Number of the records: 1

Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

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) 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. 355-368
Number 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	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

Number of the records: 1