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Exp Cell Res. 2016 May 1;343(2):97-106. doi: 10.1016/j.yexcr.2015.08.020. Epub 2015 Sep 1.

Localization microscopy of DNA in situ using Vybrant(®) DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution.

Author information

1
Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland.
2
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany.
3
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany; Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.
4
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany; Department of Physics, University of Mainz (JGU), Staudingerweg 7, 55128 Mainz, Germany.
5
Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland. Electronic address: jerzy.dobrucki@uj.edu.pl.
6
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany; Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany; Department of Physics, University of Mainz (JGU), Staudingerweg 7, 55128 Mainz, Germany. Electronic address: c.cremer@imb-mainz.de.

Abstract

Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant(®) DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of DNA in nuclei of fixed mammalian cells. This SMLM method enabled optical isolation and localization of large numbers of DNA-bound molecules, usually in excess of 10(6) signals in one cell nucleus. The technique yielded high-quality images of nuclear DNA density, revealing subdiffraction chromatin structures of the size in the order of 100nm; the interchromatin compartment was visualized at unprecedented optical resolution. The approach offers several advantages over previously described high resolution DNA imaging methods, including high specificity, an ability to record images using a single wavelength excitation, and a higher density of single molecule signals than reported in previous SMLM studies. The method is compatible with DNA/multicolor SMLM imaging which employs simple staining methods suited also for conventional optical microscopy.

KEYWORDS:

Chromatin structure; DNA dyes; Photoconversion; SMLM; Vybrant DyeCycle Violet; dSTORM

PMID:
26341267
DOI:
10.1016/j.yexcr.2015.08.020
[Indexed for MEDLINE]

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