg/kg by oral lavage 2hrs before gentamycin + gentamycin 120mg/kg i.p). Auditory brainstem response, nerve growth factor levels, TRKA mRNA in cochlear tissue, serum catalase activity and serum malondialdhyde levels were measured in all groups. Scanning electron microscopic examination of cochleal hair cells was conducted. The main findings indicated that silymarin pre-treatment produced significant decrease in auditory brainstem response (ABR) threshold with significant restoration of nerve growth factor (NGF) levels and increased Trk-A m-RNA expression in cochlear tissue as well as marked preservation of most of hair cells of the organ of Corti by scanning electron microscopy (SEM) compared to the pre-treatment by 4-methylcatechol. Silymarin caused significant amelioration in the oxidative stress state by reducing malondialdehyde (MDA) levels and increasing catalase activity. It could be concluded that silymarin was more potent than 4-methylcatechol as a protective agent against gentamicin ototoxicity.

The authors thank Mr. Ahmed Samy Elshenawy and Mr Yaseen Abdelmoneim Abdallah for their cooperation and help in dissection, tissue processing and photographing of the specimens 

Fig. 1: Scanning electron micrograph of organ of corti of normal control group showing three rows (1,2,3) of outer hair cells (OHCs) and one row of inner hair cells (IHCs). Notice stereocilia of OHCs arranged as V shape and those of IHC arranged in U shape. (Group I, SEM ×1500)

Fig. 2: Scanning electron micrograph of organ ofcorti of gentamicin-induced ototoxicity group showing loss of several inner (IHCs) and outer hair cells (OHCs) (wavy arrows). Stereocilia of these cells showing either fusion (white ►), focal loss(black ►) or complete absence (curved arrow) and irregular arrangement. (Group II, SEM ×1500)

Fig. 3: Scanning electron micrograph of organ of corti of 4-methylcatechol treated group showingpreservation of most of the hair cells except for fewinner (►) and outer hair cells (→). Notice disarrayof cilia of IHC (wavy arrow) and fusion of thestereocilia of the OHC (curved arrow). (Group III,SEM ×1500)

Fig. 4: Scanning electron micrograph of organ of corti of silymarin treated group showing preservation of nearly most of the inner (IHCs) and the outer haircell (OHCs 1,2,3). Notice also will preserved stereocilia (Group IV, SEM ×1500)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-5980 Embryonic neural stem cells survival and differentiation in cerebellar grafts and in vitro; effects of Sonic Hedgehog

Ostasov P.1,3, Cendelin J.2,3, Tuma J.2,3, Pitule P.1,3, Houdek Z.2, Purkartova Z.2, Kralickova M.1,3
1Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic, 2Department of Pathophysiology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic, 3Biomedical Centre, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic
pavel.ostasov@lfp.cuni.cz

Regeneration capability of the central neural system is very limited and therefore diseases accompanied with marked loss of neurons lead to mostly irreversible functional deficits. One of such diseases are hereditary cerebellar degenerations. Neurotransplantation represents the most promising therapy to reverse the effects of the disease. However, successful engraftment of the cells depends on the type of transplant as well as the state of the host tissue.
Adult B6CBA Lurcher mutant and wild type mice were used. Lurcher mutants carry the Grid2Lc mutation causing loss of Purkinje cells and secondary degeneration of cerebellar interneurons and inferior olive neurons (Fig. 1). Cells and tissue used for transplantation were isolated from B6CBA mouse embryos, constitutively expressing GFP.
We determined that solid transplant of embryonic cerebellum or suspension of embryonic cerebellar cells had successfully survived in the host cerebellum and differentiated into various types of cells of neuronal and glial lineage. However, such therapy would require a lot of material and is not scalable to be useful in practice. On the other hand, transplantation of neuroprogenitors derived from proliferating cells was successful only in wild type mice, while in Lurcher mouse the transplant was lost within two months after transplantation (Fig. 2A).
To overcome this problem, embryonic neural stem cells (eNSC) were used for transplantation (Fig. 2B). In addition the effects of morphogenic factor Sonic hedgehog (Shh) were studied. Shh has the ability to maintain "stem" nature of the cells and has an effect on survival, proliferation and differentiation of eNSC.
Grafts were examined 3 months after transplantation in frozen sections of the cerebellum using fluorescent microscope to detect GFP-positive cells. For the in vitro experiments, seeded cells were examined after 5 days of differentiation with or without Shh using anti-GFAP (marker of astrocytes) and anti-MAP2 (marker of neurons) immunofluorescence to evaluate lineages into which the cells differentiated. Cell proliferation activity was assessed by incubating eNSC with bromodeoxyuridine (BrdU) for 24 hours prior fixation and then counting ratio of BrdU-positive nuclei to all nuclei. The overall survival was determined by MTS test.
eNSC engrafted successfully in both wild type and Lurcher cerebellum both with and without Shh treatment (Fig. 3). Shh successfully enhanced cell "stemness" and increased eNSC proliferation and survival during differentiation in vitro.

Supported by the European Social Fund and the state budget of the Czech Republic projects no. CZ.1.07/2.3.00/30.0061, CZ.1.07/2.3.00/30.0022, GAUK 1268213 and COST no. LD12057

Fig. 1: Wild type (A) and Lurcher (B) mouse cerebellum. White arrows points to Purkinje cells.

Fig. 2: Survival of neuroprogenitors (A) and eNSC (B) after transplantation into cerebellum.

Fig. 3: Transplanted cells (green) and glial cells (red) in eNSC grafts without Shh (A) or with Shh (B)
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-6014 ULTRASTRUCTURE OF THE RETINA IN THE LAND SNAIL MEGALOBULIMUS ABBREVIATUS

Pereira N.1, 2, 3, Achaval M.2, 3, Zancan D.2, 3
1Neuroscience Graduate Program, Porto Alegre, Brazil, 2Institute of Basic Health Sciences (ICBS), Porto Alegre, Brazil, 3Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil
eyeresearch@ymail.com

Aims: In a previous study, the eye of the land snail Megalobulimus (Strophocheilus) sp. was described at light microscopic level. Some questions remain, however, about the types of photoreceptors, and about the existence of other neurons (ganglion cells or second order sensorial neurons), and glial cells in the retina. In order to clarify these questions, an ultrastructural study of the retina of M. abbreviatus was made.The existence of the glial cells in the retina was investigate by immunohistochemistry for glial fibrillary acidic protein (GFAP), which has not yet been investigated in the retina of the gastropod mollusks.


Methods and Results: Snails Megalobulimus abbreviatus were anaesthetized (menthol solution for 30 min). The isolate eyes were fixed for 2 h in 4% paraformaldehyde and 0,5% glutaraldehyde buffered with 0,1 M phosphate pH 7,4 (PB), post-fixed in 1% OsO4 in PB for 1 h, dehydrated and embedded in Durkupan ACM (Fluka) resin. Ultrathin sections were obtained on MT 6000-XL ultramicrotome and stained (2% uranyl acetate and 2% lead citrate). The sections were analyzed with Jeol JEM 1200EXII at Electron Microscopy Center of UFRGS. Two types of cells with sensorial aspects were observed in the retina of M. abbreviatus. A sensory cell (type I) possesses apical projections toward the vitreous body, which are composed of overlapped long and slender microvilli, constituting the rhabdomere (Fig. 1). The rhabdomeric portion of the inner layer of retina is thicker (28-32 µm) in the central portion than the peripher alone (2-3,6 µm) of the retina. Another sensorial cell (type II) was visualized in the peripheral areas of the retina. These cells have cilia in their apical end and few microvilli (Fig. 2). Type-I sensorial cell should be the principal photoreceptor, because of its well-organized rhabdomeric pattern and its central location in the retina. The pigmented cells are more numerous in the central portion of the retina. The nuclei of the photoreceptor and pigmented cells are located in the basal portion of the retina, the nuclear layer. The outmost layer of the retina consists of neuritic processes, the plexiform layer, that contain some axonal projections with dense-core vesicles (Fig. 3). These projections appear originate from a central ganglion of the animal. The photoreceptors may project toward the optic nerve without making synapses in theplexiform layer. Exocytosis of these dense-core vesicles could occur without specialized synapses. GFAP-immunoreaction and cells with glial aspect were observed in the nuclear and plexiform layers of the retina of the snail M. abbreviatus.

Fig. 1: Electron micrograph of central area of the retina of M. abbreviatus, showing the arrangement of microvilli with two different orientations in rhabdomere.

Fig. 2: Electron micrograph of retina in eye periphery of M. abbreviatus, showing the type-II sensorial cell with cilia (arrows). The microvilli of the type-I sensorial cells are shorter than in the central retina.

Fig. 3: Electron micrograph of the central area of retina of M. abbreviatus, showing the plexiforme layer, which is situated between the nuclear layer and the basal lamina(asterisk). The basal lamina is surrounded by a thin capsule of connective tissue. Axonal processes containing dense-core vesicles are showed (arrows).

Fig. 4: Light micrograph of the eye (demelanized) of M. abbreviatus, showing the intense GFAP-immunoreaction in the external layer of the retina.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-6032 Adult neurogenesis in the brain of the weakly electric teleost Gymnotus omarorum

Olivera-Pasilio V.1, Lasserre M. I.1, Castelló M. E.1
1Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay
mcastello@iibce.edu.uy

Adult neurogenesis is the process through which new neurons are generated and added to, or replace lost neurons of preexisting circuits. This primitive, evolutionary conserved trait was reduced along evolution from anamniotes to amniotes. In mammals is almost limited to two brain regions, but in teleosts several brain proliferating zones (PZs) with high neurogenic capacity are profusely distributed, subserving its high regenerative potential. In spite of the physiological and pathological relevance of adult neurogenesis, and the recognized need of comparative studies to achieve a better understanding of its regulation and function, few vertebrate species have already been studied (Zupanc, 2001; Lindsey & Tropepe, 2006; Grandel & Brand, 2012). Within the teleostean radiation, the wave type weakly electric fish Apteronotus, is a “model” species in which differences with non electroreceptive species in the distribution of adult brain PZs and neurogenesis are attributed to its functional specialization.. A similar distribution of brain PZs was found in the pulse-type weakly electric gymnotid Gymnotus omarorum through postnatal development (Iribarne & Castelló, 2014; Olivera & Castelló, 2014). In the present work, we evidenced the neurogenic potential of G. omarorum adult brain PZs by analyzing the co-localization of long term label retaining (30, 90 and 180 days) of the thymidine analog 5-Chloro-2′-deoxyuridine (CldU) and the expression of markers of neuronal differentiation (HuC/HuD, beta III tubulin, and tyrosine hydroxylase -TH-) or in vivo retrograde transport of the neuronal tracer neurobiotin. Our results support a rostral migratory stream, from the rostral region of the subpallial PZ to the olfactory bulb, where derived cells express HuC/HuD first, and differentiate into TH+ interneurons later (Fig. 1), and radial migration giving rise to subpallial HuC/HuD and TH+ neurons, and co-localization of CldU and b III tubulin in the caudal telencephalon (Fig. 2), and CldU and HuC/HuD in the caudal regions of the mesencephalic tectum opticum (Fig. 3) and the torus semicircularis, and the rombencephalic electrosensory lateral line lobe (ELL) and the corpus cerebelli (CCb). We further demonstrated the insertion of newborn neurons into circuits of the CCb, by co-localization of retrogradely transported neurobiotin in long CldU label retaining cells already migrated to CCb granular layer (Fig. 4). In conclusion, in this work we provide further evidences that adult neurogenesis is a conserved feature of euteleosts, giving rise to interneurons that are incorporated into preexisting neural networks. We also demonstrated for the first time a new neurogenic region at the functionally relevant ELL, the first and exclusive relay of electrosensory information.

This work was partially funded by PEDECIBA and ANII (FCE_2009_1_2246 & FCE_3_2011_1_6168).

Fig. 1: Confocal images of a stack of sequential focal planes and orthogonal images generated for the position indicated by the crossing lines for demonstrating the co-localization of CldU long term (180d) label retaining (red) and TH expression (green) in a newborn cell already migrated into the olfactory bulb (arrow in inset). Scale bars: 5 μm.

Fig. 2: Confocal images of a stack of sequential focal planes and orthogonal images generated for the position indicated by the crossing lines for demonstrating the co-localization of CldU long term (30 d) label retaining (red) and beta III tubulin immunoreactivity (green) in a newborn cell in the caudal telencephalon. Scale bars: 5μm.

Fig. 3: Confocal images of a stack of sequential focal planes and orthogonal images generated for the position indicated by the crossing lines for demonstrating the co-localization of CldU long term (90d) label retaining (red) and HuC/HuD expression (green) in a newborn of the tectum opticum (arrow in inset). Scale bars: 5μm.

Fig. 4: The integration of newborn neuron into neural circuits of the CCb was evidenced by sequential acquired confocal images of retrogradely transported neurobiotin (green, left), long term (180d) CldU label (red, middle), and the overlay (yellow, right) in a newborn cell at the granular cell layer of CCb (arrow in inset). Scale bars: 5μm.
↑ Go to the Table of content ↑
Type of presentation: Poster

LS-14-P-6056 Measuring brain repair with atomic force microscopy

Holtzmann K. J.1, Gautier H. O.1, Christ A. F.2, Franklin R.1, Karadottir R. T.1, Franze K.1, Guck J.3
1University of Cambridge, United Kingdom, 2CEA Grenoble, France, 3Technische Universität Dresden, Germany
kh428@cam.ac.uk

Atomic force microscopy (AFM) is not only an advanced imaging tool, but can also be used for exploring the mechanical properties of tissues. We are using AFM to investigate the stiffness of acute rat brain slices in demyelination, a condition causing severe functional impairment and disability.
The most common demyelinating disease is multiple sclerosis. Due to an inflammatory reaction axons lose their protective insulating layer, the myelin sheath, which in the central nervous system is formed by oligodendrocytes. To remyelinate axons and restore full functionality, recruitment and maturation of oligodendrocyte precursors cells (OPCs) is essential. As OPC function has been shown to depend on the mechanical environment [1], we are studying the tissue stiffness in remyelination.
Lesions were created by injecting ethidium bromide into the caudal cerebellar peduncle of rats and the tissue was harvested 7 and 21 days post lesion in accordance with regulations issued by the Home Office of the United Kingdom under the Animals (Scientific Procedures) Act of 1986. The resulting lesions were measured as acute slices with a JPK Nanowizard III by nano-indentation, data were analyzed using the Hertz model following a routine developed by Christ et al. [2]. AFM measurements show that at 7 days demyelinated areas of tissue are consistently more compliant than their surroundings, healthy white matter has an elastic modulus of 210 ± 150 Pa, lesion tissue of 148 ± 115 Pa. At 21 days the inner areas of the lesion are still soft at 163 ± 106 Pa, whilst the outer areas of the lesion begin to stiffen to 202 ± 105 Pa, approaching healthy tissue stifness at 238 ± 125Pa. The differences in stiffness between the two values of each healthy and lesion tissue are not statistically significant, the differences between healthy tissue and lesion tissue however are significant (Kruskal-Wallis test, p < 0.01). The migration speed of OPCs that has been reported for such lesions [3] matches the speeds observed in OPC cultures on hydrogels [1] with the same stiffness as we report for the lesion, suggesting an explanation for the perceived mismatch between OPC behaviour in lesion and OPC behaviour in culture as an effect created by the different mechanical properties of the cells' environment.
In summary, our results show that in demyelination the mechanical properties of brain tissue change and that changes in mechanics are indicative of underlying changes in biology. Further work will concentrate on harnessing these novel insights for improved therapeutic approaches to treat these devastating neurological disorders.

[1] Jagielska et al., Stem Cells Dev 21 (2012), p.2905–14
[2] Blakemore et al., J. Neuroimmunol. 98 (1999), p.69-76
[3] Christ et al., J. Biomech. 43 (2010), p.2986–2992

The authors gratefully acknowledge funding from EPSRC, the Lundgren Fund and the NanoDTC Cambridge.

↑ Go to the Table of content ↑

ID-1. Correlative microscopy in life and material sciences

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-1-IN-1629 Imaging Labeled Protein Complex Subunits in Whole Eukaryotic Cells in their Native Aqueous Environment

Peckys D. B.1, Korf U.2, de Jonge N.1
1INM – Leibniz Institute for New Materials, Saarbrücken, Germany, 2Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany
niels.dejonge@inm-gmbh.de

We have used scanning transmission electron microscopy (STEM) of cells in liquid [1, 2], so-called Liquid STEM, to study protein complex subunits. Live eukaryotic cells were grown on silicon microchips with silicon nitride (SiN) membrane windows, and incubated with specific protein labels consisting of gold nanoparticles or fluorescence nanoparticles, quantum dots (QDs). The samples were imaged with STEM. On account of the atomic number (Z) contrast of the annular dark field (ADF) detector of STEM, the nanoparticles of high-Z material can be detected within the background signal produced by the low-Z material of the cell and surrounding liquid. The highest resolution was obtained using STEM at 200 keV electron beam energy, for which the cells were placed in a microfluidic chamber with electron transparent windows (Fig. 1A). The flat parts of cells can also be imaged in a thin layer of water with environmental scanning electron microscopy (ESEM) at 30 keV with the STEM detector (Fig. 1B).

Liquid STEM was used to study several different proteins and protein complexes on intact cells in liquid, such as the epidermal growth factor receptor, and the closely related ErbB2 receptor. The particular distribution of monomers, homodimers, and heterodimers of these receptors is of relevance for basic research as well as for the analysis of drug mechanisms. We used COS7 fibroblast cells and SKBR3 breast cancer cells. The receptors were specifically labeled with nanoparticles via small, specific ligands, much smaller than antibodies used for immunogold labeling. It was observed that the expression levels and the distribution of the receptors differed significantly from cell to cell. Therefore, we used correlative fluorescence microscopy to localize cells and cellular regions where a certain type of receptor was present (see Fig. 2A). The sample was transferred into the ESEM chamber and an overview image was recorded at the same location (Fig. 2B). ESEM-STEM images were then recorded at a higher magnification to localize the individual QDs (Fig. 2C). The regions of stronger fluorescence correlated with a higher density of the QDs. Since the extensive sample preparation common for biological electron microscopy is mostly avoided, the Liquid STEM method is as simple as fluorescence microscopy. It is readily possible to acquire data of thousands of labels on dozens of cells. This advantage was used to study the distribution of EGFR monomers, dimers and clusters using a total of 1411 obtained from images of 15 cells [3].

References:

[1] N de Jonge et al., Proc Natl Acad Sci 106, 2159-2164, 2009.

[2] N de Jonge & FM Ross, Nature Nanotechnology 6, 695-704, 2011.

[3] DB Peckys et al, Scientific reports 3, 2626, 2013.

Acknowledgements: We thank Protochips Inc. NC, USA for providing the microchips and E. Arzt for his support through INM.

Fig. 1: Principle of Liquid STEM (A) The cell is fully enclosed in a microfluidic chamber with two SiN windows. Contrast is obtained on protein nanoparticle (NP) labels using the dark field STEM detector. (B) The cell is maintained in a saturated water vapor atmosphere, while a thin layer of water covers the cell.

Fig. 2: Correlative fluorescence microscopy and ESEM-STEM of a flat part of an intact SKBR3 cell in a thin liquid layer. The ErbB2 was extracellular labeled with fluorescent quantum dots (QD)s. (A) Fluorescence image of the edge of the cell. (B) The same region imaged with ESEM-STEM. (C) Boxed region in A and B at higher magnification.
↑ Go to the Table of content ↑
Type of presentation: Invited

ID-1-IN-6081 Placing molecules in a cellular context using light, electron and X-ray microscopy

Collinson L.1
1Cancer Research UK London Research Institute, London, UK
lucy.collinson@cancer.org.uk

Fluorescence microscopy is a powerful tool for localising proteins within biological samples. However, information is limited to the distribution of the tagged protein, telling us little about the ultrastructure of the surrounding cells and tissues, which may be intimately involved in the biological process under study. Electron microscopy overcomes the resolution limitation inherent in light microscopy and can reveal the ultrastructure of cells and tissues. However, protein localisation tends to be complex and is often dependent on the availability of ‘EM-friendly’ antibodies. Correlative light and electron microscopy (CLEM) combines the benefits of fluorescence and electron imaging, revealing protein localisation against the backdrop of cellular architecture.

In this talk, I will introduce several ways in which we are extending CLEM. We developed ‘correlative light and volume EM’ to enable visualisation of rare events in cells, tissues and whole model organisms, by combining correlative workflows with microscopes that automatically collect large stacks of high resolution images (Focused Ion Beam SEM and Serial Block Face SEM). We applied this technique to study disrupted nuclear envelopes in lipid-depleted mammalian cells and developing blood vessels in zebrafish. We are further developing this technique, to make CLVEM faster and more accurate, using an in-resin fluorescence (IRF) protocol for mammalian cells and tissues. GFP and mCherry are preserved through processing into resin, so that we can directly detect fluorophores and cellular structure in the same ultrathin resin section. We are now developing the next generation of integrated light and electron microscopes to image structural and functional information simultaneously. Finally we are combining cryo-fluorescence microscopy with cryo soft X-ray microscopy to study cellular events in whole mammalian cells, preserved as close to native state as possible within a vacuum.

Fig. 1: 1
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-1447 Alexa Fluor fluorescence in tissue for correlative light and electron microscopy

Röder I. V.1, Dietrich C.2, Fuchs J.3, Wacker I.4, Schröder R. R.1
1CellNetworks, BioQuant, Universitätsklinikum Heidelberg, Heidelberg, Germany, 2Carl Zeiss Microscopy GmbH, Oberkochen, Germany, 3Carl Zeiss Microscopy GmbH, Jena, Germany, 4Centre for Advanced Materials, Universität Heidelberg, Heidelberg, Germany
ira.roeder@bioquant.uni-heidelberg.de

The localization of single and rare structures within cellular volumes or tissue samples prepared for electron microscopic studies is a challenging endeavor. Correlative light and electron microscopy bridges the gap between detecting a structure and making it accessible as target for ultrastructural studies.

We are interested in the neuromuscular junction (NMJ), the synapse between a skeletal muscle fibre and the axon of a motoneuron. It is a comparably small structure within the huge volume of a muscle fibre and difficult to find in electron microscopic preparations. Therefore, we established two protocols to preserve Alexa Fluor (AF) fluorescence during the embedding of mouse muscle tissue in HM20 resin. The first protocol is based on chemical fixation. After excision and fixation of diaphragm the acetylcholine receptors (AChR) of the NMJ are labelled using alpha-bungarotoxin (BGT) conjugated to AF-555 or AF-647. Subsequently, the temperature is progressively lowered and the tissue is embedded at low temperatures [protocol modified, 1]. For the second approach based on high pressure freezing, the diaphragm is excised and immediately dissected into small pieces. During this preparation step labelling is performed. Subsequently, the samples are high pressure frozen, freeze substituted and embedded [protocol modified, 2].

We studied the final sample blocks using confocal laser scanning microscopy and used the fluorescence signal to relocate predefined regions. Furthermore, the fluorescence is still traceable in ultrathin sections by means of common fluorescence light microscopic techniques and by more sophisticated, super-resolution methods such as high numerical aperture fluorescence microscopy (Fig. 1) or localization microscopy (Fig. 2). The fluorescence signal of the commercially available organic dye imaged in light microscopy can be correlated with an electron-dense marker suitable for electron microscopy (Fig. 3). For this, additional immuno-labelling is applied on section.

Both protocols described here allow the preservation of commercially available fluorescence dyes. Thus, molecules or structures of interest can be readily identified by attached fluorescence signals simplifying the most challenging step in correlative light and electron microscopy: The labelled object can be located and analysed in electron microscopic preparations using state-of-the-art light microscopic techniques. By this means, it is possible to visualize rare events in tissue volumes using small organic dyes and to study them in their ultrastructural context. Novel details of a generally known structure, such as the NMJ, can be uncovered in its cellular environment.

1. D. Robertson et al., J Micros. (1992).

2. S. Hillmer, C. Viotti, D.G. Robinson, J Micros. (2012).

We acknowledge financial support of the German Federal Ministry for Education and Research, project NanoCombine, grant no. FKZ: 13N11401, 13N11402.

Fig. 1: Light micrograph. 70 nm HM20 section on an indium tin oxide (ITO)-coated cover slip recorded with an oil immersion objective lens (100x/1.4 Plan Apochromat, Carl Zeiss). A: Fluorescence image. AChR labelled with BGT-AF555. B: Bright field image. C: Overlay. Scale bar: 10 µm.

Fig. 2: Correlation of high resolution localization light and scanning micrograph. A: Fluorescence light micrograph (AChR labelled with BGT-AF555) recorded with an oil immersion objective lens (100x/1.4 Plan Apochromat, Carl Zeiss). B: Scanning electron micrograph recorded with a Supra 40 (Carl Zeiss). C: Overlay. Scale bar: 1 µm.

Fig. 3: Transmission electron micrograph of an immuno-labelling of AChR stained with anti-AChR-alpha-antibody (BD Biosciences) and Protein A Gold (cmc). A: Overview. B: Magnification of the boxed region in A. Scale bars: 1 µm.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-1602 3D Complex Sample and Correlative Light Immuno-Electron Microscopy: a Way of Finding « The Needle in The Haystack »

Loussert C.1, Humbel B. M.1
1Electron Microscopy Facility, UNIL, bâtiment Biophore, Quartier Sorge, 1015 Lausanne, Switzerland
celine.loussert@unil.ch

Biological organisms show a complex organization, which is already observed at the cellular level. Analysing such multi-dimensional systems is not easy and biologists have to tackle the problem of what a specific cell or cell type plays in the organism. Imaging techniques, predominantly fluorescence microscopy, have been intensively employed to dissect the complexity of 3D samples, especially since the introduction of super-resolution microscopy techniques [1]. However, this set of methods has the disadvantages that only labelled structures can be studied in relation to each other and nm-sized organelles cannot be resolved [2]. On the other hand, electron microscopy has been the method of choice to observe the molecular organization of a sample with a high resolving power. Thus specific proteins can be localized and subsequently be related within the biological context. However, the field of view in the transmission electron microscope is limited to few hundred μm2. Combining a low magnification overview and localization capabilities of light/fluorescent microscopy with the high resolution of electron microscopy, i.e., correlative light electron microscopy, appears to be the most appropriate way to dissect the complexity of biological samples. To transfer a precisely located biological region, from the light to the electron microscope, we developed workflow based on the cryo-sectioning method developed by Tokuyasu [3] for the sample preparation. The first step is based on the 3D localization of the structures of interest in the entire sample on the fluorescent microscope. The position of the fluorescence labeled structures is recorded and used during cryo-microtomy step. In order to better control the cutting area, we modified our cryo-ultramicrotome by remplacing the classical binocular by a stereo-fluorescent binocular microscope. Thus we are able to visualize the fluorescence in the tissue bloc during trimming and sectioning. The second step is based on the acquisition of a precise 2D map of the fluorescence on the cryo-section  on the TEM grid. This map will be reused to guide the electron microscope during acquisition. Finally, the last step is the imaging, at high resolution:

-in the scanning electron microscope using a STEM detector to acquire large tiles

-in the transmission electron microscope to acquire tomograms of the area of interest.

Using this workflow, we were able to characterize at high-resolution a small biological feature, a “needle”, that was spatially localized and extracted from a significantly larger biological volume, the “haystack”. Different examples of tissues will be presented to highlight the advantages of this technique along with its wide range of potential applications.

[1] Huang, B., Babcock, H., Zhuang, X. Cell, 143, 1047-1058 (2010)
[2] Loussert, C., Forestier, C., Humbel, B. Methods in Cell Biology, 111, 59-72 (2012)
[3] Tokuyasu, K. Journal of Cell Biology, 57, 551-565 (1973)

We thank Dr. Liesbeth Hekking, Dr. Matthias Langhorst, and Mr Ben Lich; the Swiss National Foundation for the R’Equipe grant and the Faculty of Biology and Medicine, UNIL.

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-1813 Targeted Nano analysis of water and ions by Cryo-CLEM in HeLa Cells

NOLIN F.1,2, PLOTON D.2, WORTHAM L.1, LALUN N.2, MICHEL J.1
11. Laboratoire de Recherche en Nanosciences, EA4682, Université de Reims Champagne Ardenne, 21 rue Clément ADER, 51100 REIMS, 22. MeDYC, UMR CNRS 7369, Université de Reims Champagne Ardenne, 51 rue Cognacq Jay, 51100 REIMS
frederique.nolin@univ-reims.fr

Cryo-EM technique avoids chemical fixation and by the way its inherent artifacts allowing the visualization and analysis of samples in a close to native state. This technique has been coupled with fluorescence imaging (cryo-CLEM). We developed correlative cryo-analytical STEM, for mapping water by dark field imaging and ions by energy dispersive X-Ray spectrometry (EDXS) within compartment previously identified by GFP tagged proteins.

Stable transfected HeLa cells expressing H2B-GFP (allowing the identification of chromatin in the nucleus) are vitrified by rapid plunging into liquid ethane without cryoprotectant. In classical cryo-CLEM methods, the grid is first placed in a dedicated cryo-setup mounted on the stage of a fluorescence microscope for recording fluorescent images then transferred in the cryo-holder of the electron microscope for imaging. However, this method suffers from an important drawback due to the transfer of the grid which can induce: i) mechanical or thermal damages, ii) grid bending, contamination or even loss of the sample during transfer. We developed an original technical protocol whereby cryo-ultrathin sections (80nm) are placed on a formvar-carbon coated indexed grid, directly and definitely mounted on the Gatan EM cryo-holder, and successively imaged by cryo-fluorescence microscopy and cryo-electron microscopy (1).

In order to demonstrate the potentiality of our method we studied effects of a cytotoxic chemotherapy drug at 2 exposure levels :

*Actinomycin D at 50 ng/mL in order to induce a nucleolar stress to inhibit RNA synthesis. We map at the ultrastructural level water and elements. Cryo-CLEM allows us to measure them specifically in rich chromatin areas that are undistinguishable by ultrustructural morphology in STEM (Figure 1). By the way we have shown that nucleolar stress induce an increase of water and a large decrease of elemental contents in all cell compartments (2).

*Actinomycin D at 500 ng/mL in order to induce an apoptosis state. Live confocal microscopy experiments allowed to define six chronological steps for apoptosis. These steps, which are not always identified by ultrustructural morphology, are fortunately easy to recognize by fluorescence microscopy. So correlative cryo-analytical STEM allows us, for the first time, to correlate water and ions content evolutions in the different cell compartments as a function of apoptosis states.

Our original process has several advantages: i) it is universal ie valid for all labeled proteins and ii) it allows the targeted nanoanalysis of water and ions in connection with fluorescent labeling.

(1) Nolin et al., JSB 180 (2012) 352-362.

(2) Nolin et al., CMLS 70(13) (2013) 2383-2394.

We acknowledge the technical support from the PICT IBiSA imaging center of Reims University.

Fig. 1: Targeted nano analysis. A) Fluorescence image of a HeLa H2B-GFP cell cryosection. B) STEM DF image of the same cell after freeze-drying. C) Merge between the strongest fluorescence areas (red) identifying clumps of condensed chromatin and the STEM image. So we identify clumps of chromatin on STEM image and we are able to target them for EDXS (D).
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-2003 Correlative Microscopy in Materials Science and Biology: TEM-SIMS based Parallel Ion Electron Spectrometry (PIES) for High-Resolution, High-Sensitivity Elemental Mapping

Eswara Moorthy S.1, Dowsett D.1, Wirtz T.1
1Department “Science and Analysis of Materials” (SAM), Centre de Recherche Public – Gabriel Lippmann, 41 rue du Brill, L-4422 Belvaux, Luxembourg
eswara@lippmann.lu

Addressing the urgent need to simultaneously acquire images with both high spatial resolution and high chemical sensitivity is of paramount importance to gain deeper understanding in physical and biological sciences. The Transmission Electron Microscopy (TEM) offers superior spatial resolution, but, the traditional analytical capabilities associated with electron microscopy such as the Energy Dispersive Spectroscopy (EDS) or Electron Energy-Loss Spectroscopy (EELS) are unfortunately inadequate for characterizing samples containing trace elements (at best 0.1 at. %) or for mapping isotopic distributions. On the other hand, Secondary Ion Mass Spectrometry (SIMS) provides extraordinary chemical sensitivity (down to ppm or even ppb) and high dynamic range, but, offers poor lateral resolution. An ex-situ combination of TEM and SIMS in an attempt to overcome the limitations of the techniques taken individually is prone to sample modifications and artefacts [1]. To overcome the intrinsic instrumental limitations, we have made an in-situ combination to complement the high-sensitivity of SIMS with the exceptional spatial resolution offered by TEM, by developing the correlative TEM-SIMS technique.

To determine the feasibility and to demonstrate the applications of the TEM-SIMS method, we have developed a prototype instrument for TEM-SIMS based correlative microscopy (Fig 1). The pole-pieces of a Tecnai F20 were specially modified to accommodate the SIMS technique. A FEI Magnum Ga+ FIB was attached to the TEM column to act as the primary ion column. The secondary ion extraction optics (extraction efficiency 90%) and a compact high-performance mass spectrometer were designed and developed in-house and are being continuously improved for optimal performance. A special sample holder which can be biased to high-voltages (±4.5 kV) was also developed in-house to enhance the collection efficiency of the secondary-ion extraction optics.

To enhance the low intrinsic yield of secondary ions for non-reactive primary ion beams such as Ga+ for the TEM-SIMS we use reactive gas flooding [2]. Specifically, the enhancement of negative secondary ion yields due to Cs flooding and of positive secondary ion yields with O2 flooding were found to be up to four orders-of-magnitude. This enhancement of secondary ion yields leads to detection limits varying from 10-3 to 10-6 for a lateral resolution between 10 nm and 100 nm respectively (Fig 2).

The utility of the TEM-SIMS based correlative method will be demonstrated with the example of samples containing low Z elements, which are particularly challenging with traditional analytical methods like EDS or EELS.


References:
[1] K. Q. Ngo et al, Surf. Sci. 606, (2012) 1244.
[2] P. Philipp et al, Int. J. Mass. Spectrom. 253 (2006) 71

Fig. 1: Schematic of the TEM-SIMS setup (left) and the photo of the TEM-SIMS prototype instrument (right).

Fig. 2: Detection limit using a Ga+ FIB with and without Cs0 flooding vs. minimum feature size: example for the detection of Si-.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-2404 Correlative super-resolution and electron tomography: A method to study colorectal cancer cells.

Cheng D.1, Henriquez J.1, Huynh M.2, Shami G.1, Braet F.1,2
1School of Medical Sciences (Discipline of Anatomy and Histology) — The Bosch Institute, The University of Sydney, NSW 2006, Australia, 2Australian Centre for Microscopy & Microanalysis, The University of Sydney, NSW 2006, Australia
delfine.cheng@sydney.edu.au

Recent developments in light/laser microscopy are pushing spatial resolution far beyond the limits of optical diffraction, ever closer to the resolving power of electron microscopy. Several studies have reported using optical super-resolution imaging techniques to observe fluorescently labelled subcellular details at the lateral resolving power of 50-75 nm.Depending on the employed optical technique, the resolving power can be pushed even further to the extreme range of 25-30 nm. Even though this range of resolution (close to the single-molecule resolution)makes the optical techniques relevant in the observation of cellular structure at the supramolecular level, transmission electron microscopy is still an inevitable technique for ultrastructural details at the nanometer scale. Hence, the Correlative Light and Electron Microscopy (CLEM) concept, which combines data collected by light/laser microscope with data collected by an electron microscope, is becoming increasingly popular. The CLEM concept has been utilised only in the recent years with the technical development in the microscopy industry.

In the present study, cultured Caco2 cells were used as model. The cells were double-labelled with GM1 for membrane rafts and Phalloidin/AF647 for actin filaments. The labelled structures were correlated with Transmission Electron Microscopy (TEM) and Tomography (TET). The decoration of actin filaments was used to assess the detection limit of different configurations of actin via ground state depletion-based super-resolution microscopy (GSD-SR). Furthermore, by applying CLEM, the direct comparison of optical data with TET data was achieved in the regions previously imaged via GSD-SR.

3-D CLEM arrays of membrane rafts and cellular actin obtained via GSD-SR and TEM or TET will be presented. A practice workflow for the sample preparation and data analysis for correlative super-resolution studies will be discussed. This study will be supported by quantitative measurements of the actin fibres from directly comparing GSD data with morphometric values from the TET models. Data on the number of fibres, their diameter, including their configuration will be discussed.

It is shown that CLEM, is a relevant tool in the study of dynamic or non-dynamic structures, allowing for the observation of fluorescently labelled structures at the EM level. When combined with super-resolution microscopy, the difference in resolving power between optical and electron microscopy is further closed down.

The authors acknowledge the facilities and the technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Australian Centre for Microscopy & Microanalysis at the University of Sydney, which houses the GSD-SR microscope.

Fig. 1: Wide-field image, TEM mosaic images,  GSD-SR image and TET model of actin filaments from the same area of cultured colorectal cancer cells. The large box represents the area imaged using GDS-SR. The smaller boxes represent the areas imaged using TET.

Fig. 2: Schematic of image analysis workflow for evaluating the resolving power of the two techniques; Ground State Depletion-Super Resolution (GSD-SR) and Wide-Field (WF) fluorescence microscopy.

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-2309 Viewing in 3D macromolecular nanomachines in their cellular and functional context

Hanein D.1, Anderson K. L.1, Volkmann N.1
1Sanford Burnham Medical Research Institute
dorit@burnham.org

Our efforts focus on developing imaging technology platform that quantitatively link macroscopic cellular outputs to molecular-resolution structural changes in the crowded cellular environment. Our central biological interest is the structures of actin cytoskeleton macromolecular assemblies; the girders and cables that control the shapes and movements of all living cells. The anchoring sites of the girders, between the cell and the extracellular matrix or other cells, are mechano-sensitive multi-protein assemblies that transmit force across the cell membrane and regulate biochemical signals in response to changes in the mechanical environment. The combined functions in force transduction, signaling and mechanosensing are crucial for cell and tissue behaviors in development, homeostasis and disease.

The heterogeneity and dynamic nature of the multi-component complexes responsible for the transmission of force between the actin cytoskeleton and integrin receptors in migrating and mechano-active cells have hindered structural studies aimed at unrevealing the underlying the molecular mechanisms of their assembly and disassembly. A large body of work carried out over the last two decades has identified key components that directly link the actomyosin system to the extracellular domain (via integrin). Here I will describe how a hybrid approach that involves a combination of correlative light and electron microscopy, cellular cryo-electron tomography, image analysis, computational docking, and biophysical tools allows us to “ “directly view” the 3D the molecular architecture of these nanomachinery in situ.

Recently, we acquired cellular cryo tomograms of whole mammalian cells using intermediate voltage TEMs equipped with various imaging devices / energy filter configurations and employed drift movie correction in conjunction with feature extraction to extend these studies and view single membrane receptors at the cell membrane extracellular matrix interface. here I will describe these results.

These studies are supported by the National Institute of General Sciences (NIGMS) Grant Number P01 GM098412 (DH and NV).

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-2618 Intravital correlative microscopy reveals tumor cell behavior in vivo at high resolution in three dimensions

Karreman M. A.1, Mercier L.2, Schieber N. L.1, Schwab Y.1, Goetz J. G.2
1European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, 2INSERM U1109, Strasbourg, France
matthia.karreman@embl.de

The main cause of cancer mortality is not the primary tumor, but metastasis: the spreading and colonization of cancer cells at a distant site. The major events in cancer metastasis involve detachment of cells from the tumor (invasion), intrusion into vessels (intravasation), circulation, exit from the vessel, and secondary site colonization. Metastatic processes involve structural and functional transformations to the invading cell, and also the tumor microenvironment is found to be an active member of the complex cellular ecosystem shaping cancer progression.

There is a strong need of the cancer research community to gain more insight into metastatic processes in vivo. Whereas fluorescence microscopy offers intravital imaging of cancer cells in tissue, it suffers from a limited resolution and is restricted to imaging of fluorescent probes. Electron microscopy, on the other hand, reveals the complete architecture of the region of interest (ROI) at the ultrastructural level. Combining these two imaging modalities results in a tool that correlates the dynamic and functional recordings of tumorigenic events in vivo to the sample’s most-detailed ultrastructure.

We have recently developed a correlative microscopy workflow that complements the advantages of intravital two-photon excitation microscopy (2PEM) of murine tumor xenografts, with volume electron microscopy (EM). Fluorescently labeled cancer cells are injected subcutaneously into a mouse ear and imaged using 2PEM. Using near-infrared branding, the position of areas of interest within the sample is labeled at the skin level, allowing for their full preservation. Concerted usage of these artificial brandings and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. Full volume correlation is then performed between the 2PEM and EM datasets. Upon retrieval of the cancer cells, their structure and microenvironment could be revealed in 3D at high resolution through electron tomography. Our approach correlates intravital microscopy to 3D electron microscopy, uniquely demonstrating in vivo formation of invasive protrusions in cancer cells and enabling visualization of cell-matrix contacts. This study therefore provides unique and unprecedented insights into tumorigenic processes, which could benefit to the cancer research community.

We thank M. Koch and P. Kessler, IGBMC imaging platform, D. Hentsch, S. Taubert and F. Egilmez and the animal facility at INSERM U1109. We are greatful to W. Hagen, R. Mellwig and the Electron Microscopy Core Facility of the EMBL Heidelberg.

Fig. 1: (A,B) Fluorescent cancer cells were xenotransplanted into mouse ear skin and imaged with intravital 2PEM. (C) An invasive cancer cells was retrieved with electron microscopy and the structural organization of invasive protrusions were revealed with electron tomography (D). Scale bars are 100 µm in B, 5 µm in C and 500 nm in D.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-3039 Quantifying Cellular Uptake of NanoDiamonds through Cathodoluminescence Spectral Imaging in a STEM

Nagarajan S.1, Tizei L H. G.1, Bertrand J. R.2, Durieu C.3, Cam E. L.3, Chang H. C.4, Treussart F.5, Kociak M.1
1Laboratoire de Physique des Solides, CNRS UMR8502, Université Paris Sud XI, 91405 Orsay, France, 2Institut Gustave Roussy, Laboratoire de Vectorologie et Thérapeutiques Anticancéreuses, 94805 Villejuif, France, 3Signalisations, Noyaux et Innovations en Cancérologie, Université Paris Sud 11, Institut Gustave Roussy, Villejuif, France, 4Institute of Atomic and Molecular Sciences, Academia Sinica, Taiwan, Taipei 106 , 5Laboratoire Aimé Cotton, CNRS, Université Paris Sud and ENS Cachan, 91405 Orsay, France
sounderya.nagarajan@u-psud.fr

Nanodiamonds (NDs) have been used for biological application in recent times. They demonstrate high Photoluminescence (PL) which can be tuned by modifying the type of defects in the NDs. For example, neutral defect Nitrogen Vacancy (NV0) have a characteristic red emission while neutral defect Nitrogen Vacancy Nitrogen (H3) have a characteristing green emission. These materials also emit light upon excitation under an electron beam which enables imaging using Cathodoluminescence (CL). This has been used to image cells with NDs in a Scanning Electron Microscope (SEM). But this method does not allow high resolution imaging and cannot be used to identify sub cellular structures. Here, the use of the CL system on a Scanning Transmission Electron Microscope (STEM)  to visualize the ND localization in mamalian cell will be discussed. The use of a STEM allows visualizing the ultrastructure of organelles in a cell and the periphery of the regions where the NDs localize after cellular uptake. The detection of CL signal from the NDs allows using multiple emitters (with distinct emissions) simultaneously. The important aspect of this imaging is that the bright field and the dark field images are aquired alongside the CL spectral image (pixel by pixel spectral data) which can be used to correlate the image of the subcellular structure and quantifying the NDs. In this study, NV0 and H3 NDs were modified with two cationic polymers commonly used in gene delivery. Cells were incubated with these NDs ,rinsed in Phosphate Buffered Saline and embedded/sectioned in epoxy resin. The ultrathin sections were loaded on collodion coated copper grids and imaged on the CL-STEM at -140oC. It was possible using CL to identify the type of polymer on the NDs and also to quantify the number of luminescing NDs in the vesicle.

Fig. 1: Bright field Images of NV0-ND and H3-ND localized in cell vesicles overlapped with the corresponding emission components extracted from the spectral image (A,C) and the respective dark field images (C,D). Scale bar : 500 nm
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-2747 Correlative Light and Electron Microscopy Without Fluorescent Probes

Arkill K. P.1, Payne L. M.2, Masia F.2, Mantell J. M.3, Langbein W.4, Borri P.2, Verkade P.1
1School of Biochemistry, University of Bristol, UK, 2School of Biosciences, Cardiff University, UK, 3Wolfson Bioimaging Centre, University of Bristol, UK, 4School of Physics and Astronomy, Cardiff University, UK
kenton.arkill@bristol.ac.uk

Correlative Light and Electron Microscopy (CLEM) combines the optical capability of imaging dynamic physiological specimens with the nanoscale three dimensional resolution of electron microscopy. Most methods for CLEM use a metal nanoparticle (mNP) bound to a fluorescent marker (Fig. 1), but the fluorescence is often quenched in the EM prep meaning that preparation artefacts can not be checked. Presented here is a novel Correlative Microscopy approach that integrates Four Wave Mixing (FWM) microscopy into a CLEM experiment only using the gold nanoparticle tag.

FWM is a multiphoton technique which exploits the nonlinear optical response of mNPs at their surface plasmon resonance (SPR) to image these mNPs as absorbers, rather than fluorescence emitters. This technique has high sensitivity, photo-stability and depth accuracy, and in addition is also background free (Fig. 2). Additional advantages are that the absorption is linear to the number of particles in the voxel, and shifts in the peak can be used to detect inter-particle distances at a macro-molecular precision. Here we demonstrate this concept by combining FWM with standard fluorescent-mNP CLEM and comment on any artefact from high pressure freezing and Lowicryl embedding.

HeLa cells were grown on 1.48mm diameter sapphire disks (with a carbon finder grids) glued into a homemade carrier suitable for high pressure freezing (Leica EMPACT2 + rapid transfer system). The cells were serum starved and then incubated (20min) in Epidermal Growth Factor-biotin bound to Alexa Fluor 488 streptavidin – 10nm colloidal gold. One cell of interest on each disk was then imaged using a confocal microscope system, high pressure frozen and Lowicryl embedded at low temperature (without additional metal staining). The embedded blocks had the sapphire disk carefully removed and were trimmed as if for EM. The 1st and 2nd 5µm sections containing the cells, placed in glycerol between a glass slide and coverslip, were imaged with FWM. After FWM imaging the sections were washed, remounted, thin sectioned and imaged with EM.

The preliminary results demonstrate the viability of FWM as a future standard CLEM technique and for testing aretfact in fluorescently labelled CLEM techniques in general.

Funding: BBI solutions and EPSRC

Fig. 1: CLEM example. HeLa Cells have internalised both EGF-Alexa488-10nm gold and Tf-Alexa594-5nm gold. Light microscopy fluorescence (left) shows an overview of the cells, One cell and structure inside can be processed and analysed at higher resolution for electron microscopy.

Fig. 2: HepG2 cells having the Golgi apparatus immunostained with GM130-Alexa Fluor 488 and 10nm gold nanoparticles (Masia et al, Opt. Lett. 34, 1816 (2009)). A) Phase contrast  B) Epi-fluorescence C) overlay and D)  Four wave mixing micrograph of a single cell
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-2760 CORRELATIVE SUPER-RESOLUTION FLUORESCENCE AND ELECTRON MICROSCOPY IN RESIN EMBEDDED CELLS USING STANDARD FLUORESCENT PROTEINS

Kaufmann R.1,2, Johnson E.3, Seiradake E.1, Davis I.2, Grünewald K.1
1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK, 2Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK, 3Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
errin.johnson@path.ox.ac.uk

In recent years various super-resolution techniques have been developed in the field of fluorescence microscopy (FM) to overcome the problem of diffraction limited resolution. Single molecule localization microscopy, which is based on the switching of fluorophores between two different states, allows fluorescently-labelled structures to be resolved down to the 10 nm range. When the same cell is imaged using transmission electron microscopy (TEM), such structures can be correlated to ultrastructure with a much higher degree of precision and detail compared to conventional FM. It is advantageous to perform correlative light and electron microscopy (CLEM) with the final resin sections, rather than performing the localization microscopy prior to embedding and sectioning, as at this resolution the TEM sample preparation steps induce visible structural and spatial changes in the sample that affect the quality of correlation. Therefore, the challenge is to maintain not only the fluorescence itself throughout the TEM sample preparation procedure, but also the switching capability of the fluorophore necessary for localization microscopy, whilst simultaneously preserving cellular ultrastructure and introducing sufficient contrast for TEM imaging.

We have compared and modified a range of different freeze substitution protocols after high-pressure freezing to optimise the TEM sample preparation protocol for performing single molecule localization microscopy of standard fluorescent proteins in ultrathin resin sections followed by TEM imaging. We present examples of correlative super-resolution in-resin fluorescence and TEM images of biological structures labelled with standard fluorescent proteins (Fig 1), and will discuss factors (such as mounting media and cryo-protectant) which influence the in-resin switching ability of the fluorophore. Using this method we achieved a structural resolution of ~ 50 nm in the FM images (mean single molecule localization accuracy: 21 nm). Not having to use special photo-activatable or photo-switchable dyes has proven extremely useful in the field of non-correlative localization microscopy in chemically fixed samples as it simplifies the fluorescent labelling procedure, making it more accessible to a wider range of researchers. We envisage that the presented method will give super-resolution CLEM a similar push forward.

Fig. 1: Overlay of FM (green) and TEM (grey) image. A: conventional FM. B: super-resolution FM image.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-2868 Correlated Optical and Isotopic Nanoscopy

Saka S. K.1, Vogts A.2, Kröhnert K.1, Hillion F.3, Wessels J.4, Rizzoli S. O.1
1Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, and and Centre for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany, 2Leibniz-Institute for Baltic Sea Research, Rostock, Germany. , 3Cameca, Gennevilliers, France, 4University of Göttingen Medical Center, Göttingen, Germany
ssaka@gwdg.de

The isotopic composition of different materials can be imaged by secondary ion mass spectrometry (SIMS). In biology SIMS is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes (15N, 13C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100 nm1. However, secondary ion mass spectrometry cannot identify specific subcellular structures like organelles, and needs to be correlated with a second technique, such as fluorescence imaging. We present here a method based on stimulated emission depletion (STED) microscopy2 that provides correlated optical and isotopic nanoscopy (COIN) images. We use this approach to study the protein turnover in different organelles from cultured hippocampal neurons. Correlated optical and isotopic nanoscopy can be applied to a variety of biological samples, and should therefore enable the investigation of the isotopic composition of many organelles and sub-cellular structures3.

References: 

1. Lechene, C. et al. High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry. J Biol 5, 1–30 (2006).

2. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19, 780–782 (1994).

3. Saka, S. K., Vogts, A., Kröhnert, K., Hillion, F., Rizzoli, S.O., Wessels, J. Correlated Optical Isotopic Nanoscopy. In press (Nature Communications).

The SIMS instrument was funded by the German Federal Ministry of Education and Research, grant identifier 03F0626A. SKS was supported by a Boehringer Ingelheim Fonds PhD Fellowship. The work was supported by grants to SOR from the German Research Foundation (grant identifiers RI 1967/2-1, RI 1967/3-1 and the Collaborative Research Center, Cellular Mechanisms of Sensory Processing, SFB 899).

Fig. 1: Correlated STED and SIMS imaging in neuronal cell bodies
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-1-O-3440 Automatic registration method to combine image sets from optical microscopy and SEM

da Fonseca Martins Gomes O.1, Galdino de Lima M. P.2, Abelha Mota G. L.2
1CETEM - Centre for Mineral Technology, 2Dept. of Informatics and Computer Science, Rio de Janeiro State University (UERJ)
ogomes@gmail.com

The combination of optical microscopy and SEM may improve their analytical capacity. However, the registration of optical and SEM images consists of a complex task, it requires corrections of translation, rotation, and non-linear and local distortions. Gomes & Paciornik [1] developed a method for this purpose that employs a standard sample for calibration. However, although this method is effective, it is not practical.
This work presents a new registration method to automatically combine image sets from optical microscope and SEM without the need of a calibration step. It was compared with different registration methods present in the literature. A set of 81 pairs of images from an iron ore sample was used for tests. The correlation coefficient was employed to evaluate the quality of registration.
The proposed method involves three stages. First, each pair of images is aligned, through cross-correlation, and then cropped to have the same size. Following, the images from SEM are registered using a transformation computed by Local Weighted Mean (LWM) [2]. At final, all images are cropped in order to represent the same field.
All image pairs are used to obtain the control points that define the transformation. They are partitioned in small sub-images, and the moments of translation of each pair are measured, determining control points covering the whole field.
Figure 1 presents the correlation coefficient for the 81 image pairs measured from (i) the acquired images; and the images registered by (ii) the method with calibration [1], (iii) SIFT [3] and Affine [4], and (iv) the new automatic method. Figure 2 shows the same results and the results obtained for the registration with bUnwarpJ [5], using a different scaling to compare them.
The lower results obtained for the original images and for the registration with Affine evidence the need of more complex transformations than a simple alignment or even rigid-body ones.
The bUnwarpJ method achieved high results for most image pairs. However, it showed up unstable, it distorted much many images, making them practically unrecognizable.
The results for the registration with calibration and for the new automatic method were similar. In fact, the new method, besides more practical, was capable of providing the best registration results.

References
[1] O.D.M. Gomes & S. Paciornik, “Multimodal Microscopy for Ore Characterization”, Scanning Electron Microscopy, InTech, Rijeka, 2012.
[2] A. Goshtasby, Image Vision Comput, 6 (1988) 255.
[3] D.G. Lowe, Int J Comput Vision, 60 (2004) 91.
[4] B. Zitova & J. Flusser, Image Vision Comput, 21 (2003) 977.
[5] I. Arganda-Carreras et al, “Consistent and Elastic Registration of Histological Sections using Vector-Spline Regularization”, Lecture Notes in Computer Science, Springer, 2006.

The authors acknowledge the support of Brazilian funding agencies CNPq, CAPES and FAPERJ.

Fig. 1: Correlation coefficient measured from the acquired images; and the images registered by the method with calibration; SIFT and Affine; and the new automatic method.

Fig. 2: Correlation coefficient measured from the acquired images; and the images registered by the method with calibration; SIFT and Affine; the new automatic method; and bUnwarpJ.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-1469 Morphology and protein composition of porcine erythrocyte ghosts altered by different buffers

Kostić I. T.1, Bukara K. M.1, Ilić V. L.2, Veljović Đ. N.1, Bugarski B. M.1
1Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11060 Belgrade, Serbia, 2Institute for Medical Research, University of Belgrade, Dr Subotica 4 (KCS) POB 39, 11120 Belgrade, Serbia
ikostic@tmf.bg.ac.rs

Recently, we reported isolation of erythrocyte ghosts from bovine slaughterhouse blood and their characterization from the aspect of morphology and structural integrity [1]. Detailed insight into ghosts morphology obtained by FE-SEM, showed slightly distortion from erythrocyte shape, an altered surface texture with increased bilayer curvature and existence of invaginations [1], probably due to impact of used hypotonic PBS buffer [2]. We extended our study to preparation of ghosts from porcine slaughterhouse blood in the presence of different buffers and correlated obtained morphology by FE-SEM with analysis of the protein retention by SDS-PAGE.
Erythrocyte ghosts from porcine blood were manufactured by hypotonic hemolysis with 35 and 65 mM sodium-phosphate/NaCl buffers, as well as 10 mM HEPES buffer and 10 mM HEPES buffer including Mg2+ (2.5 mM of MgCl2). PBS was used as resealing medium for all types of preparations. The samples for scanning electron microscopy were prepared as previously described [1] and visualized on a field emission scanning electron microscope (FE-SEM), a TESCAN MIRA 3 XMU, operated at 10 kV. Protein characterization of erythrocyte ghosts was performed by SDS PAGE procedure in 0.75 mm thick, 12% w/w polyacrylamide gel under reducing condition.
Figure 1 of FE-SEM micrographs demonstrates that different experimental conditions produced different morphological characteristics among the ghost preparations. Namely, ghost preparations obtained with 65 mM sodium-phosphate/NaCl buffer (Fig. 1b) and 10 mM HEPES buffer with 2.5 mM Mg2+ (Fig 1d) revealed at least altered surface texture. The existence of invaginations was the most pronounced in preparation obtained with 35 mM sodium-phosphate/NaCl buffer (Fig. 1a). Addition of Mg2+, led to morphology improvement and size uniformity, as it can be seen from Fig. 1d compared to Fig. 1c.
As shown in Figure 2, the SDS-PAGE analysis revealed in all erythrocyte ghosts preparations most of the major protein fractions characteristic for the intact erythrocyte membranes (marked on the left side). Spectrin, which forms the meshwork that provides animal erythrocyte shape, was slightly depleted in ghost preparation obtained with 35 mM and 65 mM phosphate buffer.
Since only slight differences in relative contents of membrane proteins in different ghost preparations were observed, the morphological alterations of porcine erythrocyte ghosts demonstrated by FE-SEM may be related to the changes of transbilayer lipid asymmetry [2].

1. Kostic I, Bukara K, Ilic V, Mojsilović S., Đorđević V., Isailović B. ,Veljović Đ., Bugarski B., Proceedings of the Microscopy Conference, Regensburg, Germany, August 25-30, 2013, MIM.4. P066, pp 520-521.
2. F.M. Harris, S.K. Smith, J.D. Bell, J. Biol. Chem. 276 (2001) pp 22722-31.

This research was funded by grants III46010 from the Ministry of Education, Science and Technological Development, Republic of Serbia.

Fig. 1: FE-SEM micrographs of porcine erythrocyte ghosts prepared in (a) 35 mM and (b) 65 mM sodium-phosphate/NaCl buffer, (c) 10 mM HEPES buffer and (d) 10 mM HEPES buffer with 2.5 mM MgCl2. The ghosts were obtained by hypotonic hemolysis followed by restoration of isotonicity.

Fig. 2: Protein retention of different porcine erythrocyte ghosts preparations. Lane 1 and Lane 2- ghosts obtained with 35 and 65 mM sodium-phosphate/NaCl buffers, respectively. Lane 3 and Lane 4 - ghosts obtained with 10 mM HEPES and 10 mM HEPES with 2.5 mM Mg2+, respectively.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-1470 Preparation of erythrocyte ghosts from slaughterhouse blood for AFM observation

Kostić I. T.1, Bukara K. M.1, lić V. L.2, Ralević U. M.3, Bugarski B. M.1
1Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11060 Belgrade, Serbia, 2Institute for Medical Research, University of Belgrade, Dr Subotica 4 POB 39, 11120 Belgrade, Serbia, 3Institute of Physics, University of Belgrade, Pregrevica 118, 11080 Zemun, Belgrade, Serbia
ikostic@tmf.bg.ac.rs

Recently, we described optimized process of gradual hemolysis for isolation of erythrocyte ghosts from slaughterhouse blood [1], which can be used for their drug loading as well. Future studies regarding the encapsulation of drugs in the ghosts will require powerful microscopy technique, such as atomic force microscopy (AFM), to provide information of relevance to cellular biophysical chemistry research. For this reason, two drying methods were examined to optimize the preparation of bovine and porcine erythrocyte and empty ghosts for AFM observation.
In order to preserve lipid and protein composition of erythrocytes and isolated ghosts, the samples were prepared for AFM by introducing next protocol: 500 µL of erythrocytes and ghosts were allowed to settle onto poly-L-lysine-coated cover glasses at 4°C overnight. Samples were fixed in 2% glutaraldehyde in PBS and 2% osmium tetroxide for 2h each, washed and dehydrated through a graded series of ethanol solutions (10, 30, 50, 70, 95 and 100%) for ten minutes each. The second procedure differed in adding dehydratation step consisting of three times washing in acetone. AFM measurements were performed in semi-contact mode at room temperature and under ambient conditions, using the NTEGRA Prima system from NT-MDT (NT-MDT Co. Moscow, Russia). NSG01 probes from NT-MDT with a typical tip curvature radius of about 6 nm and a typical force constant of 5 N /m were used.
For both samples of erythrocytes and ghosts, the preparation procedure without final dehydratation in acetone, could not allow AFM observation. Possibly, remained water induced erythrocyte membrane skeleton thermal fluctuation and/or it was deformed with the force applied by the AFM probe [2]. AFM topography images of dried bovine samples (Fig. 1 a, b) prepared with second procedure confirmed the results on the morphology and size of ghosts obtained by FE-­SEM [1]. Fig. 1 (c, d) showed the altered surface texture with invaginations in porcine ghosts as well, probably formed due to isolation process. Besides, AFM of porcine ghosts revealed the fraction of lipoprotein deposits having diameter of ~200 nm (Fig. 1 (e, f)). These fractions were not observed in the sample of bovine ghosts. Successfully demonstrated preparation procedure for ghosts AFM observation will further provide more significant qualitative and quantitative AFM measurements of single ghost mechanics, especially drug loaded ghosts, by acquisition of force-deformation profiles and extraction of Young’s moduli.

1. Kostic I, Bukara K, Ilic V, Mojsilović S., Đorđević V., Isailović B. ,Veljović Đ., Bugarski B., Proceedings of the MC 2013, Regensburg, Germany, August 25-30, 2013, MIM.4.P066, pp 520-521.
2. Takeuchi M, Miyamoto H, Sako Y, Komizu H, Kusumi A., Biophys J. (1998) pp 2171-2183.

This research was funded by grants III46010 from the Ministry of Education, Science and Technological Development, Republic of Serbia.

Fig. 1: Three-dimensional AFM images of erythrocytes and resulting ghosts attached to glass surfaces: (a) erythrocytes from bovine and (c) porcine slaughterhouse blood (b) resulting bovine and (d) porcine erythrocyte ghosts (e) fragments of lipoprotein nature derived from porcine erythrocyte ghosts (f) enlarged fragments given in (e)
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-1854 From millimetres to microns with large area EDS mapping on biological samples

Collins C. L.1, McCarthy C.1, Rowlands N.1
1Oxford Instruments Nanoanalysis
clair.collins@oxinst.com

Energy Dispersive Spectroscopy (EDS) has been used for years to analyse the chemical composition of materials, including biological matter. New large area SDD detectors offer analysts the opportunity to gather up to 15 times the counts achieved on an older 10mm2 detectors without changing any other collection conditions.

Now large area mapping (LAM) software allows analysts to map a whole sample in just one run. The analysis area is defined, the EDS map resolution and magnification set and then individual fields are mapped. When all the fields have been analysed, they are montaged into a single image which can be magnified to see details that are invisible in the larger map.

Here, we present results from a biological sample, analysed with a Oxford Instruments X-MaxN 150mm2 SDD detector at 4kV on a TESCAN Mira FEGSEM. Figure 1 shows a wheat seed which has been embedded in resin and stained with OsO4 to highlight the fatty acids surrounding the phospho-rich structures in the aleurone cells. The Os Mα and P Kα peaks overlap (Figure 2) but can be deconvoluted with ‘TruMap’ software to ensure accurate maps are obtained. Figure 3 shows the results achieved with a 10mm2 EDS detector (Fig. 3a) compared to those taken on an X-MaxN 150mm2 EDS detector (Figs. 3b & 3c).

The X-MaxN data is clearly superior in both counts & image definition. The oily bodies are clearly delineated in the Os map and easily identified as separate structures from the P-rich areas. Counting for longer yields improved signal to noise and offers sufficient structure in the Os map to enable individual oily bodies to be identified (Fig. 3c). Combining the maps into a single layered image (Fig. 3d) clearly illustrates the elemental distributions found across the sample.

Figure 4 shows a 122 individual EDS maps (each 250μm by 250μm) montaged together to make a large area map showing the chemical variation across the whole sample - a total area of 3mm by 3.5mm. The LAM can be interrogated after collection and individual maps magnified to illustrate points of interest as required.

Conclusions:

New large area SDD EDS detectors offers biological analysts a new way of collecting important information about their samples. Improved collection efficiencies mean higher count rates without changing the SEM operating conditions and large area EDS mapping allows data collection across a wide field of view.  All while retaining the detail of individual maps. Finally, truly informative EDS data across a range of scales can be collected on biological or beam sensitive materials without compromising the sample.

References:

[1] “A to Z of Technology”, S Burgess et al, Oxford Instruments. M&M 2011

[2] “High Throughput, High Quality Analysis in the EM”; A Hyde et al, Oxford Instruments, M&M 2013

The authors would like to thank Jean Devonshire of Rothamsted Research, UK for providing the wheat sample for analysis as well as the SEM image seen in Figure 1.

Fig. 1: SEM image showing the internal structure of a wheat aleurone cell.

Fig. 2: EDS sum spectrum of a wheat seed showing the overlap between Os Mα & P Kα.

Fig. 3: Comparison of P Kα and Os Mα wheat seed EDS maps taken on a 10mm2 EDS detector and 150mm2 X-MaxN SDD detector.

Fig. 4: LAM montage of 122 individual EDS maps illustrating thechemical composition of the whole wheat seed from micron scale to millimeterscale. Montaged maps include: BSE Image, C Kα, Ca Lα, Os Mα. Bottom right image shows a highlymagnified section of the Os Mα map.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-1955 Workflow for correlative light and electron microscopy

Hosogi N.1, Nishioka H.1
1EM Application department JEOL Ltd.
nhosogi@jeol.co.jp

 Temporal behavior or interaction of biomolecules is generally observed by a light microscope (LM). However, resolution of the LM is insufficient to resolve fine structural details. Although the location of target molecules can be recognized by fluorescent labeling with a fluorescent microscope (FM), other molecules or structures which are unlabeled cannot be observed in detail by an LM. In contrast, a transmission electron microscope (TEM) has much higher spatial resolution than LM. However, disadvantages of TEM include inability to observe dynamics of specimens with bioactivity and paucity of labeling methods. Correlative light and electron microscopy (CLEM) is a bridge between the two microscopic methods. The CLEM gives us advantages of both TEM and LM which are a broad overview of the specimen and temporal events with LM and high-resolution information about the specimen with TEM.

 We developed a workflow for the CLEM. The workflow utilizes a piece of software named “picture overlay program”, which shares the coordinates of TEM and LM images. The software shares the coordinates automatically, with indication of the two corresponding points in each image. The correlation of LM and TEM, which is optimized for observation of a biological sample, is realized after the sharing. The TEM used for our experiments was chosen to be JEM-1400Plus (JEOL Ltd., Japan). The correlation is realized by using a function named “stage navigation”. The “stage navigation” is realized with the “ultra low-magnification” and easy-to-use specimen drive method named “point & shoot”. The lowest magnification for the microscope is 10 x. With that magnification, a whole TEM grid can be observed in a single field of view. “Point & shoot” is a method which points the next desired destination by a mouse click and makes the point to the center of the field of view by the specimen motor-driving system of the TEM stage.

 In the workflow, first, a coordinates of LM image is shared with the corresponding ultra low-magnification TEM image by the “picture overlay program”. After the sharing of the coordinates, one can center a region as one desires by the “point & shoot” (Fig.1). And then, users can increase the magnification to see a detailed structure of the specimen. In addition, the “picture overlay program” can save a multi-layer image (Fig.2). The each layer has the detailed structural information by TEM or functional information by FM or LM. Thus, by TEM, users can observe regions of interest which are marked with LM and/or FM with high resolution. This workflow is helpful for CLEM applications.

Fig. 1: Screenshot of windows for CLEM, running software: “picture overlay program” on a TEM operation monitor. TEM and LM images are displayed simultaneously. The software superimposes the images and shares their coordinates of them. With the shared coordinates, the TEM stage can move automatically to a region of interest found in the LM image.

Fig. 2: Superimposed images of LM, FM and TEM by using the “picture overlay program”. The specimen were chemically fixed and stained by Hoechst 33342.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-1979 Correlative microscopy characterization of the interaction of magnetic nanoparticles with breast cancer cells by Soft X-ray tomography, epi-fluorescent optical and transmission electron microscopy.

Chiappi M.1, Chichón F. J.1, Conesa J. J.1, Pereiro E.2, Rodríguez M. J.1, Carrascosa J. L.1, 3
1Centro Nacional de Biotecnología (CNB-CSIC), Cantoblanco, 28049 Madrid, Spain. , 2ALBA Synchrotron Light Source. MISTRAL Beamline - Experiments division. 08290 Cerdanyola del Vallès, Barcelona, Spain., 3Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia), Cantoblanco, 28049 Madrid, Spain.
jlcarras@cnb.csic.es

We have analyzed the internalization and accumulation of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles (DMSA-SPIONs), with average diameter of 15 nm and negative surface charge, in MCF-7 breast cancer cells. Cells were incubated with 0.25 mg Fe ml-1 DMSA-SPIONs for different time intervals ranging from 0 to 24 h.
Time-dependent uptake studies showed maximum accumulation of SPIONs after 24 h of incubation. Internalized SPIONs were localized in endosomes by acidotropic probe LysoTracker and classical TEM studies. After these preliminary studies, Soft X-ray (SX) cryo-tomography was used to characterize the distribution and topological arrangement of magnetic nanoparticles (DMSA-SPIONs) in MCF-7 cells, and to define the eventual reorganization of the intracellular environment caused by the incorporation of nanoparticles. X-ray cryo-tomographic reconstructions allowed us to visualize, at nanometric 3D resolution, the whole cell without chemical fixation or staining agents. Correlative microscopy was used to facilitate the localization of those cells containing nanoparticles accumulated in endosomes labeled by LysoTracker. Vitrified cells were prepared by plunge freezing and introduced in the Transmission Soft X-ray microscope for tilted series acquisition. Reconstructed volumes show the SPION-containing endosomal as very dense bodies which accumulate in the cell cytoplasm near the Golgi area close to the nucleus. This accumulation excludes from this area other organelles like mitochondria, which are displaced to the cellular periphery.

These experiments were performed at the Mistral beamline at ALBA Synchrotron Light Facility with the collaboration of ALBA staff. We acknowledge Dr. María del Puerto Morales for providing the SPIONS. This work was partly funded by Grant BFU2011-29038.

Fig. 1: SPIONS accumulation by correlative fluorescent optical and Soft-Xray microscopy. A and B) MCF7 cells without SPIONS. C and D) MCF7 cells incubated with SPIONS for 24 hours. Yellow arrows point to the LysoTracker accumulation correlating both optical and X-ray image respectively. N Mark the position of the nucleus.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2033 SIMS Based Correlative Microscopy for High-Resolution High-Sensitivity Nano-Analytics

Wirtz T.1, Dowsett D.1, Eswara Moorthy S.1, Fleming Y.1
1Department “Science and Analysis of Materials” (SAM), Centre de Recherche Public – Gabriel Lippmann, 41 rue du Brill, L-4422 Belvaux, Luxembourg
dowsett@lippmann.lu

Nano-analytical techniques and instruments providing both excellent spatial resolution and high-sensitivity chemical information are of extreme importance in materials science and life sciences for investigations at the nanoscale. Due to the ever increasing complexity of devices and the continuously shrinking geometries in materials research, characterization tools and techniques are facing new challenges and need to anticipate future trends.

Electron Microscopy, Helium Ion Microscopy and Scanning Probe Microscopy are commonly used for high-resolution imaging. However, these techniques all have the same important drawback: they provide no or only very limited chemical information. By contrast, Secondary Ion Mass Spectrometry (SIMS) is an extremely powerful technique for analyzing surfaces owing in particular to its excellent sensitivity, high dynamic range, very high mass resolution and ability to differentiate between isotopes.

In order to get chemical information with a highest sensitivity and highest lateral resolution, we have investigated the feasibility of combining SIMS with Transmission Electron Microscopy, Scanning Probe Microscopy and Helium Ion Microscopy and developed three prototype instruments corresponding to these three combinations:

TEM – SIMS : FEI Tecnai F20 equipped with a Ga+ FIB column and dedicated SIMS extraction optics, mass spectrometer and detectorsHIM – SIMS : Zeiss ORION Helium Ion Microscope with dedicated SIMS extraction optics, mass spectrometer and detectors [1,2]SPM – SIMS : Cameca NanoSIMS 50 with integrated AFM/SPM [3-5]The results are very encouraging and the prospects of performing SIMS in combination with TEM, HIM and SPM are very interesting. In particular, excellent detection limits are reached by using reactive gas flooding techniques. This optimization of secondary ion yields leads to detection limits varying from 10-3 to 10-6 for a lateral resolution between 10 nm and 100 nm. The combination of high-resolution microscopy and high-sensitivity chemical mapping on a single instrument represents a new level of correlative microscopy.

In this talk, we will present the instruments that we have developed, give an overview of the obtained performances, present typical examples of applications and make a comparison between ex-situ and in-situ combination of these techniques.

[1] T. Wirtz et al., Appl. Phys. Lett. 101 (2012) 041601

[2] L. Pillatsch et al., Appl. Surf. Sci. 282 (2013) 908

[3] T. Wirtz et al., Surf Interface Anal. 45 (1) (2013) 513-516

[4] T. Wirtz et al., Rev. Sci. Instrum. 83 (2012) 063702

[5] C. L. Nguyen et al., Appl. Surf. Sci. 265 (2013) 489-494

Fig. 1: Prototype of a combined TEM-SIMS instrument: modified Tecnai F20 equipped with a Ga+ gun and dedicated SIMS column

Fig. 2: Detection limit using a Ga+ FIB with and without Cs0 flooding vs. minimum feature size: example for the detection of Si-.

Fig. 3: Combined SIMS-SPM 3D reconstruction of an Al (100nm) / Si sample exposed to a plasma streamer (Field of view: 15x15 µm2): (a) Al- signal. (b) Si- signal
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2484 Correlative Microscopy for DualBeamTM FIB-SEM Characterization and Sample Preparation using MAPSTM

Van Leer B.1, Passey R.1
1FEI
brandon.van.leer@fei.com

DualBeams (FIB-SEM) have been used for materials characterization and failure analysis for many years to prepare samples for cross-section analysis, TEM analysis or to image the ultrastructure [1]. The area of characterization is generally limited to 50 – 100 μm2. Often times, specimens that arrive at the DualBeam are materials that require a multi-disciplinary approach for characterization or problem solving. DualBeam characterization for ultra- or extreme high resolution imaging, analytical characterization, and sample preparation for cross-section or TEM analysis is a step that happens near the end of sample characterization lifetime since FIB analysis is generally destructive. Automated large area, high resolution imaging extends the capability of a DualBeam or SEM for sample preparation or ultrastructure determination to become a correlative tool, bridging multiple techniques to provide a context to understand a sample feature at a higher resolution than traditional optical techniques.

MAPS is a modular software application that automates image acquisition over large areas, stitches the generated images using cross-correlation algorithms and allows correlative techniques for localization, identification and characterization. Data from any external source may be used for correlative purposes, including optical micrographs, EBSD, EDS and SPM techniques. There are no restrictions in the image resolution. The software application is released on most FEI SEMs and DualBeams, as well as offline, for stitching and correlative microscopy.

A Si-Ta-Fe witness slip exposed to super heated metals by laser bombardment to better understand kinetics of laser heating was mapped in high definition using large area SEM image acquisition for exploration and navigation. The resulting image was correlated to optical and elemental data for further characterization, including cross-section, 3D reconstruction, and STEM in SEM imaging. A 8.9 mm X 4.6 mm sample was imaged using MAPS, a directional backscatter detector and a 10 nm/pixel resolution with 2 keV on a FEI Nova NanoSEM 450 (FIG. 1) [2]. Using the backscatter electron image (BSE), specific regions of interest were identified and correlated to high-resolution optical micrographs (FIG. 2). Using a FEI Helios NanoLab 660 and EDS, the regions were elementally mapped for characterization (FIG. 3). This presentation will focus on how large scale imaging is used to explore regions of interest for subsequent analytical characterization including STEM sample preparation, EDS, FIB tomography for 3D reconstruction, and the ability to correlate other 2D data, such as optical micrographs and EDS maps.

[1] F.A. Stevie, D.P. Griffis, P.E. Russel. Introduction to Focused Ion Beams, eds. L.A. Giannuzzi and F.A. Steve. Springer (2005), 52-72

[2] D.W. Phifer et al. Microsc. Microanal. 19(Suppl 2). 2013

Fig. 1: 2.6 gigapixel BSE image acquired at 2 keV

Fig. 2: High-resolution optical micrograph correlated in MAPS to 2 keV BSE image

Fig. 3: 15 keV EDS maps correlated to 2 keV BSE image revealing compositional makeup of two regions of interest
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2614 "Correlative studies on Atom Probe Tomography specimens"

Hernández-Maldonado D.1, Lefebvre W.1, Rigutti L.1, Blum I.1, Shinde D.1
1Groupe de Physique des Matériaux, UMR CNRS 6634, Normandie University, University of Rouen et INSA de Rouen, 76801 St. Etienne du Rouvray, France
david.hernandez-maldonado@univ-rouen.fr

The ultimate goal of microscopy techniques is to identify the chemistry and the position of all the atoms that a sample contains. However, up to now, there is not a single microscopy technique by itself that reaches this target. In this work we show different examples where we applied correlative studies on the same nano-objects for the analysis of Atom Probe Tomography (APT) shaped samples. One of these examples is presented here. In particular, Atom Probe Tomography (APT) analysis and High Resolution Scanning Electron Microcopy (HR-STEM) techniques on the same nano-objects. Figure1.a) is a HAADF-STEM image of an APT specimen containing a set of InGaN/GaN quantum wells synthesized by metalorganic vapor phase epitaxy (MOVPE) on the lateral m-plane sidewalls of GaN microwires. From this kind of analysis and High-resolution (HR)-HAADF images like the showed in figure1.b) we are able to characterize the structure and the presence of dislocations and staking faults. Figure 2 is a 3D reconstruction of the same specimen extracted after the APT analysis. From these analyses it is possible to study the 3D distribution of the different species present in the sample. These techniques, which are here applied on the same nano-object, yield an extremely rich and complementary set of information, which allowed us for the interpretation of the optical properties in this particular quantum well system [1].

[1] L. Rigutti, I. Blum, D. Shinde, D. Hernández-Maldonado, W. Lefebvre, J. Houard, F. Vurpillot, A. Vella, M. Tchernycheva, C. Durand, J. Eymery, B. Deconihout, “Correlation of Microphotoluminescence Spectroscopy, Scanning Transmission Electron Microscopy, and Atom Probe Tomography on a single nano-objtect containing an InGaN/GaN multiquantum well system” Nano Letters, vol. 14, p. 107-114, 2014.

The ANR (Agence Nationale pour la Recherche) is thanked for the financial support through the Programme Jeune Chercheur – Jeune Chercheuse TIPSTEM.

Fig. 1: Figure. 1. a) HAADF-STEM image of an APT sample that contains InGaN/GaN QWs perpendicular to the axis of the tip. b) HR-HAADF image of one of the QWs presented in figure 1.a).

Fig. 2: Figure. 2. Three dimensional reconstruction, obtained after the analysis by APT, of the In atoms distribution into the tip showed in figure 1.a).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2666 Biosynthesis of metallic particles and their application in material science

Avalos-Borja M.1,2, Quester K.2, Vilchis-Nestor A. R.3, Castro-Longoria E.4
1IPICyT, División de Materiales Avanzados, San Luis Potosí, S.L.P., México, 2Centro de Nanociencias y Nanotecnología, UNAM, Ensenada, B.C., México, 3Centro Conjunto de Investigaciones en Química Sustentable UAEM-UNAM, Toluca, México, 4Departamento de Microbiología, CICESE, Ensenada, B.C., México
miguel_avalos_mx@yahoo.com.mx

Nanoscience and Nanotechnology are very active fields of research in recent years. The interest in these topics is due to the fact that matter at a nanoscale reveals significantly different properties than at bulk dimensions. Therefore, modern electronics, catalysis, medicine, etc. take advantage of these properties of matter at very small dimensions, achieving the development of more powerful and smaller processors, catalysts with higher efficiency, and so on. However, rising concerns about the environmental cost of the conventional preparation methods for these nanoparticles, typically involving toxic chemicals, high pressure/temperature, etc., have pushed towards the development of more environmentally friendly methods. Some of these ‘green’ methods involve the use of microorganisms, such as bacteria and fungi, as well as plants, employing them in vivo or via plant extracts. Nevertheless, for a biological process to successfully compete with chemical and physical nanostructure synthesis, strict control over average particle size in a specific size range and uniform particle morphology is required.
We show the production of mono-metallic (Au, Ag, Pt) and bi-metallic (Au-Ag) nanoparticles using aqueous plant extracts (Citrus paradisi, Camellia sinensis, etc.) and fungi (e.g. Neurospora crassa and Alternaria solani), with very controlled dispersion. A very interesting feature of these eco-friendly procedures is that we achieved the production of various shapes like pentagons, triangles, hexagons, rods, etc., with some of these particles (triangles, hexagons) comprising peculiar aspect ratios that imply almost two-dimensional structures. Adjustment of the synthesis parameters, such as temperature, pH, and incubation time, resulted in the formation of particles with very narrow size distributions. To determine their possible applications, these particles were tested on catalytic reactions, while different-shaped nanoparticles exhibited very promising SERS (surface enhanced Raman spectroscopy) properties, as will be shown in the presentation.
A great number of particles synthesized by ‘green’ methods have been reported in the literature by other scientists; however, very little effort has been made to conduct a deep analysis. Therefore, the objective of this work was to achieve a complete characterization of the synthesized nanomaterial, including UV-vis, Raman, TEM, HRTEM, EDS, among others, and investigate their applications in materials science.

We thank, M. Antonio Camacho, Hector Silva, Nicolás Cayetano and Jennifer Eckerly for technical support, LINAN-IPICyT for access to TEM facilities, and SEP-CONACyT (grant CB2011/169154) for partial support.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2810 Correlative tomography: multiscale and multimodal 3D imaging of a specific volume of interest

Burnett T. L.1, 2, Geurts R.2, McDonald S. A.1, 4, Gholinia A.1, Slater T.1, Jazeri H.3, Northover S. M.3, Engelberg D. L.1, Haigh S. J.1, Bouchard P. J.3, Thompson G. E.1, Withers P. J.1, 4
1Materials Science Centre, University of Manchester, Manchester, UK, 2FEI Company, Achtseweg Noord, Eindhoven, The Netherlands, 3Materials Department, Open University, Milton Keynes, UK, 4BP ICAM, University of Manchester, Manchester, UK
timothy.burnett@manchester.ac.uk

In this work we present the concept of ‘Correlative Tomography’ which represents a major step forward in the level of information that can be brought to bear on a region of interest across multiple scales. The benefit of multiscale information is to find relevant areas of interest in large volumes for high resolution studies and to prove that these high spatial resolution results are representative for the macroscopic sample. Despite the rapid advance in 3D imaging techniques existing work to date has principally registered 2D images to 3D volumes, or linked
populations measured at different scales in a statistical manner. We present the integration 3D datasets from the macro to the nanoscale where the location of each new scale and modality of imaging is specifically targeted using the information from the previous scale/technique. Our example study combines macroscale X-ray tomography, high resolution X-ray tomography, focussed ion beam serial sectioning with scanning electron microscope imaging, electron backscatter diffraction and scanning transmission electron microscopy with additional energy dispersive X-ray spectroscopy all combined and spatially linked through a single workflow ensuring connectivity of the resultant data. In this way we have been able to go right to the heart of the matter by identifying the competition between the different corrosion mechanisms at play in several
example materials including corrosion of stainless steel, creep cavitation in in stainless steel and stress corrosion cracking in 7000 series aluminium. Through these examples we have revealed new insights in mature fields and we feel that correlative tomography as a technique will also bring significant insights to biological and geological sciences.

Fig. 1: Figure showing a region of interest identified from a virtual slice of the reconstructed X-ray CT data. A SEM image of the same region prepared by the FIB for extraction of a TEM sample and lastly a HAADF image and chemical maps of Fe, Cr, Ni and C of the same region recorded in the TEM.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2828 Correlative Microscopy for Luminescent Pigments

Trottmann M.1, Brunko O.1, Songhak Y.1, Pokrant S.1
1Laboratory of Solid State Chemistry and Catalysis, Empa, Überlandstrasse 129, 8600 Dübendorf, Switzerland
matthias.trottmann@empa.ch

The full text of the abstract is not available. Please contact the presenting author.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2832 Correlative light and electron microscopy in the classroom – simple protocols for undergraduate courses

Kretschmar S.1, Kurth T.1
1TU Dresden, Center for Regenerative Therapies, CRTD, Dresden, Germany
thomas.kurth@crt-dresden.de

Correlative microscopy combines the versatility of the light microscope with the high spatial resolution of the electron microscope. It is generally regarded as state-of-the-art technology and many protocols are dependent on superhuman technical skills and/or sophisticated instrumentation [1-4]. However, on-section labeling of resin or cryo-sections with fluorochrome-coupled antibodies and gold probes is a versatile and fast method for CLEM of cells and tissues [5-8]. It is easy to perform and suitable for teaching in undergraduate student courses.

Here, we describe fast and simple protocols for correlative immunofluorescence and immunogold labeling on resin- and cryo-sections successfully used in our student practicals. Ultrathin tissue sections are mounted on EM-grids and stained simultaneously with fluorescent and gold markers. The samples are analyzed at the fluorescence microscope (FLM), demounted from the microscope slide, stained with uranyl acetate and then imaged in the transmission electron microscope (TEM). Subsequently, labeled structures selected at the FLM are identified and analyzed in the TEM. This way, fluorescent signals are correlated to the corresponding subcellular structures and corresponding immunogold signals in the area of interest (Fig. 1).

Labeling and analysis can be performed within 1-2 days, depending on the number of students attending the course. In Dresden we perform a 1-week basic EM-course for master students. The CLEM experiment is used as our routine Immunogold experiment and takes 1,5 days for 12-15 students. The procedure works in 80-100%, which hopefully encourages the students to consider even sophisticated EM-methods for their future work.

Alternatively, the samples can also be processed completely, including staining with uranyl acetate and drying before imaging. Such samples can be used for demonstrations of the method without any sample preparation (Fig. 1C-E) if time is very limited.

[1] P Verkade, J. Microsc. 230 (2008), 317-328.

[2] M Grabenbauer, WJC Geerts, J Fernandez-Rodriguez, et al., Nat. Methods 2 (2005), 857-862

[3] C van Rijnsoever, VM Oorschot, and J Klumperman, Nat. Methods 5 (2008), 973-980

[4] AV Agronskaia, JA Valentijn, LF van Driel, et al., J Struct. Biol. 164 (2008) 183-189

[5] H Schwarz and BM Humbel, Methods Mol. Biol. 369 (2007), p. 229-256.

[6] T Takizawa and JM Robinson, Methods Mol. Med. 121 (2006), p. 351-369.

[7] G Vicidomini, MC Gagliani, M Canfora, et al., Traffic 9 (2008), p. 1828-1838.

[8] G Fabig, S Kretschmar, S Weiche, et al., Methods Cell Biol. 111 (2012), p. 75-93.

The authors gratefully acknowledge funding from the European Fund for Regional Development (EFRE).

Fig. 1: A,B Cryo-section through zebrafish brain with labeled endothelial cells. A Overlay of fluorescence (anti-GFP, DAPI) and EM. B Immunogold labeling in the endothelial cells (end). C-D K4M section through the retina with GFP-labeled photoreceptors. C Fluorescence in the outer segments of rod photoreceptors. D,E corresponding EM-micrographs.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2860 Integrated light and electron microscopy of ultrathin resin sections containing GFP

Yakushevska A.1, van Driel L.1, Collinson L. M.2
1FEI Company, Eindhoven, The Netherlands, 2Electron Microscopy Unit and Cell Biophysics Laboratory, London Research Institute, Cancer Research, London, UK
alevtyna.yakushevska@fei.com

Combining information from light and electron microscopy is an increasingly popular technique to study complex biological system at various levels of resolution. It adds significant value to biological imaging. Whereas fluorescence probes in fluorescence microscopy (FM) offer specificity and sensitivity, the electron microscope provides contextual information at the ultrastructural level. Until recently these correlative microscopy experiments were cumbersome due to the use of two separate microscopes for fluorescence and electron microscopy. Recent advances in imaging systems at FEI have led to the only current commercially available instrument for integrated light and transmission electron microscopy (TEM). This instrument, Tecnai with iCorrTM, enables a new era of fast, accurate integrated microscopy for the localization and analysis of ultrastructures.

The Tecnai with iCorrTM consists of a fully integrated LED based widefield FM located at the normal sample position on the TEM column. Imaging in FM and TEM mode is done sequentially without manually exchanging the sample between the two imaging modes. Using the common TEM specimen stage and specimen holder, the sample is tilted to 90˚ in order to record light microscopy images in reflection mode and fluorescence mode. The sample is then tilted to 0˚ for normal TEM imaging.

To use the advantage of the Tecnai with iCorrTM, a single sample is required that allows activity of the fluorophores and exhibits high ultrastructural preservation in TEM.

Here we will report a protocol that fits these requirements, and results in a resin-embedded sample where ultrastructure, TEM contrast and GFP fluorescence are combined in a single ultrathin section. The value of these samples is illustrated by a correlative workflow including the Tecnai with iCorrTM.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2908 Illuminating Correlative Research using Light, X-ray and Electron Microscopy

Merkle A. P.1, Gelb J.1, Lechner L.1, Schulmeyer I.2, Orchowski A.2, Fuchs J.3
1Carl Zeiss X-ray Microscopy, 2Carl Zeiss Microscopy GmbH, 3Carl Zeiss AG
arno.merkle@zeiss.com

X-ray tomography has emerged as a new powerful imaging technique that obtains 3D structural information from opaque samples under a variety of conditions and environments [1, 2]. It has rapidly become an accepted laboratory technique offering quantitative information in both the materials sciences and life sciences. We present ways in which non-destructive 3D volumetric information, obtained via laboratory nanoscale and sub-micron X-ray microscopy (XRM) are increasingly used to probe scientific questions as a complement to Electron- and Light-based microscopy methods. These correlative methods, relating to XRM, provide an opportunity to study materials evolution at multiple length scales in 3D and utilize this information to inform or guide postmortem analysis to be most efficient.

In materials research, the motivation to correlate XRM information with postmortem EM (SEM, FIB-SEM or TEM) stems from three primary reasons. First, this workflow is used to complement time dependent materials evolution (4D) studies with higher resolution imaging, diffraction or spectroscopic information. Second, hierarchical porous materials such as membranes or porous rock naturally exhibit features from mmnm, all which require characterization of a single volume in 3D with multiple imaging modalities to define performance. Finally, XRM is used as a 3D navigation system (‘Google Earth’ in 3D) for targeting and finding specific buried structures of interest for extraction or cross sectional imaging (Figure 1). We demonstrate several examples, including energy materials, automotive applications and metals (Figure 2), upon which the use of XRM and FIB/SEM information on the same specimen has contributed to a more complete understanding of a materials system.

In life sciences, correlative microscopy methods have existed for decades in various forms. One remaining challenge is to identify practical methods of localizing the same feature in multiple microscopes in 3D. XRM presents an additional opportunity to bridge the length scales between LM and EM and ease the ‘needle in a haystack’ navigation problem. Recently, XRM techniques acting as a bridge between light- and electron- microscopy have acted as an efficiency multiplier to make 3DEM methods highly efficient and targeted, be pre-defining the buried volumes of interest (Figure 3).

We conclude by offering perspectives on the future directions of the utilization of correlative microscopy techniques with respect to XRM information.

References
[1] A. P. Merkle and J. Gelb, Ascent of 3D X-ray Microscopy in the Laboratory, Microscopy Today, 21 (2013), p. 10
[2] E Maire and P Withers, Quantitative X-ray tomography, International Materials Review, 59 (2014) p. 1
[3] S Handschuh, et al., Frontiers in Zoology, 10 (2013), p. 44

Fig. 1: Target region navigation workflow, utilizing (a) XRM dataset to identify VOI, followed by (b-c) laser + FIB milling to quickly expose the feature and interface of interest on a Hall sensor device.

Fig. 2: A single Aluminum Copper eutectic sample and volume-of-interest imaged a) non-destructively with XRM at multiple resolutions b) with FIB-SEM nanotomography including c) EDS for chemical information. Sample courtesy of B. Patterson, Los Alamos National Laboratory.

Fig. 3: XRM dataset of stained (for EM) mammalian brain tissue. Such non-destructive XRM datasets are being used to navigate to specific subsurface volumes of interest quickly, thereby multiplying the efficiency of 3D EM techniques. In collaboration with the National Center for Microscopy and Imaging Research at UCSD together with ZEISS XRM.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2918 Shape tuning biosynthesis of gold nanoparticles: Structural Characterization

Vilchis-Nestor A. R.1, Silva-de-Hoyos L. E.1, Sánchez-Mendieta V.1, Avalos-Borja M.2, 3
1CCIQS, Universidad Autonoma del Estado de México, México, 2IPICYT, División de Materiales Avanzados, SLP, México, 3CNYN, UNAM, Ensenada, B.C., México
arvilchisn@uaemex.mx

The optoelectronic and physicochemical properties of nanoscale matter are a strong function of particle size. Nanoparticles shape also contributes significantly to modulating their electronic properties [1, 2]. Morphological control of gold nanostructures can be achieved with traditional (chemical and physical) methods. Furthermore, some applications of gold nanoparticles require their assembly into superstructures, and this could be difficult if the control of shape is reduced. For example, the formation of self-assembly of gold nanoparticles into thin films or 3D architectures, from anisotropic nanostructures constitutes a challenge for the nanoscience.

On the other hand, the development of ecofriendly methods for the synthesis of metallic nanostructures has become an interest of research groups [3]. Microorganisms and plants have already been used to biosynthesize metallic nanostructures, however control over the size and shape of biological routes is still poor in contrast to chemical methods. Here, we report gold nanoparticles shape tuning morphology obtained by biological methods, employing Citrus paradise (grapefruit) aqueous extract as reducing and capping agent.

Analyses include UV-Vis spectroscopy, TEM and HRTEM in order to confirm shape variation of the gold nanostructures as function of the Citrus paradise extract volume employed during the reduction process. The results presented here show that biogenic routes are able to generate a variety of shapes of gold nanostructures from spherical nanoparticles to nanotriangles, by changing only the Citrus paradise extract concentration in the synthesis stage. Hence, the optoelectronic response of the gold nanostructures could be easy tuned by the volume of the bioreducing agent employed. The present studies show that gold nanotriangles can be easily deposited on glass substrates in order to form films with potential applications as sensors.

References

1. Shiv Shankar S. et al. Nature Materials 2004;3:482-488.

2. Singh Amit et al. Nanotechnology 2006;17:2399–2405.

3. Shiv Shankar S. et al. Chem. Mater. 2005;17:566-572.

This research was supported by grant from UAEM (Program for Science and Technology 2013), project number 3467/2013CHT.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-2967 CLEM: the use of thawed cryosections and multiple labeling strategies

Vancová M.1,2, Vaněček J.1, Wandrol P.3, Nebesářová J.1,4
1Institute of Parasitology, Biological Centre of ASCR, v.v.i, Ceske Budejovice, Czech Republic, 2Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic , 3FEI Czech Republic, Brno, Czech Republic, 4Faculty of Science, Charles University in Prague, Czech Republic
vancova@paru.cas.cz

Correlative light and electron mictoscopy is gaining attention in the last years. Besides prompt development of new hardware, preparation of the sample for reliable transfer and observation in the electron microscope requires even higher focus. We present here a detailed protocols for labelling of ultrathin cryosections according to Tokuyasu as well as resin sections using either FluoroNanogold-conjugated antibody fragments followed by silver enhancement; antibodies directed to fluorescence markers and conjugated to gold particles; and finally palladium nanoconjugates (1). Fluorescence microscopy and scanning electron microscopy were performed on the same sample. We used carbon-based imprints of finder grids for accurate and reliable localization of objects of interest within a specimen for imaging using both types of microscopes (2). After fluorescence, cryosections were postfixed with osmium tetroxide, dehydrated, dried (critical point drying using carbon dioxide, alternative t-butanol drying) and finally carbon coated. Different staining protocols have been used to improve contrast of membranes. The quality and resolution of both the secondary electron and the back-scattered electron imaging have been influenced by either carbon or gold conductive coating of glass slides. We showed that metal nanoparticles as well as silver precipitates were optimally visible from an accelerating voltage 4 kV using either the back-scattered electrons or the secondary electron signal. Different atomic numbers of metal nanoparticles and back-scattered electron imaging allow multiple labelling in SEM studies.

References:

1. Vancová, M et al. (2011). Microsc Microanal. 17, 810-816.
2. Brown, E. et al. (2009). Seminars in Developmental Biology 20, 910-919.

This work was supported by the Technology Agency of the Czech Republic, project TE01020118.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3044 Positional correlative anatomy of invertebrate models organisms increases efficiency of TEM data production

Kolotuev I.1,2
1MRic-TEM, Biosit, University of Rennes 1, Rennes, France, 2IGDR, University of Rennes 1, Rennes, France
irina.kolotueva@univ-rennes1.fr

Transmission Electron Microscopy (TEM) is essential to fully understand cellular and developmental processes; it greatly complements data obtained by light microscopy. Recent technological innovations have advanced the entire field of TEM, yet classical techniques still prevail for most present-day studies. The majority of cell and developmental biology studies that use TEM do not require complicated methodologies, but rather fast and efficient data generation. A major unmet need in most TEM approaches is the ability to quickly prepare and orient a sample to identify a region of interest.


Resin embedded samples preparation (standard or high pressure frozen) is commonly used to study C. elegans and Drosophila ultrastructure. Treatment of these small model organisms is frequently complex, as it requires precisely orienting a sample to identify a region of interest. In addition, due to the size restrictions, semi-thin sections for targeting ROI cannot be performed.


I developed embedding method that permits observation of the samples after the embedding and acquisition of high quality DIC images. This positional correlative anatomy method with 2-step flat embedding is coupled to tight trimming of the sample that facilitates precise localization of ROI, permitting efficient TEM analysis of high sample numbers. It allows the recognition of cells and organs in embedded samples in a way similar to live studies, enabling spatial correlation in minimal time. Additional modifications in embedding permit simultaneous sectioning of several small samples simultaneously.


The modifications make the TEM preparation and analytic procedures faster and more straight-forward, supporting a higher sampling rate. To illustrate the modified procedures, I provide numerous examples from actual studies addressing research questions in C. elegans and Drosophila. This method can be equally applied to address questions of cell and developmental biology in other small multicellular model organisms.

I thank MRic-TEM colleagues Agnes Burel, Marie Therese Lavault, Laurence Cornevan and Ophelie Nicole for their help and support.

Fig. 1: DIC image of trachea extracted from Drosophila L3 larva embedded in epon resin by 2-step flat embedding. Different types of tubes are clearly visible in different imaging planes. White box designates the high inset from Figure 2, showing tracheal branching position.

Fig. 2: DIC image of ROI with two tubules branching. Black lines represent selected TEM regions from serial sectioning presented in Figure 3, collected for further high magnification analysis. White lines mark the extremities of branching region. Scale bar 10 µm.

Fig. 3: TEM images of selected sections selected on Figure 2. Correlative anatomy method allows a bi-directional analysis: targeting of precise region in TEM based on LM data; TEM appearance and sizes help to determine precisely the region that was sectioned. Scale bar 2 µm. [Images were rotated for presentation].
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3094 Plasmon coupling in gold and silver nanosphere dimers probed by electrons

Kadkhodazadeh S.1
1Center for Electron Nanoscopy, Technical University of Denmark
shka@cen.dtu.dk

Plasmon coupling in closely spaced noble metal nanoparticles, producing among other effects a shift in the localised surface plasmon resonance energy (LSPR) supported by the assembly, has led to their applications in several technological fields such as ultrasensitive biosensing and nanophotonics [1,2]. Despite rapid progress in these fields, direct imaging and characterisation of LSPRs in nanostructures remains a challenge, due to the insufficient spatial resolution of most optical techniques. Alternatively, the use of electron energy-loss spectroscopy (EELS) as a complementary method to probe the plasmonic and optical properties of nanostructures has become increasingly popular, owing to the Ångström spatial resolution of this technique. Here we have applied scanning transmission electron microscopy (STEM) imaging and EELS to study the plasmonic properties of individual nanosphere dimers as a function of interparticle distance, accessing both the classical and the quantum regimes. In particular, we accurately determine and compare the scaling of the LSPR energy with interparticle distance in gold and silver dimers [3]. This calibration is of great importance to nanometrology tools such as plasmon rulers, in which the shift in LSPR energy is used for distance measurement in biological and chemical systems [4]. Furthermore, we study dimers separated at subnanometer distances, monitoring their transition from classical to quantum systems as particles approach and merge, and from the onset of tunnelling to charge transfer between the particles, as a nano-bridge forms between them [5]. Finally we explore the possibility of combining EELS and optical spectroscopy by utilising TEM specimen holders with light input-output capabilities.

1. J. N. Anker, W. P. Hall, O. Lyandres, N. C. Shah, J. Zhao, and R. P. Van Duyne, Nat. Mater 7, 442 (2008).

2. V. Giannini, A. I. Fernandez-Dominguez, S. C. Heck, and S. A. Maier, Chem. Rev. 111, 3888 (2011).

3. S. Kadkhodazadeh, J. R. de Lasson, M. Beleggia, H. Kneipp, J. B. Wagner, and K. Kneipp, J. Phys. Chem. C 118, 5478 (2014).

4. C. Sonnichsen, B. M. Reinhard, J. Liphardt, and A. P. Alivisatos, Nat. Biotechnol. 23, 741 (2005).

5. S. Kadkhodazadeh, J. B. Wagner, H. Kneipp, and K. Kneipp, Appl. Phys. Lett. 103, 083103 (2013).

J. R. de Lasson, H. Kneipp, K. Kneipp, M. Beleggia and J. B. Wagner are gratefully acknowledged for valuable discussions and their contributions to this work.

Fig. 1: The fractional LSPR wavelength shift Δλ/λ0 in gold and silver dimers as a function of ratios (L/2R) and (d/2R), where λ and λ0 are the LSPR wavelengths in a dimer and in a single particle, respectively, and L, d and R are defined in the inset image in (b).

Fig. 2: STEM images and EELS spectra from a pair of silver nanoparticles with separation distances d = +1.0 to -1.6 nm, as they approach and merge, revealing the transition from classical behaviour (A) to tunnelling (B) to a contact regime (C-E).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3104 SCANNING ELECTRON MICROSCOPY OF LIVE-CELLS ENABLED BY IN-SITU FLUORESCENCE MICROSCOPY

Liv N.1, van Oosten Slingeland D. S.1, Baudoin J. P.2, Kruit P.1, Piston D. W.2, Hoogenboom J. P.1
1Imaging Physics, Faculty of Applied Sciences, Delft University of Technology, Delft, The Netherlands, 2Vanderbilt University Medical Center, Nashville, USA
j.p.hoogenboom@tudelft.nl

One of the major strengths of light microscopy techniques, and especially fluorescence microscopy (FM), is the ability to observe rare dynamic events, in vitro as well as in vivo. A main challenge, however, is to image the positions of labelled proteins with respect to cellular ultrastructure during such an event with a resolution beyond that of the light microscope. Electron microscopy (EM) of samples in a near-native (buffer) environment has recently been shown possible, but the destructive nature of EM precludes observing cellular dynamics. We present a novel approach towards dynamic bio-imaging wherein live-cell FM is carried out in-situ in a scanning electron microscope (SEM) (Figure 1). This allows us to capture structural SEM snapshots on-demand based on the FM observations.

We study the uptake and retrograde transport of EGF-conjugated quantum dots (QDots) in filopodia of fibroblasts. The cells are cultured on a thin, electron-transparent substrate, which is then placed in a light-transparent sample holder containing the EGF-QDot solution. This sample holder is mounted in an SEM with integrated high-numerical aperture fluorescence microscope. The uptake process is monitored with FM (Figure 2) and based on these observations, we determine both the desired moment for the SEM image and the location of filopodia within the SEM field of view. These SEM images show the positions of individual QDots on the cytoskeleton transport tracks within filopodia from the position of uptake up to the docking region at the microtubules frontier where Qdots accumulate before further transport towards the nucleus.

Fig. 1: (a) Schematic of the Scanning Electron Microscope with in-situ epi-fluorescence microscope. The fields of view are aligned such that an area on the sample can be observed with both microscopes. (b) Illustration showing a live cell contained in liquid in a capsule with electron and light transparent windows mounted in the in-situ microscope

Fig. 2: Snapshots from a fluorescence movie showing transport of quantum dots conjugated to epi-dermal growth factor in a cellular extension. After 51.75 seconds observation time, SEM snapshots were taken, which are shown overlaid to the fluorescence image in the main panel.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3172 Integrated Raman microscope with FIB-SEM

Timmermans F. J.1, Lenferink A. T.1, Wolferen H. A.2, Otto C.1
1University of Twente. Medical Cell BioPhysics, 7552 NB Drienerlolaan 5, Enschede, the Netherlands, 2University of Twente. MESA+, 7552 NB Drienerlolaan 5, Enschede, the Netherlands
f.j.timmermans@utwente.nl

Using multiple microscope techniques is an effective strategy to obtain more information about a samples properties. This allows researchers to gain new insights or to employ new strategies for analysis. However using different microscopes leads to new problems, moving the sample between systems often results in contaminations and deformations. Furthermore this process is often time consuming and the researcher has to take care not to lose the region of interest. Therefore we present a system employing an integrated Raman microscope in a dual beam Focused Ion Beam – Scanning Electron Microscope (FIB - SEM), as shown in figure 1.

This system combines chemical and high resolution morphological analysis with a sample processing modality. The combination of FIB with Raman allows us to investigate the redeposition of removed material and to analyse defects that occur through ion implantation. Sample analysis is performed with correlative SEM and Raman microscopy, this provides high resolution morphological information and a compound specific image. The combined system provides more information and new strategies for analysis in the material and biological sciences.

The integration of microscopes does give rise to new challenges mainly concerning engineering aspects. Implementation of the Raman objective in a confined space requires a careful design of the objective pickup system. Furthermore the vacuum pressure in the FIB chamber has to remain stable, which requires the use of coupling windows and cable feedthroughs. Ultimately the Raman microscope functions as an add on module for the FIB-SEM which means that it may not place any restrictions on its operation.

As a first project of immediate interest is the analysis of multimodal labels, i.e. labels suitable for both optical and electronic imaging. These labels consist of a gold nanoparticle with an adsorbed fluorescent dye to provide a high electron scattering and SERS signal. Multimodal labels are of interest for biological applications because the SEM contrast is limited in organic matter due to charging effects. Furthermore these labels are very promising as an aid for correlating the Raman and SEM images.

We are thankful to the STW (Stichting Technische Wetenschappen) for supporting this project. This research is performed within the STW Perspectief program Microscopy Valley.

Fig. 1: Schematic of the integrated Raman microscope in a Focused Ion Beam - Scanning Electron Microscope.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3234 Great news for integrated CLEM

Voortman L. M.1, Haring M. T.1, Effting A. P.1
1DELMIC BV, Delft, The Netherlands
voortman@delmic.com

We will present our latest innovation for integrated correlative light and electron microscopy (CLEM). During the past few years, CLEM has become an essential tool for resolving many biological questions. Nevertheless, the potential of CLEM is only accessible to a few research groups worldwide. Until recently, CLEM has been challenging, costly and time consuming. Correlative methods require two setups to be present which are traditionally distinct techniques with different facilities and sample preparation methods. Due to the differences between both microscopes, extra sample preparation steps are often required when switching from FM to EM. These steps can alter the conformation of the sample, hampering accurate correlation. In addition, it is challenging to find a region of interest identified with FM in EM since the information used to navigate in FM is not visible in EM.

At DELMIC, we aim to resolve these difficulties by providing easy to use, integrated CLEM solutions. By integrating fluorescence and scanning electron microscopy the need to transfer between two different microscopes is eliminated [1-2]. In addition, correlation is fully automated, does not need fiducials and achieves an overlay accuracy < 50 nm. Furthermore, switching between imaging modalities is instantaneous, guaranteeing that the same area of interest is imaged.

Here, we will introduce a new product (see fig. 1) which will make CLEM as accessible as FM: easy to use, easy to install, fully automated correlation, 20 nm resolution and 30 seconds from sample loading to correlative imaging. This will be the ideal research tool for experienced FM users aiming to achieve 20 nm resolution on a broad range of samples. Thanks to the quick loading, this will also be an ideal inspection or screening tool for CLEM.

Many cellular processes consist of a complex interaction between function and structure. By combining the versatility of FM with the high resolution cellular context provided by EM, CLEM is the ideal method to study these cellular processes. FM can be used to identify a region of interest such as transfected cells, specific organelles, labelled neurons or rare co-localization events. In general, multiple fluorescent labels can be correlated simultaneously with high resolution EM which provides access to a broad range of length scales and complementary contrast mechanisms, see fig. 2.

One of the challenges associated with integrated CLEM, however, is sample preparation [3]. Due to the integration, sample preparation needs to be suited for both FM and EM simultaneously. In this talk we will discuss these difficulties and how to overcome them.

[1] Zonnevylle et al., Journal of Microscopy (2013)
[2] Liv et al., Plos One (2013)
[3] Peddie et al., Ultramicroscopy (2014)

Fig. 1: New CLEM solution by DELMIC BV

Fig. 2: Simultaneous CLEM of whole uncoated cells. A,B) Fluorescence image of adenocarcinoma cells, actin is labeled with Alexa488, scale bar is 10 µm C) SEM image of boxed area, scale bar is 5 µm. Image courtesy of N. Liv & J.P. Hoogenboom
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3247 Correlative Light and Electron Microscopy – on the way from 2D towards 3D

Elli A. F.1, Hummel E.2, Boeker C.3
1Carl Zeiss Microscopy GmbH, Oberkochen, Germany, 2Carl Zeiss Microscopy GmbH, Munich, Germany, 3Carl Zeiss Microscopy GmbH, Goettingen, Germany
alexandra.elli@zeiss.com

More and more researchers have been wanting to interlink complementary information using correlative microscopy to gain insights into the interdependency of function and structure. This can involve the combination of any microscopical methods, but usually the term refers to light microscopy (LM) and electron microscopy (EM). The interest in correlative microscopy has been rapidly growing in the last decades and in 2 dimension this workflow is well established because of the ease of use of the provided solutions. For correlation of two microscopical images, regions of interest are specified and imaged in one microscope, which can be relocated easily in a different microscope by using Zeiss “Shuttle & Find”. Afterwards, the images are overlaid. However, there are still a number of challenges that have to be addressed in order to realize the full potential of correlative microscopy. One major challenge is the correlation of 3D data sets. To achieve this, it is necessary to exactly define volumes of interest (VOI) in the data of the first microscope. Further, the precise relocation of the identical VOI in the second microscope is essential as well as the registration of the 3D object in all spatial directions. Even if the correlation of 3-dimensional data from different microscopes (e.g. LSM and FIB-SEM) is feasible due to cross correlation methods it has to be stated that this workflow is not yet fully-automated [1,2]. The 3D workflow can be simplified by reducing the scale of the object in one dimension. One popular approach is to cut the sample into serial sections (correlative array tomography) [3]. Thus, the segmentation in one dimension is done mechanically and only 2-dimensional microscope images have to be correlated. Correlative array tomography allows the detection of fluorescent labels as well as the investigation of the ultrastructure of ultrathin serial sections. Regions of interest can be marked and automatically imaged within all the individual sections building up a long ribbon using a procedure according to the “Shuttle & Find” approach. The challenge of this approach is on one hand the alignment of the consecutive 2D images taken with a light microscope and a scanning electron microscope and on the other hand their subsequent registration to a correlative 3D data set. A comparison of the features in the single sections followed by an alignment of the features results in an accurate alignment of the single sections. Finally, the full volume can be reconstructed by a similar slice-to-slice stack alignment.

References [1] M Lucas et al, Imaging & Microscopy. 10(3) (2008), pp. 30-31. [2] L Blazquez-Llorca et al, J Alzheimers Dis, 34(4) (2013), pp. 995-1013. [3] KD Micheva and SJ Smith, Neuron 55 (2007), pp. 25-36.

Fig. 1:
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3298 Deciphering the ultrastructural organization of endo-melanosomal network of pigment cells in temporal and spatial high resolution by Correlative Light and Electron Microscopy.

Heiligenstein X.1,2, Delevoye C.1,2, Hurbain I.1,2, Van Niel G.1,2, Salamero J.1,3, Raposo G.1,2
1Institut Curie, Centre de Recherche, Paris F-75248 France , 2Structure and membrane compartments CNRS UMR144, 3Cell and Tissue Imaging Facility (PICT-IBiSA) CNRS UMR144
xavier.heiligenstein@curie.fr

Skin melanocytes generate a lysosome-related organelle, the melanosome, which originates within endosomal intermediates but remains distinct from lysosomes. Melanosome biogenesis and pigment synthesis requires a tightly regulated transport of melanosomal enzymes from early endosomes to the melanosome, a step affected in the Hermansky-Pudlak Syndrom (HPS) 1. Our aim is to decipher the complexity of the endo-melanosomal network in pigmented melanocytes using a multi-modal and multi-scale imaging approach, the Correlative Light to Electron Microscopy (CLEM). We internalized fluorescent transferrin (Tf) in melanocyte to load endosomal carriers and followed by live cell imaging the Tf-positive endosomes and the dark pigmented-melanosomes by “bright-field”. To highlight and preserve the connections between endosomes and melanosomes, we rapidly transfer the specimen into a high-pressure freezer to vitrify the specimen using our new technology, the CryoCapsule 2. The vitrified specimen is then processed to preserve the fluorescence for a second fluorescence imaging step on the electron microscopy sections 3,4. This secondary fluorescence is then processed for correlation with the electron micrograph to obtain a very high spatio-contextual resolution of the endosomes location and the melanosomes (figure 1). Applied to serial electron tomograms (figure 2), we have started to decipher the ultrastructural organisation of the endosomal network aiming at understanding how this organization is altered in disease such as the HPS. As a comprehensive approach, we investigate this ultrastructural organisation in three different biological conditions: in melanocyte cell cultures, in melanocyte and keratinocyte co-cultures and in reconstructed epidermis.

1. Delevoye, C. et al. AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis. J. Cell Biol. 187, 247–64 (2009).

2. Heiligenstein, X. et al. The CryoCapsule: Simplifying correlative light to electron microscopy. Traffic n/a–n/a (2014). doi:10.1111/tra.12164

3. Kukulski, W. et al. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J. Cell Biol. 192, 111–9 (2011).

4. Nixon, S. J. et al. A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos. Traffic 10, 131–6 (2009).

This work was supported by the EMBL PhD program, ANR MatetPro Microconnect, NIH grants R01 EY015625, ARC SL220100601359, the CNRS and “Institut Curie”. We acknowledge France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04, « Investments for the future »).

Fig. 1: From live cell imaging to electron microscopy.A- Transmisted light micrograph of the melanocyte. Box: two melanosome groups. B- Transferrin labeled (546) endosomal network. Box: endosomal network close to the melanosomes. C- Electron microscopy low magnification of the melanocyte. D- Overlay to locate the endosomes with respect to the melanosomes.

Fig. 2: The endomelanosomal network by electron tomography (A) and related to the fluorescently labeled endosomal network (B).A- The melanosomes stages are color coded: stage I to IV (Y, O, R, P), rough Endoplasmic Reticulum (rER, Blue). B- Overlay of the tomogram and the fluorescence to identify and segment specifically the endosomes from the rER.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3311 Searching for a single preparation to simplify comparative imaging studies involving SEM, Confocal Microscopy and Micro-CT of biological materials 

Summerfield R. A.1, Sykes D.1, Ball A. D.1, Goral T.1
1Imaging and Analysis Centre, Science Facilities, Natural History Museum, Cromwell Rd, SW7 5BD, London, UK
ras2g08@gmail.com

Biological materials present unique challenges for microscopic imaging techniques. In Scanning Electron Microscopy (SEM), uncoated biological materials may collect charge as they are scanned by the electron beam. This can reduce the overall scan quality and introduce artefacts into the dataset. This problem is largely overcome by employing variable pressure SEM. VPSEM offers nm resolution of external features, but unfortunately cannot visualise internal information. Confocal microscopy and micro-CT scanning offer avenues to visualise internal morphology, but are not without their own drawbacks. Confocal microscopy offers high resolution but can be limited with regards to sample size and penetration, and specimens may require specialist stains if they are not autofluorescent. However, when the sample is appropriate, the results are stunning! Micro-CT, whilst offering very good tissue penetration capabilities, presents problems for biological tissues, since their X-ray absorption coefficient is rather poor. As a result, the contrast displayed in the images tends to be low and thus complicates the reconstruction and subsequent interpretation of the 3D datasets that are produced. Lab based micro-CT scanning also falls well short of confocal and SEM imaging with regards to resolutions achieved.

Research ventures that combine these techniques facilitate discoveries in morphology and systematics, yet no single preparation method is currently applicable to all. Non-destructive and reversible preparations and techniques have the advantage of preserving type materials for future generations and future analyses, thus opening up the vast collections housed within regional and national museums across the world.

At the Natural History Museum (NHM) we are exploring methodologies which will allow us to employ all three techniques to investigate biological samples. Here I present the findings of a comparative study using various fixing, staining and drying techniques using simple crustacean models Artemia salina and Daphnia sp. In micro-CT, heavy metal stains like phosphotungstic acid (PTA), iodine and osmium tetroxide have been shown to differentially stain tissues and improve contrast by increasing the X-ray absorption of target tissues. But do these stains survive the drying processes necessary for SEM based work (e.g. nano-CT)? How do they compare to confocal datasets? Can the protocols be reversed to allow specimens to be returned to the Museum’s collections?

Fig. 1: Confocal micrograph of unstained female Artemia salina specimen. This is a composite image of 100 tiles, in a 10X10 grid, each scanned with a step size in z of 7.2µm for a total of 64 steps. The image was taken on the NHM’s Nikon A1SI Confocal Microscope system using a 10X objective lens to give a final resolution of 2.49µm per pixel.

Fig. 2: Rendered micro-CT data set of male Artemia salina specimen stained with iodine and PTA. Colours represent different tissue groups i.e. red-dense gut muscles; blue-finer musculature, lumen and nervous tissues. The specimen was scanned on the NHM’s Nikon HMXST 225 micro-CT system at 105kV, 90µA and 500ms exposure. Final resolution was 5µm per pixel.

Fig. 3: SEM image of the Artemia salina ciliated thoracopods. This image was acquired from an uncoated specimen using the NHM’s FEI Quata 650 ESEM FEG at 7kV and a chamber pressure of 70Pa. Final resolution is 3.3µm per pixel.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3321 Ultrastructural alterations in placenta in relation to pesticide exposure during pregnancy and delivery of low birth weight baby

Kumar S. N.1,4, Bastia B.1, Sharma S. K.2, Borgohain D.3, Raisuddin S.4, Jain A. K.1
1National Institute of Pathology (ICMR), Safdarjang Hospital Campus, New Delhi -110029, INDIA, 2Regional Medical Research Centre (ICMR), Dibrugarh, Assam, India, 3Assam Medical College, Dibrugarh, Assam, India, 4Jamia Hamdard, New Delhi, India
drakjain@gmail.com

Pesticide usage forms common agriculture practice. It is known that pesticides can reach placenta and accumulate there and have potential to cross the placental barrier and enter the foetal bloodstream. They can cause alterations in the development as well as functions of placenta resulting in adverse effects during pregnancy. The present study was undertaken to study changes in ultrastructure of placenta in tea garden workers exposed to pesticides during pregnancy and acetyl cholinesterase (AChE) activity was assessed as biomarker of organophosphate pesticide (OPP) exposure. The samples of placenta and blood (maternal and cord) were collected from singleton pregnancies from women exposed to pesticides working in tea gardens. The adverse health effects experienced by individuals were evaluated by questionnaires. Maternal & cord blood and placental tissue were assessed for traces of pesticides by GC-ECD and AChE activity was evaluated. The ultrastructure changes in placental tissue were studied with Hitachi (H-7500) transmission electron microscope (TEM).
Significantly higher levels of OPPs were observed in tea garden workers than those from house wives while the AChE activity was significantly low in maternal & cord blood and placenta of tea garden workers. In addition, ultrastructural study of placenta has revealed that the villi in placenta of tea garden workers exposed to pesticides are comparatively longer and thinner and less vascularised as compared to non exposed group.
Fibrinoid was frequently observed in villous stroma. The density of apical microvilli appeared considerably reduced and occasional microvilli-free areas were observed. The underlying trophoblastic basement membrane appeared significantly thicker than that of non-exposed workers. Occasionally fusion of cytotrophoblast and syncytiotrophoblast was also observed. Syncytial knots were numerous in exposed workers. In most of the cases of tea garden workers, trophoblasts (especially syncytial trophoblasts) showed dynamic changes in the nuclei such as increased heterochromatin content and nuclear aggregation. There was increased collagen in the villous stroma and shrunken endothelium in foetal capillaries. It is plausible that deleterious effect of pesticides on placental barrier of tea garden workers could result of impairment of placental barrier, restrict nutrient supply from mother to foetus and thus could be the cause the Low birth Weight (LBW). Therefore, it can be concluded that exposure to pesticides during pregnancy is likely to be detrimental to the growth of foetus and the extent of damage (foetal outcome) is related to the level of pesticide exposure.

