PMID- 32939606
OWN - NLM
STAT- MEDLINE
DCOM- 20210913
LR  - 20240330
IS  - 1573-6849 (Electronic)
IS  - 0967-3849 (Print)
IS  - 0967-3849 (Linking)
VI  - 28
IP  - 3-4
DP  - 2020 Dec
TI  - Prospects and limitations of expansion microscopy in chromatin ultrastructure 
      determination.
PG  - 355-368
LID - 10.1007/s10577-020-09637-y [doi]
AB  - 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 ~4.2-times physically expanded nuclei corresponding to a maximal resolution of 
      ~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 ~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.
FAU - Kubalova, Ivona
AU  - Kubalova I
AD  - Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, 
      06466, Seeland, Germany.
FAU - Schmidt Cernohorska, Marketa
AU  - Schmidt Cernohorska M
AD  - Laboratory of Adaptive Immunity, Institute of Molecular Genetics,, Academy of 
      Sciences of the Czech Republic, Prague, Czech Republic.
FAU - Huranova, Martina
AU  - Huranova M
AD  - Laboratory of Adaptive Immunity, Institute of Molecular Genetics,, Academy of 
      Sciences of the Czech Republic, Prague, Czech Republic.
FAU - Weisshart, Klaus
AU  - Weisshart K
AD  - Carl Zeiss Microscopy GmbH, 07745, Jena, Germany.
FAU - Houben, Andreas
AU  - Houben A
AD  - Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, 
      06466, Seeland, Germany.
FAU - Schubert, Veit
AU  - Schubert V
AUID- ORCID: 0000-0002-3072-0485
AD  - Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, 
      06466, Seeland, Germany. schubertv@ipk-gatersleben.de.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20200917
PL  - Netherlands
TA  - Chromosome Res
JT  - Chromosome research : an international journal on the molecular, supramolecular 
      and evolutionary aspects of chromosome biology
JID - 9313452
RN  - 0 (Chromatin)
SB  - IM
MH  - Cell Nucleus/ultrastructure
MH  - Chromatin/*ultrastructure
MH  - Fluorescent Antibody Technique
MH  - Hordeum/genetics
MH  - In Situ Hybridization, Fluorescence
MH  - Microscopy/*methods/standards
PMC - PMC7691311
OTO - NOTNLM
OT  - Chromatin
OT  - Expansion microscopy
OT  - Hordeum vulgare
OT  - Nucleus
OT  - Structured illumination microscopy
EDAT- 2020/09/18 06:00
MHDA- 2021/09/14 06:00
PMCR- 2020/09/17
CRDT- 2020/09/17 05:46
PHST- 2020/06/01 00:00 [received]
PHST- 2020/08/05 00:00 [accepted]
PHST- 2020/07/28 00:00 [revised]
PHST- 2020/09/18 06:00 [pubmed]
PHST- 2021/09/14 06:00 [medline]
PHST- 2020/09/17 05:46 [entrez]
PHST- 2020/09/17 00:00 [pmc-release]
AID - 10.1007/s10577-020-09637-y [pii]
AID - 9637 [pii]
AID - 10.1007/s10577-020-09637-y [doi]
PST - ppublish
SO  - Chromosome Res. 2020 Dec;28(3-4):355-368. doi: 10.1007/s10577-020-09637-y. Epub 
      2020 Sep 17.