Super-resolution imaging of densely packed DNA in nuclei of zebrafish embryos using stimulated emission double depletion microscopy

Abstract

Knowledge about the spatial distribution of DNA in the cell nucleus is an essential aspect of understanding how cells control gene expression. Here, we describe a protocol for the preparation of samples for super-resolution stimulated emission depletion (STED) fluorescence imaging of bulk DNA in nuclei of blastomeres within animal caps of zebrafish embryos. We evaluated different mounting media and DNA stains. With samples stained with the silicon-rhodamine dye JF646 conjugated to Hoechst 33342 as a DNA tag and mounted in glycerol, we compared confocal, STED and stimulated emission double depletion (STEDD), in particular with respect to background fluorescence and image resolution. We show that super-resolved images of lateral planes through the cell nuclei can be collected up to imaging depths of several tens of micrometers with greatly reduced background using STEDD. Acquisition of axial image stacks using both lateral and axial STED confinement was also feasible, though at reduced resolution due to photobleaching. Further optimization steps are discussed to obtain a robust experimental platform for imaging the three-dimensional distribution of DNA inside zebrafish embryo tissue.

The domain-within-domain packing of euchromatin can be described as multiplicative cascades

“…When we tested the established stain SiR-Hoechst (commercialized as SiR-DNA) in fixed embryos, we found a strong influence of the mounting medium (Fig. 1A,B), as expected from our previous results (17). Vectashield almost completely suppressed fluorescence, glycerol allowed for clearly detectable fluorescence, thiodiethanol (TDE) resulted in almost 10-fold higher fluorescence intensity than glycerol.…”

“…At the pluripotent stage of development (late blastula), these cells exhibit a euchromatinonly nuclear organization and are distributed throughout the different phases of the cell cycle (16,23,24). Both aspects have proven beneficial for the study of large-scale euchromatin organization by STED super-resolution microscopy in our previous work (16,17). To further improve image quality, we assessed the performance of STED-capable fluorogenic DNA stains that recently became commercially available.…”

“…Collection and staining of zebrafish embryos. Late blastula zebrafish embryos were collected and stained as described previously (17). Briefly, embryos of wild type zebrafish (AB, sourced from the Zebrafish International Resource Center and maintained at the European Zebrafish Resource Center) were obtained by spontaneous mating and dechorionated by pronase treatment.…”

RNA polymerase II clusters form in line with liquid phase wetting of chromatin

“…To systematically characterize cluster morphologies and their dependence on Pol II pausing and elongation, we assessed clusters by super-resolution microscopy in fixed embryos. Specifically, we applied STimulated Emission Double Depletion (STEDD) microscopy, which significantly reduces background from low frequency contributions and out-of-focus light relative to conventional STED mi-croscopy 28–30 . Here, we super-resolved the Pol II Ser5P distribution and acquired the level of Pol II Ser2P in a second color channel in the same focal plane by regular confocal microscopy.…”

Wavelet-based background and noise subtraction for fluorescence microscopy images

“…Apart from out-of-focus and scattered light, STED images always include an additional low-frequency background component that originates from incomplete depletion as well as reexcitation by the powerful depletion beam. We recently introduced stimulated emission double depletion (STEDD) as a hardware-based technique that effectively removes this component [ 14 ], which facilitates super-resolution fluorescence correlation spectroscopy experiments on solution samples, and also greatly helps remove background in 3D imaging of densely labeled structures [ 21 ]. The significant resolution enhancement between confocal and STED/STEDD modes and the effective background suppression of STEDD are obvious from visual inspection of the images taken on our 40-nm bead samples ( Fig.…”