Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe
Dominika Żurek-Biesiada,
Aleksander T. Szczurek,
Kirti Prakash,
Gerrit Best,
Giriram K. Mohana,
Hyun-Keun Lee,
Jean-Yves Roignant,
Jurek W. Dobrucki,
Christoph Cremer,
Udo Birk
Affiliations
Dominika Żurek-Biesiada
Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland
Aleksander T. Szczurek
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany
Kirti Prakash
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
Gerrit Best
Kirchhoff Institute for Physics, University of Heidelberg, Heidelberg, Germany
Giriram K. Mohana
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany
Hyun-Keun Lee
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany; Department of Physics, University of Mainz (JGU), Staudingerweg 7, 55128 Mainz, Germany
Jean-Yves Roignant
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany
Jurek W. Dobrucki
Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland; Corresponding author. Tel.: +48 12 664 63 82.
Christoph Cremer
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; Kirchhoff Institute for Physics, University of Heidelberg, Heidelberg, Germany; Department of Physics, University of Mainz (JGU), Staudingerweg 7, 55128 Mainz, Germany; Corresponding author at: Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany. Tel.: +49 6131 39 21518.
Udo Birk
Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany; Kirchhoff Institute for Physics, University of Heidelberg, Heidelberg, Germany; Department of Physics, University of Mainz (JGU), Staudingerweg 7, 55128 Mainz, Germany
Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density, and demonstrate direct proof of enhanced structural resolution. Furthermore, we compare different visualization approaches. Finally, we describe various opportunities of multicolor DNA/SMLM imaging in eukaryotic cell nuclei. Keywords: Super-Resolution, DNA, dSTORM, Localization microscopy, Fluorescence, Chromatin, Vybrant violet, DNA dye, Single molecules, Nucleus
