Correlative Light and Electron Microscopy of Early Caenorhabditis elegans Embryos in Mitosis

Abstract

The early embryo of Caenorhabditis elegans is one of the most powerful model systems to study cell division. This chapter develops a correlative light and electron microscopic approach to stage early C. elegans embryos prior to high-pressure freezing and electron tomography. Cellulose microcapillaries are used to contain the embryos in short transparent tubes. These permit the viewing of early mitotic events under a light microscope. Next step is to stage selected embryos by light microscopy (LM), to immobilize these staged embryos by high-pressure freezing, and to fix them by freeze-substitution. The chapter describes the thin-layer embedding and 3D reconstruction of spindle components by electron tomography. Using the newly developed rapid transfer system (RTS) of the Leica EMPACT2 high-pressure freezer reduces the time between light microscopic observation and cryoimmobilization to about 5 sec. The correlative approach allows systematic structure-function studies in staged early wild-type embryos and those treated with RNA-mediated interference (RNAi) to reduce the level of specific gene products.