UV-induced spectral shift and protonation of DNA fluorescent dye Hoechst 33258

DNA-bound Hoechst 33258 is readily excited with UV light and emits blue fluorescence, however, upon exposure to UV, the dye undergoes photobleaching as well as photoconversion to a blue-excited green-emitting form. We demonstrate that the UV-generated green-emitting form of Hoechst 33258 exhibits spectral properties very similar to the form of the dye that can be obtained by subjecting it to an acidic environment (pH 0.5–3.0). We also demonstrate that exposure of Hoechst 33258 to UV light (or hydrogen peroxide) leads to generation of the protonated (1+, 2+, 3+ and possibly the 4+) forms of the dye. Photoconversion of Hoechst 33258 has recently been exploited in single molecule localisation microscopy, thus understanding photophysics of this process can facilitate further development of high resolution optical imaging

Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes

Several approaches have been described to fluorescently label and image DNA and chromatin in situ on the single-molecule level. These superresolution microscopy techniques are based on detecting optically isolated, fluorescently tagged anti-histone antibodies, fluorescently labeled DNA precursor analogs, or fluorescent dyes bound to DNA. Presently they suffer from various drawbacks such as low labeling efficiency or interference with DNA structure. In this report, we demonstrate that DNA minor groove binding dyes, such as Hoechst 33258, Hoechst 33342, and DAPI, can be effectively employed in single molecule localization microscopy (SMLM) with high optical and structural resolution. Upon illumination with low intensity 405 nm light, a small subpopulation of these molecules stochastically undergoes photoconversion from the original blue-emitting form to a green-emitting form. Using a 491 nm laser excitation, fluorescence of these green-emitting, optically isolated molecules was registered until “bleached”. This procedure facilitated substantially the optical isolation and localization of large numbers of individual dye molecules bound to DNA in situ, in nuclei of fixed mammalian cells, or in mitotic chromosomes, and enabled the reconstruction of high-quality DNA density maps. We anticipate that this approach will provide new insights into DNA replication, DNA repair, gene transcription, and other nuclear processes

Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe

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