The financial support for the study received from Indian Council of Medical Research, New Delhi is gratefully acknowledged

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3360 Correlative scanning electron and confocal microscopy using an optical transparent thin coating of indium tin oxide (ITO)

Falqui A.1,2, Rodighiero S.3, Sogne E.3, Marotta R.1, Francolini M.3,4, Ruffilli R.1, Cagnoli C.3, Casu A.1, Genovese A.1
1Nanochemistry, Istituto Italiano di Tecnologia, Via Morego, 30 – 16163 Genova, Italy, 2Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST) – Thuwal 23955-6900, Kingdom of Saudi Arabia, 3Fondazione Filarete, Viale Ortles 22/4 – 20139 Milano, Italy, 4Università degli Studi di Milano, Via Vanvitelli 32 – 20129 Milano, Italy
andrea.falqui@gmail.com

Correlating the Electron Microscopy (EM) and Confocal Microscopy (CM) imaging of cells and tissues is a well-known method to understand the relations occurring between cellular structure and function. Conventional CM is capable to visualize the presence of either specific antigens by the use of immunofluorescent labelling or fluorescent proteins (FP), with resolution of few hundreds of nanometers. On the other hand, EM is capable to image the cellular ultrastructure down to nanometer scale. Putting together the information given by the two techniques on the same area of the specimen allows then to determine the antigen location on the cellular ultrastructure.
EM imaging could be carried out on biological specimens both in transmission (TEM) and in scanning (SEM) mode. In both the EM approaches, to get information on antigen distribution, cells can be labelled with antibodies conjugated with small (<20 nm) gold particles. In the case of SEM the secondary electrons (SE) are used to image the specimen surface morphology, whilst compositional contrast obtained by collecting backscattered electrons (BSE) allows to simultaneously localize the gold particles that labelled a cellular surface antigen.
A key point in the observation of the cellular ultrastructure is the preparation protocol followed. In particular, in the case of SEM imaging with surface immunolabelling, the surface of the specimen has to be rendered electrically conductive, while preserving the compositional contrast that has to be used to localize the gold nanoparticles markers. This need usually brings to exclude gold or platinum as coating agent. Enough recent literature indicates the possibility to coat the cell surface with a thin layer of chromium, but with two main limits: 1) its low atomic number giving rise to a low SE signal; 2) the lack of conductivity due to its fast oxidation if deposed under low vacuum condition or if exposed to air. [1]
In order to overcome these major limitations, we report the correlative SEM and CF microscopy imaging of HeLa cells and neurons, using an optical transparent thin layer of Indium Tin Oxide (ITO) deposed by ion sputtering, after studying the optimal ITO layer thickness. ITO was revealed to be stable, and capable to provide both suitable electrical conductivity, good SE production and preservation of the BSE signal coming from the gold immunomarkers. Finally, in order to determine if the CM imaging could be carried out after the SEM one, we also studied how both the ITO deposition and the different preparation steps for the SEM imaging affected the immunofluorescent signal.

[1] M. W. Goldberg. Immunolabeling for Scanning Electron Microscopy (SEM) and Field Emission SEM, Methods Cell Biol. 2008, 88, 109-130.

Fig. 1: (a) Fluorescence Image collected by CM on HeLa cells, due to immunolabelled CD147 membrane protein. The area indicated by the white rectangle corresponds to that reported in panel b; (b) SEM SE image corresponding to the area selected in (a). The small rectangle shows the most fluorescent zone, magnified in Figure 2 (scale bar: 10 μm).

Fig. 2: (a) High magnification SEM SE image of the area contained within the little white rectangle shown in Fig. 1b; (b) SEM BSE image corresponding to the same area shown in panel (a). The small white dots are gold particles marking the CD147 membrane protein. Scale bar: 1 μm. For SEM imaging the sample was coated with a 20 nm-thick ITO layer.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3367 Correlative in-situ microscopy in materials and life science: Bringing Raman spectroscopy into the SEM

Hoffmann B.1, Sarau G.1, Heilmann M.1, Latzel M.1, Haničinec M.2, Jiruše J.2, Christiansen S.1,3
1Max Planck Institute for the Science of Light, Erlangen, Germany, 2TESCAN Brno, s.r.o., Brno, Czech Republic, 3Helmholtz Centre Berlin for Materials and Energy, Berlin, Germany
bjoern.hoffmann@mpl.mpg.de

Novel nanostructured materials from the fields of lighting and photovoltaics as well as complex biomaterials require the combination of several local analysis methods, like EDS, EBSD, EBIC and CL to acquire a full understanding. The combination of these techniques in one single SEM is state-of-the-art nowadays. To improve such a combined analysis to a new level, Raman spectroscopy can be integrated into the SEM to add the possibility of optical characterization. While a typical CLEM (correlative light electron microscopy) procedure uses consecutive imaging in a light and an electron microscope and thus needs a precise knowledge of coordinate reproduction, our approach aims for an all-in-one solution without the need of position rekognition. In our presentation we will present two realizations of Raman integration into a multifunctional Tescan FIB/SEM system and we will show measurements on different samples from the fields of functional semiconductor devices and life sciences.
The first approach utilizes a standard cathodoluminescence system with a parabolic mirror (Horiba CLUE). Two different lasers can be guided onto the mirror and thus can be focused onto the sample. The collected signal is guided to a Raman spectrometer.
The second and new approach integrates a complete optical Raman microscope onto the SEM chamber with the objective lens inside the vacuum – so called RISE correlative microscopy by TESCAN and WITEC companies [1].
In our presentation we will discuss the advantages and drawbacks of both realizations and we will show correlative analyses of samples like graphene covered GaN nanorods (see figure 1), GaN nanowires grown on graphene, thin-film tandem silicon solar cells, and biological materials.

[1] Announced at Analytica: 24th international trade fair for laboratory technology, analysis, biotechnology and analytica conference, München, April 2014.

The research leading to these results has received funding from the European Union Seventh Framework Program [FP7/2007-2013] under grant agreement n°280566, project UnivSEM, by the DFG via the research training group 1896 “In Situ Microscopy with Electrons, X-rays and Scanning Probes”, and the DFG research group FOR1616.

Fig. 1: SEM micrograph (left) and corresponding Raman maps of GaN nanorods (center) covered with graphene (right). This high-resolution Raman map was taken in-situ with the RISE correlative microscope in less than 40 minutes.

Fig. 2: SEM micrograph of GaN nanorods grown on sapphire covered with graphene (left) and Raman maps of the GaN (center) and defective graphene (right). A clear correlation between nanowire position and defective graphene was found.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3381 The removal of fluoride from the aqueous solutions using calcium-based minerals

Kim I. W.1, Yang T.2, Lee S. S.3, Jho J. Y.2
1Soongsil University, Seoul, Republic of Korea, 2Seoul National University, Seoul, Republic of Korea, 3Korea Institute of Science and Technology, Seoul, Republic of Korea
iwkim@ssu.ac.kr

Fluoride in water is a contaminant that could harmfully affect the human hard tissues by reconfiguring the biological apatite minerals. Since fluoride could be present in groundwater in high concentrations because of both geological and industrial reasons, diverse methods have been devised to remove the problematic contaminant. In this presentation, the fluoride removal by calcium-based minerals is displayed. Calcite (calcium carbonate) and brushite (dicalcium phosphate dihydrate) were used in the form of single crystals for the atomic force microscopy (AFM). The microscopic observation was complemented by the analysis of the solid phases by X-ray diffraction. In addition, the kinetics of the fluoride removal was monitored by measuring the fluoride concentration in the aqueous solutions. The fluoride removal was through the process of dissolution-and-recrystallization, through which the calcite and brushite were transformed into fluorite and fluorapatite, respectively. AFM was especially useful to understand the surface phenomena during the transformation. The dissolution could be easily discerned by the pit formation of calcite and brushite, and the recrystallized precipitation was also found on the surfaces of the single crystals. In addition, the interference caused by organic compounds on the fluoride uptake was studied, which had practical implications during the wastewater treatment. By combining the AFM and bulk experiments, the organic interference could be understood in terms of the changes in the boundary layers on the crystal surfaces. Especially, the AFM observation on the evolution of the dissolution pits indicated the alterations in the diffusion of calcium and fluoride ions through the boundary layers. The current study could have broad implications in the wastewater treatment as well as biomedical mineralization.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3389 Modern methods of correlative light-electron microscopy and their compatibility with modern methods of the three-dimensional electron microscopy

Mironov A. A.1, Beznoussenko G. V.1
1Istituto FIRC di Oncologia Molecolare. Milan. Italy
alexandre.mironov@ifom.eu

The final goal of any morphological study is to create the average 3D model of a structure and determine its chemical composition. On the other hand, the correlative light-electron microscopy (CLEM) is the method for study of rare structures or structures formed during rare quick events. CLEM should be combined with the three-dimensional electron microscopy (3DEM). Recently, CLEM became the leading edge of EM where dozens of protocols could be combined for different purposes. For instance, CLEM could be used for examination of 1) fixed or 2) live cells with subsequent fixation and EM analysis. The goal of CLEM could be to visualize 1) the cell (after microinjection or transfection) or 2) the organelle, which could be quickly moving (we first developed CLEM suitable for the examination under EM or organelles quickly moving in living cells and labelled with a fluorescent protein (Polishchuk et al. J Cell Biol. 2000. 148(1):45). CLEM could be based on sample processing using 1) two different procedures (we first proposed to combined two different protocols for CLEM: Mironov et al. Tsitologiia. 1987. 29(4):426) or 2) direct preparation for EM with subsequent analysis of this sample under LM and EM. Fixation of cells could be based on freezing or on the use of chemicals. On the other hand, 3DEM includes analysis of samples with the help of scanning electron microscopy (as pseudo 3DEM), metal replicas; stereo-pairs examined under transmission or scanning EM; titling series obtaining during tilting of the sample and presented as a movie; serial sections obtained after sectioning outside a chamber of an EM and examined in TEM or SEM; serial images obtained using serial bloc face SEM; serial images obtained using focused ion beam SEM and finally, TEM or SEM tomography (Fig. 1). Existence of several different methods of 3DEM represents one of the main difficulties for the modern CLEM because different 3DEM need different methods of preparation. Therefore, the selection of the correct method for the 3D reconstruction and immune-EM after CLEM represents not so trivial task. In the presentation, we will compare different methods of 3DEM and their compatibility with CLEM and in particular their suitability for the CLEM of intracellular organelles using exactly the same sample (the Golgi complex, an extremely complicated membrane organelle) for all of these combinations and search the best combination of the basic configuration of EM with different EM accessories and protocols of sample preparation for the achievement of the maximal resolution and simplification of methods of preparation of samples after CLEM suitable for EM tomography, FIBSEM and SBFSEM. We present a simplified protocol for sample preparation suitable for most of 3DEM.

We thank FIRC

Fig. 1: Methods of 3DEM
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3452 Correlative 3D imaging of bat penis micromorphology - validating quantitative microCT images with undecalcified serial ground section histomorphology

Herdina A. N.1, Plenk Jr H.2, Metscher B. D.1
1Department of Theoretical Biology, University of Vienna, Vienna, Austria, 2Bone and Biomaterials Research, Institute of Histology and Embryology, Medical University of Vienna, Vienna, Austria
annanele.herdina@univie.ac.at

Shape and dimensions of the bat baculum (os penis) are established taxonomic characters. Detailed micromorphology of penis and baculum, however, have seldom been studied in bats [1]. The present preliminary study provides new insights into the 2D and 3D micromorphology of the Pipistrellus pipistrellus penis as a foundation for further functional research. Light microscopy of serial, surface-stained, undecalcified ground sections [1] of the penes of 3 P. pipistrellus specimens (1 of them sub-adult) was compared with microCT images (some penes iodine-stained [2]) of the bacula of P. pipistrellus (n=42, three of them sub-adult), P. pygmaeus (n=24), P. hanaki (n=9), and P. nathusii (n=11). The baculum in the studied species consists of a proximal base with two club-shaped branches, a long, slender shaft, and a small, forked distal tip. Proximally, entheses connect the corpora cavernosa to the branches, which consist of woven bone and contain a medullary cavity of variable size with fatty marrow. The shaft of the baculum consists of lamellar bone around a central vascular canal of variable length, surrounded by woven bone in the proximal part of the shaft. The distal end of the shaft consists of woven bone. The urethra, surrounded by the corpus spongiosum, lies ventral of the corpora cavernosa and the baculum. The dorsal half of the urethral meatus is encased by the forked distal tip of the baculum. The glans penis is made up mostly of an enlarged part of the corpus spongiosum, surrounding the baculum and urethra. In the sub-adult bats, the baculum appeared not to be fully developed. The proximal branches of the baculum where shorter and did not contain a marrow cavity, while distal tip seemed to be fully developed. The combination with histomorphological techniques enabled a more precise interpretation of the histological structures shown in microCT images from all four Pipistrellus species. The woven bone predominance in the baculum points to a tight functional connection with the surrounding erectile tissues.

References

[1] Herdina AN, Herzig-Straschil B, Hilgers H, Metscher BD, Plenk Jr. H. 2010. Histomorphology of the penis bone (baculum) in the gray long-eared bat Plecotus austriacus (Chiroptera, Vespertilionidae). The Anatomical Record 293: 1248–1258.

[2] Metscher BD. 2013.Biological applications of X-ray microtomography: imaging microanatomy, molecular expression and organismal diversity. Microscopy and Analysis, 27, 13–16.

We thank Petr Benda, the National Museum (Natural History), Prague, and Peter H.C. Lina, Naturalis Biodiversity Center, Leiden, for providing samples; and Gerd B. Müller, Department of Theoretical Biology, University of Vienna, for providing resources. Partial funding was provided by a Marietta Blau Fellowship, granted to ANH by the Austrian Federal Ministry of Science and Research and the OeAD.

Fig. 1: 3D volume renderings of microCT scan of iodine stained Pipistrellus pipistrellus penis. Left: ventral view, right: lateral view.

Fig. 2: 3D volume rendering of iodine stained distal tip of Pipistrellus pipistrellus baculum and surrounding soft tissue (ba: baculum, pr: preputium, ur: urethral meatus).

Fig. 3: Surface-stained (Giemsa stain) undecalcified ground section of distal tip of Pipistrellus pipistrellus penis (ba: baculum, gl: glans penis, pr: preputium, ur: urethral meatus).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3476 Confocal and electron microscopy for evaluation of delignification kinetics of Agave fibers by using green technology

Hernández Hernández H. M.1, Chanona Pérez J. J.1, Terrés E.2, Vega A.3, Ligero P.3, Calderón Domínguez G.1, Mendoza Pérez J. A.1
1Escuela Nacional de Ciencias Biológicas, IPN, Mexico City, 2Instituto Mexicano del Petróleo, Mexico City, 3Universidade da Coruña, Coruña España
jorge_chanona@hotmail.com

The use of organosolv pulping represents an alternative to traditional methods to obtain cellulose pulp from agro-wastes. It has been studied the use of different kinds of Agave species such as A. sisalana and A. tequilana to produce paper, but not Agave atrovirens, that is used in the “mescal” production. The wastes of this process can be a good source of cellulose and lignin. The aim of this work was study microstructural changes during kinetic of Acetosolv and Milox (green chemical methods), to produce cellulose derivatives from agave wastes. Fibers were obtained from leaves of A. atrovirens through pre-treatments that included drying at 60 °C and 3 m/s air, milling and fiber separation through mesh. A previous experimental design provide the delignification process conditions for Milox kinetics of fibers were time of 180 min, a formic acid concentration of 80% and a H2O2 concentration of 2%. Acetosolv kinetics were time of 30 min, acetic acid concentration of 80% and 0.3% of HCl. Pulps were studied by SEM (XL 30, Philips, USA) to observe overall microstructural changes during kinetic delignification of the fibers. Also it was possible to monitor the changes on lignin and cellulose in the fibers during the organosolv process by CLSM (LSM 710, Carl Zeiss, Germany). Figure 1 shows SEM and confocal images of the Acetosolv process, there, it can be observed the ordered arrangement of the bundle of the fibers with lower time treatment (Figure 1A and B). In all CLSM images, blue color is the fluorescence of stained cellulose (MR2 with calcoflour white), while green is the autofluorescence of lignin. Higher time treatment shows a collapsing of arrangement of the fibers (Figure 1C, D and E). In the case of Milox kinetic these images shown in Figure 2, it is possible to see that the fibers are separating mostly with the increasing reaction time. At the beginning of the kinetic the bundle of fibers are complete but in function of the reaction time the separation of the fibers increased until complete liberation of microfibrils at the end of the Milox kinetic (figure 2A-E). Agave fibers show a low content of lignin, even so, can be solubilized by this acidic organosolv processes. Both pulps were achieved with very high purity of cellulose and CLSM images showed that Milox was the better process for delignification of agave fibers, but at the expense of oxidizing conditions that compromise the length of the cellulose chain. Therefore, the applied microscopy techniques in this work were useful to monitor the microstructural changes occurring during the pulping processing and they were efficient for select the better process conditions for delignification of Agave fibers.

Hilda M. Hernández wishes to thanks CONACyT for the scholarship provided to stay in Coruña, Spain. Project SIP-IPN 20131864, 20130333 and 20140387. Dpto. Química Física e Enxeñería Química I. Facultade da Ciencias. Universidade da Coruña. A Coruña España.

Fig. 1: SEM and confocal images of Acetosolv pulping at different process time.

Fig. 2: SEM and confocal images of Milox pulping at different process time
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-3514 Characterization of hESC surface receptors using correlative fluorescence and electron microscopy

Jaroš J.1, Košťál V.2, Hampl A.1
1Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 3, Brno, 625 00, Czech Republic, 2TESCAN ORSAY HOLDING, a.s., Libusina tr. 21, Brno, 623 00, Czech Republic
vratislav.kostal@tescan.cz

Human embryonic stem cells (hESC) have become an important model for studying embryonic development, drug testing and disease modelling. The abilities of hESC to self-renewal and differentiation into multiple specialized cells make them promising candidates also for regenerative medicine, such as tissue regeneration in Parkinson disease, spinal cord injuries, and many more. However, all the therapies require stem cell growth in precisely controlled conditions, which influence cell behavior through the contact of cell receptors with the environment. Surface receptors are important family of proteins responsible for modulation of many stem cell functions, such as cell adhesion, differentiation and migration. Understanding of the localization, structure and function of the surface receptors is important for designing robust niche for sustained growth of hESC. Correlative light and electron microscopy (CLEM) allows correlating functional data obtained by fluorescence microscopy with a structural data collected by a high-resolution scanning electron microscope (SEM) in the same region of interest. In this work, we use the CLEM approach for visualization of receptors on the surface of hESC cells. The cells were immunolabelled with a primary antibody against specific adhesion proteins and clusters of differentiation followed by labeling with combined fluorescent and gold conjugated secondary antibody. The cells were observed in the fluorescence microscope to localize the proteins on the cell surfaces. After the collection of fluorescence images, the cells were refixed with glutaraldehyde, silver enhanced, dehydrated and carbon coated. Finally, they were loaded into an ultra-high resolution SEM. The stage was navigated to the regions of interest selected previously by fluorescence microscopy. The specificity of the antibody labeling was confirmed by observing the silver enhanced nanoparticles in the backscattered electron detector. The structural information of the receptors was obtained using the secondary electron signals. A dedicated software module for correlative microscopy (TESCAN Coral) was used to correlate the data from both sources in real time. Using this approach, changes in the structure of the receptors are being studied in respect to different extracellular conditions of the hESC culture.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-5720 FIB/SEM and correlative ArraySEM - NanoSIMS study on toxic internalization of hemoglobin by endothelial cells

Bittermann A. G.1, Schaer D.2, Ehrke H. U.3, Hillion F.4, Wepf R.1
1ScopeM ETH Zürich, Swiss Federal Institute of Technology Zurich, Switzerland, 2Division of Internal Medicine, University Hospital Zurich, Switzerland, 3CAMECA GmbH, Unterschleissheim/Munich, Germany, 4CAMECA SAS, Gennevilliers, France
annegreet.bittermann@scopem.ethz.ch

Free hemoglobin (Hb) forms under oxidizing conditions large protein aggregates, which are heavily and actively taken up by endothelial cells: Membrane ruffles reach out to grab these particles. Several membrane lamella try to wrap them independently and pull them into the cytoplasm. In the final toxic stage the aggregates fill up the cell volume, just separated by thin lamellas. As several internalization processes take place simultaneously, the definition of extracellular versus intracellular space is sometimes difficult and only 3D analysis by targeted FIB/SEM can help to answer the “in-out” question. While lamella formation at the cell surface is clearly visible on SEM-images of freeze dried cells, the internal membrane channels & cavities show up only in the FIB/SEM-cross section. The tomographic data allows to virtual blend out the hemoglobin particles for studying membrane features.

The hemoglobin precipitates are showing up in the micrographs as heavily electron dense particles. To finally proof their nature and off-spring from hemoglobin, elemental mapping for Fe was performed. While EDX, EELS and ToF-SIMS failed to detect iron in these protein-complexes due to sensitivity limitations, imaging mass spectrometry (MS) maps and local measurements became possible with a dedicated NanoSIMS tool below a Fe-concentration of 0.5mM +/- 0,03mM.

Endothelial cells in a body form normally the walls of blood vessels; in culture they are flat adherent cells, growing tight and overlapping. In order to study Hb toxicity, primary human endothelial cells were grown up to confluency on cover slips. The cultures were incubated under oxidizing conditions (GOX) with glucose and 2mg/ml hemoglobin. Under these conditions Hb aggregates heavily. The aggregates get internalized by the endothelial cells. Aggregate formation and uptake was stopped with 2,5% glutaraldehyde/PBS, followed by osmification, dehydration, resin impregnation & polymerization (thin layer plastification) and montage. By keeping the resin layer as thin as possible, hemoglobin precipitates on cells could easily be spotted in the SEM by their prominent topography and FIB/SEM tomography was performed at these preselected spots for a detailed 3D-view on internalization. Image processing allowed to uncover the membrane structures at the internalization site in silico.

Microtome ultrathin sections of block embedded cell were deposited onto ZnO-coated glass slides for SEM observation to define an area of interest for final mass spectrometry analysis. With a CAMECA NanoSIMS we were able to detect Fe (iron) within these hemoglobin aggregates and other cellular ions i.e. phosphate or chloride at subcellular resolution and physiological concentration.

Fig. 1: FIB/SEM tomography – top: principle of FIB/SEM tomography on thin layer plastified endothelial cell culture; gallery view of a selection of serial cross sections; section view of a single cell – bottom left: volume representation & bottom right: 3D-model of the internalization site after virtual removal of the precipitates.

Fig. 2: Microtome section on ZnO-coated glass – left: array scan of selected region. Endothelial cell w. ultrastructure, featuring internalized haemoglobin precipitates. – right: NanoSIMS maps of Carbonitrate, Phospate, Chloride, Iron. Concentration measurements for Fe: 1) 1,1 mM 2) 0,9mM 3) 0,5mM 4) 4,6mM 5) 3,7mM 6) 3,6mM 7) 4,8mM.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-5872 Using subcellular spatial alignment for live-cell CLEM to investigate mitochondrial degradation

Padman B. S.1,2, Bach M.1,2, Ramm G.1,2
1Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia, 2Monash Micro Imaging, Monash University, Melbourne, Victoria 3800, Australia
georg.ramm@monash.edu

Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructural dynamics of cells. However, the correlation of subcellular structures observed by live cell imaging with the EM ultrastructure remains technically challenging. In order to optimise the workflow for live-cell CLEM, we have designed a modifiable imaging surface for cellular optical imaging [1]. We first investigated the suitability of polymer films as a support film for live cell imaging. In contrast to glass coverslips, the use of polymer films not only allows for subsequent cryo-preservation, but also allowed us to directly adapt the surface of the film for correlative imaging. We made use of an office printer and laminator to mark the polymer films with a toner-based reference grid, which is already visible by eye. The toner also forms part of a multiscalar fiducial reference system which enables subcellular spatial alignment. We have used this CLEM-strategy to investigate the fate of mitochondria during induced mitochondrial degradation by mitophagy [2]. Mitophagy is a selective pathway that targets and delivers mitochondria to the lysosomes for degradation. The protonophore CCCP causes depolarization of mitochondria and induces their degradation by mitophagy. When mammalian cells overexpress the ubiquitin ligase Parkin, treatment with CCCP for more than 24 hours has been reported to trigger the clearance of all mitochondria. However, using CLEM in Parkin-expressing HeLa cells, we show that mitochondrial remnants remain present in the cell. The mitochondria were no longer easily identifiable as such due to morphological alterations, providing a possible explanation why earlier EM studies may have missed these structures. Further investigation by live-cell microscopy showed that CCCP inhibits mitophagy at both the initiation and lysosomal degradation stages. In summary, we have developed an inexpensive and robust CLEM procedure that simplifies optical imaging without limiting the choice of optical microscope. We have verified the technique by providing novel biological insights into the mechanism of mitochondrial degradation.

[1] B. S. Padman, M. Bach and G. Ramm. 2014 An Improved Procedure for Subcellular Spatial Alignment during Live-Cell CLEM. PloS one 9 (4), e95967. [2] B. S. Padman, M.Bach, G. Lucarelli, M. Prescott and G.Ramm. 2013 The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells. Autophagy 9(11), 1862-1875.

This work has been supported by the National Health and Medical Research Council under project grant 596849. We would also like to thank Stephen Firth, Alex Fulcher and July Callaghan from Monash Micro Imaging for help with optical microscopy.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-5879 Super-Resolution Microscopy Using Standard Fluorescent Proteins in Intact Cells under Cryo-Conditions

Kaufmann R.1,2, Schellenberger P.1, Seiradake E.1, Dobbie I. M.2, Jones E. Y.1, Davis I.2, Hagen C.1, Grünewald K.1
1Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK, 2Department of Biochemistry, University of Oxford, Oxford, UK
rainer@strubi.ox.ac.uk

To study the fine details of biological structures with microscopy, both the preservation of these structures during the preparation process and the achievable resolution of the imaging system are of equal importance. In the field of fluorescence microscopy, the latter has been addressed by various super-resolution methods that have been developed in recent years to overcome the diffraction-limited resolution of light microscopy. Super-resolution imaging in living cells remains very challenging. Typically, chemical fixation of the samples is required to achieve the best technical results, but unfortunately, this is associated with structural changes in the sample [1], especially at a size range that is relevant for light microscopic techniques achieving resolutions below the diffraction limit [2]. A preferable alternative is vitrification (i.e., cryo-immobilizing the structure in glasslike amorphous ice using rapid freezing techniques) that preserves the structures in a near-native state and is frequently used in the fields of electron and X-ray cryo-microscopy [3,4]. The advantages of vitrified specimens have not been fully exploited to date in fluorescence microscopy of subcellular structures. This is because one of the biggest challenges for fluorescence cryo-microscopy is currently its limited resolution of 400-500 nm due to the inherent technical challenges of the setup and in particular the lack of high NA cryo-immersion objectives [5].

We introduce a super-resolution technique for fluorescence cryo-microscopy based on photo-switching of standard fluorescent proteins in intact mammalian cells at low temperature (81 K) [6]. We demonstrate that the single molecule characteristics of reversible photobleaching of mEGFP and mVenus at liquid nitrogen temperature are suitable for the basic concept of single molecule localization microscopy. We show that single molecule localization microscopy is possible at cryo-conditions and achieve super-resolution imaging of vitrified biological samples with a structural resolution of ~125 nm (average single molecule localization accuracy ~40 nm), corresponding to a 3–5 fold resolution improvement. We expect that super-resolution cryo-microscopy will become a valuable imaging method for cryo-immobilized biological samples that is highly complementary to electron and X-ray cryo-microscopy for the study of cellular and subcellular complexity.

References:

[1] Bleck et al., J. Microsc. 2010, 237 (1), 23−38.

[2] Weinhausen et al., Phys. Rev. Lett. 2014, 112 (8), 088102.

[3] Hurbain et al., Biol. Cell 2011, 103 (9), 405−420.

[4] Schneider et al., J. Struct. Biol. 2012, 177 (2), 212−223.

[5] Briegel et al., J. Methods Enzymol. 2010, 481, 317−341.

[6] Kaufmann et al., Nano Lett. 2014, in press.

Fig. 1: Single molecule super-resolution cryo-microscopy. Photoswitching of single fluorescent molecules at low temperature (81 K) enables single molecule localization microscopy of vitrified biological samples. Compared to basic wide-field fluorescence cryo-microscopy the resolution is improved by a factor of 3-5, achieving values in the 100 nm range [6].
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-1-P-6023 Correlative Automated Particle Analysis – A New Way to Link LM and SEM/EDX in an Automated Workflow.

Hiltl M.1, Berger C.1
1Carl Zeiss Microscopy GmbH, Oberkochen, Germany
michael.hiltl@zeiss.com

Correlative automated particle analysis (CAPA) is a new software tool in the field of automated particle analysis designed to link LM (Light Microscope) and SEM/EDX (Scanning Electron Microscope/Energy dispersive X-ray) in one workflow. It is grown from correlative microscopy with the motivation to fulfill the increasing demand to analyze particles, debris or grains, which has constantly increased in fields like steel inclusion, gunshot residue, implant monitoring, mineralogical grains or environmental/pollution control over the past years. But especially automotive production/research, automotive related industry as well as component supplier have targeted automated particle analysis as an useful tool to control the technical cleanliness and to identify critical particles helping their elimination. Particle residues can cause severe damage with the consequence of additional cost, loss of reputation, wasting resources as well as harming the environment. Consequently, the overall goal is to identify the source of contamination as quick as possible. The LM investigation results in the determination of morphological parameters and in additional information about reflective and non-reflective properties of possible dangerous residues. However, another very important question cannot be answered: What is the chemical composition of the particle and, therefore, what are the mechanical and physical properties. In this context, transferring the sample from the LM to a SEM and relocating the particles of interest manually can be very time consuming and very cost intensive. The new developed correlative automated particle analysis (CAPA) makes it very easy to correlate the particles from LM to SEM leading to a quick and easy analytical characterization of critical particles and it gives the possibility to identify the source of contamination in the production chain and for eliminating quality issues. Both microscopic technologies have strengths and limitations.In conclusion, CAPA combines the strengths of both methods generating automatically a detailed and ISO confirm report.

↑ Go to the Table of content ↑

ID-2. Imaging mass spectrometry

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-2-IN-5775 Biomedical chemical imaging using atmospheric pressure imaging mass microscope “iMScope”

Setou M.1
1Hamamatsu University School of Medicine, Department of Cell Biology and Anatomy 1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan
setou@hama-med.ac.jp

Microscopy methods are used as investigative tools of the surrounding world, offering images of detailed areas that
outline morphology or spatial organization. For this purpose, external beams of particles and interaction fields are
used, such as light with different properties, X-rays, electron or ion beams, resonance magnetic fields and others.
Following a different approach, mass spectrometry methods use ions extracted from the sample itself to draw pictures
that show not only morphology, but also the chemical distribution of the sample surface. A mass spectrometer using an
optical microscope for defining the area of analysis [1] was developed in our group in cooperation with Shimadzu [2]
and released recently under the trade name iMScope [3]. A high repetition rate 1kHz, well-focused UV laser beam is used
for matrix assisted laser desorption/ionization (MALDI) of the sample which is kept at atmospheric pressure (AP) and
raster moved by a precision stage. The instrument allows obtain chemical maps with 5 µm high spatial resolution for
targeted biomedical sample regions while software-superposing histological images and other kind of information. The
imaging mass spectrometry (IMS) characterization technique, combined with different methods and materials for the MALDI
matrix deposition, has been used for molecular profiling of various tissues in animal models and human samples, for
identification of lipids, proteins and peptides, drug and metabolites, and for search of chemical compound biomarkers,
using both normal tissue and aging related/disease/cancer tissue modifications. After outlining the particular features
of the IMS instrument, the presentation will proceed to show results of the measurements and discuss their significance
as biomedical findings.


References
1. T. Harada et al., Visualization of volatile substances in different organelles with an atmospheric-pressure
mass microscope. Anal. Chem. 2009, 81, 9153–9157.
2. Mitsutoshi Setou et al, Mass spectrometer, US 7,759,640 B2 (Jul. 20, 2010).
3. Imaging Mass Microscope iMScope released in 2013 by Shimazu Corporation, http://www.shimadzu.com.au/imscope.

Acknowledgements
The research for developing the mass microscope was supported by a grant-in-aid to M.S., SENTAN program “Development of
Advanced Measurement and Analysis Systems, Development and optimization of mass microscope” of the JST Agency.

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-2-IN-6079 Mass spectrometry imaging: approaching cellular resolution for highly specific molecular information

Römpp A.1, Bhandari D.1, Huber K.1, Spengler B.1
1Institute of Inorganic and Analytical Chemistry, Justus Liebig University, Schubertstrasse 60, Giessen/D, Germany
andreas.roempp@anorg.chemie.uni-giessen.de

Mass spectrometry imaging (MS imaging) is the method of scanning a sample of interest and generating an image of the intensity distribution of a specific analyte ion. In contrast to most histochemical techniques, mass spectrometry imaging can differentiate (amino acid) modifications and does not require labelling of compounds. Our work is focused on obtaining reliable chemical information and on increasing the spatial resolution in order to detect (sub)cellular features. Here we present a number of improvements in instrumentation, sample preparation, measurement parameters and data processing.

MS imaging experiments were performed with a high resolution atmospheric-pressure imaging source (AP-SMALDI10, TransMIT GmbH, Giessen) attached to ‘LTQ Orbitrap’, ‘Exactive Orbitrap’ or ‘Q Exactive’ mass spectrometers (Thermo Scientific GmbH, Bremen). Pixel size was between 2 and 10 µm. Mass accuracy was better than 2 ppm (root mean square) under imaging conditions [1,2]. Tentative identification based on accurate mass was confirmed by on-tissue MS/MS experiments.

The capabilities and characteristics of our method will be discussed on a number of applications. Phospholipids and smaller metabolites such as nucleic acids and cholesterol were imaged in single cells at 7 µm pixel size. Phospholipids were investigated in detail in human tumor biopsies. The lateral ventricle region of a coronal mouse brain section was imaged at 2 µm pixel size. Non-mammalian tissue samples often require specific sample preparation. This will be discussed on the example of metabolite imaging in plant tissue sections.

MS image analysis for all these experiments showed excellent agreement with histological staining evaluation. In addition it provided highly specific molecular information. In many cases signals with very similar mass (∆m/z<0.1) showed distinctly different distributions, which demonstrates the need for high mass resolution in order to obtain reliable information from MS imaging experiments of complex biological samples.

Newest developments in instrumentation and methodology will be demonstrated. This includes strategies for increased measurement speed, dynamic range for MS image generation of more than three orders of magnitude and simultaneous detection of positive and negative ions in one experiment.

General trends and developments in the field of mass spectrometry will be briefly discussed. This includes strategies for flexible data analysis on the basis of the data format imzML (www.imzml.org) and activities in the framework of COST action (European Cooperation in Science and Technology) „Mass Spectrometry Imaging: New Tools for Healthcare Research” (BM1104).

[1] Römpp, Guenther, Schober, Schulz, Takats, Kummer, Spengler (2010) Angew. Chem. Int. Ed. 49 (22):3834-3838.

[2] Römpp, Spengler (2013) Histochemistry and Cell Biology 139(6): 759-783.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-2-P-1506 Imaging mass spectrometry using ultra-high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometer, SpiralTOF

Satoh T.1, Kubo A.1, Moriguchi N.2, Hazama H.2, Awazu K.2, Toyoda M.3
1JEOL Ltd., 2Graduate School of Engineering, Osaka University, 3Graduate School of Science, Osaka University
taksatoh@jeol.co.jp

Introduction
Imaging mass spectrometry (IMS) has been used for biological applications, to assess the distribution of proteins, peptides, lipids, drugs, and their metabolites in a tissue specimen. IMS has expanded during the last decade using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer, which adopted a linear and a reflectron ion optical systems. A reflectron MALDI-TOF mass spectrometer, using a delayed extraction technique, has higher mass resolution than linear MALDI-TOF mass spectrometer. However, its high mass resolution is available only within limited mass range, which isn’t sufficient for analysis in low-molecular compounds such as lipids, drugs and drug metabolites. It is necessary to extend flight path length to improve mass resolution and mass accuracy in wide mass range. However, the flight path length of a reflectron TOF mass spectrometer is limited by its instrument size, and is difficult to be extended beyond certain length restricted by the instrument dimension. We developed a MALDI-TOF mass spectrometer with a spiral ion trajectory, SpiralTOF, to solve the issue. It has 17 m flight path length within a cubic vacuum housing of approximately 0.6m x 0.6m x 0.7m.

Instrumentation
The schematic of SpiralTOF, which consists of four toroidal electrostatic sectors, is shown in Fig. 1. Each has eight stories made by nine Matsuda plates piled up inside a cylindrical electrostatic sector. The ions pass the four toroidal electrostatic sectors sequentially and revolve along a figure-eight-shaped orbit on a certain projection plane. During multiple revolutions, the ion trajectory shifts perpendicular to the projection plane every revolution cycle, thus generating a spiral trajectory. The flight path length of one revolution is 2.1 m. The total flight path of SpiralTOF was 17 m, which is 5-10 times longer than a reflectron TOF mass spectrometer.

Results & Discussion
SpiralTOF achieved ultra-high mass resolution that could separate isobaric compounds, which differed only 0.1 u each other. The advantage of isobaric separation in IMS will be shown to take the IMS for lipids distribution on mouse brain tissue section as an example. The isobaric mass separation at m/z 820 – 825 is shown in Fig. 2. Three types of lipid peaks were well separated in mass spectrum and could show the different localization respectively. The high selectivity for drawing mass image is important for understanding clear localization of compounds, especially in low mass region. Further IMS measurements for drugs distribution on mouse brain tissue sections will be reported in the presentation.

Fig. 1: Schematic of time-of-flight mass spectrometer with spiral ion trajectory (SpiralTOF). The outer electrode of the left-top electrode is not included to show the ion trajectory (red line).

Fig. 2: Ultra-high resolution mass spectrum at m/z 820–825 in imaging mass spectrometry for lipids in a mouse brain tissue section. Three types of lipids were well separated in mass spectrum and could draw different mass images from them.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-2-P-1518 Imaging mass spectrometry and depth profiling for organic thin films using laser desorption ionization.

Satoh T.1, Shima M.1, Niim H.1, Nakajima Y.2, Fujii M.3, Seki T.4, Matsuo J.3
1JEOL Ltd., 2Asahi Glass Co., Ltd., 3Quantum Science and Engineering Center, Kyoto University, 4Department of Nuclear Engineering, Kyoto University
taksatoh@jeol.co.jp

Introduction
The electrical devices composed of organic and inorganic materials such as organic light-emitting diodes (OLED) devices have been widely used. There are various techniques for surface analysis, a scanning electron microscopy/energy dispersive x-ray spectroscopy (SEM/EDS), electron probe microanalysis (EPMA), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (TOF-SIMS) and so on. However, the analytical techniques to obtain the molecular information on organic layers are limited. In this presentation, we will discuss about the availability of the imaging mass spectrometry (IMS) and depth profiling using laser desorption/ionization time-of-flight mass spectrometer (LDI-TOFMS) as for analyzing organic thin films.

Methods
Three types of OLED compounds thin films deposited on Si wafer were prepared,
(i) α-NPD/Si: α-NPD deposited on Si wafer with 600 nm thickness.
(ii) α-NPD/2-TNATA/Si: 2-TNATA was deposited on Si wafer with 700 nm thickness. The α-NPD was deposited above the 2-TNATA layer.
(iii) α-NPD/2-TNATA/Si(mesh): 2-TNATA deposited on Si wafer with 440 nm thickness. The 880 nm α-NPD was deposited making a mesh pattern of 55 lines per inch above the 2-TNATA layer. The IMS and depth profiling were performed with MALDI-TOFMS (JMS-S3000, JEOL). The α-NPD/Si was also measured with SEM/EDS (JSM-7001FTTLLV, JEOL), XPS (JPS-9010, JEOL) and TOF-SIMS (Ar gas cluster ion source developed in Kyoto Univ. was applied to JEOL’s JMS-T100LP) for supplement measurements.

Results & Discussion
The [M]+ ions of α-NPD with negligibly small fragment ions were observed from α-NPD/Si with LDI-TOFMS(Fig.1). In the case of the TOF-SIMS, not only [M]+ ions but also many kinds of fragment ions were observed. The LDI-TOFMS and TOF-SIMS had an advantage for organic compounds analysis compared to SEM/EDS and XPS which could only obtain elemental or chemical state information. The LDI-TOFMS has lower spatial resolution rather than TOF-SIMS, but clear mass spectrum obtained with LDI-TOFMS has the advantage in degradation analysis, which the measurements of minor components will be often important.
Depth profiling was estimated with two layered thin film: α-NPD/2-TNATA/Si. The accession to the boundary of two layers could understand by turnover of the ion intensities of α-NPD and 2-TNATA(Fig.2). The ionization region in depth direction was depended on the laser intensity. The several hundred nanometer layer structure was clearly observed in appropriate laser intensity. Further investigation about the IMS and depth profiling by changing the laser condition using α-NPD/2-TNATA/Si(mesh) will be given in the presentation.

Fig. 1: Mass spectrum obtained from α-NPD thin film with LDI-TOFMS. The [M]+ ions of α-NPD with negligibly small fragment ions were observed.

Fig. 2: Ion intensities variation of α-NPD and 2-TNATA obtained from two layered thin film: α-NPD/2-TNATA/Si.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-2-P-2185 Spatial and temporal resolved quantification of Gadolinium containing Magnetic Resonance Imaging Contrast Agents by MALDI Imaging Mass Spectrometry

Aichler M.1, Wildgruber M.2, Huber K.1, Kosanke K.2, Rummeny E.2, Walch A.1
1Institute of Pathology - Research Unit Analytical Pathology, Helmholtz Zentrum München, Oberschleißheim, Germany, 2Department of Radiology, Klinikum rechts der Isar, Technische Universität München, München, Germany
michaela.aichler@helmholtz-muenchen.de

Magnetic resonance imaging (MRI) has established itself as one of the best techniques for cardiovascular diagnosis. The ability of MRI of differentiating soft tissues is due to the contrast in MR images which is the result of a complex interplay of numerous physical and chemical factors. Remarkable improvements in medical diagnosis in terms of higher specificity, better tissue characterization, reduction of artifacts, and functional information have been achieved through the administration of suitable MRI contrast agents. For instance, to enhance the MR image contrast, patients are administered paramagnetic substances such as gadolinium (Gd) (III)-based contrast agents.

One of the major challenges in the use of such contrast agents is that there are still substantial uncertainties concerning the relationship between the local contrast agent concentration and the measured signal variation in MRI. Quantitative analysis of e.g. myocardial perfusion by the contrast agent can be performed but requires complex mathematical modeling.

We evaluated MALDI Imaging mass spectrometry for spatially resolved ex vivo quantification of a gadolinium-based magnetic resonance agent in correlation to in vivo MRI. Therefore, in vivo deposition of this contrast agent was investigated in a mouse model of myocardial infarction. Mice were screened by in vivo MRI at 6h, 24h and 48h after injection of the contrast agent. The animals were sacrificed after each time point and hearts were prepared for quantitative assessment by MALDI Imaging. MALDI Imaging was able to corroborate the in vivo imaging results and enabled in situ quantification of the in vivo applied contrast agent with high spatial resolution. The quantitative results of MALDI Imaging correlated well with in vivo MRI signal intensities. In this study we demonstrated that MALDI Imaging is able to provide a mass spectrometry-based quantification of gadolinium containing contrast agents in situ with high spatial resolution. We furthermore evaluate MALDI Imaging as a general useful tool for tissue kinetics for gadolinium-containing contrast agents. Our data demonstrated that MALDI Imaging may be helpful in future for a deeper understanding of tissue kinetics in contrast agent development.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-2-P-2441 NanoSIMS and Electron Microscopy: A powerful combination in Life Science research

Gilis M.1, 2, Cohen S.1, Kopp C.1, Lekieffre C.1, Escrig S.1, Loussert C.3, Mucciolo A.3, Humbel B. M.3, Meibom A.1, 2
1Laboratory for Biological Geochemistry, School of Architecture, Civil and Environmental Engineering (ENAC), École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland, 2Center for Advanced Surface Analysis, Institute of Earth Sciences, University of Lausanne, Lausanne, Switzerland, 3Electron Microscopy Facility, University of Lausanne, Lausanne, Switzerland
melany.gilis@epfl.ch

Nano-scale imaging by secondary ion mass spectrometry (NanoSIMS) is a cutting-edge method to visualize and quantify the assimilation, storage and transfer of metabolites, labeled with stable isotopes, within biological samples at subcellular scales. Pre-imaging of fixed and resin embedded samples with scanning- (SEM) or transmission- (TEM) electron microscopy allows recognition of areas of interest at the ultrastructural level.

Images of the isotope distribution obtained with the NanoSIMS instrument on the identical section of the sample can then be correlated with the electron micrographs. The choice of the sample preparation and correlative observation method, SEM or TEM, depends on the spatial resolution required and the nature of the specimen. Pre-imaging in SEM of a sample block face that was previously trimmed with an ultra-microtome is an easy and fast approach for investigation of large tissue or whole unicellular organisms (up to several hundreds of microns). In this way, even partially mineralized samples that are difficult or impossible to section can be analyzed. For higher-resolution and subcellular observations, TEM analysis of ultrathin resin sections (< 100 nm in thickness) have to be performed. Compared to SEM, TEM has a much smaller field of view, however, automatic acquisition of multiple, overlapping images in the TEM allows reconstruction of an entire TEM section at high spatial resolution.

Here, we illustrate the power of combining NanoSIMS and electron microscopy in Life Sciences to study trafficking of microbial factors in a beneficial host - bacteria symbiosis and for tracking metabolic fluxes of 13C- and 15N- labeled molecules in marine unicellular organisms or in small invertebrates.

This work was supported by European Research Council Advanced Grant 246749 (BIOCARB), FNS grant CR23I2_141048, and by the Ecole Polytechnique Fédérale de Lausanne to AM. We are grateful for access to, and expert help with electron microscopy at EMF center of Pr. Bruno Humbel from University of Lausanne.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-2-P-5810 High Resolution MALDI/LDI FTICR Mass Spectrometry Imaging of Plant Tissues

Takahashi K.1
1National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan
takahashi-k@aist.go.jp

Recently MALDI mass spectrometry imaging (MSI) has been a powerful tool to map spatial distribution of molecules on the surface of biological materials. Frequently MSI has been applied to animal tissue slices to map various biological molecules on the slice. However, most recently, it has been also applied to plant tissue analysis. We’ve been developing high resolution and high accuracy mass spectrometer dedicated for mass spectrometry imaging of plant tissues and reported the successful MSI results coming from thin slice of young leaf of Arabidopsis thaliana.

Because it’s been still difficult to make thin slices from tissues of small plants such as Arabidopsis thaliana, we tried to glue small intact tissues of plants such as leafs, roots or sprouts onto a small transparent ITO-coated slide glass instead. The intact tissues were then vacuum dried and matrix substance was applied by sublimation prior to mass spectrometry imaging experiment. The tightly focused UV-LASER beam was irradiated, inside in-house build vacuum ion source chamber, onto the matrix-coated sample surface and m/z of the produced ions were measured by commercial FTICR-MS (Bruker Daltonics Inc.; Apex-Qe-94T).

Molecular ions of various metabolites including glucosinolates and anthocyanins were observed and their spatial distribution in the tissues was mapped successfully. We also found that some of the metabolite ions were even observed when UV-LASER was irradiated onto the non-matrix coated surface of dried Arabidopsis tissues. From the LDI-MSI experiments, we found the spatial distribution of several molecules was changed in the tip of the root after auxin treatment.

This work was supported by MEXT Japan Grant-in-Aid for Scientific Research, Perceptive Plants.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-2-P-5811 LabMSI: a Software Tool to Analyze High Resolution Mass Spectrometry Imaging Data

Takahashi K.1
1National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan
takahashi-k@aist.go.jp

We’ve developed high resolution mass spectrometry imaging instrument, modifying commercial FTICR-MS with in-house build vacuum MALDI ion source. With this instrument one can acquire more than 10,000 mass spectra sequentially from small region of plant tissues glued on small slide glass. Several thousands of peaks were then picked from each spectrum, and then, line spectra were converted into imzML formatted data cube.

The imzML formatted data cube was then thin sliced according to m/z axis to make spatial map of molecules which have same m/z. When we acquired mass spectrum with the resolution higher than 10,000, the typical mass bin size the make spatial map decreases to 0.01 or less. To investigate huge number of maps to find molecules showing similar / different spatial distribution, human eye inspection is impossible to be used. When we want to see profile mass spectra without peak picking, we need to access several hundred GB data cube file. There was no software to allow to work with such huge data file.

To support the analysis of high resolution mass data cube, we’ve developed in-house software “LabMSI”. This software was developed using LabView released from National Instruments Corp. With LabMSI, one can access profile imzML file over 100GB to calculate average mass spectrum and pick peaks from average spectrum to make spatial maps corresponding to picked peaks automatically. Also one can define several ROI (Region of Interest) regions to investigate differences in average spectra of ROI. LabMSI runs on both of Windows and MacOS.

This work was supported by MEXT Japan Grant-in-Aid for Scientific Research, Perceptive Plants.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-2-P-3063 Imaging mass spectrometry of mineralized soft connective tissue

Taverna D.1 2, Boraldi F.3, Caprioli R.2, Sindona G.1, Quaglino D.3
1Department of Chemistry and Chemical Technologies, University of Calabria, Arcavacata di Rende, Italy, 2Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA , 3Department of Life Science, University of Modena and Reggio Emilia, Modena, Italy
quaglino.daniela@unimore.it

Mineralization of soft connective tissues, also known as ectopic calcification, is a pathologic process that may occur in different contexts. During aging, for instance, it could be the consequence of atherosclerotic lesions or it may be associated to osteoporosis that, through the calcification paradox, lead to increased levels of circulating ions. Hydroxyapatite can therefore precipitate within tissues mainly affecting the vascular system. Similarly, ipercalcemia- and/or iperphosphatemia-mediated calcification is present in hormonal diseases and in patients suffering from chronic kidney disorders. Furthermore, there are a group of genetic diseases in which, as a consequence of defects in different genes, some related to bone metabolism (MGP, ENPP1, GCGX) some apparently unrelated to the calcification process (ABCC6, HB), ectopic calcification takes place. Even though many key regulators have been found to be abnormally expressed in mineralized areas within soft connective tissues, pathogenic mechanisms are elusive and it is still puzzling whether calcification affects peculiar matrix components in specific organs/tissues, whereas other areas remain unaffected. Nowadays many informative data can be obtained by imaging mass spectrometry (IMS) enabling the combined identification and localization of molecules directly on tissue section in a single experiment. As an experimental model to test the potential of this approach, we have used the skin from a patient affected by Pseudoxanthoma elasticum (PXE), a rare genetic disorder mainly affecting skin, eyes and the cardiovascular system due to progressive calcification. Interestingly, calcification does not involve the whole tissue as in the skin, where mineral deposits specifically accumulate in the middle reticular dermis. IMS was performed on mineralised and non-mineralised areas of the same PXE biopsy, in order to reveal differences in protein distribution and content, and data have been also compared to those from healthy skin. Analysis of the ion density maps demonstrates that mineralized and non-mineralized areas within the PXE dermis are characterized by a distinct protein profile. Interestingly, among the proteins which are differently localised compared to the normal dermis, it has to be highlighted, for instance, thymosin-beta4 (TB4), being surprisingly absent from mineralized areas. This is a pleiotropic molecule exhibiting, within connective tissues, protective and regenerative properties. It could be suggested that tissues devoid of TB4 are more susceptible to damaging noxae, favouring mineral precipitates. Since, TB4 has been never associated to PXE, present findings could open new pathogenic pathways that should be further investigated.

Work supported by grant from FCRM-Ectocal and from PXE Italia Onlus

Fig. 1: Von Kossa staining, used to reveal the presence of calcification, confirms that mineralised areas (MA) are present only in Pseudoxanthoma elasticum (PXE) (b, arrow). Sections stained with haematoxylin eosin (c and d) underwent IMS analysis to identify proteins differently expressed and localized in MA, as in the case of thymosin-beta4 (e and f).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-2-P-3535 Sample damage during MALDI MS imaging and its impact on MSI lateral resolution

Krásný L.1,2, Strnadová M.1, Havlíček V.1, Benada O.1,3
1Laboratory of Molecular Structure Characterization, Institute of Microbiology AS CR, v.v.i., CZ14200 Prague, Czech Republic, 2Institute of Chemical Technology Prague, Technická 5, 166 28 Prague 6, Czech Republic, 3Faculty of Science, J.E. Purkinje University in Ústí nad Labem, Za Válcovnou 1000/8, 400 96 Ústí nad Labem, Czech Republic
benada@biomed.cas.cz

A real spatial resolution of any imaging approach is the most important parameter. In biology, we are usually faced with the fact that the samples suffer from beam damage, as in transmission or scanning electron microscopy. This influences the real resolution of final analyzes or images. Mass spectrometry imaging (MSI) with combinations of MALDI ionization offers a broad range of applications in biochemistry and biomedicine, mainly in the studies dealing with distribution mapping of biomolecules, drugs or metabolites in specific tissues. Reported spatial resolution of MSI analyzes reaches the order of micrometers and depends on sample prep setup and laser focus. The limiting factors are laser beam parameters, matrix crystallization and sample ablation during desorption/ionization process. Here we present the results of MALDI MSI experiments on lipid distribution in the kidney tissue sections with following preselected spatial resolution of spectra acquisitions: 50, 20 and 10 µm.
MALDI sample preparation: Sectioning of the fresh frozen mouse kidney was performed in coronal plane using Leica CM1950 cryomicrotome. Slice thickness was set to 12 µm; cutting temperature to -18 °C. Slices were cut onto ITO (indium-tin oxide) glass slides using “thaw mounting” method and vacuum-dried in desiccator for approx. 30 min. HCCA matrix (7 mg/ml in 50% acetonitrile/0.1% trifluoracetic acid) was applied by ImagePrep (Bruker Daltonics) deposition device using default method.
Mass spectrometry: Data were acquired on Solarix 12T FTICR mass spectrometer (Bruker Daltonics) equipped with a SmartBeam II UV laser. The laser diameter was set to 10 µm and laser intensity of 18% or 30%. Data processing: FlexImaging 3.0 (build 54) software (Bruker Daltonics).
Scanning electron microscopy (SEM): The tissue sections were sputter-coated with 20 nm of gold after MALDI MSI analysis. The samples were examined in Tescan Vega LSU scanning electron microscope at 5 kV.
Results: Nd:YAG laser beam intensity of 18% did not produce recordable damage of kidney tissue, however it also did not produce sufficient sample ionization. The increase of laser beam intensity of 30% overcome the problem with ionization, but the kidney tissue appeared strongly damaged. With the laser step setting of 50 µm and 20 µm we observed individual laser beam ablation paths in the scanned tissue section and obtained distributional maps of lipids with proper resolution. However, with 10 µm setting, the individual laser ablation tracks overlap resulting in large tissue areas with total ablation. The lipids distributional maps showed insufficient resolution. The SEM analysis of tissue section after MALDI MSI allowed us to properly interpret the distribution maps recorded with different different laser step setting.

This work was supported by the Ministry of Education, Youth and Sports (COST-CZ-LD13038) and the Institute of Microbiology (RVO61388971).

Fig. 1: SEM image of mouse kidney section with ablation tracks in kidney tissue after MALDI MSI analysis. MSI laser step setting: A - 50 µm, B - 20 µm and C - 10 µm. The insert is 2,8 times magnified. Scale bar represent 2 mm.
↑ Go to the Table of content ↑

ID-3. Microscopy of single molecule dynamics

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-3-IN-1939 Measuring 2D and 3D diffusion coefficient maps on lipid bilayers, vesicles and cells with light sheet based fluorescence correlation spectroscopy

Wohland T.1, 2, 3, Bag N.2, 3, Singh A. P.2, 3, Ng X. W.2, 3, Huang S.1, 3
1Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117557, Singapore, 2Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117546, Singapore, 3Centre of Bioimaging Sciences, National University of Singapore, 14 Science Drive 4, 117557, Singapore
twohland@nus.edu.sg

Fluorescence Correlation Spectroscopy (FCS) is a widely used quantitative spectroscopy technique which allows measurements of molecular parameters and interactions with single molecule sensitivity even in live samples. However, up to now FCS was used mainly in a confocal mode at single points in a sample. With the recent development of fast array detectors and light sheet based illumination, we extended this approach to Imaging FCS, i.e. the recording of FCS measurements at all pixels in an image. Despite the fact that the time resolution of Imaging FCS is lower than for single spot measurements, Imaging FCS has several advantages. The most important being that i) the illumination modes (total internal reflection (TIR) or singe plane illumination microscopy (SPIM)) expose the sample to much lower light doses and allow more measurements per sample, and ii) that it contains much more information since spatial and temporal correlations can be evaluated between any pixels or group of pixels in the image. The combination of spatial and temporal information in the same measurement allows the application of the so-called FCS diffusion law. The FCS diffusion law relates the mobility of molecules on different spatial scales to the underlying structure of the medium in which the diffusion takes place, providing information even below the diffraction limit.

Here we demonstrate the measurement of 2D diffusion coefficient maps on supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs) and live cells. We investigate the lipid organization of SLBs of different composition and live cells of different lines at different temperatures to extract diffusion coefficients and diffusion activation energies. We demonstrate that SLB organization and phase behaviour can be elucidated even blow the diffraction limit and that the corresponding live cell measurements correlate with their lipid and protein content. Finally we extend these studies to 3D diffusion maps on GUVs and live cells. Our experiments demonstrate that Imaging FCS is a quantitative imaging technique which provides a wide range of information form concentrations, diffusion and transport coefficients, to structural and organizational parameters in an imaging format.

This project was supported by a Life Science Institute – Baden Wuerttemberg grant (R-143-000-422-646) and a grant by the Singaporean Ministry of Education (R-154-000-543-112).

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-3-O-1497 Single Molecule Diffusion and Conformational Dynamics by Spatial Integration of Temporal Fluctuations

Serag M. F.1, Habuchi S.1
1Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, King Abdullah University of Science and Technology, Thuwal 23955-6900, SAUDI ARABIA.
mfserag@gmail.com

Providing unprecedented insights into diffusional processes of systems in life and material sciences, single molecule localization (SML) has been the battle-horse of single molecule fluorescence microscopy methods. Although, the long-established technology provides accurate spatiotemporal locations of single molecules, for estimating their translational speed, it fails to provide essential information about molecular conformation. Furthermore, localization of the center of mass remains a tedious and challenging task for big biomolecules. Direct and accurate access to conformational dynamics and integrating it with translational speed measurements provide valuable learning of molecular diffusion as a crucial life process and reptation as an important physical phenomenon in polymer physics and analytical science. Here, we introduce a new method that is designed to tackle the limitations of SML and to harness molecular diffusion measurements by integration of valuable information on conformational dynamics.

Our method is essentially a spatial quantization of temporal fluctuations of the cumulative area occupied by molecules. Typically, SML analyses express molecular motion in terms of accurate spatiotemporal positions of the molecule. Our method, however, expresses molecular motion in terms of the time-wise increase of the cumulative area occupied by the molecule in space (Fig. 1). Through careful adjustment of the number of pixels detected at each time frame, information on translational diffusion, molecular size and frequency of conformational changes can be obtained. We validated our approach by analyzing the statistical distribution of diffusion coefficients of dsDNA of different lengths and topological forms, a measurement that is critically sensitive to molecular size and conformational changes. Our method showed narrower and much better symmetrical distribution around the mean diffusion coefficient compared to SML (Fig. 2A). Furthermore, the chain relaxation time can be, easily, obtained via autocorrelation of the fluctuations of molecular area over time (Fig. 2B).

In conclusion, our new method holds great promise for far-reaching advancement of single molecule fluorescence microscopy of polymers that is relevant to widely differing scientific fields. From fundamental physics over material science to chemistry and biology, translational diffusion and conformational dynamics are, indeed, central to polymer multidisciplinary studies.

Fig. 1: A) Gradual increase in the cumulative area occupied by diffusing nanospheres (top) and 6 kbp DNA (bottom). B) The red-bordered region defines the area occupied by the molecule at each time frame. The dashed pixels represent the area increment at each time frame as a result of random movement of the molecules.

Fig. 2: Diffusional and conformational dynamics of 42 kbp DNA. A) Frequency histogram of diffusion coefficients. The values were determined via the area-based (green) and the SML-MSD (yellow) methods. B) Autocorrelation of molecular area fluctuations. The autocorrelation function fits to single exponential decay with mean correlation time of 350 ms.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-3-O-2586 Watching complexes and invisible states of proteins by high-precision FRET in vitro and in vivo

Seidel C. A.1
1Chair for molecular physical chemistry, Heinrich-Heine-Universität Düsseldorf, Germany
cseidel@hhu.de

So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful specific snapshots of static protein structures. Recent experiments by single-molecule and other techniques have shown the heterogeneity and flexibility of biomolecular structures and questioned the idea that proteins and other biomolecules are static structures. The visualization of transiently populated conformational states and the identification of exchange pathways are key steps to understand enzyme function.

I will present a comprehensive toolkit for Förster resonance energy transfer (FRET)-restrained modeling of proteins and their complexes for quantitative applications in structural biology [1-3] and cell biology [4, 5]. The experiments are performed by multi-parameter fluorescence detection on the single-molecule level and for complexes imaged in live cells. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. The precision and confidence levels of the models are calculated by rigorous error estimation. The accuracy of this approach is demonstrated by docking a DNA primer-template to HIV-1 reverse transcriptase. The derived model agrees with the known X-ray structure with a root mean square deviation of 0.5 Å. Furthermore, we introduce FRET-guided “screening” of a large structural ensemble created by computer simulations. Moreover we use filtered fluorescence correlation spectroscopy to characterize enzyme function and introduce a state matrix of conformational and enzyme states that assigns a functional role to conformational fluctuations. Hybrid studies of T4 Lysozyme, DNA polymerase I and the large GTPase hGPB1 using FRET, SAXS and EPR will be presented.

[1] Sisamakis, E., et al.; Methods in Enzymology 475, 455-514 (2010)

[2] Sindbert, S., et al.; J. Am. Chem. Soc. 133, 2463-2480 (2011)

[3] Kalinin et al. Nat. Methods 9, 1218-1225 (2012).

[4] Stahl, Y., et al. Current Biology 23, 362–371 (2013).

[5] Kravets, E., et. al. J. Biol. Chem. 287, 27452–27466 (2012).

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-3-P-2327 Towards the mechanism of photounbinding 

Davoudpour A.1, Heinze K. G.1
1Bio-Imaging Center and Biophotonics Group, Rudolf-Virchow Center of University Würzburg , 97080 Würzburg, Germany
amirali.davoudpour@uni-wuerzburg.de

Recent studies have shown that fluorescently labeled antibodies can be reversibly dissociated from their antigen after illumination with laser light. This unexpected phenomenon is called photounbinding, and can be both boon and bane in fluorescence imaging approaches. While photounbinding should be avoided in typical quantitative fluorescence imaging, it has also the potential to become a valuable low-invasive tool of controlling binding assays. For both avoiding and utilizing photounbinding is vital to understand its mechanism. Unfortunately, pinpointing photounbinding is not straight forward as it often remains masked in an experiment as it first glance appearing is similar to photobleaching where the fluorophore becomes dark (non-fluorescent) at some point after laser illumination. However, photounbinding is substantially different, as it additionally creates an unbound (vacant) site.

To visualize photounbinding in a confocal microscope we immobilize GFP on a glass surface, recognize it by an antibody, laser scan a region of interest to induce photounbinding and subsequently rebound with a differently labeled antibody against GFP (see figure). We hypothesize that photobleaching is most likely driven by the production of Radical Oxygen Species (ROS). To test this hypothesis, we introduce a redox sensitive GFP (roGFP2) to determine the changes in the redox potential after illumination by laser light. Our results show that redox potential increase in the roGFP2 vicinity with increasing scanning iterations or/and laser power. As ROS production is considered to be local, the distance of the local ROS production site from the antibody antigen complex should play a key role for photounbinding. By varying the effective distance of the binding partner (antigen) to its fluorescent marker, we designed a protocol that allows for comparison of distance dependent rebinding patterns. First, we modified monoclonal antibodies by adding a single heterobifunctional amino spacer with either a short or a long linker length. Second, the fluorescence dye AlexaFluor555 was bound to the functional group at one side of the linker while the antibody was bound to the other site by an active sulfhydryl group. In a Fluorescence Resonance Energy Transfer experiment we finally verified the different distances between the acceptor (AlexaFluor555) and the donor (Green Fluorescent Protein GFP) molecules by Fluorescence Lifetime Imaging Microscopy and showed that photounbinding is strongly dependent on the linker length. Thus, we can pinpoint ROS production as one of the main mechanisms of photounbinding as rebinding can hardly be observed if the local ROS production site is kept away from the antibody by the long linker.

We would like to thank Mike Friedrich, Marek Suplata and Piau Siong Tan for their assistance and RVZ and Graduate School of Life Sciences for their support.

Fig. 1: Fig.1: shows schematic setup for photoubinding, rGFP is immobilized on the surface of the coverglass by a cross linker (6,8 °A), then rGFP is incubated with the antibody which is labeled with Alexa 555 by a linker (19,2-92,5 °A) and after bleaching by laser illumination, the bleached region is filled with a second antibody during rebinding process.
↑ Go to the Table of content ↑

ID-4. High-throughput microscopy and its applications in life and material sciences

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-4-IN-6083 Introducing a Novel liquid 3 Dimensional Scaffold for Culturing and imaging Cells in 3D

Davies A. M.1
1Translational Cell Imaging Queensland (TCIQ), Institute of Health Biomedical Innovation, Queensland University Of Technology, Brisbane. Australia
anthonmitchelldavies@gmail.com

We have recently developed a new 3 dimensional cell culture technology that unlike many of the more conventional suspension cell culture systems permits the maintenance of cells in 3 dimensions in static culture hence negating the need for mixing and/or agitation. This technology offers many advantages to the more conventional suspension cell culture systems. (i) Our system comprises of a low viscosity media that can be used in any culture vessel. (ii) Our system does not require any form of continuous agitation and (iii) has been developed to permit continuous feeding of cells without the perpetuation of the culture. In this presentation we will examine the benefits of using this technology in both the manufacturing and research environments; we will also demonstrate improvements in cell growth, viability and gene and protein expression.

                                                                                                                                                                                                                                                                                                                                                                                                                                                                               

                                                                                                                                                                                                                                                                    

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-4-IN-6094 A High Content Screening Platform for Phenotypic Drug Discovery: Automated Image Analysis of Complex Cell-Based Model Systems for Oncology and Toxicology

Ahonen I.3, Akerfelt M.5, Bayramoglu N.2, Fey V.4, Schukov H. P.4, Winsel S.6, Virtanen J.1, Harma V.1, Nees M.1,5
1VTT Technical Reseach Centre of Finland, 2University of Oulu, Center for Machine Vision Research; Finland, 3University of Turku, Dept. of Statistics, Finland, 4University of Turku, Institute of Biomedicine, Finland, 5Centre for Biotechnology Turku, Finland, 6PhenoType GmbH, Berlin, Germany
matthias.nees@vtt.fi

Complex diseases such as human cancers require complex, physiologically relevant model systems that allow truly informative testing of pharmacological compounds and biosimilars in vitro. Traditionally, most model systems routinely used in drug discovery represent highly reductionist approaches, and fail to address many key aspects of genuine tissue architecture (histology). However, organotypic cell- and tissue culture models that more faithfully mimic the tumor microenvironment (TME), grapidly gain acceptance. These systems aim to recapitulate the heterotypic interaction of multiple cell types (e.g. tumor and endothelial or stroma cells) and physiological extracellular matrices (ECM, basement membranes). The main challenges are to develop a balanced approach between sample throughput and biological relevance, to provide better but accessible and affordable in vitro tools to replace animal testing and predict human risk assessment. Although such assays are increasingly being utilized for prediction of drug efficacy versus organ-specific toxicity, they often remain poorly characterized. Additional features that require more attention are the dynamics of stromal and tumor cells (tissue homeostasis versus invasion and metastasis), tumor cell plasticity (the capacity to change cellular phenotypes), and tumor heterogeneity (the complex and shifting clonal composition of tumor tissues). Very few screening platforms for drug discovery systematically address these key elements of cancer biology. In this lecture, we will introduce our approach that allows real-time, live cell monitoring of cellular dynamics using diverse microscopic techniques, automated image analyses, combined with machine learning. The major focus will be on machine vision solutions, developed specifically for tracking of dynamic stromal and tumor cell motility. This is combined with unsupervised, statistical methods to capture the intrinsic heterogeneity of tumor tissues. The final goal is to provide a high content screening platform for phenotypic drug discovery with a significantly improved experimental throughput - without sacrificing biological relevance.

Fig. 1: Schematic overview of the phenotypic drug discovery work flow

Fig. 2: Implementation of physiologically more relevant cell- and tissue culture models in the pharmacological drug discovery pipeline
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-4-O-3387 Organizing, Managing and Searching Large Collections of Images:  A New Resource To Handle High-Throughput Imaging

Morgan D. G.1, Gopu A.2, Young M.2, Hayashi S.2
1Electron Microscopy Center, Indiana University - Bloomington, 2Pervasive Technology Institute, Indiana University - Bloomington
dagmorga@indiana.edu

With the advent of digital imaging, it has become easy to record more images in less time than ever before. Individuals as well as research groups and imaging facilities can drown in the images recorded for a single project, much less those from a career or years of facility use. High-throughput methods that use imaging as recording and screening tools have made such issues worse. Archiving such large image collections can pose a problem, though university and commercial cloud storage can help. Ways to deal with storage and access have been and continue to be developed, and we present here a new resource designed with these issues in mind.

Groups from the IU-Bloomington Electron Microscopy Center (EMC) and Pervasive Technology Institute (PTI) have begun a project to deal with the tidal wave of images produced using EMC resources. The EMC PPA (Portal, Pipeline & Archive) is an offshoot of a project for handling images from the One Degree Imager on the WIYN 3.5m telescope at Kitt Peak, Arizona. The concept behind both projects is that images are uploaded from the recording instrument(s) into a database that is archival and searchable. Images are stored with calibration and reference data, instrument metadata and tag words to help users search for particular images. Access control exists for the images. The images and all functions of the database are accessible through any internet browser. Images are stored locally on the Data Capacitor shared file system and archivally in the Scholarly Data Archive (SDA). A number-crunching pipeline built on the Big Red II computing cluster is used for compute-intensive data reduction and image processing.

Fig 1 shows a typical search page. The most commonly used metadata search fields appear in the upper left, but all available fields can be accessed using the Other Fields button at the right. Tag words and anti-tag words can be search simultaneously. Search results are shown at the bottom as rows of image data starting with an image thumbnail. Sorting can be based on any column, and both actual columns and their order can be individualized.

Search results can be turned into a "collection," which can either be displayed as shown in Fig 1 or as the grid view in Fig 2. A collection can be downloaded, sent into
the pipeline for processing, or individual images can be examined (and retained or rejected from the collection). Tag words can be edited both for individual images and collections.

The EMC PPA is a work in progress. We hope it will help our users manage their image data and that it will also help the EMC track facility use and data mine for information lost in the thousands of image recorded yearly.  Since this framework now exists, the concepts can be extended to other types of image data and sources.

Fig. 1: Search Page.  Images can be searched using metadata tags (e.g., acquisition date, magnification and accelerating voltage), as well as with tags a user associates with particular images and data sets.  Metadata fields are pulled from the images themselves while other searchable tags are entered during and after uploading images into the portal.

Fig. 2: Grid View of Collection Called FirstImages_0001.  Groups of images can be assembled into permanent "collections" that are easily examined, manipulated and downloaded.  Mousing-over an image in this grid view pops up details about an image, while clicking the magnifying glass leads to an image exploration page for a single image.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-4-P-3235 High-content microscopy screening to identify novel chemical compounds modulating DNA damage response

Benada J.1, Pombinho A.2, Jenikova G.1, Bartunek P.2, Macurek L.1
1Laboratory of Cancer Cell Biology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ14200 Prague, Czech Republic, 2Center for Chemical Genetics, CZ-OPENSCREEN, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, CZ14200 Prague, Czech Republic
jan.benada@img.cas.cz

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of posttranslational modifications among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to stabilization of tumor suppressor p53, which results in a temporal arrest of cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins in DDR signaling and is essential for timely termination of the DDR.
Several key proteins of DDR machinery localize directly to the site of DNA breaks and form microscopically detectable nuclear foci. Their number reflects the level of DDR activation in a quantitative manner. The most commonly used markers of these foci are phosphorylated histone H2AX (γH2AX) and mediator protein 53BP1 which is recruited as a result of phosphorylation and ubiquitination-dependent events.
To identify novel chemical compounds that modulate DNA damage response, we set up the high-content microscopy screening of chemical compound library using Operetta system (PerkinElmer). The human osteosarcoma cell line U2OS was chosen as the well-characterized model that has been extensively used for studies of the DDR before. The cells were exposed to 3 Gy or mock-irradiated, seeded on the 396-well plates with chemical compounds, and fixed after 5 hours. Subsequently, the cells were immunostained for γH2AX and 53BP1 and analyzed by high-content fluorescent microscopy. The number, area and intensity of γH2AX and 53BP1 foci were used as readout of DDR activation. At the chosen timepoint (5h), the acute DDR is already in decline. In this setting, we can therefore score not only for compounds causing DNA damage in mock-irradiated cells, but also for the ones that interfere with timely termination of the DDR after irradiation, e.g. by inhibiting the Wip1. The identified hits will be further confirmed by dose response curve generation and studied for the mechanism of their action.

JB, GJ and LM are supported by the Grant Agency of the Czech Republic (P305-12-2485).  AP and PB are supported by the NPU1 CZOPENSCREEN (LO1220). JB is supported by the Charles University Grant Agency (GAUK 836613).

↑ Go to the Table of content ↑

ID-5. Nanoparticles: Biomedical applications and bio-safety issues

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-5-IN-2366 Characterizing bionanoparticles by STEM, EFTEM and electron tomography

Leapman R. D.1, Aronova M. A.1
1National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, MD, USA
leapmanr@mail.nih.gov

The ability to synthesize multicomponent hybrid nanocarriers with controlled architecture and chemical functionality offers great potential for developing in vivo and ex vivo medical diagnostics and therapeutics. Quantitative electron microscopy provides a tool to assess design strategies and to determine the degree of monodispersity, which are critical for controlling functionality and toxicity. Here, we illustrate that combining scanning transmission electron microscopy (STEM), energy-filtered transmission electron microscopy (EFTEM) and electron tomography (ET) gives elemental composition and 3D structure of hybrid nanocomplexes.

Fig. 1 shows how dark-field STEM tomography elucidates the structure of self-assembled, biodegradable, plasmonic gold nanovesicles that are designed for photoacoustic imaging and photothermal therapy [1]. Even at 300 kV it is not feasible to image these 200-nm diameter assemblies by bright-field TEM tomography because the strong scattering of gold gives a nonlinear signal when the nanoparticles overlap in the tilt series. However, the STEM tomogram clearly reveals a single shell of nanoparticles packed close to the vesicle membrane.

A combination of EFTEM and TEM tomography is used to characterize the flower-shaped optical nanosensor shown in Fig. 2. This nanocomplex consists of a central gold with surrounding iron oxide nanoparticles, which are attached to one end of a peptide substrate for matrix metalloproteinase enzyme, while the other end of the substrate is linked to a fluorescent dye molecule [2]. In the presence of the enzyme (expressed by cancer cells) the dye is cleaved and no longer quenched by the gold, resulting in a fluorescence signal. EFTEM (Fig. 2b) confirms that the petals of the nanosensor are iron oxide, and TEM tomography gives their 3D arrangement around the gold nanoparticle.

Fig. 3 demonstrates use of EFTEM to analyze a bionanoparticle developed for magnetic resonance imaging (MRI) of labeled cells. This nanocomplex consists of three FDA-approved drugs: heparin, protamine and ferumoxytol [3]. Each component can be mapped by its elemental composition: protamine contains nitrogen; heparin contains covalently bound sulfate groups; and fexumoxytol is a superparamagnetic iron oxide nanoparticle giving MRI contrast. The EFTEM elemental maps reveal a uniform distribution of protamine and heparin in the nanocomplex cores, whereas ferumoxytol is concentrated at the peripheries.

In summary, STEM, EFTEM and ET are valuable tools for optimizing the design and assessing the monodispersity of hybrid nanoparticles such as the ones illustrated here.

[1] P. Huang et al., Angew. Chem. Int. Ed. 52 (2013) 13958.

[2] X. Jie et al., ACS Nano 5 (2011) 3043.

[3] M.S. Thus et al., Nature Medicine 18 (2012) 463.

Research supported by the intramural program of the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health. The authors thank Drs. Xioayuan Chen, Peng Huang, Jin Xie, Joseph Frank and Henry Bryant for providing the nanoparticles used in this work.

Fig. 1: Self-assembled, plasmonic, biodegradable gold vesicle [1]: (a) annular dark-field STEM; (b) orthoslice through STEM tomographic reconstruction showing layer of nanoparticles next to membrane; (c) 3D visualization of the nanoassembly from the STEM tomogram.

Fig. 2: Hybrid Fe3O4/Au flower-shaped optical nanosensor [2]: (a) TEM; (b) EFTEM iron L2,3 map superimposed on TEM bright-field image; (c) 3D visualization of nanosensor obtained by TEM tomography.

Fig. 3: EFTEM analysis of epon-embedded and sectioned nanocomplexes composed of heparin, protomaine and ferumoxytol [3]: (a) sulfur L2,3 map; (b) nitrogen K map; (c) iron L2,3 map; (d) overlay of the elemental distributions (S in green, N in blue, Fe in red).
↑ Go to the Table of content ↑
Type of presentation: Invited

ID-5-IN-5766 Nanoparticles for targeted bio-imaging: interactions with cells, tissues and organs

Debbage P. L.1
1Medical University of Innsbruck, Innsbruck, Austria
Paul.Debbage@i-med.ac.at

The design and creation of targeted nanoparticles for use in Nanomedicine is a complex art, still in its early infancy. Ideal targeted nanoparticles would accumulate 100% exclusively in the target site. All target site(s) would accumulate the nanoparticles, no unoccupied targets would remain. The body erects numerous barriers excluding nanoparticles from their targets, notably in the form of physical and physiological barriers between the blood and the parenchyma of almost all organs. Other barriers are erected between surfaces and organ-specific epithelia, notably the mucus layers of the gut, the airway and the genito-urinary systems. The body also offers several non-target sites in which nanoparticles can accumulate, notably the liver which lacks a blood-tissue barrier, and also the lymph nodes, the spleen, and certain sectors of the vascular bed; the kidney can in some cases also provide a non-target site at which nanoparticles or their breakdown products may accumulate. The failure of nanoparticles to reach all or any their targets may compromise diagnosis and therapy, and confound drug development. Collateral accumulation of nanoparticles in non-target tissues may confuse diagnosis by generating images of unclear provenance and thus producing false positives. The accumulation of nanoparticles in high concentration in non-target organs can cause direct or indirect toxic effects. These matters will be considered in relation to experience we have gained by use of protein-based nanoparticles in a series of developmental and design steps using small laboratory animals.

This work was supported during several years by 4 grants from the Austrian National Bank Jubilee Program. The work is presently supported by an ERA-Net grant from the European Union.

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-5-O-2554 Following at high resolution the biodegradation of inorganic nanoparticles in the organism

Alloyeau D.1, Javed Y.1, Ricolleau C.1, Lartigue L.1, Pellegrino T.3, Gazeau F.2
1Laboratoire Matériaux et Phénomènes Quantiques, Université Paris 7 - CNRS, Paris, France, 2Laboratoire Matériaux et Systèmes Complexes, Université Paris 7 - CNRS, Paris, France, 3Istituto Italiano di Tecnologia, Genova, Italy
damien.alloyeau@univ-paris-diderot.fr

Given the increasing use of nanomaterials in industry and their tremendous potential in the biomedical field, human are expected to be more and more exposed to nanosized materials. A key concern raised by the development of nanotechnology is the long term fate of nanomaterials, and particularly the control over inorganic nanoparticles (NPs) full life cycle and related risks for human health. Most toxicology studies take the point of view of living organisms to examine the effects of exposure to NPs on biological functions. Here, we propose an original “Materials Science” approach to the nanotoxicology paradigm: we choose to take the point of view of nanomaterials in order to monitor the transformation and degradation of their atomic structure and physical properties in living environment and then unravel the mechanisms determining their fate. Our methodology is illustrated here with both in vivo and in vitro studies that provide new insights into the relation between the structure and the life cycle of inorganic NPs in the organism. [1,2,3] Iron oxide NPs with different forms (spherical, nanocubes, nanoflowers) and different inorganic or organic coatings (polymers, citrate, gold) were studied.


We have exploited the multifunctionality of TEM to follow the biodistribution and the structural evolution of iron oxide NPs injected into mice over a period of three months. These nanoscale investigations were complemented by the magnetic follow-up of the injected NPs to quantify iron oxide NPs at the macroscopic scale and follow the evolution of their magnetic properties. This multi-scale approach allowed characterizing for the first time, the structural and magnetic in vivo degradation of NPs and the intracellular pathway of iron transfer from NPs to endogenous ferritine [1,2] (fig. 1).


Concomitantly, we are exploiting liquid TEM to study in situ the biodegradation mechanisms of NPs in a medium mimicking the intracellular environment (fig 2), or directly into cell cultures. The use of micro-fabricated liquid-cells (Protochips) in an aberration-corrected microscope is a straightforward approach to understand how the structure of NPs and their organization in solution influence the mechanisms and kinetic of degradation. In that regards, we have already evidenced that both surface coating and aggregation state are key parameters, because they control surface reactivity in biological environment [2, 3]. Therefore, our works allows suggesting relevant strategies to modulate the degradability of NPs and to avoid the drastic annihilation of their technologically interesting properties in cells (fig 3).


[1] M. Levy et al. Biomaterials, 32, 3988 (2011), [2] L. Lartigue et al. ACS nano, 7, 3939 (2013) [3] Y. Javed et al. Small, Accepted (2014).

We are grateful to Region Ile-de-France (convention SESAME E1845), the Labex SEAM and the CNRS (projet Defi Nano).

Fig. 1: In vivo study of the biodistribution and biotransformation of iron oxide nanocubes observed in lysosome of macrophage cells (right images). HRTEM investigations (right images) allows identifying the atomic structure of the degraded nanocubes (inverse spinel structure, FFT in white frame) and ferritine proteins (hematite structure, FFT in red frame)

Fig. 2: In situ follow -up of the degradation of aggregated iron-oxide nanoflowers using liquid-TEM. The corrosion and dissolution of the NPs is directly observed in a solution mimicking the intracellular environment (ph = 4.7 + chelating agent). This experiment reveals that aggregated nanoparticles present a slower and non-linear kinetic of degradation.

Fig. 3: The structural degradation of the nanoflowers in the lysosome like medium observed in fig.2, impairs their efficiency for magnetically induced hyperthermia and magnetic resonance imaging. Decrease of the specific absorption rate in a magnetic field of 24 kA/m and 520 kHz (a) and the transverse relaxivity r2 determined at 20 Mhz (b).
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-5-O-2827 Characterizing the effectiveness of antibody labeled nanoparticle targeting of cancer cells using scanning and transmission electron microscopy

Kempen P. J.1, Kurtz D. M.2, Madsen S. J.1, Gambhir S. S.2, Sinclair R.1
1Department of Materials Science and Engineering, Stanford University, 496 Lomita Mall, Stanford, CA 94305-4034, USA, 2Multimodality Molecular Imaging Lab, Department of Radiology, Stanford University, Stanford CA 94305, USA
pkempen@stanford.edu

One of the principal hurdles to utilizing nanoparticles (NPs) for the diagnosis and treatment of cancer is the ability to target them successfully to cancer cells. NPs labeled with antibodies to specific surface proteins overexpressed by cancer cells can be utilized to target tumors for either diagnosis or treatment or both. CD47 is a transmembrane protein overexpressed in cancer cells that acts as a “don’t eat me”[1] signal for phagocytic cells. In this experiment, gold-core silica-shell NPs labeled with CD47 antibodies (CD47-NPs) were incubated with DLD1 colorectal adenocarcinoma cells to study the efficacy of this targeting approach. Due to the scale of the NPs both scanning (SEM) and transmission (TEM) electron microscopy were utilized to characterize the NP cell interactions.


CD47-NPs were incubated with both wild-type (WT) and CD47 knockout (KO), CD47 negative, DLD1 cells. Control experiments were run with both cell lines using unlabeled NPs (U-NPs). SEM samples were critical point dried. TEM samples were fixed, stained, dehydrated and embedded following standard protocols. 150 nm cell sections were cut and placed on 200 mesh formvar coated TEM grids.


SEM results show that while the percentage of cells containing NPs, figure 1A, varied little from sample to sample, the number of NPs per sample varied greatly, figure 1B. The WT cells treated with CD47-NPs had 25 NPs per cell while the KO cells contained 5. The cells treated with U-NPs had ~1 NP per cell. These results indicate that the CD47 targeting was effective. Figure 2 contains representative SEM images showing the aggregation of CD47-NPs on WT cells while the other cell samples had very few NPs.


From the TEM 32% of WT cell sections contained CD47-NPs whereas only 5% of the KO cell sections contained CD47-NPS. Additionally, the WT sample had 3 CD47-NPs per section while the KO sample only had 0.5. For the cells treated with U-NPs, there were fewer than 0.1 NP per cell section, much lower than for the CD47-NP treated cells. Figure 3 contains representative TEM images showing a large cluster of CD47-NPs on a WT cell section and far smaller NP clusters on the control groups. These results corroborate the SEM result that the CD47 targeting was successful.


SEM and TEM characterization of cells treated with antibody labeled NPs enables us to study the success of a targeting strategy by examining the number and distribution of NPs on or near the surface of a cell. These techniques showed that the CD47 antibody labeled NPs effectively targeted the DLD1 cancer cells.


[1] Willingham, S.B. et al. PNAS, 109(17) (2012) 6662-6667.

This research is supported by the Center for Cancer Nanotechnology Excellence and Translation (CCNE-T) grant funded by NCI-NIH to Stanford University U54CA161459

Fig. 1: Bar graphs showing (A) the percentage of cells with NPs and (B) the number of NPs per cell from the SEM data, and (C) the percentage of cell sections with NPs and (D) the number of NPs per cell section from the TEM data; showing higher uptake of CD47-NPs by WT cells. 1 = KO w/ U-NPs; 2 = WT w/ U-NPs; 3 = KO w/ CD47-NPs; 4 = WT w/ CD47-NPs.

Fig. 2: Representative SEM 1:1 mixed SE:BSE images from (A) KO with U-NPs, (B) WT with U-NPs, (C) KO with CD47-NPs and (D) WT with CD47-NPs. The gold core nanoparticles appear as bright spots on the surface of the cell. The WT sample with CD47-NPs has a large aggregate of NPs representative of the increased number of NPs found. Scale = 1 µm.

Fig. 3: Representative TEM images from (A) KO with U-NPs, (B) WT with U-NPs, (C) KO with CD47-NPs and (D) WT with CD47-NPs. The gold core appears dark in the TEM image. NPs were located either on the surface or within vesicles. Image (D) shows a large cluster of NPs on the surface of WT cells with CD47-NPs. Scale (A-C) = 100 nm; (D) = 500 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-5-O-3386 Localization and analysis of engineered nanoparticles in Daphnia Magna

Jensen L. S.1, Sørensen S. N.2, Thit A.3, Skjolding L. M.2, Købler C.4, Mølhave K.4, Baun A.2
1Center for Electron Nanoscopy, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark, 2Department of Environmental Engineering, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark, 3Department of Environmental, Social and Spatial Change, Roskilde University, DK-4000 Roskilde, Denmark, 4Department of Micro- and Nanotechnology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
losj@cen.dtu.dk

Engineered nanoparticles are added to a growing amount of consumer products and are considered the fastest growing nanotechnology product. They are being washed out into the waste water systems in potentially increasing amounts as the use and number of products that contain nanoparticles is expanding. There are concerns that they could be harmful to aquatic organisms in nature. The uptake mechanisms in aquatic organisms are therefore important to elucidate the toxic mechanisms.
Previous results have indicated that gold nanoparticles to a limited extent are able to pass the peritrophic membrane and in some cases enter the gut epithelial cells in the freshwater crustacean Daphnia magna. In the absence of food, the amount of gold nanoparticles is elevated compared to when the animal is feeding. It has been speculated that when gold nanoparticles are present in the gut in relatively high amounts they might be able to cross the peritropic or cell membrane during the preparation for electron microscopy, perhaps during the epon infiltration step. In the present study D. magna were exposed for 24h to 0.4 mg Au/L citrate coated gold nanoparticles (10 nm) without food and an additional control was added to the experiment; D. magna which were exposed to gold nanoparticles during the infiltration process for electron microscopy. The uptake into gut lumen and internalization into epithelial cells of exposed D. magna was examined by light microscopy, TEM, FIB-SEM and EDX. The bulk of the gold nanoparticles in the exposed animals was observed as both single nanoparticles and aggregates located in the gut lumen while relatively few gold nanoparticles were observed across the peritrophic membrane associated with the microvilli (figure 1). This is indicating that the peritrophic membrane of the gut in D. magna is able to form a barrier of low permeability to the gold nanoparticles in all cases. Very few gold nanoparticles were found to be internalized by the gut epithelial cells (figure 2). The results suggest that 10 nm gold nanoparticles are able pass the peritrophic membrane and enter the gut epithelial cells.

(1) Skjolding LM, Sørensen SN, Thit A, Købler C, Mølhave K, Baun A. Uptake of gold nanoparticle in Daphnia magna gut in the presence and absence of food using electron microscopy. To be presented at SETAC Europe 24th Annual Meeting, May 2014.

Micrographs in this presentation are recorded at Core Facility for Integrative Microscopy, Copenhagen University and Center for Electron Nanoscopy, Technical University of Denmark.

Fig. 1: Figure 1. Cross section of peritrophic membrane (PTM) and gut epithelial cells (GC) showing aggregates of gold nanoparticles (Au NPs) in the gut lumen and few gold nanoparticles associated with the microvilli (MV). Scalebar 500 nm.

Fig. 2: Figure 2. Gut epithelial cell with internalized nanoparticles. Scalebar 200 nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-5-O-3443 Three-dimensional microscopic analysis of SERS-active substrates for ultrasensitive detection of biologically significant compounds

Štolcová L.1, Ižák T.2, Petrenec M.3, Svoboda M.3, Proška J.1, Procházka M.4, Kromka A.2
1Czech Technical University in Prague, FNSPE, Břehová 7, Prague, Czech Republic , 2Institute of Physics, ASCR, Cukrovarnická 10, Prague, Czech Republic, 3TESCAN ORSAY HOLDING, a.s., Libušina třída 21, Brno, Czech Republic , 4Charles University, FMP, Ke Karlovu 5, Prague, Czech Republic
lucie.stolcova@fjfi.cvut.cz

The spectroscopy based on surface-enhanced Raman scattering (SERS) is a technique providing information on vibrational structure of molecules, and hence enabling their detection and identification at trace concentrations. The enhancement of the intrinsically weak Raman signal originates from the localized surface plasmon resonance on metal (usually gold or silver) nanoparticles or nanostructured surfaces, so-called SERS-active substrates. The enhancement can display large variations over the substrate surface, due to its high sensitivity to the surface topography. However, periodically structured substrates, yielding uniform Raman signal enhancement, overcome the problems connected with low spectral reproducibility.

Periodic arrays of close-packed polystyrene microspheres (diameters 250 nm - 4 um) were prepared using a self-assembly technique, and deep corrugations were created in the sphere surface by oxygen plasma etching. The corrugated sphere arrays coated with 20 nm of gold by magnetron sputtering displayed excellent SERS signal uniformity and low detection limits for a series of biologically significant compounds with different lipophilicity. SERS spectra of thiaclopridwere measured down to concentration of 10-8 M. The SERS signal uniformity was demonstrated by spectral mapping using Horiba JobinYvon LabRam HR800 Raman microspetrometer.

In comparison to smooth gold-coated microspheres, our measurements showed a distinct increase in SERS spectral intensity. To understand this effect, cross-sections of the polystyrene/gold nanostructures were prepared using focused ion beam (FIB). Thus, the structural features of the sputtered gold layer (at nanoscale) could be imaged and analysed together with the mesoscale architecture of the corrugated polystyrene cores. Their texture similar to a 2D labyrinth increased the surface area in a specific way, and can be viewed as an open form of pores. Due to its mesoscale dimensions, the corrugated particles behave in solutions like porous materials with all consequences for phase equilibria and analyte separation/adsorption.

The nanomilling was performed by Focused Ion Beam Equipped Scanning Electron Microscope (FIB-FESEM) TESCAN LYRA 3 with a COBRA ion column at 4 pA and 30 kV. Prior to the milling, a protective platinum layer was deposited (at 5 kV) over the corrugated spheres selected for the analysis.Using 3D tomography technique performed with FIB in 10 nm slices, the continuous gold layer verging into discrete sputtered gold grains in the corrugations could be observed for the first time. Imaging was performed with an ultra-high resolution SEM TESCAN MAIA 3 XM equipped by FEG; InBeam detectors of secondary and backscattered electrons or their mixed signals were used for observations.

This work was supported by the Czech Science Foundation grant No. P205/13/20110S.

Fig. 1: Hexagonally ordered plasma etched polystyrene microspheres coated with 20 nm of gold – top view (SEM micrograph)

Fig. 2: Individual gold-coated corrugated microsphere – tilted view, detail of the sputtered gold layer (SEM micrograph)

Fig. 3: Cross section of the gold-coated etched PS microsphere prepared by FIB nanomilling (SEM micrograph, backscattered electron detector)

Fig. 4: SERS spectra of 4-aminothiophenol obtained by spectral mapping of an ordered array of etched microspheres (raw spectra measured from 22 × 22 points separated by 2 um)
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-1505 Inhalation tests of carbon nanotubes using rats

Yamamoto K.1, Yoshida T.1, Hayashida T.1, Shimada M.2, Ogami A.3, Morimoto Y.3
1National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan, 2Hiroshima University. Higashi-Hiroshima, Japan, 3University of Occupational and Environmental Health, Kitakyushu, Japan
k-yamamoto@aist.go.jp

Industrial applications of nanomaterials are reported in many fields recently. However, the toxicity of these nano-materials for the human is not clear, so the toxicity and risk assessment of nanomaterials are important. Regulation of nanomaterials is also discussed in Organization for Economic Co-operation and Development (OECD). Authors have reported the toxicity of fullerene, multi-wall carbon nanotube (MWCNT), and single-wall carbon nanotube (SWCNT) using the intratracheal instillation in rat lung. On the other hands, an inhalation test using animal is the gold standard ones to estimate the harmful effects of inhaled chemicals. In this study, the inhalation tests using rat of MWCNT, and SWCNT were performed, and the morphology of alveolar macrophage in rat lung was examined by using transmission electron microscope (TEM).
The aerosols of MWCNT or SWCNT were generated from their liquid dispersions. The average diameter of MWCNT was 48nm. The average diameter of SWCNT was 1.8 nm, and SWCNTs were bundled. Rats were exposed to the aerosol for 6 hours a day, 5 days a week, for 4 weeks in a whole-body exposure chamber. The average mass concentrations of aerosol were 0.038 mg/m3 for MWCNT, and 0.03 or 0.13 mg/m3 for SWCNT. The lung tissues after three days, one month after the inhalation test were observed by TEM. The lung tissue was fixed using glutaraldehyde and osmium tetroxide solution, and then dehydrated in ethanol, and embedded in epoxy resin. Ultrathin sections were cut on a diamond knife with microtomy. The stained specimens were also examined using conventional biological TEM (Hitachi, H-7600). The zero loss imaging of the non-stained tissue was performed by an energy-filtering TEM with the high-resolution objective lens (Carl Zeiss, EM922HR).
TEM image of lung tissue at 3 days after inhalation of MWCNT was shown in Figure 1 (a), and the magnified image of boxed area in Figure 1(a) was shown in Figure 1(b). Fibrous objects are observed at the cytoplasm of macrophage. According to high- resolution analysis, these fibers are MWCNT, and MWCNT keeps the tube and the graphitic structures. This is because that graphite is chemically stable. In the tissue at 1 month inhalation of MWCNT, MWCNT is still remaining in alveolar macrophage. TEM image of the alveolar macrophage at 3 days after inhalation of SWCNT was shown in Figure 2(a). Black particles are observed in phagosome of macrophage. Magnified image of these black particles was shown in Figure 2(b). Fibrous structures with nanometer size are observed and it is clarified that SWCNT is taken up by macrophage. In summary, the MWCNT and SWCNT, which were inhaled in lung, were taken up in the alveolar macrophage.

Fig. 1: (a) Alveolar macrophage in the rat lung at 3 days after inhalation of MWCNT, and (b) magnified images of boxed area in (a)

Fig. 2: (a) Alveolar macrophage in the rat lung at 3 days after inhalation of SWCNT, and (b) magnified images of phagosome in (a)
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-1832 Specimen preparations for nano-particle counting by SEM and TEM– a combination method of inkjet and freeze-drying, and electrostatic aerosol deposition method

Kumagai K.1, Sakurai H.1, Kurokawa A.1
1Nanomaterial Characterization Division, National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba JAPAN
quaz.kumagai@aist.go.jp

  In response to the regulations on manufactured nanomaterials (NMs), which started in several EU countries based on the definition of NM by European Commission1, there are increasing discussions to find the solution for the size distribution measurements of NMs to test if a product meets the definition or not2. As one of approaches to the regulation, electron microscopies (EMs) such as transmission electron microscopy and scanning electron microscopy (SEM) attract attentions as NM measuring tools, due to their capability to easily and quickly observe individual nano-particles3.

  For reliable particle counting by EMs, the particles in a micrograph, should be clearly visualized and easy to count. To minimize ambiguity in particle counting, particles should be (1) uniformly placed over a specimen holder with no localization in particle size and number, or (2) placed only in a small area that allow us to count all particles in the area. For these cases, particles are expected not to overlap each other. Furthermore, it would be helpful, if there are neither agglomerating nor aggregating particles. For NMs, which are usually supplied as suspension, it is not so easy to make such specimens, although specimen preparation is the key process for particle counting by EMs.

  In this paper, we present two specimen preparation methods, which can satisfy the conditions above: a combination method of inkjet and freeze-drying, and electrostatic aerosol deposition method. In freeze-dry method, the suspension of polystyrene (PS) sphere was dropped onto a cooled Si substrate (around -20 ºC) then immediately dried in vacuum. SEM observations shown that this method put the PS spheres within small area and strongly suppressed the aggregation of the spheres (Fig. 1a), compared to one prepared with a substrate at room temperature (Fig. 1b).

  On the other hand, specimens of uniformly dispersed nano-particles were made by electrostatic aerosol deposition method. PS aerosol was generated from the suspension by an electrospray nebulizer, and then collected on a Cu grid with carbon support by an electrostatic precipitator. Figure 2 shows bright field images of PS spheres on the grid taken by SEM in transmission mode. It was observed that PS spheres in two different sizes are placed uniformly and independently over the whole grid.

  We have demonstrated that these preparation methods are promising for particle analysis by EMs. In the presentation, we also discuss how to establish particle-counting system with these preparation methods and EMs.

References:[1] European Commission, Official Journal of the European Union L 275, 20.10.2011 (2011) p. 38.  [2] A Lopez-Serrano et al, Analytical Methods 6 (2014) p. 38.  [3] EFSA Scientific Committee, EFSA Journal 9 (2011) p. 2140.

This work was performed as one of the studies conducted by the consortium for measurement solutions for industrial use of nanomaterials (COMS-NANO) in Japan.

Fig. 1: SE images of PS spheres (260 nm and 30 nm in diameter) (a) Prepared by inkjet and freeze-drying method. (b) By inkjet at room temperature. The primary beam energy was 5 keV. The lower images are enlarged images of the area indicated with square in upper images.

Fig. 2: Bright field images of PS spheres (170 nm and 70 nm in diameter) on Cu grid collected by electrostatic aerosol deposition method. (a) Overview of a hole of the Cu grid and (b) magnified image. These images were taken by SEM in transmission mode. The primary beam energy was 30 keV.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-1953 Low voltage electron microscope - new tools for the measurements of nanoparticles diameters

Kocová L.1, 2, Bílý T.1, Langhans J.1, Nebesářová J.1, 3
1Biology Centre ASCR, v.v.i., Branisovska 31, 370 05 Ceske Budejovice, Czech Republic, 2Fakulty of Science, University of South Bohemia, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic, 3Faculty of Science, Charles University in Prague, Vinicna 7, 128 43 Praha 2, Czech Republic
lucie_kocova@paru.cas.cz

The low voltage electron microscope (LVEM) is an unusual type of microscope dedicated for the observation of specimens composed of low atomic number elements [1].Its main advantage consists in the use of the accelerating voltage around 5 kV which causes nearly twenty times more image contrast enhancement than a routine transmission electron microscope working at the accelerating voltage 100 kV [2]. Its disadvantage is that such low energy primary electrons are able to pass only through extremely ultra-thin specimens with the thickness. Therefore a measurement of nanoparticles size distribution seems to be the convenient application for this microscope working in TEM mode.

In the study LVTEM (Delong Instruments, Brno) was used for measurements of size distributions of laboratory prepared palladium (Pd) cubes and spheres and commercially prepared quantum dots (QD) nanoparticles (Invitrogen), both with diameters below 20 nm. The aim was to compare the accuracy of measurements with results obtained by means of a routine transmission electron microscope working at the accelerating voltage 100 kV (TEM) and a high resolution scanning electron microscope using the accelerating voltage below 10 kV (HRSEM).

Suspensions of nanoparticles were dropped on copper grids covered by un-direct evaporated carbon layer [3] and air dried after removing of the solution excess by a small piece of a filter paper. Digitally recorded images from all microscopes were treated with the ImageJ freeware program [http://imagej.en.softonic.com/]. The counting and measuring of a nanoparticles size distribution was performed on thresholded images.

We confirmed that the LVTEM produces images with very high contrast (Fig. 1). However size distributions of measured nanoparticles were slightly shifted to the greater values in comparison with size distributions obtained by means of routine TEM (Fig. 2). The decrease of the accelerating voltage causes less resolution, which is still sufficient for the observation of nanoparticles, on the other hand, significantly increases effective cross section leading to a stronger electron scattering. This phenomenon appears mainly at the nanoparticles edges and makes them in LVTEM bigger. This explanation was proved by results of nanoparticles size distributions in HRSEM and Monte Carlo simulations of electron scattering in Pd nanoparticles (Fig. 3).
Based on our results we can conclude that the LVTEM is the excellent tool for the measurement of nanoparticles size distributions.

References
[1] LVEM5 Application Note. February (2013), www.lv-em.com
[2] Delong et al. Proc. EUREM 12, 197 (2000)
[3] MAISHEV et al. Combined ion-source and target-sputtering magnetron and a method for sputtering conductive and nonconductive materials [patent] US6214183 B1

This work was supported by the Academy of Sciences (Z60220518) and Technology Agency of the Czech Republic (TE01020118).

Fig. 1: Quantum dots nanoparticles in LVTEM (5 kV) in left and in TEM (80 kV) in right.

Fig. 2: Histogram of Pd cubes (15 nm) determined by LVTEM (5kV).

Fig. 3: The Monte Carlo model of electron scattering at Pd cubes (15 nm) simulated for 80 kV (right) and 5 kV (left) primary electrons.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-2029 Development of fluorescent plasmonic nanotags for in-flow detection of rare cancer cells

Boca-Farcau S.1, 3, Soritau O.1, Chereches G.1, Vartic O.2, Virag P.1, Susman S.2, Astilean S.3, Ciuleanu T.1
1Institute of Oncology “Prof. Dr. Ion Chiricuta”, 34-36 Republicii St., 400015, Cluj-Napoca, Romania, 2University of Medicine and Pharmacy “Iuliu Hatieganu”, 8 Victor Babeş St., 400012 Cluj-Napoca, Romania, 3Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute on Bio-Nano-Sciences, Babes-Bolyai University, 42 Treboniu Laurian St., 400271 Cluj-Napoca, Romania
sanda_c_boca@yahoo.com

Detection and discrimination of rare cells from complex mixtures require high selectivity and throughput in order to get relevant results for clinical diagnostic. Current analytical methods are often drawn back by sources of artifacts that become apparent only when large numbers of cells are acquired. Moreover, the technique should be inexpensive, facile, and accessible to researchers without specialized training in silicon microfabrication, surface functionalization, and microdevice operation. Spectroscopic detection of rare cancer cells using biocompatible nanotags developed by chemisorption of fluorophores and/or Raman reporters on gold colloid was recently demonstrated as a method of high sensitivity and enough practicability.

In this regard we synthesized gold nanoparticles of various plasmonic responses (from visible to Near-IR), we conjugated the particles with fluorophores that overlap the plasmonic band of particles (fluorescein isothiocyanate, cresyl violet perchlorate) for maximum signal intensity and capped the nanoconjugates with polymer mPEG-SH for improved stability and biocompatibility. To attain specific recognition of non-adherent colon adenocarcinoma cells Colo320 (selected as a model cell line for circulating tumor cells), the fluorophore-particle conjugates were further functionalized with epithelial cell-specific antibodies. The nanotags were characterized by transmission electron microscopy, UV-Vis-NIR absorption spectroscopy, dynamic light scattering, zeta potential, fluorescence and/or surface enhanced Raman scattering (SERS) and found to be chemically stable and detectable inside cells down to nanomolar concentrations. For quantitative validation of the method, whole blood preparations were enriched with an increasing number of Colo320 tumor cells loaded with fluorophore-particle conjugates and also stained with a vital red fluorescent membrane marker (PKH26). Stained cells were quantified microscopically and by flow cytometry.

The presented results evidence the potential of spectroscopic-active nanotags to serve as ultra-sensitive imaging tools for rare cancer cell detection.

This work was supported by CNCS-UEFISCDI, project number PN-II-RU-PD-2012-3-0111.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-2136 The use of electron microscopy in the quantification of nanoparticle dose and cellular uptake

Hondow N.1, Brown M. R.2, Summers H. D.2, Rees P.2, Brydson R.1, Brown A.1
1Institute for Materials Research, University of Leeds, Leeds LS2 9JT, UK, 2Centre for NanoHealth, Swansea University, Swansea SA2 8PP, UK
n.hondow@leeds.ac.uk

Nanotoxicology and nanomedicine are both concerned with the delivery of nanoparticles to human cells, whether this be unintentional exposure by way of, for example, occupation or use of cosmetics (nanotoxicology) or by design, such as drug-delivery vehicles or magnetic resonance imaging contrast agents (nanomedicine). Current in vitro studies are concerned with the biological impact of nanoparticles, with electron microscopy commonly employed to image the intracellular location. It is critical to quantify the absolute nanoparticle dose received per cell in a given exposure, and to understand the factors which affect this. This is challenging because of the complex and varied mechanisms of nanoparticle interactions with cells.

In this work we have developed a quantitative description of nanoparticle uptake by an in vitro cell line using protein coated CdSe/ZnS quantum dots and human osteosarcoma U-2 OS cells. TEM of thin cell sections can provide the location and number of cellular vesicles per 2-D cell slice plus the number of nanoparticles encapsulated per vesicle, while serial sectioning can provide this across the whole cell. Serial block face SEM data have been used to correlate higher resolution 2-D TEM data to high throughput, low resolution optical imaging of quantum dot nanoparticle loaded cells. This results in the determination of a calibration factor to transform flow cytometry data to a nanoparticle dose, in terms of the fundamental unit, the number of nanoparticles internalised per cell. Using a combination of these techniques we will also explore the correlation between the initial dispersion state of the nanoparticles in cell culture media and the vesicular uptake in cells. We will demonstrate that vesicle inheritance after cell division leads to an asymmetric particle dose within the daughter cells.

The authors would like to thank the EPSRC for funding (EP/H008578/1 and EP/H008683/1); A. Warley, K. Brady, and F. Winning (Centre for Ultrastructural Imaging, King's College, London, U.K.) for pelleting and sectioning the cells for TEM analysis; T. Starborg (Wellcome Centre for Cell Matrix Research, University of Manchester), A.G. Monteith and N. Wilkinson (Gatan U.K.) for 3-View imaging.

Fig. 1: (a)–(b) TEM images of a cell thin section with internalized quantum dots; (c) TEM image of a vesicle containing quantum dots from within the cell in (a) and (b); (d) automated nanoparticle counting; (e) still from a reconstruction of a cell imaged using SBF-SEM; (f) TEM image of quantum dots dispersed in cell delivery media.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-2160 Utilization of polyomavirus–based nanoparticles in cancer diagnostics and therapy

Suchanova J.1, Spanielova H.1, Sacha P.2, Forstova J.1
1Faculty of Science, Charles University in Prague, Prague, Czech Republic, 2Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic.
jirina.suchanova@natur.cuni.cz

Non-invasive imaging holds great promise for the early detection and treatment of human disease. The use of "smart" nanoparticles, that combine multiple functions of targeting, imaging and drug delivery, have tremendous potential to increase the sensitivity and specificity of therapies. We investigate the targeting potential of mouse polyomavirus based virus-like particles (VLPs) as vectors for directed cell/tissue delivery of therapeutic or diagnostic compounds. Mouse polyomavirus, type species of Polyomaviridae family, is superior for this kind of research to its human counterparts due to the absence of pre-existing immunity in human population. We intend to target the VLPs to cancer cells by changing the receptor binding site on the major capsid protein VP1. There are four predominant surface-exposed loops (BC, DE, EF and HI) in the VP1 structure, but only BC and HI outfacing loops are responsible for the attachment to the native sialic acid-containing receptor. We selected the BC loop as a candidate site for manipulation and prostate cancer cells as a model system. We used site-directed mutagenesis for insertion of a peptide ligand, which binds to prostate-specific membrane antigen (PSMA). This antigen is overexpressed in androgen-resistant human prostate cancer cells. We investigate the binding specificity of VLPs to PSMA protein in in vitro and in vivo assays. We found the discrepancy between their binding properties and internalization efficiency. The relevancy of these experiments to cell specific targeting and nanoparticle uptake will be discussed.

This work is supported by the Grant Agency of Charles University in Prague (GAUK no. 913613) and SVV-2014-260081.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-2333 Scanning electron microscopy of silver and gold nanoparticles produced by microorganisms

Kalabegishvili T. L.1, 2, Kirkesali E. I.1, Frontasyeva M. V.3, Zinicovscaia I.3, 4, Kazanskiy P. R.5, Dmitrieva T. G.5
1Andronikashvili Institute of Physics, Tbilisi, Georgia, 2Ilia State University, Tbilisi, Georgia, 3Joint Institute for Nuclear Research, Dubna, Russia, 4Institute of Chemistry of the Academy of Science of Moldova, Chisinau, Moldova, 5Systems for Microscopy and Analysis, Moscow, Russia
kazansky@microscop.ru

In recent years great attention has been paid to microbial technologies of nanoparticle production. It is important to examine a new class of microorganisms and different experimental conditions for synthesis of nanoparticles using a new high-resolution electron microscopy technique for visualization and examination of the produced nanoparticles. Novel strains of actinomycetes Streptomyces glaucus 71MD, Streptomyces sp. 211A, Arthrobacter globiformis 151B, Arthrobacter oxydans 61B and blue-green microalga Spirulina platensis were used for synthesis of silver and gold nanoparticles.
The studies were carried out using different techniques including scanning electron microscopy (SEM) and energy dispersive X-ray analysis. Microstructural images of the samples were taken on dual beam FIB/SEM FEI Quanta 3D FEG with LowVac and ESEM options. Microbial samples were studied without conductive coating in low vacuum and ESEM modes. It was demonstrated that the gold and silver granulates explicitly indicate the presence of conglomerates ~ 1 µm in size composed of much smaller particles.
Qualitative and quantitative elemental analysis was carried out by XEDS SEM electron microprobe. XEDS results confirmed the presence of gold and silver in analyzed microbial samples.

1. T.L. Kalabegishvili, E.I. Kirkesali, I. G. Murusidze, G. I. Tsertsvadze, M.V. Frontasyeva, I. Zinicovscaia, V.Y. Shklover, N.V. Shvindina. Characterization of microbial synthesis of silver and gold nanoparticles with electron microscopy techniques. Journal of Advanced Microscopy Research,6(4), 313-317,2011.
2. T. Kalabegishvili, E. Kirkesali, A. Rcheulishvili, E. Ginturi, I. Murusidze, N. Kuchava, N. Bagdavadze, G. Tsertsvadze, V. Gabunia, M. V. Frontasyeva, S.S. Pavlov, I. Zinicovscaia, M.J. Raven, N.M.F. Seaga, A. Faanhof. Synthesis of gold nanoparticles by blue-green algae Spirulina platensis. Advance Science, Engineering and Medicine (Adv. Sci. Eng. Med.), 5(1), 30-36, 2013
3. T. L. Kalabegishvili, I. G. Murusidze, E. I. Kirkesali, A. N. Rcheulishvili, E. N. Ginturi, E. Simon Gelagutashvili, N. E. Kuchava, N. V. Bagdavadze, D. T. Pataraya, M. A. Gurielidze, M. V. Frontasyeva, I. I. Zinicovscaia, S. S. Pavlov, V. T. Gritsyna. Development of biotechnology for microbial synthesis of gold and silver nanoparticles. Journal of Life Sciences, 7(2), 110-122, 2013.

Fig. 1: SEM micrograph of Spirulina Platenis cells imaged in low-vacuum mode

Fig. 2: SEM micrograph of Streptomyces sp. 211A cells imaged in native state (ESEM mode)

Fig. 3: EDS spectrum recorded from Spirulina Platensis after formation of gold nanoparticles
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-2475 The detection of metal-based particles on the bumble-bees as bioindicators of air pollution

Dědková K.1, Kašparová B.1, Kukutschová J.1
1Nanotechnology Centre, VSB-TU Ostrava, Ostrava, Czech Republic
katerina.dedkova@vsb.cz

Monitoring of the atmospheric pollution is an important part of the environmental protection. Living organisms can serve as bioindicators for observation and biomonitoring of the environmental pollution. This contribution describes the detection of metal-based particles on body of bumble-bees (Bombus terrestris) using Scanning electron microscopy (SEM) with EDX. Two groups of bumble-bees were selected for the detection. Bumble-bees caught in outdoor environment in Havířov region belonged into the first group and bumble-bees from laboratory breeding served as a reference in the second group. It was found, that bodies of bumble-bees from outdoor environment were covered with lot of micron-size particles based on dust, sand, salts and metals. These particles may come from natural but also anthropogenic sources emitting solid particles into the atmosphere. Contrary to this finding, any of these particles were found on the bodies of bumble-bees from the laboratory breeding. However, micron-sized particles of iron were detected on the head and wings of these bumble-bees. By comparison of secondary electron images and back-scattered electron images of the same area it was wound that iron particles were incorporated into the basis of hairs on the wings or cuticle on the head of the bumble-bees. The exact function of these iron-based particles in these specific bodyparts is still not well understood, whether they serve as biolocators within the magnetic field of Earth or just places of deposition of particles which entered the body after environmental exposure. SEM with EDX proved that this method could be used as one of a detection technique in the field of study of environmental pollution.

The study was supported by the projects funded by Ministry of Education, Sport and Youth of the Czech Republic No. SP2014/52 and SP2014/76.

Fig. 1: Detecterd particle on the body of bumble-bee from outdoor environment.

Fig. 2: EDX spectra confirming the presence of iron particle on the body of the bumble-bee from outdoor environment.

Fig. 3: Detecterd particles on the body of bumble-bee from outdoor environment.

Fig. 4: EDX spectra confirming the presence of nickel and iron particles on the body of the bumble-bee from outdoor environment.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-2537 Gold Nanorods with Polymers for drug delivery : Imaging the Soft/Hard Materials Interface

Bell D. C.1, Timko B. P.2, 3, Erdman N.4
1School of Enginnering and Applied Sciences, Harvard University, Cambridge MA USA, 2Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge MA.USA , 3Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Division of Critical Care Medicine, Children’s Hospital Boston, Harvard Medical School, 4JEOL USA, Peabody MA,
dcb@seas.harvard.edu

The interface between soft and hard materials is currently underexplored territory that is critical to underrated structure relationships with the hard/soft materials interface domain. You need high resolution of the hard material, but good contrast (and resolution) of the corresponding soft materials. Low-voltage High-Resolution Electron Microscopy (LVHREM) has several advantages: increased cross-sections for inelastic and elastic scattering and hence higher contrast efficiency from each atom and reduced radiation knock-on damage to samples insensitive to other damage mechanisms, which includes most metals, semiconductors and other solid state materials.  We have also conducted many experiments imaging with very low voltage SEM.

As an example: Gold nanorods can be used as photothermal converters, permitting near-infrared (NIR) light to be transmitted deep into tissues without causing damage (Fig. 1). The temperature-sensitive material poly(N-isopropylacrylamide) (pNIPAm) collapses when heated beyond 32 ºC, and has been used in drug delivery systems. Composites between these two disparate materials can be triggered with NIR light and open new avenues for triggerable drug delivery systems. Toward this end, we synthesized pNIPAm nanogel particles and conjugated them to polyelectrolyte-coated gold nanorods via electrostatic interactions. LVHRTEM enables imaging of both the polymer and gold components of these composite structures, and elucidates their interface (Fig. 2).

Although the TEM samples must be significantly thinner for low keV observation, the improvement in contrast for inorganic materials, biological samples and especially nano-biological samples in low-voltage TEM while retaining atomic resolution cannot be understated. Damage mechanisms for biological samples are complicated and very structure dependent.

D.C. Bell. gratefully acknowledges funding through the National Science Foundation award (NSF

DMR-1108382)

Fig. 1: Low Magnification TEM image of polymer spheres covered with gold nanorods

Fig. 2: Imaging the materials interface between gold nanorods and the polymer thermoplastic junction.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-2854 Magnetic-Fluid-Loaded Liposomes for Selective Targeting of Brain Tumors.

Marie H.1, Lemaire L.2, Franconi F.3, Trichet M.4, Frébourg G.4, Nicolas V.5, Ménager C.6, Lesieur S.1
1Laboratoire Physico-Chimie des Systèmes Polyphasés, Institut Galien Paris-Sud, UMR CNRS 8612, Faculté de Pharmacie, Université Paris-Sud, LabEx LERMIT, Châtenay-Malabry, France, 2INSERM UMR-S 1066, Micro et nanomédecines Biomimétiques – MINT, Université d'Angers, LUNAM Université, Angers, France, 3PRIMEX-CIFAB, LUNAM Université, Université d’Angers, IRIS/IBS, CHU d’Angers, Angers, France, 4Service de microscopie électronique, IBPS, UPMC, CNRS, Paris, France, 5Plateforme Imagerie cellulaire, IFR 141-IPSIT, Faculté de Pharmacie, Université Paris-Sud, Châtenay-Malabry, France, 6Equipe Colloïdes Inorganiques, Phenix, UMR CNRS 8234, UPMC, Paris, France
michael.trichet@snv.jussieu.fr

Hybrid devices based on the association of iron oxides with lipid nanoscale particles play an increasing role for targeted delivery of chemotherapeutics, mainly due to their recognized biocompatibility and intrinsic efficacy as contrast agents for in-vivo Magnetic Resonance Imaging (MRI). In this study, we aim to target human U87 glioblastoma, implanted into the striatum of mice, using magnetic-fluid-loaded liposomes (MFLs), sterically stabilized by hydrophilic poly(ethylene glycol) chains and loaded with a suspension of superparamagnetic nanocrystals of maghemite. MFLs targeting was achieved by applying a 190-T/m magnetic field gradient, produced by an external 0.4-T magnet placed onto the head of the mice.

In-vivo monitoring of MFLs trafficking was performed by a 7-T MRI as a function of time. MRI demonstrated a significant increase in intra-tumoral concentration of the magnetoliposomes for magnet-exposed mice, compared to not exposed ones. Animals were then sacrificed and their brains were sliced and alternatively processed for confocal microscopy or transmission electron microscopy (TEM), to perform histological and cytological analysis. Primary, detection of the rhodamine-labelling of MFLs lipid membranes in confocal microscopy revealed accumulation of magnetoliposomes in brain tumour. TEM observation of the same regions, in adjacent slices, revealed the presence of clustered electron-dense nanoparticles in the extracellular matrix space and within endosomal-structures in the cytoplasm of tumour cells and in cells lining the vascular lumen. Finally, electron energy-loss spectroscopy (EELS), coupled to energy-filtered imaging in TEM showed the iron composition of these electron-dense nanoparticles, confirming their MFLs identity.

The overall observations showed that MFLs were successfully delivered and concentrated into glioblastoma via the vasculature where they pass through the vascular endothelium as intact structures due to enhanced permeation and retention effect before to be internalized by the tumor cells. Interestingly, the magnetic field gradient does not affect the amounts of the MFLs recovered in the healthy part of brains, which comparatively remain very low according to the different imaging methods we used.

The results in their whole revealed MFLs as potent tools for selective targeting of malignant brain tumors, especially promising for therapeutic issue as it can be expected that healthy brain tissue will be spared upon treatments by deleterious anticancer drugs carried by MFLs.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-3114 The anticancer activity of binuclear Mn complex, [Mn2(bipy)2(C6H5)2CHCO2)3 (CH3O)](ClO4)],- SLNs formulation

Güney G.1, Çeçener G.1, Dikmen G.2, Kani I.3, Egeli U.1, Tunca B.1
1Uludağ University of Bursa, Department of Medical Biology, Bursa, Turkey, 2Eskişehir Osmangazi University of Eskişehir, Center Research Laboratory, Eskişehir, Turkey, 3Anadolu University of Eskişehir, Department of Chemistry, Eskişehir, Turkey
gamzeguney@anadolu.edu.tr

Inorganic compounds have played an important role in the treatment of cancer because of their activity relying on specific interactions with DNA, leading to cell damage and ultimately to cell death. However, they have several limitations for example, a lack of selective uptake in cancer cells. Solid lipid nanoparticles (SLNs) which are sub-micron colloidal carrier are attracting major attention as novel colloidal drug carrier due to unique properties including highlighted physical stability, controlled drug release, enhanced bioavailability and avoided degradation of the entrapped drug. In this study, [Mn2(bipy)2(C6H5)2CHCO2)3 (CH3O)](ClO4)] binuclear complex has been synthesized with the reaction of bifunctional 2,2′-bipyridine (bipy) and diphenyl acetic acid (C6H5)2CHCO2H). Then, SLNs formulation which included Mn complex produced by hot homogenization. The average diameter of Mn complex-SLNs formulation was about 250 nm and the size of this nanoparticle approved by using SEM. The cytotoxic effects of SLNs formulation on MCF-7 cells were determined by WST-1 test (27.4% viability at 72 h, p<0.05). In addition, the structural changes of MCF-7 cells were observed. MCF-7 cells treated with Mn-SLNs formulation were round in shape, the nuclei of cells were coarse and connection between cells was damaged. As a result, complex loaded SLNs formulations had an important inhibitory effect on MCF-7 cells and induced apoptosis. This SLNs formulation may be used as a carrier system for cancer therapy.

Fig. 1: SEM photo of [Mn2(bipy)2(C6H5)2CHCO2)3 (CH3O)](ClO4)]-SLN complex.

Fig. 2: The images of MCF-7 cells treated with Mn complex-SLNs formulations.

