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.