Chapter 10 Applications of Confocal Microscopy to Studies of Sea Urchin Embryogenesis

Abstract

The advent of confocal microscopy and three-dimensional reconstruction methods has further enhanced the capabilities of researchers in various fields of developmental biology. The chapter discusses the methodology required for microscopic analyses and applications of confocal microscopy. Protocols are described for using confocal microscopy and three-dimensional visualizations to investigate nuclear divisions in embryos that were fixed during cleavage. The chapter also describes time-lapse analyses of calcium dynamics, four-dimensional studies of embryogenesis, and supravital staining of nuclei as visualized by confocal microscopy employing the two-photon excitation technique. The methods for investigating the relationship between cell lineage and patterns of gene expression are discussed. Although the methods described in this chapter directly pertain to sea urchin embryos, many of the techniques can be adapted for use on other cellular and developmental systems. The greatest limitations of confocal microscopy at present are relatively slow acquisition rates and a limited choice of organelle- or molecule-specific probes that can be applied in conjunction with the most widely used types of laser illumination systems.