Fig. 3: The images of MCF-7 cells treated with Mn complex-SLNs formulations.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-3157 Endocytic pathways involved in polystyrene nanoparticle uptake in human gastric adenocarcinoma cells

Forte M.1, Iachetta G.1, De Falco M.1, Laforgia V.1, Valiante S.1
1Department of Biology, University of Naples Federico II
valiante@unina.it

Nanoparticles (NPs) are promising tools in medical fields, both in diagnosis and therapy (Schlorf et al., 2012; Wickline and Lanza, 2008). Despite this high applicative potential, little is known about their interaction with biological systems, almost in terms of endocytic pathways and toxicity. The first step to develop a good drug delivery systems based on NPs is to well characterize these molecular aspects. Thus, in this work, with a quantitative and qualitative approach, we studied the uptake of two representative sizes of polystyrene nanoparticles (PS-NPs), 44 nm (NP44) and 100 nm (NP100), labeled with FITC and ROD, respectively, in human adenocarcinoma gastric cells (AGS). The experiments were performed after exposure with 10µg/mL NPs for different times of incubation and temperatures (37°C and 4°C), with or without well known endocytosis inhibitor drugs (dynasore for clathrin dependent pathways and EIPA for macropinocytosis/phagocytosis). Quantitative spectrofluorimetric assays reveal a time-dependent kinetics of internalization at 37°C, with maximum values after 30 min and a decrease after 1 h for both NPs sizes. Precisely, NP44 show a high rate of uptake and a quickly internalization compared to NP100 (Fig. 1). Fluorescent images demonstrate that NPs are able to accumulate in the cytoplasm after 1 and 4 h, without reaching cell nuclei. However, NP100 tend to form aggregate after long exposition times (Fig. 3), while NP44 present an uniform cytoplasmatic distribution at all times considered (Fig. 4). Endocytosis inhibition tests show a null internalization at 4° C and a strong reduction of the uptake rate after treatment with dynasore for both NPs; EIPA, instead, partially affects NPs uptake (Fig. 2). In conclusion, in this study, we demonstrated that PS-NPs are internalized by AGS cells in a size and time dependent manner; probably, as suggest by other authors, they undergo a release process (Iversen et al., 2011). Moreover, we show that this uptake occurs through an energy dependent mechanism and that clathrin mediated endocytosis seems to be the privileged endocytic pathway for PS-NPs.

References:

Iversen TG., SkotlandT. Sandvig K. (2011). Endocytosis and intracellular transport of nanoparticles: Present knowledge and need for future studies. Nonotoday 6:176-181.

Schlorf T, Meincke M, Kossel E, Gluer CC, Jansen O, Mentlein R (2011). Biological properties of iron oxide nanoparticles for cellular and molecular magnetic resonance imaging. Int J Mol Sci. 12(1):12–23.

Wickline S.A., Lanza G.M. (2003). Nanotechnology for molecular imaging and targeted therapy. Circulation 107: 1092–1095.

The study was supported by the grant F.A.R.O. 2009: Effects of nanoparticles designed for the safe delivery on the vitality and functionality of several biological systems.

Fig. 1: Uptake kinetics of polystyrene nanoparticles in AGS cells: NP44 are faster and more efficiently internalized by AGS (1 min) compared to NP100. Both nanoparticles show the higher rate of uptake after 30 min and a release process after 1 h of incubation.

Fig. 2: Mechanism of endocytosis of polystyrene nanoparticles (PS-NPs): AGS were pre-treated with dynasore or EIPA for 30 min at 37°C or pre- incubated at 4°C for 15 min. Following pre- treatment cells were exposed to PS-NPs for 1h.

Fig. 3: Uptake of polystyrene nanoparticles 100 nm diameter labelled with Rhodamine (NP100) in AGS cells: AGS were grown in chamber slide, incubated for 4h with NP100 and analysed with epifluorescence microscopy. Nuclei were stained with Höechst. Scale bar 10 µm.

Fig. 4: Uptake of polystyrene nanoparticles 44 nm diameter labelled with fluorescein isothiocyanate (NP44) in AGS cells: AGS were grown in chamber slide, incubated 1h with NP44 and analysed with epifluorescence microscopy. Nuclei were stained with Höechst. Scale bar 10µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-3229 A cell-penetrating peptide as a tool for delivery to blood brain barrier (BBB)

Iachetta G.1, Forte M.1, Falanga A.2, Galdiero S.2, Valiante S.1
1Department of Biology, University of Naples Federico II, Naples, Italy, 2Department of Pharmacy, University of Naples Federico II, Naples, Italy
giuseppina.iachetta@unina.it

The high impermeability and selectivity of the BBB prevent the transport of many drugs into the brain, making them ineffective for the treatment of central nervous system diseases. The cell-penetrating peptides (CPPs) represent a new strategy to functionalize carriers in order to deliver therapeutic molecules to the brain. Several studies have demonstrated that gH625, a peptide derived from the glycoprotein H of herpes simplex virus 1, is able to cross the membrane bilayer and represents an ideal CPP for the delivery BBB, because of its ability to escape from the endocytic pathways1. In this study we evaluated the internalization of gH625 in human neuroblastoma (SH-SY5Y) and astrocytoma (U87-MG) cells and in rat brain. Fluorescence and spectrofluorimetric in vitro analyses show a good rate of uptake in both cell lines after 2h of treatment with gH625 labeled with 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (gH625-NBD) (Fig.1). The internalization is almost complete if higher concentration was used (5µM) and the signal is prevalently found within the cytoplasm. Immunofluorescence studies, using anti-GFAP and anti-BBB antibodies, were performed after intravenous administration (3h) of gH625-NBD (160μg/100 g bw) in rats. Five images for each experimental class were analyzed with ImageJ 1.48 software; the deconvolutionlab plugin was used to deconvolve image channels through the Tikhonov-Miller’s algorithm; the Co-localization Colormap plugin was then used to evaluate the degree of correlation between pair of pixels in the red and green channels, resulting in the distribution of the values of the normalized mean deviation product (nMDP) and the index of correlation as the fraction of positively correlated pixels in the image2. Co-localization studies produced a color scale map (from -1 to 1) where negative indexes (cold colors) represent no co-localization and indexes above 0 (hot colors) represent co-localization. The study reveals a high co-localization score with BBB and low co-localization score with GFAP protein of astrocytes; interestingly few neurons were labeled for gH625-NBD, indicating the passage through the BBB (Fig.2). The index of correlation shows poor positive correlation in gH625/anti-GFAP and high positive correlation in the gH625/anti-BBB. These data show that gH625 is up taken by neuronal cells and reaches the rat brain. Taken together, our results can be considered as a preliminary data to develop a liposome-based systems which involves the use of gH625 as an efficient drugs delivery through the BBB.

1Guarnieri D et al. 2013 Drug delivery: shuttle-mediated nanoparticle delivery to the BBB. Small 9(6): 806

2Jaskolski F et al. 2005 An automated method to quantify and visualize colocalized fluorescent signals. J of Neur Meth 146(1):42-49

The study was supported by the grant F.A.R.O. 2012: Novel mechanisms of transport through the blood brain barrier: peptides and nanoparticles

Fig. 1: SH-SY5Y (a, b) and U87-MG cells (d, e) internalize gH625-NBD; scale bars=50 µm: c, f) Degree of internalization as assessed by spectrofluorimetry for each cell line.

Fig. 2: Images of Alexafluor594, FITC, and Höechst and co-localization map (a1-f1). a,b,d scale bar=50µm; c,e,f scale bar=20µm. a) Negative control of anti-BBB on control animal; b,c) anti-GFAP on control and treated animal; d,e,f) anti-BBB on control and treated animal; g,h) Top: nMDP distributions. Bottom:The index of correlation histograms.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-3285 The role of TEM in the systematic investigation of the physicochemical factors that contribute to the toxicity of ZnO nanoparticles

Mu Q.1,2, David C. A.3, Galceran J.3, Rey-Castro C.3, Krzemiński Ł.1, Wallace R.4, Bamiduro F.4, Milne S. J.4, Hondow N.4, Brydson R.4, Vizcay-Barrena G.5, Routledge M. N.2, Jeuken L. J.1, Brown A. P.4
1School of Biomedical Sciences, University of Leeds, LS2 9JT, Leeds, UK, 2Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, LS2 9JT, Leeds, UK, 3Departament de Química and AGROTECNIO, Universitat de Lleida, 25198 Lleida, Catalonia, Spain, 4Institute for Materials Research, University of Leeds, LS2 9JT, Leeds, UK , 5Centre for Ultrastructural Imaging, Kings College London, SE1 1UL, London, UK
a.p.brown@leeds.ac.uk

ZnO nanoparticles (NPs) are prone to dissolution and uncertainty remains whether biological/cellular responses to ZnO NPs are solely due to the release of Zn2+ or whether the NPs themselves have additional toxic effects. We have addressed this concern by measuring ZnO NP solubility in dispersion media (Dulbecco’s Modified Eagle Medium, DMEM) held under identical conditions to those employed for cell culture (37 oC, 5% CO2 and pH 7.68) and by systematic comparison of cell-NP interaction for three different ZnO NP preparations. Analytical TEM has played a critical role because it has enabled visualisation and chemical analysis of individual NPs at each stage of the toxicological testing, indicating of phase and morphology of the as-purchased/produced particles, after dispersion in cell culture media and after cellular uptake.
We show that for NP concentrations up to 5.5 μg ZnO/mL, dissolution is complete in less than one hour, with the majority of the soluble zinc complexed to ligands in the medium. Above 5.5 μg/mL results are consistent with the re-precipitation of zinc carbonate, keeping the solubilised zinc fixed to 67 μM of which only 0.45 μM is as free Zn2+, i.e., not complexed to ligands. At these relatively high concentrations, NPs with a polymer-coating show slower dissolution (i.e. free Zn2+ release) kinetics compared to uncoated NPs, reaching thermodynamic equilibrium only after 48 hours. Cytotoxicity (MTT) and DNA damage (Comet) assay dose response curves for three epithelial cell lines suggest that dissolution and re-precipitation dominate for uncoated ZnO NPs.
TEM reveals the form of the as-purchased/produced NPs (Figure 1 a-c), it will also be used to assess phase and morphology of the re-precipitated material when ZnO NPs are dispersed in DMEM held under the conditions for cell culture. We will discuss quantitative assessment of ZnO NP dispersions by TEM when using plunge-freezing as a specimen preparation route (Figure 1 d+e). In addition, monitoring of intracellular Zn2+ concentrations and ZnO-NP interactions with model lipid membranes indicate that a polymer coat on ZnO NPs increases cellular uptake, enhancing toxicity by enabling intracellular dissolution and release of Zn2+. TEM confirms the cellular uptake of the coated ZnO NPs (Figure 1 f+g). Similarly, we demonstrate that needle-like NP morphologies enhance toxicity by incomplete cellular uptake. Thus, we will highlight the key role analytical TEM can play in the fields of nanotoxicology and nanomedicine.

This work was funded by the European Union (FP7-NMP-2008-1.3-2 and FP7-NMP.2012.1.3-3) under grant agreements n° 229244 and 310584.

Fig. 1: TEM images of (a) polymer coated, (b) uncoated ZnO NPs and (c) ZnO nanoneedles. TEM images of ZnO NPs dispersed in water and (d) drop-cast, (e) plunge-frozen on TEM grids. Identification of cellular uptake of polymer coated ZnO NPs, (f) partially dissolved NP within A549 cell (inset) and (g) EDX spectrum confirming the Zn-rich identity of the NP.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-5700 CHARACTERIZATION OF SPIRULINA (Arthrospira maxima) NANOPARTICLES BY MICROSCOPY AND SPECTROSCOPY TECHNIQUES

NERI E.1, CHANONA J.1, CÁRDENAS S.1, PALACIOS E.3, CALDERÓN H.4, CHAMORRO G.2, CALDERÓN G.1
1Escuela Nacional de Ciencias Biológicas-IPN, Departamento de Ingeniería Bioquímica, 2Escuela Nacional de Ciencias Biológicas Zacatenco-IPN, Departamento de Toxicología, , 3LAMEUAR. Instituto Mexicano del Petróleo, 4Escuela Superior de Física y Matemáticas-IPN
stefany_fany03@hotmail.com

The Arthrospira sp. (Spirulina) a cyanobacteria, contains a large amount of nutrients, which gives beneficial attributes for the human health. On the other hand, the mechanical milling has been used both to reduce the size of some materials and improve the potential in biological, chemical and physics systems. The aim of this work is to compare the properties of bulk Spirulina against nanostructured milled Spirulina to probe if their effects can be potentiated (Gershwin 2008).

The mechanical milling of Arthrospira maxima (Spirulina, Spex mill Sample PREP 8000D Dual Mixer) took 1 to 4 hours (named reference, 1HBC, 2HBC, 3HBC and 4HBC) the obtained Spirulina nanoparticles were carried out in 2 vials containing 3 g each. The nanoparticle characterization was divided in two stages (a) dry basis (powder) and, (b) wet basis, 0.01% dispersion of Spirulina was ultrasonicated for 20 min. The particles were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and Fourier Transformed Infrared (ATR-FTIR). The XRD analysis of the powders showed a wide peak that could be inferring amorphous zones. In general, it is possible to observe a diminishing trend in the crystallinity index, with values between XCRef= 53.3, XC1HBC = 51.7, XC2HBC = 52.9, XC3HBC = 48.1 y XC4HBC 49.1 (Segal et al. 1959). However, more studies should be done to corroborate this. Otherwise, a 3 h test of FTIR was done; the peaks suggest a large exposition of functional groups such as OH, CH2 and C-N Figure 1. (Dotto, Cadaval & Pinto 2012). Image analysis was made using AFM micrographies; the kinetic curve described a minimum average particle size of 90 nm in the first 2 hours, using a bimodal distribution. The particles ranged between 20-370 nm with an average particle size of 90±62 nm. The SEM microscopy confirms the data obtained by AFM (Figure 2). In conclusion, it was possible to obtain Spirulina nanoparticles by mechanical milling and they had more functional groups availability than the bulk Spirulina according to the studies already done.

Dotto, GL, Cadaval, TRS & Pinto, LAA 2012, 'Preparation of bionanoparticles derived from Spirulina platensis and its application for Cr (VI) removal from aqueous solutions', Journal of Industrial and Engineering Chemistry, vol 18, pp. 1925-1930.

Gershwin, ME,BA( 2008, Spirulina in Human Nutrition and Health, Taylor and Francis, London.

Segal, L, Creely, JJ, Martin, AE, Conrad, JR & Conrad, C 1959, 'An empirical method for estimating the degree of cristallinity of natice cellulose using the X-ray diffrectometer', Textile Research Journal, vol 29, pp. 786-794.

This research was funded through projects 20140387 and 20141662 from Instituto Politécnico Nacional (SIP IPN Mexico) and COFAA. The corresponding author would also like to thank CONACYT for the scholarship provided.

Fig. 1: FT-IR Spectrum for Spirulina, reference and milling time are indicated

Fig. 2: Spirulina´s nanoparticles micrographies obtained by (A) Scanning electron microscopy , (B) y (C) atomic force microscopy.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-5733 Nitrogen-vacancy centers in nanodiamond for cellular sensing

Gulka M.1, 2, Hrubý J.1, 2, Petráková V.1, Remeš Z.1, 2, Nesládek M.1, 3
1Czech Technical University in Prague, Faculty of Biomedical Engineering, Sítná sq. 3105, 272 01, Kladno, Czech Republic, 2Institute of Physics, Academy of Sciences Czech Republic, Na Slovance 5, 185 00, Prague 8, Czech Republic, 3IMOMEC division, IMEC, Institute for Materials Research, University Hasselt, Diepenbeek, Belgium
gulka@fzu.cz

Recent development of manufacturing techniques enables to create bright fluorescent nanodiamond (ND) particles of various sizes with high content of nitrogen-vacancy (NV) centres [1]. It has opened the way towards novel sensitive nanoscale biosensors that can be used for cellular imaging and in-vivo sensing of biochemical processes such as controlled drug release. NV centres exits in two charge states, both of them are luminescent with different zero-phonon lines (575 nm for NV0 and 636 nm for NV-). ND particles can be grafted with certain surface ligands for specific recognition by the cell receptors allowing targeted delivery of ND. Change in luminescence can be observed upon surface interactions of ND surface with different molecules dropped or bound to the surface leading to the change of NV0 and NV- luminescence, which can be used for molecular biosensors [2]. Moreover, detection of small magnetic field (~ 10 nT/Hz) is possible by optically detected magnetic resonance (ODMR) that can be used for nanoscale magnetic sensing.

In this work we demonstrate the capability of NV centres to be used in biological luminescence imaging and in charge sensing with nanodiamonds prepared by various manufacturing techniques and surface grafting. The single-photon confocal scanning microscope enables measuring the particle fluorescence with a high resolution allowing to detect single nanocrystals and to measure their spectra. The correlation spectroscopy measurement (anti-bunching) in biological samples allows distinguishing the particle with single NV centres and their specific interaction with the biological environment. We compare the NV centers with classical quantum dots systems (PbS) for cell imaging. Specific properties of NV centers such as surface charge sensitivity and strong a stable luminescence with no photobleaching together with diverse sensing capabilities of the single-photon confocal scanning microscope enables long term study of biomolecular processes with super-resolution techniques.

[1] J. Havlik, M. Gulka, V. Petrakova, M. Nesladek et al., Nanoscale (2013)
[2] V. Petrakova, M. Nesladek et al., Advanced Functional Materials (2012)

GAČR 14_05053S, OP VK grant CZ.1.07/2.3.00/20.0306

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-5739 Contribution of Surface Nanostructures to DNA Hybridization Efficiency

Kim H.1, Terazono H.1,2, Takei H.1,3, Yasuda K.1,2
1Kanagawa Academy of Science and Technology, Kanagawa, Japan, 2Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan, 3Department of Life Sciences, Faculty of Life Sciences, Toyo University, Gunma, Japan
ykp-kim@newkast.or.jp

Understanding how the nanoscale surface roughness contributes to the efficiency of the target-ligand reaction is important for the improvement of biomolecular sensing. Hence, we have examined the contribution of nanoscale convex/concave structures for the DNA reactions on a solid surface. Efficiency of DNA immobilization on convex surfaces has been studied using gold nanoparticles in diameter range from 10 to 50 nm [1]. Contribution of a concave structure on DNA hybridization efficiency was also examined using an inner surface of strictly size-controlled metal hemisphere nanoparticles having a spherical cup shape (nanocups) in diameter range from 140 to 800 nm [2]. In the convex experiments, thiolated DNAs were immobilized on the Au nanoparticle surfaces and the immobilized densities were evaluated with UV-vis spectra measurements (Fig.1a). In the concave experiments, thiolated DNAs were immobilized on only the inner Au layer of the fabricated nanocup, hybridized with complementary DNA-attached 20 nm Au nanocolloids, and the number of the hybridized Au nanocolloids was counted by field emission scanning electron microscopy (Fig.1b). In results, the density of immobilized DNAs on the convex surfaces increased depending on the decrease of gold nanoparticle diameter up to 130 times thicker than that on a flat surface (Fig.2a). The DNA hybridization density on concave surfaces increased gradually as the cup size decreased and reached to 394 μm−2 on a 140 nm cup, 0.88 times of a flat surface, which indicates about twice increase of the hybridization efficiency in a projection area by the fabrication of small concave structures (Fig.2b). The local density of attached Au nanocolloids within the central 25% at the bottom of the 800 nm nanocups was 444 μm−2, which was closer to that on a flat surface, and the tendency was common for all diameters of cups, indicating that the size dependency of DNA hybridization efficiency on the concave structures were mostly affected by the lower efficiency of side wall hybridization. These results suggest fabrications of both convex and concave nanostructures with suitable size contribute to the improvement of DNA reaction efficiency on a solid substrate.

References

[1] Kira, A., Kim, H., Yasuda, K., Langmuir 25 (3), 1285−1288 (2009)

[2] Kim, H., Terazono, H., Takei, H., Yasuda, Langmuir 30 (5), 1272−1280 (2014)

We greatfully thank Dr. A. Kira for his technical promotion of convex experiments. We also gratefully thank Ms. M. Murakami and Ms. M. Naganuma for their technical assistance. This work was financially supported by the Japan Prize Foundation research grants, JSPS KAKENHI grant number 24681025 and Kanagawa Academy of Science and Technology.

Fig. 1: Schematic images of the convex (a) and concave (b) experiments. (a) Thiolated DNAs (SH-DNAs) were immobilized on convex Au nanoparticles, and the densities were estimated by measuring UV-vis spectra of SH-DNAs. (b) Target DNAs were immobilized on inner surfaces of nanocups, and hybridized with complementary Au nanocolloid probes.

Fig. 2: Dependences of DNA reaction efficiencies on convex and concave surfaces. (a) Relationship between maximum number densities of SH-DNA immobilized on gold nanoparticles and diameters of particles. (b) Relationship between nanocup diameter and projection density of Au probes hybridized with target DNAs on nanocups. DFL: the density on a flat surface.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-5762 Carbon dot and photosensitizer composites fortwo-photon photodynamic cancer therapy

Chen J.1
1Fudan University, Department of Physics, China
jychen@fudan.edu.cn

The semiconductor quantum dot (QD) has become the new generation fluorescence probe widely used in bio-medical applications. However, the commonly used QDs are cadmium based so that their toxicity is a great concern. In contrast, the carbon dots are the safest dots and they become fluorescent after suitable surface modification. In this work, utilizing the high two-photon absorption cross section (TPACS) of carbon dots we explored the possibility of carbon dots for two-photon photodynamic cancer therapy (PDT). The thiol capped carbon dots were connected with positively charged photosensitizer, TMPyP, to form composites. The z-potential change from -15 mV to +5mV for un-conjugated dots and conjugated dots confirmed the successful conjugation. The absorption band of TMPyP locates at 410 nm which is just overlapped with the emission band of carbon dots, fulfilling the fluorescence resonance energy transfer (FRET) principle. The decreased fluorescence intensity of conjugated carbon dots and increased fluorescence intensity of TMPyP moiety under the excitation reflect that the FRET occurred from the donor (carbon dots) to acceptor (TMPyP). The fluorescence lifetime decrement of conjugated carbon dots relative to un-conjugated dots indicates that the FRET efficiency is about 50%, demonstrating that the TMPyP can be indirectly excited via FRET way. Under the two-photon excitation (TPE) of near-infrared (NIR) femto-second (fs) laser, the conjugated TMPyP in conjugates also can be excited via FRET and subsequently produce singlet oxygen which is the most important agent for PDT. The singlet oxygen production of carbon dot-TMPyP composites is much higher than that of TMPyP alone. Taking the Hela cancer cell as the in vitro model, the two-photon fluorescence images of carbon dot/TMPyP composites were clearly shown under NIR fs laser. Moreover, the successful cell killing via carbon dot-TMPyP composites initiated FRET mediated PDT was reached under TPE of NIR fs laser. With the composites of carbon dots-TMPyP conjugates, the TPE PDT of NIR fs laser has been achieved, expanding the action wavelength to the NIR region which is so-called tissue window region having best tissue penetration depth.

 

Financial support from the National Natural Science Foundation of China (11074053 and 31170802) is gratefully acknowledged.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-5838 mApoE-functionalized nanoliposomes delivering doxorubicin to glioblastoma cells characterized by TEM and confocal microscopy

Rodighiero S.1, Gregori M.2, Francolini M.3, Tamborini M.4, Masserini M.2, Matteoli M.5, Passoni L.1
1Fondazione Filarete, Milano, Italy, 2Università degli Studi Milano-Bicocca, Milano, Italy, 3Università degli Studi di Milano, Milano, Italy, 4Università degli Studi di Milano and Fondazione Vollaro, Milano, Italy, 5Università degli Studi di Milano and Humanitas Clinical and Research Center, Milano, Italy
simona.rodighiero@fondazionefilarete.com

To overcome the limits of current therapy for glioblastoma (GBM) [1] nanotechnology-based approaches offer attractive and innovative possibilities including improved passage of drugs across the blood brain barrier (BBB) and escaping multidrug resistance by efflux mechanisms.
Nanoliposomes (NLs) covalently coupled with a modified apolipoprotein E peptide (mApoE) have been successfully used to enhance the BBB penetration in the context of neurodegenerative diseases [2]. Here, these mApoE functionalized NLs have been modified to encapsulate the anticancer drug doxorubicin (DOXO, Fig. 1) and they have been used to target DOXO to GBM cells. The NLs preparations were characterized by transmission electron microscopy (TEM, Fig. 1) and the role of ApoE on the NLs internalization together with its mechanism have been analyzed by confocal microscopy.
GBM-derived cell lines U87-MG, A172, T98G were incubated for 4 hours with DOXO-mApoE-targeted or DOXO-non targeted NLs. DOXO intracellular uptake was significantly increased in the presence of the ApoE functionalization giving a more pronounced intracellular accumulation of DOXO in cells incubated with DOXO-mApoE-NLs compared to DOXO-NLs (Fig. 2A, B). These results suggested a mApoE-targeted NLs internalization via receptor mediated endocytosis. The presence in the incubation medium of dynasore, the inhibitor of dynamin, reduced DOXO intracellular accumulation indicating a role of clathrin-dependent endocytosis in DOXO-mApoE-NLs uptake. Conversely, a caveolae-mediated intracellular uptake did not seem to be involved since the incubation with βMCD, which selectively removes cholesterol from the membranes and disrupt formation of caveolae invagination, did not affect the DOXO-mApoE-NLs cellular uptake (Fig. 2).
Inhibition of in vitro cell growth was assayed by MTT test at 72 hours. DOXO-mApoE-NLs were found to inhibit GBM cell viability in a dose-dependent manner with IC50 values of DOXO comprised between 0,5 and 1,5 μg/ml. IC50 values of non-targeted DOXO-NLs were noteworthy higher comprised between 1,5 and 3 μg/ml. No cellular toxicity was observed upon incubation with mApoE-NLs.
Overall the data obtained support the use of mApoE-targeted nanocarriers for the delivery of chemotherapeutics and/or cytotoxic agents to GBM cells. The possibility to load mApoE-NLs with ultra-small superparamagnetic iron oxide (USPIO) nanoparticles to exploit the advantages of correlative microscopy to analyze their intracellular trafficking and the potential use of USPIO mApoE-NLs as contrast agents for Magnetic Resonace Imaging will be discussed.
[1] Tanaka et al Nat Rev Clin Oncol (2013); 10(1): 14-26.
[2] Re et al. Nanomedicine (2011); (5):551-9.

Work supported by the European Centre for Nanomedicine (CEN Foundation), Milano (Italy), in the framework of the project “Enhancement Packages” (Rif.EP017).

Fig. 1: NLs design and characterization. A) Schematic representation of ApoE- functionalized liposomes encapsulating doxorubicin and USPIO nanoparticles; B) TEM image of ApoE-functionalized liposomes.

Fig. 2: DOXO-mApoE NLs internalization in U87 cells analyzed by confocal microscopy. Confocal optical sections showing internalized doxorubicin (red) and the cell, segmented thanks to the SytoBlue 45® labelling (grey). Cells were incubated with the indicated NLs in the absence (A, B) or in the presence of endocytosis inhibitors (C, D).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-6001 IMAGING OF CELLS LABELLED BY UPCONVERTING NANOPARTICLES ON A MODIFIED FLUORESCENCE MICROSCOPE

Mrázek J.1 2, Matuška V.2, Pospíšilová M.2, Kettou S.2, Vránová J.1, Velebný V.2
1Department of Medical Biophysics and Informatics, Charles University in Prague , 2Contipro Biotech s.r.o., Dolní Dobrouč, Czech Republic
jiri.mrazek@contipro.com

Nanoparticles with upconversion luminescence (UCNP) are an emerging class of contrast agents suitable for bio-imaging. Their ability to convert infrared light to shorter wavelengths results in improved contrast and imaging depth compared to traditional fluorescent dyes and quantum dots. Unlike other multi-photon dyes, UCNP can be excited by relatively cheap continuous-wave laser diodes. However, using these light sources in common fluorescence microscopes is not straightforward and there is currently no commercially available illuminator for UCNP observation.


Here we present modification of Nikon Ti and E400 microscopes that allow wide-field epi-illumination with 980 nm fiber-coupled laser. Since the luminescence intensity of UCNP scales with powers of excitation intensity, homogenous illumination of the sample is important. We employed microlens arrays and moving diffuser to create flat-top illumination on the sample and to suppress interference artefacts caused by the coherent source.


Monodisperse upconverting nanoparticles were prepared by high-temperature decomposition of rare-earth oleates. Their luminescence efficiency was further increased by growing inert shell around the luminescent core. Hydrophilized UCNP were then used for cell labeling and samples were imaged on our modified microscopes. There was no detectable autofluorescence as the upconverting mechanism is not effective in biological samples. The UCNP displayed no significant toxicity at concentrations required for imaging.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-6040 Engineered silver nanoparticles in Pseudokirchneriella subcapitata

Jensen L. S.1, Sørensen S. N.2, Hartmann N. B.2, Baun A.2, Fleck R. A.3
1Center for Electron Nanoscopy, Technical University of Denmark, Kgs. Lyngby, Denmark, 2Department of Environmental Engineering, Technical University of Denmark Kgs. Lyngby, Denmark, 3Centre For Ultra Structural Imaging, King’s College London, London, United Kingdom
losj@cen.dtu.dk

Silver nanoparticles are postulated to be released into the sewerage systems and wider environment in increasing quantities because of an increase in the number of consumer products, often labelled as antibacterial, which contain engineered silver nanoparticles. These particles are presently considered the fastest growing nanotechnology application. Silver nanoparticles have increased cytotoxic properties compared to larger silver particles and there are concerns that they could inhibit the bacteria which are involved in the breakdown and processing of biological waste in wastewater treatment facilities and be harmful to aquatic organisms.
Whether the enhanced toxicity of silver nanoparticles is due to an increased release of silver ions or it is related to additional mechanisms for toxicity is still a matter of scientific debate since there are studies supporting both theories. Furthermore, nanoparticles are highly heterogeneous in suspension and over time undergo processes such as aggregation, sedimentation, dissolution and changes in surface chemistry [1] – thus altering the dose and posing problems in standard experimental ecotoxicology model systems. Recently a modified short-term model has been suggested, which could potentially increase the accuracy of algal growth inhibition tests with silver nanoparticles [2].

However, toxic mechanisms remain to be further elucidated and the uptake mechanism of the nanoparticles in aquatic organisms on an ultrastructural level play an important part of this.
Selenastrum capricornutum Printz (1913) CCAP 278/4 (Pseudokirchneriella subcapitata (Korschikov) Hindák 1990) is a microalgae which is routinely applied in eco toxicity tests.
In this study P. subcapitata were exposed to silver nanoparticles. They were then fixed with formaldehyde and glutaraldehyde, post fixed with osmium tetroxide, en bloc stained with uranyl acetate and dehydrated in graded series of ethanol. A number of protocols were tested to create the best contrast for FIB SEM work and for future 3View work. Finally the Samples were embedded in Spurr’s or Durcupan resin. The samples were either sectioned by ultramicrotomy and imaged by TEM, or they were imaged by serial block face sectioning in the FIB SEM for localization of the silver nanoparticles within the organisms. Furthermore, EDS was employed to analyse the silver nanoparticles. The results of these different techniques will be presented.

[1] NB Hartmann et al, Aquatic toxicology 118-119 (2012) p. 1.
[2] SN Sørensen and A Baun. Submitted to Nanotoxicology (2014).

The authors gratefully acknowledge funding from The Society of Electron Microscope Technology. Images were acquired at Centre For Ultra Structural Imaging, King’s College London and Center for Electron Nanoscopy, Technical University of Denmark.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-5-P-6061 Three-dimensional microscopic analysis of SERS-active substrates for ultrasensitive detection of biologically significant compounds

Štolcová L.1, Ižák T.2, Petrenec M.3, Svoboda M.3, Proška J.1, Procházka M.4, Kromka A.2
1Czech Technical University in Prague, FNSPE, Břehová 7, Prague, Czech Republic, 2Institute of Physics, ASCR, Cukrovarnická 10, Prague, Czech Republic, 3TESCAN ORSAY HOLDING, a.s., Libušina třída 21, Brno, Czech Republic, 4Charles University, FMP, Ke Karlovu 5, Prague, Czech Republic
lucie.stolcova@fjfi.cvut.cz

The spectroscopy based on surface-enhanced Raman scattering (SERS) is a technique providing information on vibrational structure of molecules, and hence enabling their detection and identification at trace concentrations. The enhancement of the intrinsically weak Raman signal originates from the localized surface plasmon resonance on metal (usually gold or silver) nanoparticles or nanostructured surfaces, so-called SERS-active substrates. The enhancement can display large variations over the substrate surface, due to its high sensitivity to the surface topography. However, periodically structured substrates, yielding uniform Raman signalenhancement, overcome the problems connected with low spectral reproducibility.

Periodic arrays of close-packed polystyrene microspheres (diameters 250 nm - 4 μm) were prepared using a self-assembly technique, and deep corrugations were created in the sphere surface by oxygen plasma etching. The corrugated sphere arrays coated with 20 nm of gold by magnetron sputtering displayed excellent SERS signal uniformity and low detection limits for a series of biologically significant compounds with different lipophilicity. SERS spectra of thiacloprid were measured down to concentration of 10-8 M. The SERS signal uniformity was demonstrated by spectral mapping using Horiba JobinYvon LabRam HR800 Raman microspetrometer.

In comparison to smooth gold-coated microspheres, our measurements showed a distinct increase in SERS spectral intensity. To understand this effect, cross-sections of the polystyrene/gold nanostructures were prepared using focused ion beam (FIB). Thus, the structural features of the sputtered gold layer (at nanoscale) could be imaged and analysed together with the mesoscale architecture of the corrugated polystyrene cores. Their texture similar to a 2D labyrinth increased the surface area in a specific way, and can be viewed as an open form of pores. Due to its mesoscale dimensions, the corrugated particles behave in solutions like porous materials with all consequences for phase equilibria and analyte separation/adsorption.

The nanomilling was performed by Focused Ion Beam Equipped Scanning Electron Microscope (FIB-FESEM) TESCAN LYRA 3 with a COBRA ion column at 4 pA and 30 kV. Prior to the milling, a protective platinum layer was deposited (at 5 kV) over the corrugated spheres selected for the analysis. Using 3D tomography technique performed with FIB in 10 nm slices, the continuous gold layer verging into discrete sputtered gold grains in the corrugations could be observed for the first time. Imaging was performed with an ultra-high resolution SEM TESCAN MAIA 3 XM equipped by FEG; InBeam detectors of secondary and backscattered electrons or their mixed signals were used for observations.

This work was supported by the Czech Science Foundation grant No. P205/13/20110S.

Fig. 1: Hexagonally ordered plasma etched polystyrene microspheres coated with 20 nm of gold – top view (SEM micrograph)

Fig. 2: Individual gold-coated corrugated microsphere – tilted view, detail of the sputtered gold layer (SEM micrograph)

Fig. 3: Cross section of the gold-coated etched PS microsphere prepared by FIB nanomilling (SEM micrograph, backscattered electron detector)

Fig. 4: SERS spectra of 4-aminothiophenol obtained by spectral mapping of an ordered array of etched microspheres (raw spectra measured from 22 × 22 points separated by 2 μm)
↑ Go to the Table of content ↑

ID-6. Microscopy in forensic science

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-6-IN-1762 A Forensic Mineralogy Toolbox – the next generation of instrumentation for forensic applications

Mason K. N.1
1Eastern Analytical sprl
masonkn@skynet.be

The examination of particles found in the environment, such as minerals, and particles found on a suspect or at a crime scene, such as gunshot residue (GSR) formed the basis of our search for better instrumentation to analyze these particles.

Minerals have traditionally been identified by the skilled mineralogist’s eye. The laboratory tools of XRF and XRD joined the instrumentation during the 20th century, as did the field tools of colorimetric spot tests and hand-held XRF devices. These tools work well for relatively common minerals and relatively large samples.

Over the past 20 years it has become possible to analyze crushed mineral material, as well as gunshot residue particles, using automated SEM and EDS systems. These processes are suitable for large-scale automatic analysis applications for both mineral liberation analysis (MLA) and gunshot residue analysis (GSR).

In the forensic sector, the analysis of a particular crystal or grain (and/or GSR particle) is often required because only small samples of material are available. The material is frequently millimeter or micron size, while GSR particles are often smaller. Since the 1960s it has been possible to characterize such samples using SEM/EDS/WDS. However these techniques do not work well with minerals with low concentrations of light elements such as Li, Be B, and .nH2O, or with the new heavy metal-free bullet primers (HMF).

With the advent of HMF, it is becoming more difficult to use basic SEM /EDS and GSR methods alone. Other techniques are required for the analysis of these types of ammunitions.

Consequently, a new, rapid method of mineral and inorganic particle classification has been explored using a toolbox of well-established techniques such as optical microscopy, SEM, EDS, Raman, CL and fluorescence. The SEM and optical systems, or the electron BSD signal, allow the user to find particles of interest, the EDS to determine their elements set, and the Raman to distinguish between polymorphs and to ratify non-detectable elements. All of these attachments have been mounted on the SEM. Figures 1 and 2 show an image/diagram of the combined system.

Properties such as color, density, laser-excited fluorescence and crystal morphology are among the experienced mineralogist’s tools. For this reason, an optical microscope is part of the system, not only to navigate but to allow color, fluorescence and morphology to be seen, both optically and with electrons.

Cases will be discussed that show application of these instruments to real samples, both in the mineral area and general physical evidence samples such as gunshot residue. The talk will also discuss the development and incorporation of new instrumentation on a SEM that will benefit and assist analysis for forensic applications.

Acknowledgements The author would like to thank the IMC committee for the invitation to speak at IMC2014 Prague and would also like to thank Richard Wuhrer for his helpful critique, and Lawrence Gunaratnam and Matteo Donghi for assistance providing suitable HMF samples.

Fig. 1:  FEI Quanta SEM with EDS, GSR, CL, and Raman system incorporated.

Fig. 2: Schematic of system
↑ Go to the Table of content ↑
Type of presentation: Invited

ID-6-IN-3383 Microscopy and Microanalysis of Forensic and Cultural Heritage Materials

Mansfield J. F.1
1University of Michigan, North Campus Electron Microbeam Analysis Laboratory at NCRC
jfmjfm@umich.edu

Microscopy and microanalysis techniques are applied to determine the morphology, structure and chemistry of materials in a wide range of forensic studies. Examples include the tracing of production problems in semiconductor device manufacture, identifying and characterizing gun shot residue in crimes involving firearms, identifying counterfeit components in industry and identifying the composition and source of compounds employed in works of art and sculpture. The University of Michigan Electron Microbeam Analysis laboratory has been involved in the a large number of studies of this kind, principally in the forensic analysis of cultural heritage materials. A number of examples will be discussed in the presentation and a summary of them follows.

Focused ion beam (FIB), scanning electron microscope (SEM) and X-ray energy dispersive spectrometry (XEDS) analyses were performed to determine the base composition of a series of silver based coins, with a dual phase microstructure, from the reign of King Eadberht of Northumbria. Small crevices and cracks in each of the sample’s patina were carefully enlarged with the FIB for analysis yet at a scale that did not visibly change the overall appearance of the coins, thus maintaining their value.

Analysis was performed in the environmental scanning electron microscope to identify the material trapped in the crevices and fine detail of an eighteenth century crucifix from the Kingdom of Kongo, an area that covers today's Angola and Democratic Republic of the Congo. The crucifix was mounted in its entirety inside the microscope, to avoid damage that would reduce its historical or economic value, and XEDS spectroscopy and mapping identified residual polishing compound and earthen material embedded in the carved detail. See figures.

In a collaboration with the The Detroit Institute of Arts, paint fragments extracted from a painting purported to be by Claude Monet were embedded in resin and cross sectioned prior to analysis in the environmental scanning electron microscope. XEDS revealed a number of pigments that were not typically in Monet’s palette. Further documentary research revealed that the painting had been painted by a British painter Alfred East and fraudulently attributed to Monet prior to the DIA’s acquisition of the piece.

Thanks to my collaborators in these studies, Cathy Selvius DeRoo of the DIA, Ronald Bude of the University of Michigan and Cécile Fromont of the University of Chicago.

Fig. 1: Kongo crucifix mounted for examination in the environmental scanning electron microscope. Inset outlines the area analyzed by XEDS spectra and mapping.

Fig. 2: SEM Image and accompanying XEDS maps, extracted from a spectrum image, of the various elements present in the detritus embedded in the figure's ribs.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-6-O-1663 Characterisation of Paintings for reference in Forensic Applications  

Wuhrer R.1, Dredge P.2, Sawicki M.2, Ives S.2, Allen L.2, Fania D.3, Dodd M.3, Spikmans V.3
1Advanced Materials Characterisation Facility (AMCF), University of Western Sydney, NSW, Australia, 2Art Gallery of New South Wales, Art Gallery Rd, Sydney, NSW, 2000, Australia, 3School of Science and Health, University of Western Sydney, NSW, Australia
richard.wuhrer@uws.edu.au

The determination of art fraud and trafficking frequently relies on the ability to determine the difference in paint composition based on geographical origin and era of manufacture. It therefore follows that the same analytical techniques that are used for art restoration and historical investigations into artwork can also be used for forensic investigations into art fraud and trafficking.

Recently several famous artworks were stolen from a museum in the Netherlands, and later burned in an oven to cover up the theft [Ass. Press in Bucharest, 2013]. Through the use of analytical techniques, the burned ashes were determined to have come from several of the stolen paintings. The main objective of this project is to 1) develop an analysis protocol for art history and forensic purposes, 2) get a better understanding of paint pigments, binders and powders used by artists and 3) determine the author, the date of manufacture and the amount of non-original restoration. Although many research articles focus on the analysis of paints from art works, the advantage of this project lies in the combined use of a range of techniques as an analysis protocol. An understanding of the use and availability of materials used in paintings, their development and physical characteristics could aid in an investigation of unidentified paint samples. Identification of the individual components in the paint layer can provide valuable information into the materials and techniques used.

Several projects are currently underway with the Art Gallery of NSW. The first is the investigation and conservation treatment of an oil painting on oak panel of Henry VIII thought to date from about 1535 (Fig. 1). In addition to providing detailed information regarding the condition of the painting prior to conservation treatment, forensic analysis is being undertaken in an effort to establish physical similarities to the other panel paintings of the same subject in London. It is hoped that by studying this group of works that new findings will emerge regarding authorship and the artistic practice of portrait painting in the 1530s. The second project is looking at 20th century artist material, in particular with the use of metallic paints. The artists being investigated are paintings by Roy de Maistre (1894-1968), Ralph Balson (1890-1964), Eric Wilson (1911-1946) and Charles Conder (1868-1909) (Fig. 2).

Microsamples of paint have been prepared then investigated using a range of techniques, including Optical Microscopy, Scanning Electron Microscopy and microanalysis, x-ray mapping (XRM), Fourier Transform Infrared (FTIR) and Raman Spectroscopy. The combination of these techniques allows both the inorganic and organic composition of the paints to be determined, including the paint pigments used.

The authors wish to acknowledge the Art Gallery of New South Wales and the Advanced Materials Characterisation Facility (AMCF) from the University of Western Sydney, Australia.

Fig. 1: Anglo-Flemish workshop, Henry VIII, circa 1535. Oil on oak panel, 54.5 x 38.0 cm. Art Gallery of New South Wales. Purchased 1961.

Fig. 2: Charles Conder, An Impressionist (Tom Roberts), circa 1889. Oil and metallic paint on cedar panel, 28.5 x 23.4 cm. Art Gallery of New South Wales. Purchased with funds provided by the Art Gallery Society of New South Wales 1977.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-6-O-1766 Use of Electron Microscopy for Material Analysis in Forensic Practice

Kotrlý M.1, 2, Turková I.1
1Institute of Criminalistics Prague, Bartolomejska 12, Praha 1, Czech Republic, 2Charles University in Prague Faculty of Science, Praha, Czech Republic
kup321@mvcr.cz

In forensic practice, electron microscopy and microanalysis rank among basic applications used in forensic investigation of traces and comparisons from crime scenes. These techniques allow for rapid screening and receiving essential information for a wide range of traces.

The set of materials that are analysed using electron microscopy is very extensive, practically any material produced by human and nature activities relating to the case that is solved can be delivered to a forensic lab (ranging from the fragment of an ancient vessel, to high-tech semiconductors). Therefore, materials of organic origin, plant and animal fragments are examined as well.

The current routine use of electron microscopy (including microanalyses) is in the following expert examinations:

- unknown samples (including powders from extortionate letters)

- mineralogical, petrological and gemmological objects (mineral relics, soils, precious stones and their imitations, etc.)

- gunshot residues (GSR)

- explosives, propellants and fulminating compounds

- post-blast residues (PBR), and other thermogenetic particles

- fillers and additives of paper and plastics

- pigments and paint systems, including colour layers of artworks

- cosmetic and pharmaceutical products (surfaces and coating layers of tablets, granular composition, phase analysis)

- morphological structures of textiles materials

- determination of sorts of damages to fibres (smelting, fibre fracture, tear/cut, fracture, etc.)

- expert examinations of biological materials - trichological material and its damage, shells of soil microorganisms, insect eggs to determine post mortem interval, etc.)

- intersecting lines of writing and print tools (superposition - writing tool x print toner)

- glass

- fragments of building materials

- fractures of materials (determination of the character of the fracture area)

- toolmark slipped impressions (forensic technical examinations, ballistics, etc.)

Recently, dual systems with focused ion beam have considerably extended possibilities of electron microscopy. These systems are predominantly applied in the study of the inner structure of micro-and nanoparticles, layers and composites (intersecting lines in forensic graphical examinations, analyses of functional glass layers, etc.), the study of alloys microdefects, creating 3D models of particles and aggregates, etc. Automated mineralogical analyses are a great asset for the analysis of mineral phases, particularly soils, analogously as cathode luminescence. TOF-SIMS systems and micro-Raman spectroscopy with a resolution comparable to standard analysis EDS/WDS are latest crucial innovations that are becoming to appear also at ordinary laboratories.

Microanalytical methods were supported by projects: RN19961997008, RN19982000005, RN20012003007, RN20052005001, VD20062008B10, VD20072010B15, VG20102015065, VF20112015016

Fig. 1: The structure of the inner wall of fly egg

Fig. 2: Polypropylene fibres - morphological study of damage

Fig. 3: Internal structure of natural fibers, display in secondary ion

Fig. 4: Microstructure of thermogenetic particle – FIB cut
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-6-O-1879 The Original or the Fake? The Use of Microscopy and Spectroscopy Methods in the Forensic Criminology Science in Determining the Authenticity of Artworks

Turková I.1, Kotrlý M.1, Šefců R.2
1Institute of Criminalistics Prague, Prague, Czech Republic , 2National Gallery in Prague, Prague, Czech Republic
turka@seznam.cz

The proposed contribution deals with the use of microscopic and spectroscopic forensic methods that were used in the inter-institutional cooperation of the Institute of Criminalistics Prague and the National Gallery in Prague in the detection of art forgeries. In the forensic practice, there are different types of traces and samples from the crime scene.
Legal classification of criminal offences includes a wide range of violations of law ranging from unauthorized export of artworks, determining ownership rights (copyrights), theft of artwork, to the fraudulent counterfeiting of prominent authors and their sale. We frequently encounter fakes of prominent Czech artists of the 20th century in Bohemia, such as Jan Zrzavý, Emil Filla, Josef Čapek, Václav Špála and Pravoslav Kotik.
Two paintings – artworks of Pravoslav Kotik with a similar theme that sparked controversy over the authenticity of both artworks (Fig. 1) appeared on the Czech market. In 2007 and subsequently the whole counterfeiting and its associated practices – trafficking were revealed. Works of art such as forensic traces are usually put to a detailed analysis and assessment. Particularly in the detection of forgeries, optical documentation techniques are widely used (Fig. 2) and microscopic analysis by methods of optical microscopy in a polarizing microscope (Fig. 3), scanning electron microscopy and x-ray microanalysis (Fig . 4), x-ray diffraction and fluorescence analysis, infrared microspectroscopy with Fourier transformation and Raman microscopy are frequently employed.
It is possible to accumulate significant arguments to the comparative analysis of the materials thanks to the combination of microscopic and spectral techniques, we can consider the morphological and structural characteristics of the pigments, identify organic binders and assess the painting technique execution.
Besides corpora delicti, these methods are utilized as well as for the survey of original paintings from the National Gallery, which are used to create databases as reference materials and components specifically for the assessment of works of art. Variations of materials and techniques used are very specific to each artist and based on these parameters, we can follow the author's practice.
The results obtained from the analysis are then also a significant argument for determining the authenticity from a perspective of an restorer and art historian.

The authors wish to thank Zora Grohmanová and Věra Cedlová from National gallery in Prague. Microanalytical methods were supported by projects of Ministry of Interior RN20012003007, RN20052005001, VD20062008B10, D20072010B15, VG20102015065, VF20112015016.

Fig. 1: Both paintings: a) fake, Hair style, private collection b) original, Pravoslav Kotík, Toilet, private collection. Photo© Institute of Criminalistics Prague 2014.

Fig. 2: Signatures: a) fake, b) original. Photo© Institute of Criminalistics Prague 2014.

Fig. 3: Cross-section of the green paint: a) fake, b) original. Photo©National gallery in Prague 2014.

Fig. 4: BSE image of the cross-section of the sample of fake painting. Photo© Institute of Criminalistics Prague 2014.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-6-O-3361 SEM Application in Forensic Dentistry

Eliasova H.1, Turková I.1
1Institute of Criminalistis Orague
hanaeliasova@atlas.cz

Institute of Criminalistics Prague
Dental identification has always played a key role in crime cases and disaster situations. Teeth are very high mineralized components of a human body and so they are in a certain degree resistant to flames, high temperature and chemicals. Recently forensic dentistry applies various microscopic techniques, which represent benefit for expert evidence.
Scanning electron microscopy (SEM) has been used to identify teeth by their structure, e.g. prismatic elements in enamel, dentinal tubules and evidence of previous restorations especially in diminutive fragments or incinerated remains.
SEM with energy-dispersive X-ray (EDS) analysis makes it possible to detect various restorative material residues and so indicate the ante mortem existence of a restoration. This knowledge could be valuable in a presumptive identification of the dead body.
SEM provides also evidence of tool marks and traumatic or pathological defects.
Recovered carbonized tooth fragments are not morphologically recognized as teeth. SEM can indicate special striations in tooth enamel and in addition reveal dentinal tubules. Use of SEM with EDS provides a profile of elements from special inflammable mixtures, e.g. thermit.
In some crime cases, human identity is obstructed when the bodies are destroyed partially by acid dashing or immersing in acids. Microscopic analysis can capture changes of teeth during this process and it is feasible to identify the residues of teeth on basis of recognition of the characteristic morphological features of dental tissues up to the advanced stages of the degradation. The correlation between the time of exposure to the different acid solutions was noted. Degradation proceedings recovered by prima vista are different for various acids. Moreover, SEM is able to display crystalline structures (reaction products) and detect their chemical composition typical of various acids.

Microanalytical methods were supported by projects VG20102015065 and VF20102014007.

Microanalytical methods were supported by projects VG20102015065 and VF20102014007.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-6-P-1666 Investigations of gunshot residue and environmental particles through use of focused ion beam and other characterisation techniques

Wuhrer R.1, Sarvas I.2, Green L.2, Lam R.3, Spikmans V.3, Kobus H.4
1Advanced Materials Characterisation Facility, University of Western Sydney, Sydney, Australia, 2Adelaide Microscopy, Adelaide University, Adelaide, Australia, 3School of Science and Health, University of Western Sydney, Sydney, Australia, 4School of Chemistry, Physics and Earth Sciences, Flinders University, Adelaide, Australia
richard.wuhrer@uws.edu.au

Firearms are prevalent in our society and many countries are involved in the production of firearms and munitions. It is said that approximately 8,000,000 small arms are manufactured annually [1-4].

Forensic science plays an important role in the investigation of firearm related crimes and can establish an association between firearms and assailant by detecting and identifying gunshot residue particles (GSR), which are solid microscopic particles ejected from the openings, gaps and clefts of firearms. These particles are condensed from the high temperature and high pressure gases produced by the deflagration of the primer and propellant in the cartridge case. These particles are frozen by rapid cooling and are deposited on the body and clothing of the shooter and on nearby surfaces. If the rate of cooling is sufficiently high, the solid particle should retain the structural disorder of the liquid, which is a feature anticipated in GSR morphology. GSR particles are identified by two features, composition and morphology. The presence of Pb, Sb and Ba in a particle is considered characteristic of firearms origin. The ASTM standard describes the elemental composition of GSR and classifies these elemental compositions as characteristic or consistent with GSR but the guide’s description of morphology is not extensive [5].

Recent studies have highlighted that environmental particles may exhibit similar elemental profiles and implied morphologies that may be misinterpreted as GSR particles. It has been suggested there are similar to particles to GSR in particles produced in the reaction of fireworks [6], particles produced by wear in elevated temperatures in automotive brake linings [7], cartridge tools and vehicle airbags. Adding further to the discussion, condensed species from metal free primers may be indistinguishable from environmental particles (Fig.1).

To discriminate between GSR and environmental particles, the morphological features displayed by the particle become a primary consideration in determining the particles’ origin. In order to reevaluate particle morphology and to distinguish GSR from environmental particles, FIB-SEM was utilized [8] to investigate not only the surface features, but also the interior structure of particles that had been formed in a range of high temperature environments. Through detailed morphological characterization, the source of the particle may be determined with confidence. The results from analysis including morphological characterization, SEM/EDS, Infrared analysis (IR) and Raman analysis of selected particles will be discussed within the presentation. Detailed morphological information may be the key in correctly determining the particle’s origin where elemental composition may be misinterpreted (Fig. 2).

AMCF at the UWS and Adelaide Microscopy at the Adelaide University.

Fig. 1: Comparison of normal GSR and lead free GSR. a-d) Normal GSR image, EDS spectrum, FIB image and x-ray map. e-g) lead free FIB results and x-ray maps.

Fig. 2: Typical GSR particles that can have porosity.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-6-P-1743 Forensic Scanning Electron Microscopic examination of animals Hairs from Felidae Family

HING L. H.1, TEO H. C.2, FOONG M. J.2, HUKIL S.2, WAN NUR SYAZWANI2, KASWANDI A. M.3, ZORIN S. A.4, NORMALAWATI S.4
1Environmental Health & Industrial Safety Programme, School of Diagnostics & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia , 2Forensic Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia , 3Institute of Medical Science Technology, UNIKL, 43000 Kajang, Selangorr, 4Biomedical Science Programme, School of Diagnostic & Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia
hing61@yahoo.com

The outermost cuticle of hair structure functions as a protective scales layer, where the scales overlap and arrange in the direction of hair root towards the hair tip. These scales form a unique pattern with distinctive characteristics and it is useful for forensic scientists in identification of animal species during an investigation of hair samples (1,2). Hair samples of four species from Felidae family were observed using scanning electron microscope, i.e. bengal tiger (Panthera tigris tigris), gir lion (Panthera leo), leopard cat (Prionailurus bengalensis) and sumatra tiger (Panthera tigris sumatrae). No cleaning procedure was employed to ensure the appearance of the hair samples is in its original condition. In Felidae family, hair cuticular pattern of all species showed regular wave pattern, transversal hair cuticular orientation with smooth cuticular dorsal margin (Fig 1) except gir lion (Fig 2), which possesses rippled cuticular dorsal margin. Statistical analysis showed that there is significant mean difference of average scale layer difference measured four species of animal chosen, Bengal tiger showed significant higher average scale layer difference as compared to sumatra tiger and leopard cat. We found that gir lion could be identified through its rippled cuticular dorsal margin while scale layer difference need to be measured in order to distinguish between bengal tiger, sumatra tiger and leopard cat. We also found out that cuticular scales pattern and other related characteristics could still be observed clearly even though the samples did not undergo any significant cleaning procedure. This study showed that the examination of cuticular morphology of hair samples combined with measurements are conclusive enough to draw solid identification up to between different subspecies animal level (3, 4). As compared to other group such as the deer group, no such difference could be observed.

References

B. J. Teerink, Atlas and Identification Key: Hair of West-European Mammals, Cambridge, United Kingdom: Cambridge University Press (1991). M.S. Dahiya, S.K. Yadav, Elemental Composition of Hair and its Role in Forensic Identification, Open Access Scientific Reports, 2(4), doi: 10.4172/scientificreports.721 (2013). M.S. Dahiya, S.K. Yadav, Scanning electron microscope characterization and elemental analysis of hair: a tool in identification of felidae animal, J Forensic Research, 4(1), 178, doi: 10 4172/2157-7145.1000178 (2013).H. Brunner, B.J. Coman, The Identification of Mammalian Hair, Melbourne: Inkata Press (1974).

The authors acknowledged the contribution from National Zoo of Malaysia,

Fig. 1: Hair of Bengal Tiger

Fig. 2: Hair of Gir Lion
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-6-P-1821 Monitoring your SEM and EDS Calibration and Accuracy of Analysis for Forensic Applications through use of an Automated System

Mason K. N.1, Wuhrer R.2
1Eastern Analytical sprl, 2Advanced Materials Characterisation Facility (AMCF), University of Western Sydney, Australia
masonkn@skynet.be

Gunshot residue analysis (GSR) is extensively used around the world for determining if a person has discharged a firearm. The technical data from the SEM/EDS and GSR analysis is then given as evidence in court. The operation and setup of the system have to be validated objectively, more like an industrial process than a scientific investigation. Accurate analysis results are always required for cases that have a legal consequence.

The quality of the results from automated particle search systems relies on many correctly working and calibrated subsystems, including the SEM, EDS, SEM/EDS interface and subsequent GSR and PSA systems. Consequently, there is a need for a tool to automatically validate the performance of systems.

This talk will cover the use of a system called “microValidator” which was developed to determine if the microscopy system is set up adequately to achieve consistent and reliable results when working with SEM/EDS and the forensic application automatic gunshot residue (GSR) particle analysis. The tool is used to automatically validate the performance of user’s systems and test the instrument operation and calibration (instrument health) for validation of SEM, EDS and the interface setup between SEM and EDS.

A standards block and a known GSR proficiency test samples are used to validate performance and check for measurement accuracy. The highly automated procedure enables reproducibility and automates testing of SEM, EDS and combined SEM/EDS checks. The program is also designed to differentiate between user-correctable errors and service-related errors and shows that system performance has been validated objectively. The microValidator software is compatible with EDAX and Bruker X-ray microanalysis systems.

The system uses a sequence of 20 tests that automatically checks SEM stage operation, SEM column functions, SEM beam current variation with spot size, BSD functionality, magnification calibration, stage/scan alignment, EDS system and all pulse processor settings, resolution and calibration, EDS collimation, as well as quantitative analysis of both alloy standards and light element samples.

The system developed is a hardware and software based testing and validation device (Fig. 1 & 2) that 1) checks overall system performance and diagnosis of specific faults or misalignments, 2) reduces the need to call service, as levels of faults are separated into levels of importance, 3) produces automatic validation report, 4) operates in conjunction with Plano synthetic particle test sample (system used for GSR proficiency) and 5) operates with either Edax or Bruker EDS systems in conjunction with FEI SEMs, and is currently being developed for other microscopes.

Fig. 1: The microValidator control unit and how it is connected to the instrument.

Fig. 2: Microvalidator  connections to the instrument

Fig. 3: Standards required for testing instrument performance are an important part of the validation
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-6-P-3253 Practical using of SEM in Forensic Science

Fojtášek L.1
1Institute of Criminalistics, Prague, Czech Republic
fojtasekl@volny.cz

The scanning electron microscope is one of the most versatile instruments available for the examination and analysis of many samples in various branches of forensic science.

The high resolution, large depth of field and possibility of big magnification are basic reasons for the SEM´s usefulness.

In the presentation some practical examples of using SEM are shown.

Imaging of small objects, details of the surface or structure could be visualized and proved by evidence. It is the first area of SEM utilization.

The second one is an elemental analysis. The large area of SEM using is gunshot residues analysis. The presence or absence of GSR particles on the sample could help to determine, if the suspect person shot or not. Typical GSR analysis is described together with the experiment which was joined with this real case.

But elemental analysis wins recognition also in chemistry, in toolmark examination and in anthropology. The analysis of dental filling or metal dentures material could be very helpful for identification of unknown dead bodies. The real case describes interesting finding of skull in a Czech forest with very unusual metal denture.

SEM is also very important for examination of explosives residues. On the basis of imaging and particle analysis the method can help with determination of explosive compounds. Typical examples for SEM analysis are explosions of tube bombs, when the structure of black powder residues determination can help with estimation outer packaging of explosion object. The most famous case in modern Czech history is explosion of tube bomb on the Old Town Square in Prague in 1990. This case was never explained. The second example describes very strange finding inside the sample of explosive which was discovered by microscopy analysis.

Fig. 1: Glass beads inside explosives

Fig. 2: Flax fibers in gold/silver strips from burial robe of Czech king Ladislav Pohrobek (15th century)

Fig. 3: Tooth enamel damaged by mineral acid
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-6-P-2299 Scanning Electron Microscopy with X-Ray Spectroscopy and Focused Ion Beam (FIB-SEM-EDX), an innovative tool for non-destructive analysis of Gold jewellery

Torres F. J.1, Saéz P. L.1, Bustamante L. A.1, Paiva L. A.1, Soto P. A.1
11. Sección Microanálisis, Laboratorio de Criminalística Central, Policía de Investigaciones de Chile, Santiago de Chile.
leonardo.abh@gmail.com

The marketing of counterfeit jewellery is a problem that affects at all societies in the world. Traditionally, the instrumental analysis of Gold jewellery is made by techniques such as Atomic bsorption or Inductively Coupled Plasma Mass Spectrometry, however, these studies involve  sampling methods that removing portions of the specimen. The purpose of this study was to develop a methodology that would enable the elemental chemical composition of jewels composed of a Gold alloy, without affect their integrity and monetary value.

Because the penetration depth of the primary electron beam is limited in relation to the total volume of each specimen, cross section analysis was made by FIB. The material used in this study consisted of jewellery pieces presumably composed of Gold alloys. Each specimen was subjected to a gentle cleaning process. The samples were subsequently inspected using a stereomicroscope, to verify the effectiveness of the cleaning process and the presence of singularities in their surface continuity.

Analysis by SEM-EDX-FIB, was conducted in a Dual Beam Workstation FEI QUANTA 3D 200i, equipped with a X-ray Spectrometer EDAX APOLLO XL. A section the surface of each sample was analysed by EDS. Subsequently, a portion of the sample surface was subjected to a cross section process, by FIB. and an image was obtained using the Ion Beam (SIM). Finally samples were viewed at backscattered electron and if was necessary, an EDS analysis of their layers was done and supplemented with a Bidimensional mapping.

The analytical procedure described above proved to be adequate. In several cases the observation by light microscopy, was sufficient to detect regions of samples that had different coloration on their surface, which was presumed that different layers of material were present. Further analysis of these regions, confirmed the differences in chemical composition by EDS studies. The cross-section images obtained by SIM, facilitate their interpretation, even by non-experts in the methodology (judges, prosecutors, among others), situation which is supplemented by Bidimensional mapping. These facts, together with others obtained during the criminal investigation may be useful to allow the association of various species of jewellery, which have in common the same elaboration process.

In conclusion, the implemented methodology proved to be a useful tool in the study of Gold jewellery . Being a procedure that minimally alters the evidence, allow to get a lot of information on this, without depreciating its possible economic value. Additionally, the results can be easily understood by non-experts, which facilitates submission to the court during the course of judgments.

The authors gratefully acknowledge the support of the Policía de Investigaciones de Chile, for providing the samples and instruments needed to properly develop this study.

Fig. 1: SIM image of the cross section of a sample analyzed. The red arrow show a thin film of material, coating the solid mass of specimen.

Fig. 2: Bidimensional mapping of a sample cross section. Pseudocolor representing the major chemical elements detected in the sample (gold and silver), as indicated by the corresponding X-ray spectrum. The blue area corresponds to the Platinum deposit made before cutting the sample.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-6-P-5727  SEM Image Analysis&Comparative Qualitative EDS Analysisof Screwdrivers Striations

AL-Shammari1, A., Griffin2, B.J., Dadour1, I., and Franklin1, D.
Centre for Forensic Science,Microscopy, Characterisation and Analysis, The University of Western Australia
aalshammari@hotmail.com

Characterising the marks produced by screwdrivers (using a combined SEM and LA-ICPMS approach) when they are used in forcing open metal framed windows and metal framed doors is the objective of this thesis. The current study is based on the novel approach of applying SEM analysis using SE imaging, BSE imaging, and EDS investigative techniques to screwdriver head surfaces.

Result

Cross sectional analysis

The BSE images reveal that screwdrivers have a chemically layered structure in cross-section but the imagery cannot identify these elements, only rank in average atomic number. The EDS element survey maps can identify the elements in the different layers but cannot calculate the amounts of elements present. The quantitative EDS analysis can identify and calculate how much of the different elements are present but its accuracy depends on factors such as calibration standards used, degree of polishing achieved, resolution of detector, etc.

A BSE image of a typical Chinese star screwdriver tip appears relatively homogeneous apart from some low Z contamination and possible cracking of a surface layer around the circular end of the tip (Figure 1). The tool mark associated with this screwdriver is relatively uniform with horizontal striae and surface debris (Figure 2).

In polished cross-section the BSE image clearly displays three distinct layers of different element composition. The thin outer most layer is relatively smooth and is approximately 1 micron thick. The intermediate layer is uniform, approximately 16 microns thick and the core makes up the rest of the screwdriver

Profile Plot analysis

The profile plots show the general spectrum waveform trend plus major peak and major trough trends. This data is used to calculate correlation and variance statistics.

Correlation and Variance

This line analysis obtained from Microsoft Excel is significant. It statistically compares scratch data and finds relationships where they exist. In all cases the tabulated results show high correlation between scratch points which suggest that scratches made by the same screwdriver can be identified simply by taking line plots perpendicular to the scratch direction.

Bar Code analysis

This visual image information obtained from the different scratch locations A, B and C appears to give information that cannot be easily discerned by simply inspection of the images. Therefore new approaches are required to extract relevant information – one possible approach is segmenting the area of interest into an array of smaller segments and then analysing these much smaller areas for correlation and variance based on shades of grey.

↑ Go to the Table of content ↑

ID-7. Microscopy in arts, restoration and archeology

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-7-IN-2287 The Lampshade Frame: A Study of Provenance

Vander Voort G. F.1
1Consultant - Struers A/S
georgevandervoort@hotmail.com

The lecture describes a study of the wire frame of a lampshade, found in an abandoned house in the 9th Ward of New Orleans after Hurricane Katrina devastated that area. The covering was claimed to be human skin placed on the frame at the notorious Buchenwald concentration camp during WWII by the infamous Ilse Koch. The writer was asked by Eric Gehringer, associate producer at Hoggard Films, if a study of the wire in the frame could determine when and where it was made. Hoggard Films subsequently produced a 1 hour show on the lampshade for National Geographic’s TV channel. As to where it was made, I told him that is virtually impossible to determine with any confidence for a lampshade, a commodity product. But, as to when it was made, I said that may be possible to determine, at least to a certain time frame, if there is anything unique about its manufacture. The study showed that the wire frame and the sheet steel ring which surrounded the light bulb, were not modern steels. They were heavily killed with aluminum and contained no Si. By the 1920s, it was well known that Al was a very effective deoxidizer but Si was rather weak in comparison. But, the detrimental influence of massive aluminate stringers on cold formability was not known at that time. By the late 1930s, deoxidation was conducted typically by a 4:1 addition of Mn to Si, with a smaller amount of Al (especially if the steel was to be carburized). The steels contained no incidental alloy content, so they were not melted in an electric arc furnace. The wire had a very high P and S content, suggesting it was made in an acid open hearth furnace, while the sheet steel had a very low P and S content, suggesting basic open hearth technology. The carbon and copper levels were too low to obtain using the Bessemer process, still used to some extent until ~1950. The wire surfaces were “wet drawn,” a practice used to yield a decent smooth surface, as drawing dies and the lubricants were more primitive before WWII (borax was invented in 1951). In wet drawing, the rod was soaked in a copper sulphate bath to deposit a thin layer of Cu on the wire. This practice ended in the early 1950s. All evidence indicated that the lampshade frame was made before WWII.

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-7-IN-5763 Optical Coherence Tomography (OCT) – a novel tool for examination of artworks

Targowski P.1, Iwanicka M.2, Sylwestrzak M.1, Kaszewska E.1, Szkulmowska A.1
1Institute of Physics, Nicolaus Copernicus University, Toruń, Poland, 2Institute for the Study, Restoration and Conservation of Cultural Heritage, Nicolaus Copernicus University, Toruń, Poland
ptarg@fizyka.umk.pl

Optical Coherence Tomography (OCT) is an originating from medicine diagnostic non-contact and non-invasive technique of optical sectioning. OCT employs the interferometry of light of high spatial but low temporal coherence to reveal locations of reflecting interfaces and scattering centres within internal structure of the examined object. Spectral domain version of the OCT method (SdOCT) is especially fast and sensitive. The OCT technique offers a micrometre-level in-depth resolution and therefore is well suited for investigation of fine sub-surface details of structures which absorb infrared light moderately such as varnishes, glazes and underdrawings of paintings on canvas (Fig. 1a), reverse painting on glass (Fig. 1b), glazes on porcelain and faience, jade, historic glass (Fig. 1c), and many others.
Images obtained by OCT are usually presented in convenient manner of cross-sectional views, similar to microscopic photographs of cross-sections of samples collected from the object. However, for better readability, images are usually stretched in in-depth direction as indicated by scale bars in figures and shown in false colours where cold colours indicate areas of low scatter whereas warm ones indicate areas of high scatter. Black areas correspond to either non-scattering media like air above the object or regions not reachable by the probing light - below the surface of the first opaque layer (e.g. the paint). The major advantage of using OCT is in the complete non-invasiveness of the technique (intensity of light used for examination is of order of single miliwatts), very fast data collection, and no need for any preparation of the object. Therefore the examination may be repeated as many times as necessary in many places, thus making the obtained results much more representative than obtained from sample collection.
In this contribution firstly a brief introduction to the technique will be presented and essentials of the construction of the OCT instruments will be given. Then using examples from our practice it will be shown how the OCT technique may be used to examine the structure of artworks, trace former conservation attempts and be useful for monitoring some restoration procedures. All results shown have been obtained with a spectral domain portable high resolution OCT system built by the authors especially for examination of objects of art in situ. This instrument uses IR radiation from the range 770 nm – 970 nm and its axial (in-depth) resolution is 3 μm in air (2.2 μm in varnish) whereas the lateral one is switchable between 7 μm and 13 μm. The area possible to be examined in one data collection is considerably large as for the microscopy and equals 5 x 5 mm2 and 17 x 17 mm2 respectively for both available lateral resolutions.

The research leading to these results was funded by the European Commission, FP7 Research Infrastructures Program, CHARISMA Project (grant no. 228330) and the Polish Government.

Fig. 1: Exemplary results of the OCT examination. Light approaches from the left, all scale bars are 0.5 mm, images shown in false colours; a: painting on canvas (P. Franck, "Portrait of sir John Wylie", 1815); b: reverse painting on glass ("St. Wendelin", late 19th c., Ethnographic Museum in Toruń, PL); c: atmospheric corrosion of 15th c. window glass.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-7-O-1651 Microscopy study at micro- and nano-scale in contemporary paintings treated with biocides. 

Ortiz-Miranda A. S.1, Doménech-Carbó M. T.1, Doménech-Carbó A.2
1Instituto de Restauración del Patrimonio, Universidad Politécnica de Valencia, 2Dpt. Química Analítica. Facultad de Química, Universidad de Valencia
annette.ortiz@gmail.com

The growing problems of biodeterioration undergone by commercial artists' paints have increasingly required the application of biocide treatments on contemporary artworks. In most cases, commercial biocides, which have not been created for the purpose of being used in the field of art conservation, are applied in the dosages recommended by the manufacturer without control on the effects of their application on the artwork. From this, a study has been conducted aimed to evaluate the changes induced by the biocide on contemporary paintings of acrylic and PVAc type. Two biocides hace been considered, namely, Biotin T® and Preventol RI80®. The morphological study at microscale has been performed by using optical microscopy and SEM/EDX. Chemical and morphological changes at nanoscale have been characterized by using, at first time in the field of the analysis of artworks, the novel technique of atomic force microscopy (AFM). In a second step chemical changes have been identified by using FTIR spectroscopy and UV-VIS spectrophotometry. Some of the most significant changes observed by microscopy were: appearance of spots and alteration of the brightness of the paint film, as well as, deposits of biocides. A notable delay in the coalescence phase of drying of the acrylic polymer used as binding media was recognized by means of AFM. Spectroscopic analysis results suggest that the application of the biocide causes a significant migration of addictives to the surface from the core film.

Financial support is thanked to the Spanish (MICINN) R+D Project CTQ2011-28079-C03-01 and 02 also supported with ERDF funds. Research was conducted within the "Grupo de análisis científico de bienes culturales y patrimoniales y estudios de ciencia de la conservación" Microcluster of the University of Valencia Excellence Campus (Ref. 1362).

Fig. 1: Optical microscope images for the samples: a)Liquitex® PB15 treated with Preventol RI80®; b)Liquitex® PBr7 treated with Preventol RI80®; c)Flashe® PB15 treated with Preventol RI80®; d)Flashe® PBr7 treated with Biotin T®.

Fig. 2: Scanning electron microscopy images for the samples: a)Liquitex® PB15 treated with Preventol RI80® and b)Secondary electron microphotograph of Flashe® PBr7 treated with Biotin T®.

Fig. 3: Atomic force microscopy showing substrate-film surface of the Liquitex® PB15 paint film.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-7-O-1966 Evaluation of reductive atmospheric plasma afterglow treatment on historical photographs with advanced electron microscopy techniques

Grieten E.1,2, Caen J.2, Schryvers D.1
1EMAT, Department of Physics, University of Antwerp, Antwerp, Belgium, 2Conservation Studies, Faculty of Design Sciences, University of Antwerp, Antwerp, Belgium
eva.grieten@uantwerpen.be

The quality of a TEM sample is an important factor when studying the changes in corrosion phenomena in historical photographs. This material has a complex structure made out a soft matrix with embedded image and corrosion particles. To determine the optimal sample preparation method 2 techniques are evaluated; the classical ultra-microtome and the high tech focused ion beam (FIB). Several parameters were compared such as thickness, uniformity, preservation of original structure and composition.

Classical ultra-microtome is often used for soft materials. Before the sectioning the material needs to be fixated and embedded in an epoxy. No changes to the morphology were noticed during these steps. In spite of the retained composition and achievable thickness the classical ultra-microtome sections are often deformed during section resulting in a low success rate of an intact interface between the corrosion particles and the epoxy (see fig. 1).

With FIB it is possible to directly sample with high selectivity the historical photograph. This is a great advantage when working with historical material where sampling is often restricted. Although it is possible to mill different materials several disturbing features are observed. FIB can cause preferential milling if the difference between the hard particles and soft matrix is big (see fig.2). Also the low stiffness of the gelatine results in buckling during the thinning phase. These artefacts make it difficult to make a uniform TEM lamella, which is thin enough for analytical characterization. Any Ga+ implantation during preparation does not influence or disturb the characterization since Ga can easily be distinguished from the corrosion elements (see fig 2C).

Since both techniques show artefacts making it difficult to achieve an intact thin and uniform sample a novel adaptation is suggested. Here we use the preparation steps of the classical ultra-microtome with an alternative final sectioning with focused ion beam. The difference between the classical ultra-microtome and ultra-microtome followed by FIB is the last stage or sectioning. This technique produces a TEM lamella with a clear interface and which is thin enough to determine the chemical composition or distribution of the nanoparticles in the corrosion layer (see fig.3). Although the success rate of this combined procedure is markedly better than that of the two alternatives, the main challenge remains making a thin enough sample to perform analytical characterization.

The authors thank P. Storme and O. Schalm for the plasma experiments. E. Grieten is grateful for a BOF fund of the University of Antwerp.

Fig. 1: A: Historical Daguerreotype; B: TEMcross-section of image area of figure 1A; C: STEM_EDX map of TEM lamella, withAu (R), Hg (B), Ag (G) and Pt (P).

Fig. 2: A: glass negative with silver degradation (left); B1 & 3 corrosion before plasma treatment; B2 & 4: corrosion after plasma treatment.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-7-O-3322 Ancient manuscripts, bacteria and crystals: a SEM story

Martinelli L.1, Yang H.2, Sprocati A.3, Tasso F.3, Downs R.2, Pinzari F.1,4
1Ist. Centrale per il Restauro e la Conservazione del Patrimonio Archivistico e Librario, Rome, Italy, 2Department of Geosciences, University of Arizona, Tucson, Arizona, U.S.A., 3Unità Tecnica Caratterizzazione, Prevenzione e Risanamento Ambientale, ENEA, Rome, Italy, 4Consiglio per la Ricerca e la sperimentazione in Agricoltura, Rome, Italy
livia.martinelli@yahoo.it

A severe biological attack occurred to a XVII Century document made of parchment (Fig.1a). Stained and clean samples were compared by means of Scanning Electron Microscopy (SEM). The damaged parchment surface showed the presence of needle-like crystals associated with bacterial spores. Bacterial DNA was extracted from pure cultures obtained from the document, and 16S rDNA amplification was performed using the pair of universal primers P0 (position 27 E. coli forward)/P6 (position 1495 E. coli reverse). Identification of the species was obtained after 16S rDNA sequencing, and sequence alignment with international databases (BLAST). The analysis of parchment fragments was performed with a Scanning Electron Microscope ZEISS VP-SEM EVO50, using both the VPSE and the QBSD detectors, and operating also at High Vacuum on gold sputtered samples. A qualitative chemical characterization of the inorganic constituents of parchment samples was performed by means of electronic dispersion spectroscopy (EDS. Oxford INCA 250). The ability of the bacterial strains present on the material to move salts and produce characteristic crystalline compounds was documented (Fig 1b). Large crystals with different morphologies (prismatic, platy, or needle-like) were produced in BUG agar cultures of the microorganisms (Fig 1c). Raman spectroscopy and X-ray diffraction identified the biogenic minerals as struvite. The bacteria responsible for the observed phenomenon were identified as different morphotypes of a species of Virgibacillus. The EDS analysis showed a composition of the crystals both on parchment and in vitro based on Na, Cl, P and Mg (Fig 1d). The Virgibacillus strains (one species, two different morphotypes) resulted proteolytic, moderately halophilic and showed a different ability to produce the struvite crystals. Parchment contains a high concentration of Ca, plus some NaCl, and can support the growth of microorganisms that use proteins as carbon sources. SEM-EDS study of biogenic crystals production, both in vitro and directly onto materials allowed an X-ray area scanning of what was brought into focus in SEM images, thereby creating a compositional map that documented, at a microscale level, the relationship between mineral gradients formed by bacteria, and the different crystal forms. This technique has the potential for disclosing some of the mechanisms of biogenic production of different crystal forms by bacteria.

Fig. 1: the XVII Century document showing biological damage

Fig. 2: SEM image in high vacuum mode of crystal produced by bacteria on parchment

Fig. 3: SEM-QBSD image of a crystal produced by bacteria in vitro

Fig. 4: EDS spectrum of the biogenic crystal produced by the bacterium
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-7-O-3504 Metallurgical Microstructure of the Japanese Spear Blade Manufactured in the 17th Century

Sugioka N.1, Kitada M.1, Nishijima M.2
1Tokyo University of the Arts, Tokyo, Japan, 2Institute for Materials Research, Tohoku University, Sendai, Japan
nahoppi77@ybb.ne.jp

The metallurgical microstructure and mechanical properties of the Japanese spear blade manufactured in the 17th century have been investigated. The purpose of this work is to obtain metallographic data, and to clarify the manufacturing technique and the influence of heat treatment. The specimen manufactured in the 17th century has the signature of Shinano-no-Kami Minamoto Takamichi. The spear is 33.5 cm in length and 10.7 mm in maximum width. The metallurgical microstructure and nonmetallic inclusions of the spear blade are observed using an optical microscope. The carbon concentration is determined by chemical analysis. To evaluate the hardness, Vickers hardness (Hv) is used. The microstructure is observed using an optical microscope and FE-SEM. The concentration of nonmetallic inclusions is analyzed by EDS. The crystal orientation observation in the tip of blade is analyzed by EBSD pattern. The martensite microstructure and nonmetallic inclusions of the spear blade are observed by TEM. A thin film for observing the nanostructures is prepared by the focused ion beam method.

A cross-sectional image of the spear after chemical etching is shown in Figure 1. It shows a metal flow pattern formed by deformation, possibly created by hammering. The striped structure of layers of two types of steel containing different carbon contents stacked on top of each other was observed. The bright areas after etching were cooled rapidly, forming the martensite structure. The other dark areas consist of pearlite and α-Fe (ferrite) grains. An EBSD pattern image of the tip of spear blade is shown in Figure 2. It keeps directions of many small crystal grains as the tip, and it is thought that crystallographic orientation might be aligned by hammering. Transmission electron micrograph of the precipitates existing in glassy area of nonmetallic inclusion of the specimen is shown in Figure 3(a). These areas contain various elements; O, Mg, Al, Si, K, Ca, Ti, Mn, Fe and Zr, which were detected by EDS. Typical elemental composite image of Ti and Zr is shown in Figure 3(b). High-resolution TEM micrograph of a precipitate in Figure 3(a) is shown in Figure 3(c). In the crystal interface, epitaxial growths were observed. From analysis of the diffraction patterns and lattice image, the existence of FeTiO3 and ZrO2 is certificated. It seems that a precipitate formed in glass performed epitaxial growth as fine ZrO2 on ilmenite FeTiO3.

The authors gratefully acknowledge the financial support of JSPS KAKENHI Grant Number 24680082 (Grant-in-Aid for Young Scientists (A)), 25289255 (Grant-in-Aid for Scientific Research (B)), the Moritani Scholarship Foundation, and the Kurata Memorial Hitachi Science and Technology Foundation.

Fig. 1: Cross-sectional macrograph of Japanese spear.

Fig. 2: EBSD image of martensite microstructure near the tip of spear edge.

Fig. 3: (a) TEM micrograph and (b) EDS composite image of precipitates existing in glassy area of nonmetallic inclusion. (c) High-resolution TEM micrograph of a precipitate.
Fig. 4:
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-1413 SEM-EDX study of the Pelligrini Golden Room Ensemble, The Royal Picture Gallery Mauritshuis

Haswell R.1, Pottasch C.2, van Loon A.2, van den Burg J.3, Hartman L.1, Singelenburg F.1, Genuit W.1
1Shell Global Solutions International B.V., Amsterdam, The Netherlands, 2Mauritshuis, The Hague, The Netherlands, 3Mauritshuis intern for this project
ralph.haswell@shell.com

The closing of the Royal Picture Gallery Mauritshuis for renovation in the period 2012-2014, has given the opportunity to treat and examine in detail the paintings of the Golden Room ensemble. The ensemble was painted in ca.1718 by Giovanni Antonio Pellegrini and consists of three rococo ceiling paintings, two chiaroscuro chimney paintings, four grisailles and six flower tondos. During the removal of the varnish, a haze was observed on the surface of the paint. The haze appeared to have a white colour in dark area’s and a grey colour in light area’s. It disturbed the image by working as a pall which meant that details were lost and the visual depth flattened. The haze also caused the colours in the paintings to become dull. In an effort to discover what was causing the haze, paint samples were taken and cross-sections prepared and examined with SEM-EDX. In Figure 1 a typical paint cross-section is shown where the haze is visible as a distinct crust on the paint layers. SEM-EDX mapping revealed that the main components in the crust are lead, potassium and sulphur, as shown in Figure 2. FTIR-ATR and DT-MS analysis indicates the crust consists mainly of inorganic components such as sulphates, carbonates and oxalates. In addition, the composition of the crust varies both between paintings and within individual paintings. It seems likely that the crust is the result of a reaction between sulphur from the atmosphere (from burning peat/coal in the open fireplaces) with elements that have migrated from the lead-rich ground and paint layers to the paint surface. After an extensive series of tests (to be reported separately) an aqueous-based method was developed to remove the crust without damaging the paint layer. The effectiveness and possible damage of the surface was evaluated by comparing SEM images of the paint surface before and after cleaning. An example of the effectiveness of this procedure is shown in Figure 3 which shows a paint cross-section after treatment. The paint layers have remained intact while the crust has been removed, except in degraded areas where it was only possible to partially remove it. The remarkable effect of removing the crust on the painting is shown in Figure 4 where a comparison between a cleaned and untreated region of the painting Raison d’état is shown. The complete set of paintings has now been successfully treated and will be returned to the Golden Room towards the end of 2014.

The analysis was possible due to the financial support of Shell Netherlands B.V.

Fig. 1: SEM-EDX backscatter electron image of Aurora (MH1136x15). A distinct layer is visible on the surface of the paint.

Fig. 2: SEM-EDX false colour X-ray map from Aurora (MH1136x15). Red = Sulphur, Green = Potassium and Blue = Lead.

Fig. 3: SEM-EDX backscatter electron image of Raison d’état (MH1143 x12) after cleaning. The crust layer has been removed while the paint layers have remained intact.

Fig. 4: Detail image of Raison d’état (MH1143) during removal of the crust. Details such as the fur of the lion and his teeth can now clearly be seen.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-1468 Aberration Corrected STEM to study an Ancient Hair Dyeing Formula

Patriarche G.1, Walter P.2, Van Elslande E.2, Ayache J.3, Castaing J.2
1Laboratoire de Photonique et de Nanostructures, CNRS UPR20, route de Nozay, F-91460 Marcoussis, 2Laboratoire d’Archéologie Moléculaire et Structurale, UMR 8220,, UPMC, Site ‘‘Le Raphaeël,’’ 3 rue Galilée, 94200 Ivry-sur-Seine, France, 3Institut Gustave Roussy, CNRS UMR8126, Signalisation, Noyaux et Innovations en Cancérologie, 114 rue Edouard Vaillant, 94800 Villejuif, France
gilles.patriarche@lpn.cnrs.fr

Since the Greco-Roman period, organic hair dyes obtained from plants such as henna have been used, but other unusual formulas based on lead compounds, such as the recipes describing several methods to dye hair and wool black, were also common. It is remarkable that these Greco-Roman techniques have been used up to modern times: related recipes were described by Arabian authors during the medieval period, during the Renaissance as practical application of alchemical knowledge, and by modern chemists, from the Encyclopedie of Diderot and d’Alembert [1] through to the present day [2]. In these cases, the same specific formula is provided: a mixture of lead oxide, PbO, and slaked lime, Ca(OH)2, with a small amount of water to form a paste, is applied on the hair. Successive applications on gray or light hair give rise to the black color. It is known that the blackening of hair is due to the precipitation of galena (PbS) crystals during the chemical treatment: the lead is provided from the paste deposited on the hair shafts, and the sulfur involved in the reaction comes from the amino acids of hair keratins. Here, we show that a consequence of these practices consists of synthesizing galena (lead sulfide) nanocrystals to dye hair black [3]. This very simple chemical process seems promising for the production of other nano-size semiconductor sulphides, such as HgS. Mercury sulphide is a useful material with applications in many fields such as ultrasonic transducers, image sensors and photoelectric conversion devices. We have synthesized HgS nanoparticles in the hair, two forms of HgS have been grown during the treatment, i.e. cinnabar and metacinnabar (figures 1 and 2)[4].

STEM observations were performed on thin sections prepared by ultramicrotomy, deposited on a specific TEM grids and observed at 200 kV with a Jeol 2200FS TEM/STEM microscope equipped with a Cs probe corrector. HAADF-STEM images are particularly sensitive to the presence of heavy elements as lead or mercury, until the single atoms detection possible among the light elements (C,N,O,S) forming the hair.

References

[1] D. Diderot and M. d’Alembert, Encyclopédie ou Dictionnaire raisonné des sciences, des arts et des métiers; Tome troisième: Paris, 1751; p 319.

[2] Gradual or progressive modern dyes, like Grecian Formula 16, contain lead acetate [Pb(CH3COO)2].

[3] P. Walter et al., Nano Letters 6 (2006) 2215

[4] G. Patriarche et al., Philosophical Magazine 93 (2013) 137-151

Fig. 1: Atomic resolution HRTEM image of HgS nanoparticles. Both hexagonal (cinnabar) and hexagonal (metacinnabar) crystalline phases are synthesized in the hair. The circled particle on the HRTEM image (a) reveals a cubic crystal structure with twins (Σ3 type).

Fig. 2: Atomic resolution HAADF-STEM image of HgS nanoparticles. The nanoparticle at the center of the HAADF image corresponds to cinnabar (see the Fourier Transform in insert with the six -1100 reflections of the HgS hexagonal structure).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-1596 Hand phalanx of the Denisova girl: using X-ray microscopy for nondestructive histological analysis

Mednikova M. B.1, Dobrovolskaya M. V.1, Lavrenyuk A. V.2, Kazanskiy P. R.2, Shklover V. Y.2, Shunkov M. V.3, Derevianko A. P.3
1Institute of Archaeology, Russian Academy of Sciences, Moscow, Russia, 2Systems for Microscopy and Analysis LLC, Moscow, Russia, 3Institute of Archaeology and Ethnography, Siberian Branch, Russian Academy of Science, Novosibirsk, Russia
kazansky@microscop.ru

The studies of fossil hominins’ evolution and growth are of major importance in solving problems of anthropogenesis. Particularly, multilevel macro- / microstructural studies can yield new insights regarding early ontogeny of fossil hominins as well as broader issues of human evolution. Microstructural studies revealing histological pattern of the Denisova phalanx, its osteon structure and the preservation of lamellar tissue may be extremely informative in this regard. Comparison with respective patterns in modern humans may shed new light on their genetic relationship with Pleistocene hominins like the Denisovans and establish whether these hominins are as distinct from extant people in terms of ontogenetic patterns as are Neanderthals.
In 2010, the complete mitochondrial genome of a fossil hominin from Denisova Cave, Altai was sequenced on the basis of mtDNA extracted from the hand phalanx of a girl [1]. The present study of the same phalanx describes the preservation of the bone after sampling for aDNA, analyzes 3D and 2D magnified reconstructions, and makes a comparative histological assessment of the bone’s microstructural features. The X-ray microscopy we used is a nondestructive technique, which is an important advantage given the uniqueness and the fragmentary nature of the specimen: two fragments remaining after a DNA sampling were subjected to microCT examination.
The tomographic examination was conducted on ZEISS XRADIA Versa XRM-500 X-ray microscopy system, 3D and virtual slices were generated using the system’s proprietary software.
The micro CT (X-ray microscopy) of fragments of the hand phalanx of the Denisova girl has revealed a histological pattern which corresponds with the sequence of age changes in modern children. Unlike the Neanderthal children, the Denisova child displays no contrast with modern children in microscopic indicators of bone growth and development. This may indicate certain phylogenetic affi nities, suggesting that the essentially modern pattern might have originated as early as the Lower Paleolithic. The distinctness of the Neanderthal growth pattern, then, may be an autapomorphy.

1. Krause J., Fu Q., Good J.M., Viola B., Shunkov M.V., Derevianko A.P., Pääbo S. The complete mitochondrial DNA genome of an unknown hominin from southern Siberia. Nature, 2010, Vol. 464, N 7290. – P. 894–897.

Study supported by the Russian Foundation for Fundamental Studies (Project No. 13-06-1224 ofi _m)

Fig. 1: Fragments of the distal phalanx of the hand of a girl from Denisova Cave.

Fig. 2: Virtual cross-section of the proximal metaphysis with estimated osteon size.

Fig. 3: Virtual cross-section of the diaphysis with estimated osteon size.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-1886 Non-invasive survey of metallic supports of the small-size paintings of the 18th century

Šefců R.1, Trojek T.2, Vondráčková M.1
1National Gallery in Prague, Prague, Czech Republic, 2Czech Technical University in Prague, Department of Dosimetry and Application of Ionizing Radiation, Prague, Czech Republic
sefcu@ngprague.cz

The poster summarizes the results of the all-embracing investigation of a painting technique on metallic supports used in the works of Norbert Grund (1717–1767), a foremost representative of the Rococo painting in Bohemia, from the collections of the National Gallery in Prague. Grund’s oeuvre, rich in themes conceived in a playfully lightened, idyllic spirit, draws on various European painting schools for its motifs and styles. His small-size paintings enabled the collectors, particularly those from the burgher ranks, to build up a kind of miniature picture galleries, which were diminished versions of aristocratic and monastic collections. The small-size cabinet painting on metallic supports represents a considerable part of his oeuvre (Fig. 1). The utilization of metallic supports in fine art has initiated in the 16th century. However, it has never achieved such popularity as painting on canvas or a panel painting.
More than 40 works by Norbert Grund were studied in the National Gallery in Prague. The initial research was based on application of non-invasive X-ray fluorescence analysis (XRF) with a handheld NITON analyzer (Model XL3t) due to the heritage preservation and small format. The X-ray spectra (Fig.2) were acquired for only 30 s and then the data were evaluated semi-quantitatively. Only several micro-samples were taken with the aim of the easier characterization of the surface treatment of the supports. This representative set of micro-samples was analyzed with methods of optical microscopy, Scanning Electron Microscopy and X-ray Microanalysis. Molecular analysis was done using Raman micro-spectroscopy. Analysis was performed on the individual pigment grains or in the cross sections using the mapping mode for the identification of individual components presented in the colour layers (Fig. 3). The combination of these microscopical techniques applied to such large collection enabled us to systematize obtained information and evaluate used materials. The most numerous group includes supports made of iron plates coated with tin layer, alternatively covered also with a protecting layer of an organic or a mineral basis (Fig. 3). Copper and brass supports were identified too. In addition, surface corrosion products of metallic materials were documented, see Fig.4.
Interdisciplinary cooperation has enabled us to evaluate materials used in paintings attributed to Norbert Grund and also to evoke discussion on verification and specification of the authorship of several disputable works of art.

The work was realised thanks to the means of the project Norbert Grund (1717-1767) of the programme Czech Science Foundation, GACR, Identification code: GA13-07247S.

Fig. 1: Norbert Grund, Two Nymphs bathing and a Satyr, (Inv. no. O 272, size: 18.5×15.3 cm). On the reverse side there is a copper plate. Photo©National gallery in Prague 2014.

Fig. 2: X-ray fluorescence spectra acquired with the handheld NITON analyzer, a) the copper support, b) the iron plate coated with tin layer under the protective red painting layer, c) the iron plate coated with tin layer, d) the brass support.

Fig. 3: Norbert Grund, Playing Bowls (Inv. no O 5385, size: 26.5×36.5 cm). Raman spectra of the red layer and map showing distribution of the hematite in the cross-section of the sample. Raman spectra were recorded using 780 nm laser excitation of 2 mW power and 3 m steps.

Fig. 4: Norbert Grund, Washerwoman (Inv. no. O 397, size: 17.8×19.2 cm). The degradation of iron and tin layer is visible on the surface of the support. Photo©National gallery in Prague 2014.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-2145 Utilisation of micro-optic and micro-spectroscopic methods in the identification of identical filling materials of tinned reliefs in Late Gothic panel paintings of Bohemical provenance. 

Šefců R.1, Chlumská Š.1, Třeštíková A.1, Turková I.2, Kotrlý M.2
1National Gallery in Prague, Prague, Czech Republic, 2Institute of Criminalistics Prague, Prague, Czech Republic
turka@seznam.cz

The proposed poster gives a clear summary of the results of the material investigation, which forms part of a more widely based project devoted to the historical techniques of medieval painting of Bohemical provenance and the modern microscopic and instrumental methods. It deals with the comparison of the material composition of the tinned relief applied to the surface of two important anonymous panel paintings from the nineties of the fifteenth century linked with painters active for the circle of the Jagellonian Court in Bohemia – Panels with the provincial patrons Saints Wenceslas, Sigmund and Vitus (around 1490, NG in Prague, Fig. 1) and the paintings of the large winged altarpiece, one of the most significant works of Jagellonian court art – the Ark of the Coronation of Our Lady known as the Křivoklát Ark (around 1480-1490, Palace Chapel of Křivoklát Castle). The aim of the investigation was to document the material composition of specific substances of the applied tinned relief, which in Late Medieval workshops formed part of the characteristic, unique and strictly guarded technological workshop signature. The investigation of a representative range of micro-samples utilised the methods of optical microscopy in a polarising microscope, Scanning Electron Microscopy and X-ray Microanalysis, infrared spectroscopy with Fourier transformation and the mapping of layers using the method of Micro-Raman spectroscopy. Thanks to the combined use of microscopic and spectral techniques (Fig. 2, 3) it was possible to evaluate in detail the use of the materials in the individual layers, to evaluate the morphological and structural traits of the pigments and to identify the organic binding agents. Through the scientific investigation of the material base of the decorations it was possible to evaluate the results of the elemental and molecular analyses qualitatively and quantitatively and carry out the appropriate comparison and subsequent analysis of the data acquired. The evaluation of the data on the material composition of the filler substances of the tinned relief showed similarities on parts of the clothing of St Vitus and St Sigmund on the Panel with the provincial patrons to the decoration on parts of the clothing of St Wenceslas and St Vitus on the external sides of the movable wings of the Křivoklát Ark (Fig. 4). In these substances there was shown to be similar use of pigments on the basis of minium, lead white and additional minerals and the connection was also evident in the application of the individual layers in the preparation of the relief. The recognition of the repeated use of the same materials is especially valuable and so fundamental that it is possible to confirm the assumed direct workshop connection between the two historical works.

The proposed contribution came into being thanks to the grant support of the Ministry of Culture of the Czech republic (project identification code: DF 13P010V010). 

Fig. 1: Panels with the provincial patrons Saints Wenceslas, Sigmund and Vitus, around 1490 (Inv. no. O 1360). Photo©National gallery in Prague 2014.

Fig. 2: BSE image of the tin-relief, robe of St Vitus of the Křivoklát Ark. Photo© Institute of Criminalistics Prague 2014.

Fig. 3: EDS spectrum of material of the relief, robe of St Vitus of the Křivoklát Ark.

Fig. 4: Cross-section of sample from tin-relief: a) robe of St Sigmund, Panels with the provincial patrons Saints Wenceslas, Sigmund and Vitus, b) robe of St Wenceslas, Křivoklát Ark. Photo©National gallery in Prague 2014.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-2317 Three Dimensional EDS Elemental Mapping of Nonmetallic Inclusions of a Japanese Sword with an FIB-SEM

Matsushima H.1, Kitada M.2, Mori N.1, Brunetti G.3
1JEOL Ltd., Tokyo, Japan, 2Tokyo University of the Arts, Tokyo, Japan, 3JEOL (EUROPE) SAS, Croissy-sur-Seine, France
matsushi@jeol.co.jp

 Japanese swords are made of raw steel produced by smelting iron sand. The raw steel made by the Tatara method contains less P and S, and higher concentrations of nonmetallic inclusions (NI) than modern steel. By analyzing NIs the source of the iron sand and the heat process used during processing Japanese swords have been investigated. The purpose of this study is to reveal three dimensional (3D) distribution of NIs to know solidification process. The 3D distribution was observed by an FIB-SEM. A sample was repeatedly sliced to expose a new surface for analyzing with an EDS automatically. The 3D distribution was reconstructed from the acquired EDS data to analyze solidification processes of the NIs.

 The sample was a Japanese sword with the signature of Bizen Osafune Katsumitsu (property of M. Kitada). It was made in Japan in the 16th century. An EPMA was used to draw elemental maps of NI one cross-section (CS) of the sword. An FIB-SEM, JIB-4601F (JEOL), and an EDS (Oxford Instruments) were used to determine the 3D distribution of NIs. The SEM condition was as follows; the accelerating voltage (Acc. V.): 10 kV, probe current (P.C.): 14 nA, whereas the FIB processing condition; Acc. V.: 30 kV, P.C.: 10 nA, and ion dose: 250 nC/µm2.

 An overall image of the CS after etching is shown in Figure 1. A BEI and elemental maps of O, Al, Si, and Ti in this CS are shown in Figure 2. The distribution of these elements is almost the same as show in Fig.2, which indicates that the NI was oxide. The NI in a red circle of Fig.1 was analyzed with the FIB-SEM. The 3D BEI reconstructed by the MIP method is shown in Figure 3(a). A superimposed 3D elemental map of Al, Si, and Ti is shown in Figure 3(b). The distribution of the NIs was clearly observed three dimensionally. As to the distribution of the elements in the NIs, Al rich areas were wrapped in Si rich areas, whereas Ti rich areas were relatively isolated.

 No cracks were observed in NI. This observation suggests that NI was melted during high temperature forging and then solidified during cooling. When the inclusion was cooled, Ti oxide precipitated first from molten oxide, successively the remaining molten oxide solidified as alminosilicate glass. In the area close to metallic iron with high heat-conductivity, Si rich glass solidified first followed by solidification of Al rich glass. This solidification sequence resulted in the observed Al rich areas wrapped in the Si rich areas, where Ti oxide particles were randomly distributed. The elemental distribution in the NIs suggests that the source of the iron sand was the one rich in ilumenite (FeTiO3).

Reference: M. Kitada, Fine structures of a Japanese Sword Fabricated in the Late Muromachi Era (16th Century), Uchida-Roukakuho Tokyo (2008)27-36.

Fig. 1: An etched cross-section of a Japanese sword. The 3D distribution of the NIs was analyzed in an area indicated as a red circle.

Fig. 2: Elemental maps obtained with an EPMA, JXA-8230 (JEOL), by a stage scan mapping method. 1) BEI (backscattered electron composition image), and 2) O, 3) Al, 4) Si and 5) Ti elemental map. Each scale bars were 5mm.

Fig. 3: (a): The 3D BEI showing the distribution of NIs by the MIP (Maximum Intensity Projection) method. The size of the analyzed volume was X: 20 µm, Y: 35.3 µm, and Z: 5.3 µm. The slice pitch was 100 nm. Each analysis took 23 min. (b): A superimposed 3D elemental map showing the distribution of Al (green), Si (yellow), and Ti (pink).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-2544 How do you balance the different demands for non-destructive analytical and imaging instrumentation within an interdisciplinary and multi-modal centralised research facility?

Ball A. D.1
1Imaging and Analysis Centre, Science Facilities, The Natural History Museum, London, UK.
a.ball@nhm.ac.uk

The Natural History Museum employs a centralised core imaging and analysis facility offering a wide range of imaging and analysis instrumentation to its staff, students and visitors. As the national collection for natural history, the Museum houses more than 70 million specimens covering an incredibly diverse range of subject areas and sample types, together with extensive natural history art collections and library materials. As a result, the instrumentation offered needs to be highly adaptable to suit the different needs of more than 300 annual users.

Our laboratories include electron microscopy, electron probe microanalysis, micro-CT, confocal and light microscopy, chemical and X-ray diffraction analysis and a variety of ICP-mass-spectrometry-based techniques. The practicalities of managing the varied demands of international visitors, students and experienced research staff require an in-depth knowledge of the instrumentation, how it might be used in conjunction with other techniques and to what degree the instrument might damage the samples.

As a single example, our latest variable pressure SEM was specified to have a wide range of functionality which required careful coordination between the main instrument and the accessory suppliers. This is an increasingly common situation and requires a good level of understanding and trust between all parties involved. The instrument delivered includes high resolution variable pressure and high vacuum SEM imaging; a high resolution and high speed EDX system (including the ability to perform analyses at variable pressure or very low kV and low probe currents) and high resolution X-ray micro-tomography. In addition, whilst the stage and sample handling capacity was designed around the very largest specimens (15cm x 15cm area imaging), an additional micro-tilt/rotate stage was installed for high precision work on small, demanding samples. One issue we have taken care to address is the risk posed by the rapid decompression rates which SEMs impose on samples during pump-down. This microscope has a redesigned vacuum system to allow the chamber to reach operating pressure over 20-40 minutes rather than the more common 90-120 seconds. We have also conducted extensive research to understand the potential for SEM to contaminate or chemically alter samples, particularly during X-ray microanalysis. This is of increasing importance where nanoparticle characterisation is concerned, whether these be artificial or naturally occurring.

Our experience suggests that allowing core staff free-range to explore different research ideas pays off in terms of ability to address complex research problems and with respect to job satisfaction!

Fig. 1: Large samples require large SEM chambers and stages! This statue was approximately 20cm tall. The internal camera is a vital safety feature to prevent damage to the detectors, stage or specimen.

Fig. 2: Reflection-mode CLSM image of a hubble space telescope solar cell showing damage caused by a micrometeoroid. This sample was examined as part of a feasibility study to investigate non-destructive methods for estimating volume of material lost and to measure the thickness of the various layered components making up the device. Field width 100µm.

Fig. 3: XRF mapping image of a Cambrian arthropod revealing chemical differences between the fossil and rock matrix. The iron remnants (red) are interpreted as traces of nervous system and closely match the organisation of modern arthropods. Field of view approx 10mm. Fast, large area chemical mapping techniques are invaluable for conservation research.

Fig. 4: Micro-CT scan of a glass model of a jellyfish made by in the Blaschka workshop. Soda-lime glass was used for the bell, tentacles and other details were modelled in lead glass. Different colours reflect the glass composition. Micro-CT has proven ideal for the 3D inspection of delicate articles and as a tool for preparing conservation strategies.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-2943 Black crusts on welded tuffs

Reyes-Zamudio V.1, Avalos-Borja M.2, 3, Cervantes J.1
1Departamento de Química, DCNE, Universidad de Guanajuato, México, 2Instituto Potosino de Investigación Científica y Tecnológica, División de Materiales Avanzados, San Luis Potosí, S.L.P., México, 3Centro de Nanociencias y Nanotecnología UNAM, A. Postal 2683, Ensenada, B.C., México
viridian@ugto.mx

The alteration of stones in monuments is a slow and continuous, always irreversible, natural process. Human interventions through restoration and maintenance of buildings can delay, but certainly not stop, the process. This alteration is the result of the complex interaction of several factors, mainly the rock mineralogy and composition of the surrounding environment. Monuments are more affected in urban environments, where the aggressiveness of the degrading agents can grow exponentially. Entire stone surfaces show a more or less distinctive dust and aerosol deposition; however, the formation of black crusts is an acceleration factor of this deterioration. Black crusts are formed by dry deposition on the surface; they are mixtures of airborne particles and gases from fuel combustion along with varieties of dust from the environment, including aerosols of marine salts and microbial fauna. The transformation of the stone material due to this interaction is a dynamic process involving chemical reactions, dissolution, exchange of material, and migration of soluble compounds. Current research has shown that, in general, the deterioration provoked by black crust is mainly confined to the outer surface of the different stone materials.

Welded tuffs or ignimbrites were widely used in the construction of many historic and artistic buildings in West-Central Mexico and Latin America and are present in many monuments around the world, from sculptures to buildings.

The present work has focused on the study of the origin of cracks in welded tuff samples from one of the most important historical buildings in the city of Guanajuato, Mexico: The Basilica (Figure 1). This monument, built between 1671 and 1696, is the city’s main church. The samples come from the clock tower, which was completed in 1776. This tower, like many colonial buildings, was initially covered with mortar and painted. It is not known if this coating was scratched during the movement ordered by the government at the beginning of the twentieth century, or if it has been lost little by little by reaction with the environmental acids. This is one of the most deteriorated parts of the building (Figure 2), presenting cracking, flaking, and spalling, for which reasons it was recently consolidated.

The observations and analysis by petrography and ESEM - EDX show that some cracks were formed by the physicochemical degradation of the stone material due to interactions with the black crust developed on its surface (Figures 2 and 3), and that the degradation and migration of soluble product is several centimeters deep in the stone. These results highlight the importance of cleaning and maintaining monuments.

Acknowledgements
We thank Ana Iris Peña and Jennifer Eckerly (IPICYT) for technical assistance.

Fig. 1: Panoramic view of the historic building.

Fig. 2: The Clock Tower, sampled area.

Fig. 3: Petrographic view (100X) of the black crust deposited on the outer surface of the sample and the fractures caused thereby.

Fig. 4: Morphology of carbonaceous particles forming the black crust.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-2857 Microanalytical studies of 18th century Brazilian Baroque wooden polychrome sculptures and altarpieces from Minas Gerais

Souza L. C.1, Balzuweit K.2,3, Rosado A.1, Rocha S. O.1
1Conservation Science Laboratory (LACICOR) – School of Fine Arts – Federal University of Minas Gerais (UFMG), 2Physics Department – ICEX – Federal University of Minas Gerais (UFMG) , 3Center of Microscopy (CM-UFMG) - Federal University of Minas Gerais (UFMG)
karlaweit@gmail.com

Analytical studies of works of art and cultural heritage objects are keen to contribute to: 1) the knowledge of the artists materials and techniques; 2) the understanding of their deterioration mechanisms and the evaluation of possible conservation methods; and 3) as tools to help in authentication and provenance studies.

The State of Minas Gerais, in Brazil, presents about 70% of the baroque and rococo Brazilian State listed and protected heritage. Their materials and techniques are well known, but analytical details such as the gilding alloy composition or the evaluation of trace elements in the different gilding layers are relevant to provenance and/or authorship studies.

Our team is involved with authentication and provenance studies of two particular wooden polychrome pieces, supposed to have been produced as part of one whole altarpiece. The objects are: 1) a 18th century wooden polychrome sculpture described as "Santana Mestra", found in the State of São Paulo and brought to Minas Gerais for legal identification; and 2) a wooden gilded altarpiece, nowadays located at the Chapel of the Hospital of São João de Deus, in the city of Santa Luzia, Minas Gerais, Brazil. Small gilding fragments were collected from both the sculpture and the altarpiece, through the use of a miniscalpel and a stereobinocular loupe. The fragments were further embedded in acrylic resin and polished for conventional optical microscopy, including digital photography of the polished sample surface (Zeiss Axiocam), followed by scanning electron microscopy. Equipments include a optical microscope (Olympus SZ11) from the Conservation Science Laboratory of School of Fine Arts of UFMG; and a scanning electron microscope Quanta 3D-dual FIB from FEI, equipped with a Bruker X-ray dispersive spectrometer (EDS), from the Center of Microscopy of UFMG. Both punctual and mapping energy dispersive x-ray analysis (EDS) of samples from the sculpture and the altar were performed in an area which corresponds to the sequence of layers ground - armenian bole - gilding, with a focus on the gilding composition.

On all of the metallic leaf samples we find silver as a trace element, together with the main gold component. Sometimes copper was also detected. The other elements, such as Al and Si, are characteristic of the bole argilous layer. Our expectation is to be able to compare the Ag concentration in several samples of gilding from different churches in Minas Gerais, in order to be able to prove that both gildings, from the Saint and from the Altarpiece, have been produced with the same gold leaf - or not. The set of results will be of invaluable help in determining the effective provenance of religious gilded polychrome sculpture in Minas Gerais, Brazil.

Fig. 1: Energy Dispersive X-ray point spectra of sample 2616 taken at the same region where the mapping shown below was performed.

Fig. 2: Gold Energy Dispersive X-ray mapping of a small region of sample 2616 - magnification 8.000 times

Fig. 3: Silver energy Dispersive X-ray mapping of a small region of sample 2616 - magnification 8.000 times
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-3030 Can Microscopy help in the identification of counterfeit artworks? Test on Vase of flowers, attributed to Italian artist Filippo De Pisis

Volpe L.1, Vaccaro C.1, Vaccaro C.2
1TekneHub, University of Ferrara, Dept. Physics and Earth Science, Ferrara (IT), 2University of Ferrara, Dept. Physics and Earth Science, Ferrara (IT)
lisa.volpe@unife.it

In the field of Cultural Heritage, the safeguard of artworks contends new and complex problems linked not only to conservative condition, maintenance, etc. but also to the introduction of fakes and problems related to this aspects. In the last years, dating and authentication studies, mainly based on historical-artistic-stylistic researches, have been supported by scientific world through identification of artistic techniques and materials, underlining the important role of "dating pigments".
The identification of this kind of pigments provides for in depth chemical-physical analysis, and, always more frequently, the contribute of microscopy can be fundamental, especially for artificial pigments in modern and contemporary artworks. In fact, if traditional chemical-physical analysis allows to recognize pigments, only studying the morphology of pigments’ particles is possible to understand better their origin (natural or artificial, ancient or modern, etc.).
The present studies shows results obtained by researches carried out on Vase of Flowers, a painting attributed to Filippo De Pisis (1896-1956), important and renewed Italian artist (Fig. 1a). Some doubts about the authenticity of the expertize, which accompanies the artworks, increased suspicion related to the originality of artwork too.
The comparison between this artwork and other painting made by De Pisis, through preliminary analysis carried out by optical microscope on whole artwork, already showed different artistic techniques (Fig. 1b). Moreover, even if chemical analysis identified pigments belonging to De Pisis palette, such as White Titanium Oxide, more interesting results was obtained by SEM/EDS and µRaman, carried out on µsamples taken from original area (Fig. 1c): the identification of White Titanium Oxide particles (Rutile phase) with diameter less than 0.5 µm (Fig. 1d-e) suggests the use of pigment introduced on commerce in 1957, and so after death of artist [1-3]. Therefore, considering that pigments used in this artwork are not compatible with the period, the research suggests that the analyzed artwork could be a counterfeit painting [4], highlighting how chemical-physical analysis linked to microscopy studies could help in solving doubts about artistic attribution, also for contemporary artworks.

References
[1] W. McCrone, Journal of the America Institute for Conservation, 33, no.2 (1994) 101-114.
[2] R. Leonardi, Nuclear Physics A 752 (2005) 659c-674c.
[3] P.A. Lewis, Wiley-interscience publication, II edition (1987).
[4] L. Volpe (2013), Earth Science and Modern-Contemporary art: fingerprints for the safeguard of artworks in view of Fine Arts transportation, Ph.D. Thesis, University of Ferrara (IT), extended abstract in Plinius - Italian suppl. Eur. J. Mineralogy 39 (2013) 121-125.

We would like to thank owner of artwork for the great availability and interest in this research and Prof. Leis Marilena (University of Ferrara, IT) for her kindly support.

Fig. 1: Fig. 1. Vase of Flower (oil on wood), attributed to F. De Pisis: a) paiting VIS investigation; b) microphotographs of brushstroke differently enriched in matter (OM, mag. 13.4 x); c) sample of White pigment; d) e) SEM/EDS analysis carried out on sample c) shows pigment particle which dimension are less than 0.5 µm.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-3297 Impact of micro-CT and confocal microscopy analyses on amber. A risk assessment study using optical microscopy, FTIR and Raman spectroscopy.

Bertini M.1, Ball A. D.1, Mellish C.2, Blagoderov V.1, Goral T.1, Sykes D.1, Burgio L.3, Shah B.3, Pretzel B.3, Summerfield R.1, Steart D.2, Garwood R.4, Spencer A.5
1Imaging and Analysis Centre, Science Facilities, The Natural History Museum, Cromwell Road, London SW7 5BD, UK., 2Department of Earth Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD, UK., 3Conservation Department, The Victoria and Albert Museum, Cromwell Road, London SW7 2RL, UK., 4School Of Materials / SEAES, The University of Manchester,Oxford Road, Manchester, M13 9PL, UK., 5Department of Earth Science & Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
a.ball@nhm.ac.uk

Amber represents an invaluable “time capsule” capturing fossilised residues in 3D. As such, the best material is under considerable demand by researchers. However, the analysis of amber samples and their inclusions using state of the art imaging methodologies including micro-computed-tomography (µ-CT) and confocal microscopy, which are known to yield high quality three-dimensional data, is currently restricted at many institutions. This is because both techniques have the potential to chemically alter the specimen, but the short-term and/or long-term effects of these analytical methodologies are unknown. In the course of this study, the chemical characterization of a number of samples of different types of amber was carried out using Raman and FT-IR spectroscopy, prior to and after exposure to X-rays in a µ-CT scanner and to laser illumination using confocal microscopy. Additional exposure at a synchrotron X-ray source was carried out on a few sub-samples. The results highlighted that both µ-CT and confocal microscopy do not seem to alter the specimens chemically or visually. Hard synchrotron X-rays, however, caused visible discoloration to both amber and copal samples irradiated. Although no discernible difference could be observed between the pre- and post- exposure spectra using Raman spectroscopy, FT-IR spectra showed some minor decrease of the olefin peak at 1645 cm-1 in the Baltic amber sample, and clear oxidation of the succinate esters to succinic acid could be clearly measured in the FT-IR spectra of East African copal.

Fig. 1: Stack of amber prisms wrapped in cling film and analysed via µCT (A) and rendering of insect inclusions in the matrix of 2 of the blocks (B). Scale bars 1mm.

Fig. 2: Pre and post-exposure photographs of samples of East African Copal subject to µCT scanning (A, B), confocal microscopy laser illumination (C, D) and synchrotron radiation (E, F).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-3366 A study by Analytical Electron Microscopy of Metallic Artifacts from the Chichén-Itza Mexico.

Arenas J. A.1, Contreras J.2, Ruvalcaba J. L.1
1Instituto de Física, Universidad Nacional Autónoma de México, Apdo. postal 20-364 México DF 01000, México., 2Escuela Nacional de Conservación, Restauración y Museografía "Manuel del Castillo Negrete", INAH-SEP, General Anaya 187, San Diego Churubusco, Coyoacán, 04120, México D.F.
jarenas@fisica.unam.mx

Chichen-Itzá one of the seven wonders of the world, is famous among other reasons because of its Sacred Cenote, a large flooded cavity formed in the calcareous ground typical of the Yucatan Peninsula, Mexico, mainly used for ritual acts. In the decade of 60’s some metallic objects were extracted from the Cenote. In this work Electron Microscopy Analysis of the gilding pieces from the Sacred Cenote was performed in order to identify its constituent materials and possible manufacture process. The artifacts rich in copper and gold conserve its gilding even the damage due to burial and inadequate extraction and cleaning methods (Figure 1). The analyzed artifacts are soles of sandals and other pieces of the costumes dressed by the ones sacrificed in this. Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) was used to study the surface of the samples, thickness, homogeneity, morphology and elemental chemical composition. The images were obtained with secondary and backscatter electrons (SE and BSE respectively). In figure 2 a SEM image shown an irregular surface with many roughnesses due to the corrosion occasioned by the environment in which the sample was exposed. The SEM images obtained with BSE show several contrasts that indicate an inhomogeneous chemical composition we can observe a film of high contrast mainly constituted by Au (zone 1) and other area with Cu (zone 2). It could be verified that the plating is a rich gold-silver alloy, the measurements pointed out to electrochemical deposition gilding instead of depletion gilding or other methods. Transmission Electron Microscopy (TEM) techniques were used in order to obtain information about crystals size and crystalline phases (figure 3).

The authors would like to thanks to, R. Hernández, J. Cañetas, M. Monroy, Jesús A. Lara and D. Quiterio for technical assistance.

Fig. 1: a) Some of the studied gilded artifacts. b) Light microscopy image at 10X magnification, gilded areas are observed.

Fig. 2: SEM image of the stamped sheet to 500X, we can observe a rough surface with two regions of several contrast. In both regions, EDS analysis indicate different chemical composition, the area with high contrast (1) is rich in Au. 

Fig. 3: Cuasi-spherical nanoparticle of size ~10 nm with Au as main constituent on matrix of Cupper oxide. Interplanar distance d = 0.236 nm is near to d111 = 0.2355 nm of Au.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-3371 Studies of paint materials from churches and sculptures of the eighteenth century through polarized light microscopy, x-ray fluorescence and Raman light scattering

Mendes I. C.1, Lopes F.1, Matos J. P.1, Almeida L. T.1
1Federal University of Minas Gerais, Belo Horizonte, Brazil
isoldacmendes@gmail.com

The raw material composition of wooden ceilings, walls and altarpieces paintings of Churches and polychrome sculptures of different locations of state Minas Gerais in Brazil were studied in the last two year. The work was carried to decide the appropriate intervention methods and materials to be used in the conservation of the paintings. The Churches were built in the Baroque and Rococo style in the period of 1750 to 1870.
Extensive examinations such as stratigraphic studies of cross sections, material analysis using polarized light microscopy, portable x-ray fluorescence and Raman light scattering were applied. The work revealed a number of layers of painting covering the originals (fig. 1) in most fragments, some of the wooden ceiling with the original paint, three different techniques of gilt, water gilding and oil-gilding and gold-mercury amalgam described in the Portuguese manuals of the eighteenth century (STOOTER, J. 1786 – Arte de Brilhantes Vernizes, e das Tinturas, Fazellas, e como fed eve obrar com ellas), grounds of mineral dolomites, calcium carbonate and calcium sulfate and a limited number of pigments inthe original layers commonly used as: vermillion, Prussian blue, ultramarine, chrome yellow, white lead and iron oxides (ochres). Some pigments were observed with significant variations in particle size and form suggesting the use of mineral pigments in the first layers and the synthetic ones in the later. Prototypes have been made to study the effect of multiple layers of paint on the result of the analysis and standards were used for comparison of sizes of particles encountered.

CNPq, FAPEMIG, CAPES, PRPQ, IEPHA, Grupo Oficina de Restauro, Estilo Nacional and Solo.

Fig. 1: Figure 1 – Cross section photography showing the layer sequence of a side pulpit fragment from the Church of São Gonçalo do Amarante – São Gonçalo do Rio Abaixo - Minas Gerais. (48x)
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-3399 Micro-Computed Tomography in a Museum Environment

Sykes D.1, Ahmed F.1, Summerfield R. A.1
1Natural History Museum, London, UK
D.Sykes@nhm.ac.uk

Periodically, new techniques arrive and revolutionise the working practices for conservators, researchers and curators. One such technique introduced to the Natural History Museum (NHM) in London in 2008 was the arrival of Micro-Computed Tomography. Assessment of the price of CT scanners and the increase in “affordable” computing power prompted an investment in a dedicated Micro-CT centre based in the Imaging and Analysis Centre. The CT suite at the NHM facilitates over 100 projects and typically 2000 scans per year, resulting in an incredible amount of data being produced. This non-destructive, non-invasive and exceptionally informative technique has become a key tool in the interpretation and analysis of museum specimens. This talk explores how Micro-CT can contribute to projects in a range of scientific areas by showcasing its versatility as a technique, and examines ways to share such a large, information-rich, collection.


High profile projects such as the Tissint Martian meteorite used Micro-CT to locate voids which might contain trapped Martian atmosphere [1] (Fig. 1). Other projects such as imaging the 19th century Blaschka glass models for conservators, can provide a wealth of information on the lost manufacturing techniques used to produce these delicate artworks. This is invaluable data for conservation purposes (Fig. 2), but also provided a virtual record of the condition of the specimens.

Recently, virtual collections have become a much more appealing concept for the museum environment. Micro-CT derived data can aid in producing a library of virtual specimens which can both avoid the need to loan samples, which can reduce damage or contamination to collections (Fig. 3) and be used to enable researchers to collaborate remotely. The data can be shared in various formats; raw data, mesh data, embedded models in pdf documents, numerical data, rendered images or standard 2D projections. The scope to share 3D data opens new avenues for research and takes data sharing into the future of science.


