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FLUORESCENCE SPECTROSCOPY AND MICROSCOPY: METHODS AND PROTOCOLS
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SYSNO ASEP 0506914 Document Type M - Monograph Chapter R&D Document Type Monograph Chapter Title Z-Scan Fluorescence Correlation Spectroscopy as a Tool for Diffusion Measurements in Planar Lipid Membranes Author(s) Steinberger, T. (CZ)
Macháň, Radek (UFCH-W)
Hof, Martin (UFCH-W) RID, ORCIDSource Title FLUORESCENCE SPECTROSCOPY AND MICROSCOPY: METHODS AND PROTOCOLS, Methods and Protocols. - Totowa : HUMANA PRESS INC, 2014 / Engelborghs Y. ; Visser A.J.W.G. - ISSN 1064-3745 - ISBN 978-1-62703-648-1 Pages roč. 1076 (2014), s. 617-634 Series Methods in Molecular Biology Number of pages 18 s. Number of pages 817 Publication form Print - P Language eng - English Country US - United States Keywords lifetime correlation spectroscopy ; optical saturation ; continuous-wave ; bilayers ; coefficients ; accuracy ; systems ; model ; mica Subject RIV CF - Physical ; Theoretical Chemistry OECD category Physical chemistry R&D Projects GAP208/10/0376 GA ČR - Czech Science Foundation (CSF) Institutional support UFCH-W - RVO:61388955 UT WOS 000328374800029 EID SCOPUS 84934443471 DOI 10.1007/978-1-62703-649-8_28 Annotation Studies of lateral diffusion are used for the characterization of the dynamics of biological membranes. One of the techniques that can be used for this purpose is fluorescence correlation spectroscopy (FCS), which belongs to the single-molecule techniques. Unfortunately, FCS measurements, when performed in planar lipid systems, are associated with a few sources of inaccuracy in the determination of the lateral diffusion coefficient. The main problems are related to the imperfect positioning of the laser focus relative to the plane of the sample. Another source of inaccuracy is the requirement for external calibration of the detection volume size. This protocol introduces a calibration-free method called Z-scan fluorescence correlation spectroscopy (Z-scan FCS), which is based on the determination of the diffusion time and particle number in steps along the optical (z-) axis by sequential FCS measurements. Z-scan FCS could be employed for diffusion measurements in planar membrane model systems-supported phospholipid bilayers (SPBs) and giant unilamellar vesicles (GUVs) and also in biological membranes. A result from measurements in SPBs is also presented in the protocol as a principle example of the Z-scan technique. Workplace J. Heyrovsky Institute of Physical Chemistry Contact Michaela Knapová, michaela.knapova@jh-inst.cas.cz, Tel.: 266 053 196 Year of Publishing 2020
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