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.
Highlights
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
Figures Super-resolution localization microscopy data based on blinking of Vybrants DyeCycleTM Violet dye with an affinity to nuclear DNA; 2D data constitute an optical slice ( o500 nm thickness) through cell nuclei
Data Single molecule localization microscopy reconstructions of nuclear DNA stained with the fluorescent dye Vybrants DyeCycleTM Violet
Summary
In search for optimal imaging conditions for SMLM, potentially in combination with Structured Illumination Microscopy (SIM), a number of imaging buffers with various components were tested, including: PBS, glycerol, mercaptoethylamine (MEA), glucose oxidase in combination with catalase and glucose, ascorbic acid and Prolong Golds. All of these factors may influence the number of fluorescent cycles (i.e. photons emitted by a single molecule) during an ‘ON’-state. The most prominent and efficient blinking of the green-emitting form of VdcV was observed when an enzymatic oxygen scavenging system, dissolved in glycerol, was used (Fig. 1)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
