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Article

Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

1
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, D-06466 Seeland, Germany
2
Centre of the Region Hana for Biotechnological and Agricultural Research, Institute of Experimental Botany of the Czech Academy of Sciences, 77900 Olomouc, Czech Republic
3
Carl Zeiss Microscopy GmbH, D-07745 Jena, Germany
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: George Komis
Int. J. Mol. Sci. 2021, 22(4), 1903; https://doi.org/10.3390/ijms22041903
Received: 22 December 2020 / Revised: 4 February 2021 / Accepted: 10 February 2021 / Published: 14 February 2021
(This article belongs to the Special Issue Advances in Plant Cell Imaging)
The importance of fluorescence light microscopy for understanding cellular and sub-cellular 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 restriction. 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 molecules. 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. View Full-Text
Keywords: chromatin; deconvolution microscopy; Hordeum vulgare; metaphase chromosome; nanoscopy; photoactivated localization microscopy; stimulated emission depletion microscopy; structured illumination microscopy; topoisomerase II; wide-field microscopy chromatin; deconvolution microscopy; Hordeum vulgare; metaphase chromosome; nanoscopy; photoactivated localization microscopy; stimulated emission depletion microscopy; structured illumination microscopy; topoisomerase II; wide-field microscopy
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MDPI and ACS Style

Kubalová, I.; Němečková, A.; Weisshart, K.; Hřibová, E.; Schubert, V. Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes. Int. J. Mol. Sci. 2021, 22, 1903. https://doi.org/10.3390/ijms22041903

AMA Style

Kubalová I, Němečková A, Weisshart K, Hřibová E, Schubert V. Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes. International Journal of Molecular Sciences. 2021; 22(4):1903. https://doi.org/10.3390/ijms22041903

Chicago/Turabian Style

Kubalová, Ivona, Alžběta Němečková, Klaus Weisshart, Eva Hřibová, and Veit Schubert. 2021. "Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes" International Journal of Molecular Sciences 22, no. 4: 1903. https://doi.org/10.3390/ijms22041903

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