Micro-CT data has also been providing an additional perspective on traditional 2D histology and thus has had an important impact in Taxonomy (Fig. 4) [2]. Researchers have been able to morphometrically analyse data, to process density information and obtain quantitative measurements. The combination of results enables scientists to get a better grasp of the specimen of interest. The acquisition of a Micro-CT system at the NHM also produces readily understandable, visual information that allows the public to easily understand vast range of different research projects carried out here.

[1] C. Smith and F. Ahmed. Microtomography of the Tissint Meteorite. MetSoc, 2012, Cairns
[2] S. Faulwetter et al., Zoo Keys 263 (2013) p.1-45

Fig. 1: Micro-CT rendered image of a sample from the Tissint meteorite. Colours are used to highlight different density minerals within the meteorite which can then be used to determine the different volumes of those minerals. 

Fig. 2: Micro-CT rendered image of a Blaschka glass model of a Radiolarian, which are part of the marine zooplankton. This model was created by the famous artisans Leopold and Rudolf Blaschka in the 19th century. It shows the species called Dorataspis diodon. Colours represent the different types of glass used in the production of these delicate models.

Fig. 3: Micro-CT rendered image of a holotype collection of a lizard. The skin is coloured a transparent white and the bone in a yellow-brown. An example of the building of a virtual collection of holotype material to protect the original holotype.

Fig. 4: Micro-CT rendered image of a Chrysanthemum in cross section. Colours highlight the different densities of tissue type, by examining cross sections of this material in any plane desirable many 'hidden' features can be revealed.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-3465 Microscopy in Archaeological Heritage Studies: “El Salitre” Funerary Complex, Tula (México)

Espinosa-Pesqueira M. E.1, Medina-Gonzalez I.2
1Department of Materials Technology, National Institute of Nuclear Research, 2Escuela Nacional de Conservación, Restauración, y Museografía (ENCRYM-INAH)
manuel.espinosa@inin.gob.mx

In 2003, a savage archaeological project in El Salitre –a place nearby Tula de Allende, Hidalgo, Mexico-- discovered a rich funerary complex of a single individual, whose was composed by ceramics, shells, metallic rings, and other artifacts; a context associated to Toltec Culture (800- 1250 AD)[1].Due to their opulence, variability, and physical condition, the Salitre Funerary Complex was subsequently incorporated into an interdisciplinary conservation project that involves both research and preservation strategies. One of the most interesting characteristics of this burial was that red and blue pigments covered the human remains, atrait registered both in situ and during its micro-excavation. Characterization of these pigments became relevant for investigation due to their brilliant color, coexistence, and association in the archeological context. To be true, red pigment (i.e. cinnabar and/or hematite) has been found in several Pre-Columbian funerary contexts [2,3,4]. Blue pigment is fairly common inMesoamerican mural paintings and ceramics (6,7,8,9); however, no record has been yet located regarding its use in funerary practices. Therefore, the composition, origin and possible meaning of these pigments became a matter of scientific enquiry.  Portable non-invasive techniques have proved useful forarcheological heritage analysis, but present limitations. Regardless of being considered destructive, electron microscopy is essential for heritage material analysis worldwide. In fact, the use of electron microscopy has turned into a rich venue of research in archaeology, art history, and conservation. This paper presents an interdisciplinary contribution to material analysis of Mesoamerican archaeology, which is mainly based on electron microscopy. Samples of red and blue pigments found in Salitre Funerary complex were analyzed using various microscopy techniques – optical (OM), scanning electron microscopy (SEM), Z - Contrast, SAED, HRTEM – in order to identify and characterize the components, and thus, answer questions raised by archaeologists and conservators.

References:

[1] Equihua, J. (2007) “Informe Final: Rescate Arqueológico el Salitre-Tula”, Mecanoescrito, México, INAH.

[2] Tiesler V., Cucina A. (2010) “K'inich Janaab' Pakal se Vuelve Ancestro” en L. Filloy coord. Misterios de un Rostro Maya - La Máscara Funeraria de K´inich Janaab´ de Palenque, Mexico-INAH, pp.93-94.

[3] H.W. Merwin et al.,”In the Temple of the Warriors” Eds. (Pub. 606, Carnegie Inst. of WA, WA, D.C., 1931), 60.

[4] M. José Yacamán, et al., Science Vol. 273, No. 5272 (1996) pp 223-225.

We are grateful to Arqlogo. Juan Carlos Equihua, coordinator of the El Salitre Archaeological Project (Centro INAH Hidalgo), Dr. Josefina Bautista (DAF-INAH), both of them collaborators of El Salitre Integrated Conservation Project (ENCRyM-INAH) and ININ - Electron Microscopy Facilities - Project TM-002.

Fig. 1: a) Image from the single individual burial offering at “El Salitre” Tula, Mexico; b) optical micrograph of the blue pigment; c) TEM micrograph where nanofibres can be observed and d) SAED ring pattern from region “A”-figure 1c-, interplanar distances correspond to the palygorskite crystalline phase.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-3472 Electrochemistry of copper on modified fibre and polymers – a dialogue between science and fine arts

Ploszczanski L.1, Hansal W.2, Pichler B.3, Trettenhahn G.1, Kautek W.1
1Department of Physical Chemistry, University of Vienna, Vienna, Austria, 2Happy Plating GmbH, Wiener Neustadt, Austria, 3University of Applied Arts Vienna, Department of Art and Technology, Vienna, Austria
leon.ploszczanski@univie.ac.at

Scientific investigations of historical electrochemical plating technologies as an artistic method to generate pictures on carbon coated fibrous and polymeric substrates were recently initiated. It is possible to vary the composition and nature of substrates, such as papers, textiles and polymeric surfaces. As a model system for this process served thick art paper and powdered carbon as conductive agent. The working electrode consisted of a carbon rod contacting the graphitized substrate by the cylindrical edge face. As counter electrode served a Cu bar contacting the opposite edge of the substrate laterally. The reference electrode, a Ag/AgCl electrode, could be moved to at any location above the substrate. Thus, an electric potential field mapping on the graphitized substrates could be achieved. In the course of a galvanic process in an electrolytic bath, copper atoms precipitated laterally inhomogeneously on a sheet of paper coated with adherent graphite powder. This process is inherently stochastic and can only be controlled to a limited degree. Further, a potentiostatic three electrode set up was employed which allowed the exact identification of the working electrode versus the known reference electrode. Within the parameters of the experiment, structural variations of copper morphology can be influenced by changing the voltage between working and counter electrode, applying a pulsed current instead of DC, modifying the surface texture of the substrate or by adding a brightening agent. To demonstrate the underlying mechanistic growth principles, samples are subsequently examined under the scanning electron microscope. The surface structure of the deposited copper and the nature of the various oxidic and salt coatings (Cu2O, CuO, CuSO4, CuCl, etc.) primarily determined the colour range of the images. The role of the oxidation of the surface and the detectable copper compounds needs to be investigated further.

ASEM, Dr. Tassilo Blittersdorff, Happy Plating GmbH., Wiener Neustadt

Fig. 1: DC deposited copper on carbon coated paper (40x mag.)

Fig. 2: DC deposited copper on carbon coated paper (10000x mag.)

Fig. 3: Deposited copper on FloppyDisk (500x mag.)

Fig. 4: Deposited copper on FloppyDisk (5000x mag.)
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-5725 Morphometric and chemical characterization of archeological pigments from the Teotihuacan archeological site by SEM and AFM microscopy, EDX, diffraction and RX flourescence techniques

Avelino I.1, Valadez M.2, Calderon J.3, Tovar J.4, Valdez O.5, Mendoza C.6, Garibay V.7, Leyte F.8, Pacheco U.9
1Universidad Tecnológica de Tecámac, 2Universidad Politécnica del Valle de México, 3Centro de Biotecnología Genómica-IPN, 4Instituto Mexicano del Petroleo
lizzy_130281@hotmail.com

Teotihuacan, “The place where men become gods”, was one of the most renowned  cities in the American continent during the Mesoamerican Classic Period (150-650 d. C). It was also capital of one of the most influential Mesoamerican societies in the politic, economic, commercial, religious and cultural realms thus, influencing other societies in the Mexican altiplane (INAH, 2013).
Teotihuacan mural paintings are beautiful. It is the Mesoamerican place with the largest number of frescos. Their artists used mainly materials containing hematite limonite, goethite, giobertite and malachite, applied over a supporting layer of sand and lime which enhanced the colors (Langley, 2007). In this work, a compositional and morphometric analysis of pigments sampled from the oldest paintings found in Teotihuacan will be performed, we will study the pigments particles size and distribution in order to find evidence of nanostructured particles in the pigments, since it is believed that this particle sizes have contributed to preserve the paintings in other cultures.
Also, correlative analysis will be performed by SEM and AFM imaging on samples of these pigments; chemical analyses will be completed (mapping, EDX, diffraction and RX fluorescence) in order to identify the chemical composition of these pigments. Last but not least, these results will be compared with minerals existing databases ONIONS and Mexican Geological Service, in order to both, estimate the source of these components and a potential variation at different historical times, thus shading light on the possible cultural interchange based in existing common elements present in the pigments.

References
INAH, Instituto Nacional de Antropología e Historia. Conaculta. 04 de enero de 2013. http://www.teotihuacan.inah.gob.mx.
Langley, James. "La pintura mural prehispanica en Mexico." UNAM. Teotihuacan: murals and meaning . Mexico, 2007.

We thank the following institutions for their kind support to perform this work, in alphabetical order:
Centro de Biotecnología Genómica-IPN, Reynosa, Tamaulipas, México.
Instituto Nacional de Antropología e Historia
Instituto Mexicano del Petróleo
Universidad Tecnológica de Tecámac
Universidad Politécnica del Valle de México
Their support is greatly appreciated.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-7-P-5897 Microscopic evidence of painting damage as a result of saponification

Hradilová J.1, Hradil D.2
1Academy of Fine Arts in Prague, ALMA laboratory, U Akademie 4, 170 22 Prague 7, Czech Republic, 2Institute of Inorganic Chemistry of the AS CR, v.v.i., ALMA laboratory, 1001 Husinec-Řež, 250 68, Czech Republic
hradilovaj@volny.cz

There are many reasons why pigments and binders degrade in colour layers of paintings. Some of them are chemically unstable by their nature; others interact with each other in mixtures. They could be also influenced by external factors - lighting, humidity, added chemicals (e.g. via inappropriate conservation treatment). The fatal consequences arise when degradation processes lead to colour changes or loss of mechanical integrity of painting layers. A typical representative of the second alternative is a process known as saponification of lead pigments. These pigments react with fatty acids contained in oil components of the binding media, slowly dissolve and finally form the so-called ‘metal soaps’, which are highly mobile and are able to diffuse and migrate through the layers. Therefore, the best example of saponification one can find in oil paintings containing large amounts of lead white (basic lead carbonate), e.g. in Netherlandish and Flemish paints of the 17th century (the Dutchmen were famous for their production of high-quality lead white). Other pigments, however, as red lead (Pb3O4), lead-tin yellow (Pb2SnO4 or PbSnO3) and other ones, could potentially react in similar way. Microscopy is an effective tool, how to reveal the process already in its initial stages. At an advanced stage, metal soaps aggregate and form prominent whitish or translucent lumps, which expand rapidly until they finally break through the paint surface causing paint loss and/or visual disruption of the surface. Within the extensive research we have evidenced the formation of metal soaps surprisingly also in Gothic tempera paintings where they appear accordingly with increased proportions of fatty ingredients in the binder (the so-called “fatty” tempera paints). We also observed that the process of saponification could affect the clay-based ground layers of Baroque paintings in cases where red earths were mixed with orange minium as an additional colorant. Metal soaps, indicated by their typical morphology, colour, UV luminiscence and chemical composition, thus seem to be more frequent than expected.

The authors thank restorers Mario Král and Blanka Valchářová for fruitful cooperation. Financial support of the Czech Science Foundation, project no. P103/12/2211, is kindly acknowledged.

Fig. 1: Lead soap aggregates protruding through the paint surface of the 17th century oil painting Leopards attacking deer by Carl Andreas Ruthart (1630 - 1703) (A) and detail of one individual lump in the microsection, formed by reaction of red lead with oil component of the binder in the red ground layer – in visible (B) and UV light (C), respectively.

Fig. 2: Morphology of lead soaps in back-scattered electrons – formed by reaction of lead white (A) and lead-tin yellow (B), respectively, in painting layers of 18th century paintings.
↑ Go to the Table of content ↑

ID-8. Three-dimensional reconstructions in microscopy

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-8-IN-1682 Atomic model of F420-dependent [NiFe] hydrogenase by electron cryo-microscopy.

Vonck J.1, Allegretti M.1, Mills D. J.1
1Max Planck Institute of Biophysics, Frankfurt am Main, Germany
Janet.vonck@biophys.mpg.de

It has long been recognized that electron cryo-microscopy has the potential to solve protein structures at near-atomic resolution, but until recently this was a prohibiting task, taking a huge investment in time and effort1. With the recent introduction of direct electron detection cameras with much better detective quantum efficiency and high frame rates, images with a higher signal-to-noise ratio can be obtained and alignment of video frames allows to partially correct for the effects of beam-induced particle movement and specimen drift2,3. The resulting improvement in image quality makes it possible to obtain better reconstructions with much less data. 3D reconstruction depends on the accurate determination of the relative projection angles of the images of macromolecular complexes, randomly oriented in a vitreous water layer, which is done in an iterative process. New image processing procedures make this process more robust and prevent fitting of high-frequency noise and creating spurious detail4. With these new tools we reconstructed a map of the 1.2 MDa, tetrahedrally symmetric complex of the F420-reducing [NiFe] hydrogenase (Frh). Frh is an abundant enzyme in methanogenic archaea, regenerating the reduced form of the flavin coenzyme F420 that is used by several enzymes in the methanogenesis pathway from CO2 and H25. It consists of three different subunits with several cofactors, a [NiFe] center, four [4Fe4S] clusters and an FAD forming an electron transfer chain from H2 to F420. 26,000 particle images were selected from 235 electron micrographs, collected on the Falcon II direct electron detector in video mode in a two-day microscope session. The map at 3.4 Å resolution shows all secondary structure as well as clear side chain densities for most residues and the cofactors in the electron transfer chain along with a well-defined substrate access channel. An atomic model for all but a few terminal residues could be built in the density map. From the rigidity of the complex we conclude that catalysis is diffusion-limited and does not depend on protein flexibility or conformational changes6.

References:
1 Mills, D. J., Vitt, S., Strauss, M., Shima, S. & Vonck, J. eLife 2, e00218, 2013.
2 Bai, X.-c., Fernandez, I. S., McMullan, G. & Scheres, S. H. W. eLife 2, e00461, 2013.
3 Li, X., Mooney, P., Zheng, S., Booth, C. R., Braunfeld, M. B., Gubbens, S., Agard, D. A. & Cheng, Y. Nature Methods 10, 584-590, 2013.
4 Scheres, S. H. W. J. Struct. Biol. 180, 519-530, 2012.
5 Thauer, R. K., Kaster, A.-K., Goenrich, M., Schick, M., Hiromoto, T. & Shima, S. Annu. Rev. Biochem. 79, 507-536, 2010.
6 Allegretti, M., Mills, D. J., McMullan, G., Kühlbrandt, W. & Vonck, J. eLife 3, e01963, 2014.

We thank Greg McMullan for setting up the Falcon II direct detector in video mode, Özkan Yildiz and Juan Castillo for computer support, and Stella Vitt and Seigo Shima for providing the protein. We are grateful to Werner Kühlbrandt for his support of the EM facility.

Fig. 1: Density map of the tetrahedral Frh complex at 3.4 Å resolution. Each of the twelve heterotrimers of FrhA, FrhG and FrhB is shown in a different colour.

Fig. 2: Details of the density map with fitted atomic model. Left: the FAD cofactor in its binding pocket. Right: two long alpha-helices.
↑ Go to the Table of content ↑
Type of presentation: Invited

ID-8-IN-3280 Large-scale 3D reconstructions making use of multimodal correlative light and electron microscopy markers and analytical SEM imaging – fantasy or a new paradigm?

Schröder R. R.1,2, Boese M.3, Dietrich C.4, Röder I. V.1, Wacker I.2
1Cryo-EM, CellNetworks, Universitätsklinikum Heidelberg, Heidelberg, Germany , 2Center for Advanced Materials, Universität Heidelberg, Heidelberg, Germany, 3Carl Zeiss Microscopy, Jena, Germany, 4Carl Zeiss, Oberkochen, Germany
rasmus.schroeder@bioquant.uni-heidelberg.de

While 3D reconstructions at highest resolution will undoubtedly remain the domain of TEM imaging modalities, more and more interest has been generated in SEM based techniques. This was mainly driven by the need of ultrastructural information of really large volumes such as whole cells and cellular networks in tissue (for a comprehensive review on the variations of Array Tomography [1] based applications see [2]), which via TEM could only be obtained by effusive and tedious work.

An additional facet of SEM based methods are new perspectives of imaging with low energy electrons: Low voltage TEM has proven to reduce beam damage of carbon materials and to allow analytical imaging of e.g. fluorescent polymers or other π-electron systems (cf polymer/fullerene imaging at 60keV in [3]), which uses electron energy loss spectroscopy in the optical / near optical region as readout. Because of beam damage at 60keV we did not succeed to apply low-loss TEM-EELS to typical fluorescent dyes used as markers in biological samples, even though more irradiation-resistant QuantumDots - used as multi-color LM/TEM correlative markers - can be distinguished by (S)TEM-EELS and could potentially facilitate correlative “multi-color” light and electron microscopy in biology [4]. It may thus be an attractive alternative to investigate analytical imaging possibilities at really low electron energies where less ionization damage of the sample might be expected. This will reduce the mean free path beyond usability of TEM and thus novel SEM approaches to analytical surface imaging are needed.

On the way to such a scenario we have established a hierarchical Array Tomography workflow [5], which allows super-resolution LM and SEM imaging of fluorescently labeled serial thin sections of tissue material. A typical 3D reconstruction with a super-resolution fluorescent LM modality is shown in fig. 1, which illustrates the distribution of labeled acetylcholine receptors on the post-synaptic membrane of a neuromuscular junction (mouse diaphragm). However, such samples would now need to be imaged via low-loss SEM-EELS. At present such an SEM-modality is not yet established. As a first step in this direction fig. 2 shows a comparison of a typical SE image and an energy-filtered BSE equivalent recorded on a novel high-efficiency detector. Currently we analyze how usable EEL spectra can be extracted from such images.

[1] Micheva and Smith (2007) Neuron 55, 25, [2] Wacker and Schröder (2013) J Microscopy 252, 93, [3] Pfannmöller et al. (2011) Nano Lett. 11, 3099, [4] cf abstract at IMC2014 by Pfannmöller et al. and Pfannmöller et al. in: Proceedings Microscopy & Microanalysis (2011), [5] cf abstracts at IMC2014 by Wacker et al. and Röder et al.

We thank the German Federal Ministry for Education and Research - projects “NanoCombine” FKZ 13N11401/13N11402 and “MorphiQuant-3D” FKZ 13GW0044 - for financial support.

Fig. 1: a) 3D reconstruction of 20 super-resolution fluorescence images of serial sections (thickness about 70nm, section ribbon placed on ITO-coated glass coverslip). Labeled are the acetylcholine receptors of the postsynaptic membrane of a neuromuscular junction. b) Overlay of fluorescence reconstruction and SEM-SE image of one section.

Fig. 2: a) Secondary electron (SE) image of muscle tissue (mouse diaphragm, conventionally stained and embedded, 70nm thick sections on Si wafer). b) Equivalent backscattered electron image filtered to include 0-100eV loss electrons (filtered BSE, 1.5keV prim. energy). Both images were recorded at identical initial electron dose.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-8-O-1971 A new algorithm for 3D Reconstruction in Optical Projection Tomography

Michálek J.1, Čapek M.1
1Institute of Physiology, Prague, Czech Republic
michalek@biomed.cas.cz

Optical Projection Tomography (OPT) is a computed tomography (CT) technique at optical frequencies. OPT is suitable for samples from about 0.5 mm to 15 mm in size, which fills an important “imaging gap” between confocal microscopy (useful for smaller samples) and large-sample methods such as fluorescence molecular tomography (FMT), x-ray CT or microscopic magnetic resonance imaging (µMRI). OPT can function in both fluorescence and transmission modes.
2D fluorescence or absorption images (projections) of the samples are taken over 360 degrees obtained with a fixed rotational angle along the vertical axis, e.g. 0.9 degrees, i.e. 400 projections over 360 degrees.
A standard approach to 3D volume reconstruction from 2D optical projection tomography series uses the Filtered Backpojection algorithm (FBP), whose algorithmic foundation is the Radon Transform, first published in 1917. FBP was originally used in other types of tomography, such as X-ray Computed Tomography. Other reconstruction algorithms for OPT were reported rarely, and they are not available in standard reconstruction software.
FBP may yield unsatisfactory results in cases where there are large sudden local variations in the brightness of the tomographic projections, which is often the case in OPT. Fig.1 on the left shows one of a series of 400 fluorescence tomographic projections of a 3×3×3 mm3 block cut from a rat brain, acquired by optical projection tomography (exc/em - 425nm/from 475nm). The brain was stained by Lycopersicon esculentum (tomato) lectin. Prior to acquisition by optical projection tomography (OPT) specimens were cleared using BABB (1 part of benzyl alcohol + 2 parts of benzyl benzoate).
In Fig.1 on the right, one of a total of 503 reconstructed horizontal slices of the specimen is shown. Streak artifacts at the sharp edges of the slice, as well as the halo delimiting the CCD sensor range are well pronounced.
For specimens with large regions of almost constant optical density, quality of reconstruction can be greatly improved, and the number of needed projections reduced when a reconstruction algorithm is used which - apart from aiming at cancelling the error between measured and reconstructed data - minimizes the total variation (TV), i.e. the sum of absolute brightness changes in the reconstructed sections. We solved this problem using an alternating directions algorithm.
Fig.2 shows that using the total variation minimization algorithm reconstruction quality is acceptable for as few as 100 projections (i.e. a 3.6 degree step between projections), whereas FBP reconstruction deteriorates rapidly. This allows a much higher acquisition turnover, as well as significant disk space savings when repeated acquisitions are inevitable e.g. for OPT settings optimization.

The authors acknowledge funding from the Czech Republic’s public funds provided by Academy of Sciences (AV0Z50110509 and RVO:67985823), Ministry of Education, Youth and Sports (KONTAKT LH13028), and Science foundation of the Czech Republic (13-12412S).

Fig. 1: left: fluorescence tomography projection of a block cut from a rat brain, acquired by optical projection tomography. right: one section of 3D reconstruction of structures in this block by using the FBP algorithm of the Nrecon software package.

Fig. 2: The upper row of images shows how the quality of tomographic reconstruction by the FBP deteriorates fast when a limited number of projections are used for reconstruction. The lower row demonstrates the potential of total variation minimizing algorithms. Reconstruction quality is acceptable down to as few as 100 projections.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-8-O-2915 Validation of sorted subsets for single-particle reconstruction of heterogeneous macromolecular assemblies

Xu X. P.1, Volkmmann N.1
1Sanford-Burnham Medical Research Institute, La Jolla, USA
niels@burnham.org

In single-particle reconstruction techniques, it is essential that heterogeneous populations due to conformational variability or differences in occupancy are properly accounted for in order to get the best possible representations of the underlying structures. This task is often difficult and there is a shortage of tools to detect or correct for heterogeneity in sorted data subdatasets. We developed a novel protocol to validate the homogeneity of sorted subdatasets and to correct for residual heterogeneity if present. Our approach is based on what we call the ‘inverted Einstein principle’ where the test imposes maximum bias away from the refined structure. The underlying idea is that heterogeneity in the sorted subdatasets will give rise to model dependence. We construct a test for heterogeneity by providing a starting model with maximum bias away from the main features of the reconstruction. Only if the reconstruction remains unchanged within predefined error bounds, the test is considered passed. Otherwise, sorting is repeated using specific rules defined within our procedure until convergence is achieved. The rules for constructing the new set of models for sorting and refinement, allow the models to drift away from features that are variable and to enhance features that are solid within the current sorted subdataset. We will present results for simulated data as well as for several experimental datasets that suffered from high degree of heterogeneity, showing that our procedure succeeds in each of these to generate robust sorted subdatasets that do not exhibit model bias.

Funding was provided by NIH grant P01-GM066311

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-8-O-3284 Analysis of PSF of diffracting objects from z-stack bright field microscopy images of non-stained living cells

Stys D.1, Rychtarikova R.1
1University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Institute of Complex Systems, Zámek 136, 373 33 Nové Hrady, Czech Republic
stys@frov.jcu.cz

Living cell with interior is a multifractal system, and lens system of an optical microscope during experiments introduces other multifractal characteristics to the image which is consequently discretized by a camera chip. Because each multifractal (sub)object has its probability of occurrence in the image, we extracted the information content of the z-stack image of the cell by Renyi entropy gain approach – Point Divergence Gain (PDG). By this procedure, we determine, the change of information of the image of the cell by exchange of the particular data point between two following images in z-stack at different values of dimensionless parameter α. Then each component of the multifractal (sub)object have its own generalized spectrum, i.e., each of the PDG-values at each α has its occurrence in the image explained as particular image intensity.
For 3D reconstruction of cell interior we used PDG at α = 4 with the highest amount of the occupied intensity levels, i.e., highest number of separable groups of different information contribution. The 0th intensity level of PDG4,0 represents lowest change of information between two following images of the z-stack. This identifies part of the point spread function which (a) belongs to frequent objects and (b) does not change significantly between two consecutive images in the z-stack. Points at this PDG level were used for creation of a binary mask by which the part of the image at a given z-level belonging to a distinguishable organelle image was sectioned from the original RGB image. The properties of the course of real point spread function (PSF) were examined and a model of the organelle was prepared. In that, we considered that a larger object – organelle – is filled by elementary diffracting objects. Thus, as long as certain elementary objects reside in the focus, the change of the PSF along the z-axis remains similar. Outside this region, the behaviour of the PSF changes. At the same time, in the focus the image is the darkest. We have thus two indices which we may use for determination of the position and shape of the organelle: change in the size of the spot and its coloration.
Then, the focal plane of examined nucleolus of MG63 cell corresponds to the level of the PSF with the lowest mean G-intensity and local area minimum (Fig. 1B ). This focal plane for red and green channel is the same. The course of PSF in blue channel is different as we deal with the projection of fluorescent, not diffracting, objects. The nucleolus probably occurs in the region between local area maxima around this local area minimum (Fig. 1C). The other local minima and maxima likely correspond only to light interferences along the build-up of the image projection along the optical system of the microscope.

Results of the LO1205 project was obtained during financial support of MEYS CR in the frame of NPU I programme. This work was also financed by grants Postdok JU and GA JU 134/2013/Z.

Fig. 1: Analysis of PSF of nucleolus of MG63 cell. Course of PSF in the focal region of the cell (A), its analysis (B) and course of PSF in the expected occurrence of the nucleolus (C). The colorbar below the figures shows intensity in each colour channel in Fig. A and C.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-8-O-3291 STEM tomography of blood platelets in 1.5-µm thick sections

Aronova M. A.1, Sousa A. A.1, Pfeifer C. R.1, Shomorony A.1, He Q.1, Zhang G.1, Pokrovskaya I. D.2, MacDonald L.2, Prince A. A.2, Storrie B.2, Leapman R. D.1
1National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, MD, USA, 2Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA
leapmanr@mail.nih.gov

Electron tomography in the scanning transmission electron microscope (STEM) can be performed on µm-thick plastic-embedded specimens without effects of chromatic aberration because there are no imaging lenses after the specimen [1-4]. By using a small STEM probe convergence angle of 1–2 mrad the geometrical broadening of the probe is restricted, which enables a spatial resolution of a few nanometers. Furthermore, by using an axial bright-field detector instead of the standard high-angle annular dark-field detector, image blurring due to multiple elastic scattering can be reduced in the lower part of the specimen [2].

Here, we have applied STEM tomography to elucidate the 3D ultrastructure of human blood platelets, which are small anucleate blood cells that aggregate to seal leaks at sites of vascular injury and are important in the pathology of atherosclerosis and other diseases. Of particular interest are the morphological changes that occur in α-granules, which contain important proteins released when platelets are activated [5].

Electron tomograms were acquired using an FEI Tecnai TF30 (S)TEM equipped with a field-emission gun and operating at an acceleration voltage of 300 kV. The instrument was equipped with an axial bright-field STEM detector. Specimens were prepared by immediate paraformaldehyde fixation, followed by platelet purification, then either further chemical fixation with glutaraldehyde or high pressure freezing and freeze substitution with acetone/osmium tetroxide fixative, and finally dehydration, epon embedding, and U and Pb staining of the sections. Tomograms were reconstructed from dual-axis bright-field STEM tilt series using the IMOD program [6].

Although there is extensive overlap of structure in axial bright-field STEM images of 1.5-µm thick sections even at a tilt angle of 0° (Fig. 1A), it is possible to reconstruct blood platelets from dual-axis tilt series (Fig. 1B). Orthoslices through a tomographic reconstruction of cells oriented parallel to the section (Figs. 2A-C) and oriented perpendicular to the section (Figs. 3A-C) reveal ultrastructural features including the open canalicular system of membranes, mitochondria, and α-granules. The advantage of STEM tomography is that large structures within the platelets can be imaged in their entirety. Work is in progress to compare the ultrastructure of α-granules in resting and activated platelets.

[1] A.E. Yakushevska et al., J. Struct. Biol. 159 (2007) 381.

[2] M.F. Hohmann-Marriott, A.A. Sousa et al., Nature Methods 6 (2009) 729.

[3] A.A. Sousa et al., Ultramicroscopy 109 (2009) 213.

[4] A.A. Sousa et al., J. Struct. Biol. 174 (2011) 107.

[5] J. Kamykowski et al., Blood 118 (2011) 1370.

[6] J.R. Kremer, D.N. Mastronarde, J. Struct. Biol. 116 (1996) 71.

Research supported by the intramural program of NIBIB, NIH. Work in the Storrie laboratory was supported in part by NIH grant R01 HL119393.

Fig. 1: (A) STEM image of a 1.5-µm thick section of a platelet recorded at 0°-tilt showing superposition of structures; (B) dual-axis STEM tomographic reconstruction showing 3D volume.

Fig. 2: (A-C) Three orthoslices through STEM tomographic reconstruction of 1.5-µm thick section of human platelets showing cell oriented in plane of section: open canalicular system of membranes (ocs), mitochondria (m), α-granules (αg).

Fig. 3: (A-C) Three orthoslices through STEM tomographic reconstruction of 1.5-µm thick section of human platelets showing several cells oriented perpendicular to the plane of the section: open canalicular system of membranes (ocs), mitochondria (m), α-granules (αg).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-1641 RX-TomoJ : Multimodal hard x-ray scanning tomographic software

Bergamaschi A.1, Messaoudi C.2, Somogyi A.1, Medjoubi K.1, Marco S.2,3
1Synchrotron SOLEIL, St aubin, France, 2Institut Curie, Paris, France, 3Inserm U759, Orsay, France
antoine.bergamaschi@gmail.com

Hard X-ray scanning imaging allows simultaneous acquisition of multimodal information, by absorption, phase and dark-field contrasts, providing structural and chemical details of samples. Combining these scanning techniques with the infrastructure developed for fast data acquisition at Synchrotron Soleil1 permits multimodal tomographic imaging at the Nanoscopium 155m long beamline, which will be open to users in mid-2015. A main challenge of such imaging technique is the online processing and analysis of the important amount of generated multimodal data. To this purpose we are developing a plugin for imageJ2, that we named RX-TomoJ.
RX-TomoJ will offer state-of-the-art processing and reconstruction algorithms3, adapted for multimodal scanning tomography, tuned to run on conventional computers. The software takes advantage of the multimodality to extract useful information from one mode to improve the results obtained in the others modes. For instance, the fluorescence data can be used to retrieve the number of chemical elements which might be taken as the number of classes for discrete reconstruction algorithms. In addition, to minimize the damage that can be induced by the radiation on the sample, new approaches reducing the amount of data required to compute reconstruction will be integrated.
We will present the first version of RX-TomoJ which includes: Hdf54 format reading and data management, pre-processing tools, spectroscopic analyses tools and 3D reconstruction for different modalities. This is implemented in a user friendly interface (Figure 1), allowing the user to control and perform the entire framework of image reconstruction and analysis.

1Medjoubi k et al. 2013. “Development of Fast, Simultaneous and Multi-Technique Scanning Hard X-Ray Microscopy at Synchrotron Soleil.” Journal of Synchrotron Radiation 20 (2): 293–99.

2Schneider C A et al. 2012. “NIH Image to ImageJ: 25 Years of Image Analysis.” Nature Methods 9 (7): 671–75.

3Goris, B., W. Van den Broek, K.J. Batenburg, H. Heidari Mezerji, and S. Bals. 2012. “Electron Tomography Based on a Total Variation Minimization Reconstruction Technique.” Ultramicroscopy 113: 120–30. doi:10.1016/j.ultramic.2011.11.004.

4The HDF Group. Hierarchical data format version 5, 2000-2014. http://www.hdfgroup.org/HDF5.

Fig. 1: RX-TomoJ raw data processing interface. This interface is divided into two parts, the first (on the top) allows users to have a look on absorption and fluorescence raw data, and the second contains the computation of different modalities.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-2210 EFTEM-TomoJ : software for 3D chemical mapping by EFTEM and soft X-ray NEXAFS tomography

Messaoudi C.1,2, Aschman N.1,2, Cunha M.1,2, Oikawa T.3, Sorzano C. O.4, Conesa J. J.4, Chiappi M.4, Pereiro E.5, Chichón F. J.4, Carrascosa J. L.4, Marco S.1,2
1Institut Curie, Centre de Recherche, 26 rue d'Ulm, 75248 Paris Cedex 05, France, 2INSERM U759, Campus Universitaire d'Orsay, Bât. 112, 91405 Orsay cedex France, 3JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558, Japan, 4National Center of Biotechnology (CSIC), c/Darwin, 3. Campus Univ. Autónoma de Madrid. 28049 Cantoblanco, Madrid, Spain., 5CELLS - ALBA, Carretera BP 1413, de Cerdanyola del Vallès a Sant Cugat del Vallès, Km. 3,3 08290 Cerdanyola del Vallès, Barcelona Spain
sergio.marco@inserm.fr

Electron tomography is becoming one of the most used methods for structural analysis at nanometric scale in biological and materials sciences. Combined with chemical mapping, it provides qualitative and semiquantitative information on the distribution of chemical elements on a given sample. Due to the current difficulties in obtaining 3D maps by EFTEM, the use of 3D chemical mapping has not been widely adopted by the electron microscopy community. The lack of specialized software further complicates the issue, especially in the case of data with low SNR. Moreover, data interpretation is rendered difficult by the absence of efficient segmentation tools. Thus, specialized software for the computation of 3D maps by EFTEM needs to include optimized methods for image series alignment, algorithms to improve SNR, different background subtraction models and methods to facilitate map segmentation.
Here we present a software package (EFTEM-TOMOJ), specifically dedicated to computation of EFTEM 3D chemical maps and Soft X-ray spectroscopy datasets [1]. The software includes noise filtering by image reconstitution based on multivariate statistical analysis. We also present an algorithm named BgART allowing the discrimination between background and signal and improving the reconstructed volume in an iterative way. We have uses this software for the localization of iron aggregated in the fungi F. pedrosoi (figure 1).
An alternative to EFTEM is recover X-ray absorption near-edge structure (XANES) information using synchrotron X-ray sources can be used to determine the distribution and concentration of elements in biological materials. Images at photon energies corresponding to absorption maxima and minima in different components of a specimen can be acquired with this purpose. Moreover this spectromicroscopy approach can be combined with tomography yielding a quantitative 3D map of the distribution of the interesting element within the sample. Using EFTEM Tomo-J. We have 3D studied differential soft X-ray iron cellular uptake of 15 nm nanoparticles acquiring tilted series just below and at the L3 edge (figure 3). The objective was to obtain quantitative chemical 3D maps of the iron distribution inside whole MCF7 breast cancer cells.

References:
1. Messaoudi C. Aschman N. Cunha M. Oikawa T Sorzano COS. Marco S. (2013) EFTEM-TomoJ: 3D chemical mapping by EFTEM including SNR improvement by PCA and volume improvement by noise suppression during the ART reconstruction process. Microsc. Microanal. 28:1-9.

We thanks Dr. Sonia Rozental from the Universidade Federal do Rio de Janeiro (Brazil) for the kind donation of fungal samples

Fig. 1: Example of application of EFTEM-TomoJ for study iron aggregation on F. pedrosoi. Images correspond to the central planes sections of the iron 3D-maps before MVA (panel A), after MVA (panel B) and after combining MVA and reconstruction by BgART (panel C). Delimited regions correspond to those used to compute signal-to-noise ratio (SNR). Bar 400nm

Fig. 2: 3D iron chemical map by NEXASF processed with EFTEM_TomoJ. A) 0º image projection before iron K edge (700 eV). B) 0º image projection on iron K edge (710 eV). C) 0º Edge-Map ratio. D) Central slice of a SIRT reconstructed volume. E) Same slice of a BgART implemented in TomoJ.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-2436 Use of optic microscopy in the prediction of the rheological properties of pharmaceutical´s granulates.

Amado J. R.1, Escalona-Arranz J. C.1, Da Silva C. F.2, Prada A. L.1
1Departamento de Farmacia. Universidad de Oriente, Santiago de Cuba, Cuba., 2Laboratório de Biologia Celular, Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brazil.
franca@ioc.fiocruz.br

The rheological properties of pharmaceutical´s granulates have an elevated importance in the industry, because they condition granulates’ flow through the hopper. The external form of those particles is one of the most important characteristic that conditioned their rheological properties. That is why the objective of the present work is to develop mathematical models that correlated the rheological properties of pharmaceutical´s granulates with some aspects related with the external form of those particles, using optic microscopy. By the 3-D applications of the EdN-2 software were calculated the classic variables “shape form” and “factor volume” of ten different pharmaceutical´s granulates. They were prepared mixing diverse proportions of lactose and microcrystalline cellulose. Under laboratory conditions, it was determined experimentally the “flow factor” and the “angle of repose”, being correlated with the previous particle form variables. The same proportion of five granulates with better rheological properties were considered to prepared two groups of other five formulations that includes a new ingredient: a natural extract from leaves of Tamarindus indica L. or fruits of Cassia grandis L. The mathematical models obtained can predict the rheological behavior of the ten pharmaceutical granulates without the natural extracts as well as those consisted in the same active ingredient with more of 90% of accuracy, but fails when different natural extract granulated are considered.

VLIR-UO project “Biopharmaceutical products from natural sources in the development of biotechnology” and CAPES-MES.

Fig. 1: Microphotograph of cellulose-lactose granulescontaining a natural soft extract.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-2580 The Incoherent Image for Single Particle Reconstruction

Tsai C. Y.1, Chang Y. C.2, Van Dyck D.3, Chen F. R.1
1National Tsing Hua University, Hsin-Chu, Taiwan, 2Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, 3University of Antwerp, Antwerp, Belgium
fchen1@me.com

Virus particles, purify protein, cryo-section cells and organism tissues are radiation sensitive samples and are usually embraced in amorphous ice for TEM observation known as cryo-EM. Such biological images usually exhibit low contrast and presents low signal to noise ratio due to the low electron exposure. In conventional TEM, large defocus or negative stain is necessary to gain the contrast. However, image delocalization arising from large defocus impedes is one of the key factor for the high spatial resolution for biological samples.

This research has proposed a new method to enhance the contrast in biological molecule, named incoherent image. The thermal diffuse scattering(TDS) electrons are the main contribution to formation of the incoherent image. The maximum intensity of TDS for soft material is in 1Å-1[1]. The TDS depends on the atomic number (Z), which is also called Z-contrast image. In this research, we use set of deflector coils to deflect the beam twice before illuminating on the specimen and changes the illumination azimuthal angle from 0 to 2π. By selecting the specific tilting angle and objective aperture, hollow cone illumination can collect the maximum intensity of TDS. The proof of the concept of the hollow cone illumination has been demonstrated experimentally for negative stained Chaperonin GroEL as a standard protein since it is stable, easy to obtain and the structure is well-known[2]. The three dimensional structure was reconstructed with single particle analysis in EMAN2.

As shown in Fig. 1(a), the hollow cone dark field image (HCDF) of negative stained GroEL was taken by a FEI TW20 Twin field emission gun transmission electron microscopy and a Gatan 4k by 4k CCD. In order to compare with the hollow cone dark image visusally and quantitatively, the contrast of bright field image has been inverted, as shown in Fig. 1(b). Fig. 2 shows the line profile of the same particle imaged with two individual modes, the bright field in focus mode and the hollow cone dark field mode. In conventional bright field mode, the contrast can be enhanced by defocusing the image. The contrast of soft material is relatively low at in-focus. Since the HCDF image only collects the TDS electron, it is found that the contrast is nine times as higher as that of an in focus image as shown in the line profiles in Fig. 2. And the 3D structural model of the GroEL was reconstructed by using a standard single particle analysis code.

Reference

[1] Dirk Van Dyck, Ultramicroscopy 111 (2011) 894.

[2] Wah Chu. et al., Structure 12 (2004) 1129

This work was supported by National Science Council (NSC102-2321-B-007-007 and NSC101-2221-E-007-063-MY3).

Fig. 1: (a) the HCDF image of negative stain GroEL. (b) the in focus bright field image with the contrast invert.

Fig. 2: The line profiles of individual GroEL particles with three different image mode, in focus (black line) and HCDF image (red line).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-2823 Three-dimensional animated reconstruction in the study of actin cytoskeleton rearrangement in the amoebae of the genus Entamoeba (E. histolytica and E. dispar) by laser scanning microscopy

Talamás-Lara D.1, González-Robles A.1, Chávez-Munguía B.1, Salazar-Villatoro L.1, Talamás-Rohana P.1, Martínez-Palomo A.1
1Department of Infectomics and Molecular Pathogenesis, Cinvestav, ZIP code 07360, México, D.F.
daniel_talamas@hotmail.com

Different species of Entamoeba perform similar functions such as adherence, cytolysis, and phagocytosis. E. histolytica and E. dispar share the same processes although the former is pathogenic while the second is non-pathogenic. Systematic comparison between both is an important research area. The ability to ingest microorganisms and target cells is associated with amoebic pathogenesis, and in this process, the binding of the parasite to the target cell is mediated by electrostatic forces and then, mechanisms that involved surface molecules begin to engage (Sateriale, A., et al., 2012). This binding leads to the activation of signaling pathways that promote reorganization of the actin cytoskeleton (Talamás-Rohana, P. and Ríos A., 2000).
The objective is to analyze the dynamics of the actin cytoskeleton between E. histolytica and E. dispar.
Entamoeba histolytica HM1-IMSS and Entamoeba dispar SAW 760 RR strains were used. Trophozoites were incubated on glass coverslips coated or uncoated with fibronectin (FN) at different times (15, 30, and 60 min). Parasites were fixed with 4% paraformaldehyde, permeabilized, and stained with rhodamine-conjugated phalloidin (Wehland, J., et al., 1977). Tracking of the restructuring of the actin cytoskeletal was done by laser scanning microcopy using a Carl Zeiss LSM 700 microscope.
Actin polymerization of Entamoeba histolytica begins at early times, both in glass and FN. Conversely, Entamoeba dispar presents a more limited and slower polymerization process. The structuring of actin in E. histolytica is induced in areas extremely close to the site of contact with the substrate, showing nucleation points, stress fibers and phagocytic structures; in comparison, E. dispar, even at longer times, shows a limited structuration of the actin cytoskeleton, as the fluorescent label is observed as amorphous clusters distributed throughout the cell volume without specific locations. Z-stack analysis, ortho-cuts, and quantification of fluorescence intensity allowed delimitation of specific sites where the re-structuring of filamentous actin is occurring in both species. To show with more detail, the actin structures formed as well as their location within the cell, we processed all optical images from “Z” cuts to achieve a three-dimensional animated reconstruction.
Manifestation of the pathogenic behavior requires cytoskeletal dynamics as this allows parasite movement and tissue penetration. Therefore, results obtained in this work let us conclude that differences in the pathogenic behavior of both species of amoeba might be due, at least in part, by the lower efficiency of the E. dispar actin cytoskeleton to reorganize in response to extracellular signals.

The authors are grateful to Verónica Hernández for their help with confocal studies; Anel Lagunes, César Salas, and Judith Escobedo for their technical support. This research was supported by the PNPC 2013 Program from CONACyT, México. 

Fig. 1: Actin structuring kinetics between E. histolytica and E. dispar adhered to FN. Figure 1 shows E. dispar with a cortical structure at 15 min. However, a dramatic change at 15 min in E. histolytica was seen and at 60 min, most amoebas seen in the field have well-defined new structures. E. dispar shows no significant changes even at longer times.

Fig. 2: Adherent comparison between E. histolytica (15 min) and E. dispar (60 min) using FN substrate. Figure 2A shows that structured actin of E. histolytica is totally redistributed on contact with substrate (FN), while in figure 2B, structured actin in E. dispar is redistributed through all cytoplasmic volume (Nuclei (blue) were stained with DAPI).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-3228 Algorithm for extraction of diffracting objects' PSF from z-stack of bright-field microscopy images of living cells

Rychtarikova R.1, Stys D.1
1University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Institute of Complex Systems, Zámek 136, 373 33 Nové Hrady, Czech Republic
rrychtarikova@frov.jcu.cz

We bring a segmentation algorithm for diffracting objects observed by bright-field microscopy (Fig. 1), where a binary mask for extraction of the studied objects is created by analysis of changes of information between consequent images in the z-stack. This may be in fact done by simple level-by-level intensity comparison, but the information analysis brings on top of that image point classification. Here we report information remaining unchanged at 0th digital level of green channel of two consequent PDG-subtracted images (inputs 1). In these PDG-images, borders of the objects are highlighted by subtracting of two following images in z-stack of the cell based on Renyi information entropy concept – Point Divergence Gain (PDG):
                                     PDGα,x,y = 1/(1-α)*(ln⁡∑i=1n pi,x,yα - ln⁡∑i=1n pi,x(l+1),y(l+1)α),
where pi,x,y is the probability density function of the intensity with the examined point of coordinates x, y in the previous image number l, pi,x(l+1),y(l+1) is the probability of the occurrence of the given intensity in the image where the examined point of coordinates x, y was replaced by the point at the same location in the following image number l + 1, α = 4.0 is a dimensionless coefficient, and n is a number of intensity levels (n = 256 for an 8-bit image). For each PDG-subtracted image, two images were obtained – with positive and negative change of PDGα,x,y value. Thus, information of each original image is saved in four parallel PDG-subtracted sub-images. In green channel of PDG-images, we can observe diffracting objects. In the blue channel we observe mainly the fluorescence and in the red channel the contribution of near infra-red absorption is observed. At the 0th PDG level of each channel, large objects with the minimal change of the point spread function between z levels are visible. Another intensity levels corresponds to diffraction response of less observed objects, objects surroundings as well as other parts of the point spread function belonging to large organelles.
After creation of the binary mask from PDG-images, small objects (pixels) are removed by morphological operation opening (ROI).
Fluorescent organelles and Airy discs, which are also projected at 0th intensity level of the green channel of PDG-images due to no change of information in two following microscopy images, were removed by elimination of the intensities higher than the intensity mode of the background (input 3) from the image of the cell alone (input 2).
Finally, the diffracting objects were segmented (output) by application of the binary mask to the image of the cell with deleted fluorescent objects and Airy discs. The remaining points in the segmented RGB image where removed by combination of morphological operation erosion and dilation.

Results of the LO1205 project was obtained during financial support of MEYS CR in the frame of NPU I programme. This work was also financed by grants Postdok JU, GA JU 134/2013/Z, and TA ČR TA01010214.

Fig. 1: Scheme of image processing for segmentation of diffracting objects from a bright-field microscopic image of cells.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-5749 3D atomic tomography from HRTEM –CeO2

Chen L. G.1, Kirkland A. I.2, Van Dyck D.3, Chen F. R.1
1Engineering and System Science Department, National Tsing Hua University, Taiwan, 2Department of Materials, University of Oxford, United Kingdom, 3EMAT, University of Antwerp, Belgium
liu31448@hotmail.com

Nowadays, various advance TEM hardware like Cs corrector and monochromator have been developed with sub-Å resolution. But, we still only get the two-dimensional projections from three-dimensional sample from an aberration corrected TEM. To resolve the lost information along thickness direction, TEM tomography is becoming one critical and powerful technique for analysis of three-dimension structure in materials science. (1)

<span>In this report, we demonstrate to determine the three-dimensional shape at atomic resolution positions of a nanocrystalline specimen CeO2 from an exit wave via wave back propagation and Big-Bang schemes.(2) Exit wave reconstructed from a focal series of HRTEM images of CeO2 is shown in Fig.1.<span>By back propagating the wave, the position of atomic columns can be decided. The maximum amplitude of the wave reveals the original position of the atomic columns. Next, the true positions of Ce and O atomic columns in the exit surface can be refined with Big-Bang scheme. As we can see in Fig.2,the focal maps show the de-focal values from the exit wave to atomic columns of Ce and O atoms.

<span> After the vertical positions of atoms in 3D Space are retrieved by Wave back-propagation and refined by the Big-bang theory, the thickness of atomic columns in unit of number of atom could be quantified by phase of exit wave. Once the thickness of sample known, then 3D tomography with atomic resolution can then be reconstructed. Based on these methods, three-dimensional reconstructed model with atomic-resolution was proposed.

This work was supported by National Science Council (NSC 101-2221-E-007-063-MY3).

Fig. 1: Figure1. Phase of CeO2 exit wave

Fig. 2: Figure2. Three-dimensional focal map of CeO2
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-5863 Improving temporal resolution of 3D localization microscopy using sparse support recovery

Ovesný M.1, Křížek P.1, Švindrych Z.1, Hagen G. M.1
1Institute of Cellular Biology and Pathology, Prague, Czech Republic
martin.ovesny@lf1.cuni.cz

Single molecule localization microscopy (SMLM) represents a family of methods for super-resolution imaging of fluorescence samples and is capable of very high spatial resolution. These methods separate fluorescent molecules into a sequence of hundreds or thousands of images of sparsely distributed, stochastically photo-activated molecules so that single molecules can be localized with a precision which is not limited by diffraction. The final super-resolution image is then reconstructed from all single molecules localized throughout the whole sequence.

Although SMLM methods result in high spatial resolution, they are not always suitable for live cell imaging due to limited temporal resolution. We present an algorithm for localization microscopy which is able to recover full 3D super-resolution images from a sequence of diffraction limited images with high densities of photo-activated molecules. The algorithm is based on compressed sensing and uses a Poisson noise model, a critical factor in low-light conditions. For 3D imaging we use the astigmatism method, however, the algorithm is flexible enough to allow 3D localization of molecules by other approaches, such as dual-objective, biplane or double helix methods. We demonstrate that the method performs well in low-light conditions and with high molecular density which makes it suitable for fast image acquisition in densely labeled samples. In addition we derive the upper bound of resolution of the method using tools from compressed sensing.

This work was supported by the Grant Agency of the Czech Republic [P205/12/P392, P302/12/G157 and 14-15272P], by Charles University in Prague [Prvouk/1LF/1, UNCE 204022], and by European Union Funds for Regional Development [OPPK CZ.2.16/3.1.00/24010].

Fig. 1: Comparison of (a) conventional SMLM processing and (b) our new algorithm on a sequence of 500 images of densely labeled tubulins (courtesy of Nicolas Olivier and Debora Keller, LEB, EPFL). Note the arrows which indicate high density areas which are not adequately resolved by the conventional algorithm.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-5876 Immersive visualisation of three-dimensional microscopy images using Oculus Rift headsets and the Unity3D game engine

Stephenson L. T.1, Ceguerra A. V.1, Demont R.2, Foley M.1, Ringer S. P.1
1Australian Centre for Microscopy and Microanalysis, The University of Sydney, Australia, 2École Nationale Supérieure de Mécanique et d'Aérotechnique, Pontiers France
leigh.stephenson@sydney.edu.au

Three-dimensional microscopy data offers a natural representation of material science and life science samples. Such data can be examined from all angles and often provides morphological and volumetric information without relying upon 2D stereological analysis. Using the recent innovation of the Oculus Rift (TM) headsets, we attained novel perspectives of 3D data sets usually unattainable with conventional displays that do not employ some otherwise stultifying visual trick e.g. using colour anaglyphs. Using dual Oculus Rift headsets driven by the same Unity3D software, two perspectives can be generated in tandem or with independent controls for shifting the viewpoint and manipulating the visualised data. The Unity3D engine is usually used for gaming but was found to be a versatile and accessible tool in exploiting the sophisticated rendering capabilities available in affordable laptop and desktop computers. Interfacing the engine with compiled C# scripts, it was possible to produce impressive microscopy visualisations that can either be viewed on a standard screen or through the binocular Oculus Rift headsets. Using dual Oculus Rift headsets driven by the same Unity3D software, two perspectives of the same atom-probe data can be generated in tandem or with independent controls for shifting the viewpoint and manipulating the visualised data. Providing examples, we examined the early applicability of this technology for various microscopy applications (atom probe, xray CT, EM tomography) and discuss its current limitations and any future potential as a serious research tool and novel education platform.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-5965 Delivery system, based on Internal Self-Assembled nano-structures

Demurtas D.1, Guichard P.2, Hebert C.1, Sagalowicz L.3
1Interdisciplinary center of electron microscopy (EPFL) Lausanne ( Switzerland), 2UPGON, EPFL, Lausanne ( Switzerland), 3Nestlé Research Center, Lausanne (Switzerland)
dav.bio@bluewin.ch

Amphiphilic lipids can self- assemble into a variety of structures.1 In particular, the inverse bicontinuous cubic phases and their applications and role in nature have generated strong interest.2-4 It has been shown that macroscopic properties of lipid structures depend on their fine structures. Therefore, it is of prime interest to have methods for determining those structures.
The Cryo-TEM signified a breakthrough in the imaging of hydrated specimens allowing their visualization close to native state.
In this study we have been performing Cryo-electron tomography (CET) onto these particles and characterization of a delivery systems, based on Internal Self-Assembled (ISA, Nestlé proprietary delivery systems) structures.
3D tomograms of cubosomes were obtained and this study confirms the proposed structure for cubosomes and provides novel insights in the understanding of the interface maturation.

1. Larsson, K., Cubic Lipid-Water Phases: Structures and Biomembrane Aspects. The Journal of Physical Chemistry 1989, 93 (21), 7304-7314.
2. Sagalowicz, L.; Leser, M. E.; Watzke, H. J.; Michel, M., Monoglyceride self-assembly structures as delivery vehicles. Trends in Food Science and Technology 2006, 17, 204-214.
3. Almsherqi, Z. A.; Kohlwein, S. D.; Deng, Y., Cubic membranes: a legend beyond the Flatland of cell membrane organization The Journal of Cell Biology 2006 173, 839-844
4. Siegel, D. P.; Epand, R. M., Effect of influenza hemagglutinin fusion peptide on lamellar/inverted phase transitions in dipalmitoleoylphosphatidylethanolamine: implications for membrane fusion mechanisms. Biochim. Biophys. Acta 2000, 1468, 87-98.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-5990 Progress in development of Eos/PIONE for distributed image analysis system of electron microscographs

Yasuanga T.1, 3, Ushijima M.1, 2, 3, Yamaguchi K.2, 3, Watano S.1, Tsukamoto T.1, Iwasaki A.1, Tsuruta T.1
1Kyushu Institute of Technology, Iizuka, Japan, 2JST, SENTAN, 3NaU Data Institute Inc.
yasunaga@bio.kyutech.ac.jp

  Image Processing and analysis is one of the most essential techniques to elucidate the relationship between structure and function in organisms in the life science fields. For instance,these techniques can give us the three-dimensional structure from series of 2D projection images of electron micrographs and identification of interesting structure. Recently the situations have been changed into the atomic resolved structure from sub-millions of 2D images and the new types of image detection devices, such as direct detectors. Thus the more computational resources can give us the more detailed structure. Many types of applications have been proposed and developed in progress to do so but ach of them still isn’t conclusive.

  We have also been developing an integrated environment for image processing and analysis for electron micrographs, which is called as Eos, Extensible-object oriented system for electron micrographs. We have already implement more than 400 small programs and thousands of application program interfaces, some of which GPGPU or pthread techniques can be applied to for fast processing.  Eos have been developed as an open-source  the SOURCEFORGE, https://sourceforge.jp/projects/eos/.

  Moreover, we have developed a platform to more effectively and fast process the complex tasks such as image analysis, e.g., single particle analysis, electron tomography, etc., under distributed and hetero computer resource, which is called as PIONE, Process-rule for INPUT/OUTPUT Negotiation Environment. This is a novel rule based workflow engine using forward chaining algorighm, similar to production rule systems. The defined rules for small image analysis can be executed adequately following the presence and modified times of INPUT/OUTPUT files. The rule-based processes and many types of computers can be linked by the attributes of ‘feature’ and they can work under the appropriate computers among hetero sets of computers including computers with many cores, GPGPU-based computers etc. We have implement several kinds of integrated solutions such as single particle analysis, electron tomography and so on.  In addition, we have develped a front-end for image processing using PIONE, pione-client and pione-webclient, which is a web-browser based one using AJAX techniques. PIONE have also been developed as an open source under GITHUB, https://github.com/pione/.

  Here we introduce the progress of development of Eos and PINOE and expect the feedback from uses, experts and developers for image analysis.

This development was supported by JST, SENTAN.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-8-P-6053 Three-dimensional electron diffraction of cyclic twinned nanowires

Fu X.1, Yuan J.2
1General Research Institute for Nonferrous Metals, Beijing 100088, PR China, 2Department of Physics, University of York, York YO10 5DD, UK
jun.yuan@york.ac.uk

Identification and characterization of complex nanostructures have always been a challenge. Real-space imaging through transmission electron microscopy is the conventional tool of choice to study the internal structure of materials, but it cannot be realized routinely with atomic resolution even using state-of-the-art aberration corrected microscopic techniques because of the large dimensions of the nanostructures involved in many cases. In general, the atomic resolution electron microscopy is also a destructive structural characterization technique for complex nanostructures, due to the need for preparing ultrathin cross-sectional samples, yet the knowledge on the structure of intact complex nanostructures maybe important as it could be the key behind the enhanced physical properties of the nanostructures. Non-destructive characterization of the nanostructures can be achieved through real-space electron tomography, however it often suffers from a ‘missing wedge’ problem [1]. In this paper [2], we explore the inverse relationship between the real-space nanostructrue and their diffraction to demonstrate an non-destructive study of the internal structure of a boron-rich nanowire by reconstruction of the three-dimensional diffraction. Here we will show that the analysis of 3D diffraction intensity distribution allows us not only to identify the cyclic twinned structure directly, but the technique also reveals quantitatively the orientation relationship of the internal crystallites and information about deformation and defects associated with the internal strain relaxation, all non-destructively.

[1] P. A. Midgley and R. E. Dunin-Borkowski (2009) Nat. Mater., 8, 271–280

[2] X. Fu and J. Yuan (2013) Nanoscale, 5, 9067-9072

X. Fu would like to thank National Natural Science Foundation of China for the research support (grant no. 51201015). J. Yuan would acknowledge the support of EPSRC (EP/G070474/1) and Royal Society Wolfson Foundation Laboratory Refurbishment Grant. The authors would like to thank Dr Z. Y. Yu and C. Liu for providing boron-rich nanowire samples.

Fig. 1: (a) The intensity distribution around (112) and (113) reflections from a boron carbide five-fold twinned nanowire shown at the lower left corner. The magnified view of the 3D (b) distribution of the (112) reflections (b) and the projected intensity (b) in the Ω plane defined in (a) compared with the calculated 2D intensity map (d).
↑ Go to the Table of content ↑

ID-9. Microscopic image analysis and stereology

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-9-IN-1994 Quantitative microscopy – extracting relevant information from biomedical image data

Wählby C.1
1Centre for Image Analysis, Dept. of Information Technology and Science for Life Laboratory, Uppsala University, Sweden, and Imaging Platform, Broad Institute of Harvard and MIT, Cambridge, MA, USA
carolina@cb.uu.se

Microscopes have been used for more than 400 years to understand biological and biomedical processes by visual observation. Although science is the art of observing, science also requires measuring, or quantifying, what is observed. Research based on microscopy image data therefore calls for automated methods for quantitative, unbiased, and reproducible measurements. An automated approach is further motivated by the development of scanning microscopes and digital cameras that can capture image data in multiple spatial-, time-, and spectral-dimensions, making visual assessment cumbersome or even impossible.

High-throughput screening (HTS) is a technique for searching large libraries of chemical or genetic perturbants, to find new treatments for a disease or to better understand disease pathways. We present one such study where we characterize cancer stem cells (CSCs) by quantitative microscopy, searching for therapeutically relevant regulatory differences between patients. The aim is to elucidate mechanisms of action and enable accurate targeting of disease subgroups. Modeling disease by culturing cells allows for efficient analysis and exploration. However, many diseases and biological pathways can be better studied in whole animals—particularly diseases that involve organ systems and multicellular interactions, such as metabolism and infection. The worm Caenorhabditis elegans is a well-established and effective model organism, used by thousands of researchers worldwide to study complex biological processes. Samples of C. elegans can be robotically prepared and imaged by high-throughput microscopy, and we show how novel image-analysis algorithms are capable of scoring phenotypic changes in high-throughput assays of C. elegans. In particular, we show how computational methods can identify novel anti-infectives as well as genes involved in fat metabolism.

Finally, detection, diagnosis, and severity grading of cancer are traditionally based on the visual examination under a microscope of histopathological tissue samples. The current transition from visual examination of glass slides under microscopes to whole slide scanners and computer-aided image analysis, i.e., digital pathology, holds the promise of more objective cancer grading that will lead to better prognostication while at the same time reducing the pathologist’s workload. We present recent advances in detection and spatial mapping of biomedical markers that may improve prognostication in the future.

Thanks to the research groups of Nelander and Nilsson, SciLifeLab Sweden, Ruvkun and Ausubel, MGH, Boston, MA, and colleagues at Uppsala University and the Broad Institute.

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-9-IN-5790 Characterization of advanced biomaterials and tissue engineered constructs using 3D and 4D X-ray tomography

Kurzydlowski K. J.1, Jaroszewicz J.1, Swieszkowski W.1
1Faculty of Materials Science and Engineering, Warsaw University of Technology 141 Woloska Str., 02-507 Warsaw, POLAND
wojciech.swieszkowski@inmat.pw.edu.pl

X-ray tomography becomes a widely applied tool in science and industry, with a particularly strong position in the biomedical fields. The tomography techniques show high potential in study of advanced biomaterials, both synthetic and natural. Micro and nano tomography can be used to complement existing 2D characterization methods to image specimens at the micro/nano scale, nondestructively in 3D. With such tool, it is possible to characterize newly developed biomaterial, degradation process of biodegradable materials, tissue growth into biomaterial and in situ mechanical behavior of biomaterial. The aim this study was to show how 3D and 4D imaging techniques combined with advanced image analysis could be used to characterize novel biomaterials and scaffolds for tissue regeneration. 3D imaging technique which combines X-ray absorption microtomography (uCT) and X-ray diffraction microtomography (XRD-CT) was used to examine the in vitro degradation process of polycaprolactone–based composites. The complementary information on phase distribution, changes in crystallinity and morphology during degradation process was obtained by means of image analysis of combined uCT/XRD-CT scans. The conventional uCT method was applied to examine the in vitro tissue ingrowth into biodegradable scaffolds at varying resolutions (Fig.1). Imaging of the scaffold/tissue composite at a scale of 5 mm and resolution of 5 um allows characterization of the complex ingrowth of tissue into the biomaterial. Further imaging at 1 um resolution allows for quantitative analysis of cell-tissue fine structure. These results illustrate the benefits of tomography over traditional 2D imaging techniques for the description of tissue morphology and interconnectivity. The combination of microfocus X-ray computed tomography and in-situ mechanical loading allowed us to quantify the scaffolds morphology prior and during loading, to verify the mechanical properties, to analyze fracture behavior, and to estimate the internal local deformation distributions.

The authors would like to acknowledge the following sources of funding: Polish National Science Centre (DEC-2012/07/D/ST8/02606) and BIO-IMPLANT (POIG.01.01.02-00-022/09).

Fig. 1: Polymeric scaffold seeded with Mesenchymal Stem Cells
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-9-O-2047 Mapped Textons for Tissue Microarray Classification in Digital Pathology

Fernández-Carrobles M. M.1, Bueno G.1, García-Rojo M.2, Déniz O.1, Salido J.1
1VISILAB, Universidad de Castilla-La Mancha, Spain, 2Dpto. Anatomía Patológica, Hospital General Universitario de Ciudad Real, Spain.
MMilagro.fernandez@uclm.es

Breast microscopy images contain large amounts of textures which can be used as a discriminative way to distinguish benignant or malignant breast tissue. For that reason, the selection of a valuable descriptor is essential for later tissue classifications. Frequential textons have been selected in this study to represent the texture of breast Tissue Microarray (TMA) images. Textons are textural descriptors of the spectral model. A definition of textons would be expressed as the repetitive features of the textures which humans can distinguish. Therefore, the texton concept assumes the existence of different textural components in the same texture. In our study, we used textons as discriminators to detect cancer or other sorts of tissue anomalies. Textures were represented by texton maps.

A data set of 628 microscopic images acquired by a whole-slide imaging system at 10x was selected and divided into four classes (see Figure 1).

The images are filtered by a MR8 filter bank composed by a set of filters: a Gaussian, a LoG, 18 edge and 18 bar filters with 3 basic scales and 6 orientations respectively. The MR8 filter bank is applied over the tissue images and 38 response filters are extracted. Each pixel is represented by a 38 dimensional vector. Then, a k-means clustering algorithm is applied over all the pixel vectors. Each new group extracted from the k-means algorithm is characterized by a representative vector called texton. Finally, 60 textons were selected for each class so a total of 240 textons composed the texton vocabulary. Once the texton vocabulary was extracted, each tissue image is represented by their texton histogram and its map is created (see Figure 2).

Features are extracted calculating the Haralick coefficients on the texton maps. We also considered the influence of color on the TMA images. Therefore, each feature set was extracted from eight different color models: RGB, CMYK, HSV, Lab, Luv, SCT, Hb and Lb. A total of 241 features were obtained for each color model, that is, a maximum of 1928 features were handled. Therefore we proposed a dimensionality reduction which has been performed with a correlation threshold of 97%.

The classification tests were carried out with 5 different classifiers: Fisher, SVM, random forest, bagging trees and Adaboost. The best results were obtained with AdaBoost and combination of all color models. The classification was tested by 10fcv.

We obtained 95% accuracy with 92% precision. Therefore, it is shown that texton maps are suitable to classify breast TMA images. The best results were obtained by using all color models and applying a feature correlation analysis (see Figure 3). The computational time was also reduced from 493 to 362 seconds without and with dimensional reduction respectively.

The authors acknowledge partial financial support from the Spanish Research Ministry Project TIN2011-24367 and from the EU Marie Curie Actions, AIDPATH project (num. 612471).

Fig. 1: H&E TMA images divided into four classes. Class 1) benign stromal with cellularity. Class 2) adipose tissue, Class 3) benign and benign anomalous structures: sclerosing and adenosis lesions, fibroadenomas, tubular adenomas, phyllodes tumors, columnar cell lesions and duct ectasia. Class 4) ductal and lobular carcinomas.

Fig. 2: Texton Maps. The texton histogram is created when all image pixels are classified by their nearest texton. The texton map is a representation of the image through their texton histogram. Each pixel in the new image is represented by a texton and each texton is represented by a color.

Fig. 3: AdaBoost classification error with and without dimensional reduction. The color models combination improve the classification results but also increases the size of the feature set. Feature reduction by correlation allowed a reduction of 79.2% of the initial features (from 1928 to 409 features) and improves accuracy up to 1.2%.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-9-O-2596 Automatic computation of emphysema maps on histological lung samples of treated mice

Marcos J. V.1, Muñoz-Barrutia A.2, Ortiz-de-Solorzano C.2, Cristobal G.1
1Instituto de Óptica, Spanish National Research Council (CSIC), Madrid, Spain, 2Cancer Imaging Laboratory, Center for Applied Medical Research, University of Navarra, Pamplona, Spain
jvmarcos@gmail.com

Introduction

Emphysema manifests as a component of chronic obstructive pulmonary disease (COPD), which is expected to be the third most common cause of death in 2020. Animal models such as elastase-induced emphysema mice have been used to study COPD. In this context, a method for a reliable quantification of emphysema lesions is required to evaluate the stage of the disease and the effect of treatments. The second moment of the equivalent diameter variable (D2) has shown a robust behaviour in emphysema quantification since it does not depend on the shape of the airspace and is sensible to a heterogeneous distribution of them1. However, the value of D2 by itself does not provide information about the degree of emphysema severity.

Data

All experimental protocols involving animal manipulation were approved by the University of Navarra Experimentation Ethics Committee. Lung lobe sections (H&E stained) from control and emphysema-treated mice were obtained using an automated Axioplan 2ie Zeiss microscope (Carl Zeiss, Jena, Germany). Each slide was initially acquired with a Plan-Neofluar objective (numerical aperture NA = 0.035, magnification 1.25x, pixel resolution 3.546 μm/pixel). The images of fourteen lung sections were available for the present study: 12 training images (6 control and 6 treated) and 2 test images (1 control and 1 treated). A total of 399 patches (751x751 pixels) were extracted from the training sections: 190 from normal (N) and 209 from emphysematous (E) tissue areas of control and treated mice, respectively.

Methods

Given a lung lobe image, semi-automatic segmentation based on morphological operators was applied to identify parenchyma pixels. Subsequently, a 751x751 window was centred on each pixel and the corresponding value of D2 was computed. A Bayesian approach was used to map a D2 value onto a probability index indicating emphysema severity. The posterior probability of being emphysematous was obtained as p(E|D2) = p(D2|E)p(E)/p(D2), where p(D2) = p(D2|E)p(E) + p(D2|N)p(N). The density functions p(D2|E) and p(D2|N) were obtained from samples in the training set using the Parzen’s method.

Results

Figures 1 and 2 show the emphysema map of the two lung sections in the test set (higher intensity corresponds to higher probability of emphysema). The percentage of parenchyma labelled as emphysematous, i.e., p(E|D2) > 0.5, was 17.9% and 83.3% for the control and the treated mice, respectively.

Conclusion

The proposed method could serve as an objective tool for the evaluation of emphysema severity in the context of COPD study.

[1] Parameswaran H, Majumdar A, Ito S et al., “Quantitative characterization of airspace enlargement in emphysema,” J. Appl. Physiol., 100, 186-193 (2006).

J. V. Marcos is a “Juan de la Cierva” research fellow (Spanish Ministry of Economy and Competitiveness).

Fig. 1: Left: Original lung lobe section from a control mouse. Centre: Segmented lung for the identification of parenchyma pixels. Right: Emphysema map using the proposed Bayesian inference approach.

Fig. 2: Left: Original lung lobe section from a treated mouse. Centre: Segmented lung for the identification of parenchyma pixels. Right: Emphysema map using the proposed Bayesian inference approach.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-9-O-2737 Quality control in cell image segmentation and tracking using simulated cell images

Kozubek M.1, Svoboda D.1, Ulman V.1
1Centre for Biomedical Image Analysis, Masaryk University, Brno, Czech Republic
kozubek@fi.muni.cz

Cell imaging using optical microscopy still faces the problem of the quality of cell image analysis results. The majority of acquired 2D as well as 3D cell image data are typically of not very good quality due to degradations caused by cell preparation, optics and electronics. That is why image processing algorithms applied to this data often yield imprecise and unreliable results. As the ground truth (GT) for given image data is typically not available the outputs of different image analysis methods can be neither verified nor compared to each other. This problem can be partially solved by estimating GT by experts in the field. However, in many cases such GT estimate is very subjective and strongly varies among different experts.


In order to overcome these difficulties we have created a toolbox that can generate 3D models (so-called digital phantoms) of artificial biological objects (primarily cell nuclei, see Fig. 1) along with their corresponding images virtually degraded by specific optics and electronics (using simulation of optical system and electronic detection, see Fig. 2). Image analysis methods can then be tried out on such synthetic image data. The analysis results (such as segmentation or tracking results) can be compared (e.g., using a difference) with GT derived from input models of a particular type of objects. In this way, image analysis methods can be compared to each other, their quality can be evaluated and their development can be fostered.


We started our development with simulations of cell nuclei of HL60 cells (to model simple isolated round objects) and cell nuclei of granulocytes (to model complex shapes) [1]. Then we paid attention to clusters of cells and their formations in tissues [2]. Finally, we started working on time-lapse simulations [3]. Recently, we have also generated special benchmark data sets of simulated 2D as well as 3D time-lapse series that were employed in Cell Tracking Challenges at International Symposium on Biomedical Imaging in 2013 and 2014 [4]. In this way, several state-of-the-art image segmentation and cell tracking methods could be compared [5]. The simulation toolbox is freely available on Internet and is accessible via simple web interface [6].

 

References:

[1] D Svoboda, M Kozubek and S Stejskal, Cytometry 75A (2009), p. 494-509.
[2] D Svoboda, O Homola and S Stejskal, Proceedings of 8th International Conference on Image Analysis and Recognition, LNCS 6754 (2011), p. 31-39.
[3] D Svoboda and V Ulman, Proceedings of 9th International Conference on Image Analysis and Recognition, LNCS 7325 (2012), p. 473-482.
[4] http://www.codesolorzano.com/celltrackingchallenge
[5] M. Maška et al., Bioinformatics (2014), published online, doi: 10.1093/bioinformatics/btu08
[6] http://cbia.fi.muni.cz/simulator

The authors gratefully acknowledge funding from the Grant Agency of the Czech Republic under grant number P302/12/G157.

Fig. 1: First phase of the creation of synthetic image data – digital phantom generation for a cell nucleus of granulocyte and a cell nucleus of HL60 cell.

Fig. 2: Second and third phase of the creation of synthetic image data – blurring using virtual microscopy and noise introduction using light detector simulation for digital phantoms from Figure 1.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-9-O-3081 HawkC: computer-aided 3D visualization and analysis software for electron tomography

Midoh Y.1, Nishi R.2, Shirazi M. N.3, Kamakura Y.3, Inoue Y.3, Miura J.4, Nakamae K.1
1Graduate School of Information Science and Technology, Osaka University, Japan, 2Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Japan, 3Faculty of Information Science and Technology, Osaka Institute of Technology, Japan, 4Dynacom Co. Ltd., Japan
midoh@ist.osaka-u.ac.jp

Electron tomography is widely used to obtain nanometer scale 3D structural information in cell biology and material science. Electron tomography includes four main steps: tilt-series acquisition, image alignment, 3D reconstruction and image segmentation (visualization/measurement). Especially, the image segmentation is time-consuming, and it is difficult to achieve complete automation because target objects with complex shape, low　contrast and low signal-to-noise ratio are often included.
In this paper, we introduce a computer-aided 3D visualization and analysis software for electron tomography [1]. Figure 1 shows an overview of the developed software. The software provides multiple automatic contour extraction algorithms, which are based on a class of region-based deformable contour models called R-centipedes [2]-[4] and a threshold-based approach, and manual segmentation using user-friendly user interface. We named the software “HawkC” meaning fast and accurate extraction of contours in tomographic images like a hawk. Figure 2 shows the process of contour extraction from a tomographic image of a cell nucleus using R-centipedes. It is seen that nuclear membrane and chromatin in the cell nucleus can be extracted accurately from initial curves by using the contour extraction algorithm of deflationary and inflationary modes, respectively. Figure 3 shows a 3D view of final extracted contours from tomographic volume data without manual editing. HawkC is available for free download (http://hawkc.dynacom.co.jp/?lang=en) until 31 March 2015.

[1] Takaoka, M. Cao, Y. Midoh, T. Nishida, T. Hasegawa, R. Nishi, Y. Inoue and M. Ogasawara，”Development of automatic system on electron microscopic tomography for 3D medical examination,” Korean Journal of Microscopy，38(4), pp.262-263 (2008).
[2] M. N. Shirazi and Y. Kamakura, "Restructuring centipedes and their applications to fast extraction of structures in Electron Microscope tomography images," Biomedical Engineering and Informatics (BMEI), 2, pp.518-523 (2010).
[3] M. N. Shirazi and Y. Kamakura, “PARALLELR-CENTIPEDES Fast Contour Extraction for 3D Visualization,” in Proc. International Conference on Computer Graphics Theory and Applications, pp. 713-718 (2010).
[4] Y. Kamakura, Y. Inoue, M. N. Shirazi, “R-centipede Model for Fast Contour Extraction in Electron Microscopic (EM) Tomography Image,” Medical Imaging and Information Sciences, 30(4), 95-100 (2013).

This work was supported by SENTAN, Japan Science and Technology Agency, Japan. We would like to gratefully and sincerely thank Prof. Akio Takaoka, Prof. Hirotaro Mori and Dr. Tomoki Nishida of Research Center for Ultra-High Voltage Electron Microscopy, Osaka University for for their immense help and support.

Fig. 1: An overview of the developed 3D visualization and analysis software.

Fig. 2: Examples of automatic contour extraction: deflationary and inflationary modes.

Fig. 3: 3D Visualization of a cell nucleus using HawkC (no manual editing).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-1452 The Uses of RIMAPS Technique in Microscopy: New Challenges

Favret E. A.1
1Soil Institute, CIRN, INTA; CONICET; Argentina
eafavret@cnia.inta.gov.ar

Visual observation has played a major role in all areas of natural science. Human beings perceive most of the information about their environment through their visual sense. However, quantitative evaluation of images never found widespread application until the born of personal computers. Applications in image processing have now been applied to all the sciences, e.g. linear and non-linear filter operations, fast algorithms, morphological operations to detect the shape of objects, segmentation and classification, reconstruction of three-dimensional objects and analysis of stereo images. Rotated Image with Maximum Average Power Spectrum (RIMAPS), created in 2002, is still a quite new imaging characterization technique independent of the class of microscopy and of conditions used for observation as long as they remain constant [1][2]. This technique rotates a digitized image and computes the integral of one of the two space variables for the two-dimensional Fourier transform for each value. As a consequence, averaged power spectra (APS) are obtained for each angular position. If the corresponding maximum values (MAPS) are plotted as a function of rotation angle (RI), valuable information of the surface pattern is obtained. The maxima appearing in the resulting plots indicate surface micro-pattern orientation and its characteristic topographic form. In many cases, surface patterns can be associated with simple geometrical figures as lines, squares, triangles or hexagons, distributed with a certain orientation on the surface (see Figures 1, 2 and 3). It has been used to characterize the micro-relief of metallic and biological surfaces, such as the detection of incipient damage on metallic surfaces and as a tool for describing the morphology of the trichodium net found in some grasses. Nowadays we are using RIMAPS to study the distribution pattern of ornamentation units on fungal spore surface, to describe leaf surface topography, to perform micromorphological analyses of soil samples and for quantifying microwear traces on lithic artefacts in order to identify their context of use. The present work shows a review and new uses of RIMAPS technique that could be applied on different research fields.

References

[1] Fuentes, N. and Favret, E. (2002). A new surface characterization technique: RIMAPS (Rotated Image with Maximun Average Power Spectrum). Journal of Microscopy. 206, 72-83.

[2] Fuentes, N. and Favret, E. (2009). RIMAPS and Variogram Characterization of Micro-Nano Topography. In Functional Properties of Bio-inspired Surfaces: Characterization and Technological Applications. Favret, E. and Fuentes, N. (Eds.). World Scientific Publishing. pp 155-179.

I am very grateful to my colleagues Dr. Néstor Fuentes, Dr. Adrian Canzian and Ms. Adriana Domínguez for their support.

Fig. 1: SEM micrograph of a lepidopter compound eye.

Fig. 2: A possible sketch of a lepidopter compound eye.

Fig. 3: RIMAPS spectra of figures 1 (black line) and 2 (red line). The maxima indicate the main directions of the surface pattern and its distribution.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-1646 Lamina propria of uninvolved rectal mucosa 10 cm and 20 cm away from malignant tumor: image processing/analysis of structural organization

Lalić I. M.1, Despotović S. Z.1, Milićević N. M.1, Milićević Ž. J.1
1Institute of Histology and Embryology, Faculty of Medicine, University of Belgrade, Belgrade, Serbia
ivanalalic5@gmail.com

Background: In recent years, great attention has been paid to the reciprocal influence between malignant tumor and tumor-associated stromal elements. However, it has also been shown that the changes are noticeable in distant mucosa. Connective tissue of rectal mucosa in the remote surrounding of the malignant tumor shows structural disorganization and the aim of our study was to quantify those changes using image analysis.
Materials and methods: Morphometric study of rectal mucosa was performed in samples taken 10 cm and 20 cm away from the malignant tumor during endoscopic examination of 13 patients of both sexes. The samples of rectal mucosa were obtained from 5 healthy controls during active screening for asymptomatic cancer. Tissue sections were routinely stained with Gomori's silver impregnation technique. Microphotographs were acquired with a digital camera Olympus C3030-Z connected to a light microscope Opton Photomicroscope III. Measurements of the spacing between reticular fibers were performed using plugin BoneJ within open-source software Fiji. Results were statistically analyzed with Mann-Whitney U test.
Results: At the distance of 10 cm away from the tumor lesion a significant increase in diametar of the spaces between the reticular fibers was observed (5,41±2,34µm), in comparison with both healthy controls (3,77±2,34µm) and tissue samples taken 20 cm away from the tumor (3,75±1,14µm).
Conclusion: Tumor induces structural changes of connective tissue in the remote, uninvolved lamina propria of rectal mucosa 10 cm away from the malignant lesion.

The microscope, camera, computer hardware and software are a kind donation of Alexander von Humboldt-Foundation, Bonn, Germany to N.M.M. This work was supported by the Ministry of Education and Science of the Republic of Serbia (grant no. 175005).

Fig. 1: Rectal mucosa of a healthy person. Reticular fibers of lamina propria are closely appositioned and orderly organized. Gomori's silver impregnation; x630

Fig. 2: Rectal mucosa taken 10 cm away from the malignant tumor. Reticular fibers of lamina propria show irregular organization. Spaces between the fibers are enlarged. Gomori's silver impregnation; x630

Fig. 3: Rectal mucosa of a healthy person. Graphical output from BoneJ. Spaces between closely appositioned and orderly organized reticular fibers, as presented by red rectangle in Fig. 1, measured with plugin Thickness. Yellow regions have larger diameter than blue regions.

Fig. 4: Rectal mucosa taken 10 cm away from the malignant tumor. Graphical output from BoneJ. Enlarged spaces between reticular fibers, as presented by red rectangle in Fig. 2, measured with plugin Thickness. Yellow regions have larger diameter than blue regions.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-1772 Stereological morphometric grids for ImageJ

Mironov A.1
1Electron Microscopy Core Facility, Faculty of Life Sciences, University of Manchester, Manchester, UK
Aleksandr.Mironov@manchester.ac.uk

The unbiased stereological procedures for morphometric quantifications became very popular in microscopy field and their usage is often the requirement in a number of scientific journals. The beauty of the majority of these methods lies in the simplicity of their application based on rigid mathematical framework once the major principles were followed in specimen preparation.
There are a number of ways to do stereology, but almost all modern techniques require the use of computer software to generate a grid. Many microscope control and scientific image analyzing programs have stereology modules, which can be used to display some type of stereological grid over live or recorded image from the microscope. The user will then have to count how many of grid points or grid lines intersect particular types of objects of interest. Most of stereological software is just a part of multifunctional software packages and, therefore, could incur very significant expenses that can interfere with the widespread use of computerized stereological tools.
One of the well-known exceptions is ImageJ software [1], which is a public domain Java image processing program that was designed with an open architecture that provides extensibility via plugins and macros. However, the currently implemented “Grid” and “Grid Cycloid Arc” plugins are rather limited in their functionality and applicability.
I have developed a number of macros that covers most of the well-known and widely used grid designs, which include: multipurpose grid with lines and points [2], Merz grid [3] and multi-circles grid that have built-in isotropy, grid with cycloids for vertical sections design [4] and unbiased counting frame grid [5] with the disector [6] volume feature. All of the grids have options for random offset, orientation and various densities and include help instructions. The macros display specific grid parameters (such as points/lines per area etc.) in separate window to facilitate further calculations. In combination with “Cell Counter” plugin these grids can be effectively used to count specimen features with stereological tools. Macro format allows to examine and to change the code without previous knowledge of programming language (such as Java). As an additional convenience, these macros are organized in “Tool sets” that can be added to the ImageJ toolbar as clickable icons.

References

[1] MD Abramoff et al, , Biophotonics International, 11(2004), p.36.
[2] HJ Gundersen, EB Jensen, J. Microsc. 147(1987), p.229.
[3] CV Howard, MG Reed in “Unbiased Stereology”, ed. CV Howard, MG Reed, (Garland Science/BIOS Scientific Publishers, Oxon, 2nd ed.) p.187.
[4] AJ Baddeley et al, J. Microsc. 142(1986), p.259.
[5] HJ Gundersen, J. Microsc. 111(1977), p.219.
[6] DC Sterio, J. Microsc. 134(1984), p.127.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-1898 Stereology on Electron Microscopy: make your EM count!

Vizcay-Barrena G.1, Glover L.1, Fleck R.1
1Centre for Ultrastructural Imaging, King’s College London,U.K 1
gema.vizcay@kcl.ac.uk

Qualitative electron microscopy studies coupled with expert interpretation and analysis play a valid and useful role in the initial stages of many scientific problems. However, it is the use of quantitative methods that is the hallmark of modern scientific research. Acquiring quantitative morphological data (volumes, surface areas, lengths, numbers, etc) about organelles, cells, tissues and organs should be the next step towards elucidating the exact correlation between structure and function [1]. To this end, we have used stereological approaches to investigate mitochondrial integrity/functionality.

To simplify, stereology aims to make quantitative estimates of the “amount” of a geometrical feature within the object of interest. In doing so, it manages to provide 3-D data that makes the interpretation of functional morphology more effective. Although there are more intricate stereological approaches, here we present a relatively quick and easy method to calculate the volume fraction of a component within a reference volume. This is a simple and very widely used parameter to express the proportion of a particular phase or component within the whole structure [2].

In our study, the volume fraction of cristae within the mitochondria, Vv (cristae, mitochondria), was estimated by placing a combined point counting grid (CPCG) over each mitochondrion micrograph. Thirty mitochondria from each experimental group were used for this stereological analysis. The CPCG used for this study was composed of two sets of points of different densities on the same grid; 9 fine points per coarse point. The volume of reference (mitochondria) was estimated by counting the number of coarse points that hit the reference space and multiplied by 9. The volume of the particular phase (cristae) was estimated by counting the number of fine and coarse points that hit the cristae. Thus, the volume fraction of the cristae within the mitochondria was calculated and expressed as a percentage. Student’s t-test was used to assess whether there was a significant difference between the samples at a 99% confidence level.

Our stereological study demonstrates that there is a contrasting mitochondrial morphology between female/male gametes and somatic cells. These differences in morphology are in turn linked to a functional division of labour between sperm and egg as demonstrated by other molecular approaches [3,4]. This analysis is an example that showcases how the application of stereology on Electron microscopy can be a powerful tool allowing quantification of morphological data.

References:[1] Mayhew, T.M (1991).[2] Howard, C.V. and Reed, M.G (2010).[3] Wilson de Paula et al., (2013a).[4] Wilson de Paula et al., (2013b)

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2133 Fully automated muscle quality assessment by Gabor analysis of second harmonic generation images

Paesen R.1, Smolders S.1, de Hoyos Vega J. M.1, Hansen D.2, Ameloot M.1
1BIOMED, Hasselt University, Agoralaan building C, 3590 Diepenbeek, Belgium, 2REVAL, Hasselt University, Agoralaan building C, 3590 Diepenbeek, Belgium
rik.paesen@uhasselt.be

Although structural changes on the sarcomere level of skeletal muscle are known to occur due to various pathologies, rigorous studies of the reduced sarcomere quality remain scarce. This scarceness can be mainly be accounted for by the lacking of an objective tool for analyzing and comparing sarcomere images across biological conditions. We propose a method to assess the sarcomere quality of skeletal muscle tissue imaged by second harmonic generation (SHG) microscopy. SHG microscopy is a label free technique based on the SHG signal originating from the highly ordered myosin thick filaments. This degree of ordering decreases upon muscle degradation, resulting in alterations in the regular appearance of the sarcomeres. The proposed analysis method is based on a fully automated implementation of a Gabor filter. With this Gabor wavelet approach, we can not only localize but also score sarcomere anomalies that could be related to muscle degradation. The method is rather insensitive to the signal to noise ratio and is implemented in such a way that it is independent of global intensity variations. Therefore, the resulting Gabor values allow for data comparison across various biological conditions. Using our newly introduced Gabor analysis method, we studied the effect of muscle disuse on sarcomere quality in the context of multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) rat model. Based on the gathered data, the interpretation of the Gabor values is addressed by linking them to specific anomalies (Fig. 1), and we show that a correlation exists between the sarcomere quality and EAE related muscle disuse.

Fig. 1: Interpretation of Gabor histograms for typical sarcomere structures imaged by SHG microscopy. (a) raw Gabor data of normal sarcomeres; (b) Amplitude corrected version of (a); (c)+(d) typical examples of pitchforks; (e) myocyte border effect; (f) typical transition to double band patterns; (g)+(h) respresentative examples of double band structures.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2214 High-performance Robust STEM Image Registration

Jones L.1, Yang H.1, Nellist P. D.1
1Department of Materials, University of Oxford
lewys.jones@materials.ox.ac.uk

In the scanning transmission electron microscope (STEM), there are many uses of image-series including; frame-averaging to improve signal-noise ratios, focal-series for optical-sectioning experiments / aberration studies, time-series to study dynamic processes like beam-damage, or camera-length series to study strain effects. However, at typical STEM acquisition times, stage / sample drift and low frequency distortions can become apparent. For quantitative interpretation we must correct these using rigid and non-rigid registration respectively. Recently we have developed an improved and automated registration method, customised for the challenges unique to STEM. Three modifications to the mutual-correlation function (MCF) method [1] have been designed, to specifically address problems registering images that include large areas of crystalline material, defects or edges.
Firstly, the user is able to vary the power on the MCF normalisation, changing the relative weighting of the crystal lattice in the image, between 0 (no normalisation, simple cross-correlation) and 1 (all spacings weighted equally, phase-correlation). Generally an intermediate value ≈0.4–0.75 is optimum (Fig 1, left); where crystalline information is used to provide an accurate registration but not relied upon, as this could easily introduce unit-cell ‘hops’ leading to mis-registration.
Secondly, as there are often multiple possible vectors identified when registering lattice images an iterative ‘learning mode’ has been introduced. With this option enabled the algorithm will try to iteratively learn the global drift rate. This is especially useful for time-series or focal-series data where the time between frames, and drift rate, is fairly constant. Fig 1 (right) shows an example annular dark-field STEM focal-series registered both with and without this new mode. With learning mode disabled, characteristic unit cell ‘hops’ were observed (dashed lines). While the registered images may appear reasonable, this diagnosed stage drift is not physically real. With learning mode enabled the consistent smooth drift rate is correctly determined.
Thirdly, as diagnosing non-rigid offsets can be challenging near sample edges or defects (Fig 2, middle) the user may optionally ‘lock’ the diagnosed offset vectors together in the fast scan direction (Fig 2, right). This prevents the introduction of artefacts at edges or defects whilst still allowing for effective non-rigid registration of STEM data.
Together, these refinements significantly reduce the risk of ‘crystal hops’ during rigid registration and of artefact introduction during non-rigid registration improving both the throughput and precision of automated STEM data analysis.
[1] Heel, Schatz & Orlova, Ultramicroscopy 46 (1992) 307–16

The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3).

Fig. 1: Enlargement of the central region of an example mutual-correlation function (MCF) with peaks indicating probability of image-pair offset matches (left) and right the rigid-registration results with and without the optional ‘learning mode’ (see text).

Fig. 2: Part of the non-rigid registration stage of a HAADF time-series data-set of a [110] oriented SrTiO3 nano-cube (left). The non-rigid displacement map for the y-direction is shown with pixels allowed to move freely (centre) and with the fast-scan rows locked to move together (right).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2217 Three-dimensional morphometry and optimality calculation of vascular networks examined in the SEM.

Minnich B.1, Margiol S.2, Frysak J.2, Bernroider E. W.2
1Department of Cell Biology & Physiology, Division of Animal Structure & Function, Vascular & Exercise Biology Unit, University of Salzburg, Salzburg, Austria, 2Vienna University of Economics and Business, Inst. Information Management and control, Vienna, Austria
bernd.minnich@sbg.ac.at

Scanning electron microscopes (SEMs) are potential tools for morphological research. They have a great resolution and a high depth of focus, which gives SEM micrographs pseudo three-dimensionality. Adversely, the high depth of focus prevents accurate dimensional or spatial measurements on microstructures. Macroscopic objects are viewed close up using binocular vision. Binocular vision is also used in microscopy where stereophotogrammetry and related techniques applying stereo paired images, and a variety of hardware tools calculate the third dimension (z-coordinate) using the parallax.

A method (3D-morphometry) for dimensional and angular measurements of microstructures examined in the SEM was first developed in our lab in 1999 [1]. It uses digitized stereo paired images frame-grabbed (slow scan) directly from the SEM’s photo-display, vector equation-based algorithms for the calculation of spatial coordinates and derived distance- as well as angular measurements. It comprises dynamic data exchange to MS Excel™ for further statistical analysis. Mathematical formulas for central perspective depth computation allow the overall error to be less than 1.0%. Meanwhile (2013), the method was further improved and the new java based software M3 is suitable to be also used with Windows 7 and 8™ operating systems. A modern graphical user interface (GUI) (Fig.1), a new data interface (open office) is supported and an improved usability facilitates the measurement processes [2]. Moreover, the program allows to generate anaglyphic 3D images (Fig.2).

This technique is currently used to analyse (a) the geometry of microvascular networks in terms of vascular parameters (i.e. diameters, interbranching distances, branching angles and intervascular distances) and (b) to determine bifurcation indices and area ratios, in turn to calculate optimality principles (i.e. principle of minimal lumen volume, minimal pumping power, minimal lumen surface and minimal endothelial shear force) [3-4] (Fig.3) underlying the design (in terms of construction, function and maintenance) of arterial bifurcations respectively venous mergings.

References:

1. Minnich B. et al, Journal of Microsccopy 195 (1999), p. 23-33.
2. Minnich B. et al, in “Current microscopy contributions to advances in science and
    technology”, eds. A Mendes- Vilas and J D Alvarez, Formatex, (2012), p. 191-199.
3. LaBabera M., Science 249 (1990), p. 992-1000.
4. Murray C.D., Journal of General Physiology 9 (1926), p. 835-841.

This work was supported by the University of Salzburg and by ComServ.at Austria.

Fig. 1: Figure 1. M3 graphical user interface (GUI) showing the morphometry dialog when measuring branching angles of feeding arteries of the spleen of the adult Xenopus laevis Daudin.

Fig. 2: Figure2. M3Anaglyphic 3D image representing a vascular corrosion cast of a microvascular network examined in an ESEM (FEI XL-30).

Fig. 3: Figure3. Scatterplot showing an optimality diagram of the brain microvascular bed (rhombencephalon) of the sterlet (Acipenser ruthenus). Vascular bifurcation with indications of vessel diameters and branching angles (insert).

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2378 Novel method of image analysis based on the autocorrelation function yielding lateral size distribution.

Fekete L.1, Kusova K.1, Petrak V.1, Kratochvilova I.1
1Institute of Physics, Academy of Sciences CR
fekete@fzu.cz

The lateral size of objects in microscopy images can be a useful piece of information for various purposes, but it might also be difficult to retrieve using a fast, automated approach. Although numerous ways to determine the lateral size, based on thresholding, watershedding, rolling ball background subtraction, etc., exist, they often fail because of the peculiarities of the morphology of the studied sample, especially in the case of closely packed objects (grains, cells, crystals). Using these methods, small grains are often neglected or merged with large ones, which can dramatically alter the shape of the determined size distribution. In order to circumvent these problems, we introduce a novel method for the determination of the lateral sizes (or more precisely circle equivalent diameters) of microscope-imaged objects. Our method is based on the application of autocorrelation function (ACF). Although this method requires a high-quality image, it is fast and easily automated. Moreover, the output of our method is not only a characteristic size of objects, but it allows direct fitting of the distribution of sizes. We demonstrate the capabilities of this method on a series of films composed of closely packed diamond crystals (20-450 nm in diameter) measured by AFM, because this series is a prime example of a morphology whose lateral sizes can be determined only manually, as illustrated in Figure 1a. Figure 1b then shows the radial part of the ACF of a nanodiamond layer fitted by the gamma-distribution-shape lateral sizes. Figure 1c shows the excellent agreement of the lateral size distribution determined by our method in comparison with an independently manually evaluated one. Since no theoretical constraints on the application of this method exist, its usage is directly extendable also to other types of images or objects, e.g. to images from electron or optical microscopes.

 Financial supports from projects GPP204/12/P235; TA01011165; GA-CR-13-31783S are gratefully acknowledged.

Fig. 1: An example of the application of our ACF-based analysis.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2396 Is There Possible Bias in E 112 Planimetric Grain Size Measurements?

Vander Voort G. F.1
1Consultant - Struers A/S
georgevandervoort@hotmail.com

A claim was made in 1995 and 2011 publications by a metallurgist that the Jeffries planimetric method of determining grain size in E 112, and in DIN 50601 (it is the standard method in every national and international grain size test method and is described in every text book on quantitative metallography), is wrong and produces biased grain size ratings when the counts are low, is incorrect. This claim was based upon theoretical considerations described by Saltykov who proposed using rectangles for the planimetric method, rather than circles, to minimize bias at low counts of the number of grains inside the circle. Saltykov, however, did not publish actual test data to back-up this claim. The count levels mentioned are far below those recommended by E 112 for these methods, but could be encountered in manual measurements of the size of very coarse grains (which might be performed, but is rarely done).

Actual grain size measurements using both test circles and rectangles, with a very wide range of grains within the test figures and intersecting their borders, showed that the ASTM Jeffries planimetric and the Hilliard single-circle intercept methods produced statistically identical measures of the ASTM grain size, G, down to count levels far below what is recommend – down to 30 for (ninside + 0.5 nintercepted) for the planimetric method and down to 20 grain boundary intersections, Pintersections, for the intercept method (well below the recommended minimums of 50 and 35, respectively). At levels below these limits, bias was small – mainly data scatter was observed at counts <10 for both methods. The Saltykov planimetric method using rectangles gave the best data, identical to the E 112 data, with statistically identical grain size values down to 10, and was bias free, but also exhibited data scatter at counts <10. The claim about bias by the Jeffries method has no validity. The model used in the claim did not evaluate the effect of varying the counting conditions which was the basis of the claim about bias being created. Also, they did not do actual tests to prove that their model was valid and their claim about bias was correct. Models do not have any validity if they do not test the actual conditions and are not verified by actual experimental data.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2460 Use of stereological methods for detecting destructured zones in cooked hams

Bednářová M.1, Pospiech M.1, Tremlová B.1
11 University of Veterinary and Pharmaceutical Sciences Brno, Brno Czech Republic
eliasova.martinka@seznam.cz

Destructured zones in cooked hams have recently been a major problem for meat industry where they cause significant economic losses. Occurrence of destructured zones in cooked hams is reported between 2-20 per cent. Destructured zones are typically inappropriate for cutting after the heat treatment due to reduced cohesion and they characterized by wavy structure and disrupted muscle fibers. Literature reports that these defects are caused by the quality of meat, especially when PSE (pale, soft, exudative) meat is used. It has been described that these differences in quality of raw materials are caused by different biochemical and physical properties, when muscle myofibrillar proteins are denatured, meat has poor water-holding capacity, has a lighter color, and the texture of the final product is changed. PSE in meat can be determined using a number methods, including methods of muscle fibers microstructure analysis. A possible cause of destructured zones is abnormally rapid post-mortem glycogenolysis, which is also reflected in the increased formation of lactic acid. The cause of destructured zones can also be the technology applied (Valous et al., 2009). The aim of this work was to verify the possibility of distinguishing destructured and non-destructured zones at the microscopic level using stereological methods. In this work, histological examination of cooked hams was utilized with evaluation using a point grid (Ellipse, SVK). Three samples were taken from destructured zones and three samples from non-destructured zones for this research. The samples were processed using paraffin blocks and stained with haematoxilin-eosin staining (Bancroft, 2008). The examination was performed by means of Nicon Eclipse E200 microscope (Nikon, JPN). During the examination of samples using the light microscope, significant differences in the structure of skeletal muscles were observed, namely disorganization and rearrangement of muscle fibers were found in destructured zones. Stereological examination demonstrated a significant difference in the representation of transverse and longitudinal fissures (p≤0.05). In non-destructured zones, transverse fissures were detected in the muscle fiber in the proportion of 4.39 per cent, whereas longitudinal fissures were not observed at all. Nevertheless, in destructured zones, transverse fissures in the proportion of 47.6 per cent and longitudinal fissures in 6.11 per cent were detected in the muscle fiber. Similar changes are also described by Laville et al. (2005) who evaluate them as typical for PSE meat. The results thus show that histological examination can distinguish between destructured and non-destructured zones in ham on the basis of the proportion of transverse and longitudinal fissures.

Fig. 1:  Ham – skeletal muscle (longitudinal section) without structural changes, H&E.

Fig. 2:  Ham in destructured zone – skeletal muscle (longitudinal section) with transverse and longitudinal fissures, H&E
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2524 Analysis of mitochondrial morphology and networks by Fiji

Čapek M.1, Sládková J.2, Radochová B.1, Janáček J.1
1Institute of Physiology ASCR, v.v.i., Prague, Czech Republic, 2Laboratory for Study of Mitochondrial Disorders (MITOLAB), Department of Pediatrics and Adolescent Medicine of the 1st Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic
capek@biomed.cas.cz

MITOLAB does visual analyses of microscopic images of mitochondria of child patients with mitochondrial disorders. However, due to demands on statistical evaluation, semiautomatic analysis of the data became desirable. We propose a flow that provides main important parameters of mitochondria by using freeware software package Fiji (fiji.sc).

        These parameters are of interest: 1. Number and density of mitochondria per one cell – disordered mitochondria tend to be degraded and sparsely distributed; 2. Number of mega-mitochondria, which are mitochondria collapsed into spheres instead of normal tubular shapes, which may appear in tissues of patients with disorders; 3. Directional distribution of mitochondria in cells – healthy cells have mitochondria forming a compact network of tubules in a few dominant directions distributing ATP throughout the whole cell. Disordered mitochondria networks tend to be fragmented, without dominant directions; 4. Branching of mitochondrial segments – mitochondria of healthy cells tend to form branching networks. Disordered cells contain high number of separated mitochondria, thus the branching is less observed.

        The acquired images are generally of low quality, see Fig. 1, thus the preprocessing of data is essential. It includes: I. Conversion to greyscale images (Fiji menu item Image→Color→Split Channels); II. Removing inhomogeneous background (Process→Subtract Background); III. Removing noise (Process→Noise→Despeckle); IV. Improving contrast (Image→Adjust→Brightness/Contrast); VI. Selection of an individual cell by creation its convex hull (Polygon selection); V. Thresholding MITO (Image→Adjust→Threshold), Fig. 2.

        Density and number of (mega-)mitochondria are obtained by Analyze→Analyze Particles and are expressed as Ratio AM/AC, Number of mitochondrial components and Number of mega-mitochondria, respectively, in Fig. 3. Directions of mitochondria were analyzed by generation of “directionality histogram” using Analyze→Directionality and by fitting a Gaussian function to the highest peak. Goodness of fitting directionality histogram in Fig. 3 describes partially if predominant directions are present in this histogram (it goes to 1) or not (it goes to 0). Branching was found by creation of a skeleton using Process→Binary→Skeletonize and by skeleton analysis using Plugins→Skeleton→Analyze Skeleton (2D/3D). Branchings is expressed by Ratio BP/EP in Fig. 3.

        Fig. 3 shows an analysis of images of two healthy patients and four sick patients. Although analysis of medical image data is mostly difficult and ambiguous and we present results of a restricted number of patients, we conclude that Fiji can be used for successful analysis and statistical evaluation of such data.

Supported by the Czech Science Foundation (P501/10/0340, 13-12412S), AMVIS (LH13028) and by supports of research organizations: RVO-67985823 and RVO-VFN 64165/2012.

Fig. 1: Microscopic fluorescence images of mitochondria in fibroblasts of both healthy (upper; Healthy I, see Fig. 3) and sick patients (bottom; Sick III, see Fig. 3). Magnification 600×, stained by MitoTracker Red (Invitrogen), white bar is 40 µm.

Fig. 2: Preprocessed greyscale 8 bit (left) and thresholded binary images (right) of mitochondria of cells from Fig. 1.

Fig. 3: Results of image analysis of two healthy patients and four sick patients. Mitochondria of healthy cells tend to have higher Ratio AM/AC, smaller number of fragmented parts and mega-mitochondria, higher Goodness and Ratio BP/EP than mitochondria of disordered cells.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2599 Automatic classification of brightfield microscopy pollen samples using a Tchebichef moment-based texture descriptor

Marcos J. V.1, Cristobal G.1, Bueno G.2, Nava R.1, Redondo R.2, Escalante-Ramirez B.3, Deniz O.2
1Instituto de Óptica, Spanish National Research Council (CSIC), Madrid, Spain, 2VISILAB Department, University of Castilla La Mancha, Ciudad Real, Spain, 3Dep. Procesamiento de Señales, Universidad Nacional Autónoma de México, México City, México
jvmarcos@gmail.com

Introduction

Palynology is the study of pollen grains produced by plants and spores. Pollen identification is needed in several domains (e.g., medicine, oil industry or apiculture). Currently, this task is based on visual inspection of microscopy images, which is time-consuming and costly. In this study, automated analysis of microscopic pollen images is addressed. Automatic pollen identification involves segmentation and classification tasks. Segmentation aims to localize each of the pollen grains in the slide, separating it from the rest of the content. In classification, the isolated pollen grain is assigned to one of a set of categories (taxa). The present study is focused on the latter. For this purpose, brightfield microscopy images corresponding to a subset of honey-bee pollen taxa were analyzed.

Data

Pollen images were captured using a NIKON E200 microscope and a camera NIKON DS-Fi1. Balls of pollen were prepared in slides sealed with a coverslip. A 40x magnification was used to acquire images of the slide containing several pollen grains. The acquisition consisted in stacks with 31 images of the slide in order to ensure an optimum focus. The best focused slide was identified by an expert. Subsequently, pollen grains were manually extracted from it by defining a rectangular region. Pollen samples from 15 different taxa were captured: 1) Aster, 2) Brassica, 3) Campanulaceae, 4) Carduus, 5) Castanea, 6) Cistus, 7) Cytisus, 8) Echium, 9) Ericaceae, 10) Helianthus, 11) Olea, 12) Prunus, 13) Quercus, 14) Salix and 15) Teucrium. The database was composed of 120 brightfield microscopy images per pollen taxon, resulting in a total of 1800 images. Figure 1 depicts an example for each taxon.

Methods

A pattern recognition approach (feature extraction and classification) was adopted for pollen grain classification. Figure 2 shows a scheme of the methodology. Feature extraction was carried out through pollen texture analysis using Discrete Tchebichef Moments (DTM)1. Hence, a texture signature (see Figure 3) was obtained for each grain and used as input for the classification stage. The latter was implemented by means of discriminant analysis and k-nearest neighbour algorithms.

Results

Ten-fold cross-validation was applied to estimate classification accuracy from the dataset of 1800 images. A classification performance of 92.06% was achieved. Most of the errors corresponded to ‘Citysus’ samples.

Conclusion

Our experiments show that texture is a distinctive characteristic of the pollen taxon. We propose an exhaustive analysis of texture in image-based applications pursuing automatic identification of pollen taxa.

[1] Marcos JV and Cristóbal G, “Texture classification using Tchebichef moments,” J. Opt. Soc. Am. A 30, 1580–1591 (2013)

Project “Apifresh” (Inspiralia). J. V. Marcos is a “Juan de la Cierva” research fellow (Spanish Ministry of Economy and Competitiveness).

Fig. 1: Brigthfield microscopy images from our database. One sample is provided for each of the pollen taxa analyzed in our study.

Fig. 2: Description of the methodology proposed in our study to perform automatic pollen identification. Two main steps can be identified: i) feature extraction (segmentation and image texture analysis) and ii) classification (discriminant analysis and k-nearest neighbour).

Fig. 3: Example of the texture signatures based on Discrete Tchebichef Moments obtained for three different microscopy images from our pollen database.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2784 Segmentation in phase contrast microscopy images

Soukup J.1,2,3, Lašan M.3, Císař P.1, Šroubek F.2
1University of South Bohemia, Institute of Compex Systems FFPW, Nové Hrady, Czech Republic, 2Institute of Information Theory and Automation of the ASCR, Prague, Czech Republic, 3Charles University in Prague, Faculty of Mathematics and Physics, Prague, Czech Republic
jindra.soukup@gmail.com

Time-lapse microscopy imaging is a commonly used modern method for observing the dynamics of cells and tissues. A large number of images that time-lapse microscopy generates is difficult to evaluate manually, and computer methods of image processing would be highly advantageous. Quantifying of mammalian cancer cell images captured by phase contrast microscopy is especially challenging. Cancer cells have irregular shapes that change over time and the mottled background pattern is partially visible through the cells. In addition, the images contain artifacts such as white areas around the cell edges - so called halos.

We developed a novel algorithm for segmentation of individual cells. First part separate the cells from the background and it is based on the differences in time between consecutive images and a combination of sophisticated thresholding, blurring, and morphological operations. The second part of our algorithm separates individual cells in the clusters. It uses the halos between cells (thresholding and modified skeletonization) and fills the missing parts by connecting the loose ends of the skeleton via Dijkstra algorithm.

We tested the algorithm on images of four cell types acquired by two different microscopes, evaluated the precision of segmentation against manual segmentation performed by a human operator.

We created the software which implements our segmentation method. We added the possibility to modify the resulting segmentation. User can modify the result by merging or splitting the cell regions that was found by our algorithm.

The results of the project LO1205 were obtained with a financial support from the MEYS under the NPU I program, CENAKVA CZ.1.05/2.1.00/01.0024, GA JU 134/2013/Z, GAUK grant No. 914813/2013, GAČR grant No. 13-29225S and grant SVV–2013–260103.

Fig. 2: Final segmentation of individual cells.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2852 Using Nion Swift for Data Collection, Analysis and Display

Meyer C. E.1, Dellby N.1, Dellby Z.1, Lovejoy T. C.1, Sarahan M. C.1, Skone G. S.1, Krivanek O. L.1
1Nion Co, Kirkland, WA, USA
sarahan@nion.com

Nion Swift is an open-source software platform for the collection, processing, quantification, visualization, and management of scientific data. Built on the popular Python programming language [1], Swift is written from the ground up to be extensible at every level. Although initially developed as software for controlling Nion electron microscopes and collecting and processing data from them, Swift has been designed to serve as a resource for the scientific community across a range of applications and operating systems.

Swift builds on an ever-growing library of Python tools such as NumPy and SciPy [2]. Since it is open source, users can examine the data workflow down to the source code level. All data is stored in standard formats such as TIFF or Numpy arrays.  Swift is designed to track data from collection to presentation, including relationships between data. All data is tagged with metadata such as details of the sample, the current user, and the instrument parameters. If an elemental map is produced from a spectrum image, Swift remembers from what larger data set the elemental map was produced, even if the elemental map is transferred to a colleague on another computer or network. It is then possible to return to the original data for adjustments if needed.

Processing in Swift can be performed  "live", so that the user can inspect the end results during an experimental session, and, if needed, make immediate adjustments to the experimental parameters. If an elemental map is degraded due to incorrect gain normalization of the EELS detector or poor signal-to-noise ratio, the problem can be noticed and rectified in the middle of the session.  The user can add new processing routines with Python and have multiple "live" processing and analysis items such as histograms, statistics, FFTs, or elemental maps, all visible during data collection.

The data storage and retrieval capabilities are likewise fast and automated. Swift provides powerful, extensible capabilities for sorting and filtering collected data. Data can be located from the user interface by session, sample, user, and other metadata. A community of Swift users is now contributing collection, processing and visualization modules to the software, a process that is helped by a well-defined submission procedure and guidelines for documenting the contributions. Examples of contributed modules will be shown at the meeting.

More information about Swift is available at [4].

[1] http://www.python.org/
[2] http://www.numpy.org/ and http://www.scipy.org/
[3] O.L. Krivanek et al., Ultramicroscopy 110 (2010) 935-945.
[4] http://nion.com/swift/

We are grateful to Prof. P.E. Batson for the use of the Nion UltraSTEM HERMES at Rutgers U.

Fig. 1: a) Swift acquiring a STEM HAADF image of the edge of a gold particle and of single Au atoms, b) Fourier-filtering it live with the filter shown at lower right (σ1 = 15% fN, σ2 = 7% fN, w2 = 0.14, where fN is the Nyquist freq.), c) live line profiles through (a), d) live line profile through (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2869 RAW Image Processing Software

Malakhova D.1, Nahlik T.1, Rychtarikova R.1, Stys D.1
1University of South Bohemia, Ceske Budejovice, Czech Republic
dmalakhova@frov.jcu.cz

We developed new software for image processing. This software allows us to control all steps of image formation. Typical starting dataset is the 12 bit raw data image file from the camera chip. The settings of the microscope which driven by the custom software is stored in the EXIF info in the image file (Fig.1).
Image processing starts with removing first 16 columns which used for internal calibration of the noise in the camera. RAW image data are acquired without application of the Bayer mask. This is applied in the next step. The Debayerization process produces images of ¼ resolution (doubles the point width in each axis).
Our RAW data are in 12bit format so for most of processing tools and namely display of image it is necessary to convert them to 8 bits. There are several ways for this conversion. We also developed our method which aims to maximally preserve the information carried in each of the color channels. This algorithm is called LIL (Least Information Loss) (Fig.2). We find out that not all intensity levels in 12bit images are occupied. In this case we find all unique occupied intensity levels in the whole image series, which are then rescaled to 8bit resolution. We know from our experience that non-functional camera elements produce erroneous pixels both at the lowest and at the highest levels. Settings in our software let us to remove specified percentage of the intensity levels from both sides to account for that as well as for additional very rare points in the image.
The remaining settings are connected to the settings of our microscope stored in the EXIF information. When we do the XYZ scan, Sort to Z option allows us to store XY in directories according to Z axis. Option Read and Export tags allows us to export EXIF information to the txt file. Processing of all subfolders allows us to use batch processing of the selected folder. The main correction covers cases when images are taken in wrong position or twice in the same position. Also when the microscope is setup on the limits of the device, there can be some erroneous images. The value Step of Piezo allows us to mark images as relevant or irrelevant and notify correct Z position. Marking of images as relevant or irrelevant is important for further processing, namely image information calculation and processing of 3D image structure.

CENAKVA CZ.1.05/2.1.00/01.0024, CENAKVA II (The project LO1205 was supported by the MEYS under the NPU I program); GA JU 134/2013/Z; Postdok JU CZ.1.07/2.3.00/30.0006.

Fig. 1: Fig. 1: Software GUI (Graphical User Interface) – This image shows possible settings of the software

Fig. 2: Fig. 2: Example of histogram – During LIL conversion parts marked by red arrows are removed completely and also all zero levels inside the histogram are deleted. The rest of histogram is converted to 8 bit image.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2965 Microscopy Image Browser is a new open-source tool for effective segmentation and analysis of 3D/4D volumetric datasets obtained by light or electron microscopy.

Belevich I.1, Joensuu M.1, Vihinen H.1, Jokitalo E.1
1Electron Microscopy Unit, Institute of Biotechnology, PO Box 56 (Viikinkaari 9), University of Helsinki, 00014, Finland
ilya.belevich@helsinki.fi

Rapid development of multidimensional microscopy imaging techniques during recent years has raised number of questions about effective image processing, visualization and analysis of the obtained datasets. Most universities worldwide provide access to the modern imaging techniques so that descriptive multidimensional datasets of the desired specimen can be fairly easily obtained by any researcher. After acquisition the datasets have to be analyzed and quite often the detailed analysis is impossible without segmentation (creating of a model) of objects of interest out of the multidimensional data. It seems that the segmentation is the most time consuming part of the image analysis routine. For example, it may take up to a month to properly segment a single electron tomogram. The slowness of the process is caused by two main factors: limited variety of good software tools (even commercial ones) and good segmentation algorithms that can be applied to facilitate the modeling. As a result, amount of collected and not properly processed data is much higher than the amount of produced results.


In this work we address this problem and present a free, open-source software package, Microscopy Image Browser (MIB), which can be used for image processing, analysis, segmentation and visualization of multidimensional datasets. We already utilized MIB in number of completed projects [1-3] and here demonstrate its successful application with segmentation of Golgi and other organelles of Trypanosoma brucei, and cryptomonad Rhinomonas nottbecki n. sp [4]. The program is written under Matlab environment which opens large variety of options for its extension with different tools and filters available thought the Matlab community. Even though the focus of the program is 3D segmentation of electron microscopy datasets, MIB is rather universal and can be used to perform analysis and visualization of multidimensional datasets obtained by light microscopy.


1. Puhka et al. Mol. Biol.Cell 23, (2012) 2424-.
2. Anttonen et al., Sci. Rep. 2, (2012) 978-.
3. Joensuu M et al. Mol. Biol.Cell (2014) Epub ahead of print.
4. Majaneva M et al. J Euk Microbiol (2014) accepted.

We thank Mervi Lindman and Antti Salminen for technical assistance. This work was supported by Academy of Finland (project 131650, E.J.) and Biocenter of Finland.

Fig. 1: The user interface window of Microscopy Image Browser

Fig. 2: Serial Block Face Scanning Electron Microscopy dataset of Tripanosoma brucei with one tripanosome segmented from the 3D volume using MIB. Dimensions: 11.3 x 15.4 x 6.8 μm, voxel size: 14 x 14 x 30 nm. The lower part of the figure shows visualization of individual organelles from the same model. The models were visualized using Amira.

Fig. 3: A model of Golgi apparatus and endoplasmic reticulum exit sites from a dataset obtained by Electron Tomography. The segmentation was done using MIB and the rendering done with Amira.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-2980 Microscopy Image Browser is a new open-source tool for segmentation and analysis of microscopy datasets

Belevich I.1, Joensuu M.1, Vihinen H.1, Jokitalo E.1
1Electron Microscopy Unit, Institute of Biotechnology, PO Box 56 (Viikinkaari 9), University of Helsinki, 00014, Finland
ilya.belevich@helsinki.fi

Rapid development of multidimensional microscopy imaging techniques during recent years has raised number of questions about effective image processing, visualization and analysis of the obtained datasets. Most universities worldwide provide access to the modern imaging techniques so that descriptive multidimensional datasets of the desired specimen can be fairly easily obtained by any researcher. After acquisition the datasets have to be analyzed and quite often the detailed analysis is impossible without segmentation (creating of a model) of objects of interest out of the multidimensional data. It seems that the segmentation is the most time consuming part of the image analysis routine. For example, it may take up to a month to properly segment a single electron tomogram. The slowness of the process is caused by two main factors: limited variety of good segmentation algorithms and software tools (even commercial ones) that can be used to facilitate the modelling. As a result, amount of collected and not properly processed data is much higher than the amount of produced results.


In my talk I would like to address this problem and present a free, open-source software package, Microscopy Image Browser (MIB), which can be used for image processing, analysis, segmentation and visualization of multidimensional datasets. MIB seems to be quite effective and we already utilized it in few projects [1-4]. The program is written under Matlab environment which opens large variety of options for its extension with different tools and filters available thought the Matlab community. Even though the focus of the program is 3D segmentation of electron microscopy datasets, MIB is rather universal and can be used to perform analysis and visualization of multidimensional datasets obtained by light microscopy.


1. Puhka et al. Mol. Biol.Cell 23, (2012) 2424-.
2. Anttonen et al., Sci. Rep. 2, (2012) 978-.
3. Joensuu M et al. Mol. Biol.Cell (2014) Epub ahead of print.
4. Majaneva M et al. J Euk Microbiol (2014) accepted.

Mervi Lindman and Antti Salminen are acknowledged for excellent technical assistance. This work was supported by Academy of Finland (project 131650, E.J.) and Biocenter of Finland.

Fig. 1: The user interface window of Microscopy Image Browser

Fig. 2: Serial Block Face Scanning Electron Microscopy dataset of Tripanosoma brucei with one tripanosome segmented from the 3D volume using MIB. Dimensions: 11.3 x 15.4 x 6.8 µm, voxel size: 14 x 14 x 30 nm. The lower part of the figure shows visualization of individual organelles from the same model. The models were visualized using Amira.

Fig. 3: A model of Golgi apparatus and endoplasmic reticulum exit sites from a dataset obtained by Electron Tomography. The segmentation was done using MIB and the rendering done with Amira.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-3016 Particle analysis of TEM/SEM images of nano-particles

Uematsu F.1, 3, Nakanoda S.1, 3, Kikui T.1, 3, Manabe H.1, 3, Iijima Y.1, 3, Kumagai K.2, 3, Kurokawa A.2, 3
1JEOL Ltd., 1-2 Musashino 3-Chome, Akishima Tokyo 196-8558, Japan, 2Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Ibaraki 305-8565, Japan, 3COMS-NANO, 1-1-1 Higashi, Tsukuba Ibaraki 305-8565, Japan
fuematsu@jeol.co.jp

Nano-particles are becoming widely used as functional materials. As a result, the technology to measure the size distribution of nano-particles is required. Direct observation with TEM or SEM is used for it; but, such methods are problematic because 1) the field of view of the target is limited, and 2) the measurement results depend on operator. The purpose of this study was to devise a way to solve these problems, in which samples are observed with TEM or SEM automatically (or manually), discrimination of individual nano-particles is made automatically from the image, and the size distribution is determined. As the first stage, to distinguish between adjacent particles we developed a nano-particle analysis system using the Mean Shift method, a segmentation method for image.

For the nano-particles, two types of polystyrene latex (PSL) standard spheres (30 nm, 260 nm) were used. After dispersing these nano-particles in a solution, the samples were observed using TEM and SEM. The analysis system we developed was used to determine the average particle size and size distribution from the acquired images, and the performance of the analysis system was evaluated. A JEM-1400Plus (JEOL) was used to make the TEM observations, and a JSM-7100F-TTL (JEOL) was used for the SEM observations. STEM observations were made using the SEM instrument.

