Blastomere Injection of Cleavage-Stage Zebrafish Embryos and Imaging of Labeled Cells

Abstract

INTRODUCTIONZebrafish embryos are useful for studying morphogenesis. Their foremost advantage is their optical transparency. Because the embryos are small, they can be imaged as whole-mount preparations. Crucially, this preserves both the mechanical and the molecular integrity of the embryo. This protocol describes the preparation of live zebrafish embryos for the collection of movies using four-dimensional time-lapse confocal microscopy. Methods are presented for the labeling of cells, for the orientation and immobilization of embryos, and for establishing a robust reproducible imaging environment with which to acquire high-resolution dynamic movies of cell behaviors during morphogenesis. These methods concentrate on the visualization of notochord morphogenesis in the zebrafish embryo. However, they also apply to the analysis of many other developing tissues. Factors that can influence the success of time-lapse confocal microscopy are discussed, such as the choice of cell label, the effects of genetic manipulation, and the consequences of environmental variation during imaging—all of which may influence the rate of development.