Comparing super-resolution microscopy techniques to analyze chromosomes

0545865 - ÚEB 2022 RIV CH eng J - Journal Article
Kubalová, I. - Němečková, Alžběta - Weisshart, K. - Hřibová, Eva - Schubert, V.
Comparing super-resolution microscopy techniques to analyze chromosomes.
International Journal of Molecular Sciences. Roč. 22, č. 4 (2021), č. článku 1903. E-ISSN 1422-0067
R&D Projects: GA MŠMT(CZ) EF16_019/0000827
Institutional support: RVO:61389030
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
Impact factor: 6.208, year: 2021
Method of publishing: Open access
http://doi.org/10.3390/ijms22041903

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.
Permanent Link: http://hdl.handle.net/11104/0322499