Fig. 1 shows the TEM image of the PSL standard spheres (260 nm). Fig. 2 shows the results of the segmentation of adjacent particles using the Mean Shift method. The detected average particle diameter was 258 nm, with a standard deviation of 4 nm. Fig. 3 shows the STEM image acquired with the SEM for the PSL standard spheres (30 nm). Fig. 4 shows the results of the segmentation of adjacent particles using the Mean Shift method. The detected average particle diameter was 26 nm, with a standard deviation of 7 nm. This indicates that the standard deviation differs according to the diameter of the standard sphere, suggesting that can be attributed to the classification accuracy of the standard spheres and the conditions of the particle aggregation. When using Mean Shift to perform the segmentation, the determination of the boundaries makes use of the brightness profile between the particles and substrate, which is affected by the how the particles clump together. Therefore, if a method for determining the particle-substrate boundaries that accurately reflects the true particle diameter can be established, it will be possible to use this method to measure particle diameters with a higher accuracy than the conventional methods.

References
1) D. Comaniciu, P. Meer: IEEE Trans. Pattern Analysis. Machine Intelligence, 24, 603 (2002).
2) P. Meer, B. Georgescu: IEEE Trans. Pattern Analysis. Machine Intelligence, 23, 1351 (2001).

We wish to thank Dr. M. Hayashida of AIST for TEM observations. This work was supported by the COMS-NANO Consortium.

Fig. 1: TEM image of PSL (260 nm). Acc. Volt. 120kV.

Fig. 2: Segmentation result of TEM image.

Fig. 3: STEM (SEM) image of PSL (30 nm). Acc. Volt. 20kV.

Fig. 4: Segmentation result of STEM image.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-3195 An algorithm for quantitative cytometric analysis of spatial relationships between nuclear events represented by microfoci in multicolor 3D confocal microscopy.

Berniak K.1, Bernaś T.1,2, Rybak P.1, Biela E.1, Hoang A.1, Bujnowicz Ł.1, Zarębski M.1, Zhao H.3, Darzynkiewicz Z.3, Dobrucki J. W.1
1Division of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland, 2Nencki Institute of Experimental Biology, Laboratory of Functional and Structural Tissue Imaging, Polish Academy of Sciences, Warsaw, 3Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York, USA
kberniak@gmail.com

BACKGROUND. Multicolor 3D microscopy images of various subnuclear microfoci, representing
replication sites, DNA damage response regions or other nuclear phenomena provide vast amounts
of information about localisation of these processes. However, co-occurrence of different processes
cannot be quantified by colocalisation analysis of microfoci. In order to assess potential correlations
between two processes represented by microfoci quantitative analysis of mutual distances between
their centres in 3D is required.
GOAL. This work was focused on constructing an algorithm for quantitative analysis of spatial
relations between two classes of discrete events represented by large numbers of microfoci in three-
dimensional images of cell nuclei.
METHODS. Newly replicated DNA was labelled using incorporation of a precursor (EdU) and click
chemistry. Microfoci of phosphorylation of histone H2A.X and 53BP1 were labelled by immuno-
fluorescence. 3D confocal fluorescence images were deconvolved prior to further analysis.
RESULTS. An algorithm was constructed and applied in analysis of a relationship between DNA
damage signalling and repair (H2AX histone phosphorylation, 53BP1 recruitment), and DNA
replication, in nuclei of cells treated with topoisomerase inhibitor camptothecin or hydrogen peroxide.
Relationship between spatial distributions of these two groups was quantified using distributions
(histograms) of nearest-neighbor (nn) distances. Populations of correlated and uncorrelated signals
were isolated using histogram thresholding. Analysis of spatial distributions of γH2AX and 53BP1
foci demonstrate the existence of a population of γH2AX signals associated or located afar of 53BP1
signals. The nn distance calculation was supplemented with analysis of cumulative distribution of all
possible distances (Ripley’s K functions) between signals of the same and different kinds. We found
an expected, statistically significant spatial correlation between DNA replication and damage induced
with topoisomerase I inhibitor camptothecin, but a very low correlation in cells subjected to oxidative
stress. This approach to analysis of spatial association of two nuclear events is expected to be suitable
for investigations of a relationship between any other types of cellular events represented by small
foci, in multicolor patterns found in standard and super-resolution 3D confocal images.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-3451 Quantified phenotype analysis in a cell model for Autosomal Dominant Retinitis Pigmentosa

Alghamdi R. A.1, Boguslaw O.2, South K.1, Opefi C. A.1, Reeves P. J.1, Laissue P. P.1
1School of Biological Sciences, University of Essex, Colchester, United Kingdom, 2School of Engineering and Computing Sciences, Durham University, Durham, United Kingdom
plaissue@essex.ac.uk

Autosomal Dominant Retinitis Pigmentosa (ADRP) is an inherited eye disease, which can lead to photoreceptor cell death, and result in reduced vision and complete blindness. This disorder is estimated to affect one in 3000–5000 individuals worldwide. The main causes of ADRP are mutations in rhodopsin (the light pigment protein); this glycoprotein is responsible for vision under dim light conditions. There have been more than 140 mutations identified on the rhodopsin gene which are linked to ADRP. The cellular trafficking of rhodopsin to the outer segment of vertebrate rod cells is critical for normal function; misfolding interferes with the normal trafficking pathway and may eventually lead to photoreceptor degeneration.
We have developed inducible stable HEK293S cell lines expressing wild-type and mutant rhodopsin-GFP to gain greater insight into pathways triggered by its misfolding. Using a combination of experimental procedures, standardized optimal three-dimensional image acquisition, volumetric and super-resolved localisation analysis, we characterise the phenotypes of rhodopsin mutants and show subtle but significant differences.
Mutation P23H, well researched by biochemical means in previous studies, is used as a reference. Some cells expressing mutant rhodopsin were treated with the 11-cis-retinal cofactor to see if they responded with improved folding and plasma membrane localisation; these are called ‘rescued’ cells.
Live imaging of the cells was conducted to monitor formation of aggresomes and translocation of rhodopsin-GFP to the plasma membrane. In fixed cells, variable levels of GFP expression combined with immunofluorescence showed a considerable difference in dynamic range. Wild-type and rescued mutant cells shared similar localisation and volumes of rhodopsin-GFP. Calnexin, an endoplasmic reticulum chaperone important for assisting the folding of glycoproteins, was also quantified using monoclonal antibody (mAb) labelling. We discuss the unexpected finding of lower calnexin-mAb volumes found in P23H cells.
Performances of visual experts and algorithms for overlap measurement and object-based colocalisation with high measurement accuracy are compared. They were applied to a growing database of three-dimensional multichannel images of single HEK293S cells expressing various rhodopsin-GFP mutants.

Fig. 1: Colocalisation of rhodopsin-GFP and calnexin-mAb in HEK293S cells expressing wildtype (WT) and mutant (P23A and P23H) rhodopsin-GFP (green) . Immunofluorescently labelled calnexin is shown in red and DAPI-stained nuclei in blue. Note poor plasma membrane localisation and aggresome formation of mutant P23H rhodopsin-GFP. Scalebars: 10μm.

Fig. 2: Scatterplots of rhodopsin-GFP and calnexin-mAb volumetry in cells expressing wild-type (WT), mutant (P23H and P23A) and rescued mutant (P23H resc) rhodopsin. 
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-3463 Image processing application in Mathematica™: measuring complex shaped metal nanoparticles in noisy micrographs

Novotný F.1, Proška J.1
1Department of Physical Electronics, Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Břehová 7, 115 19 Prague, Czech Republic
filip.novotny@fjfi.cvut.cz

Plasmonic noble metal nanoparticles (MNPs) retains a special place in today’s nanotechnology efforts. Their complex shapes gives them unique anisotropic optical and physico-chemical properties. Moreover, the possibility to synthesize MNPs in high yield as colloidal solution makes them cost-effective nanomaterial. The number of prospective applications for colloidal MNPs is still growing – from novel immunolabelling for both optical and electron microscopy, model nanoparticle for emerging field of theranostics, to building blocks of optical metamaterials, literally forming ordered arrays of nano-antennae. As the new technology employing MNPs emerges, the need for fast and versatile methods for their quantitative characterization is increasingly needed. Although TEM images produces superior quality with marginal noise, modern nanoparticle application often requires to characterize the particle distribution and shape purity on arbitrary substrates including non-conductive substrates as glass etc. Modern FE-SEM microscropes allows for quality images however with substantial noise. The advanced image processing filters in Mathematica™ allows to prepare the noisy micrographs for segmentation by thresholding or edge detection. The filtered images are then used for shape interactive shape detection mechanism. In this talk will be presented an image processing application in Mathematica™ designed primarily for electron microscope micrographs processing and analysis of particle shapes and size. The application combines the Mathematica™ built-in advanced image processing algorithms together with its outstanding dynamic functionality to create a fluid user interface with improved workflow of the image analysis process.

This work was supported by the Czech Science Foundation
Project P205/13/20110S.

Fig. 1: Selected screenshots from the application interface depicting (a) the image filtering part for thresholding/edge detection and (b) the component measurement with interactive histogram selection controls (left) to filter the unwanted shapes.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-3477 A Texture Based Algorithm for Analyzing Transmission Electron Micrograph Images of Nanoparticles for Size Estimation

Benitez D. S.1, Debut A.1, Guerra A.1
1Centro de Nanociencia y Nanotecnología, Universidad de las Fuerzas Armadas ESPE Sangolquí, Ecuador
dsbenitez1@espe.edu.ec

This paper presents a method for image enhancement of nanoparticles obtained from Electron Transmission Microscopy based on the two-dimensional Hurst operator for detecting edges and characterizing texture and distribution information. To measure the size of a particle, a correct identification of its edges is required; therefore for nanoparticle image analysis the edge detector to use should be able to detect weak edges and also have good noise immunity. A texture sensitive detector may help with the correct identification of each structure and assist to separate the components. In this work, we describe a new image-processing algorithm, based on the local two-dimensional Hurst operator, to improve image quality and better define the edges of the nanoparticle for later size measurement. The local two-dimensional Hurst operator uses a two-dimensional range-based neighborhood operator based on a “local Hurst operator” to extract in one operation both the edge and texture information from an image. First, a de-noising stage is performed to the original image to improve quality. Noise reduction was achieved by using wavelet transformations. Several nanoparticle samples were prepared at our Centro; these nanoparticles were composed of zerovalent or sulfate iron and carboximethyl cellulose (CMC). Images were recorded digitally with a FEI Tecnai Spirit Twin TEM operated at 80kV. Nanoparticles images samples were processed using the new image-processing algorithm in order to determine its performance. Fig. 1 shows a sample of resulting images obtained before and after image processing. Images are shown in 1024x1024 pixel resolution obtained after scaling the resulting images for visibility. As it can be seen in Fig. 1 a good improvement in image quality has been obtained. Depending of the image, the information content has been enhanced; the original structures of the particle are now clearly visible and discernible furthermore after the final stage the edges of the particle are well defined, having now a clear separation between the background and the particle. Therefore, a more precise and reliable size measurement can be performed in a next step. Experimental results show that the method is not only robust and repeatable, but it can also accommodate both nearly spherical and more irregularly shaped nanoparticles of different sizes and configurations. The results obtained suggest superior performance to previous image processing techniques and may provide a very useful tool for nanoparticle research. The algorithm was relatively easy to implement using modern software tools (such as Matlab or LabVIEW), further developments of the system will include the automated measurement of the nanoparticle size and separation of structures of interest.

Research was partially supported by the Secretary of Education, Science, Technology and Innovation of the Ecuadorian Government (SENESCYT) under the “Prometeo Program”.

Fig. 1: Figure 1. Example of images obtained after processing different nanoparticles samples. An octagonal neighborhood with 4 pixels of radio was used for the operator. Images c.1, c.2 y c.3 were obtained after applying a Hilbert-based edge detector to the imagines a.1, a.2, and a.3 obtained after applying the Hurst operator and wavelet de-nosing.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-5914 CHARACTERIZATION OF BIMODAL PARTICLE SIZE DISTRIBUTIONS (PSD) BY SCANNING ELECTRON MICROSCOPY (SEM) AND LASER DIFRACTION (LD)

Gallardo A.1, Cabrera F.1, Villar M. A.1, Bucalá V.1, Yañez M. J.2
1PLAPIQUI (UNS-CONICET), 2Laboratorio de Microscopía Electrónica (UAT-CCT BB)
mvillar@plapiqui.edu.ar

Among the more popular techniques for PSDs measurement, the SEM images analysis (IA) and laser diffraction (LD) are used alone or combined. In this work, 1 g of lactose (mean dnv= 26.5 μm, particle density = 1.36 g/cm3) and 3 g polyvinylchloride (PVC) resin (mean dnv = 142.6 μm , particle density = 1.51 g/cm3) particles were mixed to obtain a bimodal PSD. Figure 1 shows a SEM micrograph of the studied particulate system. Based on the materials properties, a relationship between the number of lactose (NL) and PVC (NPVC) particles of 46:1 is expected. The NL:NPVC ratio obtained from the LD number distributions was 43:1. However, the number passing cumulative distributions obtained from IA revealed that NL:NPVC varied from 4 to 33, being this relationship strongly influenced by the SEM sample preparation. The basic calculation and the information provided by LD (which handles thousand of particles) allowed improving the method of mounting particles on the SEM stub. Figure 2 shows the number passing cumulative functions obtained by LD and IA (using the best SEM sample preparation method). LD estimates a higher NL than that calculated from IA, probably because LD can process much more particles and it is less sensitive to segregation problems than IA.
Taking into account the mass ratio of lactose and PVC and their densities, the theoretical cumulative volume of lactose (VL) and PVC (VPVC) should be around 23 % and 77 %, respectively. LD provides directly the volume cumulative distribution of the powder that is being analyzed, instead for IA, this function has to be calculated from the number cumulative function assuming a given 3D particles shape. Figure 3 shows the volume cumulative passind distributions for LD and IA, for the last method spherical particles are assumed. These results indicate that VL for IA and LD was 28 and 18 %, respectively; values that are between the theoretical value (23 %). Eventhough IA estimates less NL, higher VL is found for this technique. For the IA data, the calculation of the volume of each class of particles assuming spherical shape requires to elevate the diameters to a power of 3. Eventhough lower NL is predicted by IA, the diameter measurement errors or the deviation of the particles shape from a sphere may lead to the observed higher VL.
The simulated bimodal PSD and its evaluation by IA and LD allows stablishing the prediction errors and modifying sample preparation procedures to optimize the PSD chacterization.

Authors express their gratitude for the financial support granted by the Consejo Nacional de Investigaciones Científicas y Técnicas and the Universidad Nacional del Sur

Fig. 1: SEM image of the bimodal PSD

Fig. 2: Comparison of number passing cumulative distributions obtained by IA and LD.

Fig. 3: Comparison of volume passing cumulative distributions obtained by IA and LD.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-5938 Comparative evaluation of image segmentation algorithms for microscopic cross-section samples

Beneš M.1, 2, Zitová B.1
1Institute of Information Theory and Automation, AS CR, Prague, Czech Republic, 2Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic
miroslav.benes@utia.cas.cz

We present results of the project aimed at comparative evaluation of image segmentation algorithms for microscopic cross-section samples. Despite the longtime effort to develop high quality segmentation algorithms, there is no universal segmentation method available. Under these circumstances, there is a dilemma which method to choose for given particular data set. Moreover, images of microscopic samples can be of various character and quality which can negatively influence the performance of image segmentation algorithms. Thus the issue of selecting suitable method for a given set of image data becomes even more prominent. We carried out a large number of experiments with a variety of segmentation methods to evaluate the behavior of individual approaches on the testing data set. We limit our study to the microscopic images that contain the sample located in the inner part of the image, mostly not reaching to the top and bottom image borders. The data may come from an analysis of painting materials used in art restoration, which is the case of the data set used in our evaluation. They can be samples of various biological materials, such as tissues, cells, or other biological structures. The task is to label an image with either foreground or background label, where the foreground is usually the inner part of the image and the background is separated and/or removed. The background is cluttered with various debris which makes the task complicated.

The set of studied segmentation methods covers various approaches such as thresholding, region growing, clustering methods and graph-based algorithms. Their results and performance were objectively evaluated by ten representative indices used for measuring the output quality of image segmentation algorithms. The main objective was to find the best average segmentation method. The method which is comparable to the best method for particular image in case of easy to segment images (majority methods can segment this image with satisfactory results) and does not completely fail in case of worse images (where most of the methods fail). Such method was found for three studied modalities (visible and ultraviolet spectra and output of scanning electron microscope) and also the lists of segmentation methods ranked according to their performances were produced through rank aggregation process. Mean Shift algorithm (EDISON) generally performed the best and thus can be considered the best segmentation method on average for related data. We verified the findings on separate testing data set and the applicability of the evaluation results was also shown on biological data.

The results of the project were submitted to the Journal of Microscopy (John Wiley & Sons).

The work has been supported by the Czech Science Foundation under project GAP103/12/2211.

Fig. 1: The image of the cross-section sample in visible spectrum. Courtesy of ALMA laboratory.

Fig. 2: The cross-section from figure 1 segmented by Mean Shift algorithm. The background is successfully removed. Courtesy of ALMA laboratory.

Fig. 3: Demonstration on biological image data -- mouse retina colored withhematoxylin-eosin. Boundary of segmented result by Mean Shift algorithm isdepicted by red line. Courtesy of Jan Cendelin, Faculty of Medicine in Pilsen.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-9-P-6042 An inexpensive system for quantitative analysis of mosaic form of Turner/Klinefelter syndrome

Schier J.1, Kovář B.1, Kuneš M.1, Honec P.2, Číp P.2, Zemčík P.3, Dubská M.3, Kočárek E.4, Tesner P.4
1Institute of Information Theory and Automation, Czech Republic, 2CAMEA, spol. s r.o. (Ltd.), Czech Republic, 3Faculty of Information Technology, Brno University of Technology, Czech Republic, 4Department of Biology and Medical Genetics, 2nd Faculty of Medicine, Charles University
schier@utia.cas.cz

In our contribution, a prototype of a simple low-cost system for automated evaluation of mosaic form of chromosome aneuploidies will be introduced. The primary goal of this system is to improve the accuracy of evaluation of simple chromosome aberrations in mosaic form, which are related to the Turner (mos 45,X/46,XX) and Klinefelter syndrome (mos 47,XXY/46,XY).

Since the frequency of the abberant nuclei is rather low in the mosaic form (up to approx. 5%), it is necessary to evaluate the counts of the fluorescence signals in a sufficient set of nuclei images (approx. 1000 interphase nuclei or more). From the point of view of image processing, this includes the standard operations of image preprocessing, segmentation, spot detection and parameter evaluation, followed by filtering based on object parameters. The prototype system is composed of hardware part, which provides automated image acquisition, and software part for image processing, image database access, statistical evaluation, vizualization, etc. To large extend, the system is composed of the COTS components: the Euromex OX.3075 fluorescence microscope with a plan fluorite lens and an Edmund Optics motorized stage have been used for image acquisition. The control protocol for the stage and the illumination system have been developed inhouse by the CAMEA company, to ensure optimum performance. The software part of the system builds partly on the open-source software: the image processing module (preprocessing, segmenation, parameter evaluation) has been designed as an ImageJ plugin, the machine learning part, which is used to filter the objects (nuclei and signals) on the basis of parameters, is implemented using the WEKA library. To store the images, the MySQL database is used. Currently, we are also testing incorporation of the OMERO client-server software, which could serve both for storing the images and for vizualition, as well as the backend system.

To test the system, samples of cultivated lymphocytes from patients with different forms of gonosomal aneuploidies have been used, as well as artificial mixed male and female samples with known ratio of cells with different karyotypes. The samples were hybridized with alpha-satellite FISH probes, which marked centromeres of the X chromosome in the individual cells (for Turner syndrome). A 40x and 60x magnification lens has been used in our experiments, resulting in images with approx. 20 nuclei. To obtain good quality images, exposition time of 500 ms was used and we have achieved processing time for one image bellow 2 s.

Currently, works are undergoing on integration of the components of the system into final prototype.

This research has been supported by the Technology Agency of the Czech Republic grant program Alfa, project TA01010931

↑ Go to the Table of content ↑

ID-10. Advances in sample preparation techniques

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-10-IN-2083 Cryo-Focused Ion Beam Sample Preparation of Biological Specimens for Cryo-Electron Tomography

Schaffer M.1, Villa E.1, Engel B. D.1, Laugks T.1, Hoffmann T.1, Ortiz J.1, Mahamid J.1, Fukuda Y.1, Schüler M.1, Baumeister W.1, Plitzko J. M.1
1Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany
schaffer@biochem.mpg.de

There has recently been a major leap forward in cryo-electron tomography (cryo-ET) of biological specimens due to the introduction of direct detection cameras, with their unsurpassed speed and sensitivity, and contrast enhancing phase plate technology. These new tools allow the acquisition of data with unprecedented clarity and spatial resolution despite the stringent restrictions of low-dose microscopy. Cryo-ET now offers the opportunity to approach a whole new range of structural biology questions.

However, the study of complex molecular processes in situ and within large organisms is still hampered by the thickness of the samples. Many biological systems of interest exceed the thickness requirements for transmission electron microscopy. Sections, thinner than 500 nm, have to be produced without compromising the intricate cellular architecture. Cryo-focused ion beam (cryo-FIB) milling of frozen hydrated samples provides a unique opportunity to generate such thin sections while minimizing preparation artifacts [1,2]. Targeted milling of electron-transparent ‘windows’ into larger cellular specimens permits access to structures buried deep inside cellular volumes [3], enabling tomography to be performed on these structures at unprecedented resolution.

In this work, we present the cryo-FIB preparation technique as a fully integrated component of the general in situ cryo-ET workflow. In situ cryo-ET requires fully vitrified sections of homogenous thickness without mechanical distortions and surface contamination. We discuss the necessary steps and techniques to achieve this goal with high fidelity and reproducibility, and then show the improvements for data acquisition with direct detectors and phase plate technology.

Examples of cryo-FIB applications on different biological systems demonstrate the versatility and power of the combined techniques.

References:

[1] M Marko et al., Nat Methods 4(3) (2007) p.215.

[2] A Rigort et al., PNAS 109(12) (2012) p.4449.

[3] E Villa et al., COSTBI 23(5) (2013) p.771.

Fig. 1: (a) Schematic of cryo-FIB preparation on a plunge-frozen hydrated cell. (b) TEM grid mounted in a modified Autogrid cryo-holder. (c) SEM image of yeast cells on a TEM grid. (d) FIB SE image of a lamella after cryo-FIB milling. (e) Cryo-TEM overview of the yeast lamella and (f) magnified view of a nucleus double membrane within one yeast cell.

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-10-IN-5888 Correlative Light and Electron Microscopy: taking snapshots of the living at the ultrastructural level

Schwab Y.1
1European Molecular Biology Laboratory, Heidelberg, Germany
schwab@embl.de

Our work is focused on the development of methods that enable high-resolution snapshots of dynamic events or of subcellular structures in cells and multicellular organisms. To achieve that, correlating light and electron microscopy is a powerful solution. By improving targeting strategies, we are successfully combining live imaging and electron microscopy on various sample types, allowing the correlation in three dimension of rare events in cultured cells, nematodes, zebrafish embryos and mouse tissues.

A generalized way to achieve the correlation and to trace the objects of interest across the switch in imaging modalities is to rely on specific landmarks that are used to navigate into the sample, to specifically collect sections at the site of interest and to register the acquired images. With cultured cells, we utilize coordinates systems etched at the surface of the culture substrate, a widespread solution that is compatible with a large variety of sample preparation techniques for electron microscopy (e.g. chemical fixation, high-pressure-freezing). On multicelluar organisms, the targeting to the region of interest is performed by the combined use of anatomical cues and of engineered landmarks that are recognizable in both light and electron microscopy. By imaging the sample in 3D, maps of its volume can be generated. These maps are then utilized to navigate the block and to target the sectioning precisely to the region of interest.

With the widespread use of easy-to-implement but efficient sample preparation methods, we believe that correlative light and electron microscopy has the potential to serve as a powerful tool to achieve single cell recordings in heterogeneous systems allowing to link functional imaging with ultrastructural analysis.

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-10-O-1591 THE NANOWORKBENCH: AUTOMATED NANOROBOTIC SYSTEM INSIDE OF SCANNING ELECTRON OR FOCUSED ION BEAM MICROSCOPES

Burkart I.1, Klocke V.1, Maynicke E.1
1Klocke Nanotechnik GmbH, Aachen, Germany
maynicke@nanomotor.de

In light microscopy it is natural to use toolsets like tweezers, knives, probes and several different measurement tools. Without this many present-day products and methods would not exist.

The operators of SEM/FIB-Systems generally work without toolsets, although the wavelength limit of light is no physical boundary. It can be imagined how technology would be pushed when a SEM/FIB Workbench reaches the same degree of practicability and utilization as toolsets for light microscopes.

The success of in-SEM/FIB-Nanorobotics depends on several important features:

• Nanomanipulators in automation, for movement of endeffectors, sample handling, and preparation,

• Endeffectors for nano- probing, cutting, cleaning, force measurement, gripping, sorting or material preparation,

• Automatic 3D tool- and sample position detection, 3D sample topography measurements,

• Precise control of all tool positions including SEM/FIB sample stage in global coordinates,

• SEM picture assisted haptic interface by “Live Image Positioning”,

• One common automation control for Nanorobotics and SEM/FIB.

Expanding the SEM/FIB to a material processing system and a nano-analytical workbench by fulfilling these upper development tasks enables new applications in research and production of material research, live sciences, tribology, environmental & forensic research and semiconductor technology.

Several examples of these new interdisciplinary research and development fields will be described, together with the invitation to participate at our research network forming further new applications.

A few examples of Nanoworkbench applications are highlighted in Fig 1. Although these examples may raise the impression of a review about different machines and their usage, this is not the case. Described is the development of the Nanoworkbench.

References:

[1] D. Morrant, EIEx Magazine of European Innovation Exchange, 1 (2009)

[2] G. Schmid, M. Noyong, Colloid Polym Sci., (2008)

[3] C.-H. Ke1, H.D. Espinosa, Journal of the Mechanics and Physics of solids, 53 (2005)

[4] Seong Chu Lim, Keun Soo Kim, Kay Hyeok An, Dept. of Phys., Sungkyunkwan University, Korea (2002)

[5] Supported by European Commission, IST and Ziel2.NRW

Fig. 1: Fig. 1. includes the rows: Nano-Probing [2]: The electrical conductivity along these gold chains is measuredNano-Tribology can be performed in high resolution [4]Nano-Cutting: fast milling of structures3D-Nanofinger: measurement of gold and EBID structuresParticle-Sorting: Gripping of a rigid CNT bundle [3]
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-10-O-2545 Changes of Materials’ Properties at the Surface of TEM-Samples Due to Low-kV Argon Ion Milling

Müller E.1
1Electron Microscopy Facility, Paul-Scherrer-Institute Villigen, Villigen/PSI, Switzerland
elisabeth.mueller@psi.ch

Abstract

The availability of Cs-corrected transmission electron microscopes (TEM) has strongly increased the quality requirements for TEM samples. Especially, the amorphous layers at the sample surface have to be as thin as possible. If Ar-ion milling is applied for cross-section TEM sample preparation, finishing the ion-milling procedure with a voltage as low as a few hundred volts may account for this [1-6]. This study, however, reveals 3 additional effects of the low-voltage Ar-ion treatment.

For the present study, a Precision Ion Polishing System (PIPS, Gatan) equipped with low voltage option was used for ion milling (final milling at 0.5kV for 5 minutes; etching angles 3 - 5°). Three specimen types (pure silicon, III-V semiconductors and SrTiO3) were investigated in a Philips CM12 (100kV) and a FEI Tecnai F30ST (300kV) TEM as well as a Hitachi HD-2700Cs (200kV) dedicated STEM.

The fine polishing at low acceleration voltage resulted in a clear reduction of the thickness of the amorphous surface layers (Fig. 1). This was, however, not the only effect observed: all three sample types reproducibly showed severe charging in all three microscopes. This effect was – especially in the dedicated STEM – present with and without subsequent plasma-cleaning for reduction of contamination. The low-voltage milling furthermore caused some re-deposition of Cu-particles onto the sample from the Cu-tube into which the material had been embedded. The Cu was observed at material boundaries and at the sample edges. It is assumed that this was due to electrostatic fields built up by charging effects already during the milling process (Fig. 2). Last but not least, another important property of the material – its oxidation behaviour – appeared to have changed drastically: While in a TEM sample the AlAs layers of an AlAs/GaAs superlattice delaminated after few hours due to oxidation, if the sample was milled at 3.5kV, the oxidation was almost fully suppressed for several weeks after low-kV milling (Fig. 3). Only along dislocations oxidation was still observed to occur.

References

[1] T. Schuhrke, M. Mandl, J. Zweck, H. Hoffmann, Ultramicroscopy 41 (1992), p. 429

[2] A. Barna, B. Pécz, M. Menyhard, Ultramicroscopy 70 (1998), p. 161

[3] A. Barna, B. Pécz, M. Menyhard, Micron 30 (1999), p. 267

[4] J.P. McCaffrey, M.W. Phaneuf, L.D. Madsen, Ultramicroscopy 87 (2001), p. 97

[5] J. Mayer, L.A. Giannuzzi, T. Kamino, J. Michael, Mrs Bulletin 32 (2007), p. 400

[6] M.J. Süess, E. Mueller and R. Wepf, Ultramicroscopy 111 (2011), p. 1224.

The support with sample preparation by Eszter Barthazy and the possibility to use the infrastructure of the Microscopy Centre at ETH Zürich, ScopeM, are gratefully acknowledged.

Fig. 1: HRTEM images of SrTiO3 show an amorphous layer at the sample edge of about 8nm after ion etching at 4kV (left). The amorphisation is reduced to below 2nm after a low-voltage etch of 0.5kV (right).

Fig. 2: HAADF STEM images of a SiGe/Ge superlattice on Si(001). Small particles are decorating the interface between the superlattice and the substrate as well as the edge of the sample (left and central image). EDX maps of the area shown in the HAADF STEM image at top right identified the particles to consist of Cu (right).

Fig. 3: GaAs/AlAs superlattice with final Ar-ion etching step at 3.5kV (left, TEM) and 0.5kV (right, HAADF STEM), respectively. While oxidation of the AlAs layers had caused a severe degradation of the sample within less than 1 day after etching at 3.5kV, the layers were still perfectly preserved 3 weeks after low-voltage milling.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-10-O-2851 Batch Preparation of Plan-View Transmission Electron Microscopy Samples by Vapor Phase Etching with Integrated Etch Stops

English T. S.1, Provine J.1, Marshall A. F.1, Kenny T. W.1
1Stanford University, Stanford, CA, USA
englisht@stanford.edu

In this abstract, we present a method to prepare plan-view transmission electron microscopy (TEM) samples using isotropic vapor phase etching with integrated etch stops. An ultrathin (5-10 nm) etch stop simultaneously serves as a support membrane, providing ideal transmission imaging conditions without the need for mechanical polishing or ion milling in sample preparation. In addition to reducing preparation time and use of consumables, vapor phase etching allows multiple samples to be prepared in parallel due to high etch selectivity (>1000:1) which accommodates over-etch and provides a large, uniformly thick area for imaging.


We highlight the benefits of this technique in high-throughput studies of nanostructures and thin films prepared by conformal deposition techniques where it is challenging the use commercially available grids with pre-thinned membranes due to front and backside membrane coating. While there are methods to prevent deposition onto both membrane faces, including mechanical clamping and masking structures, these solutions are not ideal in many studies due to limited temperature stability, outgassing, and significant topology differences introduced by the mask. The proposed vapor phase process requires consideration of etch selectivity in order to minimize the introduction of artifacts during preparation. However, selectivity against a wide range of dielectrics, metals, and polymers is possible using either XeF2 or vapor HF in conjunction with a single etch stop layer. Additionally, the sample surface is protected by a coating of Crystalbond 509 throughout the preparation process to minimize exposure to etchants.


We demonstrate the application of this technique to characterize Volmer-Weber nucleation of platinum deposited by plasma-enhanced atomic layer deposition (PEALD). Figure 1a shows a cross-section TEM image of a PEALD Pt film deposited onto an oxidized silicon wafer with a 5 nm Al2O3 seed layer. An isotropic XeF2 etch of silicon stops on SiO2 while anhydrous vapor HF is used to subsequently etch the 300 nm SiO2 down to the Al2O3 film through which the Pt film is imaged. The total tool time to complete vapor phase etching of multiple samples is typically less than one hour. Figure 1b compares 3 mm cored samples prepared by conventional (grinding, polishing, dimpling, ion milling) and vapor phase processes. Figure 2 shows a comparison of plan-view TEM images from the same 100 cycle film prepared using conventional and vapor phase methods. The schematics in figure 3a outline the preparation process. Samples ranging from 85-300 ALD cycles are prepared using the presented process and shown in figure 3b demonstrating grain structure evolution during Volmer-Weber nucleation and the transition from contiguous to continuous films.

Fig. 1: (Left) Cross-section transmission electron micrograph of sample layer structure. (Right) Comparison of samples prepared by conventional and vapor phase methods. The vapor phase sample is shown prior to HF etching. While the membrane commonly cracks, regions suitable for imaging remain along the periphery.

Fig. 2: Comparison of the same 100 cycle PEALD Pt film prepared by conventional hand and vapor phase methods.

Fig. 3: (Top) Sample preparation overview, not to scale. (Bottom) Plan-view transmission electron micrographs showing Volmer-Weber nucleation PEALD Pt films deposited on Al2O3 at 250 oC.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-10-O-2903 Cellular Electron Microscopy Methods for a New Generation: Specimen Preparation Procedures for Resin Embedding of Cryofixed Biological Samples in 6 Hours

McDonald K. L.1
1Electron Microscopy Laboratory, University of California, Berkeley, USA
klm@berkeley.edu

In the past few years my laboratory has been exploring alternatives to the conventional lengthy procedures used to fix and embed cells for cellular electron microscopy (microscopy of whole cells and tissues). We start with cells frozen by high pressure freezing, then dehydrate and stabilize the ultrastructure by freeze substitution (FS). We have reduced the time for FS from several days to a few hours. Resin infiltration and embedding is done in 2-3 hours with no special equipment. We also have a new method for on-section immunolabeling by doing FS with uranyl acetate in acetone and embedding in LR White.

FREEZE SUBSTITUTION. As described in McDonald and Webb [1] we now do FS using simple, inexpensive equipment instead of a costly automated freeze substitution (AFS) device. Briefly, frozen samples are placed in cryovials containing frozen fixative and placed in a metal block cooled to liquid nitrogen temperature. The metal block is placed in a foam box that is then put on a rotary shaker operating at 100-125 rpm. The samples are warmed up passively over 2-3 hours to room temperature at which point the fixative is rinsed out and the embedding process is begun.

RESIN INFILTRATION AND POLYMERIZATION. We used to do quick processing using a microwave oven but wanted rapid procedures that did not require this expensive equipment. We found that microwaves were not actually necessary and also discovered that rapid embedding procedures were not new [2]. Briefly, we do a stepwise increase in epoxy resin:acetone concentrations from 25, 50, & 75%, then 3 times in pure resin for 5-15 minutes each with centrifugation at 2,000 x g for 30 seconds to a minute in between changes. Polymerization is at 100 degrees C for 2 hours. While we can go go from live cells to sections in the microscope in one working day, in practice we often freeze, FS, and embed on one day and section and look at the samples the next. Results from a variety of cell and tissue types is shown in recent publications [3,4].

ON-SECTION IMMUNOLABELING. Specimens are freeze substituted in 0.2% uranyl acetate in acetone to room temperature, then rapidly embedded as above in LR White which is polymerized at 100 degrees C for 90 minutes. The advantage of not using traditional fixatives is that more antibodies are likely to work at the EM level because the fixatives are not blocking their access to antigens.

[1] McDonald, K. & R. Webb. 2011. J. Microscopy 243:227-233.

[2] Hayat, M.A. & R. Giaquinta. 1970. Tissue and Cell 2:191-195.

[3] McDonald, K. 2014. Microsc. Microanal. 20:152-163.

[4] McDonald, K. 2014. Protoplasma 251:429-448.

Fig. 1: Figure 1 (left) shows chloroplasts from a leaf of white clover (Trifolium repens) prepared by high pressure freezing followed by FS in 2.5 hours and embedding in Epon resin in 3 hours. Bar = 200 nm.

Fig. 2: Figure 2 (right) was prepared by high pressure freezing, FS in 0.2% uranyl acetate in acetone, embedding in LR White, and labeled with 10 nm gold to show actin in the microvilli of C. elegans. Total processing time about 6 hours. Bar = 200 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-1499 Towards reliable preparation of serial sections for correlative array tomography and 3D reconstruction

Spomer W.1,4, Wacker I. U.2,4, Bartels C.2, Hofmann A.1,4, Scharnowell R.1,4, Schröder R. R.3,4, Gengenbach U.1,4
1Institute for Applied Computer Science, Karlsruhe Institute of Technology, Germany, 2Center for Advanced Materials, Universität Heidelberg, Germany, 3CryoEM, CellNetworks, Universitätsklinikum Heidelberg, Germany, 4HEiKA Correlative Imaging Platform, Heidelberg-Karlsruhe Research Partnership
waldemar.spomer@kit.edu

For 3D reconstructions of biological material array tomography (AT) is a relevant method [1]. To find special regions of interest (ROI) within a large volume correlative AT (CAT) allows defining ROIs in the light microscope first which are then imaged in an SEM. For CAT serial sections are usually placed on glass coverslips coated with indium tin oxide (ITO) to render the substrate conductive. To overcome the difficulties of collecting serial sections on that substrate we developed a mechanical multi-axes support on a commercial ultramicrotome, which allows us to reproducibly create large arrays of sections (Fig. 1A). At closer inspection (Fig. 1B) it is obvious that section thickness is not uniform. For qualitative 3D reconstructions such a systematic variation is not that important, one usually assumes the nominal thickness chosen at the ultramicrotome as section thickness for the 3D volume. However, if a quantitative statistical analysis is the aim of the study, e.g. of membrane topology, such as membrane area and curvature [2] exact section thicknesses are required. Furthermore, we found that nominal thickness from the ultramicrotome’s display not necessarily agrees with actual section thickness measured using an Atomic Force Microscope (AFM, Fig. 2). Currently we are investigating these variances in more detail (e.g. temperature impact caused by environment, operator and system).

To achieve more reliable and quantitative 3D reconstructions we propose to monitor section thickness during sectioning with a digital camera. This approach utilizes the thin film interference effect, which causes the sections to be colored depending on their thickness (Fig. 3 A) [3]. A mathematical model is obtained by first measuring section thickness with an AFM (Fig. 2). These values are then correlated with the color values given by the camera (Fig. 3 B). In that way it is possible to record the thickness of every section during the cutting process. However, as shown in Fig. 3b, for the lower end of the typical ultrathin range (50-100 nm) discrimination by color information is sensitive to minimal variations of the detected spectral intensities at the camera chip. Investigations towards e.g. calibrated illumination and spectral characterization of the complete optical path (including camera) are ongoing. Due to this limitation quantitative color-based analysis is at the moment only possible for sections thicker than 100 nm. For high resolution AT of thicker sections FIB milling provides an alternative approach (cf IMC 2014 abstracts, Wacker et al., Schröder et al.).

References
[1] Wacker and Schröder (2013), J Microscopy 252, 93-99
[2] Leitte et al., EuroVis 2013, 32, No 3
[3] Parthasarathy et al., IEEE Proc. ICRA 1987, No 4

We acknowledge funding by HEiKA and the German Federal Ministry for Education and Research, project NanoCombine, grant FKZ: 13N11401.

Fig. 1: Thickness variations in serial sectioning: 220 serial sections (A) placed on ITO-coated glass coverslip for CAT between alignment markers (arrows); close-up of sections (B).

Fig. 2: Thickness profile of a section: Thickness measurement (B) obtained from the AFM image (A: along dotted line)

Fig. 3: Colors of sections on water surface of knife boat (A) - nominal section thickness grows from bottom (50 nm) to top (190 nm). Correlation between thickness and RGB signal levels (B) of digital camera.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-1594 Crystal structure of a polyvinylidene revealed by chemical nonequilibrium plasma etching and chromic attack.

Urbina de Navarro C. T.1, Diaz N. L.1
1Universidad Central de Venezuela, Caracas, Venezuela
caribay.urbina@ciens.ucv.ve

Although the existence of the so called emerging techniques, such as EELS, AFM, etc. which have allowed imaging of materials such as polymers, almost without preparation, not all electron microscopy laboratories have access to them, so using clasiccal physical or chemical techniques can not be excluded. The aim of this investigation was to determine the working condition of a homemade chemical nonequilibrium plasma device to reveal the Polyvinylidenefluoride (PVDF) morphology and compare with the results obteined by chemical etching. One of the most remarkable characteristics of polyvinylidene fluoride is its crystalline polymorphism, having identified a total of five different crystalline phases. This material is a thermoplastic polymer with excellent mechanical properties, high resistance to strong acids and alkalies and other highly corrosive solvents. Being chemically inert it is very difficult to reveal the morphology of these polymers by chemical etching. For the application of plasma chemical etching three different gases: Ar, O2 , air were used. Chemical attack was performed using a modification of the method suggested by A. S. Vaughan. Figure 1 shows the spherulites that appear in the sample after attack during 25 minutes with oxygen plasma. Polymer surface after being subjected to air plasma and argon respectively are shown in Figures 2 and 3. For these treatments PVDF spherulites were observed, but melted areas and excessive wear of the crystals also was found. In all cases, the spherulites found correspond to the alpha phase. The chemical treatment (H2SO4 /P2O5/Cr2O3) during 72 hours revealed the presence of alpha and mixed phases, figure 4. Use of oxygen plasma causes the least damage to the crystal structures of PVDF.

This research was supported by Nat Lab Project FONACIT 20010001442

Fig. 1: O2 Plasma

Fig. 2: Air Plasma

Fig. 3: Ar Plasma

Fig. 4: Chemical etching
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-1621 Advanced FIB lamella lift out technique from a TEM foil

Mueller J.1, Spiecker E.1
1Center for Nanoanalysis and Electron Microscopy (CENEM), Erlangen, Germany
julian.mueller@ww.uni-erlangen.de

To understand the mechanical properties and deformation mechanisms of crystalline materials it is important to study dislocation structures from the micrometer to the atomic scale. Conventional TEM can be used to reveal the arrangement of dislocations and determine their Burgers vectors using, e.g., the invisibility criterion (g.b=0). However, hardly any information about the core structure of the dislocation is obtained. In a sample which is suitable for high resolution imaging only dislocations oriented edge-on in very thin parts of the TEM foil (<50 nm) can be investigated and the key information about the position in the microstructure and the interconnection with other dislocations is lost. Hence, the relevance of the dislocation analysis cannot be assured since a definite correlation is generally not possible.

In this work we present an advanced approach for a FIB lamella lift out from a TEM foil (Fig. 1). Conventional TEM is used to select a specific dislocation (or any other defect or feature) and characterize it in plan-view geometry. Afterwards, the dislocation is cut in the FIB to obtain an ultrathin cross-section sample suitable for HRTEM investigation of the dislocation core.

For the specific case of superdislocations in Ni based superalloys it is particularly important to study the core structure to differentiate between the diverse superdislocation types existing. The core configuration determines if the dislocation is already mobile at lower temperatures or if it can only move at high temperatures via thermally activated processes.

The most important steps of Fig. 1 for the preparation of a superdislocation are illustrated in Fig. 2. In the first step a characteristic dislocation is selected by conventional TEM (1) and analyzed by various techniques, like the invisibility criterion, large angle convergent beam electron diffraction (LACBED) and STEM. It is important to note that, for the particular dislocation, the invisibility criterion was not applicable since it always showed residual contrast. Therefore, LACBED had to be used to determine the Burgers vector. The second step includes locating the dislocation in the SEM and deposition of Pt for protection of the dislocation during FIB milling (2). Afterwards, the FIB lamella is thinned down until it is sufficiently thin for HRTEM investigation (3). Finally, the core structure of the dislocation is analyzed (4).

The specific superdislocation studied in Fig. 2 was found to consist of two superpartial dislocations with Burgers vectors of type a0½<110> located on different {111} planes leading to an overall Burgers vector of a0[-100] in agreement with the LACBED analysis. The two superpartial dislocation dissociate further on the {111} planes forming a complex dislocation core.

The authors gratefully acknowledge the collaborative research center SFB/TRR 103 and the DFG training research group 1229 for financial support.

Fig. 1: Scheme of the advanced lift out technique from a TEM foil. Step (1) – (4) for the preparation of a dislocation for HRTEM analysis of its core structure are illustrated in Fig. 2.

Fig. 2: Important steps in the preparation and characterization of a cross section sample of a superdislocation.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-1965 Comparative study of three sample preparation techniques to prepare a TEM lamella of historical photographs for chemical characterizations

Grieten E.1,2, Leroux F.1, Caen J.2, Schryvers D.1
1EMAT, Department of Physics, University of Antwerp, Antwerp, Belgium, 2Conservation Studies, Faculty of Design Sciences, University of Antwerp, Antwerp, Belgium
eva.grieten@uantwerpen.be

The quality of a TEM sample is an important factor when studying the changes in corrosion phenomena in historical photographs. This material has a complex structure made out a soft matrix with embedded image and corrosion particles. To determine the optimal sample preparation method 2 techniques are evaluated; the classical ultra-microtome and the high tech focused ion beam (FIB). Several parameters were compared such as thickness, uniformity, preservation of original structure and composition.

Classical ultra-microtome is often used for soft materials. Before the sectioning the material needs to be fixated and embedded in an epoxy. No changes to the morphology were noticed during these steps. In spite of the retained composition and achievable thickness the classical ultra-microtome sections are often deformed during section resulting in a low success rate of an intact interface between the corrosion particles and the epoxy (fig1).

With FIB it is possible to directly sample with high selectivity the historical photograph. This is a great advantage when working with historical material where sampling is often restricted. Although it is possible to mill different materials several disturbing features are observed. FIB can cause preferential milling if the difference between the hard particles and soft matrix is big (see fig2). Also the low stiffness of the gelatine results in buckling during the thinning phase. These artefacts make it difficult to make a uniform TEM lamella, which is thin enough for analytical characterization. Any Ga+ implantation during preparation does not influence or disturb the characterization since Ga can easily be distinguished from the corrosion elements (fig 2C).

Since both techniques show artefacts making it difficult to achieve an intact thin and uniform sample a novel adaptation is suggested. Here we use the preparation steps of the classical ultra-microtome with an alternative final sectioning with focused ion beam. The difference between the classical ultra-microtome and ultra-microtome followed by FIB is the last stage or sectioning. This technique produces a TEM lamella with a clear interface and which is thin enough to determine the chemical composition or distribution of the nanoparticles in the corrosion layer (fig3). Although the success rate of this combined procedure is markedly better than that of the two alternatives, the main challenge remains making a thin enough sample to perform analytical characterization.

The authors thank the Photomuseum of Antwerp, Belgium, for providing the historical photographs and S. Van den Broeck for support with specimen preparation with FIB.

Fig. 1: Figure 1: Artefacts with ultra-microtome; A; Overlapping of corrosion and image particles, B: intensity profile from image A showing large variation in deformation, C: successful interface corrosion particles and epoxy.

Fig. 2: Figure 2: Artefacts of FIB: A: buckling and preferential milling; B: curtain artefact in STEM image C: EDX spectra of Ga+ implantation (top) and intensity profile of marked area from figure B (Bottom).

Fig. 3: Figure 3: Combination of ultra-microtome and FIB; A: STEM overview, B: intensity profile of A, C: intact interface between corrosion particles and epoxy.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-2183 Controlled TEM specimen preparation of supported nanoparticle catalysts for quantitative analysis

Pingel T.1, Skoglundh M.2, Grönbeck H.1, Olsson E.1
1Department of Applied Physics and Competence Centre for Catalysis, Chalmers University of Technology, Gothenburg, Sweden, 2Department of Chemical and Biological Engineering and Competence Centre for Catalysis, Chalmers University of Technology, Gothenburg, Sweden
pingel@chalmers.se

Obtaining meaningful statistical information from supported nanoparticle catalysts using TEM can be a challenge. Figure 1 shows an SEM image of a Pt/Pd/alumina catalyst, revealing a complicated three-dimensional mesoporous structure and alumina particle sizes of several micrometers. The specimen preparation method most commonly used for these samples involves crushing the catalyst powder in a mortar, dispersing it in alcohol and depositing some droplets onto a support film, usually carbon. While this approach has its advantages due to simplicity and speed, the obtained specimens are usually very inhomogeneous, making it difficult to find representative areas for the investigation. Also, information about differences in nanoparticle distributions along the alumina particle is lost when crushing the powder.

In this work, specimens have been prepared using the lift-out technique in a combined scanning electron / focused ion beam microscope. Prior to this, the catalyst powder has been embedded in an acrylic resin. The specimens obtained by this method contain regions which originally were inside of the alumina particle, as well as regions of the outer layer of the particle.

Figure 2 shows a STEM image of the outer edge of an alumina particle, revealing the existence of a near surface layer with a higher Pt/Pd nanoparticle density compared to the inner region, especially when looking at nanoparticles larger than 1 nm. Separate particle size distributions for these two regions have been obtained. Additionally, large nanoparticles with diameters above 10 nm are exclusively seen very close to the outer edge. This information cannot easily be extracted using the traditional crushing method.

We acknowledge financial support by the Swedish Energy Agency, AB Volvo, Volvo Car Corp. AB, Scania CV AB, Haldor Topsøe A/S, ECAPS AB, K. & A. Wallenberg FDN and Vetenskapsrådet.

Fig. 1: Secondary electron SEM image of a Pt/Pd/alumina catalyst particle. The alumina support has a complicated mesoporous structure and is several micrometers in diameter, making TEM specimen preparation challenging.

Fig. 2: HAADF STEM image of a Pt/Pd/alumina catalyst prepared by the lift-out technique, showing the outer edge of an alumina particle. Pt/Pd nanoparticles are seen as bright features. A higher density of nanoparticles with diameters above 1 nm can be seen close to the lower edge.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-2394 Short freeze-substitution protocols applied to different plant tissues

Stierhof Y. D.1
1Center for Plant Molecular Biology ZMBP, University of Tübingen, Tübingen, Germany
york.stierhof@zmbp.uni-tuebingen.de

For resin embedding of biological samples it has been shown, that cryofixation combined with freeze-substitution (dehydration at -90°C, chemical fixation between -90°C and 0°C) offers considerable advantages in regard to ultrastructure and antigen preservation in comparison to conventional chemical fixation and dehydration at room temperature. Among them are e.g., fast immobilization (within µsec to msec), simultaneous fixation throughout the whole sample, reduced shrinkage and extraction (lipids, ions, non-fixable molecules), no need for a fixation buffer. Possible disadvantages are e.g., expensive equipment for larger specimen, limited sample size, freezing artifacts, and time consuming freeze-substitution (e.g. Humbel 2009).
In general, the freeze-substitution process takes up to several days. However, it has been shown, that freeze-substitution of few hours prior to resin embedding can be sufficient for dehydration and fixation of biological samples (McDonald and Webb 2011). We have tested short protocols (in the range of 4-5 hours) in comparison to conventional protocols using different plant tissues (anthers containing pollen grains, seedling root tips) and different substitution media with and without the addition of water. Surprisingly, short protocols resulted in clear differences in the quality of ultrastructure preservation between different tissues indicating that different conditions of freeze-substitution are required for different tissues (Figs. 1-4). In some cases the addition of water to the freeze-substitution medium had a negative effect on ultrastructure preservation (Fig. 4).

References:
Humbel BM (2009) Freeze-substitution. In: Handbook of Cryo-Preparation Methods for Electron Microscopy, eds Cavalier A, Spehner D, Humbel BM, CRC Press, pp 319-341.
McDonald KL, Webb RI (2011) J. Microscopy 243, 227-233.

Fig. 1: Fig. 1: Epon-embedded pollen grain after long freeze-substitution without water.

Fig. 2: Fig. 2: Epon-embedded pollen grain after short freeze-substitution without water). Both pollen grains are well preserved.

Fig. 3: Fig. 3: Epon embedded root tip after long freeze-substitution.

Fig. 4: Fig. 4: Epon-embedded pollen grain after short freeze-substitution with water. Ultrastructural damage can be seen.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-2697 Nb-doped Sr3Ti2O7 platelets: Preparation of cross-sections thin foils for TEM observations

Gec M.1, Jerič M.1, Čeh M.1,2
1Jožef Stefan Institute, Ljubljana, Slovenia, 2Center for Electron Microscopy and Microanalysis, Jožef Stefan Institute, Ljubljana, Slovenia
medeja.gec@ijs.si

Nb-doped SrTiO3 is a promising n-type thermoelectric material. It is expected that by triggering textured grain growth one will be able to improve thermoelectric figure of merit of these materials. One of possible ways to induce textured grain growth is to add platelets seeds to the powder mixture prior to sintering. This is why we synthesized Nb-doped Sr3Ti2O7 platelets seeds via molten salt synthesis (MSS). Due to crystallographic anisotropy, when grown in an appropriate medium and conditions, Sr3Ti2O7 forms anisotropic platelets with ordered RP-type faults in [001] direction [1]. There have been several reports on synthesis of pure Sr3Ti2O7 platelets with the MSS [2], but none on Nb-doped Sr3Ti2O7. In order to study the crystallography, structure and chemical composition of synthesized platelets one has to be able to prepare thin foils for TEM observation in different orientations. This is why in our work we report on preparation of Nb-doped Sr3Ti2O7 thin foils for TEM observation in defined orientation using two preparation procedures; namely conventional ion-milling and tripod polishing, and the comparison between them. The first step in both preparation procedures was to mix Nb-doped Sr3Ti2O7 platelets with an epoxy (G1) resin and to press the mixture between Si supports in order to align as many as possible platelets parallel to the Si supports. The specimens prepared by ion-milling were then mechanically grinded to a thickness of 100 µm, followed by dimpling in a cross-section geometry down to a thickness of 16±2 µm. Afterwards, the specimens were Ar-ion thinned at 3.5 keV and at 8o incident angle to perforation in a Gatan, PIPS (without cooling) (Fig. 1). The specimens prepared by wedge-shape polishing method [3] were polished on a diamond-lapping film (DLFs) at a wedge angle of 1o and afterwards mounted on a Cu grid. The specimens were additionally thinned in PIPS at 3.5 keV for 60 min and at 0.6 keV for 20 min. During the ion-milling process the specimens were cooled using liquid nitrogen (Fig. 2). The results showed that it was possible to find electron transparent, epoxy-free regions of Nb-doped Sr3Ti2O7 platelets in cross-section geometry (c-axis parallel to the electron beam) for both specimens’ preparation procedures. However, the amorphous region in ion-milled specimens was app. 5 times as thick as compared to the specimens prepared by tripod polishing method. It was concluded that tripod polishing is far better technique for TEM specimen’s preparation of Nb-doped Sr3Ti2O7 platelets despite its difficulty to carry out.

Refrerences

[1] Ruddlesden, S. N., Popper P., Acta Cryst., 1958, 11, 54-55.

[2] Akdogan, E. K., et al, J. Electroceram., 2006, 16, 159-165

[3] Eberg, E., et al, Journal of Electron Microscopy, 2008, 57, 175-179

The financial support the research leading to these results has received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under grant agreement n°312483 (ESTEEM2).

Fig. 1: (a) SEM micrograph of Nb-doped Sr3Ti2O7platelets. (b,c) TEM micrographs of an ion-milled platelet. (d) HRTEM showinglarge amorphous layer.

Fig. 2: (a) SEM micrograph of Nb-doped Sr3Ti2O7 platelets. (b,c) TEM micrographs of a tripod polished platelet. (d) HRTEM showing almost amorphous-free region.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-2528 Clearing methods for optical projection tomography microscopy

Čapek M.1, Radochová B.1, Michálek J.1, Janáček J.1, Kubová H.1, Sedmera D.1, Kubínová L.1
1Institute of Physiology ASCR, v.v.i., Prague, Czech Republic
capek@biomed.cas.cz

Optical projection tomography microscopy (OPT) is a modern technique that makes possible to get 3D images of specimens from 1 to 12 mm in diameter. OPT is based on acquiring sets of projections of the specimen in the range of 360° and subsequent computer reconstruction of the 3D image by a filtered back projection (FBP) algorithm. Thus the principle is similar like computed tomography, but instead of using X-ray OPT uses visible light.

        Inevitable condition for getting high quality projections is optical transparency of the specimens. Under normal circumstances fixed specimens are not transparent in majority cases. Therefore, optical clearing methods are used. Its basic principle lies in tissue dehydration and subsequent immersion in a solution that has the refractive index of proteins; thus, tissues become transparent and light does not scatter.

        Standard clearing protocol is based on dehydration of the specimen in methanol and its rinsing in, so-called, BABB solution (1 part of benzyl alcohol + 2 parts of benzyl benzoate), but BABB often washes out applied fluorescence dyes and destroys GFP signal in tissues. Thereby, a number of new clearing protocols appeared in the literature lately, namely Scale (Nat Neurosci 2011), dehydration by tetrahydrofuran and clearing by dibenzyl ether (THF+DBE; PLOS One 2012), CLARITY (Nat 2013), ClearT (Dev 2013), SeeDB (Nat Neurosci 2013).

        First, to avoid washing out the fluorescence dyes, we applied BABB on a mouse brain specimen perfunded in-vivo by tomato (Lycopersicon esculentum) lectin. In this case the staining was fixed in the tissue well, and we were successful in acquisition of inner structures, especially vessels, see Fig. 1. From practical reasons we used a block of brain of the size of approx. 3×3×3 mm3. Second, to avoid disappearance of GFP signal in mouse mutant series, we successfully applied antibodies (primary anti-rabbit, secondary donkey anti-rabbit Cy5, Abcam) against GFP to preserve signal and visualized in 3D inner structures of a young mouse heart, Fig. 2. Third, we tested Scale on a young mouse heart. Scale provides worse resulting transparency of specimens than BABB, which is documented in the literature as well, but preserves GFP signal that can be visualized in small parts of tissues, see, e.g., atria of the heart in Fig. 3. Fourth, a potentially promising method is THF+DBE. Till now we applied it to mouse embryos with only partially acceptable results. According to literature, the resulting transparency of tissues is high and should be comparable with BABB, but probably due to high amount of blood in the embryo, the resulting 3D visualization is not as clear as expected, but still we can see internal structures like a backbone, etc., Fig. 4.

Supported by Czech Science Foundation (P501/10/0340, 13-12412S), AMVIS (LH13028) and by support of research organization RVO:67985823.

Fig. 1: 3D visualization of structures in a block cut from a rat brain of the size of approx. 3×3×3 mm3, acquired by optical projection tomography. Fluorescence, exc/em – 425nm/from 475nm. The brain was stained by tomato (Lycopersicon esculentum) lectin. Dhristi visualization software and a suitable transfer function were used.

Fig. 2: Combined volume and surface 3D rendering of an early stage mouse heart, acquired by OPT. Red channel: white light transmission; green channel: fluorescence exc/em – 628nm/692nm. Software VolViewer (Bangham laboratory).

Fig. 3: A Scale cleared young mutant mouse heart with GFP. Note well visible structures of atria. Fluorescence, exc/em – 425nm/from 475nm. Software VolViewer (Bangham lab).

Fig. 4: A THF+DBE cleared mouse embryo with partially visible internal structures. Red channel: white light transmission; green channel: fluorescence exc/em – 425nm/from 475nm. Software VolViewer (Bangham lab).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-2595 TEM specimen preparation of p-type oxide thermoelectric layered cobaltate ceramics by conventional ion-milling and tripod polishing

Šestan A.1,3, Gec M.2, Jančar B.1
1Advanced Materials, Jožef Stefan Institute, Ljubljana, Slovenia., 2Nanostructured Materials, Jožef Stefan Institute, Ljubljana, Slovenia., 3Center for electron microscopy and microanalysis, Jožef Stefan Institute, Ljubljana, Slovenia.
andreja.sestan@ijs.si

The most promising p-type oxide thermoelectric materials for thermoelectric generator systems are based on alkali or alkaline-earth cobaltate compounds with layered structure such as NaxCoO2 and Ca3Co4O9 [1]. The chemistry of layered NaxCoO2, however, is governed by the mobility of interlayer sodium ions, which renders the crystal structure unstable in contact with atmospheric H2O and CO2 [2], so we decided to focus on textured Ca3-xNaxCo4O9. Polycrystalline samples were prepared by solid state reaction under O2 gas flow, TEM analyses were performed in order to observe the structures. Ca3Co4O9 and NaxCoO2 share a common CoO2 layer which enables spontaneous coherent intergrowth of the two structural types.  For TEM sample-preparation two different specimen preparation techniques were employed: conventional ion milling and tripod polishing. The results of these preparation approaches are described and compared. The TEM specimens prepared by conventional method were first mechanical grinded to a thickness of 120 μm followed by dimpling in cross-section geometry down to a 20 µm at the disc center. The specimens were further thinned by at 4 keV and 10° incident angle with Ar+ until perforation using Bal-Tec RES 010 ion-mill. For the TEM-specimens prepared by the mechanical polishing method an automatic Allied MultiPrep System was used. The specimens embedded into an epoxy resin were mounted in such a way that cross-section were obtained. The specimens were mechanically polished on a diamond-lapping film (DLF) at a small wedge angle of 1.5°. The final polishing step was performed on both sides of the sample by using colloidal silica to thin the specimen until electron transparency. Afterward, the specimens were glued on a Cu-grid [3]. Additionally, the specimens were Ar+ ion thinned at low energy of 2 keV for 30 min and at 1.8 keV for 15 min while cooling the specimen using Gatan, PIPS in order to remove any possible contamination from the specimen surface. The properties of the coherent intergrowth Ca3Co4O9 and NaxCoO2 are quite different, which resulted in a preferent etching of the NaxCoO2 layer. This is a permanently damaged surface layer that decreases the feature contrast in the micrographs. The ion-milled sample also included thickness variations and surface roughness that is manifested as thickness and phase contrast variations. The specimen prepared by mechanical polishing method, have large areas of constant thickness exhibiting more information of phase contrast and provide large electron transparent area.

References
[1] Fergus J. W., (2012) J Eur Ceram Soc 32, 525-540.
[2] Vengust D., “et al”, (2013) Chem Mater 25, no. 23, 4791-4797.
[3] Voyles P. M., “et al” ,(2003) Ultramicroscopy 96, 251-273.

The authors acknowledge financial support from EU under Framework programs under agreements No. 312483 (ESTEEM2).

Fig. 1: (a) Low-mag TEM image of Ar+ ion-milled layered cobalatate ceramics, b)Extensive amorphous as a result of preferent etching , c) Atomic structure of damaged Na-layer.

Fig. 2: (a) Low-mag TEM image of the specimen prepared by wedge-shape polishing technique. (b, c) HRTEM images exhibited more information on phase contrast and shows large electron transparent area.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-2783 Sputtering thin films for high resolution scanning electron microscopy.

Rattenberger J.1, Melischnig A.1,2, Schroettner H.1,2, Letofsky-Papst I.2, Mertschnigg S.1,2, Hofer F.1,2
1Graz Centre for Electron Microscopy (ZFE), Steyrergasse 17, 8010 Graz, Austria, 2Institute for Electron Microscopy and Nanoanalysis (FELMI), Steyrergasse 17, 8010 Graz, Austria, Graz University of Technology (TU Graz)
johannes.rattenberger@felmi-zfe.at

To investigate non-conductive samples in conventional scanning electron microscopy (SEM) the sample must be sputtered or evaporated with an electrically conductive layer to prevent charging.
Evaporation with carbon is typically used for investigations with backscattered electrons or for energy dispersive x-ray spectrometry. Thin carbon layers are relatively transparent for high energetic backscatter electrons and with the exception of the carbon K line this layer does not interfere with other detected x-rays.
For acquiring high resolution secondary electron images, the sample must be metal sputtered to increase the secondary electron yield. These sputtered layers have a big drawback, the grain size within the sputtered film. The topographic information of these artefacts superimpose with information from the sample which degrades image quality especially using modern high resolution SEMs with special resolution of less than 1 nm.
All investigations were performed using a commercially available magnetron sputter coater (Leica EM ACE600) or a high frequency gas discharge apparatus (FELMI-ZFE-GEA005). The basic aim was to ensure the thinnest possible continuous film over the sample surface with the smallest grain size. Different metal targets and parameters (e.g. chamber pressure, current, layer thickness and so on…) were used to optimize the sputtered layers.
The layers were analyzed using high resolution scanning electron microscopy (HR-SEM) for topographic information, transmission electron microscopy (TEM) for crystallographic information and atomic force microscopy (AFM) for 3D information. Power spectral density analysis (PSD) was used to determine the grain size distribution quantitatively.
In figure 1 TEM bright field images of gold, gold/palladium (80/20), and chrome layers can be seen (20 nm thick, sputter coater: GEA005). The different grain sizes as well as crystalline structures can be identified.
Figure 2 shows exemplarily a 4 nm thick sputtered gold/palladium layer on glass substrate. These images were used for PSD analysis. In figure 3 the influence of layer thickness on grain size distribution using the gold/palladium target and the Leica ACE600 can be seen. The peak shifts from about 0,063 [1/nm] to 0,003 [1/nm] indicates a decreases in grain sizes from 33 nm at 6 nm layer thickness to 16 nm grain size at 1 nm layer thickness.

The author wants to thank Leica Microsystems for helpful discussions and support.

Fig. 1: TEM bright field images (left to right: 20 nm gold, gold/palladium and chrome layer)

Fig. 2: Scanning electron micrograph (sample: gold/palladium (80/20) on glass substrate)

Fig. 3: Power spectral density for different layer thicknesses (sample: gold palladium layer (80/20) on glass substrate)
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-3254 Sample preparation of heterogeneous semiconductors heterostructures for (S)TEM analyzes : the case study of III-Nitrides on Si-based substrates

Mante N.1, Feuillet G.1, Delaye V.1, Audoit G.1, Vennéguès P.2
1CEA, LETI, MINATEC Campus, 17 rue des Martyrs, 38054 GRENOBLE Cedex 9, France., 2CRHEA-CNRS, Rue Bernard Gregory, 06560 VALBONNE, France.
nicolas.mante@cea.fr

Gallium nitride is a widely used material in optoelectronic devices like LEDs. The LED structures are mostly grown by epitaxy onto sapphire substrates, but, for cost reasons, silicon is currently being studied as an alternative candidate. Transmission electron microscopy (TEM) is a powerful technique to understand the growth mechanisms of nitrides on silicon and to assess the structural quality of the epitaxial layers. For that purpose, preparing high quality cross section samples is key. The use of ion beam related techniques is well known for creating implantation and structural defects. Wedge mechanical polishing offers very satisfying results but is also quite challenging when dealing with samples composed of materials with different mechanical properties or chemical reactivity like GaN and Silicon. The problem is even more critical when the grown structure is made of different nitride materials like AlN and GaN and/or when the Si substrate is non-monolithic like for instance silicon on insulator (SOI).

The aim of this work is to present a dedicated sample preparation technique that we have developed and implemented to obtain samples from GaN-AlN heterostructures grown onto Si and SOI with good quality on large areas, typically tens of microns. The desired samples need to be as thin as possible without preparation damages, in order to make comprehensive (S)TEM studies: from high resolution to obtain interface images at the atomic scale, to larger views allowing to understand the dislocation behavior, at the micrometer scale.

The process is based on a wedge polishing method. It consists in polishing the sample with a very low angle (1-2°), in order to obtain an area on the wedge thin enough for TEM observations. The samples studied in this work were grown at CNRS / CRHEA laboratory, then prepared with an Allied Multiprep polishing tool, and observed on a FEI Titan Cs probe corrected TEM using a 300 kV tension in STEM mode, at the NanoCharacterization PlatForm (PFNC) in CEA Grenoble.

The detailed process for the preparation of nitride materials on bulk silicon and SOI based substrate with different oxide thicknesses will be presented. Typical TEM images of GaN/AlN epitaxial stacks on Si are presented in figures 1 and 2, highlighting the benefits of the described preparation technique at all scales of the TEM investigations for heterogeneous samples, and this in the different TEM modes (STEM, HRTEM, diffraction contrast).

Fig. 1: STEM HAADF image of GaN/AlGaN/AlN heterostructure, in [11-20] zone axis. A field of view of several square microns is obtained.

Fig. 2: High resolution STEM HAADF image of GaN/InGaN quantum well heterostructure, in [11-20] zone axis. Lattice resolution is obtained without any artefact induced by sample preparation.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-3266 Dry-cleaning of graphene

Algara-Siller G.1,2, Lehtinen O.2, Turchanin A.3, Kaiser U.2
1Department of chemistry, Technical University Ilmenau, Weimarer Strasse 25, 98693, Ilmenau, Germany, 2Group of electron microscopy of materials science, Ulm University, Albert-Einstein-Alle 11, 89081, Ulm, Germany, 3Faculty of physics, University of Bielefeld, Universitätsstr. 25, 33615, Bielefeld, Germany
algaragerardo@gmail.com

Graphene is thought to be the ultimate support for TEM studies with its outstanding physical and chemical properties, one-atom thickness and low background signal contribution. But contamination introduced during fabrication and transfer as well as the airborne contaminants adsorbed on graphene hinder the potential of graphene as support for the studies of inorganic, organic and biological nano-objects. Here, we present a simple and efficient table-top method for cleaning graphene using activated carbon, dry-cleaning. The dry-cleaned graphene samples were characterised by aberration-corrected high-resolution transmission electron microscopy (AC-HRTEM) using an imaging-side aberration corrector FEI TITAN 80-300 operated at 80 kV . The cleanness of graphene improved from 6% for a non-cleaned graphene to 95% for dry-cleaned graphene with activated carbon (Figrue 1 A and B) with clean areas in the micrometer range. Cleanness is defined as the ratio of the contamination-free area to total sample area. The efficient removal of carbonaceous contamiantion was confirmed by Auger Electron spectroscopy, AES (Figure 1 C). The residual contamination after dry-cleaning was observed to contain Si, as seen by the electron energy loss (EEL) spectrum in Figure 1 (D). Interestingly, bilayer and triple-layer graphene does not get clean by our dry-cleaning method nor by other cleaning methods, e.g. UHV annealing. As dry-cleaning with activated carbon was only performed in graphene, we can speculate that dry-cleaning with activated carbon can be used to clean surfaces of other TEM samples.

The authors acknowledge financial support by the DFG (SPP "Graphene" and Heisenberg Programme) and the Ministry of Sciece, Research and the Arts (MWK) of Baden-Württemberg in the frame of the SALVE (Sub Angstrom Low-Voltage Electron Microscopy) project.

Fig. 1: HRTEM images of non-cleaned and dry-cleaned graphene(A and B respectively). Inset in (B) high resolution image of clean graphene. AE spectra (C) of a TEM grid (black), non-cleaned (red) and dry-cleaned graphene (blue) and vacuum (green). (D) EEL spectrum of residual contamination on dry-cleaned graphene demonstrating its Si content.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-5867 Visualization of internal structure of banana starch granule using AFM and SEM

Peroni-Okita F. H.1, Gunning A. P.3, Kirby A.3, Simão R. A.4, Soares C. A.1, Cordenunsi B. R.1,2
1University of São Paulo, São Paulo, Brazil, 2University of São Paulo, NAPAN – Food and Nutrition Research Center, São Paulo, Brazil, 3Institute of Food Research, Norwich, United Kingdom, 4Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
fernanda_peroni@yahoo.com

Introduction: Atomic force microscopy (AFM) is a high resolution technique for studying the external and internal structures of starch granules, without complex sample preparation. Banana starch granule surface and its degradation has been studied in the last few years using microscopic techniques and, recently, it became important to study and visualize the internal structure of starch granule. The technique described by Ridout et al. (2004), where granules were embedded in a resin and sectioned allowing to visualize the inner structure of isolated granules, was used to examine banana starch. Material and Methods: Starch granules were isolated from green and mature bananas (cv. Nanicão), embedded in a non-penetrating resin (Araldite Instant Clear Syringe-90 sec), sectioned using a microtome until a flat and shiny surface is obtained. To achieve contrast, the face of the cut blocks were wetted in steam (20 sec.) and imaged in air using an AFM JPK Nano Wizard II and Alpha300 AR model. The images were obtained in an intermittent contact mode and force modulation imaging. For SEM images, the blocks were coated (10 nm thick platinum) and visualized in a FEI Quanta 600 FEG Microscope. Results: Topographic images of green banana starch (1A, C, G, K) showed large variation of height across the granule (approx. 1 µm), mainly due the absorption of water and swelling of amorphous regions. It seemed that the dark center of the granule was structurally different, with reduced absorption of water compared to the other regions. At higher resolution, the growth rings were visible close to hilum (1D, lateral deflection image and 1F, error signal image) and a different phase contrast was observed in the central region (1H). In another level of structure, the growth rings were composed of globular and uniform structures known as blocklets, hard objects embedded in an amorphous matrix, ranging from 30 to 150 nm, depending on the localization inside the granule (1B, J, N). Otherwise, images of starches from ripe bananas, showed a different level of organization, possibly due to the presence of a degradation mechanism. Granules with different sizes and elongated shapes (2A), absence of growth rings around the hilum (2I, L-M) and dark regions (2F) never reported before indicate holes created by a randomly enzymatic process. More detail is revealed by lateral deflection images (2E, G), which highlighted a dark region in 2F that was harder than the bright region. The more deformable the sample was the greater the tip-contact area becomes, hence the increase in frictional component in soft regions. Phase images (2C, K) confirmed that starches of ripe bananas had different viscoelastic properties and showed larger size of blocklets. Reference. Biomacromol, 5, 1519-1527, 2004.

The authors acknowledge FAPESP and Capes for financial support and scholarship, and I.F.R. for assistance in the samples preparation and training in AFM techniques.

Fig. 1: AFM images of the interior of green banana starch granules. (A) Topography image and (B) topography of demarcated area in A. (C, E, G, I, K, M) Topography images on the left column and complementary images on the right column: (D) lateral deflection, (F, J, L) error signal mode and (H, N) phase images. h: hilum.

Fig. 2: AFM images of ripe banana starch granules. (A) Topography of several granules inside resin. (D, F, H, J) Topography on the left column and complementary images on the right column: (E, G) lateral deflection, (I) error signal mode and (K) phase images. (B-C) Topography and phase images, respectively, on the right column. (L-M) SEM images.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-10-P-5957 Freeze-fracture technique and artefacts caused by processing conditions

Vaškovicová N.1, Valigurová A.2, Hodová I.2, Melicherová J.2, Krzyžánek V.1
1Institute of Scientific Instruments of the ASCR, v. v. i., Czech Republic, 2Department of Botany and Zoology, Faculty of Science, Masaryk University, Czech Republic
vaskovicova@isibrno.cz

Freeze-fracture technique is a method used to visualise membrane surfaces of cell organelles. This method is based on cryo-fixation that stabilizes samples. The sample is rapidly frozen in nitrogen, and cut in the chamber under a vacuum and low temperature.
Glycerol is used as a cryoprotectant preserving the fine structure of cells in their native stage. Although, cryoprotectants serve as a substitute for water and protect against ice crystal production, they could also affect the form of fracture through biological membranes. Figure 1 shows structures in a sample frozen in the presence of 25% glycerol. The temperature of the apparatus was not low enough during the process of fracturing and etching the sample. The structure of cells seems to be deformed due to melting glycerol. In contrast, figure 2 shows a replica with fine structure of frozen and proper good form of fracturing. The cells used for this study were human leukemic cells (HL-60).
Another artefact is shown in figure 3A, compare with 3B. Each sample has to be fractured with a specific speed of cut. The force used for fracturing the membranes has to be set to optimal conditions, which depend on a hardness of sample and a coherence of drops. Low speed and unstable coherence of drops resulted in sample fragmentation. High speed of cut could cause cross-section of cellular structures, similar to ultrathin sections. Figure 3A shows fragmentation of nuclear membrane. This sample was not fractured, it was fragmented due to unstable coherence of drop.
This overview shows how a combination of different conditions including the physical properties of the sample, cryoprotectants used and temperature could affect the form of fractures and hence significantly affect interpretation of morphological structures.

The research was supported by VASE GRANTY, MEYS CR (LO1212), EC (CZ.1.05/2.1.00/01.0017), ASCR (RVO:68081731), GAČR (GAP506/12/1258) and GAP 506/12/1258.

Fig. 1: Freeze-fracture of HL-60 cells in suspension: structures of fracture in the replica are affected by melting glycerol. Bar 10 um.

Fig. 2: Freeze-fracture of HL-60 cells in suspension: structures of fracture in the replica with fine frozen and optimal fracturing condition. Bar 10 um.

Fig. 3: Nuclear membrane: A) the replica of fragmented structures; B) the replica of optimally fractured structures. Different form of nuclear pores (arrows). In compare with replica of fractured structures, the fragmentation of the sample affected the form of nuclear pores and structure of the membrane. Bar 1 um.
↑ Go to the Table of content ↑

ID-11. Multidisciplinary applications of progressive light microscopy imaging techniques

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-11-IN-3271 Scanning microscopy with detector arrays

Sheppard C. J.1
1Istituto Italiano di Tecnologia
colin.sheppard@iit.it

An imaging system such as a microscope can be constructed using either an imaging detector, or by scanning using a focused illumination spot. Either approach results in the same resolution, except that for fluorescence imaging if there is a Stokes shift (typically 10%) the scanning method gives a resolution correspondingly better. Various techniques have been proposed that image using both the illuminating light and the detected light. The result is a spatial frequency bandwidth equal to the sum of those for illumination and detection. With no Stokes shift the bandwidth is doubled. If there is a Stokes shift, compared with a conventional imaging detector, the bandwidth is more than doubled.

These techniques include the confocal microscope, spinning disk microscope, structured illumination, subtractive imaging, programmable array microscope and image scanning microscopy [1, 2]. Sometimes similar techniques are known by alternative names, some other terms used being pixel reassignment [3], photon reassignment, virtually structured detection, scanning patterned detection, and scanning patterned illumination.

Structured illumination has been demonstrated to give a factor of two improvement in resolution compared with conventional microscopy. However, as structured illumination requires a demodulation step, this is usually combined with image restoration, and should therefore really be compared with a deconvolved conventional image. Confocal microscopy requires no subsequent image processing, but has the disadvantage of weak collection efficiency, as a result of the physical confocal pinhole. Pseudo random illumination and detector arrays can benefit from Fellgett’s multiplex advantage. Although illumination and detection are equivalent in imaging as a result of the principle of reciprocity, they are very different from the point of view of signal level for a given light exposure. Spinning disk and structured illumination can be seen to be in principle similar to each other.

Different designs of image scanning microscopy include optical implementations and multifocal illumination, to increase imaging speed. The approach can also be applied to multiphoton microscopy.

 

References

1. C. J. R. Sheppard, "Super-resolution in confocal imaging," Optik 80, 53-54 (1988).

2. C. B. Müller and J. Enderlein, "Image scanning microscopy," Phys. Rev. Letts, 104, 198101 (2010).

3. C. J. R. Sheppard, S. B. Mehta, and R. Heintzmann, "Superresolution by image scanning microscopy using pixel reassignment," Opt. Lett. 38, 2889-2892 (2013).

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-11-IN-6091 Advanced Light Microscopies as Tools to Reverse-Engineer the Brain

Saggau P.1
1Dept. Research Engineering, Allen Institute for Brain Science, Seattle, USA
psaggau@bcm.edu

Progress in generating, controlling and detecting photons continues to spawn new theories, technologies and innovative designs of computer-controlled light microscopes with improved spatio-temporal resolution and sensitivity.
I will present advanced microscopy schemes based on steering of laser beams with dynamic diffractive optical elements. In combination with engineered molecules, such microscopes have become powerful tools for structural and functional analysis of biological tissue including the living brain. The complexity of brain tissue requires scalable approaches which I will briefly discuss.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-11-O-2382 Full-field optical coherence microscopy with spectroscopic-based contrast enhancement

Federici A.1, Dubois A.1
1Laboratoire Charles Fabry, CNRS UMR 8501, Institut d’Optique Graduate School, Univ. Paris-Sud, 2 avenue Augustin Fresnel, 91127 Palaiseau Cedex, France
arnaud.dubois@institutoptique.fr

Full-field optical coherence microscopy (FF-OCM) is a recent optical technology based on low-coherence interference microscopy for semi-transparent sample imaging with ~ 1 µm spatial resolution [1, 2]. FF-OCM has been successfully applied to three-dimensional imaging of various biological tissues at cellular-level resolution [3]. The contrast of FF-OCM images results from the intensity of light backscattered by the sample microstructures. This contrast mechanism, based on refractive index changes, provides information on the internal architectural morphology of the sample.
Several extensions of FF-OCM have been developed including the ability to exploit the spectroscopic response of the imaged sample. The purpose of this complementary imaging modality is to enhance image contrast, permitting better differentiation of the sample structures through their spectroscopic properties and providing additional information on the sample composition.
Two different technological approaches to take advantage of the spectroscopic response of the sample are presented in this paper. A first approach involves detecting the whole interferometric signal and analyzing it using Fourier mathematics [4]. Another approach consists of imaging the sample in several distinct bands (2 or 3 are presented here):
High-resolution FF-OCM imaging is demonstrated in the 800 nm and 1200 nm wavelength regions simultaneously using a Silicon-based CCD camera and an Indium Gallium Arsenide (InGaAs) camera as area detectors and a halogen lamp as single illumination source. [5]. The setup is optimized to support the two broad spectral bands in parallel.
Three-band FF-OCM is demonstrated to image successively at 635 nm, 870 nm and 1170 nm center wavelengths using a visible to short-wavelength infrared camera and a halogen lamp [6]. Reflective microscope objectives are employed to minimize chromatic aberrations. Constant 1.9-µm axial resolution (measured in air) is achieved in each of the three bands. A dynamic dispersion compensation system is set up to preserve the axial resolution when the imaging depth is varied. The images can be analyzed in the conventional RGB color channels representation to generate three dimensional images with enhanced contrast.


1. L. Vabre et al., Opt. Lett. 27, 530 (2002).
2. A. Dubois et al., Appl. Opt. 43, 2874 (2004).
3. A. Dubois et al., Phys. Med. Biol. 49, 1227 (2004).
4. A. Dubois et al., Opt. Express 16, 17082 (2008).
5. D. Sacchet et al., Opt. Express 16, 19434 (2008).
6. A. Federici et al., Opt. Lett. 39, 1374 (2014).

The authors thank Sacchet D.

Fig. 1: Dual-band FF-OCM images of African tadpole Xenopus laevis, ex vivo, representing a field of 250 µm (x) × 1200 µm (z) at 800 nm (a) and 1200 nm (b) center wavelengths. The imaging penetration depth is larger at longer wavelength at the price of a degradation of spatial resolution.

Fig. 2: Three-band FF-OCM sections of a light (left) and a dark (right) human hair. From top to bottom : in band 1 (centered at 635 nm), band 2 (centered at 870 nm), band 3 (centered at 1170 nm) and with RGB representation. The scale bar is 20 µm in the two directions (x and z).
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-11-O-2702 Multimodal Holographic Microscope

Křížová A.1, 2, Slabý T.1, Kolman P.2, Pokorný J.1, Dostál Z.2, 3, Lošťák M.1, Kvasnica L.1, 2, 3, Antoš M.2, 3, Chmelík R.2, 3
1TESCAN Brno, s.r.o., Brno, Czech Republic, 2CEITEC BUT, Brno University of Technology, Brno, Czech Republic, 3Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology, Brno, Czech Republic
aneta.krizova@tescan.cz

Nowadays a comparison of methods is needed for complex research. Simultaneous combination of different methods and utilization of their advantages can provide new information about observed objects.

Therefore we present multimodal holographic microscope, which consist of transmission holographic microscopy, fluorescence microscopy and reflection holographic microscopy, as an interesting tool for complex research.

Holographic microscopy (HM) is becoming a popular research method. Interference is used in HM to record the whole information about the object wave, therefore intensity images and high contrast quantitative phase images can be reconstructed from a hologram. The quantitative phase images can express cell dry-mass in picograms per squared micrometres in the case of observation of live cells, or height of structure in nanometres in the case of topography studies. The holograms are processed by numerical methods that enable numerical refocusing and advanced image analysis.

The transmission holographic microscope is derived from the coherence-controlled holographic microscope [1]. It is based on off-axis interferometer and incoherent illumination. Holography as a non-invasive method enables long term observations of live cells as well as studying fast cell dynamics. Due to incoherent illumination, observation of cells in turbid or 3D scattering media is also possible.

Fluorescence microscopy is well known technique providing specific information about stained cells with molecularly specific contrast. On the other hand photochemical properties and rapid photo-bleaching can limit the period of observation.

The reflection holographic microscope is a powerful tool for studies of surface topography with nanometre axial resolution.

The possibility to combine fluorescence and holographic data in real time can provide additional useful information. Thus the multimodal holographic microscope can contribute to modern complex research in many fields of science as a powerful tool.

References:

[1] T Slaby et al, Optics Express 21, Optical Society of America (2013) p. 14747.

The authors acknowledge funding from the Ministry of Industry and Trade of the Czech Republic, project Multimodal Holographic Microscope (FR-TI4/660); and from European Regional Development Fund, CEITEC-Central European Institute of Technology (CZ.1.05/1.1.00/02.0068).

Fig. 1: MHM phase image of rat sarcoma cells, mitotic cell in right upper corner. Objective 20x/0.4, interference filter 650/10nm.

Fig. 2: MHM phase image of technical sample. Objective 20x/0.5, interference filter 547/10nm.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-11-O-2864 Advantages and limitations of spectral unmixing in the characterization of cellular autofluorescence

Chorvat D.1, Mateasik A.1, Marcek Chorvatova A.1
1International Laser Centre, Bratislava, Slovakia
chorvat@ilc.sk

Precise knowledge and characterization of molecular species underlying fluorescence signals in complex physiological conditions is a pre-requisite of their appropriate use as non-invasive tools in various biomedical applications. In this regard, monitoring of processes in living cells by detecting excited state dynamics and/or spectral characteristics of their endogenous fluorescence using latest imaging techniques is becoming an important feature of biomedical diagnostics. However, analysis of the complex multi-dimensional datasets, aimed at precise understanding of the origin and behavior of the underlying autofluorescence components, is still an unsolved problem.

NAD(P)H and flavin fingerprinting of tissues and/or isolated cells can be implemented by spectrally-resolved detection, time-resolved detection, or combination of both methods [1]. In this contribution we compare advantages and limitations of the spectral decomposition of signal derived from endogenous fluorescence by linear unmixing, classical multi-exponential data analysis and classification approaches, based on our previous experience in the field [2,3].

The study was done directly on living cells. To obtain spectrally-resolved autofluorescence images related to various states of mitochondrial metabolism and respiration, metabolic modulation was applied in combination with confocal microscopy and spectral detection. Fluorescence lifetime data were recorded using time-correlated single photon counting in single channel and multi-wavelength detection setups using pulsed laser excitation [4]. A comparison of the approaches will be presented, aiming to find and optimize accurate analytical means to label-free diagnosis of cells and tissues in their natural environment.

[1] D. Chorvat jr. and A. Chorvatova, Eur Biophys J. 36 73-83 (2006).

[2] D. Chorvat jr. and A. Chorvatova, Laser Physics Letters. 6 175-193 (2009).

[3] A. Mateasik, D. Chorvat, A. Chorvatova, Proc. of SPIE 8588, 85882J (2013).

[4] A. Chorvatova, A. Mateasik, D. Chorvat, Laser Physics Letters 10, 125703 (2013).

Authors acknowledge support from projects APVV-0302-10, LASERLAB-EUROPE III (7FP n°284464) and NanoNet2 (OPRD-RFRD fund, ITMS n°26240120018).

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-11-O-3363 Imaging G protein signaling with two-photon polarization microscopy

Bondar A.1 2, Timr S.3, Lazar J.1 2
1Laboratory of Cell Biology, Inst. of Nanobiology & Structural Biology GCRC, Academy of Sciences of the Czech Republic, Zamek 136, 37333 Nove Hrady, Czech Republic, 2Dept. of Biochemistry & Molecular Biology, Faculty of Science, University of South Bohemia, Branisovska 31, 37005 Ceske Budejovice, Czech Republic, 3Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 16610 Prague 6,Czech Republic
bondar@nh.cas.cz

Heterotrimeric G proteins play a major role in cellular signaling. They transduce signals from a multitude of extracellular stimuli (e.g., hormones, neurotransmitters, light) inside the cells. Despite a number of studies, the mechanism of G protein signaling is still not fully understood. We have now applied the technique of two-photon polarization microscopy (2PPM), developed in our laboratory, to studies of G protein signaling. 2PPM takes advantage of anisotropic absorption properties (linear dichroism) of fluorescent proteins (FPs), and allows sensitive, real time monitoring of protein-protein interactions and conformational changes in membrane proteins in living cells, through observations of changes in orientation of a fluorescent tag. 2PPM yields both structural and functional information about membrane proteins, and allows making insights into molecular mechanisms. Here we show that 2PPM allows observing GPCR interactions with extracellular ligands, interactions between GPCRs and G proteins, and interactions between G protein subunits. 2PPM allows monitoring G protein activation with higher sensitivity than resonance energy transfer methods. Using 2PPM we show that Gi/o proteins do not precouple to GPCRs, and that dissociated Gα-GTP and Gβγ subunits represent the main active form of Gi/o proteins. 2PPM requires only a single fluorescent tag, allows facile multiplexing, can utilize existing constructs, and yields insights about functional activity and structural changes in membrane protein molecules. 2PPM is a highly useful tool for studies of cell signaling processes, under conditions closer to natural than previously possible.

European Commission FP7 Marie Curie International Reintegration Grant PIRG-GA-2007-209789 “MemSensors” (to J. L.), by Czech Government Institutional Grant AVOZ60870520 (to J. L.), by Grant P205/13-10799S (to J. L.), by University of South Bohemia Grant Agency Grant 141/2013/P (to A. B.), and by a University of South Bohemia fellowship (to A. B.).

Fig. 1: 2PPM allows detection of interactions between G proteins and GPCRs. (A-B) Schematic of stable binding of activated GPCRs by a non-dissociating mutant of Gi1 protein. (C-D) Linear dichroism of non-dissociating Gαi1-FP construct increases upon GPCR stimulation, which indicates stable physical interaction of G protein molecules with activated GPCRs.

Fig. 2: 2PPM allows observation of G protein activation. (A-C) Schematics of G protein activation (D-F) Linear dichroism of Gαi1-CFP,β1,γ2 heterotrimer before activation (D), during activation (E) and after agonist washout (F). Dissociation of the G protein heterotrimer upon activation is accompanied by a disappearance of linear dichroism.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-2527 Measurement of pH micro-heterogeneity in cheese matrices by Fluorescence Lifetime Imaging

Burdíková Z.1, Švindrych Z.2, Pala J.3, Hickey C.1, Čmiel V.4, Auty M. A.1, Sheehan J. J.1
1Teagasc Food Research Centre Moorepark, Fermoy, Co. Cork, Republic of Ireland, 2First Faculty of Medicine, Charles University, Prague, Czech Republic , 3Third Faculty of Medicine, Charles University, Prague, Czech Republic, 4Faculty of Electrical Engineering and Communication, Brno University of Technology, Brno, Czech Republic
burdikova.zuzana@gmail.com

Cheese, a product of microbial fermentation may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g. pH, redox potential) due to their metabolic activity [1]. While cheese pH may be measured at macro level there remains a significant knowledge gap relating to the degree of micro-heterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns [2].

The pH of cheese samples was monitored both at macroscopic scale and at microscopic scale, using a microscopic techniques employing C-SNARF-4 fluorescent probe. The dye exhibits two emission peaks whose intensities display different pH dependencies. The results of the fluorescence ratio I580nm/I640nm have been confirmed as a reliable indicator of pH 5.3 and above [3]. In more acidic environments the fluorescence ratio is insensitive of pH (see Fig. 1a).

To extend the pH range towards more acidic values, the fluorescent lifetimes of C-SNARF-4 was explored in two spectral bands on a Leica TCS SP8 X confocal microscope with PicoQuant TCSPC equipment. Two-component fits for dye solutions buffered to different pHs revealed a clear dependence of the longer lifetime component in the red spectral band on pH in the range 4.5 to 6.5. To further explore the fluorescence properties of C-SNARF-4 and their potential for pH measurements, excitation-emission lambda scans were performed on buffered solutions of the dye (see Fig. 2). The results indicate that the pH sensitivity of the probe can be enhanced by proper choice of excitation wavelength combined with lifetime measurements of the large Stokes-shift spectral component of the emission.

As the fluorescence lifetime is an intrinsic property of given dye and does not depend on instrumentation, the calibration results obtained can be used to map the observed lifetime to local pH information (see Fig. 1b). Results indicate that for the first time localised pH measurement can be accomplished in complex foods using FLIM.

References:
[1] P L H Mc Sweeney, Cheese: Chemistry, Physics and Microbiology ed. (Elsevier, London).
[2] S Jeanson et al, Appl. Environ.Microbiol. 79 (2013), p. 6516.
[3] R C Hunter and T J Beveridge, Appl. Environ. Microbiol. 71 (2005), p. 2501.

This work was supported by the Czech Science Foundation [P302/12/G157]; by Charles University in Prague [Prvouk/1LF/1, UNCE 204022]; and by European Union Funds for Regional Development [OPPK CZ.2.16/3.1.00/24010]. Z. Burdikova is supported by the Dairy Levy Trust (RMIS 6259), Ireland.

Fig. 1: (a) The pH dependence of C-SNARF-4 fluorescence: lifetime of the longer component in the ‘red’ emission band and I580nm/I640nm ratio (as measured on Leica SP5 AOBS) . Inset shows a typical decay curve with a two-component fit, time-gated detection. (b) FLIM images of cheese matrix, scale bar = 100 µm. LUT represents local lifetimes from 0 to 7 ns.

Fig. 2: Excitation-emission lambda scans of the C-SNARF-4 solution buffered to different pH. Vertical axis shows excitation wavelengths of White Light Laser (470-670 nm), whereas horizontal axis displays emission spectra (480-730 nm) of C-SNARF-4 in different pH from left: 4.5, 5.0. 5.5, 6.0 and 6.5.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-2364 Detection of non-enzymatic browning in fruit fillings by auto-fluorescence of melanoidin precursors

Cropotova J.1, Tylewicz U.2, Romani S.2,3, Dalla Rosa M.2,3
1Practical Scientific Institute of Horticulture and Food Technology of Moldova, Chisinau, Republic of Moldova, 2CIRI Agroalimentare, Campus Scienze degli Alimenti, Cesena, Italy, 3Department of Agri-Food Science and Technology - DISTAL, University of Bologna, Campus of Food Science, Cesena, Italy
jcropotova@gmail.com

Fluorescence detection of the products of Maillard reaction under blue light excitation represents a promising technique for rapid visualizing the degree of browning of fruit fillings and sensory analysis. The main goal of this study was to evaluate the possibility of using auto-chemifluorescence of melanoidin precursors for evaluating non-enzymatic browning in fruit fillings with a wide range of soluble solids. The fruit filling samples were produced from apple puree with different amounts of sugar, inulin, low-metoxyl pectin, low acyl gellan gum and citric acid, sterilized, stored during 6 months and further observed in a thin layer by means of fluorescence microscopy without using special fluorescent substances. In confectionary products browning process generally takes place after an induction period, characterized by the creation of fluorescent uncolored intermediates. Fluorophores are considered precursors of brown pigments and permit to detect the Maillard reaction development before any visual change occurs. It was revealed that the intensive source of the autofluorescence in fillings' compositions is generally localized in polysaccharide structures partially destroyed during thermal treatment and storage, thus melanoidin precursors are mainly formed in these parts. The obtained fluorescent micrographs of the analyzed fruit fillings were compared to the results of color and hydroxymethylfurfural content. The total area of highly fluorescence compounds under blue light excitation in fruit fillings with high soluble solids and significant amount of added polysaccharides (inulin, pectin and gellan gum) was much bigger than in low-sugar samples with low amount of polysaccharides and also statistically significant differences among this value and browning process were revealed.

Fig. 1: Fluorescent micrograph of a fruit filling with 30 Brix and 8% inulin

Fig. 2: Fluorescent micrograph of a fruit filling with 70 Brix and 4% inulin
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-2480 Imaging and simultaneous biochemical analysis of intracellular bacterial pathogens using a label-free Raman-based algorithm

Grosse C.1, 2, Bergner N.2, Dellith J.2, Heller R.1, 3, Bauer M.1, Mellmann A.4, Popp J.1, 2, 5, Neugebauer U.1, 2
1Center for Sepsis Control and Care, Jena University Hospital, Jena, Germany, 2Leibniz Institute of Photonic Technology, Jena, Germany, 3Institute for Molecular Cell Biology, Jena University Hospital, Jena, Germany, 4Institute of Hygiene, University of Münster, Münster, Germany, 5Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University Jena, Jena, Germany
christina.grosse@med.uni-jena.de

Pathogenic bacteria are a main challenge to hospitals by frequently causing difficult-to-treat infections. Many of them are able to reside in eukaryotic cells for a longer time in order to establish a persistent infection. These intracellular infections are especially difficult to treat and challenging to study in their normal host cell environment. Since the host-pathogen interaction is a quite balanced state that is subject to smallest disturbances, there is an urgent need for methods which are able to study these bacteria without isolation from host cells and without introducing any modifications like labelling or destructive preparations.
Here, we present a label-free and non-invasive Raman-based imaging algorithm than can provide valuable insights into intracellular Staphylococcus aureus infection by probing the vibrational modes of molecules and thus yielding important chemical information in a spatially resolved manner. S. aureus is an important pathogen frequently found in hospitalized patients with severe invasive infections. This bacterium is able to invade, grow and persist in different host cell types. We used an in vitro infection model of S. aureus-infected endothelial cells and characterized the cells at different time points after infection. N-FINDR, a spectral unmixing algorithm, was used to generate false colour images and to specifically identify and discriminate the bacteria from other compartments of the host cell. By using a small scanning step size of 0.25 µm the shape and size of the bacteria could be well resolved. Further, recording of a z-stack allowed to determine the exact position of several individual bacteria in different layers of the host cell. As the method is non-destructive, the same sample could be used afterwards to verify the identification of the bacteria with immunofluorescence staining. Additionally, the vibrational signatures in the Raman spectra were used to derive useful information about the biochemical composition and metabolic state of the intracellular bacteria.
The presented Raman-based imaging algorithm proved to be very suitable to investigate intracellular bacteria in their natural host cell environment, because it delivers images with good resolution of the bacteria and allows label-free identification of the bacteria. As the samples are not destroyed they can be used for further analyses, which makes this method suitable to be combined with further imaging and analytical methods in order to obtain a comprehensive picture of the pathogen-host interaction.

We acknowledge the financial support by the BMBF (FKZ 01EO1002) and the EU within the Framework Program 7 (P4L, Grant agreement no.: 224014), and C. Beleites for help with “R”.

Fig. 1: Overview scan (a, c, e) and detailed scan (b, d, f) of the same endothelial cell containing intracellular S. aureus bacteria. The intensity distribution of the CH stretch band at 2938 cm-1 (c, d) only reveals the contours of the cell, whereas spectral unmixing with N-FINDR (e, f) allows detection of the bacteria.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-2569 Cell biology by Coherence Controlled Holographic Microscope (CCHM)

Collakova J.1,2, Krizova A.1,3, Dostal Z.1,2, Strbkova L.1,2, Lostak M.1,3, Kvasnica L.1,3, Slaby T.1,3, Kolman P.2, Antos M.1, Vesely P.2, Chmelik R.1,2
1Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology, Brno, Czech Republic, 2CEITEC BUT, Brno University of Technology, Brno, Czech Republic, 3TESCAN Brno, s.r.o., Brno, Czech Republic
jana.collakova@ceitec.vutbr.cz

Coherence Controlled Holographic Microscope (CCHM) [1] is a predecessor of Multimodal Holographic Microscope (MHM) [2] described elsewhere here by Krizova et al. Use of low coherence (not laser) illumination improves quality and resolution of quantitative phase images in comparison with laser-based holographic microscopes and provides for coherence gate effect to come in operation. We will present results of CCHM pilot studies in a form of videos accompanied with explanations. Emphasis will be given to exploitation of quantitative phase imaging [3] and lack of halo around phase objects in evolving a method of dynamic phase differences [4] for analysis and measurement of living cell behaviour. Moreover, in this way established coherence gate effect makes observation through and within optically diffuse (scattering) media possible. This effect will be demonstrated on visualization of cells spread on flat surface and immersed in turbid medium [5] suchlike lipid emulsion or 3D collagen gel. These qualities introduce the potential of CCHM for cell biology investigation that lies in imaging and simultaneously measuring motile activity and growth of cells.

References

[1] P. Kolman, R. Chmelik, “Coherence-controlled holographic microscope,” Optics Express 18, (2010) p. 21990

[2] T. Slaby, P. Kolman, Z. Dostal, M. Antos, M. Lostak, R. Chmelik, “Off-axis setup taking full advantage of incoherent illumination in coherence-controlled holographic microscope,” Optics Express 21, Optical Society of America (2013) p. 14747

[3] M. Mir, B. Bhaduri, R. Wang, R. Zhu, G. Popescu, “Quantitative Phase Imaging,” Progress in Optics, Chennai: B.V. (2012) p.133

[4] H. Janeckova, P. Vesely, R. Chmelik, “Proving Tumour Cells by Acute Nutritional/Energy Deprivation as a Survival Threat: A Task for Microscopy,” Anticancer Research 29, (2009) p. 2339

[5] M. Lostak, R. Chmelik, M. Slaba, T. Slaby, “Coherence-controlled holographic microscopy in diffuse media,” Optics Express 22(4), (2014) p. 4180

The research has been supported by Ministry of Industry and Trade of the Czech Republic, project Multimodal Holographic Microscope (FR-TI4/660), internal project of the BUT (FSI-J-13-2139) and CEITEC – Central European Institute of Technology (CZ.1.05/1.1.00/02.0068) from European Regional Development Fund.

Fig. 1: Two frames from series showing rat sarcoma cell in mitosis: a) metaphase and b) cytokinesis. Quantitative phase image permits measuring cell mass in picograms (see graphs). Images were adjusted by non-linear filtration to enhance visualisation of condensed chromatin. However, measurements were done on raw data obtained with objective lens 40x/0.65
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-2883 Wide-field Fluorescence Lifetime Imaging using Multi-Anode Detectors

Zuschratter W.1, Prokazov Y.1, Turbin E.1, Weber A.1, Hartig R.2
1Leibniz Institute for Neurobiology, Magdeburg, Germany, 2Otto von Guericke University, Magdeburg, Germany
zuschratter@lin-magdeburg.de

Fluorescence Lifetime Imaging Microscopy (FLIM) has become a powerful tool to monitor inter- and intra-molecular dynamics of fluorophore-labeled proteins in living cells. Among other FLIM techniques only single photon counting in the time-domain is capable to record the detected photons as efficiently as it is physically possible.That means, a sample is illuminated with short laser pulses and the time difference between the emitted laser-pulse and the appropriate fluorescent photon at the detector is measured resulting in a histogram of arrival times. Unfortunately, most FLIM-systems work at high illumination intensities that might induce photodynamic reactions and ROS during the experiment. Consequently, visualisation of dynamic processes within living cells requires a minimal-invasive approach.

Here, we present a positional sensitive wide-field single photon counting detector system to measure fluorescence lifetimes in the time domain and demonstrate its usage in Förster Resonance Energy Transfer (FRET) applications, to study metabolic changes by NADH imaging and for long term observation of living cells. The time and position sensitive camera consists of a photocathode, a stack of microchannel plates (MCPs) for amplification and a position sensitive anode. The mechanism of data acquisition can be described as follows: an incident photon interacting with the photocathode results with certain probability in an initial electron that is driven towards the MCP amplification stack where it is multiplied. The resulting electron avalanche carries up to ten millions electrons following an applied electrical field and falls on the position sensitive anode. The widefield FLIM camera system provides everything required for robust and reliable single photon counting based imaging. Realtime event selection logic processes registered photons to avoid artifacts like multi-photon events, MCP noise and pile-up effect. In addition to position and picosecond time information (< 50 ps) absolute arrival time (AAT) is acquired with ns precision. The system features a number of additional digital and analog I/O lines available for the user. The values are read and stored along with every detected photon or in custom protocol basis that allows one to extend the set of acquired parameters. The whole setup consists of an inverted fluorescence microscope equipped with a white light laser and/or other short-pulse lasers that illuminate the entire field of view at minimal invasive illumination intensities thereby enabling long-term experiments of living cells. The system is capable to acquire images from single molecules and measures directly the transfer rate of fast photo-physical processes as FRET, in which it can resolve complex fluorescence decay kinetics.

This work was supported by collaborative research grants: BMBF Novel Optics, FKZ 13N10077; BMBF Biophotonic V, FKZ 13N12675; DFG SFB 854 TPZ; DFG FOR 521

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-3150 Microscopy under pressure: Optical micro-spectroscopy technique to study nanomaterials under high hydrostatic pressure

Valenta J.1
1Charles University in Prague, Department of Chemical Physics & Optics, Faculty of Mathematics & Physics, Prague 2, Czechia
jan.valenta@mff.cuni.cz

Changes of external conditions, like electric field or pressure, provide additional information on a studied material, for example semiconductor nanoparticles in colloidal suspensions. Under increased hydrostatic pressure of the order of GPa changes of crystalline lattice parameters, reconstruction of surface states and even structural transitions take place which substantially modify optical properties of the studied material. Here we present a special microscope configuration which enables us to study luminescence spectra and kinetics of nanoparticles under pressure up to or even above 20 GPa. The pressure is generated by lever acting on a diamond anvil cell (Fig. 1). The diamond cullet diameter is 0.4 mm and the sample volume is only 0.01 l. Illumination and imaging takes place trough one of the diamonds. The signal is detected by a special micro-spectroscopy apparatus with parallel detection branches for visible (500-1000 nm) and near-infrared (1000-1600 nm) region and detection by 2D focal-plane arrays (based on Si and InGaAs, respectively) or photomultipliers (for time resolved detection). Pressure is monitored using the shift of red luminescence lines from ruby microparticles mixed with a sample. Application of this setup is demonstrated with silicon based nanoparticles whose investigation shed light on the role of surface passivation or embedding matrix on the energies and the direct/indirect character of optical transitions [1,2].

References
[1] K. Kůsová et al.: Luminescence of free-standing versus matrix-embedded oxide-passivated silicon nanocrystals: The role of matrix-induced strain, Appl. Phys. Lett. 101 (2012) 143101.
[2] K. Kůsová et al.: Direct bandgap silicon: Tensile-strained silicon nanocrystals, Adv. Mater. Interfaces 1(2) (2014) in print, DOI: 10.1002/admi.201300042.

The author thanks Dr. Kalbac and Dr. Frank for providing the DAC cell. This research received support from the EC FP7 programme (No.245977 NASCEnT) and the Czech-Japan collaborative project LG14246.

Fig. 1: The mounting of the diamond anvil cell on an inverted microscope which is the central part of our multi-purpose VIS/NIR micro-spectroscopy set-up.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-3357 Determination of Orientational Distributions of Fluorescent Molecules in Phospholipid Membranes by Polarization Fluorescence Microscopy

Lazar J.1,2, Timr S.3, Melcr J.3, Bondar A.1,2, Cwiklik L.4, Kevorkova A.1,2, Stefl M.4, Vazdar M.5, Jungwirth P.3,6
1Institute of Nanobiology and Structural Biology, Nove Hrady, Czech Republic, 2University of South Bohemia, Budweis, Czech Republic, 3Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic, 4Heyrovsky Institute of Physical Chemistry, Prague, Czech Republic, 5Rudjer Bošković Institute, Zagreb, Croatia, 6Tampere University of Technology, Tampere, Finland
lazar@nh.cas.cz

Orientation of fluorescent molecules (fluorescent dyes or fluorescent proteins) associated with a biological membrane can report on the molecular environment within the membrane, but also on molecular processes involving membrane proteins. In our other work we have demonstrated that changes in fluorescent molecule orientation, detected by two-photon polarization microscopy (2PPM), can be used to report on binding of ligands to G protein coupled receptors (GPCRs), interactions between GPCRs and G proteins, G protein activation, changes in the cell membrane voltage, changes in intracellular calcium concentration, and other molecular processes taking place in living cells. Apart from simply reporting a non-specified change in molecular orientation, 2PPM and related techniques should also be able to yield detailed, quantitative information on molecular orientation and its changes, and therefore provide quantitative insights into membrane protein structure, directly in living cells. In order to develop the quantitative structural capabilities of 2PPM, we have now investigated simple model systems that allow conclusions drawn from our microscopic observations to be independently verified by computational molecular dynamics simulations. Here we present the results of our microscopic observations and molecular dynamics simulations of simple fluorescent dyes and fluorescent protein constructs associated with artificial lipid membranes, demonstrating the quantitative capabilities of 2PPM and related techniques.

Czech Science Foundation grants P205/13-10799S (J.L.) and P208/12/G016 (M.S., M.H., P.J.); University of South Bohemia graduate research scholarship 093/2009/P (A.B.) and 155/2014/P (A.K.); FiDiPro award (P.J.), Praemium Academie award (P.J., M.H.)

Fig. 1: Giant unilamellar phospholipid vesicles stained with a fluorescent dye (F2N12S), imaged by two-photon polarization microscopy. Fluorescence excited by light polarized horizontally and vertically is colored red and green, respectively. The red-green pattern is indicative of the dye molecules being present in a restricted orientational distribution

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-5783 Molecular interaction between insect lipid membrane and Bacillus thuringiensis cytolytic toxin

Tharad S.1,3, Krittanai C.1, Promdonkoyb B.2, Toca-Herrera J. L.3
1Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakhonpathom, 73130, Thailand., 2National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand., 3Institute for Biophysics, Department of Nanobiotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna 1190, Austria.
sudarattharad@gmail.com

Bacillus thuringiensis is a biological control agent producing the insect larvacidal proteins including cytolytic protein (Cyt). Cytolytic protein has larvacidal activity against Dipteran insect

larvae. Moreover, Cytolytic protein synergize activity with Crystal (Cry) protein and act as Cry receptor to overcome the Cry-resistance larvae stains. The cytolytic mechanism was proposed to pore forming and detergent-like mechanisms. However, the mechanism is still unclear. The Cyt protein-lipid membrane interaction was proposed as a critical step for the mechanism. To demonstrate the protein - lipid membrane interaction on Aedes albopictus cell membrane, Cyt protein was labeled by Texas red fluorescent dye and observed under fluorescent microscopes. Confocal and TIRF microscopy reveal that Cyt bind and aggregate on Aedes albopictus cell membrane that cause cell swelling after that Cyt pass into cytoplasm and bind to nuclear membrane and nucleolus. The mass adsorption on lipid membrane of Cyt was determined by quartz crystal microbalance with dissipation. The artificial insect cell membrane was deposited on silicon-coated crystal sensor. The result reveal that the mechanism of Cyt protein is not the pore-forming and detergent-like. The Cytolytic protein adsorption on lipid membrane could explain by the combination of adsorption and diffusion equations. These data suggest that the aggregation complex of Cyt protein compose of two processes. Firstly, Cyt adsorb on the cell membrane nearly complete coverage of the cell membrane, the protein adsorption behavior was changed to diffusion.

This work was supported by Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (PHD/0116/2551).

Fig. 1: (A) and (B): Cytolytic protein translocation into Aedes albopictus cytoplasm. (C): Mass adsorption of Cytolytic protein on lipid membrane.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-5784 Influence of HepG2 Cell Shape on Nanoparticle Uptake

Prats-Mateu B.1,2, Toca-Herrera J.1
1Department of Nanobiotechnology, Institute for Biophysics, University of Natural Resources and Life Sciences (BOKU), Muthgasse 11, 1190 Vienna, Austria, 2Department of Material Sciences and Process Engineering,Institut für Physik und Materialwissenschaft,University of Natural Resources and Life Sciences (BOKU), Peter-Jordan-Straße 82, 1190 Vienna, Austria
jose.toca-herrera@boku.ac.at

Cell mechanics tries to understand cell responses to external stress, which can influence a variety of cell functions. Understanding the relation between mechanical stimulus, cell shape and cell function is essential in controlling cell behavior in different environments and conditions. In this poster, we present first results on the effect of cationic and anionic interfaces on cell shape of hepatocellular carcinoma cells (HepG2) and nanoparticle uptake activity. On one hand, HepG2 cells incubated on cationic poly-ethylene imine exhibited a spread-like form with lamellar protrusions. On the other hand, hepG2 cells adopted a more round-like form when hosted on polystyrene sulfonate. The difference in cell shape did not influence the uptake of 49-nm polystyrene particles (driving by diffusion). However, the internalization of 240-nm diameter polystyrene particles was larger on cells seeded on cationic polyelectrolyte (suggesting an active uptake process). The particle uptake was measured at 4ºC and 37ºC and the optimal incubation time was found to be 6 h. Cell shape and particle uptake were monitored by fluorescence and confocal microscopy. Quantification of particle internalization was carried out with flow cytometry. In summary, the results indicate that cell shape can be changed by using ionic interfaces, and that such change influences nanoparticle uptake. It seem that in this context, the mechanical properties of the plasmatic membrane are crucial for cellular uptake, and that possibly the Young modulus could be a physical indicator of such fact. Therefore, more experiments with different functional nanoparticles should be carried out to get more insight about the uptake mechanism.

BPM thanks FFG and AIT.  The authors acknowledge Monika Debreczeny and Darren Tan Cheng-Weng for technical help.

Fig. 1: Fluorescence microscopy graphs of HepG2 cells on three different substrates: control polystyrene (PS), polystyrene sulfonate(PSS), and polyethylenimine (PEI). Adherent cells on PEI show atendency to spread and cover higher surface area than cells on PSS orPS.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-5854 Ultramicroscope based on the application of an axicon lens

Cesal J.1
1Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University in Prague
jancesal@seznam.cz

Ultramicroscope (UM) was invented and developed more than 100 years ago by Zsigmondy and Siedentopf. The basic idea was a visualization of objects smaller than the resolution limit of classical microscopes exploiting light scattering by particles under the perpendicular (dark field) illumination. Nowadays, some of the novel techniques breaking the diffraction resolution-limit (so called super-resolution techniques) remind principles of the ultramicroscope - for example STORM (Stochastic Optical Reconstruction Microscopy) – based on the localization of single emitting centers (molecules, nanoparticles).

This contribution presents our innovative home-built ultramicroscope, which contains a special dark-field illumination system based the axicon lens (lens with one conical and one flat surface, often used to generate Bessel beams). The axicon transforms the illumination laser beam into cone focused on the sample image plane by an objective. This provides oblique illumination of the sample which acts as a dark-field illumination if combined with an objective lens with the appropriate numerical aperture. The setup is assembled from commercially available optomechanical components, which makes the construction of the ultramicroscope relatively easy and adaptable to various applications. We demonstrate the properties of our ultramicroscope by performing scattering and fluorescence measurements of semiconductor nanoparticles. In our assembly is possible qualitative and quantitative analysis of the sample.

I would like to express my deep gratitude to Professor Jan Valenta, my research supervisors, for your patient guidance, enthusiastic encouragement and useful critiques of this research work.

Finally, I wish to thank my parents for their support and encouragement throughout my study.

Fig. 1: Simplified schematic diagram of the ultramicroscope
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-5935 Multimodal holographic microscopy for cell applications

Fukalová J.1, Křížová A.2, Hozák P.1, Chmelík R.2
1Institute of Molecular Genetics, Dept. of Biology of the Cell Nucleus, Academy of Sciences of the Czech Republic, Prague, Czech Republic , 2CEITEC - Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic
fukalova@img.cas.cz

The cooperation between TESCAN, a.s., Brno University of Technology and Institute of Molecular Genetics led into the first prototype of a Multimodal holographic microscope (MHM) - a unique instrument using the incoherent holography for the quantitative phase imaging (1). This microscopic methodology is suitable for non-invasive imaging and characterization of cellular mass and growth dynamics. This provides a powerful quantitative tool for label-free biological cell investigation, without using any adverse markers and toxic substances. MHM enables imaging in dispersion solutions, suppresses the coherent noise significantly and therefore provides high imaging sensitivity. The microscope allows quantitative imaging of the phase difference between the reference and object wave, which monitors dry mass distribution within the cells. In addition, it provides the combination of unique holographic properties with other conventional microscopic techniques and maintains the optimal conditions for investigation of living cells during long-term observations (2).
Our study demonstrates the applicability of a multimodal holographic microscope as a suitable tool for quantitative analysis of the cell cycle. First of all, we compared measurements of the same biological samples using the conventional fluorescence technique and the off-axis holographic technique. We obtained similar information about cellular behaviours at different phases during a full lifecycle of HeLa cells from these two different methods. We calculated distribution of intracellular dry mass and its changes in synchronous cells.
References:
(1) Slabý T., Kolman P., Dostál Z., Antoš M., Lošťák M., Chmelík R.: Off-axis setup taking full advantage of incoherent illumination in coherence-controlled holographic microscope, Optics Express, Vol. 21, Issue 12, pp. 14747-14762 (2013).
(2) Kolman P., Chmelík R.: Coherence-controlled holographic microscope, Optics Express, Vol. 18, Issue 21, pp. 21990 - 22003 (2010).

This study was supported by the grant MPO-Tescan (Reg. No. FR-TI4/660).

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-11-P-5978 Dual-mode microviscosity measurements using a molecular rotor

Vysniauskas A.1, Kuimova M. K.1
1Imperial College London, London, UK
a.vysniauskas12@imperial.ac.uk

Viscosity of the cellular membrane has a significant impact on cellular processes, including on the diffusion of biomolecules within or through the membrane. Although measurement of viscosity on a microscopic scale is a challenging task, we have demonstrated1 that it can be achieved with the aid of molecular rotors, which are fluorophores with viscosity dependent fluorescence intensities and lifetimes. Microviscosity measurements are usually performed via Fluorescence Lifetime Imaging Microscopy (FLIM), which produces a spatial map of the lifetime of a molecular rotor in the object of interest. Alternatively, ratiometric imaging can be used if the molecular rotor exhibits a viscosity dependent intensity ratio between two peaks in its fluorescence spectrum.

In this work we have examined a molecular rotor MB, constructed as a porphyrin dimer, which is capable of measuring viscosity via both of the methods described above. Only a few such molecular rotors are reported in the literature, however, this provides a useful opportunity as it allows us to independently double check measured viscosity values. We performed the calibration of the rotor in methanol/glycerol mixtures of varying viscosity using both FLIM and ratiometric imaging (Figure 2). MB was then employed for measuring viscosity in (i) lipid monolayers made by coating water droplets in dodecane with the lipid of choice (Figure 1). Viscosity change upon irradiation was measured in saturated (DPhPC) and unsaturated (DOPC) lipid monolayers. (ii) Viscosity was measured in SK-OV-3 cells upon irradiation.

Prof HL Anderson group for the synthesis of MB
EPSRC Doctoral Prize Studentship
EPSRC Career Acceleration Fellowship to MKK

Fig. 1: Figure 1. FLIM (a) and ratiometric (b) images of lipid monolayers around water droplets in dodecane. Viscosity values corresponding to the lifetimes and ratios given in the colorbar are shown in brackets.

Fig. 2: Figure 2. Calibration data of MB in methanol/glycerol mixtures. Normalized fluorescence decays (a) and normalized fluorescence spectra (b) in solvent mixtures of different viscosities. (c) Lifetime (blue) and ratiometric (red) calibration curves obtained from the data in (a) and (b).
↑ Go to the Table of content ↑

ID-12. In-situ and environmental microscopy of processes in materials and material reactions

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-12-IN-1851 High-speed atomic force microscopy for dynamic nano-visualization of biomolecular and cellular processes

Ando T.1
1Kanazawa University, Kanazawa, Japan
tando@staff.kanazawa-u.ac.jp

Structural biology created in 1950th has revealed in detail static three dimensional structures of many proteins using techniques represented by X-ray crystallography. However, protein molecules are dynamic in nature; the molecules fluctuate, undergo conformational changes, bind to and dissociate from the partner molecules and transverse a range of energy and chemical states. Therefore, we have had limitations in understanding how proteins function from their static structures. To make it possible to study the dynamic behavior of proteins, single-molecules biophysical techniques including single-molecule fluorescence microscopy and optical trap nanometry have been created and successfully used. However, the protein molecules themselves are invisible in the single-molecule observations even with super-resolution bypassing the diffraction limit. Thus, simultaneous assessment of structure and dynamics has long been infeasible, which is the main cause for the difficulty in understanding the functional mechanism of proteins.

To break this long-standing situation, high-speed atomic force microscopy (HS-AFM) has been developed (T. Ando et al., PNAS, 98, 12468, 2001; T. Ando et al., Prog. Surf. Sci. 83, 337, 2008). It enables direct visualization of dynamic structural changes and dynamic interactions occurring in individual proteins molecules at sub-100 ms temporal resolution. The revolutionary power of this new microscopy has recently been demonstrated in the studies of bacteriorhodopsin responding to light (M. Shibata et al., Nat. Nanotechnol. 5, 208, 2010), myosin V walking actin filaments (N. Kodera et al., Nature 468, 72, 2010), rotor-less F1-ATPase (T. Uchihashi et al., Science 333, 755, 2011), and others (see reviews: T. Ando et al., 42, 393, 2013; T. Ando et al., Chem. Rev., in press). The molecular movies with sub-molecular resolution have yielded significant findings, providing new insights into how the proteins function. The very recent progress in this microscopy has also been making it possible to observe dynamic molecular processes on live bacterial cells (H. Yamashita et al., J. Mol. Biol. 422, 300, 2012) and dynamic phenomena occurring in live eukaryotic cells (H. Watanabe et al., Rev. Sci. Instrum. 84, 053702, 2013). In this talk, I will review these studies and discuss future prospects of HS-AFM studies.

