Chapter 11 Resolution of Subcellular Detail in Thick Tissue Sections: Immunohistochemical Preparation and Fluorescence Confocal Microscopy

Abstract

This chapter focuses on fluorescence confocal microscopy techniques for resolution of subcellular detail. Gentle procedures for specimen preparation and techniques for removing the out-of-focus fluorescence signal are equally important for maximizing resolution. The chapter illustrates the substitution of optical sectioning with the laser scanning confocal microscope (LSCM) for mechanical sectioning to generate the thin sections necessary for high contrast and resolution. LCSM allows the shallow depth of field of the light microscope to be used effectively. There are two other benefits of confocal microscopy: (1) resin or paraffin embedding is no longer necessary and consequently structural alteration and autofluorescence caused by these procedures is eliminated, and (2) three-dimensional information can be gathered with ease. The combination of immunofluorescence and LSCM makes the goal of viewing cytoskeletal arrays and other subcellular detail in three dimensions in intact tissues not only theoretically possible for the cell biologist, but also feasible.