Because tissues are generally soft and fragile, even after fixation, they need to be either frozen or embedded in a supporting medium before being sectioned. The usual embedding media are waxes or resins. In liquid form, these media both permeate and surround the fixed tissue; they can then be hardened (by cooling or by polymerization) to form a solid block, which is readily sectioned with a microtome. This is a machine with a sharp blade, usually of steel or glass, which operates like a meatslicer (Figure 9–9). The sections (typically 0.5–10 μm thick) are then laid flat on the surface of a glass microscope slide.
There is little in the contents of most cells (which are 70% water by weight) to impede the passage of light rays. Thus, most cells in their natural state, even if fixed and sectioned, are almost invisible in an ordinary light microscope. We have seen that cellular components can be made visible by techniques such as phasecontrast and differentialinterferencecontrast microscopy, but these methods tell us almost nothing about the underlying chemistry. There are three main approaches to working with thin tissue sections that reveal differences in types of molecules that are present.
First, and traditionally, sections can be stained with organic dyes that have some specific affinity for particular subcellular components. The dye hematoxylin, for example, has an affinity for negatively charged molecules and therefore reveals the distribution of DNA, RNA, and acidic proteins in a cell (Figure 9–10). The chemical basis for the specificity of many dyes, however, is not known.
movement of microtome arm ribbon of ribbon of sections on glass slide, stained and mounted under a glass cover slip
Figure 9–9 Making tissue sections.
this illustration shows how an embedded tissue is sectioned with a microtome in preparation for examination in the light microscope.
Second, sectioned tissues can be used to visualize specific patterns of differential gene expression. In situ hybridization, discussed earlier (see Figure 8–34), reveals the cellular distribution and abundance of specific expressed RNA molecules in sectioned material or in whole mounts of small organisms or organs. This is particularly effective when used in conjunction with fluorescent probes (Figure 9–11).
A third and very sensitive approach, generally and widely applicable for localizing proteins of interest, also depends on using fluorescent probes and markers, as we explain next.
Fluorescent molecules absorb light at one wavelength and emit it at another, longer wavelength (Figure 9–12A). If we illuminate such a molecule at its absorbing wavelength and then view it through a filter that allows only light of the emitted wavelength to pass, it will glow against a dark background. Because the background is dark, even a minute amount of the glowing fluorescent dye can be detected. In contrast, the same number of molecules of a nonfluorescent stain, viewed conventionally, would be practically indiscernible because the absorption of light by molecules in the stain would result in only the faintest tinge of color in the light transmitted through that part of the specimen.
The fluorescent dyes used for staining cells are visualized with a fluorescence microscope. This microscope is similar to an ordinary light microscope except that the illuminating light, from a very powerful source, is passed through two sets of filters—one to filter the light before it reaches the specimen and one to
Figure 9–10 Staining of cell components.