Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F68378050%3A_____%2F20%3A00538175" target="_blank" >RIV/68378050:_____/20:00538175 - isvavai.cz</a>

Result on the web

<a href="https://link.springer.com/article/10.1007/s10577-020-09637-y" target="_blank" >https://link.springer.com/article/10.1007/s10577-020-09637-y</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1007/s10577-020-09637-y" target="_blank" >10.1007/s10577-020-09637-y</a>

Result language

angličtina

Original language name

Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

Original language description

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.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

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OECD FORD branch

30102 - Immunology

Project

<a href="/en/project/GJ17-20613Y" target="_blank" >GJ17-20613Y: BBSome and actin interplay in ciliogenesis and formation of the immunological synapse</a><br>

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2020

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Chromosome Research

ISSN

0967-3849

e-ISSN

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Volume of the periodical

28

Issue of the periodical within the volume

3-4

Country of publishing house

NL - THE KINGDOM OF THE NETHERLANDS

Number of pages

14

Pages from-to

355-368

UT code for WoS article

000571228600001

EID of the result in the Scopus database

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