Distribution of staining agents in samples of mice soft tissues prepared for Serial Block Face SEM

0568500 - ÚPT 2023 CZ eng A - Abstrakt
Nebesářová, Jana - Ďurinová, Eva - Kitzberger, František - Skoupý, Radim - Týč, Jiří
Distribution of staining agents in samples of mice soft tissues prepared for Serial Block Face SEM.
16th Multinational Congress on Microscopy, 16MCM, 04-09 September 2022, Brno, Czech Republic. Book of abstracts. Brno: Czechoslovak Microscopy Society, 2022 - (Krzyžánek, V.; Hrubanová, K.; Hozák, P.; Müllerová, I.; Šlouf, M.). s. 189-190. ISBN 978-80-11-02253-2.
[Multinational Congress on Microscopy /16./. 04.09.2022-09.09.2022, Brno]
Grant CEP: GA TA ČR(CZ) TN01000008; GA MŠMT(CZ) LM2018129
Institucionální podpora: RVO:68081731 ; RVO:60077344
Klíčová slova: SBF SEM * biological sample preparation * chemical reagents * electron microscopy
Obor OECD: Cell biology
https://www.16mcm.cz/wp-content/uploads/2022/09/16MCM-abstract-book.pdf

Serial block-face scanning electron microscopy (SBF SEM) uses an ultramicrotome installed inside the microscope to cut away ultrathin slices together with backscatter imaging to visualise
each newly exposed layer. Such arrangement brings the possibility to acquire a series of images allowing reliable and precise volume reconstruction. The biggest challenges we face in SBEM are sample preparation, finding the region of interest (ROI) within the sample, sample charging during the imaging, and finally, data processing and storage. Insufficient contrast in specimens often seriously limits the visualisation and makes the sample preparation one of the crucial steps in the SBEM analysis. The sample preparation is based on procedures standardly used for transmission electron microscopy. It starts with chemical fixation followed by dehydration, including en bloc staining. The process is finished by sample embedding in a hard epoxy resin. Several protocols have been developed by modifying the chemicals used, incubation times or temperatures. This study compared these protocols and evaluated the resulting contrast for SBF SEM analysis of soft animal tissues like mouse brain, liver and kidney. The samples prepared according to these protocols were analysed using light microscopy, SBF-SEM examination, and EDX analysis. We found out that the frequently used protocol suggested by Deerinck et al. resulted in an unevenly contrasted sample, where only thin outer regions approx. up to 100 µm below the surface were osmificated sufficiently. The protocol suggested by Hua et al. provided the homogenous stained layer with an almost double thickness. The protocol using tannic acid as a mordant gave an excellent result with minimal difference in contrast between the samples central and edge areas. In contrast, a protocol called fBROPA, which omits uranyl acetate, led to weak and inhomogeneous contrast mainly in the central parts of the specimen.
Trvalý link: https://hdl.handle.net/11104/0339801