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Comparing super-resolution microscopy techniques to analyze chromosomes
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SYSNO ASEP 0545865 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Comparing super-resolution microscopy techniques to analyze chromosomes Author(s) Kubalová, I. (CZ)
Němečková, Alžběta (UEB-Q) ORCID
Weisshart, K. (DE)
Hřibová, Eva (UEB-Q) RID, ORCID
Schubert, V. (DE)Number of authors 5 Article number 1903 Source Title International Journal of Molecular Sciences. - : MDPI
Roč. 22, č. 4 (2021)Number of pages 19 s. Language eng - English Country CH - Switzerland Keywords Chromatin ; Deconvolution microscopy ; Hordeum vulgare ; Metaphase chromosome ; Na-noscopy ; Photoactivated localization microscopy ; Stimulated emission depletion microscopy ; Structured illumination microscopy ; Topoisomerase II ; Wide-field microscopy OECD category Biochemistry and molecular biology R&D Projects EF16_019/0000827 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Method of publishing Open access Institutional support UEB-Q - RVO:61389030 UT WOS 000623902300001 EID SCOPUS 85100752070 DOI 10.3390/ijms22041903 Annotation The importance of fluorescence light microscopy for understanding cellular and sub-cel-lular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this re-striction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single mole-cules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution. Workplace Institute of Experimental Botany Contact David Klier, knihovna@ueb.cas.cz, Tel.: 220 390 469 Year of Publishing 2022 Electronic address http://doi.org/10.3390/ijms22041903
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