Specimen Preparation

Abstract

A key difference between confocal microscopy and widefield microscopy is that the aim of confocal is to explore the structure and structural relationships along the optical (Z) axis as well as in the X-Y plane. In other words, the investigation of spatial relationships is being evaluated in at least three dimensions. Often, this involves the collection of a series of planar images along the Z-axis and the reconstruction of the data into an image that depicts all three dimensions. However, even when the interest is solely in a single X-Y plane, the goal is to separate as thin a plane of information as possible from the planes directly above and below. Obviously, in order to acquire and analyze three-dimensional (3-D) structural information, the 3-D structural relationships must be preserved during the preparation of the sample. In contrast, in standard widefield microscopy, information along the Z-axis is merged into a single 2-D image. Because of the added need to preserve the 3-D structure for confocal images, methods suitable for preparing samples for widefield microscopy must often be altered to accommodate the additional demand of preserving relationships in the third dimension. Additionally, since in confocal the specimen is often thicker than for conventional widefield microscopy, the excitation and emitted light beams must traverse greater depths. In this chapter we review preparation methods that maximize preservation of specimen depth and minimize loss of signal during imaging.