Chapter 6 Nuclear Ultrastructure: Transmission Electron Microscopy and Image Analysis

Abstract

Publisher Summary This chapter provides an overview to both standard and advanced methods for the sample preparation, staining, and structural analysis of nuclear architecture using transmission electron microscopy (TEM). It describes standard TEM techniques or specific three-dimensional reconstruction methodologies. The applications of light microscopy are severalfold. A very powerful application, especially in situations in which in vivo observations are feasible, is the use of light microscopy as a check on sample preparation conditions. In principle, requiring that the fixation and sample preparation conditions used for a TEM project at least preserve the appearance of the sample as observed at lower resolution by light microscopy is a natural and obvious quality check. In practice, this type of control is rarely performed, and many studies of nuclear and chromosome structure have used buffer conditions that produce striking alterations in ultrastructure, obvious even at light microscopy resolutions. The availability of a variety of fluorescent stains specific for various cell organelles and macromolecules, and the recent introduction of the green fluorescent protein (GFP) as a fluorescent epitope tag for the in vivo imaging of specific proteins, facilitates this approach.