1 The role of scanning electron microscopy in cell and molecular biology: SEM basics, past accomplishments, and new frontiers

New developments in scanning electron microscopy (SEM) have resulted in a wealth of new applications for cell and molecular biology as well as related biological disciplines. New instrument developments coupled with new sample preparation techniques have been key factors in the increasing popularity of this versatile research tool. The desire to view biological material in native states at high resolution has stimulated new approaches to cryo-SEM applications and environmental SEM (ESEM). In addition, new staining techniques and novel processing applications that combine embedding and resin-extraction for imaging with high-resolution SEM has allowed new insights into structure–function relationships. Advances in immuno-labeling have further enabled the identification of specific molecules and their location within the cellular microenvironment. It is now possible to analyze macromolecular complexes within their three-dimensional cellular environment in near native states, and this in many cases has provided advantages over twodimensional imaging with transmission electron microscopy (TEM). New instrument developments include helium ion microscopy that allows imaging greater details of cellular components; new technology approaches include automated block-face imaging combined with serial sectioning inside an SEM chamber that in recent years has increasingly been utilized for a variety of biological applications. Focused ion beam milling (FIB) combined with block-face SEM is among the newer approaches to analyze cellular structure in three dimensions. The present chapter introduces the basic features of SEM and sample processing for SEM, and highlights several advances in cell and molecular biology that have greatly benefited from using conventional, cryo-, immuno-, and high-resolution SEM.