HIGH-RESOLUTION IMAGING AT THE CELLULAR AND SUBCELLULAR REVELS IN FLATTENED WHOLE MOUNTS OF EARLY ZEBRAFISH EMBRYOS

We describe a rapid and sensitive method for high-resolution imaging a t the cellular and subcellular levels in the whole-mount zebrafish emb ryo. The procedure involves fixing and staining the embryo, followed b y deyolking and flattening it under a cover slip, to produce a planar mount that is 20 to 100 mu m thick. Such a flattened whole mount allow s imaging with a spatial resolution of similar to 500 nm in the x-y pl ane and does not require the use of embedding, sectioning, confocal mi croscopy, or computational deblurring procedures. We can resolve all i ndividual nuclei and chromosome sets in the embryo, up to the late gas trula stage (10 000 cell stage). In addition, older embryos (through t he segmentation stage) can also be examined, with the preservation of significant morphological detail. Because of its ability to resolve su bcellular detail, the flattened whole-mount method can provide signifi cant biological information beyond what can be obtained from conventio nal (three-dimensional) whole mounts. We have used the flattened whole -mount method to study subcellular events related to progression throu gh the cell cycle or to apoptosis, in cells of the early zebrafish emb ryo. A specific DNA-binding dye (Hoechst 33258) or an antibody against a chromosomal protein (histone H1) was used to stain the nuclei of in dividual cells in the embryo. This allowed us to determine the spatial positions of all the individual cells, and also their stages in the c ell cycle. A terminal transferase (TUNEL) assay was used to detect apo ptotic cells. This combination of specific stains allowed us to study the behaviors of groups of cells in situ, within the developing zebraf ish embryo.