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Prospects and limitations of expansion microscopy in chromatin ultrastructure determination
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SYSNO ASEP 0538175 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Prospects and limitations of expansion microscopy in chromatin ultrastructure determination Author(s) Kubalova, I. (DE)
Schmidt Černohorská, Markéta (UMG-J) ORCID, RID
Huranová, Martina (UMG-J) ORCID
Weisshart, K. (DE)
Houben, A. (DE)
Schubert, V. (DE)Number of authors 6 Source Title Chromosome Research. - : Springer - ISSN 0967-3849
Roč. 28, č. 3-4 (2020), s. 355-368Number of pages 14 s. Publication form Online - E Language eng - English Country NL - Netherlands Keywords Chromatin ; Expansion microscopy ; Nucleus ; Structured illumination microscopy ; Hordeum vulgare Subject RIV EB - Genetics ; Molecular Biology OECD category Immunology R&D Projects GJ17-20613Y GA ČR - Czech Science Foundation (CSF) Method of publishing Open access Institutional support UMG-J - RVO:68378050 UT WOS 000571228600001 DOI https://doi.org/10.1007/s10577-020-09637-y Annotation Expansion microscopy (ExM) is a method to magnify physically a specimen with preserved ultrastructure. It has the potential to explore structural features beyond the diffraction limit of light. The procedure has been successfully used for different animal species, from isolated macromolecular complexes through cells to tissue slices. Expansion of plant-derived samples is still at the beginning, and little is known, whether the chromatin ultrastructure becomes altered by physical expansion. In this study, we expanded isolated barley nuclei and compared whether ExM can provide a structural view of chromatin comparable with super-resolution microscopy. Different fixation and denaturation/digestion conditions were tested to maintain the chromatin ultrastructure. We achieved up to similar to 4.2-times physically expanded nuclei corresponding to a maximal resolution of similar to 50-60 nm when imaged by wild-field (WF) microscopy. By applying structured illumination microscopy (SIM, super-resolution) doubling the WF resolution, the chromatin structures were observed at a resolution of similar to 25-35 nm. WF microscopy showed a preserved nucleus shape and nucleoli. Moreover, we were able to detect chromatin domains, invisible in unexpanded nuclei. However, by applying SIM, we observed that the preservation of the chromatin ultrastructure after the expansion was not complete and that the majority of the tested conditions failed to keep the ultrastructure. Nevertheless, using expanded nuclei, we localized successfully centromere repeats by fluorescence in situ hybridization (FISH) and the centromere-specific histone H3 variant CENH3 by indirect immunolabelling. However, although these repeats and proteins were localized at the correct position within the nuclei (indicating a Rabl orientation), their ultrastructural arrangement was impaired. Workplace Institute of Molecular Genetics Contact Nikol Škňouřilová, nikol.sknourilova@img.cas.cz, Tel.: 241 063 217 Year of Publishing 2021 Electronic address https://link.springer.com/article/10.1007/s10577-020-09637-y
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