I thank my colleagues and collaborators for their enthusiasm to pioneer HS-AFM studies. This study was supported by Grant-in-Aid for Basic Research S from the MEXT, Japan (20221006), and JST/CREST

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-12-IN-6087 In-situ SEM studies of working catalysts

Schlögl R.1, 2
1Fritz Haber Institute of the Max Planck Society, Berlin, Germany , 2Max Planck Institute for Chemical Energy Conversion, Mülheim a.d. Ruhr, Germany
acsek@fhi-berlin.mpg.de

The study of heterogeneous catalysts by in-situ methods has compiled solid evidence that their function is intimately connected to a dynamic response of their precursor structure to the chemical potential of the reacting environment. This is also valid for the supra-molecular or mesoscopic dimension at which the performance of catalysts is decided critically by transport phenomena of molecules and energy. This mesoscopic dimension is well accessible by modern field emission SEM instrumentation. With advent of the ESEM technology it became conceivable to observe catalysts at truly near-ambient pressure conditions at work. As it turns out there are still obstacles to be overcome having to do with imaging conditions and the sample environment. We succeeded in modifying a commercial instrument such that we can observe simultaneously the catalytic activity of a specimen and its morphology. This is possible for complex reactions such as the epoxidation of ethylene over silver and for harsh conditions such as the CVD synthesis of grapheme over metal catalysts. The contribution uses these two examples to illustrate the value of insights one can obtain from such kind of in-situ microscopy. The results will be combined with insights gained from NAP XPS experiments that can be conducted under identical pressure-and temperature conditions. Together one obtains a combined picture of the geometric and electronic structure of working catalysts illustrating the hitherto barely detectable structural dynamics.

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-1778 In situ SEM observation of electrode reactions in ionic liquid-based lithium-ion secondary battery

Tsuda T.1, Sano T.1, Kanetsuku T.1, Oshima Y.2, Ui K.3, Yamagata M.4, Ishikawa M.4, Kuwabata S.1
1Department of Applied Chemistry, Osaka University, Osaka, Japan, 2Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, Osaka, Japan, 3Department of Frontier Materials and Function Engineering, Iwate University, Iwate, Japan, 4Faculty of Chemistry, Materials and Bioengineering, Kansai University, Osaka, Japan
ttsuda@chem.eng.osaka-u.ac.jp

Ionic liquid (IL) generally exhibits attractive physical and chemical properties, e.g., negligible vapor pressure, relatively-high ionic conductivity, wide electrochemical window. We have focused on IL’s negligible vapor pressure to create novel technologies and analytical methods combined with vacuum technology and ILs.1) Based on our first report that IL can be observed by a common SEM,2) we have established several novel SEM observation and EDX analysis methods using ILs.3) The aim of this study is to directly observe electrode reactions in IL-based lithium-ion secondary battery (LIB), which is expected to be a next generation energy storage device.

     IL-based LIB used in this investigation was composed of a Si negative electrode, a LiCoO2 positive electrode, a glass fiber membrane, and an IL electrolyte with lithium bis(trifluoromethanesulfonyl)amide (Li[(CF3SO2)2N]). We mainly used 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)amide ([C2mim][(FSO2)2N]) as the IL electrolyte because the cell performance of the LIB with the [C2mim][(FSO2)2N] electrolyte is comparable to present LIB system using organic electrolytes.4) Figure 1a shows a photograph of an IL-based LIB cell employed for in situ SEM observation. The LIB configuration is basically the same as commercially-available LIB cells. The detailed information on the configuration is given in Figure 1b. Silicon micro/nanoparticles, which have extremely-high theoretical capacity (4199 mAh g-1) compared to graphite electrode (372 mAh g-1) widely used in common LIBs, was exploited as the active material for the negative electrode. Si particle volume become up to 4.0 times when the electric energy is fully stored. Although the deterioration of the Si negative electrode is believed to be due to the drastic volume variation, there is insufficient information on the deterioration mechanism. Figure 2 shows typical SEM images of the Si negative electrode in the IL-based LIB during charge/discharge process. We could directly observe the variation in the Si electrode by our in situ SEM observation technique. The detailed results and discussion will be given in our presentation.

Part of this research was supported by Grant-in-Aid for Scientific Research B, Grant No. 24350071, from Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT), from Advanced Low Carbon Technology Research and Development Program (ALCA), Japan Science and Technology Agency (JST), and from Japanese Ministry of Economy, Trade and Industry (METI).

Fig. 1: (a) Photograph of one of IL-based LIB cells used for in situ SEM observation and (b) explanation of the cell depicted in Fig. 1a.

Fig. 2: Variations in morphology of the Si negative electrode observed by the in situ SEM observation system during charge/discharge processes at 0.2 C in the IL-based Li-ion secondary battery prepared in this study. The used IL was [C2mim][(FSO2)2N].
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-1782 New possibility of ETEM studies of thick samples by HVEM

Tanaka N.1, Fujita T.2, Takahashi Y.3, Arai S.1
1Facility for High-Voltage Electron Microscopy, Ecotopia Science Institute, Nagoya University, Nagoya, 464-8603, Japan , 2WPI of Tohoku University, Sendai, 980-8577, Japan, 3Kansai University, Suita, 564-8680, Japan
n-tanaka@esi.nagoya-u.ac.jp

Environmental transmission electron microscopy(ETEM) recently attracts a strong interest of many materials researchers, because the observation of chemical reaction process with gases and liquids becomes important for the practical use. However, the samples to be observed are only thin edges of films and particles due to the limitation of electron transmission in ordinary TEM up to 300 kV accelerating voltage. ETEM observations of samples of a few micron thickness in gas environment of hydrogen or oxygen are crucial for development of practical fuel cells and solar batteries, and various kinds of chemical and structural materials. 3D observation of samples is also necessary for clarifying the morphologies related to the chemical properties such as catalysis.

     We have developed 1MV TEM/STEM with an open-type environmental cell which enables observation in 100 Torr atmosphere of gases, named "Reaction Science HVEM(RSHVEM)"[1]. High-transmission of electrons insures the point-to-point resolution less than 0.2 nm even in gas-environment of 100 Torr. High-speed beam blanking system reduces irradiation effects of incident electrons on the samples.

     Figure 1 shows an external view of the RSHVEM with a box-shaped environmental cell inserted in the pole-piece-gap of an objective lens from the left-hand side. Figure 2 shows in-situ atomistic observation of oxidation process of a copper(Cu) particle followed by reduction by hydrogen. The repeated reaction process is monitored also by in-situ EELS. Figure 3 shows in-situ observation of porous gold (Au) catalyst in reaction, whose inner surface with zigzag atom arrangement enhances the catalytic reaction with carbon-mono oxide[2,3]. Figure 4 shows the world-first in-situ observation of hydrogen brittleness of a copper(Cu)/silicon(Si) interface as well as in-situ measurement of stress/strain curves in a mixed gas of hydrogen and nitrogen[4]. The present microscope can be used also for in-situ high-resolution STEM-EELS mapping[5] for elemental, chemical and physical analysis, and 3D electron tomography of large inorganic particles and biological cells[6] is possible.

[1]N. Tanaka et al., Microscopy, 62, 305 (2013).

[2]T. Fujita et al., Nature Mater., 16, August, (2012).

[3]T. Fujita et al., Nano lett., in press, (2014).

[4]Y. Takahashi et al., to be submitted (2014).

[5]S. Muto et al., Material Trans., 50, 964 (2009).

[6]Y. Murata et al., to be submitted (2014), and in this conference.

The present authors would like to acknowledge many collabolators and graduate students for help to the present studies.

Fig. 1: Front-view of RSHVEM in Nagoya University.

Fig. 2: In-situ observation of oxidation of a copper particle           followed by the reduction.

Fig. 3: In-situ HRTEM of reaction change of a (011) surface            of gold with CO gas.

Fig. 4: In-situ TEM of fracture of a Cu/Si interface in H2 +           N2 gas.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-1974 Single atoms in AC ESTEM catalysis studies

Boyes E. D.1, Lari L.2, Ward M. R.2, Martin T. E.2, Wright I.2, Gai P. L.3
1The York Nanocentre and Departments of Physics and Electronics, University of York, UK, 2The York Nanocentre and Department of Physics, University of York, UK, 3The York Nanocentre and Departments of Chemistry and Physics, University of York, UK
ed.boyes@york.ac.uk

We have recently developed [1] aberration corrected environmental scanning transmission electron microscopy (AC ESTEM) with a controlled gas atmosphere and hot stages, a full range of STEM (and TEM) imaging modes with <0.1nm resolution and single atom sensitivity, electron diffraction and other analyses including uncompromised EELS and EDX in gas at >600oC. We have modified for purpose a JEOL 2200 FS 200kV FEG system equipped with Cs correctors for STEM probe and TEM image. The new studies reveal single Pt atoms (FWHM = 0.11±0.01nm in H2) (Fig.1) on the carbon support surface between larger entities. The latter start out as loosely assembled 'rafts' (Fig.1) of lower density and somewhat ragged outline with single atom high edges. On heating at 500oC in H2 they become more densely packed and finally grow into crystalline nanoparticles with sharply defined cuboid {200} cliff faces typically 4 or more atoms high (Fig.2). Different forms of metal atom neighbourhoods include individual atoms isolated on the substrate surface; rafts of differently assembled structures and densities; progressively more structured 2D forms; and then the fully crystalline 3D nanoparticles. In each case the local bonding and electron energy states of the Pt atoms will be different and they can be expected to have distinct chemical reactivity and catalytic selectivity. The accessibility and activity for reactions of each type of Pt atom site will be different: isolated on the support surface, at raft edge or centre, on a crystalline face of buried deep inside an essentially impervious crystalline 2nm nanoparticle. The forms and properties of a catalyst can change radically, and often rapidly, during initial reactor operations. The new data show a population of single Pt atoms persists on the support between the larger particles and they should be highly active species for reaction. Mobility seems to be intermittent and this aids their detection. Multiple line scans, typically 5-10 per atom, reinforce the imaging of each atom over 0.1sec. Initial studies have been at 1-10Pa gas pressure measured at the sample, and we use a modified furnace type hot stage (Gatan 628 special) with a Keithley 2614B power supply and a DENS MEMS heater. A reconfigured column vacuum system uses multiple TMPs and additional ion pumping to create differential pressure zones across existing and newly introduced fixed apertures up and down the column. With the new atom-by-atom analysis E-tool we can assess better the conflicting requirements for high initial activity and selectivity with the attendant need for stability of nanostructures for more consistent outcomes in the management of reactor operations.

Reference
[1] E Boyes, M Ward, L Lari and P Gai, Ann Phys, 525 (2013) 423

The AC ESTEM project at York is supported by EPSRC grant EO/J018058/1, the University of York and ERDF

Fig. 1: Pt on C model catalyst system after initial room temperature treatment in H2.  Imaged in the H2 gas atmosphere in the York AC ESTEM, showing single atoms and different forms of raft structures, and hence of atom neighbourhoods, physical and chemical properties.   Insert A shows a line trace over a single Pt atom image = 0.11±0.01nm FWHM. 

Fig. 2: Pt on C model catalysts after heating in H2 at 500oC and forming cuboid and fully crystalline nanoparticles of Pt up to 2nm in size.  This figure includes illustrative data for how their shapes can be analysed atom by atom with quantitative nZ HAADF STEM image contrast for the initial and final forms of the Pt in these experiments.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-2049 High resolution in-situ study of reactions and bio-structures in graphene liquid cells

Wang C.1, Qiao Q.1, Shokuhfar T.2, Klie R. F.1
1Dept. of Physics, University of Illinois at Chicago, Chicago, United States, 2Dept. of Mechanical Engineering, Dept. of Biomedical Engineering, Michigan Technological University, Houghton, United States
cwang70@uic.edu

Chemical reactions or biochemical activities often occur in the presence of a liquid. To study liquid sample in an electron microscope, several liquid cell designs have become commercially available in recent years that enable materials to be imaged in a carefully controlled liquid environment within the vacuum of a TEM. However, the Si3N4 layers (50-500 nm thick) used as electron transparent windows in these liquid cells have limited the imaging resolution to nanometers [1], also degraded the electron energy-loss spectroscopy (EELS) signal [2]. On the other hand, radiation damage is a fundamentally limiting factor when examining beam sensitive materials and/or hydrous samples in TEM. Traditional coating and cryo techniques [3, 4] have shown positive effects against radiation damage and the results suggest that it is possible to reduce radiation damage to below breakage of covalent bonds. However, further reduction of radiation damage is needed for characterization of biological samples, since many biological structures and functions are related to the much weaker hydrogen bonds.
We introduce a biocompatible approach of encapsulating liquid containing samples in monolayers of graphene. This not only allows biological samples to be directly imaged at atomic resolution in a native liquid state without limitations from the window thickness, but also enables nm-scale analysis using EELS to quantify biochemical reactions in an aqueous environment (Fig. 1 and 2) [5]. We show that the graphene encapsulation provides a radiation damage reduction mechanism, allowing for high resolution imaging and spectroscopy of beam sensitive materials. Details, such as individual Fe atoms or polypeptides of unstained protein, are resolved in a liquid environment. EELS elemental identification of ferritin molecules with 1 nm resolution is achieved showing both the iron core and the protein shell. We also show that beam induced reactions can be initiated in-situ and monitored in real time at nm precision inside graphene liquid cells (GLC) (Fig. 3). By carefully controlling the induced electron dose rate, we show that radiation damage can be limited to be within hydrogen bond breakage level, preserving the functionality of the ferritin protein while observing the valence change of the iron core showing initial stages of iron release by ferritin.
References:
[1] de Jonge, N. & Ross, F. M. Nature nanotechnology 6, (2011), 695-704.
[2] Holtz, M. E., et al, Microscopy and Microanalysis 18, (2012), 1094-1095.
[3] Salih, S. & Cosslett, V. Philosophical Magazine 30, (1974), 225-228.
[4] Fryer, J. & Holland, F. Proceedings of the Royal Society of London. A. (1984), 353-369.
[5] C. Wang, Q. Qiao, T. Shokuhfar, R. Klie, Advanced Materials, (2014), doi: 10.1002/adma.201306069.

This work is funded by Michigan Technological University, as well as an MRI-R2 grant from the National Science Foundation (DMR-0959470).

Fig. 1: Schematic diagram, as well as HAADF images (A and B) of ferritin molecules in graphene sandwiches (A) and GLCs (B). (B) shows individual Fe atoms in a liquid environment. (C) is a EELS map of ferritin molecules sandwiched between graphene sheets with 1 nm resolution, showing both the protein shell and the iron core.

Fig. 2: EEL spectra of ferritin (1, 2, 4, 5) and water (3). (1) and (2) are taken from the iron core (1) and protein shell of ferritin (2) in graphene sandwiches where the liquid has dried. (3, 4, 5) are taken from water (3), at the center of the iron core (4), and at the edge of a ferritin molecule (5) inside a graphene liquid cell.

Fig. 3: Stills from a video showing controlled beam induced local bubble formation and condensation process in GLC. The video is recorded via repeat scanning in HAADF mode. In selected areas a bubble is formed at 00:06s, 00:15s, and 00:22s respectively by temporary switching to small area scanning.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-2096 Capturing the dynamics of pulsed laser induced melting and crystal growth during rapid solidification of Al-Cu alloys with unprecedented spatio-temporal resolution in-situ TEM

Zweiacker K. W.1, McKeown J. T.2, LaGrange T.2, Reed B. W.2, Liu C.1, Campbell G. H.2, Wiezorek J. M.1
1University of Pittsburgh, Pittsburgh, PA, USA, 2Lawrence Livermore National Laboratory, Livermore, CA, USA
kwz3@pitt.edu

The unprecedented spatiotemporal resolution offered by the dynamic transmission electron microscope (DTEM) facilitates unique studies of irreversible transitions in solids [1]. Prior DTEM work using single-shot ns-temporal resolution bright field imaging and diffraction established feasibility of studies of rapid solidification (RS) in Al, Cu, Ag and Al-Cu alloy [2-5]. Advances in DTEM instrumentation have enabled acquisition of multiple-image sequences following a single drive event, which promises to greatly reduce quantitative uncertainties in measurements and increase by about one order of magnitude the data density obtained from direct TEM observations of the dynamics of the irreversible transformation processes [6]. Here, we present and discuss results of in-situ DTEM investigations of RS in Al-Cu alloys ranging in composition from Al-3at%Cu to the eutectic Al-17at%Cu. Figure 1 shows the global microstructural changes in a time-delay of image sequence recorded as an 80nm-thick Al-3at%Cu thin film solidifies after pulsed-laser (PL) melting. The rows of images were recorded from two separate laser induced melting experiments. Each experiment spans 20.4µs and produces nine images using a 2.5µs interframe spacing and a 50ns frame exposure time. The liquid-solid interface velocity, averaged along the complete interface can be related to the RS microstructure evolution (Figure 1). In the initial stages, ≤ 7µs after PL melting, the liquid-solid interface is essentially stagnant. At about 10µs after PL melting solidification commences, producing a columnar grain structure behind a planar transformation interface that accelerates up to >2m/s. Solidification completes by ~31µs after PL melting. Figure 2 shows an example time-delay sequence of images recorded during for an 80nm-thick Al-17at%Cu thin film. Higher magnification imaging permits study of local morphology development on the individual columnar grain level for the RS in the eutectic Al-Cu alloy. Acquisition of several such image-series with systematically varied pre-delay times, ∆t, enabled direct observation of the dynamics of crystal growth mode changes for the different alloy composition and measurements of growth mode depended, instantaneous, and average transformation interface velocities. Microstructural evolution and solidification kinetics for the alloy compositions investigated have been analyzed and will be discussed.

References
[1] King, W.E., et al., J Appl Phys (2005) 97: 111101.
[2] Kulovits, A.K., et al., Microsc Microanal (2011) Vol. 17 (Suppl. 2): 506.
[3] Kulovits A, et al., Philos Mag Lett (2011) 91: 287.
[4] McKeown, J.T., et al., Microsc Microanal (2012) Vol. 18 (Suppl. 2): 602.
[5] McKeown, J.T., et al., Acta Mater (2014) 65: 56.
[6] LaGrange T., et al., Micron (2012) 43: 1108.

Funded by the U.S. DOE and LLNL, Grant No. DE-AC52-07NA27344. Work at University of Pittsburgh was supported by the U.S. NSF, Grant No. DMR 1105757.

Fig. 1: Dynamic time-delay image sequences of re-solidification in 80-nm-thick Al-3at.%Cu. Delays (in µs) between the laser pulse for melting and the 50-ns electron pulse for exposure are indicated above each image. Letters S and L mark solid and liquid state of the alloy.

Fig. 2: Image sequence of re-solidification in 80-nm-thick Al-17at.%Cu. Delay times for image #1 (prior to melting), images #2-#8 (solidification), and image #9 (post-solidification) are shown. Letters S and L mark solid and liquid state. Measured over the 7µm diameter field of view the discernible individual grains solidified at ~0.27m/s.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-2236 Measurements of local chemistry and structure in NiO/YSZ composites during reduction using energy-filtered environmental TEM

Jeangros Q.1, Hansen T. W.2, Wagner J. B.2, Dunin-Borkowski R. E.3, Hébert C.1, Van herle J.4, Hessler-Wyser A.1
1Interdisciplinary Centre for Electron Microscopy, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland, 2Center for Electron Nanoscopy, Technical University of Denmark, Kgs. Lyngby, Denmark, 3Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons and Peter Grünberg Institute , Jülich Research Centre, Jülich, Germany, 4Fuelmat Group, School of Engineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
quentin.jeangros@epfl.ch

The activation of a solid oxide fuel cell anode, a process that involves the reduction of the as-sintered nickel oxide (NiO)/yttria-stabilized zirconia (YSZ) composite to the electrochemically active Ni/YSZ state, is assessed using energy-filtered transmission electron microscopy images acquired in an H2 atmosphere at elevated temperature in an environmental transmission electron microscope (ETEM). Quantitative measurements of both reaction kinetics (using oxygen K edge images) and evolution of thickness (using total-inelastic mean free path images) are obtained during NiO/YSZ reduction to Ni/YSZ in 1.3 mbar of H2 up to 600 °C (Fig.1) [1]. In addition, structural and crystallographic changes are monitored using bright-field (BF) imaging and selected area diffraction.
Measurements of the relative changes in thickness highlight the formation of voids within Ni particles to compensate the volume loss induced by oxygen removal. In regions not affected by diffraction effects, local measurements of volume shrinkage induced by NiO reduction to Ni agree with the theoretical prediction of -41% [2]. The sequence of oxygen maps allows the extraction of reaction kinetics localized at the pixel/nm scale and demonstrates the initiation of the reaction at grain boundaries with the YSZ phase. Density functional theory calculations suggest that this process may result from oxygen ion transfer from NiO to YSZ at these grain boundaries [3], which creates oxygen vacancies in the NiO phase and in turn triggers the reduction reaction [4]. The YSZ backbone remains stable throughout the reduction of the NiO phase at these temperatures.
While factors related to sample preparation, spatial drift and the presence of the high-energy electron beam must be considered, with care energy-filtered imaging in a gas atmosphere at elevated temperature has the ability to provide quantitative new insight into the activation of SOFC anodes with a spatial resolution in the nm range. Differences in reaction rate as a result of local features can be investigated in detail using the present methodology, paving the way for the development of detailed reaction/activation models.

[1] Q. Jeangros, et al., Chemical Communications, 2014, 50, 1808-1810.
[2] A. Faes, et al., Membranes, 2012, 2, 585-664.
[3] Q. Jeangros, et al., Acta Materialia, 2010, 58, 4578-4589.
[4] J. A. Rodriguez, et al., Journal of the American Chemical Society, 2002, 124, 346-354.

The authors wish to thank U. Aschauer, A. Faes, F. Bobard, M. Cantoni, D. Laub, G. Lucas, D. Alexander, E. Oveisi, P. Stadelmann and the Swiss National Science Foundation.

Fig. 1: Reduction of NiO-YSZ in H2 up to 600 °C. Selection of bright-field (BF), thickness maps (t/λ) and oxygen K edge (O K) images acquired at 300 °C, 428 °C and 604 °C. Depletion of oxygen in NiO occurs at NiO-YSZ grain boundaries (arrows at 428 °C). Measurements of volume shrinkage in regions A and B yield -40% and -42%, respectively.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-2296 Catalytically active surfaces revealed by In Situ Measurements of Localized Surface Plasmon Resonance

Lin P. A.1 2, Kohoutek J. M.1 2, Winterstein J.1, Lezec H.1, Sharma R.1
11Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Gaithersburg, MD, USA, 22University of Maryland – IREAP,College Park, MD, USA
pinann.lin@nist.gov

During heterogeneous catalysis, gas adsorption on the active metal nanoparticle (NP) surface results in electron density changes.  These can be measured by localized surface plasmon resonance (LSPR) shifts.  However, nanometer spatial resolution and an energy resolution sufficient to detect LSPR shifts of a few meV is needed to resolve the active NP surfaces. This requirement has been met by using a monochromated high-energy electron source. Therefore, we have employed a monochromated 80kV electron source in an environmental scanning transmission electron microscope (ESTEM) to measure, in situ, the LSPR response of different surfaces on individual triangular Au NPs in vacuum, H2, and CO at various pressures. The local LSPR energies are obtained by electron energy-loss spectroscopy (EELS) in STEM mode with a beam size of ≈ 1 nm and 100 meV  energy resolution. Our results confirmed by Finite Difference Time Domain (FDTD) simulation .
Figure 1a shows the STEM dark-field image of a triangular Au NP on TiO2 support. LSPRs measured at two locations, at the corner (b) and side (c), of the Au NP, were observed to shift to lower energy when the H2 pressure was increased to 100 Pa (Fig. 1b and c). However, the extent of the energy shift at the corner (Fig. 1b) was more than that at the side (Fig. 1c) of the NP. This shift indicates a site-specific change in the local electron density that may be due to preferential hydrogen interaction/adsorption on the corners. We use two models for our FDTD simulations: (i) we decrease the electron density due to adsorption of electronegative H2, for a 1 nm surface layer over the entire particle and interrogate 1 nm regions at the corner (Fig. 2a) and on the side (Fig. 2b). In this case we find that the same shifts occur both at corner and side surfaces. (ii) We change the electron density only at the corners and find that the simulated energy only shifts at the corners (Fig. 2c) – matching with our experimental data.
Therefore, we can conclude that the H2 is preferentially adsorbed/interacts at the corner of the Au NP. We have employed these techniques to interrogate the adsorption sites for other gasses, including CO, and a detailed discussion of our findings will be presented.

Fig. 1: (a) STEM image (scale bar is 20 nm) and a stack of EELS spectra displaced vertically aquired (b) at the corner and (c) the side of Au NP on TiO2 in high vacuum (HV), 10 and 100 Pa H2, and after H2 pumped out (HV-after). The red dashed line is aligned at the LSPR energy in HV-before, and the black dashed line indicates the extent of energy shift.

Fig. 2: (a) Simulated LSPR spectra at the corner and (b) the side, in the case of gas- interaction with entire NP surface (insets). (c) Simulated LSPR spectra at the corner and (d) the side, in the case of gas-interaction restricted to the corner of NP (insets). Red arrows indicate the direction of the energy.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-2635 Studying the in situ growth and degradation of inorganic nanoparticles by liquid-cell aberration corrected TEM

Javed Y.1, Ricolleau C.1, Ammar S.2, Belkahla H.2, Gazeau F.3, Alloyeau D.1
1Laboratoire Matériaux et Phénomènes Quantiques, UMR 7162 CNRS/Université Paris Diderot, 10 rue Alice Domon et Léonie Duquet, F-75205 Paris Cedex 13, France. , 2ITODYS, Universite Paris Diderot, Unite Mixte de Recherche Paris 7-CNRS 7086, 15 rue Jean de Baif, F-75205 Paris, France, 3Laboratoire Matières et Systèmes Complexes, UMR 7057 CNRS/Université Paris - Diderot, 10 rue Alice Domon et Léonie Duquet, 75013 Paris, France
yasir.javed@univ-paris-diderot.fr

Using liquid-cell TEM holder in an aberration corrected TEM is a major technological rupture for understanding the complex phenomena arising at the liquid/solid interface.[1] Recent MEMS-based technology developed by Protochips inc., allows imaging the dynamics of nano-objects in an encapsulated liquid solution within an electron-transparent microfabricated cell. The environmental conditions are finely controlled with a micro-fluidic system which enables to mix different reaction solutions at the observation window. Here, we performed the direct in situ study of two crucial phenomena for the design of efficient nanomaterials in biological environment: (i) the growth mechanisms of gold nanoparticles (NPs) and gold nanoshells over magnetic iron oxide NPs. (ii) The degradation mechanisms of iron oxide NPs in a solution mimicking cellular environment.
(i) We have studied the growth of gold NPs via the reduction of metal salt. These growth mechanisms observed with a resolution below 0.2 nm, is indirectly induced by the electron beam. Indeed, 200kV incident electrons radiolyze the water, creating free radicals and aqueous electrons that reduce metallic precursors. We have evidenced that the growth mode of gold NPs highly depends on the electron dose. High electron dose results in a diffusion limited growth mode leading to large dendritic structures, while low electron dose allows the formation of facetted NPs due to a reaction-limited growth. These latter conditions enable the fascinating study of the size-dependant equilibrium shape of Au nano-polyhedra in water (Fig. 1). Similarly, we have also evidenced the Volmer-Weber growth mode (3D mode) of gold-shells over iron oxide NPs.
(ii) If the understanding of the formation mechanisms of inorganic NPs is very important for controlling upstream their shape related-properties, studying their reactivity and transformation mechanisms in cellular environment is essential for evaluating their long-term efficiency as diagnostic or therapeutic agents. Here we demonstrated for the first time that liquid-cell TEM is a relevant method to follow the (bio)degradation of iron oxide NPs in a solution mimicking the intra-cellular environment to which they are exposed during their life-cycle in the organism (fig.2).[2]
[1] Liao et al. ChemComm 49, 11720 (2013). [2] L. Lartigue et al. ACS nano, 7, 3939 (2013)

We are grateful to Region Ile-de-France for convention SESAME E1845, The CNRS (defi nano project) and the lebex SEAM for financial support.

Fig. 1: In situ growth of facetted gold NPs observed by low dose STEM-HAADF. We observe a shape transition between two nano-polyhedra.

Fig. 2: In situ follow-up of the degradation of iron-oxide NPs. The corrosion and dissolution of a single NP (white arrow) is directly observed in a solution mimicking the intracellular environment. Observation time: (a) 0 s, (b) 220 s, (c) 540 s (an additional NP appeared in the field of view by diffusion), (d) 1080 s.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-2655 Structure and deformation processes of nanocrystallinemetals and alloys characterized by ACOM-STEM in combination with in-situ straining

Kübel C.1,2, Kobler A.1,3, Hahn H.1,3
1Institute of Nanotechnology, KIT, Karlsruhe, Germany, 2Karlsruhe NanoMicro Facility, KIT, Karlsruhe, Germany, 3Joint Research Laboratory, TUD, Darmstadt, Germany
christian.kuebel@kit.edu

Nanocrystalline metals have attracted a lot of interest due to their modified (mechanical) properties compared to the corresponding bulk materials. In particular, their increased strength is promising for technical applications. However, currently their practical use is limited especially due to their low ductility. Understanding the dominant deformation mechanisms active in nanocrystalline metals is crucial for improving their performance and stability as needed for technical applications. A lot of insight has already been gained, e.g. from in-situ deformation experiments by XRD [1]. However, it is difficult to understand the local processes based on bulk measurements. Local processes are traditionally investigated using BF/DF-TEM. However, varying contrast due to local orientation changes, bending and defects during in-situ BF-TEM straining experiments make an accurate interpretation for nanometer sized grains a difficult task. Therefore, we have been developing a quantitative approach to characterize nanoscale structures ex-situ and in-situ during mechanical deformation in the TEM using Automated Crystal Orientation Mapping (ACOM). ACOM-STEM allows for the identification of the crystallographic orientation of all crystallites with crystal sizes of a few nm, well below the limit of the more established EBSD techniques.

We established ACOM (ASTAR by NanoMegas) on a FEI Tecnai F20 in micro-probe (up) STEM mode, that allows us to acquire (fast) STEM reference images, and combined it with in-situ straining using Hysitron’s TEM Picoindenter [2]. This combination was the key to a new data evaluation approach based on orientation maps. By tracking individual crystallites throughout an entire straining series, not only changes in size could be identified, but also the crystal orientation could be followed to directly image grain rotation and distinguish this from overall sample bending during straining.

These measurements and our evaluation routines were applied to magnetron sputtered Au and Pd pure metal and alloy films. Deformation of these films inside the TEM revealed grain growth (Fig. 1) and grain rotation (Fig. 2b) with increasing strain as well as twinning and Σ9 type lattice flips (Fig 2a) during deformation. Despite this strong plastic response to the deformation, we could show that the structural changes induced during plastic deformation are partially reversible during relaxation of the film indicating a noticeable Bauschinger effect in the nanocrystalline materials.

References:

[1] J. Lohmiller et al., Acta Materialia, 2014, 65, 295-307.

[2] A. Kobler, A. Kashiwar, H. Hahn, C. Kübel, Ultramicroscopy, 2013, 128, 68-81.

This work was supported by DFG under grant FOR714

Fig. 1: ACOM-STEM crystalorientation maps and stress-strain curves of a straining series of annealed nc-Pd.

Fig. 2: Crystal rotationduring straining of annealed nc-Pd (left high-angle rotations due to Σ3 and Σ9 flips, right: low-angle rotations).

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-2691 A Novel Method of Studying Small Particles in Wet Environmental-Cell TEM (WETEM)

Kuwamura Y.1, Chiou W.2, Minoda H.1
1Department of Applied Physics, Tokyo University of Agriculture and Technology, Naka cho, Koganei, Tokyo 184-8588, Japan, 2NISP Lab, NanoCenter, University of Maryland, College Park, MD 20742-2831, USA
hminoda@cc.tuat.ac.jp

Wet environmental-cell TEM (WETEM) is at the forefront of providing new observational capabilities and technology in a myriad of research science and engineering capabilities. WETEM is capable of imaging wet materials and resolving real-time dynamic environmental processes at micro- and nano-scales. Much attention has been devoted to the development of the TEM sample holder over the last decade. However, the relatively high cost of the commercial environmental TEM holder has prevented many researchers from studying wet materials in TEM. This paper presents a simple, cost-effective method of constructing a wet environmental cell (EC) for WETEM research.

To construct a wet environmental-cell (EC), amorphous carbon films with very fine ridges were fabricated on a special Cu grid that has only a few small holes. To prevent possible breakage of carbon film, the hole that allows the electron beam to penetrate is rather small (~ 0.1 mm). An enclosed EC was built by sealing two pieces of carbon film/grids with carbon side facing each other in a regular TEM holder (Fig. 1). A tiny drop of smectite clay suspension was pipetted onto the bottom side of grid, and then covered with another carbon grid on the top. A very small amount of vacuum grease was applied to seal both grids. Special care was exercised to prevent adhesives from entering into the grids and contaminating the sample. The EC with wet sample was evacuated in a pre-pumping chamber to ensure the integrity of the wet cell before inserting into the TEM (modified JEOL JEM 2010 operated at 200 kV) equipped with a CCD camera for examining wet samples.

Conventional TEM image (Fig. 2a) shows typical smectite aggregates and well-defined particle outlines. Electron diffraction of dry particles reveals a stacking of smectite particles (i.e., aggregate) in a very clear dot pattern. The WETEM micrograph, as shown in Fig. 2b, reveals a blurred image indicating electron scattering by water molecules in the sample and EC. SAD pattern obtained from WETEM also depicts strong diffused electron scattering in the sample/EC. Analysis of SAD patterns obtained from dry (Fig. 2a*) and wet state (Fig. 2b*) illustrates lattice expansion of (h k 0) (Fig. 3). The small lattice expansion in (h k 0) probably resulted from the expansion of (0 0 l) plane due to addition of water molecules in the crystal along c-axis. This unique method provides a simple and affordable method for preparing and investigating wet samples in TEM. These experiments signify the opening of new opportunities in fundamental “in-situ” dynamic processes research.

Conventional TEM work performed at NISP Lab was partially supported by NSF-MRSEC (DMR 05-20471, Shared Experimental Facility) and UMD. WETEM was carried out at TUAT. Smectite samples were provided by Drs. S. Kaufhold and R. Dohrmann.

Fig. 1: Schematic diagram shows the basic construction of an EC in a TEM holder (left), and the concept of the nano-wet environmental-cell (right).

Fig. 2: Conventional TEM micrograph reveals typical morphology of dried particles and aggregates (a), whereas blurred image of smectite particles was observed in WETEM (b). The corresponding SAD patterns (a* and b*) also depict electron scattering due to water molecular in the sample/EC.

Fig. 3:  A comparison of calculated d-spacings of the dried (top) and fully hydrated samples (bottom). Lattice expansion in different (h k l) probably resulted from the intercalation of water molecular in the smectitie crystal.

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-2798 In situ Observation of the Growth of Two-Dimensional Palladium Dendritic Nanostructures in the Liquid Cell Transmission Electron Microscopy

Zhu G.1, Jiang Y.1, Jin C.1, Zhang H.1, Yang D.1, Zhang Z.1, Lin F.2
1State Key Laboratory of Silicon Materials, Key Laboratory of Advanced Materials and Applications for Batteries of Zhejiang Province, Department of Materials Science and Engineering, Zhejiang University, Zhejiang, China, 2College of Science, South China Agricultural University, Guangzhou, China
gmzhu@zju.edu.cn

    Recent advances in the construction of environmental cells for transmission electron microscope have enabled directly observing the dynamic process in liquid, including electrochemical reactions[1], nanocrystal growth[2], biomaterials imaging[3], corrosion[4] etc. We have adopted a technique[5,6] to get a good resolution and contrast to directly observe the growth of two dimensional palladium dendritic structure growth in the liquid cell TEM.
    We present a study on in situ observation of the growth of palladium dendritic structure based on the bubble induced ultrathin liquid layer in the liquid cell TEM (Tecnai G2 F20). Figure 1 presents a time sequence of HAADF images of a growing palladium dendritic nanostructures (DNS) , the nucleation of which is controlled by the focused electron beam. We directly observe the growth and give a dynamic picture of the dendritic fractal dimension and radius which suggest that diffusion limited aggregation could be applied to account this. Figure 2 presents a randomly nucleation induced palladium DNS growth. There is limited nucleation in the field of view. Compared with the traditional DLA controlled DNS which has an open structure, in our case, all DNSs have a compact dendritic structure with most of the gaps filled which can be explained by direct atomic deposition, as palladium atom can be easily captured by inner branches. All in all, we have described the whole growth mechanism in Figure 3 based on the DLA and direct atomic deposition. This study suggests that we can use liquid cell TEM to directly observe the growth of complex dendritic nanostructure and further our understanding about these nanostructure.

References
(1) Radisic A, Vereecken PM, Hannon JB, Searson PC, Ross FM. Nano Lett. 2006; 6(2): 238-42.
(2) Liao HG, Cui L, Whitelam S, Zheng H. Science. 2012; 336(6084): 1011-4.
(3) de Jonge N, Peckys DB, Kremers GJ, Piston DW. PNAS. 2009; 106(7): 2159-64.
(4) Chee S, Hull R, Ross FM. Microsc. Microanal. 2012; 18(S2): 1110-1.
(5) Zhu G, Jiang Y, Huang W, Zhang, H, lin F, Jin C. Chem. Commun. 2013, 49, 10944;
(6) Zhu G, Jiang Y, lin F, Zhang H, Jin C, Yang D, Zhang Z. 2014 submitted

This work is financially supported by the National Science Foundation of China (51222202,), the National Basic Research Program of China (2014CB932500), the Program for Innovative Research Team in University of Ministry of Education of China (IRT13037) and the Fundamental Research Funds for the Central Universities (2014XZZX003-07).

Fig. 1: (A) Schematic of the liquid cell device (B-F) A time lapse series of HAADF-STEM images following the growth of palladium DNSs. Scale bar = 50 nm. (G) Calculated fractal dimension vs. growth time. The blue dots and black line represent the experimental and fitting values, respectively. (H) The average radius of the DNSs versus growth time.

Fig. 2: Series of HAADF-STEM images recorded during the formation of a few randomly nuclei (A to C). Adjacent DNSs meet and penetrate each other, as indicated by the arrows in green. Scale bar = 50 nm. (D) Image series highlighting individual DNS marked with a red circle in A, scale bar = 10 nm.

Fig. 3: Schematic of the growth of palladium DNSs in a liquid cell under the illumination of an electron beam with high energy. There are two ways contributing to the dendritic formation, direct atomic deposition from reduction of palladium ion and aggregation between cluster and the edge.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-12-O-3099 In-situ environmental TEM studies using MEMS based devices

Malladi S. K.1, Basak S.1, Wu M. Y.1, Xu Q.1, Erdamar A. K.1, Tichelaar F. D.1, Zandbergen H. W.1
1National Centre for HREM, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, Zuid Holland, Netherlands
S.R.K.Malladi@tudelft.nl

Investigating dynamic changes in specimen while applying a stimulus inside a transmission electron microscope (TEM) has always been an exciting field of study. With the advancements in TEMs and microelectromechanical systems (MEMS), in-situ TEM has progressed extensively over the last decade. Here, we show the application of MEMS based devices developed in-house to carry out in situ heat-treatment and environmental TEM studies1. For environmental TEM studies, a controlled environment is achieved inside the TEM by one of the following approaches: the open type, using a differentially pumped vacuum system where the reactive gases are spread around the specimen area of the TEM; and the closed type, using a windowed environmental cell. Our studies are based on the closed environmental cell called the nanoreactor, Fig.1. The nanoreactor consists of two silicon chips facing each other with thin electron-transparent silicon nitride membranes. One half of the nanoreactor (bottom half) is embedded with a Pt coil for resistive heating2, Fig. 2. These MEMS based devices in combination with specially developed TEM holders, Fig.3, make it possible to carry out a wide range of experiments, a few of which are mentioned here.
Using one half of the nanoreactor, we have demonstrated the three-dimensional compositional and structural evolution during heat treatment at 100–240°C in a FIB specimen of a commercial aluminium alloy AA2024, revealing in unparalleled detail where and how precipitates nucleate, grow or dissolve3. On closing the nanoreactor with the other half, we have shown that it is also possible to carry out gas-liquid-material interactions. One such example is the room temperature corrosion studies of AA2024-T3 exposed to oxygen bubbled through aqueous hydrochloric acid of pH = 3 at a pressure of ~ 1.5 bar4. In the present study, we investigate the corrosion behaviour of AA2024 after growing the needle-like S-phase type precipitates by in situ heat-treatment. These sort of experiments are critical to understand the performance of engineering materials in service conditions. Furthermore, will present some of our preliminary results on metal-liquid electrolyte interfaces to extend the environmental-cell based approach for introducing liquids inside the TEM.
References:
1 Malladi, S. R. K. In-situ TEM Studies: Heat-treatment and Corrosion. (2014).
2 Creemer, J. F. et al. Atomic-scale electron microscopy at ambient pressure. Ultramicroscopy 108, 993-998 (2008).
3 Malladi, S. K. et al. Real time atomic scale imaging of nanostructural evolution in aluminium alloys. Nano letters (2013).
4 Malladi, S. et al. Localised corrosion in aluminium alloy 2024-T3 using in situ TEM. Chemical Communications 49, 10859-10861 (2013).

Part of this research was carried out under project number MC6.05222 in the framework of the Research Program of Materials innovation institute M2i in the Netherlands (www.m2i.nl). The authors also acknowledge the ERC project 267922 'In situ NanoElectrical Measurements in TEM' for support.

Fig. 1: (a) Conceptual sketch of a nanoreactor. (b) Photographs of the top and bottom chip, with ~ 400 nm SiXNY window in 800 X 800 µm2 area; inlet and outlet for reactive gases in the bottom chip.

Fig. 2: Optical micrographs of the 400 nm SiXNY window showing (a) top and (b) bottom chips respectively with 10 µm circular holes over spanned with ~ 20 nm SiXNY membranes for electron transparency. The bottom chip is embedded with a Pt spiral for heating. The red encircled region in (c) showing aligned assembly.

Fig. 3: Holders for in-situ TEM studies: (a) Gas holder with Pt tubing; (b) A single-high-tilt heating holder in which the bottom chip of the nanoreactor can be used for in situ heating experiments. This holder has a capability to tilt through ±70°, which makes it possible to carry out tomography experiments too.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-1501 Gas environment study of Fe nanoparticles using in-situ aberration corrected E-(S)TEM

Lari L.1,4, Carpenter R.1, Lazarov V.1, Boyes E. D.1,2,4, Gai P. L.1,3,4
1Department of Physics, University of York, YO10 5DD, United Kingdom , 2Department of Electronics, University of York, YO10 5DD, United Kingdom , 3Department of Chemistry, University of York, YO10 5DD, United Kingdom , 4The York JEOL Nanocentre, University of York, YO10 5BR, United Kingdom
leonardo.lari@york.ac.uk

Fe and Fe-oxide nanoparticles have a series of promising potential applications in physical and medical sciences. These include magnetic storage devices, catalysis, sensing, contrast enhancement in magnetic resonance imaging and magnetic hyperthermia [1-3]. Understanding of the Fe-Oxide NPs reduction to metal and the oxidation processes down to atomic scale is paramount for the control of the quality and the optimization of their applications.

A recently modified double aberration corrected JEOL 2200FS (S)TEM [4] has demonstrated the possibility of the analysis of metallic nanoparticles in gas environment at temperature allowing single atom visualisation by HAADF STEM in controlled gas reaction environment [5].

In this study, thin films of iron were deposited by sputtering on C films supported by standard TEM Cu grids as in Figure 1a). Nanoparticles were produced by annealing in-vacuum the films within the microscope column (pressure ~1.0 * 10-5 Pa) at temperatures from room temperature to up to 600 °C using an in-house designed Gatan heating holder (Fig 1b). Nanoparticle formation and size distribution was monitored in-situ as a function of time and temperature by HAADF STEM imaging. After annealing nanoparticles were shown to consist of single crystal metallic Fe, composition confirmed by EDX analysis. The Fe nanoparticle samples interaction with Hydrogen (Fig 2a) and Oxygen (Fig 2b) gases were studied in-situ at 300 °C with a differential pressure at the specimen in the range of 2.5-3.0 Pa. The interaction of the nanoparticles with the gases, as well as the substrate, will be discussed in terms of the changes in nanoparticle geometry, composition, size distribution, crystallinity and microstructural defects.

Fe to FexOy phase change was identified by HAADF/BF STEM imaging, and EDX line scans (Fig 3). These show uniform Fe/O composition within each particle with comparable particle sizes, but complex morphologies for the metallic and the oxide phases. The mechanism is being developed with single atom sensitivity.

References:
[1] B D Terris and T Thomson, J. Phys. D 38, R199–R222 (2005)
[2] H. Galvis et al. Science 335, 835–838 (2012)
[3] Q A Pankhurst, N T K Thanh, S K Jones, and J Dobson, J. Phys. D 42, 224001 (2009)
[4] P L Gai and E D Boyes, Microscopy Research and Technique 72, 153 (2009)
[5] E D Boyes, M R Ward, L Lari, and P L Gai, Annalen der Physik 525, 423 (2013)

We thank the EPSRC (UK) for research grant EP/J018058/1

Fig. 1: (a) Initial sputtered Fe film, left in air for two days before annealing in the microscope up to 600 °C to form nanoparticles (b).

Fig. 2: (a) Overview of Sample A after exposure to H2 gas environment with a pressure at the specimen of 3 Pa and with a sample temperature of 300 °C. (b) Overview of Sample B after exposure to O2 gas environment with a pressure at the specimen of 2.5Pa and with a sample temperature of 280 °C.

Fig. 3: (a) EDX line scan of Fe nanoparticles at of 300 °C in vacuum showing O signal at background in the particle as well as on the support. (b) EDX line scan of a FexOy nanoparticle done in O2 environment at 2.5 Pa and 300 °C (scale bar 10nm).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-1529 In-situ observation of facet-dependent heterogeneous catalysis in an environmental TEM

Wang Y.1
1Center of Electron Microscopy and State Key Laboratory of Silicon Materials, Department of Materials Science and Engineering, Zhejiang University, Hangzhou 310027, China
yongwang@zju.edu.cn

Heterogeneous catalysis is of paramount significance in chemical processes, where efficiency relies on the characteristics of the nanocrystal-catalyst surfaces [1-3]. Quantitative in situ study of the catalytic reactions on different crystallographic planes could provide a great insight to improve the performances of heterogeneous catalysts. Great efforts have been made through spectroscopy and microscopy studies; however, a direct observation at the atomic level of the facet-dependent activity of nanocrystal-catalysts remains challenging. In this report, we present a direct experimental observation of a crystal facet-dependent catalysis on a single nanocrystal-catalyst through an in situ study of a model catalysis platform, platinum-catalyzed oxidation of graphene layers, in an environmental transmission electron microscope [4]. Our experimental setup for the first time implements a simultaneous recording of diverse catalytic activities on different facets on a single particle at atomic resolution, revealing that Pt{100} is more active for the graphene oxidation than Pt{111}. Density functional theory calculation shows that the Pt{100} facets favor both the adsorption and dissociation of oxygen species (O2 and O) compared to the Pt{111} facets, and this facilitates C-C bond breaking, promoting a faster oxidation of graphene to CO2 on Pt{100}. Our study not only directly demonstrates crystal facet-dependent catalysis in situ but also offers a novel approach for the dynamical study of the catalytic mechanism of heterogeneous catalysts at the atomic level.

References
1.Somorjai, G. A. Science 227, 902-908 (1985).
2.Tao, F. & Salmeron, Science 331, 171-174(2011).
3.Ertl, G. Angew. Chem.-Int. Edit. 47, 3524-3535(2008).
4.W. Yuan, Y. Wang, and Z. Zhang et al., submitted

 

We acknowledge the support of NSFC (51390474，11234011) and Key Science and Technology Innovation Team of Zhejiang Province (2010R50013).

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-1550 In situ TEM/EELS study of structural changes of Pt/GC electrocatalyst in CO gas

Yaguchi T.1, Shimizu T.2, Kamino T.2, 3
1Hitachi High-Technologies Corp., Ibaraki, Japan, 2Japan Automobile Research Institute, Ibaraki, Japan, 3University of Yamanashi, Yamanashi, Japan
yaguchi-toshie@naka.hitachi-hitec.com

 Improving the long-term performance of catalyst is one of the most important issues in polymer electrolyte fuel cell (PEFC) technology. Environmental transmission electron microscopy (ETEM) has proven to be an effective characterization tool for understanding the degradation mechanism of the catalytic materials. Our goal is to optimize the instrumentation which can simulate the working environment of a catalyst inside the microscope. For this purpose, we have developed an ETEM based on a conventional analytical TEM and a specimen heating holder equipped with a gas injection nozzle 1). Different from conventional ETEM techniques, the specimen heating holder is designed to introduce a gas locally to the specimen. The inner diameter of the gas nozzle is 0.5 mm, and the distance between the nozzle and the specimen mounted on a heating element is 1.0 mm. This unique design of the holder allows a gas pressure up to 100 Pa near the specimen, while maintains a vacuum of 10-5 Pa in the electron gun chamber. In order to study the effect of gas on the structural changes during heating, it is important to be able to switch gas quickly. In our design, gas bottles are directly connected to the specimen heating holder with a path length of 20 cm, which is much shorter than that of conventional ETEM. Thus, we can switch a gas in several seconds as it has been verified by electron energy-loss spectroscopy (EELS) gas analysis. The advantage of the quick gas switch benefits our in situ TEM study of energy-related nanomaterials.
 In this paper, we report an in situ TEM/EELS study of the degradation mechanism of a Pt/graphitized carbon (GC) electrocatalyst, using a Hitachi H-9500 ETEM equipped with a Gatan GIF EELS which has an energy resolution of 1.0 eV at 300 kV. The gas pressure was maintained at 60 Pa near the specimen which has been heated up to 200 oC. Fig. 1 presents the EELS analysis of the CO gas, showing the characteristic sharp π* peak of C-K and high signal-noise ratio. Fig. 2 shows the EELS spectrum of a carbon support of Pt/GC catalyst heated to 100 oC in 60 Pa of CO gas atmosphere. A series of TEM images obtained during the in situ dynamic TEM observation are shown in Fig. 3. Nucleation and growth of Pt particles occurred when the CO gas pressure was increased to 60 Pa. Other structural changes such as formation of PtO2 were characterized by high-resolution TEM and selected nano area electron diffraction analysis. The results from the improved ETEM demonstrated the applicability of this technique for the evaluation of the state of the graphitized carbon black-supported Pt catalysts.
1) Kamino T, et al, J. Electron Microsc.54, (2005) pp.497–503.

Fig. 1: EELS spectrum of the CO gas at 60 Pa: the characteristic sharp π* peak of C-K and high signal-noise ratio.

Fig. 2: EELS spectrum of the carbon support of the Pt/GC catalyst heated to 100 oC in 60 Pa of CO. It shows the convolution of π* peak from CO gas and ρ* peak from solid carbon support.

Fig. 3: Sequential TEM images recorded during in situ dynamic ETEM study. Nucleation and growth of Pa particles occurred when the CO gas pressure increased to 60 Pa.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-1569 Combining ESTEM and Kinetic Monte Carlo simulations to investigate sintering of Cu

Martin T. E.1, Lari L.1, Gai P. L.2, Boyes E. D.3
1The York Nanocentre and Department of Physics, University of York, UK, 2The York Nanocentre and Departments of Chemistry and Physics, University of York, UK, 3The York Nanocentre and Departments of Physics and Electronics, University of York, UK
tm526@york.ac.uk

Combining an improvement in the environmental sustainability of energy solutions, whilst also remaining economically feasible, is obviously of great importance. Catalysis could provide part of the solution for realising environmentally friendly, economical processes for the conversion of fossil fuels to energy. Methanol synthesis has attracted interest due to its potential for use in fuel cells and because it is one of the most important basic components in the chemical industry (worldwide production ~45 million tons in 2010). Methanol synthesis is catalysed by Cu based systems and an improved understanding of thermal deactivation mechanisms (Ostwald Ripening (OR) and Particle Migration and Coalescence (PMC), Figure 1) is of importance to improve both catalyst efficiency and lifetime.

Development of new catalysts is inhibited by a limited understanding of these processes. Through knowledge of the atomic scale mechanisms that govern catalytic properties it is possible to improve control of catalyst sintering. Computer simulation and theoretical models have increasingly been used in combination with experiments in order to further understand known catalytic processes and in the design of new ones[1].

The development of a double aberration corrected environmental scanning transmission electron microscope (ESTEM) which has the ability to image single atoms in situ allows for the exploration of deactivation mechanisms (such as OR) of heterogeneous catalysts in a gas environment [2]. Imaging of the OR mechanism at an atomic scale requires understanding of the interplay of temperature, gas pressure and the metal-support interaction with the imaging capability of the electron microscope. Prediction of the number and visibility of single atoms undergoing OR is achieved using Kinetic Monte Carlo simulations.

ESTEM experiments in the 200-350˚C range with 3Pa of H2 have shown that deactivation of the Cu catalyst on a C support occurs primarily via OR (Figure 2). There is a general lack of atomic scale understanding of the OR mechanism, but important observations have recently shown [1]that sintering particles undergo periods of size stability followed by rapid decay. This is hypothesised to be due to particle morphology. The effect of particle shape and degree of perturbation from the minimum surface energy state can be modelled using KMC to further understand the OR mechanism.

The ability of KMC to provide atomic detail (and macroscopic trends) combined with Angstrom level resolution of ESTEM at York [2] allows a different approach to investigating catalyst sintering. Experiments have allowed some of the first images of catalyst single atoms in situ and this provides a different perspective in terms of verifying theoretical models at the atomic scale.

The authors thank the EPSRC for support from grant EP/J018058/1
References:

1. Hansen, T.W., et al., Accounts of chemical research, 2013.

2. Boyes, E.D., et al., Annalen der Physik, 2013. 525(6): p. 423-429.

Fig. 1: : Diagram of 2 sintering mechanisms: (a) Particle Migration and coalescence and (b) Ostwald Ripening where single atoms/small clusters migrate from smaller to larger particles. Sintering is driven by reduction of free surface energy.

Fig. 2: Overlay showing that facetted particles have increased in size, but not changed position with temperature and pressure increase, suggesting OR mechanism. Overlay (red): after heating at 312˚C at 2Pa H2, Underlay (green) after heating at 361˚C at 3Pa H2.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-1649 In-situ Studies of Ceria Supported Copper Nanocatalysts at the Atomic Level Using Wet- Environmental TEM

Yoshida K.1, Boyes E. D.2 3, van de Water L.4, Watson M.4, Gai P. L.2 5
1Institute of Advanced Research, Nagoya University, Nagoya, Japan, 2The York Nanocentre, Department of Physics, University of York, UK, 3The York Nanocentre, Department of Electronics, University of York, UK, 4Johnson Matthey Technology Centre, UK, 5The York Nanocentre, Department of Chemistry, University of York, UK
ky512@esi.nagoya-u.ac.jp

Ceria supported catalysts are expected to exhibit better stability than other supports in a variety of processes including automotive emission control and water gas shift reactions. Ceria is an important support because of its key role in oxygen storage with oxygen uptake in oxidising conditions, releasing oxygen in reducing conditions. Ceria supported copper catalysts are of interest in water gas shift reactions involving the oxidation of CO and the production of hydrogen [1].
The in-situ direct observation of the surface structural and chemical evolution and the dynamic reaction mode of operation of catalysts under reaction conditions at the atomic scale is crucial in understanding and controlling catalytic reactions and the catalyst performance [2-6]. However there is a general lack of in-situ studies of Cu/ceria catalysts at the atomic level and therefore the dynamic behaviour of the nanocatalysts in the reactions are not well understood. We have therefore examined the copper/ceria catalyst in various reaction environments to obtain the direct evidence of dynamic processes at the catalyst surface.

We prepared electron microscopy samples on both copper and titanium grids and carried out in-situ studies in vacuum and in environments of carbon monoxide (CO) and CO and H2O, using spherical-aberration corrected environmental transmission electron microscope with a humidifier system (Wet-ETEM) [6]. The studies in CO and water have demonstrated changes in the Cu particle morphology as well as in the ceria support as a function of the reaction environment. The experiments have further revealed that under the reaction environments copper oxide in the samples is reduced to copper metal. The existence of highly dispersed copper clusters was observed in CO and water. Examples in Figure 1 and 2 show time resolved sequences of copper/ceria nanocatalysts in vacuum. In the presentation, we will discuss the effect of the different reaction environments as well as the presence of copper metal on the extent of reducibility of ceria substrate and the role of anion vacancies in ceria on the mechanism.

References
[1] Li Y et al; Appl catal. B 27 (2000) 179.
[2] Boyes E.D. and Gai, P.L. , Ultramicroscopy 67 (1997) 219.
[3] Gai P.L. and Boyes E.D., Microscopy Res and Tech. 72 (2009) 153.
[4] Boyes E.D., Ward M, Lari L and Gai, P.L., Ann.Phys. (Berlin) 525 (2013) 423.
[5] Yoshida K. et al, Nanotechnology 24 (2013) 065705.
[6] Yoshida K. et al, Invited Paper, MMC 2014 Conf. Proceedings, and Nanotechnology (sub). 

The authors thank the EPSRC (UK) for Critical mass grant EP/J018058/1.

Fig. 1: Time resolved studies of Cu supported on ceria showing no changes in the interface of ceria particles.

Fig. 2: Surface atomic movement in ceria in the catalyst system.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-1733 ELECTRON RADIATION DAMAGE IS A VERY LONG DYNAMIC PROCESS

Massover W. H.1
1Department of Biological Sciences (retired), Rutgers University –Newark, 100 University Ave., Newark, N.J. 07102, USA.
bill.massover@comcast.net

    All biomedical, chemical, and physical specimens suffer radiation damage from the electron beam during imaging, diffraction, or analysis [1].  Electron microscopists often view radiation damage as a short process involving only a few very rapid steps.  Dried sodium phosphate buffer shows structural changes during high-dose electron microscopy, with bubbling, bubble fusions and fissions, shrinkage, and thinning [2].  X-ray energy-dispersive spectroscopy reveals that the several atomic species in these inorganic materials have quite different rates of mobility/stability [3].  This study investigates new radiation-induced changes occurring very long after the early damage. 

    Thin specimens on supports of carbon were prepared with 100mM sodium phosphate buffer (Na2HPO4/NaH2PO4; pH 7.0), as described earlier [2].  Data from a FEI Tecnai-12 electron microscope (100kV) was recorded on a Gatan cooled 4x4kB CCD camera.  Each identical exposure (1 s) produced a dose of around 50–250 e-/Ų. 

    Serial high-dose imaging shows that already damaged areas of dried buffer [2] develop new changes after much additional dosage.  Irregular dark deposits (initial diameter = 7-15nm) form independently at sites always associated with a flattened bubble (Fig. 1).  Deposits must be either inside a bubble lumen, or in the thin matrix between the support film and a bubble’s lower surface.  Many bubbles do not form any deposits, and deposits never arise in the matrix between bubbles (Fig. 1).  Smaller bubbles only have a single deposit, while bubbles fusing together can form several (Fig. 2a).  Dynamics induced by further irradiation include growth, shrinkage, movements, aggregation, and coalescence (Figs. 1, 2b-d).  Huge flattened bubbles created by many compound fusions have numerous deposits (Fig. 2e), suggesting that a component needed for deposition is present at limiting concentration in small bubbles.  Some aggregated deposits persist with further dosage to over 100 exposures (Fig. 2e).  Chemical composition of these deposits is not yet known. 

    In-situ results indicate that high-dose electron irradiation of a dried inorganic buffer causes new very late changes in already damaged specimens.  The early and late changes are not the same.  The late deposits indicate that the electron beam is interacting with one or more products from the earlier dosage.  The concept of electron radiation damage must be expanded to also include different multi-step dynamic physical/chemical processes occurring over very prolonged times. 

[1]  Reimer, L. & Kohl, H. (2008) In: Electron Microscopy, 5th Ed., Chp. 10, pp. 456-487.   
[2]  Massover, W.H.  (2010) Microsc. Microanal. 16:346-357.
[3]  Massover, W.H. & Zaluzec, N.J. (2012) In: Proc. Microsc. Soc. America, CD abst. 

I thank Prof. Edward M. Bonder for access to the electron microscopy facility.  Supported in part by private funds; the author declares there is no conflict of interest.

Fig. 1:   Serial high-dose images of beam-induced late changes.    Panels 1a,e: after previous damage, but before deposits form; 1b,f: same, showing new deposits (arrows); 1c,d,g,h: with more dosage, these deposits can grow or shrink.  Exposure numbers are at upper left.  Bar = 50nm for all panels. 

Fig. 2:   High-dose images of multiple deposits.  Panel 2a: one fused bubble has 2 separate deposits (arrows); 2b: several deposits aggregate; 2c: same after more dosage; 2d: same after 55 more exposures; 2e: numerous aggregates at this giant bubble. The 4 very dark dots are 15nm colloidal gold particles.   Exposure numbers are at upper left.  Bar = 50nm.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-1864 Towards pure and compact FEBID nanostructures via e-beam assisted H2O purification at room temperature

Geier B.1, Gspan C.2, Fowlkes J. D.3, Rattenberger J.2, Fitzek H.2, Rack P. D.4, Plank H.1
1Institute for Electron Microscopy and Nanoanalsis, Graz University of Technology, Graz, Austria, 2Center for Electron Microscopy, Graz, Austria , 3Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN, United States, 4Department of Materials Science and Engineering, University of Tennessee, Knoxville, TN, United States
harald.plank@felmi-zfe.at

During recent years, focused electron beam induced deposition (FEBID) has made significant progress with respect to potential applications ranging from passive devices, such as plasmonic gratings, towards active concepts like mechanical, magnetic or chemical sensing. Beside the undoubted advantages of FEBID as direct write tool with spatial nanometer resolution the limitations due to low material purity still remain. Successful attempts of purifying metal containing deposits have been demonstrated, mostly using a purification gas together with elevated temperatures although another issue remained: an often porous deposit morphology which complicates the fabrication of very small but still compact structures as often needed for nano-applications. Recently, it has been demonstrated that the key for high fidelity morphologies lies in the application of low temperatures during purification. It has been shown that electron beam assisted purification of Pt-C deposits with 50 °C oxygen gas leads to highly compact and nominally pure deposits with purification rates of about 6 min per µm2 deposit footprint. In this study we push this concept even further and demonstrate that water vapor can be used as purification gas at room temperature together with the e-beam. For constant pressure conditions an environmental scanning electron microscope has been used and the results show that very high rates better than 1 min per µm2 deposit footprint can be achieved together with nominally pure Pt structures (EDX based). Furthermore, the deposits are found to shrink by about 60 vol. % but still maintain their original footprint shape (AFM based). The surface roughness of such high fidelity is found to less than 1 nm and no holes or cracks appear on the surface. The study is complemented by a systematic variation of e-beam parameters during purification (beam current, dwell time, and pixel point pitch) revealing that a localized pressure of 10 Pa is sufficient to provide close to reaction rate limited conditions with respect to the water adsorbates. Additionally, transmission electron microscopy and electron energy loss spectroscopy has been used to identify the purification as bottom up process. After demonstrating the purification capabilities on 3D structures, high resolution FEBID single lines with widths down to 30 nm have been subjected to ESEM based purification. It is found that ideal curing conditions can lead to pure and compact Pt single lines with line widths down to 15 nm, overcoming classical resolution limitations of FEBID processes. By that a new and comparable simple purification strategy is introduced which does not require gas and / or substrate heating while water, as purification gas, is almost straight-forward in terms of technical implementation.

Fig. 1: (a) energy dispersive X-ray spectroscopy characterization of as-prepared Pt-C (red) and fully purified Pt deposits (blue) together with substrate (grey) and Pt reference spectra (green); (b) shows the dynamic evolution of the carbon peak during purification.

Fig. 2: atomic force microscopy height images of as-prepared Pt-C (a) and fully purified Pt deposits (b) revealing highly compact morphologies while maintaining the footprint geometry.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-2957 In-situ observation of Fe precipitation from Fe2O3 by SiO2 and electron irradiation

Ishikawa N.1, Kimura T.1, Takeguchi M.1, Kurokawa D.2, Inami T.2
1National Institute for Materials Science, 2Ibaraki University
ISHIKAWA.Nobuhiro@nims.go.jp

Introduction

The steel industry emits much amount of carbon dioxide because coke is used as reducing agent of iron-ore reduction and heat source in blast furnace. Of course the innovation in ironmaking process in steel industry is urgently required and there are many researches for the reduction of energy consumption.

On the other hand authors have been carried out the in-situ analysis of the reaction of carbon and iron-oxides in TEM1). Recently the iron precipitation in iron-silicon composite oxide without any reducing agent was found in TEM2). And it was found that iron precipitated at the contact of hematite(Fe2O3) and Silica (SiO2) successively. This paper describes about these results.

Experimental procedure

The reagent of α-hematite(99.9%) and silica (99.99%) as rod were used in this study. The TEM specimens were prepared by FIB using pick up method. Hematite and silica was put on top of one another on the Aduro E-chip which is made for heating holder made in Protochips. Figure 1 shows the photograph of the specimen and the heating holder taken in optical microscope. Another hematite put on next place as a reference specimen. TEM analysis was carried out using JEM-2100F with EDS and video recording system.

Results

It was difficult to find the location of the nucleation of iron precipitation. But small particles were found during the keeping 873K. Figure 2 is the captured photos in the continuous observation of the growth of the precipitates at the edge of hematite. Growth of such precipitates were seen in only electron irradiated area and no precipitates were found in another hematite without silica even done similar condition. Figure 3 is the final precipitates irradiated after a few hours and the spectrum of the EDS measured the place pointed by an arrow. The component was almost iron.

Conclusion

The iron precipitation fromα-hematite without reducing agent needs the condition as follows,

200kV electron irradiation, degree of vacuum: 10-5Pa, temperature 873K, additive: SiO2.

References

1) N. Ishikawa, T. Ogiwara, M. Takeguchi, Y. Oba and T. Inami;

MICROSCOPY AND MICROANALYSIS, 16(2010), 352-353

2) N. Ishikawa, M. Takeguchi and T. Inami; ICSRI2013 Proceedings, 2013

Ibaraki University

This research was supported by JSPS KAKENHI Grant 25550075.

Fig. 1: TEM specimen put on the E-chip for heating holder.

Fig. 2: Continuous observation of precipitates. Elapsed time is after finding the precipitates showed in a), b) 130s, c) 430s, d) 2050s passed respectively.

Fig. 3: Final stage of precipitates and its EDS spectrum.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-2438 In-situ TEM reduction of core-shell Ag@In2O3 nanoparticles

Langlois C. T.1, Cottancin E.2, Epicier T.1, Piccolo L.3, Pellarin M.2, Ramade J.2, Santos Aires F.3, Aouine M.3, Blanchard N.2
1MATEIS Laboratory, INSA Lyon, France, 2Light Matter Institute, Claude Bernard University of Lyon, France, 3IRCELYON, Lyon, France
cyril.langlois@insa-lyon.fr

Under gaseous operating conditions, the structure of bimetallic catalysts is highly dependent on the pressure, temperature, exposure time and composition of the surrounding atmosphere. Their catalytic properties are therefore prone to large variations during a catalytic experiment, due to the evolution of the morphology, chemical arrangement, and eventually oxidation reduction, of the two metals. For the same reasons their optical properties, particularly the frequency of the surface plasmon resonance (SPR), are very sensible to these environmental parameters. It follows that structural and optical characterization techniques must be implemented in an environment as close to the catalytic conditions as possible.
We present an environmental TEM study on Ag@In2O3 nanoparticles. These particles exhibit a reversible shift of their SPR, after air exposition and after hydrogen reduction at 300°C [1]. The synthesis was made using a physical route at the Low Energy Cluster Beam Deposition source of PLYRA in Lyon (France). With this kind of source, the size and the composition of the particles can be monitored independently and the obtained nanoparticles are very clean, with no chemical ligand around the particles.
The microscope used is a TITAN FEI Environmental Transmission Electron Microscope (ETEM) operating at 300 kV, with a Cs corrector of the objective lens; this instrument is installed at CLYM (www.clym.fr). The sample was mounted on a GATAN TEM heating holder with Inconel oven; HRTEM images and movies of the bimetallic nanoparticles were recorded up to 500°C and 10 mbar [2].
The starting chemical configuration of the particles is a core-shell structure, with silver cores of ~4 nm and In2O3 amorphous shell with a thickness between 1 and 2 nm. These nanoparticles have been exposed in the ETEM to successive (H2 pressure; temperature) couples, from (1 mbar H2 ; 25°C) to (10 mbar H2 ; 500°C). The structural changes observed at the atomic scale range from the formation of Janus Ag-crystalline In2O3 nanoparticles to the complete reduction of indium oxide, and the incorporation of indium into the remaining silver nanoparticles to form an InAg solid solution. Results are followed by a discussion about the precautions to be taken in the interpretation of such results and the complementary experiments needed to conclude about the effective role of each experimental parameter.
[1] Plasmon spectroscopy of small indium–silver clusters monitoring the indium shell oxidation, E. Cottancin, C. Langlois, J. Lerme, M. Broyer, M-A. Lebeault and M. Pellarin, Phys.Chem.Chem.Phys. (2014) 16 5763-5773
[2] Advances in the environmental transmission electron microscope (ETEM) for nanoscale in situ studies of gas–solid interactions , J.R. Jinschek, Chem. Commun. (2014) 50 2696-2706

Fig. 1: Core-shell nanoparticles after air exposition

Fig. 2: Conditions 450°C – 1,5 mbar H2

Fig. 3: Nanoparticles after total reduction showing an InAg solid solution, with indexation and EDX measurements
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-2518 Faceted nanopores in magnesium: fabrication and electron-beam assisted healing

Wu S.1, Sheng H.1, Liu C.1, Cao F.1, Liu Y.1, Zheng H.1, Zhao D.1, Wang J.1
1School of Physics and Technology, Center for Electron Microscopy and MOE Key Laboratory of Artificial Micro- and Nano-structures, Wuhan University, Wuhan 430072, China
wang@whu.edu.cn

Solid state nanopore-based sensing has emerged as a promising candidate for the detection and characterization of biomolecules. To date, most of the fabricated nanopores in various materials including SiO2,1 Si3N4,2 Al2O3,3 etc., are circular with no obvious facets, as a result of the isotropic property in amorphous materials. While faceted nanopores can be potentially employed in rapid electrical detection and analysis of biomacromolecule with various shapes, the relevant investigation has been rarely reported. Herein, we show the successful fabrication of faceted nanopores in magnesium via focused e-beam inside transmission electron microscope (TEM). By manipulating the e-beam irradiation direction, the as-fabricated nanopores exhibit different shapes as observed along different typical orientations, e.g., [0001], [11-20], etc (Fig. 1).4

Surprisingly, when the e-beam is spread out, the nanopores would continuously shrink and finally disappear. Such atomic-scale healing dynamics are directly recorded by the in situ high-resolution transmission electron microscopy (HRTEM) techniques, as evidenced by the layer-by-layer growth of atomic planes at the nanopore periphery (Fig. 2). Meanwhile, it is noted that the proposed healing process was attributed to the e-beam-induced anisotropic diffusion of Mg atoms at the nanopore edges.5 When the e-beam was turned off, the nanopore would retain its shape. Hence, using TEM images, which provide real-time feedback during the healing process, allows for the precise control of pores with sub-nanometer sizes along different directions. The size-controllable synthesis of faceted nanopore does not only broaden its potential applications but provide an important insight into the nanopore patterning in metallic materials. The direct observation of atomic diffusion process indicates that TEM may serve as an alternative to other techniques, such as scanning tunneling microscopy, in the race towards comprehensive investigations of surface science.

1. A. J. Storm, et al. Nat. Mater. 2, 537 (2003).

2. U. F. Keyser, et al. Nano Lett. 5, 2253 (2005).

3. B. M. Venkatesan, et al. Adv. Mater. 21, 2771 (2009).

4. S. Wu, et al. Appl. Phys. Lett. 103, 243101 (2013).

5. H. Zheng, et al. Sci. Rep. 3, 1920 (2013).

This work was supported by the 973 Program (2011CB933300), the National Natural Science Foundation of China (51071110, 51271134, 40972044, J1210061), the China MOE NCET Program (NCET-07-0640), MOE Doctoral Fund (20090141110059), and the Fundamental Research Funds for the Central Universities.

Fig. 1: Fabrictiaon of facted nanopores in Mg. (a-c) A chematic illustration and (d-f) HRTEM images of nanopores along the (a) [0001], (b) [11-20], and (c) [11-23] zone axes, correspondingly. The insets in (d-f) show the SAED patterns of the corresponding regions, respectively.

Fig. 2: E-beam assisted healing of facted nanopores in Mg along the [0001] zone axis. (a-c) A chematic illustration and (d-f) time-lapsed experimental images showing the healing of an individual nanopore under wide-field e-beam irradiation.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-2713 In situ characterization of Pd2Ga catalysts during particle formation and methanol synthesis

Fiordaliso E. M.1, Sharafutdinov I.2, Hansen T. W.1, Chorkendorff I.2, Wagner J. B.1, Damsgaard C. D.1,2
1Center for Electron Nanoscopy, Technical University of Denmark, 2Center for Individual Nanoparticle Functionality, Technical University of Denmark
e.fiordaliso@cen.dtu.dk

Environmental transmission electron microscopy (ETEM) provides a window to catalyst formation as well as catalytic reactions. In conjunction with complementary techniques such as in situ XRD, new insight into the various steps of reactions can be obtained. In this study, Pd2Ga/SiO2 is characterized during nanoparticle formation and methanol synthesis. In situ XRD is used to investigate the phase at four stages along the catalyst life cycle, i.e. drying, calcination, reduction, and CO2 hydrogenation to methanol. TEM images of identical locations (IL TEM) are acquired after each stage of the life cycle to monitor Pd2Ga nanoparticle formation and evolution ex situ, whereas ETEM is used to monitor the development of the catalyst in situ. Pd2Ga/SiO2 (23 wt.%) catalysts are prepared by impregnation of Pd and Ga nitrates in nitric acid into high surface area SiO2. The catalyst life cycle is carried out at the XRD setup at 105 Pa, using an Anton Paar XRK-900 furnace cell connected to a gas handling system. Drying and calcination are carried out in air at 120°C and 260°C, respectively. Reduction is performed in a flow of 25 % H2 in Ar at 550°C. Methanol synthesis is carried out between 175°C and 250°C in a mixture of CO2 (25%) and H2 (75%). In situ XRD measurements are acquired along the life cycle and TEM images of identical locations are recorded at the end of each step of the cycle. In order to follow the evolution of the catalyst in situ and in real time, the catalyst life cycle is reproduced at the ETEM (Titan, FEI) at 400 Pa, where the silica supported Pd and Ga nitrides precursors are deposited on a SiN membrane of MEMS heaters. XRD patterns reveal the PdO crystallographic phase of catalyst during drying and calcination. No distinct peaks are observed in the XRD patterns for Ga and Ga2O3, indicating amorphous gallium compounds. The Pd2Ga phase is formed upon reduction and the in situ XRD pattern of the reduced catalyst is shown in Fig. 1 (a). IL TEM images show particle formation upon calcination, which determine the distribution of the Pd2Ga nanoparticles formed during reduction. A TEM image of the reduced catalyst is shown in Fig. 1 (b). ETEM images acquired along the catalyst life cycle are consistent with the ex situ images. In Fig. 2 (a) an image of the catalyst acquired at the ETEM under reduction conditions is shown together with a high resolution image of a single Pd2Ga nanoparticle (b) and the corresponding FFT (c). The combination of in situ techniques such as XRD and ETEM together with IL TEM imaging proves a valuable route for studying the life cycle of heterogeneous catalysts.

This work was supported by the Catalysis for Sustainable Energy (CASE) research initiative, funded by the Danish Ministry of Science, Technology and Innovation.

Fig. 1: (a) In situ XRD pattern of the reduced Pd2Ga/SiO2 catalyst and (b) TEM image acquired after reduction.

Fig. 2: ETEM image of the Pd2Ga/SiO2 catalyst acquired under reduction conditions (400 Pa H2, 550°C). (b) High resolution image of a in situ reduced Pd2Ga nanoparticle and (c) corresponding FFT.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-2742 Charge density distribution in an atom probe needle measured using electron holography without mean inner potential effects.

Migunov V.1, London A.2, Farle M.3, Dunin-Borkowski R. E.1
1Ernst Ruska Centre for Microscopy and Spectroscopy with Electrons and Peter Gruenberg Institute, Forschungszentrum Juelich, Juelich, Germany, 2Department of Materials, University of Oxford, Oxford, UK, 3Fakultaet fuer Physik and Center of Nanointegration (CeNIDE), Universitaet Duisburg-Essen, Duisburg, Germany
v.migunov@fz-juelich.de

Since atom probe tomography (APT) utilizes atom-by-atom field evaporation of a sharp needle by an applied voltage, it is important to know the electric field distribution around the tip with nm spatial resolution for successful reconstruction. Here, we use off-axis electron holography to measure the electric field around an electrically biased Fe needle that contains yttrium oxide nanoparticle inclusions. The electric field was generated by applying a voltage between the needle and a counter-electrode that was placed coaxially with the needle at distance of ~400 nm from it. The results (Fig. 1) were interpreted both by fitting the recorded phase shift to a simulated phase image modeled using two lines of constant but opposite charge density [1] (Figs. 1 b, c) and by using a model-independent approach that involves contour integration of the phase gradient to determine the charge enclosed within the integration contour [2] (Fig. 2a). Both approaches required subtraction of the magnetic contribution to the recorded phase shift, which was achieved by calculating the difference between phase images recorded at applied bias voltages of 0 and 5 V. This approach also automatically resulted in elimination of the mean inner potential (MIP) contribution to the phase shift, which was found to be essential for the latter (model-independent) approach for the present sample. Figure 2 shows cumulative charge profiles along the needle measured using the two approaches, which are consistent with each other, with the model-independent approach revealing the presence of charge accumulation at the apex of the needle. (The black line has a steeper slope in the figure). On the assumption of cylindrical symmetry, the three-dimensional electrostatic potential and electric field around the needle could be inferred from the results, as shown in Fig. 3.

[1] G. Matteucci et al. Ultramicroscopy, 45(1): 77 – 83, 1992.

[2] C. Gatel et al. Phys. Rev. Lett., 111: 025501, 2013.

We are grateful to Jan Caron for help with the development of the contour integration algorithm and to Giulio Pozzi and Christian Dwyer for fruitful discussions.

Fig. 1: Equiphase contours corresponding to a) an original phase image recorded from the needle; b) the difference between phase images acquired at two different bias voltages; c) a best-fitting model-dependent simulation to the result shown in b).

Fig. 2: a) Illustration of the use of an integration contour in the model-independent approach. b) Cumulative charge profiles measured using the model-independent (black) and model-dependent (red) methods.

Fig. 3: Central slice of the three-dimensional distribution of electrical potential (colours) and electric field (white lines) around the needle, inferred from the results shown in Fig. 2.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-2797 In situ Study of Oxidative Etching of Palladium Nanocrystals by Liquid Cell Electron Microscopy

Jiang Y.1, Zhu G.1, Lin F.2, Zhang H.1, Jin C.1, Yang D.1, Zhang Z.1
1State Key Laboratory of Silicon Materials, Key Laboratory of Advanced Materials and Applications for Batteries of Zhejiang Province and Department of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, P. R. China, 2College of Science, South China Agricultural University, Guangzhou, Guangdong 510642, P. R. China
stellajiang915@gmail.com

      The development of liquid flow holder incorporated within a transmission electron microscope (TEM) enables the possibility to observe the size and shape change of nanocrystals in liquid through the electron-transparent viewing window[1,2]. In this talk, we will report our observations on the dissolution process of palladium (Pd) nanocubes within a TEM (FEI Tecnai F20).

      Figure 1A shows time consequential HAADF-STEM images of a dissolving palladium nanocube during the oxidative etching process. At the beginning, the nanocube exhibited well-defined cubic structure terminated with {100} facets (Figure 1A, 0 s). As the chemical etching proceeded, the nanocube started to shrink firstly from its apexes and edges rather than the side facets, and then transformed itself into a round-shaped NC. This was an energy-favored process on considering the fact that those atoms located at the apexes and on the edges have lower coordination and therefore possess higher chemical reactivity. The mechanism of oxidative etching here was similar to that for conventional synthesis by introducing Br- ions and O2, where palladium was oxidized to Pd2+ ions. The introduction of Br- ions could further promote such an oxidizing reaction by forming the complexes such as [PdBr4]2-. Here instead of dissolved oxygen in the solution, the oxidizing agents such as OH•, HO2•, O could be produced in the liquid solution via the interaction between high-energy electrons and the water molecules. The whole process can be summarized as illuminated in Figure 2.

References
(1) Niels de Jonge*, Diana B. Pekys et al. PNAS. 2008, VOL 106, NO. 7, 2159-2164
(2) Edward R. White, Scott B. Singer, Brian C. Regan* et al. ACS NANO. 2012, VOL 6, NO. 7, 6308-6317

This work is financially supported by the National Science Foundation of China (51222202,), the National Basic Research Program of China (2014CB932500), the Program for Innovative Research Team in University of Ministry of Education of China (IRT13037) and the Fundamental Research Funds for the Central Universities (2014XZZX003-07).

Fig. 1: (A) Time sequential color-enhanced HAADF-STEM images showing the dissolution of an individual palladium nanocube during the oxidative etching. The scale bar is 10 nm in all panels. (B) The corresponding 3D geometric models of the dissolving nanocube shown in A.

Fig. 2: A schematic illumination for the process of oxidative etching of palladium nanocubes in liquid as triggered by electron beam irradiation.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-2834 Nanoscale imaging of oxygen assisted reconstructions over Pt and Rh catalysts and their effects over local catalytic activity

Barroo C.1, Lambeets S. V.1, Kruse N.1, Visart de Bocarmé T.1
1Université Libre de Bruxelles - CPMCT
cbarroo@ulb.ac.be

Field emission techniques are used to study the morphological and structural changes occurring over rhodium and platinum nanosized model catalysts after oxygen exposure. The effects of reconstructions over local catalytic activity of O2 and NO2 hydrogenation reactions are also investigated. Field ion microscopy (FIM) and field emission electron microscopy (FEM) were used to characterize the apex of tip samples before, during and after the catalytic reactions.

On rhodium samples, the exposure of less than 10 Langmuir of O2 is sufficient to induce significant morphological changes. Higher exposures leads to the formation of dissolved oxygen, known as subsurface oxygen, which can assist a morphological reconstruction upon a short heating of the sample. This is illustrated with atomic resolution by FIM at 50 K. Similar pattern is also visible at high temperature (505 K) in the presence of a reactive mixture of H2 and O2. This reaction shows a structure sensitivity towards surface oxidation at temperature from 505 K and lower and occur first along the <001> zone lines. This effect is less and less observed at increasing temperatures. A cross comparison of the influence of the temperature and the tip morphologies indicates that reconstructions have only a limited influence on the reactivity at temperature above ~500 K

On platinum, the kinetic instabilities of the NO2-H2 reaction are followed by FEM at 390 K starting from a hemispherical tip sample. The most active facets for the reaction appear to be the {110} and {012}. Inversely, {113} and {111} appear and remain dark and inactive during the process. The dormancy of the {113} and {111} facets is due to the presence of oxygen adsorbates exhibited by the dark regions on FIM images at low temperature. The instabilities are expressed as surface explosions occurring randomly in time, but synchronized over {011} facets. These instabilities expand along the <001> lines over the (001) pole and exhibit self-sustained kinetic oscillations. Control experiments by FIM at different temperatures show that the FEM pattern during the reaction at 390 K is very similar to the FIM pattern at 145 K, but cooling the sample down to 29 K and imaging it in helium allows the observation of {113} and {111} facets with atomic resolution. From these control experiments, we conclude that Pt undergoes only limited structural changes during the ongoing NO2-H2 reaction.

Nanoparticle dynamics must be accounted in models describing the non-linear features of catalytic reactions and more generally included in the description of catalytic properties of nanosized particles.

C.B. and S.V.L. thanks the Fonds de la Recherche Scientifique (F.R.S.-FNRS) for financial support (PhD grant from FNRS and FRIA respectively). The Wallonia-Brussels Federation is gratefully acknowledged for supporting this research (Action de Recherches Concertées n°AUWB 2010-2015/ULB15).

Fig. 1: Field emission pattern during catalytic NO2 hydrogenation over Pt a) 200 ms before the maximum intensity of the surface explosion. b) At the highest intensity. c)  400 ms after. d) brightness signal over a few cycles of reaction for the (011) and (001) facets (from DOI: 10.1016/j.apsusc.2014.01.158)
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-2836 Catalytic hydrogenation of CO2 on rhodium surfaces: field emission techniques approach

Lambeets S. V.1, Barroo C.1, Kruse N.1, Visart de Bocarmé T.1
1Université Libre de Bruxelles - CPMCT
slambeet@ulb.ac.be

The valorisation of CO2 gas into useful products for the industry seems to be one key to address the issue of global warming. Yet, its application has to be economically viable. For instance, heterogeneous catalysis is used to produce methanol by CO2 hydrogenation. In order to improve the efficiency of such catalysts, a deep understanding of the reaction processes at the molecular level is needed. Catalytic particle shape, its size and its surface composition are some of the features that fill influence the activity and that should be determined with time so as to unravel the detailed mechanism the this reaction. This paper reports the study of CO2 adsorption as well as the interaction of H2/CO2 gas mixtures on nano-sized rhodium crystals. The systems have been studied at the nanoscale using field emission techniques including Field Ion Microscopy (FIM) and Field Emission Microscopy (FEM). The FIM/FEM device is able to image in real time the surface of a conductive material, conditioned as a tip, at the nanoscale with 0,2 nm lateral resolution in FIM mode and 2 nm in the FEM mode. The structure of the rhodium nanocrystals have been characterised by FIM with atomic lateral resolution, whereas CO2 adsorption and dissociation have been followed by FEM. Brightness analysis is used to monitor the reaction in while it proceeds. The brightness intensity of the FEM pattern depends on the amount of electrons emitted from the nanocrystal and thus, on the work function of the surface. The introduction of pure CO2 gas during the imaging causes the brightness to decrease due to the dissociative adsorption of CO2 gas to O(ads) and CO(ads) species. Upon increase of the hydrogen pressure, reaction phenomena were observed from 650 K to 700 K. The increasing brightness reflects the occurrence of a reaction between adsorbed hydrogen and adsorbed oxygen. Results from the brightness analysis have been compared to literature data which allow to propose a chemical scenario explaining these observations and identify the reaction as the Reverse Water Gas Shift. These assumptions are in line with direct local chemical analysis performed by atom probe techniques. The latter consist to the coupling of a FIM device with a Time of Flight mass spectrometer operated during the ongoing processes using field pulses to desorb surface species as ions.

S.V.L. and C.B. thanks the Fonds de la Recherche Scientifique (F.R.S.-FNRS) for financial support (PhD grant from FRIA and FNRS respectively). The Wallonia-Brussels Federation is gratefully acknowledged for supporting this research (Action de Recherches Concertées n°AUWB 2010-2015/ULB15).

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-2873 The response of nanofibrous mats to mechanical stress

Hadraba D.1,3,4, Lopot F.1, Suchy T.2, Moravek M.1, Bacakova M.3, Zaloudkova M.2, Ameloot M.4, Jelen K.1
1Dept. of Anatomy and Biomechanics, Charles University, Czech Republic, 2Institute of Rock Structure and Mechanics, AVCR, Czech Republic, 3Institute of Physiology, AVCR, Czech Republic, 4Dept. of Biophysics, UHasselt, Belgium
daniel.hadraba@uhasselt.be

Nanofibrous materials are being utilised in various industries, for example, biomedicine, pharmacy, filtration, cosmetics, etc. Even though commercial nanofibers are produced as nonwoven fabric mats, a single fibre is usually tested for mechanical properties and not the whole mat. As far as the final products differ, for instance, in the fibre density, sheet thickness or surface finishing process (plasma treatment, etc.), the mechanical properties differ, too. Unfortunately, there is an absence of a reliable method that provides sufficient mechanical feedback on the nanofibrous mats for the users and manufacturers.
Mainly the fragility, inhomogeneity and geometrical characteristics are the major pitfalls for assigning the results to standard mechanical constants. The home-built device solves these problems and correlates the stress and strain states with the changes in the nanofibrous sheet. The stress is applied to the nanofibrous mat inside the chamber of a SEM microscope and simultaneously the mat is imaged from the top and from its side at different magnifications. The results that include the changes in the thickness of the mat, thickness of the fibres, density of the fibres and fibre orientation, are directly related to the mechanical properties of the mat. The approach reveals the true stress in the mat, the level of isotropy while stress is applied, brittleness and the inner interactions of fibres within the mat. Even the mechanical constants such as elastic modulus are determined with higher precision. The conclusions based on this analysis can help to target specific mechanical properties in the process of manufacturing or to choose the material which is most suitable for certain application (cell seeding, filtering, etc.).

Project supported by the grants: GAUK 956213, GAUK 545312.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-3004 Real time observations of collector droplet oscillations in scanning electron microscope

Kolíbal M.1,2, Vystavěl T.3, Šikola T.1,2
1Institute of Physical Engineering, Brno University of Technology, Technická 2, 616 69 Brno, Czech Republic , 2CEITEC BUT, Brno University of Technology, Technická 10, 61669 Brno, Czech Republic, 3FEI Company, Podnikatelská 6, Brno 612 00, Czech Republic
tomas.vystavel@fei.com

Semiconductor nanowires (NW) are intensively studied for their promising properties in nanoelectronics, photonics, gas and bio-sensing etc. The nanowire shape (cross-section, sidewalls’ orientation etc.) plays a major role in determining electrical transport or sensing properties of nanowire-based devices. The ability to fully control the nanowire morphology is, of course, based on our understanding of the growth process. In this respect, in-situ Transmission Electron Microscopy (TEM) studies have provided vital information [1] on this issue.
In our contribution, we will present our results on the vapor-liquid-solid (VLS) germanium nanowire growth by evaporation inside a SEM vacuum chamber. Compared to TEM, scanning electron microscopy (SEM) can give three-dimensional information of the growth scenario. As the group IV nanowires grown by evaporation are highly faceted, we will focus on effects where SEM can give substantial information. In particular, the initial formation of the growth interface between the droplet and the substrate is decisive on the nanowire orientation (see Figure 1). We will show that it is dependent on the evaporation rate and, hence, by this parameter one can control the growth orientation [2.3]. In another example, we will demonstrate that the droplet on top of a nanowire is not necessarily pinned to the growth interface. Instead, under certain growth conditions it slides down the sidewalls and then climbs up again to the top. Therefore, the growth interface is not planar, but dynamically changes (Figure 2), which results in very complex nanowire morphology [4].

References
[1] Ross F. M., Rep. Prog. Phys. 73 (2010) 114501.
[2] Kolíbal M., Vystavěl T., Novák L. et al., Applied Physics Letters 99 (2011) 143113.
[3] Kolíbal M., Kalousek R., Vystavěl T. et. al., Applied Physics Letters 100 (2012) 203102.
[4] Kolíbal M., Vystavěl T., Varga P., Šikola T., Nanoletters, accepted.

We acknowledge Libor Novák for technical help. This work was supported by the Grant Agency of the Czech Republic (P108/12/P699) and by European Regional Development Fund – (CEITEC - CZ.1.05/1.1.00/02.0068). M. K. acknowledges the support of FEI Company.

Fig. 1: a-c) Initial stage of Ge NW growth from eutectic Au-Ge droplet. The droplet gets filled with evaporated Ge atoms and its volume virtually increases as the excess Ge nucleation takes place at the droplet/substrate interface, c)  the droplet unpins from the substrate and dewetts the nanowire base (c-k, 3 minute steps). The scale bar is 200 nm.

Fig. 2: a) Sequence of SEM images taken during in-situ observationof Ge nanowire growth in the <111> direction on Ge(100) substrate at400°C. a), b) The droplet mostly resides on top facet, butalternatively the triple phase line, slides down and wets {111}-orientedsidewalls. The scale bar is 200 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-3078 In situ TEM study on redox of Pd nanoparticles

Wu M.1, Shen C.1, Dam B.2, Zandbergen H.1
1Kavli Institute of NanoScience, HREM, Delft University of Technology, Delft, The Netherlands, 2MECS, Chemical Engineering, Delft University of Technology, Delft, The Netherlands
m.y.wu@tudelft.nl

Palladium-based catalyst has proven to be an excellent catalyst in the automotive catalyst and for the combustion of natural gas. Due to the oxygen involvement and the temperature range of catalytic reaction, PdO and/or Pd can play an important role in catalytic reaction. Understanding of the redox of Pd is essential for manipulation and control of the catalyst.

Abundant studies have been done on redox of palladium by different techniques: thermogravimetric analysis(TGA), surface techniques such as: X-ray Photoelectron spectroscopy; Auger-Electron Spectroscopy(AES); Low Energy Electron Diffraction (LEED); Scanning tunneling Microscopy(STM), and Transmission Electron Microscopy(TEM), but most of this information is obtained under conditions far from the actual catalytic reactions in automotive applications because these techniques require for instance a limited gas pressure, limited temperature, limited resolution, limited detection depth etc. An analytical tool that can operate at realistic gas pressures, reaction temperatures, and can monitor the changes of particles morphology, nanostructure and chemical composition measurement at the same time, will give us no doubt unique information on the catalyst. Here we present the in-situ TEM results on reduction and oxidation of ~20 nm Pd nanoparticles in different gases O2, H2 and He with pressure of 0.5-0.65 bar, temperature range from room temperature to 800 °C using a static nanoreactor. When heating from room temperature to 800 °C in O2, the morphology of initial metal particles changes in different way depending on their initial structures. Void formation, wetting, de-wetting and sintering of particles occur. All the changes demonstrate that, during heating, there exists a drastic interaction at gas-solid and solid-solid interface (figure 1). Particles, which were first hydrogenated to the beta phase under 0.55bar, released hydrogen in an oxygen atmosphere first at around 130 °C and oxidized at a higher temperature. Reduction and oxidation of Pd nanoparticles can also be induced by the electron beam depending on oxygen gas pressure and temperature.

This work is supported by ERC NEMinTEM Project 267922.

Fig. 1: Image sequences of Pd nanoparticles oxidation during heating in O2 0.6 bar. Heating rate: 13K/min. (a)RT. (b) 195 °C .(c)265 °C. (d) 540 °C. In general, during heating, particles show de-wetting, facet formation, voids appearance and disappearance, wetting and growth. 4 particles numbered show typical type of changes in their morphologies.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-3105 From Dry to Wet: Dynamics of Water Transport Through Phase-Separated Polymer Films

Jansson A.1, Boissier C.2, Marucci M.2, Nicholas M.2, Gustafsson S.1, Hermansson A. M.3, Olsson E.1
1Chalmers University of Technology, Gothenburg, Sweden, 2AstraZeneca R&D Mölndal, Mölndal, Sweden, 3Chalmers University of Technology, Gothenburg, Sweden
eva.olsson@chalmers.se

Drug release from oral pharmaceutical tablets or pellets can be modified by the application of a polymer coating with controlled mass transport properties. When the coating film is made from a blend of polymers, the polymers can phase-separate and form domains enriched in either one. It is common to use a blend of a water-soluble and a water-insoluble component. Exposure to water may then cause the water-soluble polymer to leach out and create water-filled pores through which the drug can be released[1]. Thus, the pore network evolves with time and the permeability toward water and drug increases. The size and connectivity of domains in the phase-separated system affect the dynamics of this process as well as the structure of the evolving pore network. We recently studied the water transport through phase-separated polymer films made from a blend of water-insoluble ethyl cellulose (EC) and water-soluble hydroxypropyl cellulose (HPC)[2]. Using a novel ESEM-based method[3] for in situ controlled wetting, we obtained visual information about the water transport through such films for the first time. Local variations in permeability could be detected and correlated with the phase-separated microstructure, as shown in Fig. 1 and 2. Moreover, varying the blend ratio of the polymers significantly affected the film microstructure and water transport properties. Current studies focus on the transport mechanisms involved in the initial stages of wetting of EC/HPC films. Using a variety of techniques such as in situ ESEM, FIB-SEM and TEM, we investigate the dynamics of water transport through the films as well as the structure of the phase-separated system on the micro and nano scale. An important theme is the swelling and dissolution of HPC and its role in the water transport observed in the in situ ESEM wetting experiments. Our approach differs from traditional methods, where diffusion measurements and microstructural investigations are performed in separate and the main focus is the transport through the pore network after leaching of the HPC. It provides new and otherwise inaccessible information about the processes occurring as the material goes from dry to wet, which may increase the understanding of the function of EC/HPC films as oral controlled release coatings on tablets or pellets.

[1] Siepmann, F., Siepmann, J., Walther, M., MacRae, R.J. and Bodmeier, R. (2008). Journal of Controlled Release 125(1), 1-15.

[2] Jansson, A., Boissier, C., Marucci, M., Nicholas, M., Gustafsson, S., Hermansson, A.-M. and Olsson, E. (2014). Article in press: Microscopy and Microanalysis 20(2), April 2014.

[3] Jansson, A., Nafari, A., Sanz-Velasco, A., Svensson, K., Gustafsson, S., Hermansson, A.-M. and Olsson, E. (2013). Microscopy and Microanalysis 19(1), 30-37.

The authors gratefully acknowledge VINNOVA for financing the project and the VINN Excellence Center SuMo Biomaterials, the Materials Science Area of Advance and the SOFT Microscopy Centre at Chalmers for support and collaboration.

Fig. 1: ESEM image of the surface of an EC/HPC film containing 70% EC and 30% HPC. Dark regions are HPC domains and bright are EC.

Fig. 2: ESEM image of the same EC/HPC film as in Figure 1, a few minutes after the opposite surface was exposed to water. Droplets of water and dissolved HPC have formed on the film surface and appear as bright regions in the image, indicating water transport through the film.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-3158 Real-time Visualization of Phagocyte-like Migration and Coalescence of Si3N4 Supported Pd Nanoparticles under O2 Atmosphere

Lu P.1, Ai F.1, Xie D.1, Shan Z.1
1Center for Advancing Materials Performance from the Nanoscale (CAMP-Nano), State Key Laboratory for Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an 710049, China
penghanlu@gmail.com

The stability of metal nanoparticles is pivotal for their extensive use as heterogeneous catalysts for many industrial applications. The high temperature and reactive gas environment encountered during catalysis, however, often exacerbate the tendency of nanoscale particles to coalesce into larger aggregates, which results in a reduction of active surface area and thus an undesired catalyst deactivation [1-4]. Here we use the differential pumping-based Hitachi H-9500 environmental TEM (Fig.1a) and the MEMS-based Protochips heating holder (Fig.1b-c) to reveal a real-time visualization of the oxygen-induced migration and aggregation behavior of Pd nanoparticles, which plays a crucial role in CO oxidation as well as a variety of catalytic reactions under oxidative atmosphere.

In this study, Pd nanocubes with the size of ~10 nm were dispersed in ethanol under sonication and dropped onto the Si3N4 membrane support (Fig.1c) for the in situ TEM experiments. The samples were then heated up to 300 °C followed by immersed in the oxygen environment. A typical example of the dynamic behavior of Pd nanoparticles is illustrated in Fig. 2. After oxygen injection, some of the particles were firstly activated and moved around, with dramatic shape variations during the migration. These pioneers tried to ingest other particles nearby (Particle IV in Fig.2a-c), like the phagocytes prefer to surround bacteria and swallow them. This led to a domino effect that an increasing number of particles served as active “phagocytes” after they contacted or coalesced with the original pioneers (Particle I-IV in Fig.2d-e, Particle I-II-IV in Fig.2e-f, Particle III-IV in Fig.2d-f). In response to this chain reaction, most of the small particles finally aggregated into larger ones with an average diameter of ~30 nm.

Although the sintering behavior of metal particles on carbon or hydrocarbon has been found as a catalytic gasification process [1,5,6], such phagocyte-like migration and coalescence dynamics in metal/Si3N4 model systems have not been reported before, especially considering that Si3N4 cannot be as easy to be oxidized as carbon at this temperature range. Detailed analysis of the trajectories of particle motion coupled with the inspection of the composition and microstructure of the samples would be presented further to clarify the mechanism behind this intriguing phenomenon.

References
[1] R.-J. Liu et al. Microsc. Microanal. 10, 77-85 (2004).
[2] M. A. Newton et al. Nat. Mater. 6, 528-532 (2007).
[3] K. Paredis et al. J. Am. Chem. Soc. 133, 13455-13464 (2011).
[4] G. S. Parkinson et al. Nat. Mater. 12, 724-728 (2013).
[5] R. T. K. Baker et al. J. Catal. 41, 22-29 (1976).
[6] K. Yoshida et al. Nanotechnology 24, 065705 (2013).

This work was supported by the Grants from NSFC (50925104 and 51231005). The authors would like to thank Dr. Mingshang Jin from XJTU for providing palladium nanoparticles samples, and also appreciate the fruitful discussion with Prof. Ju Li from MIT.

Fig. 1: Schematic of experimental setup. (a) Differential pumping-based Hitachi H-9500 ETEM. (b) Front end of Protochips heating holder with four electrical leads connecting a clamped MEMS heater chip. (c) Cross-section view of the chip shown in (b), illustrating Pd NPs were supported on the Si3N4 membrane in this study.

Fig. 2: Time-lapsed TEM images of Pd NPs during exposure to oxygen after heating up to 300 °C, showing the phagocyte-like migration and coalescence dynamic behavior. The times relative to the start of the oxygen injection are indicated.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-3163 In-situ TEM study of cathode-electrolyte interface structure change in a LiMn2O4 nanowire battery

Lee S.1,2, Oshima Y.2,3, Takayanagi K.1,2
1Tokyo Institute of Technology, Tokyo, Japan, 2JST-CREST, Tokyo, Japan, 3Osaka University, Ibaraki, Japan
slee@surface.phys.titech.ac.jp

For development of lithium ion batteries, one of important issues is long lifetime. Charge-discharge cycles have been reported to change the interface structure between electrode materials and electrolyte irreversibly [1], accompanying the degradation of the battery lifetime. For detecting such structural change, transmission electron microscopy (TEM) is one of powerful methods [2]. Especially, in-situ TEM observation during the charge-discharge cycles provides the information of real-time structure change.
We developed a new ‘nanowire-battery’, consisting of LiMn2O4 nanowire cathode, ionic liquid electrolyte (ILE) and Li4Ti5O12 (LTO) crystal as shown in Fig.1 (a). The nanowire-battery was loaded in our home-made TEM holder, and the structure change was observed at the cathode-electrolyte interface during charge/discharge process (Fig.1b). Fig. 1(c) shows the cyclic voltammetry curve of the nanowire-battery. A pair of the “double-peaks” marked by arrow heads characterizes the 4V reaction of the LiMn2O4. Transmission electron diffractions (TEDs) revealed that the cubic structure changed into tetragonal phase at the interface area of a LiMn2O4 nanowire covered by (dipped in) the ionic liquid electrolyte during discharge process, and the tetragonal phase returned into cubic phase reversibly in the sequential charge process. The tetragonal phase is interpreted to have formed due to high concentration of lithium ion at the interface area. Despite of the cubic-tetragonal transformation which resulted in distortion, c/a=1.11, the LiMn2O4 nanowire shows no fracture. This robustness of a LiMn2O4 nanowire is directly seen by in-situ TEM-TED. Aa shown in Fig. 2, the cubic structure (Fig. 2a) was changed into orthorhombic phase (splitting of TED spots in Fig. 2b) and then into the tetragonal one (Fig. 2c). It is considered that the orthorhombic phase mediates cubic-tetragonal transformation, so as to avoid the fracture and help reversible cycle of battery reaction. Present in-situ study clarified that LiMn2O4 nanowire battery can work reversibly without fracture, which promise that a LiMn2O4 nanowire is useful for the long-life-time battery [3]

[1] D. Aurbach, et al., J. Power Sources 68. (1997) 91-98
[2] M. M. Thackeray, et al., Electrochem. Solid State Lett., 1 (1998) 7–9
[3] S. Lee, et al., J. Phys. Chem. C 117 (2013) 24236–24241

The authors are grateful to Dr. Hosono and Dr. Zhou in AIST for providing LiMn2O4 nanowires, and to Dr. Kim and Dr. Kanno in TITECH for fruitful discussion.

Fig. 1: (a) The illustration of a ‘nanowire-battery’. It consists of LiMn2O4 cathode, ionic liquid electrolyte (ILE) and Li4Ti5O12 (LTO). The observed area is marked by a broken-line square. (b) The TEM image of the nanowire-battery. (c) The cyclic voltammetry curve of the nanowire-battery. Arrow heads indicate current peaks due to battery reaction.

Fig. 2: The TED patterns obtained during the initial stage of discharge process. The nanowire axis was the horizontal direction of all TED patterns which is the [440] direction in (a), (b) and is the [224] direction in (c). White arrows in (b) shows splitting of TED spots.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-3524 Novel functionalized MEMS devices for in-situ Electron Microscopy

Bormans B.1, Xu Q.1, Zandbergen H. W.2
1DENSsolutions B.V., Delftechpark, 2628 XH Delft, the Netherlands , 22Delft University of Technology, Kavli Institute of Nanoscience, 2628 CJ Delft, the Netherlands
q.xu@tudelft.nl

MEMs-based in situ TEM holders for heating experiments do provide a large flexibility in performing heating experiments [1], because the virtually zero drift, very fast temperature changes (in millisecond) and accurate measurement of the real sample temperature. There are still questions on these heaters that have to be addressed: 1. How to get regular sample onto the MEMS chip ? 2. How to further reduce the drift induced by large temperature changes to study real dynamics 3. What is the temperature distribution? 4. How to improve accuracy of the temperature? 5. How to overcome bulging of the membrane that carries the sample ? Since a microheater in a MEMS device is small, e.g. 300 µm diameter, the sample has to be also small. Thus sample preparation has to be unconventional, if one want make full use of the microheater advantages. One approach is the “lift-out” method, developed for Dual Beams, in which a lamella is cut out from specimen and placed on the MEMS heater. Another approach is to cut by FIB lamella-like samples from a conventional TEM specimen prepared by PIPS or electrochemical polishing methods (Hui Wang, this conference). The drift of the MEMS-based heaters is in general very low, but if the temperature is changed over more that 50°C a significant drift can be present for several minutes, hampering HREM imaging. We will discuss novel designs of the heater chip in which this this problem can be reduced such that HREM recording remains possible throughout the jump in T. The temperature distribution over the heater cannot be completely prevented and it is in quite a number of experiments quite useful, for instance to repeat an experiment at a certain temperature with a particles that are further to the outside of the heater and thus at lower temperature. Thus if the exact temperature profile is known, then the temperature gradient is actually quite useful. The temperature gradient can be determined very well with Raman spectroscopy. Bulging of the membrane occurs due to the thermal expansion. For the SiN membranes that we are using, this bulging can be very significant, resulting in a change in focus if the temperature is changes. Such bulging can be calibrated and by coupling of the heater and TEM software a automatic correction can be made. We will discuss novel MEMS based heater chips, in which the bulging can be almost completely overcome in a specific temperature range. Also we will discuss additional functions designed into the MEMS based heater chips like contact lines for biasing experiment and gas and/or fluid in- and outlets. Also we will discuss a technology road map for MEMS based sample carriers enabling very sophisticated in-situ dynamic experiments in TEM. References 1. Van Huis, MA., et al, Adv. Materials, 21 (2009)

Fig. 1: (a) Dark field (with blocking of only the central beam; shown here because of the long exposure time) HREM image of 4-5 layer graphene taken with a FEI Titan at 300 kV with exposure time of 10 seconds and the sample at 600 C. (b) FT of the image given in (a) showing that the information is better than 1Å (indicated by circle).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-5768 Investigation of gold nanoparticle movement in liquid by scanning transmission electron microscopy

Pfaff M.1, de Jonge N.1, 2
1INM – Leibniz Institute for New Materials, Saarbrücken, Germany, 2Vanderbilt University School of Medicine, Department of Molecular Physiology and Biophysics, Nashville, TN, USA
marina.pfaff@inm-gmbh.de

We have used scanning transmission electron microscopy (STEM) in liquid, so-called Liquid STEM [1], to study the motion of gold nanoparticles [2]. The liquid containing nanoparticles was enclosed in a micro-chamber consisting of two Si microchips with electron transparent SiN windows (Fig. 1), and imaged with STEM (Fig. 2) at 200 keV. The nanoparticles were visible with high contrast in the background signal caused by the liquid, on account of the Z-contrast of the used annular dark-field (ADF) detector. The spatial resolution on the gold nanoparticles amounted to 1.5 nm [2]. We have also used an alternative experimental approach employing environmental scanning electron microscopy (ESEM) equipped with a STEM detector. Nanoparticles were imaged in a thin layer of cooled water on a SiN window. The gas pressure in the microscope chamber was adjusted to maintain a stable water layer. There was thus no need of enclosure of the liquid in a microfluidic chamber. At the SEM electron energy of 30 keV the resolution was reduced compared to STEM at 200 keV.

The nanoparticles were applied onto the SiN windows either from solution or via sputtering prior to STEM imaging. The liquid used for the experiments contained water and 50% glycerol to increase the viscosity. After scanning a sample area with a high magnification for a few times, the immobilized nanoparticles detached from the membrane and started to move. This movement was then visualized in a movie based on the running average of subsequent STEM images. The movement did not correspond to the expected Brownian motion of particles moving freely in liquid. On the one hand, the nanoparticles did not move randomly but the movement showed a preferred direction either in one corner of the image (Fig. 3a) or radially outwards from the image center (Fig. 3b). On the other hand, the displacement of the nanoparticles was three orders of magnitudes smaller than the result of calculations on the basis of Brownian motion. Possible explanations involve the occurrence of van der Waals forces or electrostatic interactions between the nanoparticles and the SiN membrane [2]. Although the deviation from Brownian motion has not been understood yet, it is beneficial for the imaging of nanoscale objects in liquid by STEM, since the spatial resolution obtained for imaging nanoparticles in proximity of the supporting film is not degraded by their displacement within the image acquisition time. Therefore, this technique is well suited for the investigation of a wide range of processes occurring in liquid – either in material science or in biology [3].


[1] DB Peckys & N de Jonge, Microsc Microanal 20, 346-65, 2014.
[2] EA Ring & N de Jonge, Micron 43, 1078-1084, 2012.
[3] N de Jonge & FM Ross, Nat Nanotechnol 6, 695-704, 2011.

We thank E.A. Ring for research work and E. Arzt for his support through INM.

Fig. 1: SEM image of enclosure of two Si microchips with electron transparent SiN windows sealed together with epoxy. Reproduced with permission from [2].

Fig. 2: Principle of STEM imaging of nanoparticles in liquid enclosed between the SiN windows. Images are obtained by scanning a focused electron beam over the sample and detecting the transmitted electrons for each pixel. Depending on the imaging conditions bubbles are formed under electron irradiation. Reproduced with permission from [2].

Fig. 3: Trajectories of selected nanoparticles reconstructed from time series of ADF STEM images taken at 200 keV. Two different movements were observed: (a) a linear movement and (b) a radial movement outwards the illuminated sample area. Reproduced with permission from [2].
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-5774 In-situ observation of graphene growth dynamics by environmental scanning electron microscopy

Wang Z. J.1, Weinberg G.1, Schlögl R.1, Willinger M. G.1
1Department of Inorganic Chemistry, Fritz Haber Institute of the Max Planck Society, Berlin, Germany
zhujun@fhi-berlin.mpg.de

Most promising approaches for industrial scale production of graphene are based on metal catalysed chemical vapour deposition (CVD). Although improvements in graphene quality and yield have been achieved, there remains a lack in the mechanistic understanding of graphene formation. This lack of understanding is due to the fact that most insights on graphene growth have been derived from post growth characterizations, which are in principle incapable of capturing the dynamics of a CVD process. As we have learned from heterogeneous catalysis, a mechanistic insight can (in most cases) only be obtained on the basis of in-situ techniques that are capable of capturing the interaction of the catalyst with the environment while the product is formed. Here we report on in-situ graphene growth on nickel, copper and platinum catalysts inside a modified environmental scanning electron microscope. Using this method, we are able to visually follow the complete CVD process involving substrate annealing, graphene nucleation and growth and finally, substrate cooling. Due to the high sensitivity of the secondary electron signal to changes at the surface, we are able to visualize the formation and growth of single atom thick graphene sheets.
The in-situ experiments presented here reveal the dynamic nature of the process in an unparalleled way and provide important insights on the growth kinetics and the substrate-film interactions at the micron to nanometer scale (Figure 1). In the case of growth on nickel, temperature and atmosphere induced dissolution and precipitation dynamics can be observed. For the case of copper, it is found that graphene growth above 850°C occurs on a pre-melted, highly mobile surface.
The nucleation and growth behaviour will be discussed and the influence of grain dependent surface dynamics presented. Furthermore, we show that graphene induced copper surface reconstructions occur during cooling.

Fig. 1: a) shows colorized snapshots taken during low-pressure CVD growth of graphene on copper at 1000°C. The growth and nucleation behaviour can directly be abstracted from the recorded images as shown in b).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-5843 Nanostructures of bimetallic Pt-Sn fuel cell catalysts

Ward M. R.1, Sharman J.4, Ozkaya D.4, Martinez Bonastre A.4, Boyes E. D.1,2, Gai P. L.1,3
1The Nanocentre, Department of Physics, University of York, UK, 2The Nanocentre, Department of Electronics, University of York, UK, 3The Nanocentre Department of Chemistry, University of York, 4Johnson Matthey Technology Centre, Sonning Common, Reading, UK
michael.ward@york.ac.uk

For direct ethanol fuel cells (DEFCs) to become viable, the efficient oxidation of ethanol at the anode is required, such that all twelve of the possible electrons are captured in converting the chemical energy of the ethanol molecule. Carbon supported Pt can work (1), but tends to become poisoned by intermediate reaction products (2). It is therefore necessary to develop multifunctional catalysts with reduced Pt content (1-3). One possible solution is to combine Pt with Sn which overcome these limitations (1-3). Reactivity measurements show favourable trends with Pt-Sn bimetallic catalysts (2, 3). Pt-Sn can form a variety of different phases, which are usually cubic or hexagonal, but in general, only the Pt3Sn (cubic) and PtSn (hexagonal) are reported in fuel cell catalysts (4).

From a TEM point of view, distinguishing the different Pt-Sn phases is challenging because the spatial frequencies (and atomic plane spacings) of Pt-Sn bimetallic phases are similar to Pt (4). We use a mixture of HRTEM, HAADF-STEM and electron diffraction techniques to investigate the nanostructures of fresh Pt3Sn/C and PtSn/C electrode catalysts supplied by Johnson Matthey.

Figure 1 shows HAADF-STEM images of a Pt3Sn/C and PtSn/C catalyst. The nanoparticles are on average approximately 4 nm in diameter on the Pt3Sn/C catalyst and appear to be separate crystals. By contrast, nanoparticles on the PtSn/C catalyst form an almost continuous layer of metal on the surface of the carbon support. The nanoparticles on the PtSn/C catalyst are approximately 2 nm diameter on average. Figure 2 shows higher magnification HAADF-STEM images. Clusters were observed in both electrode catalysts. Intensity variations in the nanoparticles such as the central nanoparticle in Figure 2(b) could be explained by the random occupation of Sn within the Pt lattice since HAADF-STEM is sensitive to atomic number. Figure 3 shows HRTEM images of the two electrode catalysts, which show clearly the difference in nanoparticle morphologies between the two catalysts. Measuring the lattice spacings from most of the nanoparticles suggests Pt or Pt3Sn in the Pt3Sn/C catalyst, in addition to some SnO2 crystals. A wider range of lattice spacings in the PtSn/C catalyst were measured suggesting the presence of different Pt-Sn phases and SnO2.

References

1. M. Z. F. Kamarudin, S. K. Kamarudin, M. S. Masdar, W. R. W. Daud, Int J Hydrogen Energ 38, 9438 (Jul 26, 2013).

2. C. Lamy, S. Rousseau, E. M. Belgsir, C. Coutanceau, J. M. Leger, Electrochim Acta 49, 3901 (Sep 15, 2004).

3. E. Antolini, E. R. Gonzalez, Catal Today 160, 28 (Feb 2, 2011).

4. V. Radmilovic, T. J. Richardson, S. J. Chen, P. N. Ross, J Catal 232, 199 (May 15, 2005).

The authors thank the EPSRC for support from critical mass EPSRC grant EP/J018058/1

Fig. 1: HAADF-STEM images of the (a) Pt3Sn/C and (b) PtSn/C electrode catalysts

Fig. 2: High magnification HAADF-STEM images of the (a) Pt3Sn/C and (b) PtSn/C electrode catalysts.

Fig. 3: HRTEM images of the (a) Pt3Sn/C and (b) PtSn/C electrode catalysts
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-5925 In-situ TEM observation of crystal precipitation in liposomes filled with sodium chloride aqueous solution

Ai H.1, Moriya N.1, Harumoto T.1, Ishiguro T.1
1Department of Materials Science and Technology, Tokyo University of Science, Tokyo, Japan
8214601@gmail.com

Observation of chemical reactions using transmission electron microscope (TEM) reveals the details of the reaction, such as reactive sites and/or elementary process of the reaction. For instance, J. M. Yuk et al. had succeeded in in-situ observation of coalescence of two platinum nanocrystals in liquid encapsulated in graphene cells [1]. However, the graphene cells may not always suit for aqueous solutions since graphene is hydrophobic. Thus, another cell for aqueous solution is demanded and a liposome, which is composed of dipalmitoylphosphatidylcholine (DPPC) bilayers, is one of such cells. In this study, the precipitation process in liposomes filled with aqueous solution is observed using TEM.

The liposomes were synthesized using Bangham method [2]. In brief, DPPC layer reinforced with cholesterol were fabricated and the ultrasonication was conducted in sodium chloride aqueous solution to form the liposomes filled with the aqueous solution.

The prepared liposomes were directly observed without staining using conventional TEM (JEM-2000FX, JEOL) operated at 200 kV. TEM image of the synthesized liposomes is shown in Figure 1 and it is confirmed that the liposomes are successfully fabricated. Figure 2 is a continuous observation of the liposome. After electron irradiation, fluctuation on the contrast of the liposome takes place as shown in (a) and (b). In the final state (Fig. 2(c)), the precipitation of square nanocrystals is observed. According to the selected area electron diffraction pattern, the precipitated nanocrystals are sodium chloride single crystal. Thus, the chemical reactions in aqueous solution can be observed using liposome cells filled with water and solute.

[1] J.M. Yuk, et al. Science 336 (2012) 61.

[2] A.D. Bangham et al. J. Mol. Biol. 13 (1965) 238.

This research is supported by a Grant-in-Aid for Exploratory Research (No. 25630269) from the Japan Society for the Promotion of Science (JSPS) and a Grant-in-Aid for imaging technology from Tokyo University of Science.

Fig. 1: TEM image of the prepared liposomes filled with sodium chloride aqueous solution. Precipitation of sodium chloride crystals takes place partially.

Fig. 2: Precipitation of sodium chloride crystals in the solution-filled liposome by electron irradiation: (a) first exposure with 1.0×103 electrons/nm2, (b) after irradiation of 4.1×104 electrons/nm2, and (c) precipitated sodium chloride.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-12-P-5942 In situ Transmission Electron Microscopy Studies of Cathode Materials for Lithium Ion Batteries

Schrader F.1, Iskandar R.2, Noyong M.1, Mayer J.2, Simon U.1
1RWTH Aachen University, Institute of Inorganic Chemistry, Aachen, Germany, 2RWTH Aachen University, Central Facility for Electron Microscopy, Aachen, Germany
felix.schrader@ac.rwth-aachen.de

The understanding of the fundamental processes during charging and discharging in a lithium ion battery (LIB) is of great interest for tailoring future electrode materials. To elucidate e.g. aging effects on a microscopic length scale, analysis is limited to a few methods. Post mortem-analysis, such as transmission electron microscopy (TEM) on battery sub-units, isolated from their electrochemical environment, is commonly applied, whereas the structural and compositional changes during cycling remain widely unexplored. Therefore, it would be high desirable to study these processes with spatial and temporal resolution in situ, i.e. under charging and discharging conditions. Here we report first analyses by means of in situ TEM in liquid environment to monitor structural changes with sub-10 nm resolution of lithium iron phosphate .

Therefore we have synthesized crystalline and carbon coated lithium iron phosphate nanoparticles (NPs) in a size range of 10‒50 nm. This material is characterized by means of scanning electron microscopy, X-ray diffraction, infrared spectroscopy and elemental analysis. To evaluate its properties as a cathode material, a LIB cell with a lithium anode and an electrolyte (lithium hexafluorophosphate in a 1:1 mixture of ethylene and diethyl carbonate) was assembled. The material is not optimized regarding its specific capacity, but showed typical cycling behavior compared to literature.

By using an in situ TEM liquid flow sample holder, the degree of lithiation was analyzed by selected area electron diffraction (SAED) and electron energy loss spectroscopy (EELS). As a first set of experiments the particles were investigated inside these liquid cells under inert gas atmosphere. Despite of gaseous layers of several micrometers, high resolution TEM as well as SAED and EELS in the low loss region were feasible. In the second set of experiments the cell was filled with water. We found that particles with diameters down to 2 nm could be imaged with a 200 nm thick water layer. The opportunities and limitations, which arise from these in situ measuring conditions, will be discussed.

[1] N. de Jonge, F. M. Ross, Electron microscopy of specimens in liquid, Nature Nanotechnology, 2011, 6, 695–704.

[2] Y. Wang, Y. Wang, E. Hosono, K. Wang, H. Zhou, The Design of a LiFePO4/Carbon Nanocomposite With a Core-Shell Structure and Its Synthesis by an In Situ Polymerization Restriction Method, Angew. Chem., 2008, 120, 7571–7575.

[3] R.F. Egerton, Electron Energy-Loss Spectroscopy in the Electron Microscope, Springer New York, 2011.

We thank the Federal Ministry of Education and Research (BMBF) for financial support.

Fig. 1: Lithium iron phosphate in H2O.

Fig. 2: Magnification of the blue framed area in Fig. 1.

Fig. 3: Electron energy loss spectrum for thickness determination of the red circled area in Fig. 1.
↑ Go to the Table of content ↑

ID-13. Materials for medicine and biomaterials

↑ Go to the Table of content ↑
Type of presentation: Invited

ID-13-IN-1430 MESOPOROUS SILICA/APATITE NANOCAPSULES FOR DRUG DELIVERY

Gonzalez G.1, Sagarzazu A.1, Cordova A.1
1Venezuelan Institute for Scientific Research, Center of Materials Engineering and Nanotechnology
gemagonz@gmail.com

Mesoporous silica materials, with pores with diameters between 2 and 50 nm, have a highly organized porous structure with uniform pore size and vast surface area, being excellent candidates for drug delivery carriers. Due to their bioactive characteristics the walls of mesoporous silica can be coated on their surface with a bonelike apatite layer by immersion of the material in simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma. The tuneable mesopore structure and modifiable surface of mesoporous silica nanoparticles allows incorporation of apatite and of various classes of drug molecules and controlled delivery to the target sites.

