title: Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe.
abstract: 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.
authors.affiliation: Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland.
authors.forename: Dominika
authors.initials: D
authors.lastname: Żurek-Biesiada
authors.affiliation: Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany.
authors.forename: Aleksander T
authors.initials: AT
authors.lastname: Szczurek
authors.affiliation: 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.
authors.forename: Kirti
authors.initials: K
authors.lastname: Prakash
authors.affiliation: Kirchhoff Institute for Physics, University of Heidelberg, Heidelberg, Germany.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Best
authors.affiliation: Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany.
authors.forename: Giriram K
authors.initials: GK
authors.lastname: Mohana
authors.affiliation: Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany; Department of Physics, University of Mainz (JGU), Staudingerweg 7, 55128 Mainz, Germany.
authors.forename: Hyun-Keun
authors.initials: HK
authors.lastname: Lee
authors.affiliation: Institute of Molecular Biology (IMB), Ackermannweg 4, 55128 Mainz, Germany.
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Roignant
authors.affiliation: Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland.
authors.forename: Jurek W
authors.initials: JW
authors.lastname: Dobrucki
authors.affiliation: 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.
authors.forename: Christoph
authors.initials: C
authors.lastname: Cremer
authors.affiliation: 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.
authors.forename: Udo
authors.initials: U
authors.lastname: Birk
keywords: Chromatin; DNA; DNA dye; Fluorescence; Localization microscopy; Nucleus; Single molecules; Super-Resolution; Vybrant violet; dSTORM