Imaging Subcellular Details Often Requires That Specimens Be Fixed, Sectioned, and Stained

As we have seen, live cells and tissues generally do not absorb light, so they are nearly invisible in a light microscope. Although cells can be visualized by the special techniques we have just discussed, these methods do not reveal the fine details of structure.

Specimens for light microscopy are commonly fixed with a solution containing chemicals that cross-link most proteins and nucleic acids. Formaldehyde, a common fixative, cross-links amino groups on adjacent molecules; these covalent bonds stabilize protein-protein and protein–nucleic acid interactions and render the molecules insoluble and stable for subsequent procedures. After fixation, a tissue sample for examination by light microscopy is usually embedded in paraffin and cut into sections about 50 µm thick (Figure 4-11a). Cultured cells growing on glass coverslips, as described above, are thin enough so they can be fixed in situ and visualized by light microscopy without the need for sectioning.

image
FIGURE 4-11 Tissues for light microscopy are commonly fixed, embedded in a solid medium, and cut into thin sections. (a) A fixed tissue is dehydrated by soaking in a series of alcohol-water solutions, ending with an organic solvent compatible with the embedding medium. To embed the tissue for sectioning, the tissue is placed in liquid paraffin. After the block containing the specimen has hardened, it is mounted on the arm of a microtome, and slices are cut with a knife. Typical sections cut for light microscopy are 0.5 to 50 µm thick. The sections are collected on microscope slides and stained with an appropriate agent. (b) Sections of normal (top) and cancerous (adenocarcinoma, bottom) human colon stained with H&E stain. Notice the disorganization of the cells in the cancer tissue.
[Part (b) courtesy of Dr. Alexander Nikitin, Cornell University.]

A final step in preparing a specimen for light microscopy is to stain it so as to visualize the main structural features of the cell or tissue. Many chemical stains bind to molecules that have specific features. For example, histological samples are often stained with hematoxylin and eosin (“H&E stain”). Hematoxylin binds to basic amino acids (lysine and arginine) on many different kinds of proteins, whereas eosin binds to acidic molecules (such as DNA and side chains of the amino acids aspartate and glutamate). Because of their different binding properties, these dyes stain various cell types sufficiently differently that they are distinguishable visually (Figure 4-11b). If an enzyme catalyzes a reaction that produces a colored or otherwise visible precipitate from a colorless precursor, that enzyme can be detected in cell sections by their colored reaction products. Such staining techniques, although once quite common, have been largely replaced by other techniques for visualizing particular proteins, as we discuss next.

143