In the present work the formation of the apatite layer in two mesoporous materials with different pore configuration, and the effect on drug delivery has been studied.

The synthesis of the mesoporous materials was performed using supramolecular templates, Triblock copolymers: (EO20PO70EO20) (Pluronic P123), MW=5800 and (EO106PO70EO106) (Pluronic F127) and Tetraethylorthoxysilane (TEOS) as silica source. The former generates a hexagonal mesoporous structure of aligned channels (SBA-15) and the latter a structure of channels and cavities with a cubic arrangement (SBA-16). Once the mesoporous structures were obtained, the materials were soaked in inorganic body fluid solution for different periods, resulting in the formation of a thin layer of apatite over the internal and external surfaces of the mesoporous material. HRTEM, HAADF and EDS analysis were used to characterize the apatite formation. Also, FTIR, XRD and surface area measurements complemented the structural characterization. Drug adsorption and delivery was study using HPLC, UV and TGA

Fig. 1 shows a HRTEM image of the mesoporous structure after 1 and 3 weeks (Fig. 1a and 1b) immersion in SBF. It can be observed that the arrangement of ordered mesoporous is broken (1c) after only one-week immersion, due to the apatite deposition that modifies the structure. Convoluted concentric cylinders coated with nanocrystals of apatite are observed (Fig 1d). The EDS analysis of these samples showed the presence of Si, O and small amounts of Ca and P, corroborating the presence of apatite forming the coating. These morphological characteristics give these materials unique features for drug carriers due to the high superficial area and therefore adsorption capabilities and on the other hand, the formation of the nanocrystaline apatite coating increases the biocompatibility of the materials enhancing osseointegration.

To FEI Co. for the use of the Tecnai instrument, for the High resolution TEM images. To Fonacit for the financial support through project G-200100900.

Fig. 1: Fig. 1a HAADF image of Mesoporous SBA-apatite after1 week SBF immersion

Fig. 2: Fig. 1b HRTEM showing the effect of apatite deposition breaking the regular mesoporousarrangement

Fig. 3: Fig.1c Apatite nanocrystals on mesoporous structure forming a concentric arrangement

Fig. 4: Fig.1d Apatite nanocrystals formed outside of the mesoporousmaterials after 3 weeks immersion in SBF. The inset is an EDS spectrum showing Caand P presence
↑ Go to the Table of content ↑
Type of presentation: Invited

ID-13-IN-2072 Interaction of cells with biomaterials for tissue engineering in vitro – a review

Bacakova L.1
1Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4 - Krc, Czech Republic
lucy@biomed.cas.cz

Tissue engineering is defined as an interdisciplinary field that applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function [1]. These substitutes contain a cell component and a material component, which serves as a carrier for the cells. In advanced tissue replacements, the material component should actively promote the adhesion, growth, differentiation, phenotypic maturation and other cell functions in a controllable manner. In other words, the material component should act as an analogue of the native extracellular matrix.

In the first part of our studies, we focused on endothelialization of clinically-used knitted polyethylene terephthalate prostheses. The inner surface of these prostheses is rough and highly hydrophobic, and this hampers the adhesion and growth of endothelial cells. However, when covered with fibrin-based films (Fig. 1A), this surface allows the formation of a confluent and shear stress-resistant endothelial cell layer with well-developed cell-matrix contacts, namely talin-containing focal adhesion plaques (Fig. 1B) and intercellular contacts, visualized by staining of VE-cadherin (Fig. 1C) [2, 3].

In the second part of our studies, we concentrated on bone implants. Metallic materials are still unavoidable in load-bearing applications, e.g. as substitutes for big joints (hip, knee, shoulder), although these material are too stiff, too weighty, and often release cytotoxic and immunogenic ions. In order to increase their chemical stability and their attractiveness for cell colonization, the surface of these materials is modified by various approaches, such as electric discharge machining, acid etching, shot peening or deposition of various films (Fig. 2A). Simultaneously, three-dimensional degradable porous or fibrous synthetic polymeric scaffolds have been developed, enabling the ingrowth of cells and the formation of newly-regenerated bone tissue (Fig. 2B, C) [4, 5].

The third part of our studies focused on structures for skin reconstruction and regeneration, based on degradable polymeric nanofibrous meshes. These meshes enable the adhesion, growth, phenotypic maturation of keratinocytes (Fig. 3 A, B), and enable them to communicate with the underlying dermal fibroblasts (Fig. 3C) [6].

[1] Langer R, Vacanti JP: Science 260: 920-926, 1993 1993

[2] Filova E, Brynda E, Riedel T et al: J Biomed Mater Res Part A: 102A: 698–712, 2014

[3] Chlupac J, Filova E, Riedel T et al: Physiol Res, in press

[4] Havlikova J, Strasky J, Vandrovcova M et al: Mater Sci Eng C, in press

[5] Pamula E, Filova E, Bacakova L et al: J Biomed Mater Res 89A: 432-443, 2009

[6] Bacakova M, Lopot F, Hadraba D et al: J Biomater Appl, submitted

Supported by the Grant Agency of the Czech Republic (grants No. P108/10/1106, P108/11/1857 and P107/12/1025).

Fig. 1: A nanofibrous fibrin layer for inner modification of vascular prostheses (A), and immunofluorescence of talin (B) and VE cadherin (C) in endothelial CPAE cells in cultures on these layers. Bar 10 μm (A) or 25 μm (B, C).

Fig. 2: Human osteoblast-like Saos-2 cells immunostained for talin (A) and MG-63 cells (B, C) in cultures on Ti-6Al-4V alloy modified by electrical discharge machining and shot peening (A), and on porous (B) or fibrous (C) polylactide-co-glycolide scaffolds. Bar 30 μm (A) 400 μm (B) and μm (C).

Fig. 3: Immunofluorescence of cytokeratin 5 (A) and filaggrin (B) in human HaCaT keratinocytes in cultures on polylactide nanofibrous membranes treated with plasma (power 75 W, exposure time 30 s). C: Layers of HaCaT keratinocytes (green) and human dermal fibroblasts (red) separated by a nanofibrous membrane. Bar 25 μm (A, B) or 100 μm (C).

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-13-O-1443 Gelatin microsphere-collagen hydrogel composite scaffolds for tissue engineering with human adipose derived stem cells

Tong Y. W.1, Kodali A.1
1National University of Singapore
chetyw@nus.edu.sg

Microsphere scaffolds provide a unique three dimensional micro-environment for the cells cultured on their surfaces through their curvature. Many studies have shown previously that the topographical cues like curvature have an effect on the differentiation abilities of the stem cells. Similarly, studies have also shown that mechanical cues like matrix stiffness and various other biomolecular signals can play a crucial role in determining the stem cell fate. In this study, we designed a three dimensional composite scaffold by encapsulating gelatin microspheres in collagen hydrogels, in order to present a combination of topographical, mechanical and biomolecular signals to human adipose derived stem cells (ADSCs) and studied their osteogenic differentiation abilities. Topographical cues are provided in the form of microsphere curvature by culturing ADSCs on gelatin microspheres to form cell-microsphere aggregates. Further, varying amounts of these cell-microsphere aggregates are then encapsulated into collagen hydrogels to fabricate microsphere-hydrogel composite scaffolds with varying mechanical properties. The mechanical properties of such scaffolds were studied using rheometry. ADSCs encapsulated in such composite scaffolds with varying mechanical properties were induced towards osteogenic lineage and further characterized using gene expression studies of osteogenic marker genes and by measuring the alkaline phosphatase activity. We found that encapsulating increased amounts of microspheres in collagen hydrogels increases the storage modulus of the gels and favors the osteogenic differentiation of ADSCs. To further accentuate the osteogenic differentiation of ADSCs, we then provided the biomolecular cues by encapsulating basic fibroblast growth factor (bFGF) into the scaffolds and releasing it in a controlled manner. Gene expression of osteogenic marker genes and alkaline phosphatase activity of ADSCs upon differentiation in the bFGF encapsulated scaffolds seem to be enhanced compared to the scaffolds with bFGF supplementation directly in the media. Overall, this study shows that, osteogenic differentiation of ADSCs can be enhanced by culturing them in microsphere – hydrogel composite scaffolds which can subsequently be used as effective injectable delivery vehicles for ADSCs as well as various biomolecules.

The authors would like to acknowledge funding from the National University of Singapore.

↑ Go to the Table of content ↑
Type of presentation: Oral

ID-13-O-1625 Wood nanocellulose – Characterization and potential application as barrier against wound bacteria

Chinga Carrasco G.1, Powell L. C.2,3, Khan S.2, Hill K. E.2, Thomas D. W.2
1Paper and Fibre Research Institute, Høgskoleringen 6b, NO-7491 Trondheim, Norway., 2Tissue Engineering and Restorative Dentistry, Cardiff University School of Dentistry, Cardiff, UK., 3Centre for NanoHealth, College of Engineering, Swansea University, Swansea, UK.
gary.chinga.carrasco@pfi.no

Wood nanocellulose is a novel biomaterial for wound dressing applications. Nanocellulose can be manufactured using various pre-treatments, which facilitate the effective fibrillation from micrometre-sized cellulose fibres to nanofibrils. In addition to facilitating the fibrillation of cellulose fibres, chemical pre-treatments modify the surface chemistry of cellulose nanofibrils, which can be applied for functionalization purposes [1]. The purpose of this work was to quantify the morphology of nanocellulose surfaces by applying various complementary microscopy techniques. In addition, the colonisation of the nanocellulose surfaces with bacteria was assessed in detail.

Wood nanocellulose was produced from never-dried P. radiata pulp fibres. The applied pre-treatment was 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) mediated oxidation. To characterise bacterial growth, P. aeruginosa PAO1 biofilms were grown in Mueller Hinton broth (37oC for 24-48h) on air-dried films. Various microscopy techniques, including atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM) and field-emission scanning electron microscopy (FESEM), were applied to characterise the nanocellulose material and the bacterial-nanocellulose interactions [2]-[4]. The FESEM analysis was performed on areas without metallic coating, applying the capabilities of in-lens detectors. The images were thus acquired with low acceleration voltage (<1 kV) and short working distance (<1mm).

Multiscale assessments, including FESEM and AFM, revealed the effective fibrillation of the fibre wall structure, yielding nanofibrils with diameters less than 20 nm and lengths in the micrometre-scale (Figure 1). Importantly, we have demonstrated that the growth of PAO1 was inhibited in the presence of the nanocellulose suspensions when compared to the control. Additionally, SEM imaging revealed distinct clusters of PAO1 cells growing on the surfaces of nanocellulose films (Figure 2). This work highlights the potential usefulness of novel nanocellulose materials in wound dressings with optimized characteristics.

[1] K. Syverud et al., Nanoscale research letters 6 (2011) 626

[2] G. Chinga-Carrasco et al., Microscopy and microanalysis 17(4) (2011) 563-71.

[3] G. Chinga-Carrasco et al., Micron 56 (2014) 80-84.

[4] S.L. Percival et al., Wound Repair and Regeneration 19(1) (2011) 1-9

This work has been funded by the Research Council of Norway through the NANO2021 program, grant no. 219733 – NanoHeal: Bio-compatible cellulose nanostructures for advanced wound healing applications.

Fig. 1: FESEM of the surface of a P. radiata kraft pulp fibre.

Fig. 2: FESEM of the surface of a nanocellulose film composed of individualized cellulose nanofibrils.

Fig. 3: SEM imaging of P. aeruginosa PAO1 grown on nanocellulose surfaces for 24 h.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-13-O-2972 TEM investigation of the temperature dependence on nucleation and growth of hydroxyapatite on arc-deposited TiO2 coatings

Gustafsson S.1, Lilja M.2, Stromme M.3, Olsson E.1
1Chalmers University of Technology, Gothenburg, Sweden, 2Sandvik Coromant Sverige AB, Stockholm, Sweden, 3Uppsala University, Uppsala, Sweden
stefan.gustafsson@chalmers.se

Hydroxyapatite (HA) is the most commonly used bioactive ceramic material due to its excellent affinity to bone. The Biomimetic coating process allows to produce HA coatings with excellent biocompatible and bioactive properties at low temperatures having good adhesion and step coverage. Biomimetic HA coatings are also considered promising for functionalization of metallic implants. HA crystallizes spontaneously on both anatase and rutile phases when being immersed in simulated body fluid. However, for polycrystalline samples the HA nucleation process remains not clearly understood. In this work we investigate the nucleation of HA on arc-deposited anatase phase dominated TiO2 coatings, exposed to phosphate buffered saline (PBS) up to 24 h at two different temperatures, 37 °C and 60 °C. The aim is to contribute towards understanding the mechanism of HA coating growth behavior, thereby facilitating the development of tailor-made HA implant coatings with optimized structures for improved biological performance. Cross sectional samples for TEM were prepared by the lift-out technique using a FEI DB 235 Strata and TEM analysis was performed using a FEI Tecnai F20 operating at 200 kV. The HA precipitation layers after 24 h exposure to PBS are shown in Figure 1 for 37 °C and 60 °C, respectively. At 37 °C, HA crystals form a continuous precipitation layer with a thickness of 700-800 nm after 24 h exposure to PBS. This is in contrast to 60 °C, where the precipitated HA layer after 24h is discontinuous with an average thickness of around 500 nm. On the other hand, the nucleation rate at earlier time points was seen to be much higher at the elevated temperature. Energy dispersive X-ray analysis (EDX) of the precipitation HA layers after 12 h exposure at 37 °C shows a Ca/P ratio gradient; around 1.3 close to the TiO2 interface and decreasing to around 1 in the outer parts. At early time points for 60 °C this trend is reversed; the interface area now showing the lowest Ca/P ratio. Differences in morphology and nucleation rate as seen at the two temperatures may be attributed to differences in ion mobility and Ca-ion consumption as well as surface potential.

Fig. 1: TEM images of the HA precipitation layer after (a) 24 h at 37 °C and (b) 24h at 60 °C.
↑ Go to the Table of content ↑
Type of presentation: Oral

ID-13-O-3487 Small, bumptious, dangerous?  Confocal microscopy analysis of nanoparticles accumulation and migration to mesenchymal stromal cells and intracellular organels.

Skopalik J.1,5, Cmiel V.2, Solar J.2, Havrdova M.3, Magro M.4, Justan I.1, Polakova H.1, Hruskova D.1, Polakova K.3
1Dep. of Pharmacology, Masaryk University, Brno, CZ, 2Dep. Of Biomedical Engineering, Faculty of Electrical Engineering and Communication, BUT, Brno, CZ, 3RCPTM, Department of physical chemistry, Faculty of Science, Palacky University, Olomouc, CZ, 4Department of Comparative Biomedicine and Food Science, University of Padua, IT, 5Internal Hematology and Oncology Clinic, Masaryk University, Brno, CZ
j.skopalik@gmail.com

Backround: Magnetic resonance tracking of magnetically labeled cells is non-invasive and suitable for longtime studies of patients after cell transplantation /1/. The usual way to incorporate many types of nanoparticles smaller than 100 nm into the cell in vitro is their direct addition to the cultivation medium. The incorporation process of the SPIO nanoparticles involves adsorption to cell membrane that is followed by translocation through membrane /1/. The changes in physiology or viability of SPIO labeled cell were tested for many types of  SPIO in last years. Nevertheless, only a few SPIO particles were precisely evaluated from the view of localization in cytoplasm and internal organels of cells.

Aim and methods: “SAMN“  are modern nanoparticles which produce significant MRI contrast /2/ and do not disturb cell physiology, as we published previously. In this study, the SPIO nanoparticle „SAMN“ were conjugated with rhodamine to obtain fluorescent complexes. Our aim was investigation, if these complexes are detectable by confocal microscopy (outside/inside the cells, ) and if we can quantify nanoparticles distribution in cell nucleus and mitochondria. Mesenchymal stromal cells (MSC) were exposed to SAMN-rhodamine nanoparticles (concentration 50 ug/ml, time of labeling 12h and 24h). The cells were then labeled with the mitochondrial marker MitoTracker Green (Invitrogen), and arranged to confocal microscope sample chamber (Leica TSC SP8 X). Spectral overlap of rhodamine and MitoTracker was evaluated. Scaning mode of MSC was optimized and semiautomatic software utility for analysis of confocal scans was developed (in Matlab R2010a).

Results and conclusion: SPIO-rhodamine complexes showed similar excellent uptake by MSC as previously tested  SAMN without rhodamine. Confocal microscopy scans displayed SAMN-rhodamine particles, which were accumulated in clusters in all parts of cytoplasm. Our  Matlab utility exhibited that nanoparticles was not localized in the mitochondria or cell nucleus. This localization was not significantly change after 1  week of cultivation. Our Matlab utility also quantified mitochondria volume and shape in labeled/unlabeled MSC, statistical results demonstrated very minimal pathophysiological effect of SPIO. Our study conclude that our modified SAMN-rhodamine nanoparticles are safety and do not invide to cell nucleus and mitochondria, also they do not desintegrate the mitochondria and metabolism.

/1/Berry (2003) J Phys D Appl Phys,36:198–206.  /2/Magro (2012) Acta Biomater,8:2068–76

Supported by Czech Ministry of Education (project LM2011017) and EU Operational Program Research and Development for Innovations  (CZ.1.05/2.1.00/03.0058)

Fig. 1: MSC cell in 3D visualization after confocal microscopy scanning. Green – mitochondria, Red – clusters of nanoparticles.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-1577 Assessing fractures resistance and pattern of over-flared root canal in vitro: Applications of focus variation 3D scanning microscopy

Masudi S. M.1, Abdo S. B.1, Masudi N. S.1
1School of Dental Sciences, Universiti Sains Malaysia, USM Health Campus, 16150 Kubang Kerian
sam@usm.my

Introduction
     It has been postulated that endodontic treatment results in reduction of fracture strength of teeth. Brittleness of the dentin in the endodontically treated teeth has been attributed to dehydration and loss of collagen cross-linking.  However, more recent studies concluded that neither dehydration nor endodontic treatment caused degradation of the physical or mechanical properties of the dentin. One of the aims of root canal filling is to reinforce the dentin and increase the fracture resistance.
     Studies suggested that filling the coronal and radicular loss tooth structure with bonded restorative materials, such as glass ionomer cement or composite resin, could reinforce the compromised teeth.
     Mineral trioxide aggregate (MTA) is a fine biocompatible hydrophilic material that hardens in the presence of moisture or blood. The aim of this study was to measure the fracture resistance of over-flared root canals filled with different materials (gutta-percha-nano HA, resilon-epiphany, composite and MTA) using the Instron machine test and 3D focus-variation scanning microscopy images were used to illustrate the type of fracture patterns of the specimens
Materials and Methods
     One hundred and twenty extracted human mandibular single- rooted premolars were selected. A total of 105 out of the selected teeth were prepared to the working length and over-flared, leaving the apical 5 mm undisturbed. Fifteen samples had no treatment and were used as a positive control group (Group +ve). The 105 test teeth were further divided into 7 groups of 15 samples each. One of the 7 groups was designated as negative control (Group -ve) where teeth were over prepared and left without obturation. Remaining groups were filled with gutta-percha-nanoHA (Group1), gutta-percha-nano HA+composite (Group 2), gutta-percha-nano HA+MTA (Group 3), resilon-epiphany (Group 4), resilon-epiphany+composite (Group 5), and resilon-epiphany+MTA (Group 6). Fracture resistance of all samples was measured using the Instron testing machine. Two samples from each group had their surface topography and fracture pattern of the specimens were evaluated with a (Alicona Imaging, Graz, Austria).
Results and Discussion
     Statistical analysis for root fracture resistance showed highly significant difference between all groups with p value < 001. Micro CT Scan and 3D focus-variation scanning microscopy analysis indicated the ability of MTA to withstand vertical force. The fracture pattern for roots filled with MTA is only one vertical line, which is initially wide and becomes progressively narrower. Whereas roots filled with composite and resilon show several cracks all over the root in addition to the vertical root fracture.

This study was supported by Universiti Sains Malaysia research grant.

Fig. 1: Image of root fracture filled with gutta-percha/ Nano HA+MTA showing a fracture line across the MTA, becoming narrow and thin. The 3D image shows the surface topographic information in combination with its true color information on tooth 34 (5x Objective lens).

Fig. 2: Similar images of root fracture on tooth 34. The mixture of pseudo and real colors has constructed the rough surfaces of the sample to be more contrast in combination with color gradients. This sample is captured by 5x Objective lens.

Fig. 3: Image of root fracture filled with gutta-percha/Nano HA+resin composite showing a complete vertical line of fracture line. The 3D image shows the surface topographic information in pseudo+real colors in combination on tooth 25 (5x Objective lens).

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-1578 A confocal microscopic evaluation of resin-dentin interface using adhesive systems: in vitro study

Masudi S. M.1, Mahmoud Z. K.1, Masudi N. S.1, Luddin N.1
1School of Dental Sciences, Universiti Sains Malaysia, USM Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia.
sam@usm.my

Introduction
     One of the key functions of a dental restoration is to seal the exposed dentin from the oral environment, to prevent pulpal damage and further decay. Therefore, the microleakage at the tooth- restorative interface is a major concern influencing the clinical longevity of composite resin restorations. The integrity and durability of the marginal seal has always been of prime concern in the investigation of dental restorative materials performance.
     The aims of this study was to compare the effect of self-etch, one-step, one-component adhesive system and etch and rinse adhesive system as well as the nanocomposite and microhybrid composite on the microleakage of class II composite restorations located in dentin.
Materials and Methods
     Fifty-two upper permanent premolar teeth were used and two class II cavities (3 millimeter (mm) width x 1.5 mm depth) with gingival margins ended 1mm below CEJ were prepared and filled in each tooth. Two adhesive systems: self-etch, one-step, one-component adhesive system (G Bond, GC, Japan), etch and rinse adhesive system (Adper Single Bond 2, 3M ESPE, USA) and two composite materials: nanocomposite (Filtek Z350, 3M ESPE, USA), microhybrid composite (Filtek Z250, 3M ESPE, USA) were used and applied in this study according to the manufacturers instructions. The 104 cavities were divided randomly into four groups (n=26). The first two groups were restored with Filtek Z350 (3M ESPE, USA) while the last two groups were restored with Filtek Z250 (3M ESPE, USA). All the cavities in group 1 and 3 were bonded with G Bond while the cavities in group 2 and 4 were bonded with Adper Single Bond 2.
     The specimens were thermocycled between 5° to 55° C with 30 second dwell time for 500 cycles. The samples were then immersed in 0.5% Rhodamine B dye for 10 hours and sectioned longitudinally. Dye penetration at the gingival margin was quantified under confocal laser scanning microscopy/CLSM (Leica,TCS SP2) at 10x magnification. Data were analyzed using Two-Way ANOVA and results with p<0.05 were considered statistically significant
Results and Discussion
      No significant difference (p>0.05) in dye penetration was discovered between self-etch, one-step, one-component adhesive system (G Bond) and etch and rinse adhesive system (Adper Single Bond 2). No significant difference (p>0.05) was also found in dye penetration between the composite materials used. No significant difference (p>0.05) in dye penetration was observed on the gingival margin among all study groups. Self-etch, one-step, one-component adhesive system and nanocomposite restorative materials produced similar results to those of multi-step adhesive systems and microhybrid composites in microleakage of class II composite restorations.

This study was supported by Universiti Sains Malaysia research grant.

Fig. 1: CLSM images of dentinal tubules.  A. Without Rhodamine B fluorescent dye.

Fig. 2: CLSM images of dentinal tubules. B. Stained with Rhodamine B fluorescent dye.

Fig. 3: CLSM images of dentinal tubules. C. Showing micro leakage alone.

Fig. 4:  CLSM images of dentinal tubules. D. Showing microleakage dye in interface with restoration.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-1579 Synthesis and characterization of porous biphasic calcium phosphate scaffold using different porogens: FESEM study

Masudi S. M.1, Alkaisi A.1, Masudi N. S.1, Ahmad Z. A.2
1School of Dental Sciences, Universiti Sains Malaysia, USM Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia., 2School of Materials & Mineral Resources Engineering, USM, 14300 Nibong Tebal, Penang, Malaysia.
sam@usm.my

Introduction
     Biphasic calcium phosphate (BCP) a biomaterial consists of a mixture of hydroxy apatite (HA) and ß-tricalcium phosphate (ß-TCP) which belong to calcium phosphate ceramics (CaPCs). It is biocompatible, biocontactive material and possesses reasonable biodegradable properties, depending on HA: ß-TCP ratio. In addition to it osteoinduction properties in the micro-macroporous BCP forms, BCP is also used in bone grafting due to its similarity in chemical composition to that of bone’s, for drug delivery in association with therapeutics agents (bone morphogenetic proteins, human growth hormone, antibiotics, anti-osteoporotic, anticancer drugs, and as a scaffold in tissue engineering to ensure stem cells remain in the recipient site.
     The minimum recommended pore size for a scaffold is around 100 μm, but subsequent studies have shown better osteogenesis for implants with pores >300 μm. For fabricating biomaterial scaffolds that can meet the requirements set by the specific site of application, there has been an attempt to create a porosity gradient in BCP scaffolds both in the macroporous (>100 μm) as well as in the microporous (<1μm) scales.
Materials and Methods
     By using the wet precipitation method, Biphasic calcium phosphate granules were synthesized with Ca/P ratio1.52 and controlled porosity, pore size distribution, and granule size. Microporosity was then obtained by adjusting sintering temperature while macroporosity was prepared by adding 1:3 wt% ratio of two normally used porogens (naphthalene and sugar) and 2 newly introduced porogens (sago and lentil). Samples from each ratio were pressed into pellets and were fired at 500oC for 2 hours with 0.5°C/minute heating rate (for removal of porogens) and further sintered at 850C for 2 hours with 5°C/minute before cooling down to room temperature. The granules were prepared by crushing and sieving BCP sintered pellets to get granules of sizes ranging from 250-500μm. X-rays diffraction (XRD), field emission scanning electron microscope (FESEM), particle size and porosity analyses were employed in order to characterize the granules.
 Results and Discussion
      In this study, prior to compaction process, BCP powder was mixed with the commonly used (naphthalene and sugar, respectively), and their effect was compared with the newly studied porogens (lentil and sago, respectively). In this method, pores were created during sintering, porogen burned out leaving pores identical to its size and shape. These pores were found to be in the range of trabecular bone pore size of 200-400 μm. This approach gives the desirable properties near to normal bone leading to a perfect osteogenesis for the purpose tissue engineering.

This study was supported by Universiti Sains Malaysia research grant.

Fig. 1: FESEM images on the formation of macroporosity and microporosity, when BCP powder were added with different porogens. Samples were sintered at 850°C for 2 hours. (a) Sugar at 50X, (b) Sugar at 20KX, (c) Naphthalene at 50X, (d) Naphthalene at 20KX, (e) Sago at 50X, (f) Sago at 20KX, (g) Lentil at 50X, and (h) Lentil at 20KX.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-1580 Morphology of glass ionomer cement by incorporating hydroxyapatite-silica nano-powder composite: Sol–gel synthesis and SEM evaluation

Masudi S. M.1, Shiekh R. A.1, Rahman I. A.1, Luddin N.1
1School of Dental Sciences, Universiti Sains Malaysia, USM Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
sam@usm.my

Introduction
      Glass ionomer cement (GIC), classified as acid–base reaction cement, consists of an aqueous solution of polyacrylic acid and an acid-decomposable fluoro-aluminosilicate glass powder. GIC  is widely used in clinical dentistry as tooth colored restorative material. Hydroxyapatite (HA) possesses a chemical composition and crystal structure similar to bone thus suitable for bone substitution and reconstruction. HA has improved the compressive strength, flexural strength, diametral tensile strength, toughness, bonding and fluoride release properties of GIC, has been reported. However, incorporation of HA-silica nano-composite to GIC is not yet reported.
      The present study aims to synthesize HA-silica nano-composite by one-pot sol-gel technique and to assess the effect on the hardness of GIC.
Materials and Methods
       Nanohydroxyapatite was produced by using the in situ sol-gel technique. Calcium hydroxide and phosphoric acid were the main sources of calcium and phosphorus.
                                               5Ca(OH)2 + 3H3PO4    à     Ca5(PO4)3OH + 9H2O
The suspension was stirred 48 hour to get a white viscous sol.  5 ml of TEOS (99%, Fluka) diluted in 10 ml of ethanol was added drop wise after 12 h of stirring. The sol was filtered; freeze dry and calcinated at 600 0C for 1 h. The same procedure was repeated for addition of 10ml and 20ml TEOS. The percentage of silica in each 5 ml, 10 ml, 20 ml addition of TEOS were found to be 11%, 21%, 35 % respectively and were labeled as HA-11SiO2, HA-21SiO2 and HA-35SiO2.
     Nano-HA–silica was mixed with a commercial GIC (Fuji IX GP, GC International Japan) at various percentages (by wt): 1%, 3%, 5%, 7%, 9%, 15%, and 20% of each respectively and were mixed according to the manufacturer instructions.  Cements were covered with moisten gauze after completion of initial reaction and left undisturbed for 24 hours to enable complete setting reaction. Three specimens were made for each percentage of material.
Results and Discussion
      SEM characterization revealed that the morphology of HA-silica nano composite was a mixture of spherical silica particles embedded within elongated HA.  Silica particles not only fill the void between the elongated shaped of HA particles, but they also occupy the empty spaces between the glass ionomer glass particles and act as a reinforcing material in the composition of the GIC.
    The new Sol-Gel method provides a simple route for synthesis of HA-silica nanocomposites powder.  Higher the content of nanosilica, resulted in denser cement and produced a stronger GIC. Application of HA-silica-GIC with improved hardness property might lead to extended clinical indications, especially in stress bearing areas.

This study was supported by Universiti Sains Malaysia research grant.

Fig. 1: SEM images showing different pattern of HA-Silica with different solvents (Ethanol).

Fig. 2: SEM appearance when solvent is Ethanol (A and B).

Fig. 3: SEM appearance when solvent is Methanol (C).

Fig. 4: SEM appearance when solvent is Methanol (D).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-1759 Nanoparticles made of amphiphilic biotransesterified cyclodextrins: ultrastructure and thermal behavior

Putaux J. L.1, Lancelon-Pin C.1, Choisnard L.2, Gèze A.2, Levilly D.2, Charrat C.2, Rochas C.1, Nishiyama Y.1, Jean B.1, Wouessidjewe D.2
1CERMAV, UPR CNRS 5301, ICMG FR 2607, BP 53, F-38041 Grenoble Cedex 9, France, 2Département de Pharmacochimie Moléculaire, UMR CNRS 5063, ICMG FR 2607, UFR de Pharmacie, Université de Grenoble 1, BP 53, F-38041 Grenoble Cedex 9, France
christine.lancelon.pin@cermav.cnrs.fr

One major challenge of nanomedicine is to design nanocarriers that deliver active compounds to a target site, at a sufficient concentration and without premature degradation, in order to maximize the efficiency of the substance while limiting secondary effects. In this context, we have developed colloidal nanovectors based on cyclodextrin (CD) amphiphilic derivatives. βCDs were acylated on their secondary face using thermolysin to catalyze the transesterification. After dissolution in acetone, a series of βCD-Cn (n = 6 to 14) derivatives were nanoprecipitated in water [Gèze et al., Mater. Sci. Eng. C29 (2009), 458]. The resulting nanoparticles were observed by transmission electron microscopy (TEM) after negative staining and by cryo-TEM. Small-angle X-ray scattering (SAXS) patterns were collected from concentrated suspensions at the BM02 beamline at ESRF (Grenoble, France). The thermal evolution of the systems was monitored by recording SAXS patterns of suspensions in sealed glass tubes every 10°C from 25 to 130°C. After cooling, the suspensions were observed by cryo-TEM as well.
The SAXS patterns of freshly prepared suspensions revealed periodic structures in the particles when the grafted alkyl chains contained at least 8 carbon atoms. In most cases, 3 to 5 diffraction rings were observed whose distribution was consistent with a hexagonal structure when the degree of substitution (DS) of the parent derivative was higher than 5 (Figs. 1a,d). βCD-Cn (n = 8, 10 and 12) particles had a barrel-like morphology, exhibiting two different sets of longitudinal lattice fringes depending on their orientation in the embedding ice film (Fig. 1b). For the smallest particles, axial projections of the hexagonal lattice were sometimes observed. βCD-C14 particles had tortuous shapes and a multidomain structure. Lattice images showed longitudinal and axial projections of the hexagonal structure (Fig. 1e). The particles obtained from βCD-C10 (Figs. 1g,h) and βCD-C14 derivatives with a DS lower than 5 were spherical, exhibiting a multilamellar structure with concentric bilayers of amphiphilic CDs.
Upon heating to 130°C, the repeating distance of the multilamellar systems slightly increased but no structural transition was observed (Fig. 1i). The hexagonal structure of the βCD-C8 system disappeared at 95°C, a lamellar organization forming upon cooling. Hexagonal-to-hexagonal transitions were detected at 80-100°C in βCD-Cn systems with n = 10, 12 and 14. Upon cooling, βCD-C10 particles were converted to multilamellar nanospheres (Fig. 1c). βCD-C12 particles became spherical too but no clear structure was recognized. βCD-C14 particles exhibited a bulkier prismatic morphology and were constituted of hexagonally-packed hollow hoops (Fig. 1f).

The authors gratefully acknowledge funding from Agence Nationale de la Recherche, ESRF and Institut de Chimie Moléculaire de Grenoble.

Fig. 1: SAXS patterns (a,d,g) and cryo-TEM images of nanoparticles prepared from βCD amphiphilic derivatives: βCD-C10 with DS 7 (a-c), βCD-C14 with DS 8 (d-f) and βCD-C10 with DS 4 (g-i). a, b, d, e, g and h correspond to the systems nanoprecipitated at room temperature while in c, f and i, the suspensions have been heated at 130°C.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-2080 On the trail of strong iron enrichment in hard dental tissues

Srot V.1, Salzberger U.1, Bussmann B.1, Pokorny B.2,3, Jelenko I.2, van Aken P. A.1
1Stuttgart Center for Electron Microscopy, Max Planck Institute for Intelligent Systems, Stuttgart, Germany, 2ERICo Velenje, Ecological Research and Industrial Cooperation, Velenje, Slovenia, 3Environmental Protection College, Velenje, Slovenia
srot@is.mpg.de

A great diversity of biominerals formed inside the living organisms show a high variety of structures and composition. Many of such biominerals are highly complex composite materials possessing excellent physical and mechanical properties [1,2] which cannot be reproduced in laboratory. These exceptional combinations of organic matrix linked together with crystalline or amorphous minerals are formed under conditions of moderate temperature, pressure and pH. Their unconventional architectures show advanced materials properties compared to their mineralogical varieties [2,3]. Rodents possess opposing long pairs of continuously growing incisors that are worn down by gnawing. The front surface of the incisors is enamel, which is the hardest tissue of the body, consisting of 96 wt% of inorganic material. The innermost part is softer dentine that forms the bulk of the teeth [4]. The surface of the incisors of several rodent species is identified with the presence of iron [5].

In our study, upper and lower incisors of the feral coypu (Myocastor coypus Molina) were investigated. The microstructure and chemical composition of the enamel and dentine were studied in detail by using energy-dispersive X-ray spectroscopy (EDX) and electron energy-loss spectroscopy (EELS) combined with scanning transmission electron microscopy (STEM) at high spatial and high energy resolution. Our investigations uncovered the layer with a variable thickness present on the outer surface of the incisors which has not been reported before in rodent teeth. An annular dark field (ADF)-STEM image of a cross-sectional view showing the interface between the Fe-rich surface layer (Fe-SL) and Fe-rich enamel (Fe-E) is presented in Figure 1. O-K energy-loss near-edge structures (ELNES) measured from Fe-SL and from Fe-E show distinctly different shape, peak number and peak positions (Figure 2). On the other side the Fe-L2,3 ELNES (not shown here) measured in Fe-SL and Fe-E appears similar where Fe is in predominantly 3+ valence state. Considering our EDX measurements (Figure 3) the amount of Fe in the Fe-SL is considerably higher compared to the concentration values reported by now.

The function of iron in hard dental tissues is still not adequately understood. Present research will enlighten the understanding of Fe incorporation in the enamel at the nanoscale level.

References:

[1] UGK Wegst and MF Ashby, Philos Mag 84 (2004), 2167.

[2] AP Jackson and JFV Vincent, J Mater Sci 25 (1990), 3173.

[3] PUP Gilbert et al., Rev Mineral Geochem 59 (2005), 157.

[4] BA Niemec in “Small animal dental, oral & maxillofacial disease” (2010), Manson Publishing Ltd, London.

[5] EV Pindborg JJ Pindborg and CM Plum, Acta Pharmacol 2 (1946), 294.

The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under grant agreement n°312483 (ESTEEM2).

Fig. 1: ADF-STEM image of the interface between Fe-rich surface layer (Fe-SL) and Fe rich enamel (Fe-E).

Fig. 2: O-K ELNES measured from Fe-SL and Fe-E showing distinct difference in the edge fine structures.

Fig. 3: EDX spectra acquired from Fe-E and Fe-SL. The amount of Fe in Fe-SL is much higher compared to the amount measured in Fe-E. In addition, minor amounts of Na, Mg and K were detected in Fe-SL.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-2109 STRUCTURAL TEM STUDY OF THE HYDROXYAPATITE SINTHESIZED BY POLYMERIC PRECURSORS METHOD

Vargas-Becerril N.1, Téllez-Jurado L.2, Rodríguez-Lorenzo L. M.3, Reyes-Gasga J.4, Álvarez-Pérez M. A.1
1Laboratorio de Bioingeniería de Tejidos; División de Estudios de Posgrado e Investigación de la Facultad de Odontología, UNAM. Circuito Exterior s/n. Cd. Universitaria, 04510 Coyoacán México D. F., México., 2Departamento de Metalúrgía y Materiales, E.SI.Q.I.E. - I. P. N., México D. F., México. , 3Nanomateriales Poliméricos y Biomateriales, ICTP-CSIC, Madrid, España. , 4Instituto de Física, UNAM. Circuito de la Investigación Científica s/n. Cd. Universitaria, 04510 Coyoacán México D. F., México.
nancyvb09@gmail.com

In this work we present the structural TEM characterization of the hydroxyapatite (HAP) synthesized by the Pichini method. The Pichini method is a polymeric precursor method which utilizes the ability of somes α-hydrocarboxylic acids to produce chelates with metallic ions. The chelates can undergo polyesterification when heated with a polyhydroxy alcohol [1,2]. In this work the ethylene-glycol and the citric acid were used as polymeric precursors, whereas the calcium hydroxide and the phosphoric acid were used as precursors of HAP.
To obtain the polymeric matrix (polyester resins), the pH was set 8 at 140ºC by 2 h with stirring. Then the polyester resin was heat treated from 200 to 600 °C in order to obtain the HAP powders. HAP powders were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM) thermogravimetric analysis (TGA), infrared spectroscopy (FTIR) and X-ray powder diffraction (XRD).
SEM and TGA results indicated the polymeric matrix decomposition leads to formation of the HAP (figure 1). In TEM, the selected area electron diffraction (SAED) patterns of the powder obtained at 600 ºC display the HAP characteristic planes (211), (210) and (002) (figure 2).
The TEM results showed a decrement of the a-axis and a increment of the c-axis of the HAP unit cell parameters. This indicates the PO43+ ion substitution by the CO32- ion in the structure of the HAP. Also the FTIR results suggested that the obtained HAP was B-type in a carbonated HAP. This last result was also supported by the XRD analysis.
HAP powders presented nanometric and irregular grains. Particle size distribution, measured from the TEM dark field images, indicated the crystal size between 10 to 25 nm (figure 3).
References
[1] M. P. Pechini, “Method of preparing lead and alkaline earth titanates and niobates and coating method using the same to form a capacitor”, 3,330,697 Patented. July 11, 1967.
[2] C. L. Chu, p. H. Lin, y. S. Dong, d. Y. Guo, “Influences of citric acid as a chelating reagent on the characteristics of nanophase hydroxyapatite powders fabricated by a sol-gel method”. J. Materials Science Lett. 21 (2002) 1793-1795.

Authors want to thank to UNAM-DGAPA for postdoctoral scholarship supporting to NVB during the course of this study and P. Mexia and S. Tehuacanero for technical help. This research was financially supported by funds from CONACyT, ESIQIE-IPN, DGAPA-UNAM (project IN106713) and ICTP-CSIC.

Fig. 1: SEM image of the HAP obtained powder by the Pichini method.

Fig. 2: TEM dark field image, HRTEM image and SAED pattern of the obtained HAP powder.

Fig. 3: TEM dark field image and the particle size distribution. Note that particles size is between 10 to 25 nm.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-2113 Structure and Biomechanics of Phragmites Australis used for Japanese Double Reed Wind Instrument “Hichiriki”

Nobuchi T.1, Nakafushi Y.2, Nose M.3, Kawasaki M.4, Shiojiri M.5
1Professor Emeritus of Kyoto Univ., 1-68 Kunobe, Shiga 520-2353, Japan, 2School of Sci. & Engin., Univ. of Toyama, Toyama 930-8555, Japan, 3Faculty of Art & Design, Univ. of Toyama, Toyama 933-8588, Japan, 4JEOL USA Inc., 11 Dearborn Road, Peabody, MA 01960, USA, 5 Professor Emeritus of Kyoto Inst. of Tech., 1-297 Wakiyama, Kyoto 618-0091 Japan.
nose@tad.u-toyama.ac.jp

Hichiriki is a traditional Japanese double-reed wind instrument (Fig. 1), which has been used in Japanese Gagaku, ancient imperial court music, since the 7th century. The best reeds for hichiriki are made of stems of P. australis that are harvested only from a limited reed bed of Udono in the Yodo River near Kyoto. This resembles that the best reeds for clarinet, oboe and bassoon are produced from stems of Arundo donax L. grown only in a few areas of the Var in France.
We examined the structure of stems of P. australis which were sampled as desirable materials of hichiriki reeds from reed beds along different rivers. Specimens for light microscopy were prepared by cutting the sample pieces to transverse sections about 30 μm thick with a sliding microtome, followed by double staining with safranin and fast green FCF. The hardness and Young’s modulus of different parts on the cross-sectioned surfaces of four hichiriki reeds were measured with a Vickers indentation load of 5 mN using a nanoindentation system (Fischerscope, H100C-XYp). The cross section of the sample was polished using wet sheets of finer grades of sandpaper. Fig. 1c shows the section of the stem from Udono. P. australis almost resembles Arundo donax L in plant anatomical structure but not in outer shape and size of cells. This is the reason why Arundo donax L. cannot be used for hichiriki reeds. Fig. 2 shows the structure of the samples from different reed beds. T is the wall thickness measured from the image. Sample #5 (Udono), to be the best material for hichiriki reed, has a structure where harder materials such as epidermi, hypodermis, and vascular bundle sheath with thick cell walls and softer materials such as parenchyma cells are orderly intermingled, and satisfied the shape condition of an outer diameter of about 11 mm and a wall thickness of about 1 mm. In Fig. 3 and Table 1, the hardness measurements of two hichiriki reeds are illustrated. The reed shown in Fig. 1b, assessed as excellent in musical performance, made from Udono stem is much harder over all the parts than a reed made from a stem sampled from Kitakami River reed bed.

We thank Ms. Hitomi Nakamura, the Reigakusha Gagaku Ensemble, for providing samples and valuable comments from a view of musical performance.

Fig. 1: (a) Japanese double-reed wind instrument ‘Hichiriki’. (b) Hichiriki’s reed (#R1). (c) Light micrograph of Udono’s P. australis. Ep: epidermis. Hp: hypodermis. Vb: vascular bundle. Bs: vascular bundle sheath. Cor: cortex. Pa: parenchyma cell. Ct: Cells with thin cell wall. Sc: Sclerenchymatous cells. Pc: pith cavity.

Fig. 2: Light micrographs of P. australis stems from different reed beds. Kitakami Riv. near Sendai, Watarase Riv. near Nikko, Turumi Riv. in Tokyo, Uji Rev. in Kyoto.

Fig. 3: Crosssections of Udono reed (#R1) (a) and Kitakami reed (#R4) (b). Table 1 Indentation hardness HIT and Young’s modulus EIT of different parts shown in (a) and (b).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-2241 BIOCOMPATIBILITY OF A COMBINED SCAFFOLD WITH POLY (VINYL ALCOHOL), POLYURETHANE AND HYDROXYAPATITE

Rodrigues A. D.1, Andrade S. C.2, Bastos G. T.3, Dias C. B.4
1Instituto Evandro Chagas, Laboratório de Microscopia Eletrônica, Belém, Pará - Brazil, 2Instituto Federal do Pará, Belém, PA – Brazil., 3Universidade Federal do Pará, Laboratório de Neuroinflamação, Belém, Pará - Brazil, 4Universidade Federal do Pará, Faculdade de Engenharia Mecânica, Belém, Pará - Brazil
apdrodrigues@gmail.com

Numerous efforts are going to develop functional scaffold to regeneration and bone repair application. Scaffolds that combine the bioactivity of the hydroxyapatite (HA) and the adjustable degradability of the polyurethane (PU) matrix obtained from the Poly(vinyl alcohol) (PVAl) were developed in this research and submitted to morphological and biological characterizations. This new kind of scaffold is non toxic, has interconnected pores and microporous at the wall, great mechanical resistance and great cellular growing activation. These properties are essential to the clinical use requirements. In vitro and in vivo assays were performed to determined biocompatibility of PVAl-PU/HA scaffolds and analyze by scanning electron microscopy (SEM). Firstly, for in vitro assay, NIH3T3 cells were cultured for 7 days under scaffolds. A dense and continuous layer of fibroblasts under and inside of macroporous and microporous of scaffold were observed (Figure 1B and 1C – arrows). Cells covered by spreading themselves most of the outer surfaces of scaffold, due to the presence of lamellipodia and filopodia (Figure 1D – arrowheads). The PVAl-PU/HA promoted a great template for cell migration and spreading in vitro. For in vivo assays, PVAl-PU/HA discs were implanted in the subcutaneous tissue of Wistar rats for 24 hours, 7 and 14 days. After 24 hours of insertion (Figure 2), the biomaterial PVA1-PU/HA presented adherent cells, probably fibroblast, spreading forming compact and homogeneous cellular layer. Groups with PVA1-PU/HA inserted for 7 days, evidenced that cell adhesion colonization and infiltration were strongly affected by macro-architecture of the scaffold pore structure (Figure 3B-C). It was also observed an intense interlaced fibrous network formation inside of macroporous (Figure 3D). Finally, the implants maintained for 14 days presented fibrous capsule surround and inside of scaffold (Figure 4A and 4C). The scaffolds interact directly with tissue compounds growing through the scaffold's interconnected pores, without causing any inflammatory or rejection process (Figure 4B and 4D). Therefore, the PVA1-PU/HA scaffolds improving cell adhesion, showing greater biocompatibility and indicating promising expectations to osseous implants.

CAPES, CNPq, Ministério da Saúde-MS

Fig. 1: SEM of NIH3T3 cells cultured for 7 days in PVAl-PU/HA scaffold. A. Scaffold general view. B. Scaffold general view with NIH3T3 cells. C. Macroporous details showing tightly attachment of NIH3T3 cells with dense and continuous layer formation (asterisk). D. Details of cells spreading themselves outer surfaces of scaffold.

Fig. 2: SEM of the PVAl-PU/HA scaffolds into subcutaneous space for 24 hours. A. Scaffold general view. B. Details from inset in A. Observe cell migration and spreading (thin arrows) and early layers formation (arrows) in the scaffold. C. Cells suggestive of fibroblasts recovery all the scaffold surface.

Fig. 3: SEM of the PVAl-PU/HA scaffolds into subcutaneous space for 7 days. A. Scaffold general view. B. Details from 1A. Cells suggestive of fibroblast with extensive spreading ability. C. Details from 2A. Cells with high spreading ability (arrow). D. Details from 3A. Structures like collagen fibers. IN: internal area; * external area; C: fibrous material

Fig. 4: SEM of the PVAl-PU/HA scaffolds into subcutaneous space for 14 days. A. Scaffold general view. B. Details from A. Prolonged structures like collagen fibers. C. Scaffold general view. Innermost conection with tissue. D. Details from C. Invasion process of fibrous inner into scaffold. C: fibers; BM: biomaterial; CL: fibrous layer; CC: corneous layer.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-2243 IN VITRO AND IN VIVO BIOCOMPATIBILITY OF PHEMA-PLLA SCAFFOLDS

Rodrigues A. D.1, Dias D. C.2, Passos M. F.3, Dias C. B.4, Bastos G. T.3, Maciel Filho R.3
1Instituto Evandro Chagas, Laboratório de Microscopia Eletrônica, Belém, Pará - Brazil, 2Universidade Federal do Pará, Laboratório de Neuroinflamação, Belém, Pará - Brazil, 3Universidade Estadual de Campinas, Faculdade de Engenharia Química, LOPCA/LDPS, Campinas, SP – Brazil, 4Universidade Federal do Pará, Faculdade de Engenharia Mecânica, Belém, Pará – Brazil
apdrodrigues@gmail.com

Polymers development as biomaterial has a prominent position in medical implants, pharmacological and biotechnology. Poly 2-hydroxy ethyl methacrylate (PHEMA) and poly lactic acid (PLLA) represent two polymeric materials with well known biocompatibility. However, the combination of PHEMA and polyester generally has not been widespread in literature, especially for medical applications. In order to increase the hydrophilicity of PLLA and improve the cell adhesion to PHEMA, in vitro and in vivo biological assays were performed to analyze the biocompatibility of this combination (PHEMA-PLLA scaffold). Firstly, for in vitro assay, human fibroblast cell (MRC-5) was cultivated as 3D-culture for 7 days. MTT assay were performed to determine cell proliferation and citotoxicity and was observed 99% of cell viability. The material also, promoted a proliferation in its surface. Adhesion and cell morphology of MRC-5 cultivated under PHEMA-PLLA disks for 72h was analyzed by scanning electron microscopy. The cells were shown a tightly attachment to the PHEMA-PLLA scaffold after 72 hours. For in vivo biocompatibility assay, PHEMA-PLLA disks were implanted in the subcutaneous tissue of Swiss mice for 4 days. The PHEMA-PLLA scaffold the scaffold promoted a great template for cell migration and spreading in vivo and also induced early layers formation. A fibrous capsule with a large number of cells suggestive of fibroblasts was surrounding of PHEMA-PLLA discs. The results suggest that the PHEMA-PLA biomaterials did not promote cytotoxicity activity in vitro and did not promote an inflammatory processes in vivo, improving cell adhesion and showing greater biocompatibility.

CAPES, CNPq, Ministério da Saúde-MS, FAPESP

Fig. 1: MRC-5 fibroblasts viability by MTT assay.

Fig. 2: Scanning electron microscopy of MRC-5 fibroblasts upon PHEMA-PLLA after 72 hours. A. Broad view of PHEMA-PLLA disc without fibroblasts cells. B. Broad view of half part of PHEMA-PLLA disc with MRC-5 for 72h. C. High magnification of B, showing fibroblasts cells overspread and well attached to PHEMA-PLLA scaffold. Bars: A-B: 1.0 mm; C: 100 µm.

Fig. 3: SEM of PHEMA-PLLA scaffolds inserted into mouse subcutaneous space for 4 days. A. PHEMA-PLLA general view. B. High magnification of A. Observe the presence PHEMA-PLLA involved by connective tissue and large number of fibroblasts. C. High magnification of C. Observecell association forming a connective tissue. Bars: A: 1.0 mm; B: 200 µm; C: 40 µm.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-2293 TEM characterization of calcium phosphate particles grown on a polymeric surface

Silva L. C.1, Bertran C. A.1, Gonçalves M. C.1
1Chemistry Institute, UNICAMP, Campinas, Brasil
laucaetano@gmail.com

Calcium phosphates are a class of bioactive bioceramics widely used in the field of biomaterials. There are several known morphologies and crystalline structures for calcium phosphates [1]. These structures, dependent on calcium phosphate synthesis conditions and chemical environment, usually control the solubility and biocompatibility of these bioceramics.
Since the 80´s there is an increasing trend in coating implantable devices with calcium phosphate films in order to enhance its biocompatibility. The effectiveness of this method is recognized, however it is still very challenging to predict the behavior in vivo of these devices, due to the difficulty of characterizing calcium phosphates attached to a surface [2]. Therefore, the aim of this work is to characterize by TEM calcium phosphate particles grown attached to a polymeric surface. The morphologies of the samples were also investigated in a Carl Zeiss Libra 120 transmission electron microscope (120 kV) equipped with an in-column OMEGA energy filter and a Olympus CCD camera 14 bits with 1376 x 1032 resolution.
Poly(ε-caprolactone) (PCL) thin films containing a nucleating agent were prepared by spin coating. The nucleating agent is a molecule that has a negative charge and a nonpolar carbonic chain. These negative charges migrate to the surface in order to act as a nucleation site to calcium phosphate growth, while the carbonic chain acts as a polymer anchor.
Figure 1a shows the bright field image of a semi-crystalline calcium phosphate particle, attached on the PCL film surface, produced by 10 cycles of alternating soaking in calcium and phosphate solutions. Figure 1b shows the diffraction pattern of the calcium phosphate particle. Figure 1c shows the bright field image of another particle and Figure 1d the corresponding diffraction pattern, showing that the latter is much more crystalline. This demonstrates that even at the same synthesis conditions and chemical environment it is possible to obtain different calcium phosphate crystalline structure and morphology.
These particles were also characterized by ESI-TEM (Figure 1e) and EELS (data not shown). Figure 1f shows a magnified bright field image of this crystalline particle, showing it has an amorphous layer surrounding the inner crystalline structure.
This work shows that it is possible to characterize these particles by ESI-TEM in spite of polymer and particle low resistance to beam damage. Determining the particle structures at different growth stages is essential to understand their formation mechanism and also to predict in vivo behavior.
REFERENCES
[1] Vallet-Regi, M., & González-Calbet, J. M. (2004). Prog in Solid State Ch, 32 (1), 1-31.
[2] Surmenev, R. A., Surmeneva, M. A., & Ivanova, A. A. (2014). Acta Biomater, 10 (2), 557-579.

The authors would like to thank FAPESP, CAPES and CNPq for funding and Marcelo Alexandre de Farias and Douglas Soares for technical support.

Fig. 1: Bright field images (a, c and f) of calcium phosphate particles attached to a PCL thin film surface, diffraction patterns (b and d) and calcium map (e) of the same particle showed in (c).
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-2294 Physicochemical characterization by electron microscopy of the hydroxyapatite nanoparticles obtained by co-precipitation in presence of tannic acid.

Santana M M.1, Ramirez A.2, Zorrilla C.3, Reyes J.3, Herrera R.3
11 Posgrado en Ciencia e Ingeniería de Materiales, Instituto de Física, UNAM Circuito Exterior, Ciudad Universitaria, Coyoacán, 04510, México., 22 Unidad de Posgrado de Odontología, UNAM Circuito Exterior, Ciudad Universitaria, Coyoacán, 04510, México., 33 Departamento de Materia Condensada, Instituto de Física UNAM Circuito de la Investigación Científica Ciudad Universitaria, Coyoacán, 04510, México.
cristina@fisica.unam.mx

Numerous are the reports in literature on the synthesis of hydroxyapatite (HAP) highlighting characteristics as particle size and morphology, which can be associated with the complexity of the particular method of preparation thereof [1, 2]. Most of the synthesis reports include reactions which must take place at relatively high temperatures and/or longer heat treatment times.

The synthesis of HAP nanoparticles obtained by the co-precipitation method at room temperature in presence of tannic acid, and varying by two different species of calcium, is reported in this work. The electron microscopy characterization was performed using the electron microscopes SEM JSM5600 and JEOL FEG 2010.

The first synthesis reaction was carried out from (CaCl2•2H2O) 0.1M, (N(C3H7)4OH), 0.1M and (H3PO4) 0.06M using 2.17% tannic acid. The reaction is performed in a round-bottomed flask of three necks maintained in constant reaction stirring of 455 rpm for 30 min. After this, the H3PO4 is added with continuous stirring for two hours. The obtained product was washed 3 times with methanol-distilled water (1:1) to maintain a neutral pH.

The procedure is similar for the second synthesis variation. This was carried out using the reagents (Ca(OH)2) 0.1 M and (H3PO4) 0.06M into a concentration of 1.79% tannic acid.

Structural and chemical analysis by scanning electron microscopy (Figure 1) and transmission electron microscopy (figure 2) confirms the obtaining of the HAP particles in nanometer size, with sizes of 20nm approximately. Figure 3 shows the EDS spectrum from the sample shown in Figure 1.

[1] M.P. Ferraz, F.J. Monteiro, C.M. Manuel, Journal of Applied Biomaterials & Biomechanics 2 (2004) 74-80.

[2] H. Alobeedallah, J. L. Ellis, R. Rohanizadeh, H. Coster and F. Dehghani, Trends Biomater. Artif. Organs 25, (2011) 12-19.

We thank Roberto Hernandez Reyes for technical assistance. We also thank the DGAPA-UNAM for the financial support through the grant PAPIIT IN105112.

Fig. 1: Figure 1 SEM secondary electron image of the HAP grains synthesized by co-precipitation in presence of tannic acid.

Fig. 2: Figure 2 TEM bright field image of the HAP grains synthesized by co-precipitation in presence of tannic acid. Note their nanometric size.

Fig. 3: Figure 3. EDS spectrum of the HAP grains shown in figure 1.

↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-2298 Characterization of the porous coatings with incorporated silver deposited by micro-arc oxidation

Karbowniczek J.1, Cempura G.1, Muhaffel F.2, Cimenoglu H.2, Kayali S.2, Czyrska-Filemonowicz A.1
1AGH University of Science and Technology, Al. A. Mickiewicza 30, 30-059 Kraków, 2Istanbul Technical University, 34469 Istanbul, Turkey
jkarbow@agh.edu.pl

The development of implants over last eighty years includes improvement of known materials as well as designing new ones to increase their mechanical properties, corrosion resistance in body fluids and biocompatibility. Despite of the significant progress still high number of implants failure is connected with bacterial infections. To reduce the risk of microbial infection during surgery different approaches are proposed. One of them is the modification of the biomaterials surface to prevent bacterial attachment and biofilm formation. Another is the incorporation of antimicrobial agents (e.g. silver, NO, antibiotics) into the coating structure to inhibit bacterial proliferation.

This study shows microscopic characterization of the porous coatings with incorporated silver deposited on commercially pure titanium (CP-Ti) and two-phase (a+b) Ti6Al4V alloy by micro-arc oxidation (MAO) process. The process parameters and electrolyte composition were the same for both materials. Solution used for deposition contained: (CH3COO)2Ca·H2O, Na2PO4 and the addition of 0.001 mol/l of CH3COOAg. Microstructure and chemical composition of the coatings were investigated utilizing SEM- and TEM-EDS techniques.

SEM investigation of the samples plain view showed high porosity and surface development of the coatings (Fig. 1a). The complex microstructure, including specific platelets (red circle in Fig. 1b) and nanometer sized particles (yellow circle in Fig. 1b) was visible around the pores. EDS point analysis of different areas of samples surface showed that platelets contained only Ca, P and O while particles area was composed of around 40 at.% of Ag and additionally Ti, Ca, P and O. Detailed TEM investigation, including phase identification by electron diffraction is in progress. The analyses of sample cross-sections revealed the differences in the coatings thickness between both materials. The thickness of the coating formed on CP-Ti was measured as around 12 μm, while that on Ti6Al4V was around 22 μm thick (Fig. 2). Both coatings had the layered structures. Directly at the substrates, titanium dioxide phases were present, while outer layer was composed of calcium phosphates. The Ag distribution within both coatings was determined using EDS mapping. In the case of CP-Ti, Ag was present around the pores in the TiO2 layer, while in the coating formed on the Ti6Al4V alloy it was accumulated at the interface between titanium dioxide and calcium phosphates layers.

Many reports are proving excellent antibacterial activity of silver releasing materials, however in some case also harmful effect on human cells was observed. To verify biological properties of prepared coatings in vitro cytotoxicity tests with cell cultures are in progress.

The study was realized within COST MP1005 (NAMABIO) and OPTYMED project (nr 2013/08/M/ST8/00332) financed by NCN.

Fig. 1: SEM images of the microstructure of the coating (plain view) deposited on CP-Ti, a) general view, b) high magnification view showing specific platelets (red circle) and nanometer sized particles (yellow circle).

Fig. 2: SEM images of the coatings’ cross-sections deposited on: a) CP-Ti substrate, b) Ti6Al4V substrate.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-2493 Determination of AFM surface roughness parameters and topography of contemporary dental nanocomposites after artificial saliva storage

Lainović T.1, Blažić L.1, Vilotić M.2, Kukuruzović D.2, Kakaš D.2
1Faculty of Medicine, School of Dentistry, University of Novi Sad, Novi Sad, Serbia, 2Faculty of Technical Sciences, University of Novi Sad, Novi Sad, Serbia
tijana.lainovic@gmail.com

Smooth surface of a dental restoration is necessary to obtain clinical durability and aesthetic appearance, as well as to prevent discolorations and dental biofilm retention [1]. Therefore, surface of the restoration has to be stable in the oral environment where it is constantly exposed to saliva humidity. Natural saliva is an aqueous hypotonic solution of water, containing inorganic ions, organic constituents and proteins [2]. There are many artificial saliva products, which can serve as a replacement for natural saliva in experiments that simulate oral environment conditions in vitro. The aim of this study was to determine the effect of artificial saliva storage on AFM roughness parameters and topography of dental composites.
Four representative dental resin-based composites were tested in the study: nanofilled and nanohybrid (Filtek Ultimate Body, and Filtek Z550, 3M ESPE), microfilled (Gradia Direct, GC) and microhybrid composite (Filtek Z250, 3M ESPE). Each polymerized specimen was polished according to dental polishing protocol (Shofu). Topography and roughness of each specimen was examined by AFM before and after artificial saliva storage (aqueous solution of Sinclair saliva supporting tablets, in dark containers, at 37 ºC in a water bath, for three weeks).
The AFM images of both testing groups show surfaces with furrowed topography, created by the abrasive wear mechanism of the conducted polishing method. However, the artificial saliva storage did not qualitatively change the topography of the tested materials (Fig. 1). Calculated AFM average roughness parameter (Ra) showed a reduction of roughness after storage in saliva for both nanofilled and nanohybrid composites, as well as for microfilled composite (Fig. 2). Only the microhybrid material showed different results with increased roughness values after the storage. AFM peak-to-valley distance roughness (Rp-v) also decreased for nanohybrid and microfilled material after the medium; for nanofilled the influence of storage solution did not show a significant difference; and again only the microhybrid material had higher roughness values after the saliva storage (Fig. 3). The artificial saliva has not negatively affected surface topography of the tested nanocomposite materials. However, inorganic filler particles can be removed or leached by the artificial saliva medium, leading to positive or negative changes in roughness values, depending on the size of filler particles.

1. Antonson SA et al. Comparison of different finishing/polishing systems on surface roughness and gloss of resin composites. J Dent 2011; 39s:e9-e17.
2. Gibson J et al. Natural and synthetic saliva. Biotechnol Genet Eng Rev 1994;12:39-61.

Supported by Project TR 035020 and Project III-45006 of the Ministry of Education, Republic of Serbia; and 3M (East) AG company and Mikodental – Shofu, Japan for Serbia.

Fig. 1: AFM images of surface topography of the tested materials

Fig. 2: Comparison of AFM average roughness values (Ra) among the tested materials

Fig. 3: Comparison of AFM peak-to-valley roughness values (Rp-v) among the tested materials
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-2921 Novel nanocomposites biosynthesis based on bovine-bone: Electron Microscopy Characterization

Vilchis-Nestor A. R.1, López-Téllez G.1, Sánchez-Mendieta V.1, Becerril-Juárez I. G.2, Jose-Yacamán Y.3
1CIQS, Universidad Autónoma del Estado de México, México, 2Centro de Investigación y de Estudios Avanzados del IPN, México, 3University of Texas at San Antonio, USA
arvilchisn@uaemex.mx

Nature is source of a great number of biomaterials, such as bovine bone, capable of being used as template in the synthesis of biocomposites. Hydroxyapatite, being represented by the formula Ca10(PO4)6(OH)2 [1], is the most ubiquitous compound of the family of calcium phosphates, well-known for being the main compound in bones and teeth structure. Furthermore, bovine bone is a natural, cheap, very resistant and waste material. Even more, it is biodegradable and renewable material. [2] In other hand, mono- and bimetallic Au, Ag, Pt, Pd, Fe and Cu nanostructures can be synthesized by reduction of their corresponding ions assisted by aqueous extract green tea (Camellia sinensis) [3]. Bio-reduction technique involves biomolecules present in green tea extracts for reducing the metallic precursor to obtain different kind of nanostructures, in size, shape and composition. The phenolic compounds and terpenoids are responsible for the formation and stabilization of nanoparticles, thus the concentration of these metabolites can serve as control of the size and shape of the nanostructures formed. This ecofriendly bioreduction method allows the formation in solution and in-situ support of metallic nanostructures on bovine bone and others biomaterials.

In this work, bovine bone is employed as a template to direct the nucleation and growth of mono (Au, Ag, Pt, Fe and Cu) and bi-metallic (Ag-Au and Au-Fe) nanostructures (micro-, submicro- and nanostructures to obtain, thus, the low cost and renewable metal/bovine bone nanocomposite. Novel biocomposites were analyzed by SEM, TEM and STEM (HAADF and BF) techniques. Microscopy studies of metal nanoparticles/bovine bone composite show their average size is under 10 nm for Au, Pt and Ag (Figure 1). Diffuse reflectance UV-Vis spectroscopy (DRS) was used to probe surface plasmon resonance behavior in the biocomposites,

One of the most promising area of application for nanotechnology is that related to environmental sciences, it is well known that the focus is towards green and viable methods that allow the remediation and treatment of wastewater, based on this, this project intends to obtain biocomposites build from metallic nanoparticles supported on bovine bone and use them as catalysts in the reduction of phenolic compounds presents in contaminated water. Bionanocomposites obtained have also promising future applications as catalyst, sensor and medical bone replacements.

References

[1] Kannan S., et al. Chem Mater 2006;18:2181–6.

[2] Becerril-Juárez I. G. et al. Materials Letters 2012;85:157–160

[3] Vilchis-Nestor A. R. et al. Materials Letters 2008;62(17):3103–3105.

Author Vilchis-Nestor thank to CUMex Mobility Program 2012. This work was partially supported by grant from the National Institute on Minority Health and Health Disparities (G12MD007591) from the National Institutes of Health

Fig. 1: Figure 1.- HAADF and BF-STEM images of platinum nanoparticles supported on bovine bone.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-3085 TEM study of the structural transformations of fluorapaite obtained from shark teeth.

Solla E. L.1, Balboa E.2, Coladas P.2, Rodríguez-Valencia C.2, López-Álvarez M.2, Serra J.2, González P.2
1Servicio de Microscopía Electrónica, CACTI, Universidade de Vigo, As Lagoas-Marcosende, 36310 Vigo, Spain. , 2New Materials Group, Applied Physics Department, School of Industrial Engineering, Institute of Biomedical Research of Vigo, University of Vigo, As Lagoas-Marcosende, 36310 Vigo, Spain.
esolla@uvigo.es

The shark teeth present a great similarity in composition to the mammalian teeth and bone tissue, which consists of a mineral phase of calcium-deficient carbonated hydroxyapatite together with fluorapatite, where hydroxil is partially replaced by fluorine [1]. Thus, this fishing by-product has a potential application in the medical field as a bone filler material in orthopedical surgery, enamel regeneration and other dental and maxillofacial treatments.

In the present work, a TEM analysis was performed to study the structural transformations during the fabrication process of these materials to obtain the final bioceramic product. Teeth from two species of shark (Isurus oxyrinchus and Prionace glauca) were boiled in water to remove organic remains from the jaw. Then, the whole natural pieces were subjected to a ball mill (Retsch MM2000) during 5 minutes and with an oscillation frequency between 3-35 Hz to obtain a powder. This powder was pyrolyzed at 950 ºC during 12 hours with a heating ramp of 2 ºC/min and an ending cooling ramp of 20 ºC/min to remove the organic matter.
In order to assess the composition of the starting material, SAED patterns were obtained from the non-pyrolized powder (Fig.1). The analysis of the electron diffraction pattern (Fig.2) allowed for the indexing of the (002), (121) and (1-12) reflections as found in the crystal structure resolved by Hughes et. al. [2], thus confirming the presence of fluorapatite on the shark teeth. However, when the same analysis was conducted on the pyrolized sample (Fig. 3), the SAED diffraction spots could no longer be attributed to fluorapatite but to the formation of whitlockite, as indicated by the indexing of the (223), (0-14) and (131) corresponding to the crystal structure described by Calvo et. al. [3]. It has already been reported that the thermal treatment of biological hydroxyapatites induces the formation of whitlockite [4], that albeit having a different crystal structure, it has also shown excellent biocompatible properties [5].

REFERENCES
[1] J. Enax, O. Prymak, D. Raabeb, M. Epple. J. Struct. Biol. 178 (2012), 290-299.
[2] J.M. Hughes, M. Cameron, K.D. Crowley, Am. Mineral. 74 (1989), 870-876.
[3] C. Calvo, C. Gopal 60 (1975). Am. Mineral. 120-133.
[4] A. Kohutová, P. Honcová, L. Svoboda, P. Bezdička, M. Maříková, J. Therm. Anal. Calorim. 108 (2012), 163–170.
[5] H.L. Jang, K. Jin, J. Lee, Y. Kim, S.H. Nahm, K.S. Hong, K.T. Nam, ACS Nano 8 (2014), 634-641.

FEDER MARMED Atlantic Area Transn. Prog., Xunta de Galicia GRC2013-008, Fundación Mutua Madrileña 2013/14. M. López-Álvarez thanks FP7/ REGPOT-2012–2013.1 n° 316265, BIOCAPS.

Fig. 1: TEM image of a particle of shark tooth powder prior to pyrolization. The circle indicates the area where the SAED aperture was placed.

Fig. 2: SAED pattern showing the reflections indexed as fluorapaite.

Fig. 3: TEM image of a particle of 1000 ºC pirolyzed shark tooth powder. The circle indicates the area where the SAED aperture was placed.

Fig. 4: SAED pattern showing the reflections corresponding to the presence of whitlockite.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-3281 Local chemistry and structure of the human tooth by a low-voltage CS-corrected TEM

Zhang Z. L.1, Dehm G.2
1Erich Schmid Institute of Materials Science, Austrian Academy of Sciences, Austria, 2Max-Planck-Institut für Eisenforschung GmbH, Düsseldorf, Germany
zaoli.zhang@oeaw.ac.at

Dentin is the most abundant mineralized tissue in the human tooth. It is composed of collagen fibrils and nanocrystalline apatite minerals. Recent advances in TEM, i.e spherical aberration techniques and low-voltage capability available with sufficient spatial resolution, allow us to characterize the dentin structure in a more easily way with considerable less damage and a high resolution. These advances definitely help to gain insights into the dentin, and dentin tubule structure and chemistry in tooth, and provide a deeper understanding as the material remains unaltered for quite a long time at a low voltage. The damage energy threshold is supposed to be less than 100 kV [1-2].

In this study, we have used an image-side CS-corrected TEM (TEM/STEM JEOL 2100F), working at 80kV to closely examine the dentin microstructure and atomic structure of a healthy human tooth, and to analyze the effects of electron beam induced damage at different accelerating voltages on the dentin structure. During the experiment, a cooling stage was employed. TEM specimens were carefully prepared by wedge Tripod polishing, and followed by a final low energy ion milling for a few minutes.

The very local chemistry of dentinal tubule (DT), peritubular dentine (PD) and intertubular dentine (ID) have been carefully analyzed (Fig.1). By means of intensive EDX and EELS spectrum-image analysis, it is demonstrated that the local chemical compositions in DT, PD and ID are different, particularly, the Ca/P atomic ratio. It is found that peritubular dentine (PD) shows a relative low the Ca/P atomic ratio as compared to DT and ID. Energy loss near-edge structure analysis reveals that the fine structures of P-L2,3, O-K and low-loss spectra in DT, PD and ID are dissimilar, which are supposed to relate to the change of local chemistry and coordinates. Fig.2 shows one EELS measurement on the intertubular mineral region.

At 80 KV, atomic structure of hydroxyapatite (HA), such as nanocrystalline apatite mineral can be imaged very clearly. It shows a well-defined crystalline structure and reveals additional grain boundaries and interfaces. It is noted that under a certain amount of beam doses the plate-like crystal starts growing into a big crystal within a few minutes (Fig.3 and Fig.4).

Reference

[1] E F Brès et al, Ultramicroscopy 35(1991), p. 305.

[2] A E Porter et al, Biomaterials 26(2005), p. 7650

Gabriele Moser and Herwig Felber are gratefully acknowledged for their help with sample preparation.

Fig. 1: The bright-field image shows the plate-like intertubular mineral structure morphology in the normal dentin and intratubular mineral regions much dark areas at left side. The numerous voids are obvious and distributed among the intertubular structures.

Fig. 2: A ADF image showing a crack starts at DT and normally ends at IDs. The tubules are thus believed to be the sites of microcrack nucleation

Fig. 3: EELS spectrum taken from the intertubular mineral region, where C, Ca and O peak are visible, and corresponding low-loss spectrum inserted.

Fig. 4: A typical HRTEM image acquired from the crystallite in intertubular mineral region after exposed to a certain electron beam doses shows the formation of a CaO –type crystal with multiple twins
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-3473 BaFe12O19 as drug delivery vehicle: A morphological and structural studio

Torres S.1, Bravo A.2, López V. S.1, Contreras M. E.1
1Instituto de Investigaciones Metalúrgicas, UMSNH, Morelia, Michoacán, México, 2Centro Multidisciplinario de Estudios en Biotecnología, UMSNH, Morelia, Michoacán, México
storres_c@hotmail.com

Over the last years, the synthesis and application of biomaterials has become one of the most active and promising areas of research. A broad range of organic and inorganic materials have been explored as biomaterials. Recently, porous magnetic materials are of great interest due to their potential applications such as magnetic resonance imaging [1], magnetic hyperthermia [2] and as drug carriers [3]. Herein we report a morphological ans structural studio on M-type barium ferrite  with hexagonal structure (BaFe12O19). Shape and cristalline structure of the as-synthesized BaFe12O19 were characterized by scanning electron microscopy (SEM, FE-SEM, JEOL JSM-7600), transmission electron microscopy (TEM, Tecnai, FEG Phillips f20), and X-ray difraction (XRD, SIEMENS, D-5000) respectively. BaFe12O19 porous nanostructured materials was synthesized by sol-gel method via spray drying (Mini-Spray Dryer SDL31 Yamato). Chemicals used for the synthesis of BaFe12O19 were iron (III) nitrate nonahydrate (Fe(NO3)3·9H2O), barium carbonate (BaCO3), ammonium hydroxide (NH4OH) and tween-20 as surfactant. All reagent-grade commercial products were used without further purification. Polystyrene (PS) spheres were used as templates in order to obtain the porousness. Characterization of BaFe12O19 magnetic nanoparticles shows that the chemical composition proposed here for the synthesis of BaFe12O19 coukd be used as a good approach in engineering magnetic nanoparticles as drug delivery vehicles owing to their great potential.

keywords: Biomaterials, Drug delivery, Barium hexagonal ferrite

[1] Tina Lam, Phiippe Pouliot, Pramod K. Avti, Frédéric Lesage, Ashok K. Kakkar, "superparamagnetic iorn oxide based nanoprobes for imaging and theranostics", Advances in colloid and Interface Science, 199-200 (2013) 95-113

[2] Caroline de Montferrand, Ling Hu, Irena Milosevic, Vincent Russier, Dominique Bonnin, Laurence Motte, Arnaud Brioude, Yoann Lalatonne, "Iron oxide nanoparticles with sizes, shapes and compositions resulting in different magnetization signatures as potential labels for multiparametric detection", Acta Biomaterialia, 9 (2013) 6150-6157

[3] Il Keun Kwon, Sang Cheon Lee, Bumsoo Han, Kinam Park, "Analysis on the current status of targeted drug delivery to tumors", Journal of Controlled Release, 164 (2012) 108-114 

This project was supported by CONACyT, México

Fig. 1: SEM image of BaFe12O19 showing a spherical shape.

Fig. 2: TEM image of BaFe12O19 showing a spherical shape.

Fig. 3: EDS spectrum showing the characteristics peaks of BaFe12O19.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-3500 Comparative study of loss dental enamel after debonding the braces by Scanning Electron Microscopy (SEM)

Rodríguez J. A.1, Monrroy M.2, Zorrilla C.2, Arenas J. A.2
1División de Estudios de Posgrado e Investigación, FO-UNAM Mexico, 2Departamento de Materia Condensada. IF-UNAM. D.F. México
jarch17@hotmail.com

The clinical procedures when braces fixation suggest adhesion forces between 2.8 and 10.0 MPa as appropriates. In this work the dental enamel edge was evaluated by SEM before and after debonding the braces and measure the bond strength and enamel loss after the brackets debonding with the help of AutoCAD software. 30 bicuspids with prophylaxis were used and observed with SEM, metallic braces (Roth Inovation .022 GAC) were bonded with Transbond Plus SEP 3M Unitek adhesive and Transbond XT 3M resin. The samples were colocated to 37°C during 24 hours and submitted to tangential forces with the Instron Universal machine with speed load sheading 1.0mm/min to obtain the strength resistance when debonding. ARI test was made, the base of the braces and bicuspids were observed. All the SEM images of the braces were processed with AutoCAD program was used to measure area of enamel lost, resin over the bracket base and the metal base free of resin (mm2) over the SEM images. In the shear bond strength test was obtained an average value of 6.8MPa (SD±3.2MPa). The  63.3%  of the samples presented value 1 ARI, the 20% value 2, the 13.3% value 3 and 33% presented value of 0. All those samples with dental enamel lost presented different situations as fractures, steps, horizontal lost, and vertical in some cases, and little lines of scratches. There is no association between the debonding resistance and enamel presence. When the resin area increases is also increasing the debonding resistance.

The authors would like to thanks to Roberto Hernández and Jacqueline Cañetas for their technical assitance. CONACYT Master's and Doctoral Program in Medical Sciences and Dental Health UNAM 

Fig. 1: Presence of enamel prisms Transbond Plus® SEP

Fig. 2: Different situations as fractures, steps, horizontal lost, and vertical in some cases, and little lines of scratches.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-3508 Preparation, characterization and functionalization of cellulose nanoparticles from agave waste and wood pulp.

Ponce C. E.1, Chanona J. J.1, Garibay V.2, Palacios E.2, Terres E.2, Sabo R.3
1Instituto Politécnico Nacional, ENCB, Mexico City, Mexico., 2Instituto Mexicano del Petróleo, Mexico City, Mexico., 3USDA Forest Products Laboratory, Madison, Wisconsin, USA.
ericka1a@gmail.com

Cellulose is being widely studied as nanomaterial due to its reinforcement capacity, strength and thermal expansion. However, metallic nanoparticles are more studied as reinforcers than thus from organic sources [1]. To purify cellulose from agave waste and wood pulp could be the basis to obtain organic nanoparticles that can be functionalized with different compounds to improve their properties. The crystalline properties of cellulose make it stable; however, the amorphous part is the most important for the functionalization due to its free functional groups [2]. The aim of this work is to produce cellulose nanoparticles (CNP) from the purification of agave waste and wood pulp, characterize and functionalize them for possible use as reinforcers. The nanoparticle synthesis was made using dry agave fibers and wood pulp, [2] which were washed in DI water. Then, the fibers were pretreated with a NaOH solution and washed to remove the lignin and hemicelluloses. The pretreated fibers were hydrolyzed with a H2SO4 and HCl solution in an ultrasonic bath. Finally, the functionalization was made by adding Congo red solution (0.01%) and silver nitrate 250mM dropping meanwhile heating. Some images were taken using SEM and TEM; these images were analyzed to obtain morphology, particle size, distribution, crystallinity and atomic distribution. In this work, an image analysis was made, using micrographs taken with SEM, to calculate particle size of the pure CNP, obtaining sizes between 20 ± 12 nm with a quasi-spherical morphology meanwhile, CNPF sizes are between 2-20 nm and more disperse than CNP (Fig.1). Analyzing high resolution TEM images (Fig.2) allowed the observation of the atomic columns, the difference between the arrangements of the crystalline regions and the amorphous regions in a nanoparticle, these results were similar to other reports [3]. Also, some diffraction patterns were taken to measure and obtain some information about the cellulose crystalline structure, which is triclinic (Fig.3). The CNPF have different diffraction patterns due the presence of silver (Ag) and Congo red. Also, the amorphous regions of the CNP disappear on the CNPF TEM images, which indicate a more stable structure. This work demonstrated that it was possible to produce cellulose nanoparticles, and that its amorphous and crystalline regions are useful to functionalize them with silver and Congo red to improve and develop some properties so that they could have a potential use in the reinforcement of films with potential applications in the food industry.

1.Hepworth, D. (2000). Composites. Part A. 31: 283–285. 2.Liitiä, et al. (2003). Cellulose. 10: 307–316. 3.Nishiyama, et al. (2003). Crystal Structure and Hydrogen Bonding System in Cellulose. JACS.

The authors wish to thank CONACyT for the scholarship provided. We want to thank the financial support of the Mexican Government (COFAA, EDI, and SIP project 20130333, 20140387 of IPN and CONACYT)

Fig. 1: A) SEM image with measures of the nanoparticles, notice the size that goes from 20 to 80 nm. B) SEM of CNPF, notice the roundness and the smaller sizes.

Fig. 2: TEM. A) Cellulose nanoparticle micrograph and diffraction pattern on top. B) Silver-cellulose nanoparticle micrograph and diffraction pattern on top. C) Cellulose-silver-Congo red nanoparticle micrograph.

Fig. 3: A) Nanoparticle zooming. B) Diffraction pattern of the nanoparticles. C) Drawing of the triclinic diffraction pattern. D) Measuring of the angles and shapes.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-5877 Electron Microscopy: A helpful tool in the development of original soft materials based on organogels

FRANCESCHI S.1, PEREZ E.1
1Laboratoire des I.M.R.C.P. UMR5623 CNRS, Université Paul Sabatier, Toulouse, France
sfrances@chimie.ups-tlse.fr

Organogels are soft materials, which result from the immobilisation of an organic liquid or oil in a three dimensional network by a gelator. The organogels in this study were obtained from an organic liquid and a low molecular mass organic gelator (LMOG). In solution, an organogelator as 12-hydroxystearic acid (HSA) self assemble by non-covalent interactions and form fibrous structures responsible for the gelation phenomena. We elaborated original soft materials from organogels, and at all the development steps, electron microscopy (SEM, LV-SEM and TEM) have been used to characterize and optimize these materials.

Gelation of the oil:
In order to visualize the self-organization of the low molecular-mass organogelator (HSA), SEM observations were carried out (Fig.1a, 1b) and it clearly shows the nanofibrillar organization in a vegetable oil like soybean oil.

Microporous organogels:
This soft material was prepared from HSA as organogelator, and soybean oil as organic liquid. Particulate leaching technique with sugar, salt and powdered sugar templates were used to introduce a controlled porosity inside the organogels. The obtained organogels are used as artificial matrices for cell cultures, or as new adsorbent porous material to collect or remove pollutants. Scanning electron microscopy was intensively used to characterize the morphologies and the porosity of the materials (Fig.1c)

Water dispersion of gelled nanoparticles:
Gelled particles of soybean oil and HSA were obtained by hot emulsification (T > Tgel), with a stabilizing agent (surfactant or polymer), and cooling at room temperature (T < Tgel). The TEM observations of the dispersions show spherical particles with a mean diameter of 200 nm (Fig.1d). The gelled particles are able to encapsulate hydrophobic drugs for a controlled delivery.

All of these results underline the importance of electron microscopy in the characterization and optimization of these original soft materials based on organogels.

References:
L. Lukyanova, R. Castangia, S. Franceschi-Messant, E. Perez, I. Rico-Lattes, ChemSucChem, 1, 6, 514, (2008)
L. Lukyanova, S. Franceschi-Messant, P. Vicendo, E. Perez, I. Rico-Lattes, R. R. Weinkamer, Colloids and Surfaces B. Biointerfaces, 79, 105 (2010)
A. Boudier, P. Kirilov, S. Franceschi-Messant, H. Belkhelfa, E. Hadioui, C. Roques, E. Perez, I. Rico-Lattes, J. of Microencapsulation, 27 (8), 682 (2010)

Fig. 1: Figure 1: Electron microscopy images of organogels. a,b) TEM observations of fibrils and fibers of HSA self-assembled in soybean oil. c) LV-SEM of a microporous organogel obtained with sugar porogen and HSA/soybean oil as gel phase. d) TEM of organogel nanoparticles HSA/soybean oil.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-5993 A Novel Platform for Electrical and Optical Cell Sensing

Oyman G.1, Geyik C.2, Ak M.3, Odaci Demirkol D.2,4, Timur S.2,4, Coskunol H.2,5
1Ege University, Graduate School of Natural and Applied Sciences, Biotechnology Dept, Turkey , 2Ege University, Institute on Drug Abuse, Toxicology and Pharmaceutical Science, İzmir, Turkey, 3Pamukkale University, Faculty of Arts and Science, Chemistry Dept., Denizli, Turkey, 4Ege University, Facılty of Science, Biochemistry Dept., İzmir, Turkey, 5Ege University, Faculty of Medicine, Psychiatry Dept., İzmir, Turkey
gizemoyman@gmail.com

The mechanisms of life and their effects to diseases form the basis of biological researches. Due to the dynamic nature of the biological organizations, monitoring of living cells and effects of drugs and chemicals on mammalian cells has very essential importance. In recent years, lab-on-a chip systems have been introduced to detect for cellular organizations quickly and accurately [1]. The aim of this study is designing biofunctional conductive electrode surfaces which will allow long-term cultivation of mammalian cells for lab-on-a chip systems. However, most of the electrodes (gold, platinum, etc.) are not transparent and thus, not suitable for many light microscopy techniques. ITO-glass surface is most advantageous for light microscopy techniques due to its transparent nature; it is also conductive and can be used for electrochemistry.
Here we showed that 4-(2,5-di(thiophen-2-yl)-1H-pyrol-1-l)benzene meta-amine (RMF) monomer can be electropolimerized on ITO electrode surfaces without effect on the surface transparency. We also modified surfaces with RGD peptide to investigate cell adhesion and proliferation. Morphology of the surfaces was analyzed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Biocompatibility was determined by in vitro tests, the African green monkey kidney cell line (Vero), human keratinocyte cell line (HaCaT) and human neuroblastoma cell line (SH-SY5Y) were cultivated on the biofunctionalized electrode surfaces. Their adhesion, proliferation, spreading and homogeneous distribution on polymers was monitored by fluorescence microscopy. Our results showed that RGD modified electroactive surfaces showed better cell adhesion than non-modified electrode surfaces and conventional polystyrene surfaces. We were able to conduct cell imaging studies up to 72 h on these surfaces with comparing all of the cell lines. We also determined that polymer thickness effects the cell adhesion. The polymers were deposited on the ITO working electrode with scans of 5, 10 and 25 cycles which correspond to 16±2.14, 26±5.14, 31±0.69 nm respectively. The 10 cycle polymer deposited surfaces were the best effective substrates for cell adhesion.
In conclusion, we determined optimum conditions for bio-electronic platforms. Proposed electrode was successfully used for monitoring cell adhesion and viability via microscopic techniques. In future studies, we plan to test cell adhesion and proliferation on this bio modified surfaces by using holographic microscopy in a label-free manner.
References
[1] Primiceri, E., Chiriaco M. S., Rinaldi, R., Maruccio, G., “Cell chips as new tools for cell biology – results, perspectives and opportunities” Lab on a Chip,13, 3789-3802, (2013)

This work was supported by Research Foundation of Ege University. Grant #: 2014-FEN-023 and TUBITAK #:113Z918

Fig. 1: Schematic representation of electrode preparation for cellular imaging platform.
↑ Go to the Table of content ↑
Type of presentation: Poster

ID-13-P-6021 Microscopic study of adipose tissue-derived mesenchymal stem cell adhesion on matrix-mimetic peptides covered surfaces

Kristóf Z.1, Tóth S.2, Tátrai P.3, Szepesi Á.3, Matula Z.3, Mező G.4, Uher F.5, Markó K.6, Német3,7
1 Dept of Plant Anatomy, Eötvös L. University, Budapest, Hungary , 2Ligeti Dental Clinic, Érd, Hungary, 3Research Center for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary, 4Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Budapest, Hungary, 5Stem Cell Laboratory, National Blood Transfusion Service, Budapest, Hungary, 6Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary, 7Creative Cell Ltd, Budapest, Hungary
kristofz@caesar.elte.hu

Background and aims Seeding of dental implants or bone substitutes with mesenchymal stem cells (MSCs) may improve osseointegration and bone regeneration. MSCs are multipotent stem cells capable of differentiating toward osteogenic, adipogenic and condrogenic lineages. Apidose tissue derived MSCs can be easily harvested from donor fat tissue with liposuction. In our model system different surfaces like titanium alloy meshes, plastic and bovine bone substitutes (Bio-Oss) were covered with modified matrix-mimetic oligopeptide motifs, RGD. The arginyl- gliycyl-aspartate (RGD) have been broadly applied for biomimetic functionalization of various coating polymers.
Materials and methods Cell adhesion, and differentiation of Ad-MSCs was followed by immunohistochemical staining of osteogenic progression indicating proteins, GFP expression and calcium deposition staining with alizarin. Adhesion of Ad-MSCs on covered and uncovered surfaces was visualized by scanning electron microscopy.
Results Alizarin and ALP staining were sensitive indicators of osteogenic progression. However titanium alloy and bovine bone substitutes itself exhibit excellent biocompatibility, cell adhesion as well as osteogenic induction was highly increased by functionalization of surfaces with our polypeptide conjugates. The better adhesion of Ad-MSCs to coated surfaces resulted proliferation and differentiation. The stronger attachment of MSCs to coated surfaces were visualized by SEM as well.
Conclusions A reliable system was established for the follow-up of in vitro ossification and testing various scaffold surfaces. The effect of the peptides is promising, and will be further tested in animal models.

