What is microscopy?

Indications and Procedures

Conventional microscopy uses a beam of light to illuminate a thin slice of material to be viewed. The material may be stained to provide contrast among its components. The visible light is aimed through the material and collected in a lens. Additional lenses magnify the image until it is visible to the eye of the viewer.

Electron microscopy is a procedure in which a beam of electrons, rather than visible light, is projected toward an object to be viewed. The object is prepared by coating it with a fine layer of metal, frequently gold, that is one or two atoms thick. The electrons reflect off the coated object and hit a screen. The image on the screen is magnified and becomes visible to the human eye. An alternative procedure involves a very thin section of material that is dried and put into a vacuum chamber. A beam of electrons is directed through the prepared specimen. A coated screen receives the electron beam and transforms the image into one visible to the human eye.

Fluorescence microscopy is a procedure that is based on the fact that fluorescent materials emit visible light when they are irradiated with ultraviolet light, which is outside the spectrum visible to the human eye. Some materials manifest this property naturally; others may have to be treated with fluorescent solutions in a process similar to staining. When the absorption of the specimen is in the relatively long ultraviolet range, two filters are also used. The first is placed over the light source to eliminate all but the desired long ultraviolet rays. The second is placed over the eyepiece. The result is a field that becomes dark and allows any red or yellow fluorescence to be visible.

Uses and Complications

Microscopy has extended the range of understanding for physical objects. Intracellular organelles are routinely made visible. Microscopy has made possible the science of microbiology. Fluorescence allows immunoglobulins to be routinely assayed. Viruses are too small to be seen with a light microscope, but electron microscopy has enabled virologists to view and classify viruses.

The drawbacks of current microscopy techniques are primarily that only small samples of material can be viewed at a time. Usually, the material must be destroyed while it is being prepared. Living tissue may be viewed, but only at levels of magnification below those possible at the limits of normal microscopy and far below those possible with electron microscopy.

Electron microscopy allows the greatest amount of magnification for objects. Because the wavelength of electrons is thousands of times shorter than that of visible light, the resulting magnification is several thousand times greater than that possible with visible light. The use of subatomic particles theoretically increases the potential of magnification, but a continuous source of subatomic particles is difficult and quite expensive to supply.

Bibliography

Black, Jacquelyn G., and Laura J. Black. Microbiology. Hoboken, N.J.: Wiley, 2013.

Hawkes, Peter W., and John C. H. Spence, eds. Science of Microscopy. New York: Springer, 2007.

Madigan, Michael T., and John M. Martinko. Brock Biology of Microorganisms. 13th ed. San Francisco: Pearson/Benjamin Cummings, 2012.

Murray, Patrick R., Ken S. Rosenthal, and Michael A. Pfaller. Medical Microbiology. 6th ed. Philadelphia: Mosby/Elsevier, 2009.

Singleton, Paul, and Diana Sainsbury. Dictionary of Microbiology and Molecular Biology. Rev. 3d ed. Hoboken, N.J.: John Wiley & Sons, 2007.

Spector, David L., and Robert D. Goldman, eds. Basic Methods in Microscopy: Protocols and Concepts from Cells—A Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2006.