Samples for Microscopy Must Be Fixed Sectioned and Stained to Image Subcellular Details

Live cells and tissues lack compounds that absorb light and are thus nearly invisible in a light microscope. Although such specimens can be visualized by special techniques to be discussed shortly, these methods do not reveal the fine details of structure and require cells to be housed in special glass-faced chambers, called culture chambers, that can be mounted on a microscope stage. For these reasons, cells are often fixed, sectioned, and stained to reveal subcellular structures.

Specimens for light and electron microscopy are commonly fixed with a solution containing chemicals that crosslink 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

Specimen holder

Specimen block

Specimen holder

Specimen block

Sections

Microscope slide

Copper mesh grid

Copper mesh grid

▲ EXPERIMENTAL FIGURE 5-43 Tissues for microscopy are commonly fixed, embedded in a solid medium, and cut into thin sections. 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 for light microscopy or in liquid plastic for electron microscopy; after the insoluble and stable for subsequent procedures. After fixation, a sample is usually embedded in paraffin or plastic and cut into sections 0.5-50 ^m thick (Figure 5-43). Alternatively, the sample can be frozen without prior fixation and then sectioned; such treatment preserves the activity of enzymes for later detection by cytochemical reagents.

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, 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 aspartate and glutamate). Because of their different binding properties, these dyes stain various cell types sufficiently differently that they are distinguishable visually. If an enzyme catalyzes a reaction that produces a colored or otherwise visible precipitate from a colorless precursor, the enzyme may 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 or structures as discussed next.

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