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One of the most attractive features of light microscopy is the ease with which most specimens can be prepared for examination. Preparation often involves nothing more than mounting a small piece of the specimen in a suitable liquid on a glass slide and covering it with a glass coverslip.
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The slide is then positioned on the specimen stage of the microscope and examined through the ocular lens, or with a camera. Magnification can be changed simply by rotating a turret that holds different objective lenses.
Objective lenses capable of high magnification (40 x and greater) generally require the application of a small drop of immersion oil to the coverslip for optimal viewing. Specimens that are pathogenic or potentially pathogenic are often killed chemically prior to viewing by the application of a small amount of fixative (such as an aqueous buffered aldehyde solution) to the specimen suspension.
Technique # 1. Specimen Processing – Fixation:
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Often the first step in preparing the specimen is primary fixation, generally in a buffered aldehyde fixative. Fixation kills the cells, stabilizes their chemical components, and hardens the specimen in anticipation of further processing and sectioning. One way to fix a specimen is simply to immerse it in the fixative solution.
An alternative approach for animal tissues is to pass the fixative through the blood stream of the animal before removing the organs. This technique, called perfusion, may help reduce artifacts, false or inaccurate representations of the specimen that result from chemical treatment or handling of the cells or tissues.
Technique # 2. Embedding and Sectioning:
In most cases, the next step is to prepare thin sections of the fixed specimen. For this purpose, the specimen is embedded in a medium that will hold it rigidly in position while sections are cut. The usual choice of embedding medium is paraffin wax. Since paraffin is insoluble in water, any water in the specimen must first be removed (by dehydration in alcohol, usually) and replaced by an organic solvent such as xylene, in which paraffin is soluble.
The processed tissue is then placed in warm, liquefied paraffin and allowed to harden. Dehydration is less critical if the specimen is embedded in a water-soluble medium instead of in paraffin. Specimens may also be embedded in epoxy plastic resin.
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Next, the embedded specimen is sliced into thin sections, usually a few (1-10) micrometers thick. Sections are cut with a microtome, an instrument that operates somewhat like a meat slicer.
The paraffin or plastic block containing the specimen is mounted on the arm of the microtome, which advances the block by small increments toward a metal or glass blade. As successive sections are cut, they usually adhere to one another forming a ribbon of thin sections. These sections can be mounted on a glass slide, stained (if desired), and protected with a coverslip.
Technique # 3. Staining:
The purpose of staining is to give distinctive colour characteristics to different kinds of cellular components. The general approach is to mount the fixed tissue on a microscope slide and then treat it with any of a variety of dyes and stains that have been adapted for this purpose. Sometimes the tissue is treated with a single stain, but more often a series of stains is used, each with an affinity for a different kind of cellular component.
Technique # 4. Autoradiography:
Autoradiography is a technique that uses photographic film to determine where within a cell a specific radioactively labeled compound is at the time the cell is fixed and sectioned for microscopy. Autoradiography can therefore be used to localize cellular processes to specific structures and to provide information about cell function.
In essence, the process involves incubating cells, organisms, or tissue with a radioactively labeled compound, fixing the specimen, sectioning it in the conventional way, and mounting the fixed sample on a microscope slide.
The slide is then covered with a thin layer of photographic emulsion and placed in a sealed box for the desired length of time, often for several days or even weeks. During this time, the radioactivity in the sample exposes the emulsion directly above it, providing a photographic record of precisely where in the cell the radioactive compound is located.
Thereafter, the slide containing the specimen and the emulsion is developed in much the same way as conventional black-and-white film. It is then ready for examination under the microscope. Autoradiography can be applied to both light microscopy and electron microscopy. In either case, it provides valuable information about the localization of specific molecules, structures, or processes within the cell.
