The Electron Microscope

The study of cells using the compound microscope in a little over 100 years contributed immensely in the development of modern science. The compound microscope uses the nature of light in such a way that it is not possible to get a clear image when the magnification of the object for study is greater than 2000 times. The limit of resolution of the compound light microscope, even with the best of lenses, is about one half the wavelength or 200 nm. or 2 um. of the visible light used, which has a minimum wave length of about 400 nm. Hence many minute submicroscopic structures within cells were not visualized until the electron microscope was, directed to cytologic study.

One of the most powerful research tools nowadays is the electron microscope. It differs from the compound microscope in such a way that instead of light, electrons pass through the specimen to be focused there, whereas in the electron microscope, electrons pass through the specimen to be focused on a viewing screen from which a photograph is made an electron micrograph. With the most modern electron microscopes the dimensions of the object under observation may be magnified 1 million times. If careful photographic techniques are used, a further enlargement can be accomplished, so that today a potential magnification of 2 million or more times exist. .With this magnification, a human hair viewed in its entirety in the electron microscope would appear twice the size of a California redwood.

Another electron microscope is the scanning electron microscope. It provides a three-dimensional quality to study of specimens at magnifications up to 100,000 times the actual size. It sweeps a very narrow beam of electrons back and forth across a specimen, revealing its surface features rather than its internal structure.

Ocular Micrometer

A unit even smaller than the micrometer must be used in designating the size of objects too small to be visualized in the light microscope. This unit is the nanometer. The sizes of microscopic and ultramicroscopic objects are compared. An ostrich egg and the mature human ovum are added to illustrate the magnitude of the size discrepancy.

The ocular micrometer is a device used in the measurement of microbes microscopically. It consists of a scale on a glass disk that fits between the lenses of the eyepiece. The spaces between the lines cm this scale represent arbitrary units. Actual values are obtained by calibrating the ocular micrometer against a stage micrometer, a glass slide with a true measurement scale on it; the lines on the scale are either exactly 10 or 200 µm. apart. By placing the stage micrometer in the position normally occupied by a smear of cells and by looking through the ocular of the microscope, one may superimpose the scale of the ocular on that of the stage micrometer. GIM can then determine the actual unit length that the distance between the lines of the ocular micrometer represents.

Fluorescent Microscope

A fluorescent substance, such as a fluorescent dye, or fluorochrome, is one that absorbs ultraviolet light, and then, after it has absorbed energy, emits visible light. Fluorescence microscopy is the instrument designed to visualize the microscopic preparation stained or in some way tagged with fluorescent material. To do this, the microscope must be specially equipped with a source of ultraviolet light, various filters, and a condenser.

For illumination, the commonly used source of ultraviolet light is either a high-pressure mercury lump contained within its own housing unit, or a halogen quartz lamp. Filters are an integral part of the system. Both light sources require an exciter filter to select the indicated wavelength for exciting the fluorescent material, and both require the right barrier filters to allow the longer fluorescent wavelength to pass while eliminating the shorter wavelengths. The condenser used is the dark-field condenser, which spreads the exciting light at such a great angle that negligible amounts enter the objective and only the fluorescent light off the specimen is viewed against a dark background.

The most intense fluorescent dyes for practical application are fluorescein with a yellow-green fluorescence and rhodamine with a reddish orange fluorescence. Of the two, fluorescein is more useful because the human eye is more sensitive to its yellow green color than to the reddish-orange color of rhodamine. Also, in nature, red autofluoresence is more prevalent than green autofluorescence. An example of the application of fluorescent microscopy to the visualization of bacteria is the fluorochrome staining of the tubercle bacillus known as Mycobacterium tuberculosis. When a specimen containing tubercle bacilli, such as sputum, is stained with the fluorescent dye, auramine, the tubercle bacilli readily take up the fluorochrome whereas most other bacteria present do not. When the specimen is observed in the fluorescent microscope, the tubercle bacilli are nicely identified as brightly fluorescent objects against the black background.