↑ Go to the Table of content ↑

Index of authors

* D' Avila H.
A.
ABE S.
ADDAD A.
ADRIEN J.
AKBI M.
AL-Shammari
ALLAIN S.
ALLEGRI P.
ALLOYEAY D.
ALTIN E.
ALTIN S.
AMARA H.
AOUINE M.
ARAB M.
ARSLAN G.
AUTRET-LAMBERT C.
AVCI S.
Aarholt T. M.
Ababei G.
Abakumov A.
Abart R.
Abdeen A. A.
Abdel Rehim R. A.
Abdel-Aziz A. A.
Abdillahi S. M.
Abdo S. B.
Abe E.
Abe Y.
Abelha Mota G. L.
Abellan P.
Abou-Ras D.
Abrahamyan L.
Abrishami V.
Acar M.
Acebedo B.
Acharya S.
Achaval M.
Acikel Elmas M.
Ackermann J.
Ackermann M.
Acosta D.
Acosta M.
Adair-Kirk T.
Adam J.
Adam R.
Adamec L.
Adamo C.
Adams M.
Addad A.
Adikimenakis A.
Adlassnig W.
Aebersold A. B.
Aeimlapa R.
Afonso C. R.
Afzan M Y
Afzan MY
Agari M.
Agero U. B.
Agrawal R. K.
Agredano-Moreno L. T.
Agudelo C. G.
Aguilar Torres A. T.
Aguirre M. H.
Agulló-Rueda F.
Ahishali B.
Ahmad Z. A.
Ahmed F.
Ahmed M. S.
Ahn T.
Ahonen I.
Ai F.
Ai H.
Aichler M.
Aitouchen A.
Aizawa S.
Ajayan P. M.
Ak M.
Akamine H.
Akano T.
Akase Z.
Akash R.
Akashi T.
Akbal C.
Aken P. A.
Akerfelt M.
Akiba E.
Akimoto Y.
Akin D.
Akita T.
Akiyoshi R.
Aktaa J.
AlAfeef A.
Alarcón H.
Alassaad K.
Albers S. V.
Albert S.
Albonetti C.
Albrecht M.
Albrechtová J.
Albu M.
Alekseev A.
Alekseev M. V.
Alem N.
Alexander D. T.
Alexander G. Anisimov
Alexandrov S.
Alexe M.
Aley P.
Alfonso C.
Algara-Siller G.
Algarabel P. A.
Alghamdi R. A.
Algorta G.
Aliaga C. R.
Alican I.
Alivisatos A. P.
Alix K.
Alkaisi A.
Alkan F.
Alknes P.
Allegretti M.
Allen C. S.
Allen L.
Allen L. J.
Allen V.
Alloyeau D.
Almeida D. B.
Almeida L. T.
Almeida M. S.
Almeida T. P.
Alomari M.
Alonas E.
Aloni S.
Alonso J.
Alonso J. M.
Alonso-Murillo C. D.
Altantzis T.
Althumayri K.
Altin E.
Altin S.
Altman M. S.
Altmann F.
Altoe V.
Alves A. P.
Alán L.
Amado J. R.
Amano T.
Amaral K. B.
Amato M.
Amatucci G.
Ambro L.
Amdor G.
Ameloot M.
Ameneiro Prieto O.
Amin-Ahmadi B.
Ammar S.
Amodeo J.
Amos C. D.
Amutkan D.
Anachkov M.
Anada S.
Anamthawat-Jonsson K.
Anantasomboon G.
Anaya J.
Andersen S. J.
Anderson K. L.
Anderson J. R.
Anderson P. A.
Anderson T. J.
Ando T.
Andrade H. R.
Andrade I. F.
Andrade S.
Andrade S. C.
Andreazzi A.
Andrieux A.
Andrzejczuk M.
Angelakeris M.
Angeles-Chavez C.
Angelini A.
Angeloni L.
Anger M.
Angulo B. E.
Ankah G. N.
Annand K. J.
Annunziata M. G.
Anoufa M.
Antoniou N.
Antonoaea R.
Antos M.
Antoš M.
Antreich S.
Antunez-Flores W.
Antúnez-Flores W.
Aoki T.
Aouine M.
Aoyama K.
Aoyama M.
Aoyama T.
Appel C. C.
Apperley M.
Apperley M. H.
Aqua J. N.
Arabasz S.
Arai M.
Arai S.
Arai Y.
Aramaki S.
Aranda-Cstillo M. G.
Araujo L.
Araullo-Peters V. J.
Arbak S.
Arbak S.
Arbiol J.
Arbouet A.
Archanjo B. S.
Arciniegas M. P.
Arda O.
Arellano M.
Arenal R.
Arenas J.
Arenas J. A.
Argun-Kurum G.
Arias D. C.
Arie A.
Ariese F.
Arima T.
Arina V. Ukhina
Arizpe-Zapata J. A.
Arkill K. P.
Armstrong D.
Arnaudas J. I.
Arnold J.
Aronova M. A.
Aronovitch E.
Arpaia R.
Arranz R.
Arredondo M.
Arriortua M. I.
Arroyo Rojas Dasilva Y.
Arslan I.
Artemov V. V.
Arzel B.
Arzola-Rubio A.
Arıcan G.
Asahata T.
Asahina S.
Asano H.
Ascencio A.
Ascencio-Aguirre F. M.
Aschl T.
Aschman N.
Ashokkumar A. E.
Asif S.
Aso R.
Assenmacher W.
Assis A. A.
Assy A.
Astilean S.
Ates M.
Ateş C.
Atienza-Guillén E.
Atsız A.
Attané J. P.
Attias M.
Atıkeren P.
Aubry D.
Audiffred M.
Audoit G
Audoit G.
Augustine T. N.
Aungsuchawan S.
Aurbach D.
Aursand M.
Autthawong T.
Auty M. A.
Auzelle T.
Avalos-Borja M.
Avci S.
Avelino I.
Avril L.
Awadova S.
Awazu K.
Ayache J.
Ayadi A.
Ayday A.
Aydin M. S.
Aydınlar Ilgaz E.
Ayla S.
Azough F.
Azpiroz F.
B.J.
BASTIN P.
BATISTA N. V.
BAUBET B.
BAVENCOFFE M.
BAYRI A.
BAZAN-DIAZ L. S.
BENDAHAN M.
BERNARDINI S.
BLANCHARD N.
BOSMAN M.
BOUCHOU A.
BOUYANIFIF H.
BRACKX E.
BROURI D.
BRÈS E. F.
Baaziz W.
Babchenko O.
Babiker M.
Babonneau D.
Baburin I. A.
Bacakova L.
Bacakova M.
Bach M.
Bachelet R.
Bachmatiuk A.
Backes C.
Badjeck V.
Badri N.
Badro J.
Baek S. H.
Baer B.
Baeumler W.
Baffa A. F.
Bag N.
Baggio-Saitovitch E.
Bagot P.
Bahena Uribe D.
Bahri M.
Bahrig L.
Bai X.
Baiazitova L.
Baik H. S.
Baimpas N.
Baiutti F.
Bajer J.
Baji Z.
Bajčetić M.
Bakardjieva S.
Baken E.
Bakhteeva N. D.
Bakulina A.
Balachandran S.
Balades N.
Balakrishnan G.
Balan A.
Balazsi C.
Balazsi K.
Balboa E.
Balboni R.
Balci H.
Ball A. D.
Ball S. C.
Ballarino A.
Baller J.
Ballif C.
Balog M.
Balos S.
Bals S.
Balzuweit K.
Balázsi K.
Bambach M.
Bamiduro F.
Bammes B. E.
Banciu A.
Banciu D. D.
Bando Y.
Banerjee A.
Banerjee D.
Banerjee R.
Bang C. W.
Bangert U.
Banhart F.
Bansal V.
Bar Sadan M.
Baran V.
Baranov A.
Barbat-Rogeon A.
Barbeito L.
Barbero C. A.
Barbosa M. S.
Barbosa P. F.
Barbosa S.
Barbosa S. E.
Barboza A. P.
Barboza V. A.
Baris A.
Barjon J.
Bark C. W.
Barker D.
Barnard D.
Barnes J P
Barnett M. R.
Baron C.
Baronnet A.
Barraud S.
Barreau N.
Barrero Sanchez J. M.
Barrier N.
Barroo C.
Barroso M. S.
Barrow C. J.
Barrow S. J.
Barski A.
Bartels C.
Barteri M.
Barthel J.
Barthel J.
Barthélémy A.
Barti B.
Bartlett P. N.
Bartolomé F.
Bartolomé J.
Barton B.
Bartova B.
Bartova E.
Bartunek P.
Barus V.
Basak S.
Basile L.
Baslar Z.
Basnar B.
Bassan F.
Bassani S.
Bastia B.
Basto r.
Bastos G. T.
Basu J.
Basyuk E.
Batakliev T.
Batenburg K. J.
Bathula V.
Batista D. G.
Batista D. J.
Batista M. M.
Batista R. J.
Battirola L. C.
Battistelli M.
Battistig G.
Bau H. H.
Bauch T.
Baudin T.
Baudoin J. P.
Bauer A. J.
Bauer B.
Bauer F.
Bauer M.
Baum J.
Baum M. M.
Baumeister W.
Baumgarten M.
Baumgartner Y.
Baun A.
Baus Lončar M.
Bayesteh S.
Bayle-Guillemaud P.
Bayliss P. A.
Bayramoglu N.
Bazioti C.
Başıbüyük C. S.
Beanland R.
Becerril-Juárez I. G.
Beche A.
Bechteler A.
Becker K.
Bedel-Pereira E.
Bednářová M.
Begin D.
Begin-Colin S.
Behrens M.
Behzad A. R.
Bei H.
Beigmohamadi M.
Beitlschmidt D.
Bejtka K.
Bekker S. M.
Beladi H.
Belarre F. J.
Bele M.
Beleggia M.
Belevich I.
Belić D.
Beljakov V.
Belkahla H.
Bell D. C.
Bell G.
Bellet-Amalric E.
Bellido E. P.
Bello U. M.
Bellová J.
Belmonte M.
Belovari T.
Beltowska-Lehman E.
Beltran A. M.
Beltrán A. M.
Bemis J.
Ben Jemaa N.
Benabbas C.
Benada J.
Benada O.
Benaissa M.
Benchimol M.
Benckiser E.
Bender H.
Benecke M.
Benedetti A.
Benesik M.
Beneš M.
Bengoechea-Encabo A.
Benguedouar M.
Benitez D. S.
Benito A.
Benjamin H.
Benke J.
Benner G.
Bennett W.
Benrezzak S.
Bentley J.
Bentzen J. J.
Berbezier I.
Berecz T.
Bergamaschi A.
Bergen M.
Berger C.
Berger D.
Berger J. E.
Bergmann K.
Bergner N.
Bergter E. B.
Berkels B.
Bermanec V.
Bernardi J.
Bernaś T.
Berniak K.
Bernier N.
Bernroider E. W.
Bernthaler T.
Bertagnolli E.
Bertali G.
Bertin F.
Bertin N. I.
Bertini M.
Bertoni G.
Bertram F.
Bertran C. A.
Bertrand E.
Bertrand J. R.
Beránek J.
Bese N S.
Besser M.
Bessho Y.
Bessolov V. N.
Best G.
Best G.
Bester J.
Bester M. J.
Betancourt P.
Bettini J.
Beyazoglu O.
Beyer A.
Beyer V.
Bezdička P.
Beznoussenko G. V.
Bhadare S.
Bhandari D.
Bhardwaj R.
Bharti S. K.
Bhattacharya A.
Bhattacharyya D.
Bhowmick S.
Bi X.
Bi Z.
Bianchini P.
Biazik J.
Bibes M.
Bichoutskaia E.
Bichoutskaja E.
Biedermann K.
Biegalski M. D.
Biela E.
Bietz S.
Biggiogera M.
Bijeesh M. M.
Bijelic N.
Bikiaris D.
Bilhorn R. B.
Bilir A.
Bimberg D.
Binek A.
Binev P.
Bingöl Özakpınar Ö.
Biniek L.
Birch J.
Biren S.
Biren S.
Birk U.
Biró L. P.
Biscarini F.
Bischoff J. L.
Biskup N.
Biskupek J.
Bisseling T.
Bittencourt C.
Bittermann A. G.
Bitzek E.
Bizarro-Nevares P.
Bizzarri R.
Björklund U.
Bladt E.
Blagoderov V.
Blakenship S.
Blanc W.
Blanchard N.
Blanco D.
Blanco E.
Blanco G.
Blanco R.
Bland P.
Blank H.
Blank V. D.
Blasco J.
Blaskov V.
Blat-Yrieix M.
Blavette D.
Blazit J.
Blažić L.
Bliokh K. Y.
Blon T.
Blot X
Blum I.
Blumstengel S.
Bobynko J.
Boca-Farcau S.
Bocek D.
Bocher L.
Bock B.
Boeker C.
Boese M.
Bogicevic C.
Bogomaz D.
Boguslaw O.
Boissier C.
Boivin G.
Bok J.
Bolaños Burgos I. C.
Bolfarini C.
Bolkent S.
Bolognini G.
Bonchio M.
Boncz D.
Bondar A.
Bondarenko V. I.
Bonetti L. V.
Bonfante P.
Bonifacio C. S.
Bonmassari R.
Bonollo F.
Bonomi E.
Boons G.
Booth R. A.
Boothroyd C.
Boothroyd C. B.
Boraldi F.
Borba-Santos L. P.
Bordini B.
Borgatti F.
Borgohain D.
Boris B. Bokhonov
Borisenko K. B.
Borisevich A.
Borisevich A. Y.
Borkovec J.
Bormann R.
Bormans B.
Bornhoefft M.
Bornschlegl M.
Borojevic R.
Borowik Ł.
Borri P.
Borysiuk J.
Botero A.
Botero L.
Botta W. J.
Botta W. J.
Bottega Peripolli S.
Botton G.
Botton G. A.
Bottura L.
Bouchard P. J.
Bouche P.
Bouchear M.
Boucher F.
Bouchou Akbi
Bouet C.
Bougeard J.
Boughorbel F.
Boukhari M.
Boulat L.
Boullay P.
Bourgeois S.
Bourgeois L.
Bourrellier R.
Bouzon Z. L.
Bouřa E.
Bowden D.
Bowen J. R.
Bowen P.
Bowers M. L.
Bowman J. W.
Bowman W. J.
Boyd A.
Boyd R. W.
Boydens E.
Boyer L.
Boyes E. D.
Boykin D. W.
Boyraz C.
Boz İ. B.
Bozbay E.
Bozza P. T.
Bozzano P. B.
Bozzini B.
Bozzuto G.
Bočina I.
Brackx E.
Bradby J. E.
Bradley S. A.
Braet F.
Brajpuriya R. K.
Branowska D.
Brault J.
Braun S.
Bravo A.
Breczewski T.
Breen A. J.
Breitenbach T.
Brescia R.
Bresque M.
Breuer T.
Bridel J. S.
Brinkmann M.
Brisset F.
Brito C.
Broad G. R.
Brodusch N.
Brohan L.
Brorson M.
Brown A.
Brown A. P.
Brown A. S.
Brown E. S.
Brown H. G.
Brown M. R.
Browning N. D.
Browning N. D.
Brož A.
Bruckova L.
Bruggeman K. F.
Brun N.
Brun R.
Brunetti G.
Brunko O.
Brunner K.
Bruno F.
Bruno G.
Brydson R.
Brydson R. M.
Brzakova A.
Bréard Y. o.
Bublíková P.
Bucalá V.
Bucher E.
Budd P.
Budeanu L.
Bueno G.
Bueno G.
Buffat P. A.
Buffière M.
Bugarski B. M.
Bugarski B. M.
Bugnet M.
Buhmann H.
Buhse T.
Buijsse B.
Buiķis I.
Bujnowicz Ł.
Bukara K. M.
Bulan Ö.
Bulou H.
Bulusheva L. G.
Bunker R. D.
Buontempo F.
Buranova Y. S.
Burattini S.
Burda K.
Burdet P.
Burdikova Z.
Burdíková Z.
Burgess J. K.
Burghammer M.
Burgio L.
Burkart I.
Burke M. G.
Burlaka L.
Burnett T. L.
Burns A. R.
Buršík J.
Busch K.
Bussmann B.
Bustamante L. A.
Butz B.
Buys A. V.
Buzás E. I.
Bußmann J.
Bučinská L.
Byrne H. J.
Bárcena G.
Bárcena-González G.
Bárány I.
Béché A.
Bílý T.
Böhm K.
Börner A.
Börrnert F.
Büchele P.
Büchner B.
Bürgler D. E.
Bērziņš J.
C.
CADETE SANTOS AIRES F. J.
CALDERON G.
CALDERÓN G.
CALDERÓN H.
CANHASSI G. S.
CARA D. C.
CARDENAS S.
CAZOTTES S.
CEH M.
CHAMORRO G.
CHANONA J.
COJOCARU C S.
COOLEY W. A.
COURTOIS B.
CUVILLY F.
Caballero-Hernández J.
Cabero M.
Cabie M.
Cabot A.
Cabrera F.
Cabrera H.
Cabrera J.
Cacho-Nerin F.
Caciagli A.
Caciagli P.
Cacucci A.
Cadel E.
Caen J.
Cagnoli C.
Cai R.
Cai R. F.
Cai T.
Cai Z.
Cairney J.
Cairney J. M.
Cal K.
Calcabrini A.
Caldas L. A.
Calderon G.
Calderon H. A.
Calderon J.
Calderón Domínguez G.
Caliskan T.
Callahan C.
Calleja E.
Callisti M.
Calvino J. J.
Calvié E.
Cam E. L.
Cam F. S.
Camacho A.
Camacho M. A.
Camara F.
Camargo P. H.
Camin B.
Campanini M.
Campbell G. H.
Campeanu R. A.
Campos A. P.
Campos A. P.
Campos G.
Camus P. P.
Can O.
Canalias C.
Canavan M.
Candal R.
Candan S.
Cangani C. C.
Cano V.
Cano-Sarabia M.
Canton P.
Cantoni C.
Cantoni M.
Cantu-Valle J.
Cao F.
Cao L. Y.
Cao M.
Caola I.
Capani F.
Caprioli R.
Carasevici E.
Carazo J.
Carballo-Carballo A.
Carbone F.
Cardarelli F.
Cardosa J.
Cardoso F.
Cardoso K. R.
Cariño-Calvo L.
Carlino E.
Carlson G. M.
Carlsson A.
Caroff P.
Caron J.
Carpenter R.
Carpenter R. W.
Carr A.
Carraro M.
Carrascosa J. L.
Carrétéro C.
Carter C. B.
Carter R.
Carvalho J.
Carvalho V. E.
Carvalho W. A.
Casandruc A.
Casanova G.
Casanova G.
Casanove M. J.
Casas- Mejía O.
Casas-Cabanas M.
Casavola M.
Casiraghi C.
Cassette E.
Castaing J.
Castano E.
Castellanos-Gomez A.
Castelló M. E.
Castiella M.
Castilla R.
Castillo L. A.
Castillo-Rodriguez M.
Castro C.
Castro Riglos M. V.
Castro-Longoria E.
Castán-Guerrero C.
Castón J. R.
Casu A.
Catalano M.
Cataldi A.
Catta-Preta C.
Cattin J.
Cattle J. E.
Caumanns T.
Cauqui M. A.
Cavadini S.
Cavaillé J. Y.
Cavalcanti D. P.
Cavalieri F.
Cavallini M.
Cavosie A. J.
Cazaux J.
Ceballos Mendoza A. J.
Cebecauer M.
Cedeño M.
Ceguerra A.
Ceguerra A. V.
Ceh M.
Celichowski G.
Celik A. F.
Celik M.
Celik N.
Celis A.
Cellet T. S.
Cempura G.
Cendelin J.
Cerna R.
Cernohorsky P.
Cerutti L.
Cervantes J.
Cesal J.
Cesar C. L.
Cha E. J.
Chabaud M.
Chacham H.
Chacón Hernandez U. D.
Chae B. G.
Chae B. K.
Chaffron L.
Chagoyen M.
Chairuangsri T.
Chakif M.
Chakravadhanula K. V.
Chamberlain T.
Chamberlain T. W.
Champliaud J.
Chan G.
Chan T.
Chandran N.
Chang W. T.
Chang C. S.
Chang H.
Chang H. C.
Chang H. J.
Chang J.
Chang L.
Chang M. T.
Chang S.
Chang W. T.
Chang Y. C.
Chang Y. L.
Changklungmoa N.
Chanona J.
Chanona J. J.
Chanona Pérez J. J.
Chantree P.
Chaparro O.
Chapuis P. O.
Charaï A.
Chari A.
Charles F.
Charlie Johnson A. T.
Charoenphandhu N.
Charrat C.
Chateigner D.
Chatterjee D.
Chattopadhyay K.
Chavan D. C.
Chayaburakul K.
Chbihi A.
Chelpanov V.
Chemeris A.
Chen F. R.
Chen Y. W.
Chen D. H.
Chen F. J.
Chen F. R.
Chen G.
Chen H.
Chen J.
Chen J. H.
Chen K. F.
Chen L. G.
Chen M W.
Chen M. J.
Chen M. W.
Chen M. Y.
Chen P.
Chen P. Y.
Chen T.
Chen W. L.
Chen X.
Chen X. Q.
Chen Y. S.
Chen Y. T.
Chen Y. Y.
Chenevier B.
Cheng D.
Cheng J.
Cheng S. L.
Cheng X. Y.
Cheng Z.
Cheng Z. Y.
Cheng-Hsuan Chen
Cheniclet C. I.
Chereches G.
Cherezova V.
Cherkashin N.
Chernenkaya A.
Chernev B.
Chernov S.
Chernov S. V.
Chernyavskiy O.
Chevalier J.
Chevalier N.
Cheynet M.
Chiang C. L.
Chiang C. L.
Chiappi M.
Chiarini-Garcia H.
Chicano A.
Chichon F. J.
Chichón F. J.
Chien Y. C.
Chinchilla L. E.
Chinga Carrasco G.
Chiodini S.
Chiodoni A.
Chiou W.
Chiou W. A.
Chiriac H.
Chirico G.
Chirila C.
Chisaka S.
Chisholm M. F.
Chiu K. A.
Chiussi S.
Chlumská Š.
Chmelik R.
Chmelík R.
Choi S. Y.
Choi C. H.
Choi J.
Choi J. H.
Choi J. K.
Choi K.
Choi M.
Choi P. P.
Choi S.
Choi S. Y.
Choisnard L.
Chomsaeng N.
Chorkendorff I.
Chorvat D.
Chotigkrai N.
Chou Y. C.
Choudhary O. P.
Chow F.
Christ A. F.
Christen H. M.
Christen J.
Christiansen S.
Chu M. W.
Chu A.
Chu Y.
Chu Y. H.
Chuang Y. J.
Chudapongse N.
Chundak M.
Chung K.
Chuvilin A.
Chávez-Munguía B.
Chávez-Rosasles R.
Ciancio R.
Cieslar M.
Cieszyński K.
Cigáň M.
Cimenoglu H.
Cintan S.
Ciolino A. E.
Cirera A.
Cisterna B.
Ciston J.
Ciuleanu T.
Cizek P.
Cizek P.
Cizkova D.
Claramunt S.
Clare D. K.
Clark L.
Clemens H.
Cleveland C.
Cleveland J.
Clifton P. H.
Clode P. L.
Clough R. N.
Clément L.
Cmiel V.
Coban I.
Cobden D. H.
Cocho G.
Cockshott W. P.
Coehlo J.
Coelho J.
Coelho Neto P. M.
Cohen H.
Cohen Hyams T.
Cohen L. B.
Cohen S.
Cohen-Bouhacina T.
Cohen-Hyams T.
Coimbrão D. D.
Coladas P.
Colas des Francs G.
Coleman J. N.
Coles B. C.
Coll M.
Colla M. S.
Collaert N.
Collakova J.
Colliex C.
Colligon J.
Collins C. L.
Collins S. M.
Collins-Martínez V.
Collinson L.
Collinson L. M.
Colone M.
Colone M.
Colsmann A.
Colín-Barenque L.
Conesa J. J.
Conesa J. J.
Conrad E. W.
Contreras J.
Contreras M. E.
Cook D.
Cook R.
Cooper D.
Cooper D
Cooper D.
Cordenunsi B. R.
Cordova A.
Corma A.
Cormary B.
Cornet A.
Coronas J.
Correa-Duarte M. A.
Corrina L.
Cortes-Jacome M. A.
Corticelli F.
Cortés-Gil R.
Cosandey F.
Coskunol H.
Cossio P.
Costa J. P.
Costa A.
Costa C. A.
Costa G.
Costa G. B.
Costanzo M.
Costello S.
Cott D. J.
Cottancin E.
Coue M.
Coufalova E.
Coufalová E.
Couillard M.
Cours R.
Courtois-Manara E.
Coutino-Gonzales E.
Couvant T.
Covre da Silva S. F.
Cowin G.
Cowman A. F.
Craven A. J.
Craven A. J.
Crawford K.
Cremer C.
Cremer C.
Crespo-Monteiro N.
Cretoiu S. M.
Cretoiu D.
Cretoiu S. M.
Cretu O.
Cribb B.
Cristiano F.
Cristobal G.
Cropotova J.
Cross B. J.
Croxall S. A.
Crozier P. A.
Crozier A. P.
Crozier P. A.
Cruz H.
Cruz R.
Cruz Viana Neto B.
Cruz-Höfling M. A.
Cruz-Rivera J. J.
Cruz-Silva E.
Cryan J. F.
Csóré A.
Cuellar L. K.
Cuellar N.
Cuenca J.
Cuervo A.
Cuestas C.
Cufino V.
Cui C.
Cui J. Z.
Cumbal L.
Cumbul A.
Cumings J.
Cummings C.
Cunha M.
Cunha M. M.
Cuniberti A.
Curzi D.
Custardoy L.
Cwiklik L.
Cyrklaff M.
Czarnik C.
Czarnik C. M.
Czerwiński A.
Czigány Z.
Czyrska-Filemonowicz A.
CÁRDENAS S.
Céspedes M. V.
Císař P.
Côrte-Real S.
D'Alfonso A. J.
D'Angelo E.
D'Avila H.
D'Este E.
D'Hulst C.
D'Imprima E.
D'addato S.
D.
DAIO T.
DANOIX F.
DAUMONT C.
DE FRUTOS M.
DEMIREL S.
DEVERS E.
DOLDER H.
DONZEL-GARGAND O.
DUBREUIL A. C.
DUGNE O.
Da Silva C. F.
Daban J. R.
Dabrowski J.
Dachraoui W.
Dadour
Dadras M.
Dag I.
Dagan M.
Dagdelen M.
Dagdeviren O.
Dagotto E. R.
Dahint R.
Dahmen T.
Dahmen W.
Dalili N.
Dalla Rosa M.
Dalmaschio C. J.
Dalui A.
Daly L.
Dam B.
Damm C.
Damsgaard C. D.
Daneu N.
Danev R.
Daniel R.
Danielsson K.
Danilina N.
Dankházi Z.
Danylyuk S.
Daraspe J.
Darbal A.
Darmostuk M.
Darzynkiewicz Z.
Das P.
Daudin B.
Daumont C. J.
Dauzou F.
David C. A.
David M. L.
David T.
David V.
Davies A. M.
Davis D. M.
Davis I.
Davoudpour A.
Davydova O. K.
Dawson M.
Daza J.
De Angelis F.
De Backer A.
De Caro R.
De Clercq A.
De Colli M.
De Falco M.
De Graef M.
De Knoop L.
De Leo G.
De Leon-Covian L. M.
De León E C.
De Matteis L.
De Meri M.
De Pietro L. G.
De Souza E. M.
De Souza W.
De Spirito M.
De Trizio L.
De wael A.
Debbage P. L.
Debut A.
Dechkrong P.
Dedja A.
Degenhard S.
Degterev I. A.
Dehlinger A.
Dehm G.
Dehonor M.
Deiana D.
Dekiwadia C.
Del Marro M.
Delacotte C. h.
Delaye V.
Delaye V.
Delehouze A.
Delevoye C.
Delgado L.
Delille D.
Delimitis A.
Della Mea V.
Dellby N.
Dellby Z.
Dellith J.
Delmelle R.
Delpuech N.
Delsim-Hashemi H.
Delsing P.
Demers H.
Demirci S.
Demirci-Tansel C.
Demirci-Tansel C.
Demirel S.
Demko V
Demoli N.
Demont R.
Demurtas D.
Den hertog M. I.
Deneke C. F.
Deng Q. S.
Deng S.
Deniz O.
Dennenwaldt T.
Denneulin T.
Dentel D.
Deretzis I.
Derevianko A. P.
Derivaz M.
Descamps-Mandine A.
Descoins M.
Descrovi E.
Despeisse M.
Despotović S. Z.
Dessapt R.
Detavernier C.
Devadig P.
Devred A.
Dey G. K.
Dhanalakshmi P.
Dhanasekaran V.
Dhar A.
Dhote B. S.
Dhungana D. S.
Di Bella M. A.
Di Luca M.
Di Valerio V.
Dias C. B.
Dias D. C.
Dias F. F.
Diaspro A.
Diaz Barriga E.
Diaz I.
Diaz N. L.
Diaz Perez L. D.
Diederichs A. M.
Diehle P.
Dieker C.
Dierkes R.
Dietrich C.
Dietrich J.
Dijkstra H.
Dijkstra M.
Dijon J.
Dikmen G.
Dilahire S.
Dille J. A.
Dimitrakopulos G. P.
Dina V. Dudina
Ding L.
Ding R. F.
Diniz J. P.
Diniz V.
Diop D. K.
Distaso M.
Dithmar S.
Dittmann C.
Divakar R.
Divinski S. V.
Divitini G.
Dix N.
Djaziri S.
Dkhil B.
Dlouhý A.
Dlouhý I.
Dluhoš J.
Dluzewski P.
Dmitriev R. I.
Dmitrieva T. G.
Do H.
Do Nascimento J. M.
Dobashi T.
Dobbie I. M.
Dobrik G.
Dobrovolskaya M. V.
Dobrucki J.
Dobrucki J. W.
Dodd M.
Dodony E.
Dodt H. U.
Dogru-Abbasoglu S.
Doherty E.
Doherty E. M.
Dohr J.
Dohrmann R.
Dolder M. H.
Dolezelova E.
Dolle C.
Domenichini B.
Dominici F.
Doménech-Carbó A.
Doménech-Carbó M. T.
Dona P.
Donati S.
Dong Z. L.
Donnadieu P.
Donoval D.
Dorfman S.
Dorval N.
Dos Santos Neves J.
Dostal Z.
Dostál Z.
Douglas A. M.
Douhéret O.
Douillard T.
Doupal A.
Downing C.
Downs R.
Dowsett D.
Drahokoupil J.
Dramicanin M.
Drasar P.
Draxl C.
Draz E. I.
Drazic G.
Drašar P.
Dražić G.
Dredge P.
Dreo T.
Drev S.
Driehorst I.
Drillien R.
Drndić M.
Drsticka M.
Druckmüllerová Z.
Drutsa V. L.
Drábek M.
Drštička M.
Du H.
Du H. C.
Du K.
Du Prez F.
Duan B.
Duan C.
Duan S. Y.
Duan X F.
Duan X.
Dubertret B.
Dubois A.
Dubourdieu C.
Dubrovin E. V.
Dubská M.
Ducastelle F.
Ducati C.
Duchamp M.
Dudeck K. J.
Dudák M.
Dugne O.
Duim W.
Duishoev N.
Dujmović I.
Duncan H.
Dunin-Borkowski R. E.
Dunin-Borkowski R. E.
Durieu C.
Durman M.
Duscher G.
Dussi S.
Dusza J.
Dvořák J.
Dwyer C.
Dycus J. H.
Dyson M. A.
Dzijak R.
Däbritz S.
Déniz O.
Díaz G.
Díaz-Amarilla P.
Dörfel I.
Dědic R.
Dědková K.
EKTER KANTEN G.
ELKABLAWY M. A.
EPICIER T.
ERSEN O.
EVEREST D. J.
Ebihara T.
Ebina Y.
Ebner C.
Ebrahim S.
Ecija A.
Ecke M.
Ecker R.
Eckert J.
Eckhardt R.
Eder F. R.
Eder J.
Eder K.
Edgcombe C. J.
Edström A.
Effting A. P.
Eftychis S.
Egami M.
Egeli U.
Egerton R.
Egerton R. F.
Eggebrecht T.
Eggeler G.
Eggeler Y. M.
Eggeman A. S.
Eggert F.
Egido N.
Egoavil R.
Egusa D.
Ehrenberg H.
Ehrke H. U.
Eibinger M.
Eichhorn S.
Eichner A.
Eifert A.
Eils N.
Einsle J.
Ek M.
Ekmekci H.
El Kousseifi M.
El hajraoui K.
El-Gomati M. M.
ElMandouh Z. S.
ElMeleegi H. A.
Elbe H.
Eliasova H.
Eliseev A. A.
Eliyahou Z.
Eliyas A.
Eliášová K.
Eljarrat A.
Ellenberg J.
Elli A. F.
Ellinger A.
Elmers H. J.
Elsnicova B.
Elías A. L.
Emon Tural S.
Emons H.
Emura Y.
Endo T.
Engel B. D.
Engelberg D. L.
Engelhard M. H.
English T. S.
Engstová H.
Entrup M.
Enyashin A. N.
Eom C.
Eom C. B.
Epherre J. F.
Epicier T.
Epishin A.
Epp S. W.
Epperlein H. H.
Erben D.
Erbudak M.
Ercan F.
Ercan F.
Ercius P.
Erdamar A.
Erdamar A. K.
Erdani Kreft M.
Erdman N.
Erdogan A.
Erdoğan A.
Ergin B.
Eric Cekic O.
Eriksson M.
Eriksson F.
Erk P.
Ernestová M.
Erni R.
Ernst W. E.
Erozenci L. A.
Ersen O.
Ersen O.
Erturkoglu S.
Erturkuner S. P.
Erzin Y. Z.
Esaki M.
Escalante-Ramirez B.
Escalona-Arranz J. C.
Escarré J.
Escrig S.
Eser M.
Esfandiarei M.
Eskazan A. E.
Eskelinen E. L.
Esnouf C.
Espinosa-Pesqueira M. E.
Espinosa-Villanueva J.
Esquinca C.
Esrefoglu M.
Esteves A.
Esther U.
Estivill R
Estrade S.
Estrader M.
Estradé S.
Eswara Moorthy S.
Etheridge J.
Eun Kyung Choi
Evans J. E.
Evans K.
Evans K. L.
Evans R.
Evstaf'eva E. N.
Ewels C. P.
Ewels P.
Eychmüller A.
Ezeasor N. D.
Ezersky V.
FARIAS L. S.
FARRUSSENG D.
FERREIRA A. V.
FEUILLARD G.
FLOREA I.
FONSECA R. C.
FOONG M. J.
FORAY G.
FORTINEAU J.
FOURDRINIER L.
FRANCESCHI S.
FU X.
FU Q.
Faber P.
Fabrega C.
Fabretti A.
Fabritius T.
Fabrizi A.
Faccioni-Heuser M. C.
Facey S. J.
Faes A.
Fahey K.
Fahl A.
Fahrenson C.
Falanga A.
Falcieri E.
Falcieri F.
Falcony C.
Falke M.
Fallqvist A.
Falqui A.
Fan H.
Fan Y.
Fan Z.
Fanali C.
Fang J. M.
Fang J. M.
Fania D.
Farcau C.
Farias L. S.
Farias L. S.
Farina M.
Farjami S.
Farle M.
Farokhipoor S.
Fauske V. T.
Fave L. G.
Favero G Franceschetti L Rossini C Stacchiotti A Rodella L F Rizzoni D Rezzani R
Favero G.
Favia P.
Favre-Nicolin V.
Favret E. A.
Fawey M. H.
Fay M. W.
Faye N. R.
Fazzini P. F.
Fecher G. H.
Feder D.
Federici A.
Fedorenko I.
Feierstein C. E.
Feist A.
Fejfar A.
Fekete L.
Felber M.
Felfer P. J.
Felix M.
Felix R.
Felser C.
Feng L.
Feng S.
Feng W.
Feng Z. D.
Fernandes L. S.
Fernandes T. F.
Fernandéz A.
Fernández A.
Fernández A. S.
Fernández J. F.
Fernández J. J.
Fernández J. R.
Fernández M. M.
Fernández-Carrobles M. M.
Fernández-López C.
Ferrarezzi M. F.
Ferreira N. C.
Ferreira F. O.
Ferreira J.
Ferreira P.
Ferreira P. J.
Ferreira S. O.
Ferrer-Miralles N.
Ferri M.
Ferro G.
Ferroni M.
Feser M.
Festy F.
Feuillet G.
Fey V.
Fichtner M.
Fidler J.
Field M. R.
Figueiredo F. C.
Figueiredo e Silva R. C.
Filatov A.
Filimon M.
Filimonenko V.
Filion L.
Filip S.
Filippousi M.
Filonov G. S.
Filova E.
Findlay S. D.
Fink H-W
Fink R.
Fiordaliso E. M.
Fischer E. S.
Fischer N.
Fisher C. A.
Fisslthaler E.
Fitzek H.
Flannigan D. J.
Flechsler J.
Fleck R.
Fleck R. A.
Fleming Y.
Floettmann K.
Florea I.
Florea I.
Flores-Santos L.
Florini N.
Flötenmeyer M.
Fogarassy Z.
Foitová I.
Fojtášek L.
Folci A.
Foldyna M.
Foley M.
Fongalland D.
Fonseca B. B.
Fontana M.
Fontcuberta J.
Foray G.
Fordsmand H.
Formanek P.
Forstova J.
Forte M.
Fortea M.
Fortelný I.
Fortis A. M.
Fortoul T. I.
Foschi F.
Fossard F.
Fouillen A.
Fowlkes J.
Fowlkes J. D.
Frabboni S.
Frade P. R.
Fraefel C.
Fragal V. H.
Fragoso- Soriano R.
Fraiberk M.
Francois-Saint-Cyr H.
Francolini M.
Franconi F.
Frangis N.
Frank L.
Frankel P.
Franklin R.
Franklyn P. J.
Frankowski K.
Franze K.
Fraser H.
Frechero M. A.
Freeman H. M.
Freer R.
Freire-de-Lima C. G.
Freitag B.
Freitas E. F.
Freitas K. M.
Freitas S. C.
Freivalds T.
Frenkel A.
Frenkel A. I.
Friedrich D.
Friedrich H.
Friend R. H.
Fries S. G.
Friesland M.
Frischknecht F.
Fritz S.
Froech J. E.
Frontasyeva M. V.
Frost C.
Frysak J.
Frébourg G.
Fréty N.
Fréty N.
Frómeta M.
Fu X.
Fua P.
Fuchs J.
Fujii H.
Fujii M.
Fujii T.
Fujikane R.
Fujimoto T.
Fujino T.
Fujioka H.
Fujita K.
Fujita N.
Fujita S.
Fujita T.
Fujiyoshi Y.
Fukalová J.
Fukalová J.
Fukuda T.
Fukuda Y.
Fukushima K.
Fukutomi T.
Fukuyama M.
Fulin J.
Fung C.
Funston A. M.
Furtado R. R.
Furukawa H.
Furutsu T.
Furuya M.
Fusil S.
Fuzik T.
G Tizei L. H.
G Tizei L. H.
GANASH M. A.
GAVARRI J. R.
GAY A. S.
GERVAIS F.
GIRLEANU M.
GOHIER A.
GOUNE M.
GULGUN M. A.
GUO L.
Gaal R.
Gaberscek M.
Gabr T. A.
Gabrielli A.
Gaceur M.
Gacevic Z.
Gadelha A. P.
Gaginskaya E.
Gai P. L.
Gai P. L.
Gailhanou M.
Gajović A.
Gal L.
Galceran J.
Galceran M.
Galdiero S.
Galdino de Lima M. P.
Galembeck F.
Galiev G. B.
Galimberti V.
Galindo P.
Galindo P. L.
Galkina S.
Gallais Y.
Gallardo A.
Gallego J.
Galli R.
Galloway S.
Galopin E.
Galvita V.
Gama G. R.
Gamalier J. P.
Gambardella A.
Gambhir S. S.
Gameiro A.
Gammer C.
Gammoudi I.
Gan L.
Gan Y.
Ganapathy S.
Gandolfi D.
Gangopadhyay P.
Ganguly A.
Ganner T.
Gannott F.
Gao Z. W.
Gao P.
Gao Q.
Gao W. P.
Gao X.
Gao Y.
Gao Z.
Gaona D.
Garbaczewska G.
Garbin D.
Garbrecht M.
Garcia Junior A. J.
Garcia L. R.
Garcia M. A.
Garcia R.
Garcia S.
Garcia V.
Garcia-Garcia J.
Garcia-Gutierrez D. F.
Garcia-Gutierrez D. I.
Garcia-Moreno F.
Garcia-Sierra F.
García L.
García L. M.
García-Castelló N.
García-Doval C.
García-Lepetit N.
García-Negrete C. A.
García-Rojo M.
García-Verdugo J. M.
Garfield D.
Gargarella P.
Garibay Febles V.
Garibay V.
Garibay-Febles V.
Garitagoitia Cid A.
Garling J.
Garner A.
Garnier J.
Garnier V.
Garrido B.
Gartnerova v.
Garwood R.
Gaspar A. R.
Gasparini L.
Gass M.
Gastaminza P.
Gatel C.
Gates B. C.
Gatica J. M.
Gattaneo G.
Gatzmann J.
Gatzmann J. G.
Gault B.
Gauquelin N.
Gautier H. O.
Gautreau A.
Gautron E.
Gauvin R.
Gavagnin M.
Gavendová P.
Gay A. S.
Gayral B.
Gazeau F.
Gazquez J.
Gazzadi G. C.
Gačević Ž.
Gec M.
Gehrke H.
Gehrke T.
Geier B.
Geiger D.
Geim A. K.
Geipel C.
Geisse N.
Geitmann A.
Gelb J.
Gelissen A.
Gellert M.
Gelnar M.
Gemeiner P.
Genc E.
Gengenbach U.
Genovese A.
Genre A.
Gentili R.
Genuit W.
Genz F.
Genç A.
Geobaldo F.
Georgakilas A.
George E. P.
Georgiev V.
Gerard M.
Gerganova T.
Gergely-Fülöp E.
Gerisch G.
Gerlach J. W.
Gernert U.
Gerthsen D.
Geurts R.
Gevko P. N.
Geyer S. H.
Geyik C.
Ghaith S. M.
Ghallab A. M.
Ghanbari M.
Ghegin E.
Ghica C.
Gholinia A.
Ghosh C.
Ghosh S.
Gi-Yeop K.
Giacomini C.
Giagnacovo M.
Giammanco F.
Giannazzo F.
Giannopoulos G.
Giannuzzi L. A.
Gibson M.
Gidcumb E.
Giddings A. D.
Gil A.
Gilardi E.
Gilis M.
Gilks D.
Gillet P.
Gimmler C.
Giorgetti C.
Giorgetti E.
Giorgio S.
Giorgis F.
Giovannetti I.
Giraldez l.
Girard C.
Girard-Dias W.
Girgsdies F.
Girleanu M.
Giusca G.
Gkanatsiou A.
Gladkov P.
Glas F.
Glatzel T.
Gloter A.
Gloter A.
Glover L.
Gluch J.
Gnanasekaran K.
Gocmez S. S.
Godec M.
Godet S.
Godinez-Salomon F.
Godinho V.
Goetz J. G.
Gogendeau d.
Gogneau N.
Gohara K.
Gokbuget A. Y.
Goldie K. N.
Golla-Schindler U.
Gollmer A.
Golubeva A. V.
Gomes M. E.
Gomes S.
Gomes S. A.
Gonnissen J.
Gontard L. C.
Gonzalez E.
Gonzalez G.
Gonzalez J.
Gonzalez S.
Gonzalez-Posada F.
Gonzalez-Treviño M. T.
Gonzalez-Villalva A.
González G.
González L.
González P.
González-Calbet J. M.
González-Castro A.
González-García V.
González-Merchante D.
González-Montiel A.
González-Robles A.
González-del Carmen M.
Gonçalves C. S.
Gonçalves F. L.
Gonçalves M. C.
Goode B.
Goodenough J. B.
Goosen W. E.
Gopu A.
Goral T.
Gorantla S.
Gorecki A.
Gorgojo P.
Goris B.
Gorkunov M. V.
Gornostayev S.
Goscinski W. J.
Goto S.
Gould C.
Gourrier A.
Goux L.
Govorcin Bajsic E.
Grab A. L.
Grabher C.
Grafe C.
Graff A.
Graham A. C.
Graham B. S.
Granados-Hernández M. V.
Grandjean N.
Grauby S.
Grauby-Heywang C.
Grauer S.
Grazu V.
Graça L. M.
Greczynski G.
Green J. B.
Green L.
Greene J. E.
Gregg J. M.
Gregori D.
Gregori G.
Gregori M.
Greibe T.
Grenier A
Grey C. P.
Grieb T.
Gries K. I.
Grieten E.
Griffin
Griffin B. J.
Griffiths G.
Grillet N.
Grillo V.
Grimley E. D.
Grinan E.
Grinblat J.
Grobelny J.
Grochocka E.
Groenewald H. B.
Grogan J. M.
Grogger W.
Groma I.
Groom D. J.
Grosche A.
Grosche J.
Grosse C.
Grovenor C.
Grovenor C. R.
Grubhoffer L.
Grundler P. V.
Grushko B.
Grünewald K.
Grym J.
Grzanova P.
Grzelakowski K. P.
Grönbeck H.
Gröning P.
Grünewald K.
Gspan C.
Gu C.
Gu G.
Gu G. H.
Gu H.
Gu L.
Gu M.
Gu X.
Guadarrama L.
Gualda E. J.
Gualtieri A.
Guardia P.
Guastamacchia M.
Gubicza J.
Guck J.
Gudheti M.
Gudrā D.
Guenthert M. M.
Guerra A.
Guerra C.
Guerra C. R.
Guerra F. D.
Guerrero E.
Guerrero M.
Guerrero-Ferreira R. C.
Guerrero-Lebrero M. P.
Guerrero-Vargas J. A.
Guevara P.
Guha D.
Gui J.
Guiblin N.
Guichard P.
Guida L.
Guillermier C.
Guilloy K.
Guimaraes M. H.
Guimarães de Oliveira L.
Guise O.
Gulec A.
Guleken Z.
Guliants V. V.
Gulka M.
Gull K.
Gumbsch P.
Gummalla M.
Gumusel A.
Gunawardana M.
Gunkel M.
Gunnarsson K.
Gunning A. P.
Gunning P. W.
Gunnæs A. E.
Guo Y.
Gurbán S.
Gustafsson S.
Gutiérrez H. R.
Guyomard D.
Guzel E.
Guzel E. E.
Guzman R.
Guzzinati G.
Gysin U.
György B.
Gärtnerová V.
Gèze A.
Gómez D. M.
Gómez M. M.
Gómez R. M.
Gómez-Cadenas A.
Gómez-Cortés J. F.
Götte M.
Göttfert F.
Gülgün M. A.
Güney G.
Gürsoy D.
Güzel E.
HAN X.
HERRERA-BECERRA R.
HING L. H.
HOMBOURGER C.
HUGON A.
HUKIL S.
Haas B.
Haberfehlner G.
Haberl B.
Habermeier H.
Habermeier H. U.
Habibi A.
Habler G.
Habuchi S.
Hada M.
Hadad C.
Hadraba D.
Haehnel V.
Haenen K.
Haeussler D.
Hage F.
Hage F. S.
Hagen C.
Hagen C. W.
Hagen G. M.
Haghi-Ashtiani P.
Hagmann M.
Hahn C.
Hahn H.
Haider M.
Haigh S.
Haigh S. J.
Halabi M.
Hall A. N.
Haloda J.
Halodova P.
Halsall M.
Hamanaka T.
Hambach R.
Hamburger M.
Hammer E. R.
Hamochi M.
Hamon A. L.
Hampl A.
Han
Han X. D.
Han H.
Han K.
Han S. S.
Han W.
Han X. D.
Han Y.
Han Z.
Hanein D.
Hangleiter A.
Haničinec M.
Hanlon S. D.
Hansal W.
Hansen D.
Hansen L. P.
Hansen T. W.
Hanssen E.
Hansson E.
Hantschel T.
Hanusová P.
Hanyu H.
Hapis S.
Hara M.
Hara T.
Harada K.
Harada T.
Haraguchi T.
Harata M.
Harcuba P.
Hardcastle T.
Hards G.
Hardtdegen H.
Hargitai H.
Haring M. T.
Harju L.
Harma V.
Harmand J. C.
Harris J.
Harris P.
Harrison W.
Hart G. W.
Hart J.
Hartel P.
Hartfield C.
Hartig R.
Hartl F. U.
Hartman L.
Hartmann J.
Hartmann N. B.
Hartmann R.
Harumoto T.
Haruta M.
Haruta T.
Harzer T. P.
Hasebe Y.
Hasegawa Y.
Haselbach D.
Hashimoto A.
Hashimoto N.
Hashimoto T.
Haswell R.
Hatemi H.
Hatina J.
Hatzopoulos Z.
Haung T. W.
Hausmann M.
Haušild P.
Havancsák K.
Havelka M.
Havlíček V.
Havrdova M.
Hawes C.
Hawthorne F. C.
Hayashi K.
Hayashi S.
Hayashida M.
Hayashida T.
Hayat A.
Hayun S.
Hazama H.
Hazirah NH
He Q.
He Y.
He Z.
Hebert C.
Hector A. L.
Heczko O.
Hedley G. J.
Heeren R. M.
Hefel B.
Heger D.
Heggen M.
Heggie M.
Heidari H.
Heiligenstein X.
Heilmann M.
Heimerl T.
Heinig K. H.
Heino J.
Heintzmann R.
Heinze K. G.
Hekking L.
Helbert A. L.
Held A.
Helgesen G.
Hell S. W.
Heller R.
Hellested M. D.
Helmich L.
Helveg S.
Henneberger F.
Henrard L.
Henriquez J.
Henry C. R.
Herbert S.
Herbst M.
Herdina A. N.
Hermansson A. M.
Hermes W.
Hernandez H.
Hernandez R.
Hernandez-Casillas L. P.
Hernandez-Ramirez F.
Hernandez-Rivera J. L.
Hernández F.
Hernández Hernández H. M.
Hernández J.
Hernández S.
Hernández-Cristobal O.
Hernández-Garrido J. C.
Hernández-Maldonado D.
Hernández-Mariné M.
Hernández-Velasco J.
Herranz G.
Herre P.
Herrera Becerra R.
Herrera R.
Herrera-Becerra R.
Herrero-Albillos J.
Herrmann J.
Herzer G.
Herziger F.
Herzog D.
Hesser J.
Hessler-Wyser A.
Hetaba W.
Hetherington C.
Heuser J. E.
Heux L.
Hibara A.
Hickey C.
Hickey S. G.
Hida S.
Higashi R.
Higashida K.
Higl J.
Higuchi K.
Hildenbrand G.
Hill K. E.
Hillion F.
Hiltl M.
Hinderhofer K.
Hinderks D.
Hipkaeo W.
Hipólito-García M.
Hiraga K.
Hirahara K.
Hirai Y.
Hirakawa K.
Hirakawa M.
Hiramatsu K.
Hirano K.
Hirano R.
Hirata A.
Hirayama T.
Hirayama Y.
Hirlimann C.
Hirohata Y.
Hirokawa N.
Hiroki K.
Hirosawa S.
Hiroyama T.
Hirsch P. B.
Hirscht J.
Hirvonen L.
Hiscock M.
Hisinuma Y.
Hitchcock A. P.
Hizrri M A
Hjelen J.
Hjorvarsson B.
Hjörvarsson B.
Hladík L.
Hmaid A.
Hnilica F.
Hoang A.
Hobisch J.
Hoch R.
Hodgson P. D.
Hodgson P. D.
Hodnik N.
Hodoroaba V. D.
Hodová I.
Hoelzenbein T.
Hof M.
Hofer F.
Hoffie K.
Hoffmann A. K.
Hoffmann B.
Hoffmann L.
Hoffmann M.
Hoffmann T.
Hofkens J.
Hofmann A.
Hofmann S.
Hofstaedter F.
Hohenester U.
Hohlbach S.
Holl J. M.
Holl P.
Hollricher O.
Holmestad R.
Holsgrove K. M.
Holtzmann K. J.
Holubiec M.
Holzner C.
Holý V.
Hombourger C.
Honda K.
Honda Y.
Hondow N.
Honec P.
Hong J.
Hong K.
Hong P.
Hong S.
Hong W.
Hono K.
Hoogenboom J. P.
Hoque S.
Horibuchi K.
Horita Z.
Hormann K.
Hornikova D.
Hornikova L.
Horodyský P.
Hort N.
Horvitz D.
Horváth Z. E.
Horák M.
Horáček M.
Hosogi N.
Hosokawa F.
Hosono E.
Hostounský Z.
Houben A.
Houben L.
Houdek Z.
Houdellier F.
Hoummada K.
House M.
Houtepen A. J.
Hovnanyan K. O.
Hovnanyan N. L.
Hovorka M.
Howe J.
Howie A.
Hoyle D.
Hozák P.
Hrachovec V.
Hradil D.
Hradilová J.
Hrnčíř T.
Hromadkova J.
Hrubanova K.
Hrubý J.
Hruskova D.
Hsieh C. Y.
Hsieh H. Y.
Hsu W. H.
Hsu C. C.
Hsu W. H.
Hu J.
Hu L.
Hua X.
Huang T. W.
Huang B. M.
Huang C.
Huang C. Y.
Huang H. L.
Huang J.
Huang J. Q.
Huang K. Y.
Huang M. R.
Huang R.
Huang S.
Huang T. W.
Huang W.
Huang W. Q.
Huang X.
Huang Y.
Huang Y. C.
Huang s.
Huber D.
Huber K.
Hubmann J.
Hubálek Kalbáčová M.
Hubálková P.
Huchim E.
Hudec J.
Hudoklin S.
Huellen K.
Huerta A.
Huerta E. F.
Huerta M. J.
Huh U.
Huh Y. H.
Huhn S.
Huisken J.
Huismans H.
Hulicius E.
Hultman L.
Hultman L. G.
Humbel B. M.
Hummel E.
Hummer G.
Humphreys C. J.
Hungria T.
Hungría A. B.
Huo Z.
Hurbain I.
Hussaini Z.
Huth M.
Huttunen S.
Huynh M.
Hwang I. S.
Hwang C. S.
Hwang H. S.
Hwang I.
Hwang I. S.
Hwang J.
Hyde T.
Hyono A.
Hytch M. J.
Hájek M.
Hála J.
Häusler I.
Hébert C.
Hébert S.
Hébert S. y.
Höfle S.
Hübner R.
Hübner S.
Hüe F. l.
Hühne S. M.
Hütten A.
Hývl H.
Hÿtch M.
Hÿtch M. J.
I.
ICHIKAWA T.
ISHII Y.
ISHIKAWA Y.
ISSA I.
Iachetta G.
Iannuzzi D.
Ibarra A.
Ibarra M. R.
Ibarra R.
Ibáñez M.
Ichikawa T.
Idrissi H.
Idrissi H.
Idrobo J.
Idrobo J. C.
Igbokwe C. O.
Iglič A.
Igor S. Batraev
Ihiawakrim D.
Ihiawakrim D.
Ihle S.
Iida S.
Iijima H.
Iijima S.
Iijima Y.
Iizumi Y.
Ikai T.
Ikarashi M.
Ikatura N.
Ikeda A.
Ikeda M.
Ikeda U.
Ikeno S.
Ikeuchi S.
Ikuhara Y.
Ikuhara Y. H.
Ikuta T.
Ilenok S.
Ilha J.
Ilieva L.
Iliopoulos E.
Iliopoulou E. F.
Ilić V. L.
Illig S.
Illés L.
Ilvan S.
Ilvan S.
Ilyas M.
Im J.
Imadate K.
Imaeda N.
Imamura D.
Imani R.
Imbert F.
Imhof A.
Imhoff L.
Imurai S.
Inada H.
Inami T.
Inamura T.
Inan G.
Inan S.
Inayoshi K.
Ince C.
Incesulu A.
Indyka P.
Infante I. C.
Ingham N.
Inoue K.
Inoue Y.
Inui H.
Iraz M.
Irban A.
Iribarnegaray V.
Iriyama Y.
Irsen S.
Isa F.
Isakozawa S.
Isella G.
Ishida T.
Ishiguro T.
Ishii M.
Ishii T.
Ishikawa A.
Ishikawa M.
Ishikawa N.
Ishikawa R.
Ishikawa T.
Ishikawa T. T.
Ishino S.
Ishino Y.
Ishiwata S.
Ishizuka K.
Iskandar M. R.
Iskandar R.
Islam A.
Ismail B.
Ismail M. A.
Isobe S.
Isoda S.
Ispánovity P. D.
Issa I.
Isıldar B.
Itakura M.
Ito H.
Ito T.
Ito Y.
Ivan A. Bataev
Ivanov S.
Ivanov I.
Ivanov Y. P.
Ivanova A. G.
Ivanova M. E.
Ivanovic N.
Ives S.
Ivshina I. B.
Iwahori T.
Iwama T.
Iwan H.
Iwane T.
Iwanicka M.
Iwasaki A.
Iwasawa Y.
Iyison N. B.
Iñiguez J.
Ižák T.
J Kashtiban R.
J P Arul Mozhi Varman
JABER N.
JI G.
Jackson C. A.
Jackson S.
Jackson S. S.
Jacob D.
Jacob D. E.
Jacobsson D.
Jacques P. J.
Jacquet E.
Jacquot J. F.
Jaeger W.
Jafri H.
Jagadish C.
Jager A.
Jain A.
Jain A. K.
Jaiswal P. K.
Jamet M.
Jan V.
Janda P.
Jandieri K.
Jandová D.
Janek J.
Jang H. W.
Jang J.
Janish M. T.
Jankovic A.
Janowska I.
Janoš P.
Janse van Vuuren A.
Jansen J.
Jansen S.
Janssen A.
Janssens S. D.
Jansson A.
Jany B. R.
Janáček J.
Jančar B.
Jara A.
Jardim P. M.
Jarero-Basulto J. J.
Jarić S.
Jarolimek K.
Jaroszewicz J.
Jaroš J.
Jarulertwathana B.
Jasmin A.
Javed Y.
Javon E.
Jazeri H.
Jašek O.
Je A. R.
Jean B.
Jeangros Q.
Jefferson D. A.
Jelen K.
Jelenko I.
Jenei P.
Jeng Y.
Jenikova G.
Jenko D.
Jenko M.
Jensen J.
Jensen L. S.
Jeong Hun Kim
Jeong J.
Jerič M.
Jesbains K.
Jeschke S.
Jeuken L. J.
Jezierska E.
Ježek P.
Jha S. K.
Jho J. Y.
Jia Z. H.
Jia C.
Jia C. L.
Jia Z. H.
Jiang Y.
Jiang Y.
Jiao C.
Jimenez L.
Jimènez-Garcìa L. F.
Jiménez de Haro M. C.
Jiménez-García L. F.
Jiménez-Ramírez J.
Jiménez-Riani M.
Jin C Q.
Jin C.
Jin C. H.
Jin K.
Jin L.
Jin X.
Jing Zhu
Jinnai H.
Jinschek J. R.
Jiruše J.
Jirásek M.
Jirásková Y.
Jiwajinda S.
Jo J.
Jo W.
Jochen M. C.
Joensuu M.
Johann F.
Johnson D. D.
Johnson E.
Jokisaari J. R.
Jokitalo E.
Jokitalo E.
Joly-Pottuz L.
Jomaa M. H.
Jonas A. M.
Jones C. G.
Jones E. Y.
Jones L.
Jones L. B.
Jooss C.
Jordan V.
Jordão E.
Jorge A. M.
Jorge Jr A. M.
Jose Yacaman M.
Jose-Yacamán Y.
Joshi V.
Joti Y.
Jovanovic P.
Joy D. C.
Jozinovic B.
Juchtmans R.
Junesch J.
Jung W.
Jungbauer M.
Jungjohann K. L.
Jungwirth P.
Jurasek M.
Jurczak M.
Jurek K.
Jurášek M.
Juschkin L.
Justan I.
Jäger A.
Jönen H.
Jørgensen P. S.
Jůda P.
KALEMTAS A.
KARA F.
KASWANDI A. M.
KASWANDI M. A.
KAWAI K.
KAWASAKI S.
KAYA P.
KERVANCIOĞLU E.
KIM C. Y.
KIM H. S.
KIM J. G.
KOTİL T.
KUMAR M.
Kabiri Y.
Kable E. P.
Kable S. H.
Kaderabek L.
Kadkhodazadeh S.
Kado M.
Kaeppel A.
Kaewsai K
Kafexhiu F.
Kagesawa K.
Kahl F.
Kaimakamis G.
Kainer K.
Kainuma R.
Kaiser U.
Kajimura N.
Kakanakov R.
Kakas D.
Kakaš D.
Kakeshita T.
Kakimi S.
Kalabegishvili T. L.
Kalaga K.
Kalasova I.
Kalasová I.
Kalbac M.
Kalendová A.
Kaliappan S.
Kalina M.
Kalinin S. V.
Kalmykov A. E.
Kalok D.
Kalácska S.
Kamakura Y.
Kamalnath K.
Kamarás K.
Kameda T.
Kamimura O.
Kamin D.
Kamino T.
Kaminska A.
Kamp V.
Kamran M. A.
Kan D.
Kanaji A.
Kanazawa N.
Kandil A.
Kaneko O.
Kaneko T.
Kaneko Y.
Kanetsuku T.
Kaneyama T.
Kang Y.
Kani I.
Kania B.
Kanngießer B.
Kano E.
Kantiranis N.
Kapaca E.
Kapatenakis M. D.
Kapetanakis M. D.
Kaplan W. D.
Kaptay G.
Kapucu A.
Kapush D.
Kapusta P.
Karabulut E.
Karaca C.
Karaca O.
Karadottir R. T.
Karakostas T.
Karayannis V.
Karbaum C.
Karbowniczek J.
Karczewski G.
Kardynal B.
Karimi E.
Karimi E.
Karlsen M.
Karlsson L. H.
Karlík M.
Karnthaler H. P.
Karolak F.
Karreman M. A.
Kasai H.
Kasama T.
Kasetty G.
Kasetty K.
Kashtiban R.
Kashtiban R. J.
Kasim M.
Kasim M.
Kasl J.
Kassa H. G.
Kast A. K.
Kasuya K.
Kaszewska E.
Katan A.
Kato H.
Kato T.
Katsikini M.
Katz A.
Kauffmann Y.
Kaufhold S.
Kaufmann R.
Kaufmann W. A.
Kauko H.
Kautek W.
Kavalakkatt J.
Kavouras P.
Kawabata R.
Kawai A.
Kawai K.
Kawai S.
Kawakami H.
Kawamoto S.
Kawamura Y.
Kawasaki M.
Kawasaki T.
Kawashima Y.
Kawata H.
Kawata M.
Kawata S.
Kawauchi K.
Kaya Dagistanli F.
Kaya Dağıstanlı F.
Kaya E.
Kaya-Dagistanli F.
Kayacan S.
Kayali S.
Kayunkid N.
Kazanskiy P. R.
Kazda T.
Kazumori H.
Kazzazi A.
Kašiarová M.
Kašparová B.
Ke X.
Kearsley A. T.
Keawdee J.
Keckes J.
Kehagias T.
Keilová E.
Keimer B.
Kell D. B.
Kelly T. F.
Kelsch M.
Kempen P. J.
Kemper B.
Kenny T. W.
Kepaptsoglou D.
Kepaptsoglou D. M.
Kepinski L.
Kepinski L.
Kerckhove G.
Kereïche S.
Kerschensteiner M.
Kerstin V.
Kervancıoğlu G.
Kerwin J.
Keskin S.
Keskinbora K.
Kestens V.
Kettou S.
Kevorkova A.
Keyhani E.
Keše D.
Khalakhan I.
Khalil A. S.
Khan S.
Khlobystov A.
Khlobystov A. N.
Khmelenin D. N.
Khodak Y. A.
Kholmogorova S.
Khoshouei M.
Khouchaf L.
Khranovskyy V.
Kiat J. M.
Kiener D.
Kienle M.
Kießling F.
Kikkawa J.
Kikkawa M.
Kikuchi K.
Kikuchi N.
Kikuchi S.
Kikui T.
Kikuta J.
Kildahl P.
Kim C. S.
Kim M. J.
Kim A.
Kim C. I.
Kim D. H.
Kim G.
Kim H.
Kim H. G.
Kim H. J.
Kim H. R.
Kim I. W.
Kim J.
Kim J. J.
Kim J. S.
Kim J. Y.
Kim K.
Kim K. C.
Kim M.
Kim M. J.
Kim M. K.
Kim M. R.
Kim M. S.
Kim S.
Kim S. D.
Kim S. G.
Kim S. J.
Kim S. K.
Kim T. H.
Kim W. T.
Kim Y.
Kim Y. C.
Kim Y. T.
Kiminami C. S.
Kimizuka H.
Kimoto K.
Kimura A.
Kimura T.
Kinoshita H.
Kinoshita T.
Kinyanju M. K.
Kinyanjui M. K.
Kioseoglou J.
Kirby A.
Kirilenko D. A.
Kirkesali E. I.
Kirkland A. I.
Kirmse H.
Kirmse R.
Kirschner J.
Kirves J.
Kis V.
Kiselev N. A.
Kishida K.
Kishimoto M.
Kishita K.
Kisielowski C.
Kisielowski C. F.
Kiss G.
Kiss Á. K.
Kister T.
Kistler J. D.
Kitada M.
Kitajima T.
Kitamura S.
Kitaura R.
Kitta M.
Kittel Á.
Kiyonari S.
Kizilyaprak C.
Klautau M.
Kleebe H. J.
Klein O. D.
Klementová M.
Klie R.
Klie R. F.
Klimenkov M.
Klimša L.
Kling J.
Klingauf J.
Klinger M.
Klingl T.
Klocke V.
Klupp Taylor R. N.
Klán P.
Kmoníčková E.
Knappett B. R.
Knez D.
Kniep R.
Knoke I.
Knott G. W.
Knyrim P.
Knápek A.
Ko H.
Ko H. C.
Kobayashi T.
Kobe S.
Kobler A.
Kobus H.
Kocagoz A.
Kocak-Toker N.
Kocevski V.
Koch C.
Koch C. T.
Koch M.
Kociak M.
Kocová L.
Kocyigit A.
Kodali A.
Kodama T.
Kodrík D.
Koga G. Y.
Kogoj R.
Koguchi M.
Koh A. L.
Kohama K.
Kohashi T.
Kohda D.
Kohiki S.
Kohl H.
Kohn A.
Kohn E.
Kohno H.
Kohno Y.
Kohoutek J. M.
Kohyama M.
Koirala P.
Kokkonen T.
Kokoskova M.
Kolaklieva L.
Kolar F.
Kolarik V.
Kolařík V.
Kolb D.
Kolbasi B.
Kolenc M.
Kolgazi M.
Koller G.
Koller-Peroutka M.
Kolman P.
Kolobylina N. N.
Kolosov V. Y.
Kolotuev I.
Kološová J.
Kolíbal M.
Komelj M.
Komninou P.
Konczak C.
Kondo H.
Kondo R.
Kondo Y.
Kong C.
Kong X.
Kongtawelert P.
Konings S.
Konno M.
Konno T. J.
Konomi M.
Konsolakis M.
Konstantinidis S.
Konvalina I.
Konyuba Y.
Kopecek J.
Kopecký V.
Kopeček J.
Kopp C.
Kopáček P.
Korac A.
Korac B.
Korf U.
Korkmaz E.
Kormout K. S.
Korneev D V Generalov V M Zaitsev B N
Korniyenko K.
Korobchevskaya K.
Korobkov D. A.
Koroleva O. N.
Korshunova I. O.
Korsunsky A. M.
Korytov M.
Kosanke K.
Kosel J.
Koshikawa T.
Kostanyan A.
Kostina O. V.
Kostić I. T.
Kostić J.
Kostromina E.
Kotakoski J.
Kotani M.
Kothleitner G.
Kotil T.
Kotova S. L.
Kotrlý M.
Kotula P. G.
Koukoula T.
Koukouzas N.
Kouri J. B.
Koutsogeorgis D. C.
Kovacheva D.
Kovacovicova K.
Kovacs A.
Kovács A.
Kováčik L.
Kovář B.
Kowada T.
Kowal A.
Kowalsky W.
Kowshik M.
Koyama M.
Koyuturk M.
Kozek W. J.
Kozeschnik E.
Kozieł E.
Kozlova T.
Kozubek M.
Kozák J.
Koçak H.
Kočka J.
Kočárek E.
Kočí P.
Kočík J.
Košťál V.
Krakow R.
Kralickova M.
Kramer M. J.
Kranz C.
Kraschewski S. M.
Kratochvilova I.
Kraus T.
Krause F. F.
Kraxner J.
Krehl J.
Kreiliger T.
Krekeler T.
Krenz G.
Kretschmar S.
Kreusch M.
Kripnerová M.
Krishna YGR
Krishnamra N.
Kristofikova Z.
Kristóf Z.
Krittanai C.
Krivanek O. L.
Krizik P.
Krizova A.
Krok F.
Kromka A.
Kroon R. E.
Kroupa A.
Kruit P.
Kruk A.
Krumeich F.
Kruse J.
Kruse N.
Kruska K.
Kryazhov A.
Krylov V. N.
Krzemiński Ł.
Krzyzanek V.
Krzyžánek V.
Král P.
Král R.
Krásný L.
Krátká K.
Krátký S.
Krämer A.
Kröhnert K.
Kuball M.
Kubec A.
Kubelka M.
Kubesova A.
Kubi J. A.
Kubiak J.
Kubiak J.
Kubo A.
Kubová H.
Kubínová L.
Kubínová Z.
Kudaba I.
Kudo M.
Kudo T.
Kudryashev M.
Kuebel C.
Kuhn R. J.
Kuimova M. K.
Kukuruzović D.
Kukushkin S. A.
Kukutschová J.
Kula A.
Kulnitskiy B. A.
Kulovits A. K.
Kulzick M. A.
Kumagai K.
Kumakura H.
Kumar A.
Kumar B.
Kumar D.
Kumar K.
Kumar S. N.
Kumkrai P.
Kumlehn J.
Kumskov A. S.
Kun A.
Kundu P.
Kundu S.
Kunert B.
Kuneš M.
Kuniaki T.
Kunieda T.
Kuo J. R.
Kuo C. H.
Kupreenko S. U.
Kupsta M.
Kurashima K.
Kurata H.
Kurczynska E.
Kurko S.
Kurobe H.
Kurokawa A.
Kurokawa D.
Kuroki K.
Kurosaka D.
Kurosawa C.
Kurth T.
Kurttepeli M.
Kurtz D. M.
Kurushin V.
Kurzydlowski K. J.
Kuskova A. N.
Kusova K.
Kusunoki S.
Kutejová E.
Kutleša P.
Kuwabara A.
Kuwabata S.
Kuwahara M.
Kuwahara R.
Kuwamura Y.
Kuwano M.
Kuwano N.
Kuwauchi Y.
Kuyukina M. S.
Kuzmina N. V.
Kučerová V.
Kužmová E.
Kvasnica L.
Kvasov A.
Kweon H. S.
Kwon Y. K.
Kylberg G.
Kyratsi T.
Károly Z.
Käch A.
Käppel A.
Kóvacs A.
Köroğlu M. K.
Köroğlu M. K.
Köroğlu P.
Købler C.
Kübel C.
Küpper L.
Küçükali B.
Kılıç A. D.
Kıran D.
Křivánek L. O.
Křivánek O. L.
Křivánková D.
Křížek P.
Křížová A.
LAIK B.
LALUN N.
LANDON-CARDINAL O.
LANGLOIS C.
LE BOUAR Y.
LEE J. H.
LEFORESTIER A.
LEIFER K.
LEROUX C.
LEVEAU L.
LI S.
LIAO Z.
LIU C.
LIU J.
LIVOLANT F.
LONGUET J. L.
LYTH S. M.
La Fontaine A. J.
La Magna A.
La Torre A.
LaGrange T.
Labacevski B.
Labacevski N.
Labuda A.
Labudović-Borović M.
Labus D.
Lacerda R. G.
Lacroix B.
Lacroix L. M.
Ladeira L. O.
Lafond D.
Laforgia V.
Lagaňa R.
Lagoeiro L. E.
Lagoutte J.
Lahaye M.
Lai C. C.
Lai M. W.
Lai S. H.
Lai W. C.
Laibl L.
Laik A.
Laimbacher A. S.
Lainović T.
Laissue P. P.
Lajaunie L.
Lalinsky O.
Lalić I. M.
Lam R.
Lam Y. M.
Lamb R. D.
Lambeets S. V.
Lambrichts I.
Lancelon-Pin C.
Landa M.
Landa-Cánovas A. R.
Landi S. M.
Landin T.
Landini G.
Lang C.
Lang I.
Langbein W.
Langenwalter P.
Langer E.
Langhans J.
Langhorst M.
Langlois C. T.
Lanzer M.
Lanzoni E. M.
Lapmanee S.
Lara R.
Lara-Marrtínez R.
Lara-Martínez R.
Largeau L.
Lari L.
Larqué A.
Larson D. J.
Larsson A.
Lartigue L.
Lasserre M. I.
Latychevskaia T.
Latzel M.
Lau D. W.
Laue M.
Laugks T.
Lauridsen E.
Laurinavicius A.
Lavazza A.
Lavenus P.
Lavkova J.
Lavrenyuk A. V.
Lawrence D. F.
Lazar J.
Lazar S.
Lazarov V.
Lazarov V. K.
Lazzaroni R.
Lačković V.
Lašan M.
LeBeau J. M.
LeCoustumer P.
Leapman R. D.
Lebedev O. I.
Lebert R.
Lebrun L.
Lechner L.
Leclère P.
Ledig J.
Ledinský M.
Lee C. Y.
Lee H. H.
Lee M. E.
Lee B. H.
Lee C.
Lee C. H.
Lee C. S.
Lee C. Y.
Lee H.
Lee H. H.
Lee H. K.
Lee H. Y.
Lee J.
Lee J. H.
Lee J. K.
Lee J. Y.
Lee K. W.
Lee M. E.
Lee M. R.
Lee S.
Lee S. P.
Lee S. S.
Lee S. W.
Lee S. Y.
Lee V.
Lee Y.
Lee Z.
Lees M R.
Lefebvre W.
Lefèvre S.
Legall H.
Lehmann M.
Lehtinen O.
Lei C.
Lei M.
Leifer K.
Leitinger G.
Leiva D. R.
Lekieffre C.
Lemaire L.
Lemgruber L.
Lemmer U.
Lenaerts S.
Lenart A.
Lendvai A.
Lenferink A. T.
Lenk S.
Lenrick F.
Lenser C.
Lentijo-Mozo S.
Lentijo-Mozo S.
Lentz M.
Lentzen M.
Leon C.
Lepinay K.
Lepoittevin C.
Lercher J. A.
Lereah Y.
Leroux C.
Leroux F.
Lesieur S.
Leskovšek V.
Leson A.
Lestriez B.
Leszczynski M.
Letertre L.
Letofsky-Papst I.
Letouzey A.
Levi G.
Levilly D.
Levin A. A.
Levrat J.
Lewandowska M.
Lewandowska-Łańcucka J.
Lewis E. A.
Lewys J.
Leyte F.
Leyte F.
Lezec H.
Lhotáková Z.
Li Z. Q.
Li C.
Li H.
Li J. H.
Li K.
Li L.
Li M.
Li Q.
Li R.
Li R. K.
Li S. W.
Li T.
Li W.
Li W. F.
Li X.
Li X. S.
Li Y.
Li Z.
Li Z. A.
Liakakos N.
Liang D.
Liang Q.
Liang Y. M.
Liao H. S.
Liao Z.
Liberti E.
Lich B.
Licha K.
Lichte H.
Lichte H.
Lichtscheidl I.
Lichtscheidl I. K.
Liddicoat P.
Lidorikis E.
Lidzey D. G.
Liebel A.
Liedke B.
Liepniece-Karele I.
Lifland A. W.
Ligas A.
Ligero P.
Lilach Y.
Lilja M.
Lim C.
Lim T. W.
Lima J.
Lin C. Y.
Lin F.
Lin W. T.
Lin C. L.
Lin C. Y.
Lin F.
Lin P. A.
Lin W. T.
Lin Wei Yu
Lin Y.
Linck M.
Lindén M.
Lingaraju G. M.
Lingelbach K.
Link T.
Lionel J.
Liou G. G.
Liou S. C.
Lioutas C. B.
Lipinski B.
Lipińska-Chwałek M.
List-Kratochvil E.
Litvinov D.
Liu M. H.
Liu S. Y.
Liu A. C.
Liu B. Y.
Liu C.
Liu C. H.
Liu H.
Liu H. B.
Liu J. L.
Liu J. Z.
Liu K.
Liu L.
Liu L. Y.
Liu M.
Liu M. P.
Liu P.
Liu P. T.
Liu Q.
Liu R.
Liu S. S.
Liu S. Y.
Liu X.
Liu Y.
Liu Z.
Liv N.
Liz-Marzan L. M.
Liz-Marzán L. M.
Lloyds S.
Lo S. C.
Lo Schiavo V.
Lobato I.
Lobo B.
Lobotka P.
Loch R. A.
Logar M.
Logica Tornatore T.
Logvenov G.
Logvinenko O.
Lohner T.
Loiseau A.
Lolivier J.
Lombardi F.
London A.
Long E.
Long E.
Long E. A.
Long H. B.
Long L.
Longo E.
Longo G.
Longo J. F.
Longo P.
Loosen P.
Lopatin S.
Lopes F.
Lopes-Bezerra L. M.
Lopes-Moriyama A. L.
Lopez E. O.
Lopez O. V.
Lopez-Echarri A.
Lopez-Haro M.
Lopez-Iglesias C.
Lopot F.
Lorsuwannarat N.
Lorut F.
Losquin A.
Lostak M.
Losurdo M.
Lotnyk A.
Lotsari A.
Lottini E.
Lotz M.
Lounis B.
Lourie O.
Louro R. P.
Loussert C.
Lovejoy T. C.
Lovrincic R.
Lozano J. G.
Lozano-Perez S.
Lošťák M.
Lu J.
Lu L.
Lu P.
Lu S. R.
Lu T. M.
Lu W.
Lu Y.
Lu Y. G.
Lu Z.
Lubk A.
Lucas G.
Lucchese Marcia M.
Lucchetti D.
Lucci C. M.
Luddin N.
Lugg N. R.
Lugstein A.
Luini A.
Lukes J.
Lukinavičius G.
Luna C.
Lunkenbein T.
Lunn J. E.
Lunt A. J.
Luo J.
Luo X. T.
Lupini A. R.
Luppo M. I.
Lupu N.
Lutterotti L.
Lutz G.
Lux A
Lux A.
Lux J.
Luysberg M.
Lv D.
Lyu X. W.
Lábadi z.
Lábár J. L.
López C.
López G. A.
López A.
López G. A.
López M. F.
López V. S.
López-Conesa L.
López-Díaz D.
López-Ferreño I.
López-Haro M.
López-Ortega A.
López-Romero D.
López-Téllez G.
López-Vidrier J.
López-Álvarez M.
Löffler M.
Löffler S.
M. Casinelli
M. Kozuka S. Otani Y. Aruga
MADIGOU V.
MAIRE E.
MARCO S.
MARTIN J. J.
MASENELLI-VARLOT K.
MASSIANI P.
MENDEZ J.
MESSAOUDI C.
MICHEL J.
MOREAU P.
MUTO S.
Ma E.
Ma T. W.
Ma Y. H.
Maaß T.
MacArthur K. E.
MacDonald L.
MacLaren I.
MacLellan-Gibson K.
Maccagnani P.
Macedo A. M.
Macedo M. L.
Macedo-Silva R.
Machado G.
Machado G. S.
Machado I. F.
Machoke A.
Maciel Filho R.
Macit Ç.
Mackay D. T.
Mackovic M.
Macurek L.
Mader W.
Madigou V.
Madina Neto A.
Madiomanana K.
Madras G.
Madsen S. J.
Maeda H.
Maeda S.
Maeda Y.
Maegli A.
Maehara Y.
Maehata K.
Mafakheri E.
Mafakheri E.
Magalhaes M. S.
Magaña C.
Magen C.
Maghraoui S.
Magnusson J.
Magro M.
Magyar A. P.
Magén C.
Mahamid J.
Mahmoud Z. K.
Mahr C.
Maia L.
Maier A. G.
Maier J.
Maier U.
Maier-Flaig F.
Mainz R.
Maisch T.
Majert S.
Majetich S.
Majimel J.
Major R.
Majzlíková P.
Makarov V. V.
Makineni S.
Makino A.
Makkonen H.
Malac M.
Malachias A.
Malachov M.
Malakhova D.
Malaplate J.
Malatesta M.
Malchère A.
Maldener I.
Maldonis J.
Malecka M. A.
Malet L.
Maletta M.
Malher B.
Malindretos J.
Malladi S. K.
Mallinger B.
Malta K. K.
Malterre D.
Mamoňová M.
Man Gil Yang
Man K. L.
Manabe H.
Mancarella F.
Mandl M.
Mangel S.
Mangelinck D.
Mangler C.
Mangues R.
Maniv T.
Mank A. J.
Manna L.
Mannocci F.
Mansfield J. F.
Mante N.
Mantei J. R.
Mantell J. M.
Mantl S.
Mantlikova A.
Mantovani D.
Mantsevich V. N.
Manz S.
Manzo A.
Mao S. C.
Mapelli J.
Marabini R.
Maraloiu V. A.
Marangoni S.
Maraschin M.
Marcek Chorvatova A.
Marcelot C.
March K.
Marco S.
Marco s.
Marconi G. D.
Marcos J. V.
Mardhiah M
Margeat O.
Margheri G.
Margiol S.
Marianecci C.
Marie H.
Marikovsky Y.
Marin G. B.
Marini D.
Marinova M.
Marioara C. D.
Mariolle D.
Mariolle D
Mark A. F.
Markelic M.
Marko D.
Marko M.
Markovsky B.
Marks L. D.
Marks S.
Markurt T.
Markus K.
Markó K.
Marnellos G. E.
Marotta R.
Marques P. P.
Marques W. A.
Marquina C.
Marras S.
Marsalek L.
Marshall A. F.
Marsili P.
Marteleur M.
Martelli A.
Martens W.
Martin F.
Martin Gonzalez M.
Martin I. Y.
Martin M.
Martin N.
Martin T.
Martin T. E.
Martin Y.
Martin-Fernandez M. L.
Martinelli L.
Martinez A.
Martinez Bonastre A.
Martinez C.
Martinez E.
Martinez G.
Martinez G. T.
Martinez-Alonso E.
Martinez-Amesti A.
Martinez-Macias C.
Martinez-Martinez N.
Martinez-Menarguez J. A.
Martinka M
Martinka M.
Marty A.
Martín G.
Martín R.
Martín-Benito J.
Martínez A.
Martínez- Calleja A.
Martínez-Alonso E.
Martínez-Martínez N.
Martínez-Menárguez J. A.
Martínez-Palomo A.
Marucci M.
Marugan j.
Maruno S.
Maruta S.
Maruyama Y.
Marx A.
Maryasov A.
Marzec M.
Marzocca A. L.
Marín L.
Masaaki Sugiyama and Akira Taniyama
Maschmeyer T.
Masenelli-Varlot K.
Maser W.
Mashukov A. V.
Mashukova A. E.
Masia F.
Masini L.
Mason K. N.
Masova S.
Maspoch D.
Masseboeuf A.
Masserini M.
Massote D. V.
Massover W. H.
Massé R. C.
Masters A.
Masters R. C.
Mastromatteo F.
Masuda H.
Masuda T.
Masudi N. S.
Masudi S. M.
Mateasik A.
Materna-Morris E.
Mathelié-Guinlet M.
Mathes F.
Matko I.
Matolin V.
Matolinova I.
Matos J. P.
Matsuda J.
Matsuda K.
Matsuda T.
Matsui Y.
Matsumoto A.
Matsumoto H.
Matsumoto J.
Matsumoto S.
Matsumura S.
Matsunaga K.
Matsuo J.
Matsuoka Y.
Matsushima H.
Matsushita M.
Matsutani T.
Matsuura K.
Matsuura M.
Matsuzaki K.
Matteoli M.
Matula Z.
Matuška V.
Matyssek C.
Matějka M.
Matějková-Plšková J.
Mauguin O.
Maultzsch J.
Maurer B.
Maximova E.
Mayall B.
Mayall B.
Mayanagi K.
Mayanagi K.
Mayer A.
Mayer F.
Mayer J.
Mayer S.
Mayer T.
Mayes E. L.
Maynicke E.
Mayoral A.
Mayrhofer C.
Mazaj M.
Mazet L.
Mazzer E. M.
Mazzoni M. S.
Mačković M.
Mačukanović-Jocić M.
McAllister D. A.
McCarthy C.
McCreary A.
McCulloch D. G.
McDonald K. L.
McDonald S. A.
McDougall N. L.
McGrouther D.
McGuiness P.
McGuire E. K.
McIntyre P. C.
McKenna K.
McKenna K.
McKeown J. T.
McLeod R.
McLeod R. A.
McVitie S.
Medeiros B. B.
Medhekar N. V.
Medina-Campos O. N.
Medina-Gonzalez I.
Medjanik K.
Medjoubi K.
Medlin R.
Medlín R.
Medlín R.
Mednikova M. B.
Mee M.
Meeldijk J. D.
Mehdi B. L.
Meher S.
Mehrtens T.
Mehta V.
Meibom A.
Meier A. F.
Meier S.
Meinke M.
Meisnar M.
Melcr J.
Mele C.
Mele L.
Meledina M.
Melgarejo Z. H.
Melicherová J.
Melin T.
Melinte G.
Melinte G.
Melischnig A.
Melle A. K.
Mellish C.
Mellmann A.
Melo R. C.
Melville A.
Melz G.
Melzer M.
Melzer K.
Melzer M.
Mendel B. J.
Mendes I. C.
Mendes M. C.
Mendez Mendez J.
Mendiuk O.
Mendoza B.
Mendoza C.
Mendoza Cruz R.
Mendoza J.
Mendoza M. E.
Mendoza Perez J.
Mendoza Pérez J. A.
Mendoza-Sanchez B.
Mendoza-Santoyo F.
Mendoza-Sánchez B.
Meng Q.
Meng X.
Meng X. M.
Meng Y. F.
Menna-Barreto R. S.
Menyhárd M.
Mercade E.
Mercier L.
Merkle A.
Merkle A. P.
Merlo A. F.
Mertens T.
Mertschnigg S.
Meshi L.
Meslin E.
Mesman R.
Mesquita O. N.
Messaoudi C.
Messaoudi c.
Mestre M.
Metscher B. D.
Meulenberg W. A.
Meunier V.
Meuret S.
Meuret S.
Meyer B.
Meyer C. E.
Meyer E.
Meyer J. C.
Meyer zu Heringdorf F. J.
Meza-Rocha A. N.
Mező G.
Mhamdi M.
Mi S. B.
Mi Z.
Miao X. P.
Michael J. R.
Michail A. Korchagin
Michalcová A.
Michalička J.
Michalková M.
Michalska M.
Michel S.
Michl M.
Michler J.
Michálek J.
Micuda S.
Midgley P.
Midgley P. A.
Midlej V.
Midoh Y.
Migliori A.
Migunov V.
Mika F.
Mika J.
Mikmekova S.
Mikmeková Š.
Mikula R. J.
Mikulics M.
Mikulčić-Pavlaković S.
Milagres T.
Milat O.
Mildner K.
Milićević N. M.
Milićević Ž. J.
Miller R. J.
Miller B. K.
Mills D. J.
Mills M. J.
Milne S. J.
Min B. K.
Minakata S.
Ming W. Q.
Ming-Hao Lee
Ming-Wen Chu
Minnich B.
Minoda H.
Minoda M.
Minor A. M.
Minoux D.
Minárik P.
Miquita D. R.
Miranda - Sánchez M. M.
Miranda K.
Miranzo P.
Mironov A.
Mironov A. A.
Mironov B. E.
Mirzayev R.
Mishra R.
Misják F.
Misra S.
Misteli T.
Miszta K.
Mitard J.
Mitome M.
Mitsche S.
Mitsuda K.
Mitsuhara M.
Mitsutaka H.
Mittelberger A.
Mitterer C.
Miura J.
Miura Y.
Miyake H.
Miyata T.
Miyatake K.
Miyayama M.
Miyazaki N.
Miyazawa A.
Mizaikoff B.
Mizoguchi T.
Mlayah A.
Moeen Uddin G.
Moerman D.
Moffatt P.
Moghimian P.
Mogstad T.
Mogwitz B.
Mohallem N. S.
Mohamed W. I.
Mohandas E.
Mohn M.
Mohun T.
Mokhtari P.
Mokry J.
Moldovan S.
Moldovan G.
Moldovan S.
Molenkamp L. W.
Molina S.
Molina S. I.
Molina-Boisseau S.
Molina-Luna L.
Molinari A.
Moliner M.
Mollard N.
Mondragon R.
Monge N.
Mongillo M.
Monroy E.
Monrroy M.
Monsalve G.
Montanari B. G.
Monteagudo J.
Montellano-Lopez A.
Monthioux M.
Moodie A. F.
Moody M.
Moody M. P.
Moody S.
Moon S. Y.
Moore K. L.
Morales G. M.
Morales Masis M.
Morales-Luckie R. A.
Morandi V.
Morante J. R.
Morat F.
Moratis K.
Moravek M.
Moreau P.
Moreira B. B.
Moreira D. A.
Morel R.
Morellón L.
Morelos-Gómez A.
Moreno M. S.
Moreno M.
Moreno O.
Morgado-Vargas M.
Morgan D. G.
Mori H.
Mori N.
Mori Y.
Moriguchi N.
Morikawa A.
Morikawa D.
Morikawa K.
Morimoto Y.
Morishita S.
Morita C.
Morita H.
Moriwake H.
Moriya N.
Morone N.
Moros A.
Moroté F.
Morris H.
Mortada H.
Mortet V.
Morthomas J.
Mory C.
Mosecker L.
Moser D. E.
Moser T.
Moses P. G.
Moshar A.
Moshnyaga V.
Mosquera Sanchez L. P.
Mostaed A.
Mostovoy M.
Motai K.
Motlik J.
Motohashi H.
Motomura S.
Motta C.
Motta M. C.
Mou C. Y.
Mouillard-Stéciuk G.
Mousley M.
Moynet D.
Mrak P.
Mrigesh M.
Mrázek J.
Mu Q.
Mu X.
Mucciolo A.
Muciño Gutierrez O. M.
Muehle U.
Muehlmann L. A.
Mueller J.
Mueller P.
Mueller V.
Muhaffel F.
Muhling J.
Mukai M.
Mukai T.
Mukhitov A. R.
Mukhitov A. R.
Muniz E. C.
Muniz-Miranda M.
Munnangi A. R.
Munoz Rojo M.
Munoz V. A.
Munroe P. R.
Murakami T.
Murakami Y.
Muraleedharan K.
Muranaka Y.
Murase H.
Murata H.
Murata K.
Muratovic A.
Muravnik L. E.
Murdoch B. J.
Muroga T.
Murru L.
Music D.
Muszynska A.
Mutlu H. S.
Muto S.
Mutoh M.
Muxworthy A. R.
Muñiz Díaz de León M. E.
Muñoz M.
Muñoz M. A.
Muñoz-Barrutia A.
Myasoedov A. V.
Myers D.
Myers D. J.
Mynář M.
Mytnik-Ejsmont J.
Mácová P.
Málek J.
Mädler L.
Ménager C.
Mörgelin M.
Möslang A.
Mølhave K.
Mørtsell E. A.
Müller E.
Müller F.
Müller H.
Müller J.
Müller K.
Müller M.
Müller M. G.
Müller M. M.
Müllerová I.
Münzenberg M.
NASCIMENTO J. M.
NEGULESCU B.
NERI E.
NOLIN F.
NONNET H.
NORMALAWATI S.
NYSTEN B.
Na Y.
Nabiei F.
Nachtnebel M.
Nadeau O. W.
Nagai T.
Nagakubo Y.
Nagami T.
Nagao M.
Nagaoki I.
Nagarajan S.
Nagase T.
Nagashima S.
Naghavi M.
Naglic T.
Nagoshi M.
Nagy K.
Nagy K. H.
Nahlik T.
Naidoo S. R.
Nair M.
Nair V.
Najafi E.
Nakafushi Y.
Nakahira A.
Nakajima Y.
Nakamae K.
Nakamichi H.
Nakamichi T.
Nakamura J.
Nakamura K.
Nakamura M.
Nakamura R.
Nakamura Y.
Nakanishi R.
Nakanishi S
Nakanishi S.
Nakanoda S.
Nakashima P. H.
Nakashima P. N.
Nakazawa E.
Nalawade S.
Nam Hyun Jung
Nam K.
Nam K. T.
Nambo Y.
Namburi H. K.
Namekawa R.
Nanci A.
Nanova D.
Napchan E.
Narajczyk M.
Narajczyk M.
Narayanan A.
Narrandes A.
Nascimento V. B.
Nasilowski M.
Nasirov M.
Nasybulin E. N.
Nau S.
Nava R.
Navarro G.
Navratilova L.
Ndjindji O.
Neagu A.
Nebel C.
Nebesarova J.
Nebesarova J.
Nebesářová J.
Nebesářová J.
Nedela V.
Nedelkoski Z.
Neděla V.
Neelamraju S.
Nees M.
Neethling J. H.
Neethling J. H.
Negrea R. F.
Negreira C.
Neiner T.
Neish M. J.
Neisius T.
Neitz A. W.
Neklyudova M.
Nelayah J.
Nellist M.
Nellist P. D.
Nelson C. T.
Nemecek D.
Nemeth I.
Nemoto T.
Nemouchi F.
Neogy S.
Nepijko S. A.
Nepomuceno T.
Nerl H. C.
Nesládek M.
Neto S. F.
Neugebauer U.
Neumann S.
Neumüller J.
Neusser G.
Neusser G.
Neuville D.
Neves A. M.
Neves B. R.
Nevius M. S.
Nevoralova M.
Nevoralová M.
Ng X. W.
Ngernsoungnern A.
Ngernsoungnern P.
Nguyen D. T.
Nguyen H. P.
Nguyen Hung T.
Nguyen N.
Nguyen N. Q.
Nguyen N. T.
Nguyen P.
Nguyen P. D.
Nguyen V. D.
Ni N.
Ni S.
Niarchos D.
Niazi S.
Nicholas M.
Nicholls A.
Nicholls A. W.
Nicholls R.
Nicholls R. J.
Nichterwitz M.
Nicolai J.
Nicolas V.
Nicolay S.
Nicoletti O.
Nicolopoulos S.
Nicolosi V.
Nicotra G.
Niculae A.
Nidetzky B.
Nie J. F.
Nie J. Q.
Nie Z.
Nie Z. R.
Niedoba K.
Niehle M.
Niekiel F.
Niermann T.
Niese S.
Niewczas M.
Niewolak L.
Niidome Y.
Niim H.
Niimi H.
Nik S.
Nikiel M.
Nikiyan H. N.
Nikolsky E. E.
Nilsson H.
Ninago M. D.
Niquet Y. M.
Nisaratanaporn E.
Nisbet D. R.
Nishi R.
Nishida H.
Nishida M.
Nishida T.
Nishihara S.
Nishijima M.
Nishimura S.
Nishino Y.
Nishioka H.
Nishiuchi T.
Nishiyama H.
Nishiyama Y.
Nissen J.
Nistor L. C.
Niu H. Y.
Niwa T.
Noack J.
Nobuchi T.
Nofz M.
Nogueira R. P.
Noguez-Hernández R.
Nogués J.
Noheda B.
Noisternig S.
Nokuo T.
Nollo G.
Norberg P.
Nordström R.
Nori F.
Northover S. M.
Norton M. G.
Norton M. M.
Nose M.
Noseda Grau V.
Notelaers K.
Notkins A. L.
Novacek J.
Novak P.
Novakova N.
Novakova P.
Novakovic J. G.
Novakovic N.
Novara C.
Novotný F.
Novák P.
Novák L.
Novák P.
Nowakowska M.
Nowell M. M.
Noyong M.
Nshingabigwi E. K.
Ntampegliotis K.
Nuhfer N. T.
Nunes C. L.
Nurcahyo W.
Nurul Munirah A
Nutarelli D.
Nuzzo R.
Nuzzo R. G.
Nysten B.
Német
Nó M. L.
Nó M. L.
Nó M. L.
Němec M.
Němeček D.
O'Brien P.
O'Connell J. H.
OZ E.
Oberdick S. D.
Oberholzer H. M.
Obradors X.
Ocelic Bulatovic V.
Odaci Demirkol D.
Odnodvorets L. V.
Oe Y.
Oelsner A.
Oestlund F.
Ogai K.
Ogami A.
Ogashiwa T.
Ogawa T.
Ogawa N.
Ogumi Z.
Ogura T.
Oh S.
Oh H.
Oh S.
Oh S. H.
Ohgaki M.
Ohkubo T.
Ohkura Y.
Ohlsson J.
Ohnishi I.
Ohnuki S.
Ohta K.
Ohta S.
Ohtska M.
Ohtsuka M.
Ohtsuka T.
Ohwada M.
Oikawa T.
Okada S.
Okayama S.
Okazaki T.
Oki H.
Okiji T.
Okotrun A. V.
Oktem G.
Okubo M.
Okunishi E.
Okuno H.
Okura Y.
Oleg I. Lomovsky
Oleynikov P.
Oliveira A L R
Oliveira A. B.
Oliveira C. F.
Oliveira C. K.
Oliveira J. A.
Oliveira L. P.
Olivera S.
Olivera-Bravo S.
Olivera-Pasilio V.
Olivier E. J.
Olmos C.
Olsen A.
Olson D. P.
Olsson E.
Olsson S.
Oláh N.
Omori T.
Omoto K.
Onal T.
Oner Y.
Oner-Iyidogan Y.
Ongchai S.
Oni A.
Onishi I.
Ono S.
Onoe J.
Onose Y.
Opefi C. A.
Ophus C.
Ophus C. L.
Oppeneer P. M.
Oppo C. I.
Orai Y.
Orchowski A.
Ordavo I.
Orenstein M.
Oreshkin A. I.
Oreshkin S. I.
Orger M. B.
Orgiani P.
Orlandi M. O.
Orlanducci S.
Orlov I.
Orlova E. V.
Ornelas C.
Ortega N.
Ortega F.
Orthacker A.
Ortiz J.
Ortiz J. O.
Ortiz-Miranda A. S.
Ortiz-de-Solorzano C.
Ortolani L.
Oruganti R.
Oshima Y.
Ospina C. A.
Ospina C. A.
Ostafińska A.
Ostasevicius T.
Ostasov P.
Oster M.
Ostrovská L.
Ostrowski A.
Otal E. H.
Otasevic V.
Otsuka T.
Ott A.
Ott R. T.
Otto C.
Otto F.
Otubo J.
Otulak K.
Ou P.
Ougazzaden A.
Oveisi E.
Ovesný M.
Ow-Yang C.
Ow-Yang C. W.
Oxley M. P.
Oyman G.
Oz E.
Ozbek E.
Ozbilgin K.
Ozdas Beyhan S.
Ozdemir I.
Ozdemir M.
Ozdemir U.
Ozer O. F.
Ozerol E.
Ozgur F.
Ozin G.
Ozkan S.
Ozkaya D.
Ozpinar A.
Ozsoy Keskinbora C.
Oztatlici M.
Ozturk M.
Ozturk Z. B.
Oztürk M.
Ozyaman S.
P.
PALACIOS E.
PARK G. S.
PARK H. S.
PARK S. Y.
PEREA M.
PEREIRA DE SOUZA C.
PEREIRA R. V.
PEREZ E.
PERRET A.
PLOTON D.
Pacakova B.
Pacaud J.
Pachatouridou E.
Pacheco J. P.
Pacheco S. U.
Pacheco U.
Pacherová O.
Paciornik S.
Packirisamy M.
Padman B. S.
Paesen R.
Page C.
Pages S.
Pailloux F.
Paiva L. A.
Pakzad A.
Pala J.
Palacio I.
Palacios E.
Palasse L.
Palatinus L.
Palenicek T.
Palisaitis J.
Pallocca G.
Palmieri V.
Palus M.
Pan C. T.
Pan M.
Pan Q. X.
Pan X.
Pan X. Q.
Pan Y. C.
Panciera F.
Pandey P.
Pangrác J.
Pantaleo G.
Pantanella F.
Pantelic R. S.
Pantelides S.
Pantelides S. T.
Pantnaik s.
Pantucek R.
Pantzas K.
Pantzer A.
Panzirsch B.
Papadomanolaki E.
Papageorgiou C.
Papapolymerou G.
Papet P.
Papila M.
Papista E.
Papkovsky D. B.
Papworth A. J.
Paraguay-Delgado F.
Parashuraman R.
Pardini L.
Pardoen T.
Parent L. R.
Park C.
Park C. G.
Park D.
Park E. S.
Park H. S.
Park J.
Park J. B.
Park J. C.
Park J. H.
Park M.
Park P.
Park S. J.
Park S. M.
Parker J. E.
Parlinska M.
Parlinska-Wojtan M.
Parmenter C. D.
Partoens B.
Partridge J. G.
Parvizi A.
Pasos F.
Passafaro M.
Passeri D.
Passey R.
Passoni L.
Passos M. F.
Patarroyo J.
Patincharath N.
Patommel J.
Patriarche G.
Patsalas P.
Pauc N.
Paudel T. R.
Paul G.
Paul J.
Paulauskas T.
Pauly S.
Pautrat A.
Pavel P.
Pavelka M.
Pavlidou E.
Pavlisic A.
Pavlova E.
Payne L. M.
Peach A.
Pearce J. T.
Pearce J. T.
Pechnikova E. V.
Pecinar I.
Pecinar I. M.
Peckys D. B.
Pedetti S.
Pedraza-Chaverri J.
Pedrazzani M.
Peiró F.
Pekic Quarrie S.
Pekić Quarrie S. V.
Pekny M.
Pelc R.
Pelekanos N. T.
Pellarin M.
Pellegrino T.
Pellicciari C.
Pelloquin D.
Pelloquin D. e.
Pelzer K.
Peng F.
Peng G.
Peng H. J.
Peng X.
Penha L.
Pennington R. S.
Pennycook S.
Pennycook S. J.
Pennycook T. J.
Peplinski B.
Pepperkok R.
Peral I.
Perea-López N.
Pereira A. F.
Pereira D.
Pereira D. T.
Pereira H.
Pereira J. L.
Pereira N.
Pereira de Souza C.
Pereiro E.
Perera R.
Perez-Berna A. J.
Perez-Herrero G.
Perezhogin I. A.
Perillat-Merceroz G.
Peroni-Okita F. H.
Perret A.
Persiano A. C.
Persson A.
Persson J. M.
Persson P.
Persson P. O.
Peréz Estébanez M.
Pesanova D.
Pescia D.
Pesquera D.
Peterek M.
Peterka M.
Peterková L.
Peterlechner M.
Peters J.
Peters J. J.
Peters P. J.
Peters T. A.
Petersen T. C.
Petr M.
Petrak V.
Petrenec M.
Petrov I.
Petrova P.
Petrushevska M.
Petráková V.
Petráňová V.
Pettersson H.
Petyukh V. M.
Peukert W.
Pevala V.
Pezza A.
Peřina P.
Pešta M.
Pfaff M.
Pfannmöller M.
Pfeifer C. R.
Pflegerová J.
Pfrieger F. W.
Pham-Huu C.
Phifer D. W.
Philimonenko A. A.
Philimonenko V. V.
Philipp P.
Philips P.
Phillips J. B.
Phillips P.
Phillips P. J.
Piatkevich K. D.
Piazolo S.
Piazzetta M. H.
Picanço-Diniz C. W.
Piccoli F.
Piccolo L.
Picher M.
Pichler B.
Pierce D.
Pierce D. T.
Pierce W.
Pieribone V. A.
Pierret A.
Pietra F.
Pilloni A.
Pimentel E. R.
Pimentel V.
Pinakidou F.
Pingel T.
Pintilie L.
Pinto S. O.
Pinto A. L.
Pinto C.
Pinzari F.
Pippan R.
Piraux L.
Pires M. P.
Pirri C. F.
Piston D. W.
Pitonak R.
Pitule P.
Pizzagalli L.
Pizzarossa C.
Piña Angeles J.
Piňos J.
Planck H.
Plank H.
Plecitá - Hlavatá L.
Plemmons D.
Plenk Jr H.
Plevka P.
Plitzko J.
Plitzko J. M.
Plodinec M.
Ploszczanski L.
Pochet P.
Poddar D.
Podlogar M.
Podor R.
Poelman H.
Poelt P.
Pofelski A.
Pohl D.
Pohl U. W.
Pokle A. S.
Pokorny B.
Pokorná Z.
Pokorný J.
Pokorný P.
Poková M.
Pokrant S.
Pokrovskaya I. D.
Polakova H.
Polakova K.
Polanski F.
Polat I.
Polat M.
Polcar T.
Poletti M. C.
Poliani E.
Poljšak-Prijatelj M.
Pollard S.
Pollastri S.
Polsan Y.
Polster J.
Polsterová H.
Polychroniadis E. K.
Polychroniadis E. K.
Polzer F.
Pombinho A.
Ponce A.
Ponce C. E.
Ponce-Soto L. A.
Ponchet A.
Pongratz P.
Ponomarev I. I.
Ponomareva S. V.
Ponticelli G. S.
Pontoire B.
Popescu L. M.
Popov M. Y.
Popovici M.
Popp J.
Popp M.
Poppitz D.
Porcu M.
Porret C.
Porro S.
Porzionato A.
Pospiech M.
Pospíšilová M.
Potapov L.
Potapov P.
Pothacharoen P.
Potin V.
Potocek P.
Pottasch C.
Pouch S.
Pouchon M. A.
Poulsen H. F.
Poulsen K.
Powell A.
Powell L. C.
Pozzi G.
Pozzi P.
Prabhudev S.
Prada A. L.
Pradeep K. G.
Prades J. D.
Prado P.
Prager A.
Prakash K.
Prang R.
Prange M. P.
Praro M.
Prasanta D.
Prati L.
Prato M.
Pratoomthai B.
Prats-Mateu B.
Pratsinis S. E.
Preat T.
Prescott S.
Presniakov M. Y.
Prestat E.
Pretorius E.
Pretzel B.
Preuss M.
Preziosi M.
Pribat D.
Priesol J.
Prieto J. L.
Prima F.
Prince A. A.
Prinz F. B.
Prochazka J.
Prochazkova B.
Prochazkova M.
Procházka M.
Proctor J.
Prokazov Y.
Proksch R.
Promdonkoyb B.
Proost J.
Prosa T. J.
Provaznik I.
Provazník I.
Provine J.
Proyer A.
Proška J.
Prunier H.
Pruthi V.
Prystawko P.
Prytz Ø.
Przybilla F.
Przybilla T.
Przyborski J. M.
Prášek J.
Puig T.
Pukahuta C
Pulickal Rajukumar L.
Pulido M.
Puntes V.
Purkartova Z.
Puschenreiter M.
Pushkarev S. S.
Puster M.
Putaux J. L.
Puškaš N.
Pálóczy K.
Pécz B.
Pérez A.
Pérez-Juste J.
Pérez-Omil J. A.
Pérez-Salinas P.
Pétrier C.
Pírez M. C.
Pöhl A.
Pölt P.
Qi Z. J.
Qi H. Y.
Qian J.
Qian W.
Qiao L.
Qiao Q.
Qiu G.
Qiu Y.
Quadakkers W. J.
Quaglino D.
Queralto A.
Quester K.
Quinn P.
Quintana A.
Quintana M.
R. Selleri
REYES-GASGA J.
RICOLLEAU C.
RODRIGUES A. D.
ROIBAN L.
RUYTER A.
Raabe D.
Rachel R.
Rachid R.
Rack P. D.
Radaev A.
Rademann K.
Rades S.
Radi Z.
Radnóczi G.
Radnóczi G. Z.
Radochová B.
Radtke G.
Radu B. M.
Raghavan S.
Raghavan R.
Raghavan S.
Rago I.
Rahman I. A.
Raisuddin S.
Rajendran S.
Rajnovic D.
Rak S.
Rakovsky S.
Ralević U. M.
Ralph S.
Ram F.
Ramachandra R.
Ramachandran D.
Ramade J.
Ramanathan R.
Ramar A.
Ramasse Q. M.
Ramasse Q.
Ramasse Q. M.
Ramesh R.
Ramirez A.
Ramlov F.
Ramm G.
Ramsperger U.
Ramírez C.
Ran K.
Rancan F.
Rancic D.
Rancic D. V.
Randhawa G. S.
Ranftler C.
Rapino M.
Raposo G.
Rapôso C.
Rashkova B.
Raska I.
Raskin J. P.
Raskovic – Lovre Z.
Rasouli S.
Ratera I.
Rattenberger J.
Rau E. I.
Rauch E. F.
Rauch E. R.
Rauschenbach B.
Ravaszová F.
Ravaux F.
Raveendran-Nair R.
Ravisankar N.
Ravishankar N.
Ravnikar M.
Ravot D.
Raya A. M.
Razorenov D. Y.
Raška I.
Read N. D.
Reaney I. M.
Rebillat F.
Rebled J. M.
Reddy A. L.
Redondo R.
Reed B. W.
Rees P.
Reeves P. J.
Reggente M.
Rego R.
Reguera E.
Rehbein S.
Rehberg M.
Rehr J.
Reiche I.
Reichelt R.
Reichenbach A.
Reifler E. S.
Reiger E.
Reinauer F.
Reinhard D. A.
Reining L.
Reipert S.
Reischig P.
Reisinger E.
Rek V.
Rellinghaus B.
Remeš Z.
Remiášová J.
Remmele T.
Remy E.
Ren Y.
Renault L.
Rendon L.
Renfigo C. E.
Renfigo E.
Renk O.
Rentenberger C.
Repko A.
Repnik U.
Resende C. P.
Respaud M.
Reséndiz-Avendaño S.
Rethore J.
Reuter K.
Reuter M. C.
Revelo N. H.
Revenko I.
Rey F.
Rey-Castro C.
Reyes G J.
Reyes J.
Reyes M.
Reyes Ábalos A. L.
Reyes-Gasga J.
Reyes-Gutiérrez P. E.
Reyes-Zamudio V.
Reyes-Ábalos A. L.
Reykhard L.
Rez P.
Rezaei Mianroodi J.
Rezek B.
Rezzani R.
Rečnik A.
Rhyim Y.
Rhyim Y. M.
Ribeiro Andrade R.
Ribera-Calzada A.
Riccardi E.
Ricci A.
Rice P.
Richard O.
Richardson
Richert M.
Richtering W.
Ricolleau C.
Riedel R.
Riedel T.
Riedinger A.
Riekehr L.
Riese M.
Riethmüller C.
Rigo J.
Rigutti L.
Rimpelová S.
Rinck J.
Rincon J.
Rind F. C.
Ringe E.
Ringer S. P.
Ripova D.
Risueño M. C.
Ritter M.
Ritter S.
Rivera Calzada A.
Rivera-Calzada A.
Rivero G.
Rivolo P.
Rizzi A.
Rizzo R.
Rizzoli S. O.
Ro Y.
Roberts S. G.
Robertson
Robertson V.
Robin E.
Robino L.
Roca i Cabarrocas P.
Rocci M.
Rocha S.
Rocha S. O.
Rochas C.
Rocquefelte X.
Roddatis V. V.
Rodella L. F.
Rodemeier R.
Rodenburg C.
Rodighiero S.
Rodnina M. V.
Rodrigues A. D.
Rodrigues A. D.
Rodrigues C. A.
Rodrigues C. D.
Rodrigues G. S.
Rodrigues W.
Rodrigues W. N.
Rodriguez A. L.
Rodriguez J. B.
Rodriguez M. J.
Rodriguez-Cruz F.
Rodríguez J.
Rodríguez J. A.
Rodríguez L. A.
Rodríguez M. J.
Rodríguez-Gomez Y.
Rodríguez-González B.
Rodríguez-Gómez Y.
Rodríguez-Lorenzo L. M.
Rodríguez-Manzo J. A.
Rodríguez-Sanz H.
Rodríguez-Valencia C.
Roebben G.
Roeffaers M. B.
Roehling J. D.
Roemer R. A.
Rogov O. Y.
Roguska A.
Rohnke M.
Rohožková J.
Rohrer J.
Roiban L.
Rojas O. I.
Rojas-Lemus M.
Rojo T.
Rokuta E.
Roland F.
Roldan M. A.
Roldán M.
Roleder K.
Rolfe D. J.
Roling B.
Roman B.
Roman E.
Romani S.
Romanova K.
Romeis S.
Romeo C.
Romero J.
Romero-Ibarrra Josue E.
Romero-Núñez A.
Ronda A.
Rong Yu
Rongeat C.
Rooch H.
Ropers C.
Roque N. R.
Rosado A.
Rosalie J. M.
Rosario A. M.
Rosas-Aburto A.
Rosciano F.
Rose H.
Rose J.
Rosenauer A.
Rosenkranz R.
Rosillo J. C.
Rosillo J. C.
Rosnecký V.
Ross F. M.
Ross U.
Rossberger S.
Rosseeva E.
Rossi M.
Rossi A. L.
Rossi Junior W. C.
Rossi M.
Rossi S.
Rossinyol E.
Rossmann M. G.
Rosso G.
Rossouw D.
Rossow U.
Rothenstein D.
Rothländer T.
Rotunno E.
Roussel L.
Roussel X.
Routledge M. N.
Rouveire J. L.
Rouviere J.
Rouviere J. L.
Rouvière J. L.
Roven H. J.
Rowlands N.
Rowlands N.
Rozental S.
Roŝker S.
Ruamsook S N.
Rubanov S.
Rubiano da Silva N.
Rubino S.
Rubira A. F.
Rudi S.
Rudmin M.
Rudneva M.
Rudolf D.
Rudolf E.
Rudolf M.
Ruffilli R.
Rug M.
Ruiz Perez A.
Ruiz-González L. M.
Ruiz-González L.
Ruiz-Larrea I.
Ruml T.
Rummeny E.
Rumé-Strobl J.
Runštuk J.
Russell G. T.
Russo G.
Russo G.
Rusz J.
Rutgers M.
Rutkowski B.
Rutten T.
Ruvalcaba J. L.
Ruzicka F.
Ryabchenko B.
Ryazantsev S.
Rybak P.
Rychtarikova R.
Rye M. J.
Ryll H.
Ryu S.
Rzchowski M. S.
Rämö O.
Réthoré J.
Rödel J.
Röder F.
Röder I. V.
Röder M. S.
Römer R.
Römpp A.
Rösner B.
Rösner H.
Røyset J.
Rümmeli M. H.
Růžek D.
SAHALAN A. Z.
SANTOS AIRES F. C.
SASAKI K.
SATO K.
SAUVAGE T.
SHAWUTI S.
SHI J.
SILVA B. M.
SILVA E. O.
SOLAKOĞLU S.
SONOBE S.
STAYKOV A.
STURM S.
SUETOMO Y.
SUNG B.
Saberi-Moghaddam R.
Sabo R.
Sabyasachi Saha
Sacha P.
Sader K.
Sadofev S.
Sagalowicz L.
Sagarzazu A.
Saggau P.
Saghafi S.
Saghi Z.
Saidi W.
Saidi w.
Saifitdinova A.
Saify M. T.
Saikawa S.
Saino T.
Saino T.
Saint-Antonin F.
Saint-Girons G.
Saito H.
Saito M.
Saito T.
Saito Y.
Saitoh K.
Saitow A.
Saka S. K.
Sakaebe H.
Sakaew W.
Sakaguchi M.
Sakakibara Y.
Sakaue M.
Sakuda Y.
Sakultanchareonchai S.
Sakurai H.
Salafranca J.
Salama N. M.
Salameh S.
Salamero J.
Salamon K.
Salazar-Alvarez G.
Salazar-Villatoro L.
Sales D. L.
Salgado C. M.
Salge T.
Saliba M.
Salido J.
Salih J.
Sall M.
Sallez N.
Salmeron M.
Saltzmann T.
Salucci S.
Salvador V.
Salviati G.
Salvo-Romero E.
Salzberger U.
Samardžija Z.
Sameshima K.
Samudram A.
Samuel I. D.
Samuelson L.
San German-Perez S.
San Juan J.
Sanati Nezhad A.
Sanchez F.
Sanchez A. M.
Sanchez F.
Sanchez M.
Sanchez S. I.
Sanchez-Sanchez M.
Sanchez-Santolino G.
Sanctuary R.
Sanders T.
Sandoval Jimenez A. s.
Sanen K.
Sang X.
Sangregorio C.
Sani M.
Sannomiya T.
Sano R.
Sano T.
Santamaria J.
Santamaría J.
Santana M M.
Santangelo P. S.
Santiago L. M.
Santiago N.
Santiago P.
Santiago U.
Santler B.
Santos A. S.
Santos Aires F.
Santos G.
Santos J.
Santos L. P.
Santos R. W.
Santos V. R.
Santos W. M.
Santos-Mallet J. R.
Sanz O.
Sanz-Ochotorena A.
Saowakon N.
Saraga Babić M.
Sarahan M. C.
Sarakonsri T.
Sarau G.
Sargsyan K. A.
Sarhan N. I.
Sarma D. D.
Sarto M. S.
Sartorel A.
Sartori A. F.
Sartory B.
Sarvas I.
Sarıoglu T.
Sasaki H.
Sasaki K.
Sasaki T.
Sasaki Y.
Sasaki Y. C.
Sasam F.
Sasam F. C.
Sass M.
Sassmann S.
Sathapathy S.
Sato C.
Sato K.
Sato M.
Sato T.
Sato Y.
Satoh Y.
Satoh K.
Satoh T.
Sauerová P.
Saunders M.
Sauvage X.
Savinov S. V.
Savkova J.
Savoie S.
Sawada H.
Sawicki M.
Sax S.
Sayed S. S.
Saéz P. L.
Saïdi W.
Sberveglieri G.
Scaramuzzo F.
Scavone Guillermo P.
Scavone P.
Scenini F.
Schaan G.
Schabes -Retchkiman P. S.
Schabes-Retchkiman P. S.
Schachinger T.
Schaedle T.
Schaefer G.
Schaer D.
Schaff O.
Schaffer B.
Schaffer M.
Schamm-Chardon S.
Schaper A. K.
Scharnowell R.
Schattschneider P.
Schauer P.
Schaumberg C. A.
Schauss J.
Schellenberger P.
Schenk A.
Schenk D.
Scherer T.
Scherrer B.
Schertel A.
Schertz F.
Scheu C.
Schieber N. L.
Schier J.
Schierholz R.
Schilling S.
Schimpke T.
Schindler P.
Schinnerl M.
Schippa S.
Schlarb H.
Schliwa A.
Schloffer M.
Schlom D. G.
Schlögl R.
Schlörb H.
Schmid H.
Schmid H. K.
Schmidt E. C.
Schmidt B.
Schmidt E. C.
Schmidt J.
Schmidt K.
Schmidt N. H.
Schmidt O.
Schmidt R.
Schmidt S. S.
Schmidt V.
Schmidt W.
Schmidtchen A.
Schmied R.
Schmitt L. A.
Schneider A. K.
Schneider C.
Schneider C. M.
Schneider J. M.
Schneider N. M.
Schneider R.
Schneider S.
Schnick W.
Schock F.
Schoenen F.
Scholz G.
Scholz M.
Schowalter M.
Schrader F.
Schraner E. M.
Schreck M.
Schreiber D. K.
Schrenker N.
Schrenková J.
Schreyeck S.
Schroeder J.
Schroeder J. A.
Schroeder S.
Schroeder W. A.
Schroettner H.
Schryvers D.
Schröder R. R.
Schubert J.
Schukov H. P.
Schulmeyer I.
Schultz L.
Schultz P.
Schulz R.
Schulze J. H.
Schulze W.
Schumacher C.
Schumacher P.
Schumann J.
Schué L.
Schwab Y.
Schwamm C. L.
Schwartz N.
Schwarz B.
Schwarz H.
Schwarz-Finsterle J.
Schwedt A.
Schweizer P.
Schwertner M.
Schwickert A.
Schwieger W.
Schäfer A.
Schäfer N.
Schäfer S.
Schäublin R.
Schöberl T.
Schönhense G.
Schühly W.
Schüler M.
Sciacca E.
Scigaj M.
Scott A. J.
Scott A. J.
Scotto M.
Scuderi M.
Scullion A.
Seabourne C. R.
Seabourne C. R.
Seabra S. H.
Sealey M.
Seara L. M.
Searle S.
Sebaihi N.
Seckin I.
Seckin İ.
Seddio S. M.
Sedlakova Z.
Sedlaříková M.
Sedlmaier S. J.
Sedlák P.
Sedláček L.
Sedmera D.
Sedov A.
Segers D.
Segura L.
Segura-Valdez M. L.
Seidel C. A.
Seidel F.
Seim C.
Seiner H.
Seiradake E.
Seiter J.
Seki T.
Sekiguchi H.
Sel S.
Selek S.
Selve S.
Semsari Parapari S.
Senna C. A.
Senturkoglu S.
Seo J. W.
Seog-Young Y.
Sepitka J.
Serag M. F.
Seral-Ascaso A.
Seras-Franzoso J.
Serga V.
Serin V.
Serra J.
Serra R.
Serrano-Ramón L.
Serényi M.
Sesé J.
Setou M.
Setter N.
Sever O.
Severa L.
Seveyrat L.
Sevim S.
Seyithanoglu M.
Sgambato A.
Sha G.
Shaburova O. V.
Shah B.
Shaikh T. R.
Shami G.
Shan Z.
Shan Z. W.
Shang L.
Shapter J.
Sharafutdinov I.
Sharath K.
Sharma M.
Sharma P.
Sharma R.
Sharma S. K.
Sharma s. K.
Sharman J.
Shaw J.
Shawrav M. M.
Shawrav M. M.
Shcherbakova D. M.
Sheehan J. J.
Shemet V.
Shen C.
Shen H. H.
Shen Y.
Sheng H.
Sheppard C.
Sheppard C. J.
Shi J.
Shi L.
Shi S.
Shibata K.
Shibata M.
Shibata N.
Shibuya S.
Shichiji T.
Shiekh R. A.
Shigeta M.
Shiloh R.
Shima M.
Shimada M.
Shimakawa Y.
Shimizu T.
Shimogori T.
Shimoyama H.
Shimoyamada A.
Shimura N.
Shin K.
Shin Y.
Shinde D.
Shindo D.
Shinkawa T.
Shinohara H.
Shinohara K.
Shiojiri M.
Shiono M.
Shipochka M.
Shirai T.
Shirazi M. N.
Shklover V.
Shklover V. Y.
Shokuhfar T.
Shomorony A.
Shtrikman H.
Shukla A. K.
Shunkov M. V.
Si-Young C.
Siciliano J. A.
Sickmann J.
Sicot M.
Siddheswaran R.
Sidjanin L.
Sieger M.
Siegmund H.
Siemons W.
Sigalas I.
Sigle W.
Sijbers J.
Sikora T.
Silke B.
Silva E. O.
Silva A. M.
Silva E. O.
Silva E. R.
Silva Gomes L.
Silva J. B.
Silva L. C.
Silva L. F.
Silva P. B.
Silva R.
Silva R. C.
Silva T. P.
Silva-de-Hoyos L. E.
Simaõ D.
Simioni C.
Simke J.
Simon P.
Simon U.
Simon-Najasek M.
Simonetti A.
Simonot L.
Simsek A.
Simsek E.
Simson M.
Simsone Z.
Simão R. A.
Simão T. P.
Simões G. F.
Simões L. A.
Sina M.
Sinclair R.
Sindona G.
Singelenburg F.
Singh A.
Singh A. P.
Singh G. K.
Singh I.
Singh V.
Sintorn I.
Sintorn I. M.
Siozios A.
Siqueira C. A.
Sirringhaus H.
Sitte W.
Situm A.
Sitzman S.
Sivakumar V.
Sjoman P. O.
Sjöström C.
Skalicky J.
Skjolding L. M.
Sklenička V.
Skoglundh M.
Skone G. S.
Skopalik J.
Skowron S. T.
Skurat E. V.
Slaby T.
Slabý T.
Slater T.
Slater T. J.
Slimak J.
Sloan J.
Slouf M.
Sloufova I.
Slusallek P.
Sládek Ľ.
Sládková J.
Smallenburg F.
Smarsly B. M.
Smetana K.
Smirnov E.
Smisdom N.
Smith A. E.
Smith C.
Smith C. L.
Smith D.
Smith D. C.
Smith D. J.
Smith K.
Smith N. I.
Smith T. L.
Smolders S.
Snaith H. J.
Snoeck E.
Snyders R.
Soares C. A.
Soares E. A.
Sobczak K.
Sobhon P.
Sobhon P.
Sobol M.
Sobotka R.
Sodek M.
Soeiro M. C.
Soejima Y.
Sofuoglu K.
Sogne E.
Sojref R.
Sokolova E.
Sokolova O.
Sokolova O. S.
Solange P. Y.
Solar J.
Solc P.
Soliwoda K.
Solla E. L.
Solorza-Feria O.
Solorzano I. G.
Solovyev A. G.
Soltau H.
Soltau J.
Solís J. L.
Solís M. T.
Solórzano-Naranjo I. G.
Somehara K.
Somekawa H.
Somogyi A.
Somsen C.
Sonawane M.
Soner B. C.
Song B.
Song D. S.
Song J.
Song K.
Song M.
Song T. E.
Song X.
Songhak Y.
Songkoomkrong S.
Songmuang R.
Sonobe S.
Soon L.
Soonklang N.
Sorbier L.
Soritau O.
Sorokin L. M.
Sorrentino A.
Sorzano C.
Sorzano C. O.
Sotelo J. R.
Sotiriou G. A.
Soto A. B.
Soto D. A.
Soto M.
Soto P. A.
Sougrat R.
Soukup J.
Soukup M.
Soulantica K.
Sousa A. A.
South K.
Southall N.
Souza D. S.
Souza E. B.
Souza L. C.
Souza P. L.
Souza R.
Sowa R.
Soysal T.
Spachholz E.
Spadaro M. C.
Spadoni T.
Spanielova H.
Spannenberger S.
Sparenberg M.
Spasevski L.
Specht P.
Spehner D.
Spektor G.
Spence J. C.
Spencer A.
Spengler B.
Spicuzza M. J.
Spiecker E.
Spiegelberg J.
Spikmans V.
Spiliotis X.
Spilman M.
Spinella C.
Spinella S.
Spomer W.
Spoutil F.
Sprocati A.
Spuch-Calvar M.
Srismith S.
Srivastava C.
Srivastava D.
Srivastava S.
Srivibool Rattanaporn. -.
Srot V.
Srot v.
Stacchiotti A.
Stach E. A.
Stadelmann P.
Stadlober B.
Stahlberg H.
Stakveev D.
Stambolova I.
Stambula S.
Stamps R. L.
Stange M.
Stanković N.
Stapel H.
Stark H.
Stark M.
Starsich F.
Staszewska M.
Statham P. J.
Stauffer D.
Stašková T.
Steart D.
Steel K. P.
Stefanov S.
Stefl M.
Steib F.
Steigenhöfer D.
Steiger-Thirsfeld A.
Steinacher R.
Stemmer S.
Stepan P.
Stephan O.
Stephens C. J.
Stephens I.
Stephenson L. T.
Steven A. C.
Stewart D.
Stewart P.
Steyer A.
Steyer A. M.
Stiel H.
Stierhof Y. D.
Stikic R. I.
Stipcich M.
Stitt M.
Stixová L.
Stoeckel D.
Stoeger-Pollach M.
Stoemenos J.
Stoian G.
Stolz W.
Stone D. S.
Storrie B.
Stowe D. J.
Strachota B.
Stracke G.
Stramer B.
Strassberger L.
Strassburg M.
Strasser P.
Straub A.
Straubinger R.
Strauch S.
Strbkova L.
Strenger C.
Stringaro A.
Strittmatter A.
Strnad M.
Strnadová M.
Stromme M.
Stroppa D. G.
Strueder L.
Stráský J.
Strüder L.
Stuhlmueller M.
Sturm S.
Sturm T.
Stys D.
Stéphan O.
Stöger-Pollach M.
Su D.
Su D. S.
Suarez E.
Subkorndej P.
Suchanova J.
Suchy T.
Sudfeld D.
Suenaga K.
Sueters J.
Sueyoshi H.
Suga M.
Sugahara D.
Sugawara A.
Sugioka N.
Sugita H.
Sugiyama A.
Suguihiro N. M.
Sui T.
Sui M. L.
Sui T.
Sukedai E.
Suksatu A.
Suludere Z.
Sulyok A.
Suman P. H.
Sumimoto N.
Summerfield R.
Summerfield R. A.
Summers H. D.
Sumpownon C.
Sumpter B. G.
Sun J.
Sun L.
Sun T.
Sunada S.
Sunaoshi T.
Sundaram S.
Sunding M. F.
Sung K. T.
Sung Jin Kang Sung-Hwan Kim Heung Nam Han Min-Ho Park Cheol-Woong Yang Hu-Chul Lee Yoon-Uk Heo Miyoung Kim
Sung Sik
Sung-Dae K.
Suntornsaratoon P.
Surrey A.
Susaki T.
Susan D. F.
Suski T.
Susman S.
Susor A.
Sutachan J.
Sutlupınar N.
Sutrova V.
Sutta P.
Suvorova A.
Suvorova A. A.
Suyetin M.
Suzuki K.
Suzuki M.
Suzuki S.
Suzuki T.
Suzuki Y.
Svedlindh P.
Svegliati S.
Svelto M.
Svendsen B.
Svidenská S.
Svoboda D.
Svoboda M.
Swaminathan S.
Swanson D.
Swieszkowski W.
Syahmeiyah WI
Sygusch J.
Sykes D.
Sylwestrzak M.
Szabó P. J.
Szabó T. G.
Szajna K.
Szczurek A.
Szczurek A. T.
Szepesi Á.
Szkudlarek A.
Szkulmowska A.
Szumska D.
Szívós J.
Sáfrán G.
Sánchez F.
Sánchez-Chardi A.
Sánchez-Chardi A.
Sánchez-García M. A.
Sánchez-Gil J. J.
Sánchez-Hidalgo R.
Sánchez-Mendieta V.
Sánchez-Mendieta V.
Söderström T.
Sönmez H.
Sørensen S. N.
Süzer O.
TALEB A. L.
TANAKA M.
TATSUMI K.
TEO H. C.
THERSLEFF T.
THOMAZINI B. F.
TIZNADO-OROZCO G. E.
TREPOUT S.
TUEL A.
TUNALI G.
TURAN S.
Tabasum M. R.
Tacke S.
Tagantsev A. K.
Taguchi N.
Taheri M.
Tai C. W.
Tainoff D.
Taipina M. O.
Tait E. W.
Takahashi C.
Takahashi K.
Takahashi Y.
Takaki S.
Takano A.
Takatori S.
Takayanagi K.
Takebe M.
Takeda M.
Takeda S.
Takeda Y.
Takeguchi M.
Takei H.
Takenaka K.
Takeno Y.
Takeshita H. T.
Talamás-Lara D.
Talamás-Rohana P.
Talbot E.
Talbot M. J.
Taleb-Ibrahimi A.
Talebi N.
Talele H.
Talgorn E.
Tallarek U.
Tamai T.
Tamborini M.
Tamburri E.
Tamotsu S.
Tamura K.
Tan G T.
Tan H.
Tan H. H.
Tan R.
Tan Z. Q.
Tanaka E.
Tanaka K.
Tanaka M.
Tanaka N.
Tanaka S.
Tanaka T.
Tanaka Y.
Tanbek K.
Tang W. X.
Tang Y.
Tang Y. X.
Tanigaki T.
Taniguchi T.
Taniguchi Y.
Tanji T.
Tanriverdi G.
Tanrıkulu-Kucuk S.
Tantale K.
Tanwar U. K.
Tapaszto O.
Tapul L.
Tarakina N. V.
Tararan A.
Targowski P.
Tarleton E.
Tarábková H.
Tasci A. I.
Taslidere E.
Tassin a. m.
Tasso F.
Tasyurekli M.
Tatarintsev A. A.
Tatematsu T.
Tatkiewicz W.
Tatlybaeva E. B.
Tatsumi K.
Taurino A.
Taute H.
Tavabi A. H.
Taverna D.
Tazaki A.
Tchenio P.
Tedde S. F.
Tedin K.
Tehrani A. Y.
Teichert N.
Teissier R.
Teixeira L.
Teixeira R.
Teixeira-Neto E.
Tejeda A.
Tekaya L.
Temel M.
Tencé M.
Tendeloo G. V.
Teodorescu V. S.
Teplý F.
Terada D.
Terasaki O.
Terasawa T.
Terauchi D.
Terauchi M.
Terazono H.
Terborg R.
Terranova M. L.
Terres E.
Terrones H.
Terrones M.
Terrés E.
Tesař K.
Tesner P.
Tessarek C.
Tessarolo F.
Tessier M. D.
Tessier P. Y.
Testillano P. S.
Tewari A.
Tewari R.
Thackery J.
Thakur I. S.
Thaler P.
Thamm C.
Thanachayanont C.
Tharad S.
Thelander E.
Thersleff T.
Thirunavukkarasu G.
Thiry M.
Thit A.
Thomas D. W.
Thomas P. A.
Thombare S. V.
Thompson A.
Thompson G. E.
Thompson K.
Thomsen E.
Thomä N. H.
Thorner G.
Thukral A. K.
Thust A.
Tian C. M.
Tian G.
Tian G. L.
Tichelaar F.
Tichelaar F. D.
Tihlarikova E.
Tilley D.
Timashev P. S.
Timischl F.
Timko B. P.
Timmermann J.
Timmermans F. J.
Timmermans J.
Timr S.
Timur S.
Tinoco M.
Tiribilli B.
Tissot N.
Titus s.
Tizei L H. G.
Tizei L.
Tizei L. G.
Tjin G.
Tlahuice Flores A.
To Baben M.
Toake Y.
Tobler K.
Tobler K.
Toca-Herrera J. L.
Toca-Herrera J.
Toda T.
Todorova E. V.
Todt J.
Togano K.
Tognolina M.
Togo A.
Tok O. E.
Tokimatsu R. C.
Tokunaga Y.
Tokura Y.
Toledo D. A.
Toledo-Antonio J. A.
Tolley A.
Tomancak P.
Tomasi C.
Tomastik C.
Tomašić N.
Tomizawa J.
Tomkinson T.
Tone S.
Tong W.
Tong Y. W.
Tongpeng S.
Topuria T.
Topuzoov A.
Tornos J.
Torrents O.
Torres F. J.
Torres M.
Torres S.
Torres-Cruz F. M.
Torres-Pardo A.
Torruella P.
Tort M.
Tortora M.
Torun H.
Toshimori K.
Tournié E.
Tovar J.
Tovar J. E.
Towe E.
Townley H. E.
Toyoda M.
Toyoura K.
Trampert A.
Tran C. M.
Trasobares S.
Trava-Airoldi V.
Travassos de Lima R.
Travers L.
Travessa D.
Trebbia J. B.
Treguer-Delapierre M.
Tremiliosi F. G.
Tremlová B.
Trepout s.
Trettenhahn G.
Treussart F.
Trewhitt H.
Trichet M.
Trimby P. W.
Trimmel G.
Triozon F.
Tripathi S.
Tripp P.
Troadec D.
Trojacanec - Pavlovska J.
Trojek T.
Tromp R. M.
Trottmann M.
Trtica M.
Truckenbrodt S.
Trudeau M.
Trujillo J. U.
Trunkin I. N.
Trunschke A.
Tréguer-Delapierre M.
Trías E.
Trügler A.
Tsai C. Y.
Tsai K. C.
Tsai A. P.
Tsai C. T.
Tsai C. Y.
Tsai H. C.
Tsai K. C.
Tsai S. P.
Tsai Y. T.
Tseng F. G.
Tseng F. G.
Tsiaoussis I.
Tsivoulas D.
Tsuchiya K.
Tsuchiya M.
Tsuda K.
Tsuda T.
Tsujimoto M.
Tsukamoto T.
Tsuneta R.
Tsuno K.
Tsuruta H.
Tsuruta T.
Tsymbal E. Y.
Tucuch J.
Tugcu V.
Tuková J.
Tuma J.
Tuma J. V.
Tuma L.
Tunca B.
Tuncdemir M.
Tunckan O.
Tung S. Y.
Tunçdemir M.
Tur V. A.
Turan S.
Turbin E.
Turchanin A.
Turková I.
Turkoz Uluer E.
Turner S.
Tusche C.
Tusek Znidaric M.
Twesten R. D.
Twesten R. D.
Tylewicz U.
Tyls F.
Tytko D.
Tyukalova E. V.
Tyurnina A.
Tyutyunnikov D.
Tátrai P.
Tändl J.
Téllez J L.
Téllez-Jurado L.
Tóth A. L.
Tóth L.
Tóth S.
Tóth-Kiss R. J.
Tögel S.
Třeštíková A.
ULRICH L.
Uchida M.
Uchiyama N.
Ueda M.
Uegaki K.
Uematsu F.
Ufheil J.
Uher F.
Uherek F.
Uhlemann S.
Ui K.
Ujihara T.
Ujimine S.
Ukraintsev E.
Ulfig R. M.
Ulicna L.
Uličná L.
Ulman V.
Ungermann J.
Unocic R. R.
Unzueta U.
Uppström M.
Uras F.
Urban K. W.
Urbina de Navarro C. .
Urbina de Navarro C.
Urbina de Navarro C. T.
Urbánek M.
Ushijima M.
Ushikubo T.
Uslu S.
Uslu E.
Uslu S.
Uslu U.
Usui T.
Usukura J.
Utke I.
Uysal Tüten P.
VILLAIN s.
Vaccaro C.
Vackova T.
Vacková T.
Vacík T.
Vaibhav K.
Vajtai R.
Vakahy A.
Valach J.
Valadez M.
Valdez O.
Valdés A.
Valenta J.
Valery A.
Valiante S.
Valiante S.
Valigurová A.
Valley J. W.
Valpuesta J. M.
Van Aert S.
Van Aken P. A.
Van Boxem R.
Van Cappellen E.
Van Dyck D.
Van Elslande E.
Van Helvoort A.
Van Leer B.
Van Marcke P.
Van Niel G.
Van Rooy M.
Van Swaaij R.
Van Tandeloo S.
Van Tendeloo G.
Van Wilpe E.
Van de Moortele B.
Van de Moortèle B.
Van den Broek W.
Van der Schoor C.
Van der Zant H. S.
Van herle J.
Vancova M.
Vancová M.
Vander Voort G. F.
Vandervorst W.
Vanmaekelbergh D.
Vannucci L.
Vanover D.
Vaněček J.
Vardi N.
Varea A.
Varela J. A.
Varela M.
Varfolomeev I. A.
Varga G.
Vargas B N.
Vargas J.
Vargas-Becerril N.
Varier G. K.
Varitis S.
Vartiainen M.
Vartic O.
Vasconcelos T. L.
Vasilenko I.
Vasiliev A. L.
Vassilev S.
Vats N.
Vavrova J.
Vazdar M.
Vazquez E.
Vazquez C.
Vaškovicová N.
Veciana J.
Vega A.
Vega- López M. A.
Veith L.
Vejpravova J.
Velebný V.
Veleva L. V.
Velikanova T. Y.
Veljović Đ. N.
Velázquez M. M.
Venezia A M.
Venien-Bryan C.
Venit T.
Venit T.
Venkatesan S.
Vennéguès P.
Ventrella V. A.
Veranič P.
Verbeeck J.
Verbruggen S. W.
Verdánová M.
Veretennikov L. M.
Verezhak M.
Vergari C.
Verkade P.
Verkhusha V. V.
Versaci R. A.
Vesely P.
Vesely T.
Veselý J.
Vetterlein M.
Vetushka A.
Veziroglu L.
Vezzoli E.
Večko Pirtovšek T.
Vianello E.
Viani A.
Vicarelli L.
Vicario M.
Vicente T. M.
Vidal H.
Vidal K.
Vidal L. O.
Vieira C. P.
Vieira C. S.
Viennois R.
Vieweg B.
Vignado C.
Vignoles G.
Vignoli R.
Vigouroux M.
Vigouroux M.
Vigouroux M. P.
Vihinen H.
Viita T.
Vila L.
Vila T.
Vila T. V.
Vilanova-Martínez P.
Vilchis-Nestor A. R.
Villa A.
Villa E.
Villalejo-Fuerte M.
Villar Arias S.
Villar M. A.
Villar S.
Villaverde A.
Villinger C.
Vilotic M.
Vilotić M.
Vinciguerra P.
Vindigni A.
Virag P.
Virga A.
Virtanen J.
Visart de Bocarmé T.
Vishnyakov V.
Vishwanath T.
Vissers R.
Vittori-Antisari M.
Vizcay-Barrena G.
Vladimir Yu. Ulianitsky
Vlassiouk I.
Vlckova B.
Vlugt T. J.
Vodopivec F.
Vogelgesang R.
Vogts A.
Volk A.
Volkenandt T.
Volkmann N.
Volkmann N.
Volkmmann N.
Volpe L.
Volz K.
Vonck J.
Vondrák J.
Vondráková Z.
Vondráčková M.
Voortman L. M.
Vorokhta M.
Voskanian N.
Vourlias G.
Vouroutzis N.
Voyles P. M.
Vranjes N.
Vránová J.
Vukojević K.
Vyacheslav I. Mali
Vysniauskas A.
Vystavel T.
Vystavěl T.
Vázquez-Vázquez C.
WAN NUR SYAWANI W.
WAN NUR SYAZWANI
WANG J.
WOLFMANN J.
WORTHAM L.
Waag A.
Wacey D.
Wachsmuth P.
Wachter S.
Wacker C.
Wacker I.
Wacker I. U.
Wagemaker M.
Wagener R.
Wagenknecht T.
Wagner J.
Wagner J. B.
Waitz T.
Wakasugi T.
Waku Y.
Wakui S.
Walch A.
Waldmann D.
Walford S.
Walia A.
Waligórski P.
Walker C. G.
Wall D.
Wall D. C.
Wall G. V.
Wallace R.
Wallenberg L. R.
Wallisch W.
Walls M.
Walmsley J. C.
Walse B.
Walsh M. J.
Walter P.
Walters D.
Walther P.
Walther R.
Walther T.
Walz T.
Wan W.
Wandrol P.
Wang H. W.
Wang X.
Wang X. L.
Wang C.
Wang C. H.
Wang D.
Wang G.
Wang G. Q.
Wang H.
Wang H. W.
Wang J.
Wang L J.
Wang L.
Wang L. H.
Wang L. Q.
Wang P.
Wang S. J.
Wang X.
Wang X. L.
Wang X. M.
Wang Y G.
Wang Y M Ge B H Che G C
Wang Y.
Wang Y. C.
Wang Z.
Wang Z. J.
Wang Z. Q.
Wang c.
Wanichanon C.
Wanner G.
Wanzenboeck H. D.
Wanzenboeck H. D.
Ward M. R.
Warner J. H.
Warot-Fonrose B.
Warren G.
Warren O.
Watanabe H.
Watanabe K.
Watanabe M.
Watanabe T.
Watano S.
Waterhouse P. M.
Watson M.
Wattebled F.
Wazen R. M.
Webb S. E.
Weber A.
Weber H. B.
Weber K.
Webster P.
Wege C.
Wegele T.
Wegscheider R.
Wehmann H. H.
Wei F.
Wei H.
Wei L. L.
Wei X. K.
Weidinger M.
Weinberg G.
Weinert M.
Weirich T.
Weisbecker P.
Weiss E.
Welk B.
Wells L.
Wells T.
Wen C. Y.
Wen J.
Wen M.
Wen S. P.
Wen T.
Wendt C.
Wendt F.
Weninger W. J.
Wenner S.
Wentz S. C.
Wepf R.
Wepf R. A.
Werckmann J.
Werner K.
Wernitznig S.
Wessels J.
Westermark G.
Westraadt J. E.
Weyda F.
Weyland M.
Wheatley A. E.
Whetten R.
Whitby J.
White R. G.
White T.
Whiting J.
Whitmore L.
Whitmore L. C.
Whyte J.
Wiengmoon A.
Wierzbińska M.
Wiesemann U.
Wiezorek J. M.
Wild J.
Wild P.
Wilde G.
Wilde S. A.
Wildgruber M.
Wilfried V.
Wilkin G.
Wilkinson A. J.
Wilkinson N.
Willhammar T.
Williams G. M.
Williams J. S.
Williams R. J.
Williams W.
Willinger M. G.
Wilson C. M.
Wilson D.
Wilson R.
Windl W.
Winkler R.
Winsel S.
Winter B.
Winterstein J.
Wirth R.
Wirtz T.
Wisnet A.
Wisnivesky Rocca Rivarola F.
Withers P. J.
Withers R.
Withers R. L.
Witherspoon K. C.
Withyachumnarnkul B.
Witte G.
Witters L.
Wittig J.
Wittig J. E.
Wittmann T.
Woehl T. J.
Wohland T.
Wojcik T.
Wolf W.
Wolf D.
Wolferen H. A.
Wolfert M. A.
Wolfgang S.
Wollgarten M.
Wollschläger N.
Womack C.
Wong A. B.
Wong Y. H.
Wong-Leung J.
Wongdee K.
Woo J.
Woo P.
Woo S. Y.
Woodward D. I.
Worman C. O.
Wouessidjewe D.
Wouossaju S.
Wright E. R.
Wright I.
Wright S. I.
Wu C. L.
Wu C. Y.
Wu D.
Wu H.
Wu L.
Wu M.
Wu M. Y.
Wu S.
Wu T.
Wuhrer R.
Wuttig M.
Wählby C.
Wéber F.
Xanthakis J. P.
Xi L.
Xia J. H.
Xia K.
Xia Y.
Xiang S. S.
Xiao J.
Xiaoyan Zhong
Xie D.
Xie J.
Xie L.
Xie Y.
Xie Z. L.
Xing H.
Xing Y.
Xing Y. T.
Xu B. B.
Xu H.
Xu P.
Xu Q.
Xu W.
Xu X.
Xu X. P.
Xu Z.
YAMANE H.
YAMAOKA N.
YASUHARA A.
YIN J.
YOO S. J.
YOSHIDA Y.
YRIEIX B.
YU D.
YURDAKUL H.
Yada C.
Yada K.
Yagi K.
Yaguchi T.
Yakimchuk I. V.
Yakimova R.
Yakushevska A.
Yakıncı Ö. F.
Yalcin A. O.
Yamada H.
Yamada K.
Yamada S.
Yamagata M.
Yamagishi K.
Yamaguchi K.
Yamaguchi M.
Yamakami W. J.
Yamamoto A.
Yamamoto H.
Yamamoto K.
Yamamoto M.
Yamamoto N.
Yamamoto T.
Yamamoto Y.
Yamanaka M.
Yamanaka Y.
Yamane M.
Yamasaki J.
Yamasaki K.
Yamasaki M.
Yamasaki Y. Y.
Yamashita K.
Yamashita T.
Yamazaki S.
Yaminsky I. V.
Yanase R.
Yang J. R.
Yang D.
Yang B.
Yang C.
Yang C. W.
Yang D.
Yang H.
Yang J. C.
Yang J. R.
Yang L.
Yang M.
Yang S.
Yang T.
Yang Y.
Yang Y. S.
Yang Z.
Yankovich A. B.
Yao Y.
Yapislar H.
Yaprak Sarac E.
Yarmak A. V.
Yarwood A.
Yassin A.
Yasuanga T.
Yasuda H.
Yasuda K.
Yasuda M.
Yasue T.
Yasuhara A.
Yasunaga T.
Yavari A. R.
Yañez M. J.
Ydenberg C.
Ye H. Q.
Ye S. J.
Yedra L.
Yelken G. O.
Yen H. W.
Yentekakis I. V.
Yeoh C. S.
Yeom J.
Yeste M. P.
Yetmen O.
Yeğen B. Ç.
Yi G. C.
Yildirim S.
Ying S.
Ying S.
Yohda M.
Yonemaru Y.
Yoo H.
Yoo M. W.
Yoon K. B.
Yoon S.
Yoshiasa A.
Yoshida K.
Yoshida H.
Yoshida K.
Yoshida R.
Yoshida S.
Yoshida T.
Yoshinari M.
Yoshioka T.
Yotsuji T.
You C. J.
Young M.
Young Mok R.
Young N. P.
Young Suk Yu
Yu D.
Yu K.
Yu K. M.
Yu R C.
Yu R.
Yu X.
Yu X. Z.
Yu Y.
Yu Y. D.
Yuan H.
Yuan J.
Yuasa S.
Yucel D.
Yucel D.
Yue Y. H.
Yuk J. M.
Yuksel M.
Yumoto S.
Yun J. D.
Yurdakul H.
Yüksel M.
Yılmazer S.
ZAPOLSKY H.
ZHANG J.
ZORIN S. A.
Zach J.
Zaefferer S.
Zafirov D.
Zagar K.
Zahel T.
Zaitsev S. V.
Zakel J.
Zakharov D.
Zakhozheva M.
Zalazar M.
Zaletel I.
Zaloudkova M.
Zaluzec N. J.
Zamani R. R.
Zamora Rangel L. z.
Zamora-Cura A.
Zan R.
Zanaga D.
Zancan D.
Zancanaro C.
Zandbergen H. W.
Zandbergen H.
Zandbergen H. W.
Zanella R.
Zanfoni N.
Zanin D. A.
Zankel A.
Zapata Peñasco I.
Zara S.
Zaramella P.
Zarantonello V.
Zaroui A.
Zarsky S.
Zarębski M.
Zaumseil J.
Zavala G.
Zavaleta-Mancera H. A.
Zavašnik J.
Zbarsky V.
Zechmann B.
Zeiner C.
Zellnig G.
Zeman M.
Zemčík P.
Zeng L. J.
Zeng Y. W.
Zepeda A.
Zettl A.
Zeuschner D.
Zha X.
Zhan Q.
Zhan W.
Zhang H.
Zhang Z.
Zhang B.
Zhang B. S.
Zhang C.
Zhang D.
Zhang D. F.
Zhang G.
Zhang H.
Zhang J.
Zhang J. G.
Zhang J. P.
Zhang J. Y.
Zhang L.
Zhang P.
Zhang Q H.
Zhang Q.
Zhang S.
Zhang W.
Zhang X.
Zhang Y.
Zhang Y. F.
Zhang Z.
Zhang Z. L.
Zhao D.
Zhao H.
Zhao M.
Zhao M. Q.
Zhao S.
Zhdanov A. V.
Zheng C. L.
Zheng H.
Zheng H. T.
Zheng J.
Zheng K.
Zheng L.
Zheng w.
Zhigalina O. M.
Zhigalina V. G.
Zhiying Cheng
Zhong X. L.
Zhong X. Y.
Zhou D.
Zhou H.
Zhou K. G.
Zhou O.
Zhou W.
Zhu C.
Zhu G.
Zhu G. Q.
Zhu G. Z.
Zhu J.
Zhu Y.
Zhu Y. M.
Zhuang X.
Zickler G.
Zickler G. A.
Ziemer K.
Zila V.
Zillmann D.
Zimina M.
Zimmer C.
Zimmermann S.
Zimprich M.
Zinicovscaia I.
Ziqiang Wang
Zitová B.
Ziętara M.
Zobelli A.
Zoberbier T.
Zonnevylle A. C.
Zorko M.
Zorrilla C.
Zorrilla-Cangas C.
Zou J.
Zou X.
Zou X. D.
Zrník J.
Zschech E.
Zuber G.
Zuma A.
Zuma A. A.
Zunino P.
Zuo B.
Zuo J M.
Zuo J.
Zuo J. M.
Zurmanova J.
Zuschratter W.
Zweck J.
Zweiacker K. W.
Zytkiewicz Z. R.
adeyemo k. a.
and Franklin
balogun w. g.
cobham a. e.
da Fonseca Martins Gomes O.
de Almeida L. H.
de Araújo M. R.
de Azevedo R. B.
de Barroa A. T.
de Carlan Y.
de Heer W. A.
de Hoyos Vega J. M.
de Jonge M. D.
de Jonge N.
de Julián Fernández C.
de Kloe R.
de Knoop L.
de Lima C.
de Marco A.
de Nijs B.
de Souza W.
de Souza W.
de Teresa J. M.
de With G.
de la Fuente J. M.
de la Mata M.
de la Peña F.
deOliveira A. P.
den Dekker A.
den Hertog M. I.
dos Santos G. R.
enaibe b. u.
imam a.
ishola a. o.
kumar S.
lić V. L.
olajide o. j.
van Aken A. P.
van Aken P. A.
van Benthem K.
van Blaaderen A.
van Blaaderen A.
van Breemen C.
van Dijk-Moes R.
van Driel L.
van Driel R. R.
van Duinen G.
van Helvoort A. T.
van Hoorn C. H.
van Huis M.
van Huis M. A.
van Loon A.
van Niftrik L.
van Oosten Slingeland D. S.
van Raaij M. J.
van Rooy M. J.
van Staden V.
van Teeseling M.
van Tendeloo G.
van Vilsteren C.
van de Water L.
van den Boogaard R.
van den Bos K.
van den Burg J.
van der Merwe C. F.
van der Spuy W. J.
von Harrach H. S.
von Känel H.
von Wezyk A.
wang X. M.
Álvares da Silva G. H.
Álvarez P M. A.
Álvarez-Gregoso O.
Álvarez-Pérez M. A.
Åkesson J.
Çadırcı S.
Çetinel Ş.
Çeçener G.
Çilingir Ö. T.
Ögüt B.
Österle W.
Özdemir İ.
Özer S.
Özyurt N.
Özyılmaz Yay N.
Üstüner K.
Čapek J.
Čapek M.
Čeh M.
Černá M.
Červinka M.
Čmiel V.
Čobić A.
Čudek P.
Čupera J.
Čupera J.
Číp P.
Čížek J.
Ďurkovič J.
Łuszczek D.
Şehirli A. Ö.
Şener G.
Şener G.
Şener T. E.
Şimşek F.
Şirvancı S.
Šajgalík P.
Šantić A.
Šatka A.
Šašek P.
Šefců R.
Šest I.
Šestan A.
Šesták J.
Šetina Batič B.
Šikola T.
Šipušić J.
Šlouf M.
Šmigová J.
Šmilauerová J.
Šmíd O.
Šolić S.
Šoufek M.
Špaček T.
Šroubek F.
Štolcová L.
Štrus J.
Šturm S.
Štěpán P.
Šubrt J.
Švancárová M.
Švindrych Z.
Żurek-Biesiada D.
Żurek-Biesiada D. J.
Žagar K.
Žnidaršič N.
Žužek B.
↑ Go to the Table of content ↑